BMB & Pharm Research Conference 2012 ___________________________________________________________________

RETREAT MISSION STATEMENT

The Verna and Marrs McLean Department of Biochemistry and Molecular Biology

and The Department of Pharmacology

We take pride in the enormous breadth of biomedical research carried out within our two departments. This broad scope provides unique opportunities for cross-field education and intra- and inter-departmental collaboration. It is in the spirit of this cooperative venture that we hold the annual retreat, the goals of which are threefold: 1. To inform the departments as a whole about the state of research in the specific fields represented

within the departments. 2. To provide a congenial atmosphere where department members can present their research in a

manner conducive to collaboration. 3. To allow scientists at all levels the opportunity to hone their professional skills in an informal setting.

1

BMB-PHARM RESEARCH CONFERENCE

OCTOBER 10 & 11, 2013

SCHEDULE OF EVENTS

Thursday, October 10 8:00 am – 8:45 am Continental Breakfast Salon A & B

8:00 am – 8:45 am Registration East and West Mainsail Lobby

8:50 am Opening Remarks East and West Mainsail

9:00 am – 10:00 am SESSION 1 East and West Mainsail

10:00 am – 10:15 am Departmental Photo

10:15 am – 10:30 am Break

10:30 am – 11:00 am Poster Ranking Salon C & D

11:00 am – 12:00 pm SESSION 2 East and West Mainsail

12:00 pm – 1:00 pm Lunch Salon A & B

1:00 pm – 2:00 pm SESSION 3 East and West Mainsail

2:10 pm – 3:00 pm Life of a Scientist East and West Mainsail

3:00 pm – 4:00 pm POSTER SESSION 1 (Odd) Salon C & D

4:00 pm – 5:30 pm Free time

5:30 pm – 7:00 pm Cocktails & Costumes Poolside

7:00 pm – 8:30 pm Dinner Salon A & B

8:30 pm – 11:00 pm Dancing Salon C & D

Friday, October 11 8:30 am – 9:30 am Breakfast Buffet Salon A & B

9:30 am – 10:30 am SESSION 4 East and West Mainsail

10:30 am – 10:45 am Break

10:45 am – 11:45 am POSTER SESSION 2 (Even) Salon C & D

11:45 am – 12:45 pm Lunch Salon A & B

12:45 pm – 1:45 pm SESSION 5 East and West Mainsail

1:45 pm – 2:00 pm Break

2:00 pm – 3:00 pm SESSION 6 East and West Mainsail

3:00 pm Awards and Announcements

2

BMB-PHARM RESEARCH CONFERENCE

OCTOBER 10 & 11, 2013 THURSDAY, OCTOBER 10

8:00 am – 8:45 am CONTINENTAL BREAKFAST Salon A & B

8:00 am – 8:45 am Registration East & West Mainsail Lobby

8:50 am Opening Remarks: John Wilson East & West Mainsail

SESSION 1 Session Moderator: Justin Cordill [GS] (Kuspa Lab)

9:00 am – 9:15 am Lynn Zechiedrich [F] Multidisciplinary Approaches Converge to

Reveal the Active Structures of DNA

9:20 am – 9:35 am Carolyn Adamski [GS] (Palzkill Lab) BLIP-II: A Useful Model for

Investigating Specificity of Protein Interactions

9:40 am – 9:55 am Shigenori Hirose [PD] (Kuspa Lab) Allorecognition-based

Cooperation is Required for Morphogenesis in Social Amoeba

10:00 am – 10:15 am Departmental Photo

10:15 am – 10:30 am Break

10:30 am – 11:00 am Poster Ranking Salon C & D

SESSION 2 Session Moderator: Michele Darrow [GS] (Chiu Lab)

11:00 am – 11:15 am Yufeng Gou [GS] (Wang Lab) Identification of a Novel PRC2/TrxG

Recruiter in Mammalian Cells

11:20 am – 11:35 am Suman Maity [PD] (Sreekumar Lab) Pathway-Centric Integration

of OMICS Datasets in Breast Cancer

11:40 am – 11:55 am Lin Tian [GS] (Xiang Lab) The Role of Adaptive Immune System

in the Escape of Breast Cancer Cells from Primary Tumors

3

12:00 pm – 1:00 pm Lunch Salon A & B

SESSION 3 Session Moderator: Leonardo Sepulveda [GS] (Golding Lab)

1:00 pm – 1:15 pm Liya Hu [PD] (Prasad Lab) Structural Basis for Diverse Glycan

Recognitions by Rotavirus Capsid Protein VP8

1:20 pm – 1:35 pm Lauren Figard [GS] (Sokac Lab) Plasma Membrane Unfolding

Fuels Surface Growth During Cell Shape Change

1:40 pm – 1:55 pm Jesus Galaz-Montoya [GS] (Ludtke Lab) Automation of Single

Particle Cryo-electron Tomography to Study the TRiC

Chaperonin's Inhibition of Mutant Huntingtin Aggregates

2:10 pm – 3:00 pm Life of a Scientist East & West Mainsail

3:00 pm – 4:00 pm POSTER SESSION 1 (Odd) Salon C & D

4:00 pm – 5:30 pm Free time

5:30 pm – 7:00 pm Cocktails & Costumes Poolside

7:00 pm – 8:30 pm Dinner Salon A & B

8:30 pm – 11:00 pm Dancing Salon C & D

FRIDAY, OCTOBER 11

8:30 am – 9:30 am Breakfast Buffet Salon A & B

SESSION 4 Session Moderator: Liuliu Zheng [GS] (Sokac Lab)

9:30 am – 9:45 am Bo Chen [GS] (Chiu Lab) Binding to Human γD-crystallin

Causes Partial Closure of Cis-ring and Symmetry-broken

Features of Type II Chaperonin

9:50 am – 10:05 am Fengyun Ni [PD] (Ma Lab) Structural Basis of Membrane

Fusion Induced by Influenza B Virus Hemagglutinin

4

10:10 am – 10:25 am Hye Jin Kang [GS] (Wensel Lab) Selectivity and Evolutionary

Divergence of Metabotropic Glutamate Receptors for Endogenous

Ligands

10:30 am – 10:45 am Break

10:45 am – 11:45 am POSTER SESSION 2 (Even) Salon C & D

11:45 am – 12:45 pm Lunch Salon A & B

SESSION 5 Session Moderator: Michael Evangelista [GS] (Zechiedrich Lab)

12:45 pm – 1:00 pm Qian Shen [GS] (Z Zhou Lab) Clathrin-actin Crosstalk Plays a

Novel Role in Phagocytosis

1:05 pm – 1:20 pm Ilya Novikov [GS] (Lichtarge Lab) Functionally Important

Nucleotides in Non-Coding RNAs Evolve In Highly Compact

Clusters: Sequence-Structure Analysis of RNA-Based Machines

1:25 pm – 1:40 pm Jiaming Sun [GS] (Barth Lab) Computational De Novo Design

of Transmembrane Peptides Inhibitors Targeting Oncogenic

Receptor Associations

1:45 pm – 2:00 pm Break

SESSION 6 Session Moderator: Zheng Zhou [F]

2:00 pm – 2:15 pm Koen Venken [F] Genetic Manipulation, Genome Engineering,

and Human Disease Modeling in Drosophila melanogaster

2:20 pm – 2:35 pm Jin Wang [F] Biodegradable Polypeptides for In Vivo Non-Viral

Gene Delivery

2:40 pm – 2:55 pm Ming Zhou [F] Mechanism of Gating in the TrkH K+ Channels

3:00 pm AWARDS CEREMONY: Ted Wensel

5

THE JOSHI

MEMORIAL PRIZE Dr. Vadusev C. Joshi was born in Gujarat, India. He

received his B.S. degree in pharmacy from Madras

Medical College in 1959, ranking among the top 5%

of students in the state. After earning his M.S.

degree in pharmacy from Andra Pradesh University in

1961, Dr. Joshi began his Ph.D. studies in the

Department of Biochemistry at the Indian Institute of

Science in Bangalore. Under the tutelage of Dr.

T. Ramasarma, he studied ubiquinone and related

compounds, earning his doctorate in biochemistry in

1964. While a postdoctoral fellow at the Indian

Institute of Science, Dr. Joshi was awarded a Ford

Foundation Research Fellowship. In 1966, he was

awarded the prestigious Fulbright Fellowship, which

allowed him to come to the United States to further his education at Duke University in the

Departments of Pediatrics and Biochemistry. There, he continued his studies of ubiquinone

and eventually became interested in fatty acid synthesis. In 1972, Dr. Joshi accepted a

position as an assistant professor in the Department of Biochemistry at Baylor College of

Medicine under the leadership of Dr. Salih J. Wakil. While at BCM, he continued his

investigations of fatty acid synthesis. He was promoted to associate professor in 1977 and

was honored with a prestigious Research Career Development Award from the National

Institutes of Health the same year. During his tenure at BCM, Dr. Joshi published numerous

papers and was an active member of the Admissions Committee. In 1980, Dr. Joshi spent a

year in the laboratory of Dr. Hargovind Khorana in the Department of Chemistry at the

Massachusetts Institute of Technology. After returning to BCM in late 1981, he continued his

research in fatty acid synthesis and gene regulation until his untimely death in 1982. Dr.

Joshi not only was a committed and hardworking scientist; he was an active member of the

Indian community and a strong patron of the arts. Most importantly, Dr. Joshi was a devoted

husband to his wife Yogini and a doting father to his three daughters, Harshini, Yamini, and

Meena.

As a tribute to Dr. Joshi for his dedication to excellence in education and research, his family,

friends, and colleagues established a memorial fund in his honor in 1986. The Joshi

Memorial Prize is granted to students and postdoctoral trainees within the Department of

Biochemistry in recognition of their outstanding oral and poster presentations at the annual

research conference.

6

JUDGING CRITERIA Oral Presentations

I PRESENTATION (30%)

Organization of talk, clarity of presentation

Quality of slides, legibility & clarity of labeling, color & contrast

Engaging, entertaining, style

Time management

II RESEARCH (40%)

Significance for biomedical or basic science

Quality of data, difficulty of experiments

Interpretations justified, conclusions sound

Innovative approach or techniques

Clear acknowledgment of work by others

III INTERACTION (30%)

Did the talk stimulate questions

Ability to respond to questions

Display knowledge outside prepared presentation

7

JUDGING CRITERIA Poster Presentations

I POSTER (30%)

Organization of poster, clear flow, aesthetics

Abstract, hypothesis or question, conclusions, acknowledgments

Legible figures, clearly & concisely labeled

II RESEARCH (40%)

Significance for biomedical or basic science

Quality of data, difficulty of experiments

Interpretations justified, conclusions sound

Innovate approach or techniques

How much work is done by the presenter

III INTERACTION (30%)

Was the poster presented clearly

Ability to respond to questions

Knowledge outside prepared presentation

8

POSTER RANKING CRITERIA

I POSTER (40%)

Organization of poster, clear flow, aesthetics

Abstract, hypothesis or questions, conclusions, acknowledgments

Legible figures, clearly & concisely labeled

II RESEARCH (60%)

Significance for biomedical or basic science

Quality of data, difficulty of experiments

Interpretations justified, conclusions sound

Innovative approach or techniques

How much work is done by the presenter

9

10

Table of Contents

Name Type Page

Carolyn Adamski Talk 11

Melina A Agosto Poster #1 12

Kannan Alpadi Poster #2 13

Ivan A. Anastassov Poster #3 14

Justin Anglin Poster #4 15

Salil Kumar Bhowmik Poster #5 16

William G. Bornmann Poster #6 17

William G. Bornmann Poster #7 18

William G. Bornmann Poster #8 19

Alex Brewer III Poster #9 20

James Campbell Poster #10 21

Berenice Carrillo Poster #11 22

Jianwei Chen Poster #12 23

Kuang-Yui Chen Poster #13 24

Bo Chen Talk 25

Jinxuan Cheng Poster #14 26

Zachary C. Conley Poster #15 27

W. Justin Cordill Poster #16 28

Michele Darrow Poster #17 29

Michael Evangelista Poster #18 30

Xiang Feng Poster #19 31

Lauren Figard Talk 32

Jesus Galaz-Montoya Talk 33

Monica Galaz-Montoya Poster #20 34

Yufeng Gou Talk 35

Feng He Poster #21 36

Shigenori Hirose Talk 37

Corey Hryc Poster #22 38

Teng-Kuei Hsu Poster #23 39

Liya Hu Talk 40

Gilbert Huang Poster #24 41

Hye Jin Kang Talk 42

Akash Kaushik Poster #25 43

Giedre Krenciute Poster #26 44

Name Type Page

Aditya Kulkarni Poster #27 45

Zao Li Poster #28 46

Suman Maity Talk 47

Jennifer McGehee Poster #29 48

Shrenik Mehta Poster #30 49

Phillip J. Minnick Poster #31 50

Jessica Moore Poster #32 51

Zana Muhaxhiri Poster #33 52

Fengyun Ni Talk 53

Ilya Novikov Talk 54

Nagireddy Putluri Poster #34 55

Sam Regenbogen Poster #35 56

Soung-Hun Roh Poster #36 57

Ramon Roman-Sanchez Poster #37 58

Susmita Samanta Poster #38 59

Beatriz A. Santillan Poster #39 60

Sreejesh Shanker Poster #40 61

Qian Shen Talk 62

Lynn Zechiedrich Talk 63

Jiaming Sun Talk 64

Xianzhou Song Poster #41 65

Vlatko Stojanoski Poster #42 66

Yun-Min Sung Poster #43 67

Lin Tian Talk 68

Koen Venken Talk 69

Maria Viskovska Poster #44 70

Jin Wang Talk 71

Sara Wright Poster #45 72

Xiaowei Xu Poster #46 73

Zenghui Xue Poster #47 74

Zhixian Zhang Poster #48 75

Yan Zhang Poster #49 76

Liuliu Zheng Poster #50 77

11

CHARACTERIZATION OF A NOVEL INTERACTION BETWEEN BLIP-II AND

STAPHYLOCOCCUS AUREUS PBP2A

Carolyn Adamski, Dar-Chone Chow, Nicholas G. Brown, Timothy Palzkill

The prediction and manipulation of protein-protein interactions remains a difficult task.

Model systems such as the β-lactamase inhibitory protein II (BLIP-II) and β-lactamases

have been used to investigate the principles of protein-protein interactions. Previous

studies focused on the determinants of binding affinity and specificity between BLIP-II

and class A β-lactamases. However, interactions between BLIP-II and other proteins have

yet to be explored. In this study, we characterized the novel interaction between BLIP-II

and penicillin binding protein 2a (PBP2a) from methicillin resistant Staphylococcus

aureus (MRSA). Mutagenesis and surface plasmon resonance experiments were used to

determine how specificity is achieved between BLIP-II and its binding partners. The

results suggest that an outer ring of residues on the BLIP-II interface plays a critical role

in binding PBP2a while an inner ring of residues on the binding surface is primarily

responsible for the binding of β-lactamases. Interestingly, changes in BLIP-II binding

affinity for PBP2a were due to changes in both on and off rates while changes in binding

affinity for β-lactamases were primarily mediated by changes in off-rates. In summary,

the results of the study indicate BLIP-II binds PBP2a in addition to β-lactamases and

provide insights into how BLIP-II binds a wide range of target proteins.

12

CHARACTERIZATION OF TRANSIENT RECEPTOR POTENTIAL CHANNEL TRPM1 PURIFIED

FROM INSECT CELLS

Melina A. Agosto, Zhixian Zhang, Feng He, Theodore G. Wensel

TRPM1, a 180 kDa protein which likely forms a multimeric channel complex, is required

for the depolarizing light response in rod and cone ON bipolar cells. Little is known

about the structure of TRPM1 or any of its close relatives. Blue-native PAGE and size

exclusion chromatography of purified protein both indicate an apparent molecular weight

(MW) of ~550 kDa. Since both of these techniques typically over-estimate the MW of

membrane proteins, this may be consistent with either a dimer or trimer of TRPM1. After

addition of an amine-directed cross-linking reagent, SDS-PAGE indicates that the major

cross-linked species are dimers, though a small amount of higher-order species were also

detected. Single particle reconstruction revealed that the major population of particles are

of a size consistent with a dimer. Furthermore, reconstruction with two-fold symmetry

imposed bears resemblance to a model made without symmetry constraints. The three-

dimensional structure is characterized by a small putative transmembrane domain and a

larger domain with a hollow cavity. A dimer is unlikely to have channel activity, raising

the possibility that in the retina, TRPM1 may assemble as hetero-oligomers with as-yet

unidentified subunits.

To study native TRPM1, eleven new monoclonal antibodies were generated against full-

length TRPM1. All of the clones detect a band consistent with the MW of TRPM1 by

western blot of retina extract, and label the outer plexiform layer and the distal part of the

inner nuclear layer, consistent with the expected TRPM1 localization in bipolar cells.

Blue native PAGE of retina extract followed by TRPM1 western blot revealed two

distinct bands, both migrating more slowly than purified recombinant TRPM1. These

results suggest that in the retina, TRPM1 forms higher-order oligomers and/or complexes

with other proteins. The new monoclonals will aid in future studies aimed at identifying

novel TRPM1 interacting proteins by immunoprecipitation and mass spectrometry.

13

IN VITRO AUTOPHAGOCYTOSIS ASSAY: A BIOCHEMICAL ASSAY TO INVESTIGATE THE

AUTOPHAGIC PROCESS IN YEAST

Kannan Alpadi, Thirupathaiah Sirupangi, Aditya Kulkarni, Christopher Peters

Organelle degradation via lysosomes or vacuoles is known as autophagy. It is an

evolutionarily conserved catabolic process that involves the sequestration and transport of

organelles and macromolecules to the lysosomes or vacuole for degradation and

recycling. Autophagy is now known to be involved in multitude of cellular processes

including tumerigenesis, immunity, development and aging. Two different forms of

autophagy have been described in yeast depending on the mechanism of delivery of

cargoes or organelles to the vacuole: macroautophagy and microautophagy. Initially it

was believed that the autophagic process occurs as a non-selective process but later, the

concept of preferential degradation of organelles or macromolecules evolved, known as

selective autophagy such as, mitophagy (selective degradation of mitochondria) and

pexophagy (selective degradation of peroxisomes). Yeast genetics has been vital for the

elucidation of the molecular machinery involved in autophagy processes. Understanding

the molecular mechanism of selective organelle degradation and recycling requires in-

depth biochemical analysis. Although many core components of the autophagocytic

machinery have been identified, little is known about their control and interplay. This is

mainly due to the fact that most of the discoveries in the field have been obtained using in

vivo systems. To date, no in vitro system is available allowing detailed analysis of the

autophagic uptake mechanism. Quite similar to the in vitro vacuole fusion system, which

has proven to be very effective over the last 10 years in regard to the understanding of the

membrane fusion process, an in vitro autophagocytosis assay would allow us to

investigate the molecular mechanism of the autophagic uptake in more detail.

14

DEVELOPMENT OF AN ASSAY FOR LOCALIZATION AND CHANNEL ACTIVITY OF TRANSIENT

RECEPTOR POTENTIAL CHANNEL TRPM1 IN CELL CULTURE AND ISOLATED RETINAL

BIPOLAR CELLS

Ivan A. Anastassov, Melina A. Agosto, Theodore G. Wensel

Recently the transient receptor potential channel TRPM1 has been identified as the ion

channel responsible for the sign-inverting response of ON-bipolar cells in the vertebrate

mammalian retina. TRPM1 is believed to be a constitutively open, mostly nonselective

cation-permeable channel, which is kept closed in darkness by a G-protein signaling

cascade coupled to the glutamate receptor mGluR6. The light response requires TRPM1

and capsaicin was found to activate the channel, while capsazepine inhibited activity.

Capsaicin and capsazepine also activate and inhibit the activity of TRPV1, respectively.

However, still little is known about what elements are required for proper TRPM1

function.

HEK-293 cells were transfected with full length murine TRPM1 cDNA plasmid fused at

the C-terminus to a 1D4 epitope tag. Expression was confirmed by Western blot and

immunocytochemistry with a 1D4 antibody. Almost complete lack of co-localization

with the membrane marker wheat germ agglutinin was evident. Calcium flux assays were

performed on cells transiently transfected with TRPM1 and TRPV1. Changes in

intracellular Ca2+

in response to various combinations of capsaicin, capsazepine,

resiniferatoxin, and ruthenium red were interpreted as changes in channel activity. The

agonist/antagonist couples affected channel activity of TRPV1 transfected cells, but not

TRPM1 transfected cells.

Since native bipolar cells from the mouse retina should contain all the necessary

components for proper TRPM1 localization and function, the development of a reliable

primary culture from these cells could be considered a viable alternative. To that end,

bipolar cells were enzymatically dissociated and kept in culture. Bipolar neurons were

identified based on morphology, which was confirmed later by immunocytochemistry

with monoclonal TRPM1 antibodies and bipolar cell markers.

15

INHIBITORS OF THE H3K79 METHYLTRANSFERASE DOT1L KILL MLL-REARRANGED

LEUKEMIC CELLS

Justin Anglin, Lisheng Deng, Yuan Yao, Zheng Liu, Jiang Hong, Gang Cheng, Pinhong

Chen, Yongcheng Song, Shuo Dong

MLL-translocated leukemias comprise 70% of infant leukemias and 10% of adult

leukemias. This leukemia has less than a 40% survival rate and it is therefore imperative

that new drugs are needed. DOT1L, a histone3-lysine79 methyltransferase, plays a

crucial role in the initiation and maintenance of MLL-translocated leukemia and

knocking down DOT1L activity results in the loss of transforming ability of MLL-

oncogenes. Therefore, DOT1L represents a target for intervention.

Our lab has developed novel small molecules with the goal of competitively inhibiting

DOT1L and inhibiting the growth of MLL-translocated leukemic cell lines. We show

that, by retaining the adenosine moiety of SAM but substituting the 5’ position, we can

obtain inhibitors with inhibition constants as low as 0.5 nM. A urea moiety in the 5’

substituent is critical for high binding activity. These compounds are specific for DOT1L

compared to three other SAM-dependent histone methyltransferases. Also, isothermal

titration calorimetry studies show that two representative inhibitors bind to the DOT1L:

nucleosome complex and only compete with SAM but not the nucleosome substrate. In

addition, potent inhibitors of DOT1L showed selective activity against the proliferation

of the MLL-translocated cell lines MV4; 11 and THP1 with EC50 values of 4-11 μM.

One selected inhibitor is shown to downregulate expression of the leukemia-relevant

genes Hoxa9 and Meis1, induce differentiation, and reduce the population of leukemic

stem cells.

Supported by a training fellowship from the Keck Center for Interdisciplinary Bioscience

Training of the Gulf Coast Consortia (NIGMS Grant No. 1 T32 GM089657-02).

16

METABOLIC PROFILING OF CANCER STEM CELLS USING LIQUID CHROMATOGRAPHY/MASS

SPECTROMETRY: PATHWAYS AND BIOMARKERS

Salil Kumar Bhowmik, Esmeralda Ramirez-Peña, Arnold James Michael, Nagireddy

Putluri, Akash Kumar Kaushik, Sendurai A. Mani, Arun Sreekumar

Breast cancer is the most common cancer in women worldwide. The development of new

technologies for better understanding of the metabolic changes involved in breast cancer

progression is essential. Epithelial to mesenchymal transition (EMT) is considered an

essential process in the metastatic cascade. Relatively little is known regarding common

metabolic pathways altered in induced EMT by different transcription factors. By

integrating metabolomics and transcriptomics data, we hope to better nominate the

pathways which are changing during EMT and identify potential targets for the control of

metastasis. Breast Cancer Stem Cells (CSC) generated by the ectopic expression of Snail,

Gooscoid, TWIST and TGFB in immortalized human mammary epithelial cells (HMLEs)

results in the possession of mesenchymal traits and an increased ability to form

mammospheres, a property associated with mammary epithelial stem cells. First, non-

vector control (HMLE) and cancer stem cell (HMLE-Snail) have been used for the

metabolomic studies. Metabolomics studies were carried out using Liquid

chromatography Mass Spectrometry coupled to reverse and aqueous normal phase

separation of compounds. Gene expression analysis was done using the ‘Affy’ package

from Bioconductor and GSEA from the Broad Institute. Differences in metabolism and

gene expression between control and induced cancer stem cells have been identified.

Different metabolic pathways including glycolysis, TCA, methionine, polyamine

biosynthesis, pentose phosphate and carnitine shuttle have been found to be altered in

cancer stem cells. Gene expression data identified changes to mitochondrial oxidative

respiration and ATP synthesis. Therefore, ongoing studies should help to answer

important questions relating to the use of metabolomics and CSC evaluation as new

strategies to monitor the mechanism and to identify markers for cancer progression and

toxicity.

17

SAR STUDY OF NEW RHAZINILAM ANALOGUES

William G. Bornmann, Zhenghong Peng, Duoli Sun

(-)-Rhazinilam has been reported to have a unique activity of profile. This antimitotic

compound induces spiralization of tublin such as that observed for Vinblastine and also

inhibits the cold-induced disassembly of microtubules as that described for Taxol. In the

present study, novel racemic rhazinilam derivatives by modification of the D-ring size

and the substituents on the juncture of D and B rings have been prepared from aldehydes

and indoloazepine. X-ray structure study of three different D-ring size rhazinilam

analogues is reported. Structure-activity relationship studies suggest that changing the

ring size of the D-ring or moving the ethyl group is not desirable, but that the ethyl group

can be replaced with other substituents.

18

OPTIMIZED SYNTHESIS OF Ψ- RHAZINILAM

William G. Bornmann, Zhenghong Peng, Duoli Sun

Rhazinilam is an antimitotic agent mimicking the action of both Vinblastine (by inducing

spiralization of tubulin) and Taxol (by inhibiting the cold-induced disassembly of

microtubules). Rhazinilam has been isolated from Melodius australis, Rhazya stricta and

Kopsia singapurensis. Rhazinilam biogenetically derived from natural Aspidosperma

indole alkaloid Vincadifformine. This prompted us to consider the possibility that Ψ–

Rhazinilam could potentially be a naturally occurring compound derived from Ψ–

Vincadifformine which is a known alkaloid. Here we report the synthesis of four possible

stereoisomers of a new Ψ-Rhazinilam analogs.

19

NEW AND IMPROVED TOTAL SYNTHESIS OF 15,20-DIHYDROSECODINE,

TETRAHYDROPRESECAMINES AND TETRAHYDROSECAMINES.

William G. Bornmann, Zhenghong Peng, Duoli Sun

The indole alkaloids tetrahydropresecamine and tetrahydrosecamine were first isolated in

1968 by G. F. Smith from the leaves of Rhazya orientalis and Rhazya stricta. G. A.

Cordell and G. F. Smith were able to show in a brilliant series of experiments that the

tetrahydropresecamine alkaloids were in fact dimers of 15,20-dihydrosecodine.

Subsequent simple acid catalysis gave the corresponding tetrahydrosecamine alkaloids.

While the basic skeletal structure was tentatively established, the absolute

stereochemistry of these alkaloids has remained elusive. In 1981 G. A. Cordell reported

anticancer activity for tetrahydrosecamine in P-388 (ED50 0.4 mg/ml) and KB carcinoma

of the nasopharynx (ED50 0.69 mg/ml). The combination of cytotoxic properties,

scarcity, difficulty in isolation and lack of stereochemical assignment, made these

alkaloids attractive synthetic targets. Here we report the total asymmetric synthesis of

15,20-Dihydrosecodine, Tetrahydropresecamine and Tetrahydrosecamine which has been

achieved in 13 steps with an overall yield 15.33%. It also has been clearly established

from total asymmetric synthesis that the absolute stereochemistry of natural 15,20-

Dihydrosecodine is S.

20

GENETIC VARIANTS OF THE DOPAMINE TRANSPORTER (DAT1) MEDIATE THE ACUTE

SUBJECTIVE RESPONSES TO COCAINE IN COCAINE-DEPENDENT VOLUNTEERS

Alex Brewer III, David A. Nielsen, Catherine J. Spellicy, Sara C. Hamon, Justin

Gingrich, James J. Mahoney III, Thomas R. Kosten, Thomas F. Newton, Richard De La

Garza

AIMS: The dopamine transporter (DAT) has been implicated in the subjective and

reinforcing effects produced by cocaine, and polymorphisms within the DAT gene

(DAT1, or SLC6A3) have also been linked to variations in the response to cocaine. The

aim of the present study was to identify candidate gene variants of DAT1 that modulate

subjective responses to cocaine in cocaine-dependent volunteers.

METHODS: Non-treatment seeking, cocaine-dependent volunteers received a single

bolus infusion of saline and cocaine (40 mg, IV) in randomized order. Subjective effects

questionnaires (visual analogue scales: VAS) were administered before (-15 min) and up

to 20 min after infusion. VAS scales ranged from zero (no effect) to 100 (greatest effect).

Subjective effects ratings were normalized to baseline by subtracting saline infusion

values. Data was analyzed using repeated measures ANOVA. DNA from subjects was

genotyped for the DAT1 intron 8 and 3′ UTR VNTRs.

RESULTS: Participants (N=47) had a mean age of 43.5 ± 6.6 (mean ± standard

deviation) years, were mostly black (78%) and mostly males (87%), and had a mean of

12.8 ± 1.8 years of education. The primary route of cocaine use was smoking (94%), and

the participants used a mean of 2.1 ± 2.3 grams of cocaine per day, and 16.8 ± 7.7 days

out of the last 30. Carriers of the 9-allele of the DAT1 3’ UTR (9/9 and 9/10) exhibited

significantly greater responses to cocaine for “High” (p = .0002), “Any Drug Effect” (p =

.00005), and “Stimulated” (p = .0004). In addition, individuals with the 6/6 genotype for

the DAT1 intron 8 polymorphism exhibited significantly greater responses to cocaine for

“Desire” (p = .0005).

CONCLUSIONS: The data presented here support the hypothesis that individual genetic

differences of DAT1 contribute to variation of individual responses to cocaine among

dependent participants.

21

NOVEL CYSTIC FIBROSIS THERAPEUTICS THROUGH ACTIVATORS OF CYCLIC GMP

DEPENDENT PROTEIN KINASE II

James Campbell, Choel Kim

Cyclic GMP (cGMP) is a second messenger that is known to relay extracellular signals to

a variety of downstream targets. cGMP dependent protein kinase (PKG), one of the main

receptors for cGMP, is a central mediator of the Nitric Oxide-cGMP signaling cascade,

which regulates smooth muscle and vascular tone, platelet aggregation, nociception and

electrolyte/water secretion. There are 3 isoforms of PKG, the cytosolic PKG Iα and β and

the membrane bound, PKG II. Structurally PKGs have two domains, a regulatory (R)-

domain at the N-terminus, and catalytic (C)-domain at the C-terminus. The R-domain

contains tandem cGMP binding domains (CNB-A and B) each with different affinities for

cGMP. PKG II is expressed in intestine and kidney tissues where it plays a role in the

regulation of water and electrolyte flux. While PKGs represent an important drug target

for treating hypertensive diseases, erectile dysfunction and cystic fibrosis (CF),

developing specific activators and inhibitors has been difficult due to little structural

information. A cGMP pathway activator Sildenafil (Viagra) is currently in phase 2 trials

to treat CF, however due to nonspecific interactions it has many side effects. Structure

based design to generate selective PKG II activators would be a great benefit in the

treatment of CF. The goal of this proposal is to elucidate the mechanism of cGMP

induced activation of PKG II and identify candidate compounds that will isoform

specifically target PKG II in order to develop therapeutic agents to treat CF. To do this

we will be characterizing and targeting the cyclic nucleotide binding (CNB) domains of

PKG II. Our first aim will be to determine which CNB domain provides cGMP

selectivity in PKG II. Secondly, we will biophysically characterize CNB:cGMP

interactions, and determine their role in C-domain activation PKG. Finally, we will carry

out high-throughput drug screening.

22

X-RAY STRUCTURES OF INFLUENZA FULL-LENGTH NS1 SHOW DETERMINANTS OF

STRUCTURAL POLYMORPHISMS AMONG NS1S

Berenice Carrillo, Jae-Mun Choi, Andre P. Rice, B.V.V. Prasad

Influenza A virus is the causative agent of seasonal flu epidemics, and currently the

highly lethal H5N1 avian strains pose a threat for a worldwide pandemic. Increased

lethality observed in the H5N1 strains is partly attributed to the NS1 protein. NS1

contains a RNA binding domain (RBD), a linker region (LR) and an effector domain

(ED). Variability in the size of the LR has been observed between NS1s of H5N1 and

non-pathogenic strains. NS1 performs multiple functions, which include inhibiting

various interferon-induced pathways, many are strain specific, and the structural basis for

the highly multifunctional nature of NS1 is poorly understood. The only full-length (FL)

structure of NS1 to date is that of the VN/04 H5N1 strain which was found to be involved

in inter-molecular interactions leading to the formation of tubular structures with a

possible role in dsRNA sequestration. This structure, like most recent isolates of H5N1

influenza NS1, contains a five amino acid deletion (aa80-84) within the LR; this deletion

is not found in non-H5N1 NS1s. To examine whether the length of the LR affects the

relative orientations of the ED and RBD and whether similar inter-molecular interactions

are formed by other strains, we determined the structure of the FL H6N6 NS1 as a

representative example of NS1s with a longer LR as well as the structure of H6N6 NS1

containing a H5N1 LR deletion. Our crystal structures reveal that the length of the LR as

well as a naturally occurring E71G mutation in some H5N1 NS1 strains, affect the

relative orientation of the ED with respect to the RBD. These results highlight the LR as

a significant component of NS1, allowing NS1 to adopt several conformations important

to the functional diversity of NS1 and virulence of influenza virus.

23

TUNABLE THIOESTERS AS “REDUCTION” RESPONSIVE FUNCTIONALITY FOR TRACELESS

REVERSIBLE PROTEIN PEGYLATION

Jianwei Chen, Mingkun Zhao, Fude Feng, Antons Sizovs, Jin Wang

Disulfde has been the only widely used functionality to serve as a reduction responsive

trigger in drug delivery. We introduce thioester as a novel thiol responsive chemistry for

drug delivery, whose reactivity can be conveniently modulated by choosing the

appropriate steric environment around the thioester. Compared with disulfides, thioesters

are facile to synthesize and have an order of magnitude broader kinetic tunability. A

novel traceless reversible protein PEGylation reagent is developed based on thioester

chemistry

24

REPROGRAMMING THE CONFORMATIONAL REGULATION OF G PROTEIN-COUPLED

RECEPTOR SIGNALING

Kuang-Yui Chen, Patrick Barth

G protein-coupled receptors (GPCRs) are one of the largest families of membrane-

embedded receptors that transduce extracellular stimuli into cytoplasmic responses.

Increasing evidence indicate that these receptors signal through long-range

conformational changes. However, the atomic-level mechanisms by which such allosteric

transitions are propagated remain poorly understood, thereby hindering the development

of more effective therapeutics targeting GPCRs. We have developed an integrated

homology modeling/multistate design/experimental approach to reprogram the

conformational regulation of the signaling properties of structurally uncharacterized

GPCRs by redesigning conformational switches in transmembrane and loop regions. The

method was applied to switch the function of structurally uncharacterized dopamine D2

receptor (DRD2) towards the active state. Inactive and active states of DRD2 were

modeled by conformational ensembles generated using the homology modeling mode of

RosettaMembrane. Multistate design of the DRD2 transmembrane region resulted in

receptor variants exhibiting up to an eight-fold increase in basal activity compared to

wild type receptor. Starting from homology models of a DRD2/Gi active state complex,

we redesigned the 140-residue-long native intracellular loop 3 (ICL3) into a five-residue

loop in an active state conformation. Predicted conformational shifts toward the active

state and increased active state stability of the designed DRD2 variants were confirmed

by fluorescence spectroscopy and functional assays in vitro. Our successful redesign of

residue interactions switching the receptor’s conformation and function indicate that

conformational switches play a key role in regulating the signaling properties of GPCRs.

This method should prove useful for designing variants of membrane receptors stabilized

in specific conformations and predicting the effect of mutations on receptor function.

This work was partially supported by a grant from the NIH (1R01GM097207-01A1) to

P.B. and by a training fellowship to K.M.C. from the Keck Center of the Gulf Coast

Consortia, NIGMS (T32GM008280).

25

CRYO-EM STUDY OF TYPE II CHAPERONIN ASSISTED FOLDING OF HUMAN GAMMAD-

CRYSTALLIN

Bo Chen, Oksana Sergeeva, Daniel Goulet, Kelly Knee, Joanita Jakana, Wah Chiu,

Jonathan King

Chaperonin is a class of protein that plays an essential role in protein folding for all cells

from bacteria, archaea and eukaryotic cells. Recently it is reported that a type II

chaperonin from archaea Methanococcus Marapaludis (Mm-Cpn) could assist refolding

of human γD-crystallin to its native conformation. Human γD-crystallin is a lens protein

which is associated with the onset of cataract when it partially unfolds. We are interested

in understanding the structural mechanism of how type II chaperonin recognizes the γD-

crystallin.

Here, cryo-electron microscopy (cryo-EM) single particle analysis method was applied to

resolve the structure of Mm-Cpn and human γD-crystallin during their initial recognition

step. We used a multi-model refinement protocol to sort out particle images according to

their structural uniformity. Our analysis showed three conformations: 33% of the

particles (Subset II) resembled the apo state Mm-Cpn conformation; 39% of particles

(Subset I) had one-ring less open. The control cryo-EM map from the Mn-Cpn showed

both ring open which was similar to the map from the subset II of the Mm-Cpn and

human γD-crystallin complex.

Based on the results above, we conclude that the subset I corresponds to the Mm-Cpn

population with substrate binding. 3D variance analysis also validated that the variance of

subset II was mostly caused by flexible N- and C-terminal tails, while subset I showed

higher variance level inside the top ring. Subsequent symmetry-free reconstructions of

subset I particle images converged to one-ring less open and one-ring open conformation.

Furthermore, differential conformations of each of the 8 subunits in the less open cis-

rings were observed to form a tetramer of dimers while both rings in subset II appeared to

have a good 8-fold symmetry.

In conclusion, our results demonstrate the conformational changes and symmetry broken

features of type II chaperonin upon binding to γD-crystallin.

26

THE ROLE OF THE RAC GTPASE REGULATORY PROTEINS TIAM1, TIAM2 AND BCR IN

ADULT NEUROGENESIS

Jinxuan Cheng, Sanyong Niu, Karen Firozi, Kimberley R. Tolias

Adult Neurogenesis is the process of generating functional neurons from adult neural

precursor cells throughout life. It is thought to happen in two discrete brain regions, the

dentate gyrus of the hippocampus and the subventricular zone of the lateral ventricle.

Defects in adult neurogenesis correlate with impairments in learning, memory and mood

regulation, and are often seen in neurological disorders such as Alzheimer’s disease,

bipolar disease and schizophrenia. Thus, adult neurogenesis has been considered as a

potential therapeutic target for the treatment of mental disorders. Our lab is interested in

studying the factors that regulate adult neural precursor cells proliferation and

differentiation. Emerging evidence indicates that the Wnt pathway regulates both adult

neurogenesis and neuronal development. Our lab has also demonstrated that the Rac

guanine nucleotide exchange factor (GEF) Tiam1 and the Rac GTPase-activating protein

(GAP) Bcr cooperate to control dendrite and excitatory synapse development in the

hippocampus. Interestingly, these Rac GTPase regulatory proteins remain highly

expressed in the adult dentate gyrus and appear to function in the Wnt pathway.

Preliminary data indicates that Tiam1 and closely related Tiam2 regulate dendrite and

spine development in hippocampal dentate granule cells and loss of Tiam1 and/or Tiam2

decrease adult neurogenesis. Our results suggest that these Rac regulatory proteins play

important roles in adult neurogenesis. Ongoing studies are focused on determining how

Tiam1, Tiam2 and Bcr regulate adult neurogenesis and newborn neuron development in

the dentate gyrus in response to Wnt signaling.

27

THE ANTIFUNGAL DRUG CICLOPIROX AFFECTS SIDEROPHORE PRODUCTION IN GRAM-

NEGATIVE BACTERIA SUGGESTING AN ADDITIONAL MECHANISM FOR ITS ANTIMICROBIAL

EFFECT

Zachary C. Conley, Kimberly M. Carlson-Banning, Andrew Chou, Yongcheng Song,

Richard J. Hamill, Lynn Zechiedrich

New antibiotics are needed because antibiotic resistance has rendered most existing

antibiotics ineffective. We discovered that the antifungal drug, ciclopirox, is effective

against problematic multidrug-resistant clinical isolates. Even though ciclopirox has been

used clinically for over thirty years, how ciclopirox works is poorly understood. Using E.

coli, we showed that both sugar metabolism and the availability of free iron in the growth

medium affect ciclopirox activity. Additionally, iron- acquiring siderophore production in

P. aeruginosa was altered in the presence of ciclopirox, suggesting a connection to iron

utilization. From these fungi and bacteria data, general iron chelation is a proposed

mechanism of action for ciclopirox. To examine which iron utilization pathways might be

affected by ciclopirox, we used single-gene deletion strains to screen for enhanced

sensitivity to ciclopirox. Strains with deletions of any gene encoding a function in the

biosynthesis (entB, entC, entE, or entF) or uptake (tonB, exbB, exbD, fepA, fepB, fepC,

fepD, fepG) of the siderophore enterobactin had at least 2-fold lower ciclopirox minimum

inhibitory concentrations (MICs) compared to the isogenic parent strain. We are currently

testing whether this increased susceptibility is specific to ciclopirox by measuring MIC

changes of these mutant strains in response to general iron chelators. Although different

siderophores are used in P. aeruginosa, our previous P. aeruginosa data combined with

these enterobactin data suggest that siderophore production may be altered when gram-

negative bacteria are exposed to ciclopirox. Together, these data show that ciclopirox is

not a general iron chelator, but specifically affects siderophore pathways. These data are

an important step toward developing ciclopirox or new derivatives of ciclopirox against

multidrug-resistant infections by gram-negative pathogens for which few therapeutic

options exist currently.

28

TGRM1 IS A DEVELOPMENTALLY REGULATED, GPI-ANCHORED CELL ADHESION PROTEIN

THAT FUNCTIONS IN THE POST-AGGREGATIVE STAGE OF DICTYOSTELIUM DISCOIDEUM

DEVELOPMENT

W. Justin Cordill, Gad Shaulsky, Adam Kuspa

Dictyostelium discoideum is an amoeboid eukaryote that leaves its unicellular vegetative

state upon starvation to undergo aggregative development into a mature multicellular

fruiting body. Its simplicity and genetically tractable properties make it an excellent

model of multicellular development. An important component of all multicellular

organisms is the use of a cell adhesion system for organizing and anchoring cells and

tissues. Dictyostelium cell adhesion has been studied during the aggregation stage of

development: the cadA gene product is a calcium-dependent cell adhesion protein that

functions during streaming and aggregation, and gp80 and tgrC1/tgrB1 cell-adhesion

proteins function in an EDTA-resistant manner during streaming and the mound stage of

development. However, cell adhesion has not been studied after this stage when the

expression of these genes falls dramatically, though development continues with apparent

increasing complexity. We now have data that suggests that a paralog of tgrC1 and tgrB1,

tgrM1, may function as a cell adhesion protein during late development. A strain which

overexpresses tgrM1 shows development-independent aggregation in shaking culture,

suggesting that tgrM1 is a hemophilic adhesion molecule. tgrM1 mRNA levels rise as

gp80 mRNA levels fall after the streaming and stage of development. Like gp80, tgrM1

is predicted to be GPI-anchored, and may exist in lipid microdomains such as the triton-

insoluble floating fraction. tgrM1 knockout cells exhibit an EDTA-resistant cell adhesion

defect during mid- to late development and produce thinner slugs which frequently break

apart. When tgrM1-null cells are developed under a layer of soil, fewer spores are

produced at the surface, resulting in a fitness disadvantage compared to wildtype cells.

Additionally, a tgrM1 ortholog exists in another Dictyostelid, D. purpureum, and has a

similar transcriptional trajectory through development, suggesting that the role of this

gene product has been conserved for 400 million years. Together these data suggest that

tgrM1 is an important component of the cell adhesion machinery. Currently we are

producing a tagged version of tgrM1 to follow its gene product spatially and temporally

during development.

29

VISUALIZING SICKLE RED BLOOD CELLS

Michele Darrow, Bertrand Cinquin, Yujin Zhang, Rosanne Boudreau, Mark LeGros,

Yang Xia, Carolyn Larabell, Wah Chiu

Sickle cell disease is an autosomal recessive genetic disease caused by a single point

mutation in the β-globin subunit of hemoglobin. This point mutation changes the

hydrophilic glutamate to a hydrophobic valine, resulting in non-covalent polymerization

of the full hemoglobin protein under low-oxygen conditions. Formation and elongation of

these fibril bundles leads to red blood cell distortion, with repeated sickling episodes

causing damage to the cell membrane, decreasing the cell’s elasticity and its ability to

return to a normal biconcave disc shape when normoxia conditions are restored.

Current techniques for imaging red blood cells provide limited and potentially misleading

information because they are restricted to two-dimensional data collection. Our goal is to

obtain a three-dimensional structure of sickle red blood cells and the hemoglobin

aggregates inside in order to gain a better understanding of the sickling process. To do

this we use soft X-ray cryo-tomography in conjunction with scanning electron

microscopy of plastic-embedded red blood cells.

Upon visualization of sickled red blood cells, it became clear that the route from a

biconcave disc to a fully sickled cell involves multiple stages. We have identified and

begun characterizing four distinct morphological stages in the severity of a sickled cell.

Additionally, we have created a processing protocol to identify and isolate densities that

we believe correspond to hemoglobin internal to the cell. Lastly, we have tested four

inhibitors of sphingosine-kinase-1 as potential drugs for treatment of sickle cell disease.

30

THE NAD-DEPENDENT LIGASE, LIGB, FUNCTIONS IN THE BASE EXCISION REPAIR

PATHWAY IN E. COLI

Michael Evangelista, Huan Ting Change, Lynn Zechiedrich

NAD-dependent ligases are a family of proteins found only in bacteria that use

nicotinamide adenine dinucleotide (NAD) as a substrate instead of ATP, which is used by

most bacteria and all eukaryotic ligases. Escherichia coli and the members of the family

of Enterobacteriaceae encode two NAD-dependent ligases denoted as LigA and LigB.

LigA, an essential protein, has been well-studied; it is highly expressed and is responsible

for ligating Okazaki fragments during replication. LigB, however, is not essential and has

less than 1% the ligase activity of LigA in vitro. No role for LigB has been found despite

its strikingly high amino acid, chromosomal location, and sequence conservation across

the entire family of Enterobacteriaceae. Because of this conservation and our recent

discovery of a SNP in the ligB gene associated with fluoroquinolone-resistance (Swick,

Evangelista et al. 2013 PLoS One 8:e65961), we hypothesized that LigB has a role in

DNA repair that affects fluoroquinolone resistance. Indeed, comparing a ligB deletion to

the parent strain, we uncovered that the deletion strain was 50-fold more susceptible to

hydrogen peroxide than the isogenic parent strain. Comparing these strains after

treatment with UV irradiation, cumeme hydroperoxide, mitomycin C, and bleocin, we

found that the ligB deletion strain was only more susceptible to treatments that resulted in

the oxidation or alkylation of DNA (hydrogen peroxide, cumene hydroperoxide, and

mitomycin C). Because oxidized and alkylated bases are removed and repaired through

the base excision repair (BER) pathway, we propose a model whereby LigB is the

terminal ligase for the repair of these types of DNA damage. LigB is, thus, not essential

under ideal laboratory growth conditions but becomes essential in the presence of

oxidating and alkylating agents, which includes the fluoroquinolones and many other

antibiotics.

31

A SIMPLIFIED SEQUENCE/STRUCTURE ALPHABET FOR MULTIPLE TRANSMEMBRANE

HELICES SASEMBLIES

Xiang Feng, Patrick Barth

Helical transmembrane (TM) proteins play critical roles in diverse physiological

functions and are thus important drug targets. The lack of structural information,

however, hinders our understanding of their functional regulation and prevents the

rational design of selective therapeutics. Computational modeling techniques represent

important alternative approaches but currently lack the efficiency and accuracy required

to consistently predict membrane protein structures at atomic resolution. Understanding

the sequence/structure determinants controlling the packing of TM helices (TMH) is a

major step in accurately modeling and designing TM proteins but such determinants has

not yet been characterized for multiple TMH assemblies.

We conducted an exclusive bioinformatics analysis on the sequence and structure motifs

defining the packing of three TMH. We generated a library of more than 800 closely

packed TMH trimer structure elements by dissecting available crystallographic structure

of TMH domains. Based on structure similarity, we found that more than half of the

structure elements can be classified into 6 major clusters with distinct

geometrical/topological features, highlighting the limited conformational space sampled

by TMH trimers. The two most abundant clusters contain TMH trimers that have all left-

handed pairs and all right-handed pairs, respectively. With a sequence pattern search

method (TMSTAT), we also found frequently occurring sequence motifs that correlate

with the geometric feature of major classes, suggesting that the identification of specific

sequence motifs may simplify the prediction of TMH trimer and larger structures.

Further, we demonstrate that sequence motifs that are enriched on trimer assembly

interface also correlate with interaction patterns, indicating the convergent rule of

multiple TMH packing.

Our results indicate that a limited number of local sequence/structure motifs can

recapitulate a large fraction of TMH trimer structures. The results provide important

novel insights in convergent rules of TMH interaction, multi-pass TMH domain modeling

and a simplified alphabet to design complex TMH structures.

32

MICROVILLI ARE DISASSEMBLED TO FUEL CLEAVAGE FURROW INGRESSION

Lauren Figard, Heng Xu, Anna Marie Sokac, Ido Golding

Cell shape change requires cell surface growth, but the source of new membrane fueling

this growth is in many cases unknown. A decades-old hypothesis proposes that unfolding

microvilli and other cell surface specializations can provide new membrane for cell

surface growth during cytokinesis, phagocytosis, wound healing, and cell spreading,

although this has never been definitively demonstrated. Here we show that microvilli

serve as a membrane source for cleavage furrow growth during cellularization, the first

complete cytokinetic event in the Drosophila embryo. Using scanning electron

microscopy, we have calculated that microvilli at the start of cellularization contain

approximately half the membrane required for furrow ingression. Accordingly, we see

many microvilli before cellularization, but almost none after. Using 3D imaging of live

cells over time, we find that microvillar membrane is depleted slowly at the start of

cellularization and rapidly later in the process. Remarkably, these microvillar depletion

kinetics follow the biphasic kinetics of furrow ingression. Microvillar membrane is

depleted linearly with increasing furrow length, suggesting a mechanistic link between

depletion of the microvilli and growth of the furrows. We find that experimental

manipulations preventing full microvillar disassembly disrupt furrow ingression, and

conversely, disrupting furrow ingression kinetics has a similar effect on microvillar

depletion kinetics. Together, these results indicate that microvilli provide membrane for

furrow ingression and that furrow ingression itself drives disassembly of the microvilli.

Furthermore, we find that pulse-labeled apical microvillar membrane moves along the

plane of the cell surface into the furrows during furrow ingression, suggesting that

microvillar membrane is translocated predominantly by a pulling mechanism, rather than

by endocytosis. This work shows that microvilli are disassembled to provide membrane

for furrow ingression and suggests that their disassembly is fueled by a pulling force

originating at the ingressing furrows.

33

AUTOMATION OF SINGLE PARTICLE CRYO-ELECTRON TOMOGRAPHY TO STUDY THE TRIC

CHAPERONIN'S INHIBITION OF MUTANT HUNTINGTIN AGGREGATES

Jesus Galaz-Montoya, Sarah Shahmoradian, Boxue Ma, Christoph Spiess, Judy

Frydman, Michael Schmid, Wah Chiu, Steve Ludtke

Single particle cryo-electron tomography (SPT) offers a powerful alternative approach to

traditional 2-D single particle analysis cryoEM (SPA) to study macromolecules in

solution that undergo substantial motion, or assemblies in-situ. Indeed, SPT is the only

technique that can yield nanometer resolution structures of complexes inside the cell. By

providing a 3-D view of individual macromolecular complexes, SPT minimizes the

problem that arises in SPA of distinguishing between different conformations and

different orientations. As such, SPT can address the study of macromolecular structures

that are not readily amenable to other techniques, such as amyloid aggregates with no

regular structures.

Although SPT has gained much popularity over the last few years, more reliable and

efficient methodologies are still under active development. We have developed a

complete processing pipeline for SPT and applied it to controls (e.g., the GroEL

chaperonin and the e15 virus) and to study the structure of mutant huntingtin (mhtt)

aggregates interacting with the TRiC chaperonin.

An expanded polyglutamine tract (Q>36) in the huntingtin protein leads to Huntington's

disease, a deadly neurodegenerative disorder. Incubation with TRiC in vitro, and over

expression of TRiC in cells, inhibit mhtt's aggregation and restore cell viability. We

applied SPT to mhtt (Q >51) aggregates + TRiC in vitro, and found that the chaperonin

inhibits mhtt aggregation both by 'capping' the tips of growing mhtt fibrils and by

'capturing' smaller mhtt oligomers. Since mhtt oligomers and fibrillar-aggregates are

strongly implicated in the pathogenesis of Huntington’s disease, the interactions that we

describe provide a structural mechanism for the chaperonin’s inhibition of mhtt

aggregation in vitro, and suggest a potential for TRiC-inspired therapeutics.

34

AN ENDOGENOUS CA2+

RELEASE PATHWAY ACTIVATED BY BETA-2 ADRENERGIC

RECEPTORS

Monica Galaz-Montoya, Gustavo Rodriguez, Olivier Lichtarge, Theodore G. Wensel

Beta adrenergic receptors are important for cardiovascular regulation and for

physiological responses to adrenaline and noradrenaline throughout the body and are the

targets of numerous widely used drugs. The downstream signaling of these receptors was

long thought to depend on cAMP-dependent protein kinase (PKA) and to be distinct from

those downstream of G-protein coupled receptors which activate phospholipase C (PLC)

and elevate intracellular Ca2+

. By monitoring of intracellular Ca2+

levels in real time we

found that an endogenous receptor in HEK-293 cells responds to the adrenergic agonist

norepinephrine, by a delayed rise in intracellular Ca2+

. The response is blocked by ICI

118,551, a selective antagonist for beta-2 adrenergic receptor (β2-AR), and the relative

potency of agonists is isoproterenol > epinephrine > norepinephrine, consistent with the

pharmacological profile of β2-AR.Treatment with cholera toxin indicated that activation

of Gαs is sufficient for Ca2+

release. Increasing intracellular cyclic AMP levels by

treatment with phosphodiesterase inhibitors IBMX and rolipram, potentiates the

response. However, treatment with PKA inhibitors H-89 and KT5720 had no effect on

the Ca2+

signal, nor did treatment with the cAMP analogue 8-bromo-cAMP which

strongly suggests that the cAMP effector PKA is not involved in this signaling pathway.

Treatment with thapsigargin (an inhibitor of the SERCA Ca2+

pump of the endoplasmic

reticulum) and chelation of extracellular Ca2+

revealed that the Ca2+

is released from

intracellular stores. The release is sensitive to PLC and InsP3 receptor inhibition. These

results reveal a novel pathway by which β2-AR can trigger the release of Ca2+

from

intracellular stores in a PKA independent manner. Preliminary evidence suggests that the

cAMP-activated guanine nucleotide exchange factor (GEF), EPAC couples cAMP

production to PLC activation and Ca2+

release. These results point to previously

unrecognized modes of action of beta agonists and blockers.

35

IDENTIFICATION OF A NOVEL PRC2/TRXG RECRUITER IN MAMMALIAN CELLS

Yufeng Gou, Jia Zeng, Qinghua Wang, Jianpeng Ma

Polycomb repressive complex (PRC) 2 and Trithorax Group (TrxG) proteins are master

epigenetic regulators that function antagonistically to regulate thousands of target genes

involved in important processes such as stem cell differentiation and carcinogenesis.

However, how PRC2/TrxG are recruited to specific regions of their target genes remains

elusive. Here, we identified a new transcription factor MSANTD3 that can recruit both

PRC2 and TrxG complex to their target genes. Gel shift assays indicated that MSANTD3

binds to a specific DNA sequence via its N-terminal DNA binding domain. MSANTD3

physically interacted with the components of PRC2 or TrxG to trimethylate H3K27 or

H3K4. Chip-seq and Chip-qpcr experiments indicated the co-localization of MSANTD3

and PRC2 or TrxG components on human and mouse genomes. Together, these findings

revealed the molecular mechanism of MSANTD3 in regulating expression of thousands

of its target genes through selective recruitment of PRC2 or TrxG complex.

36

CHARACTERIZATION OF PHOSPHOINOSITIDES IN MOUSE RETINA

Feng He, Melina A. Agosto, Theodore G. Wensel

The phosphoinositides display altered levels in response to numerous signal transduction

pathways and play the role in ion channel regulation, phagocytosis, endocytosis, and

other membrane trafficking processes, as well as in production of the second messenger

InsP3. It has been reported that PI(4,5)P2 enhances the interactions between PDE6 and

transducin. We have developed a sensitive new assay based on recombinant epitope-

tagged phophoinositide binding domains and an enzyme-linked immunosorbent assay

(ELISA) with chemiluminescence detection. It allows quantification of phosphoinositides

with a detection limit of 0.004 pmol. When we applied this assay to preparations of

mouse rods containing the outer segment and distal portion of the inner segment, we

observed a striking increase in phosphorylated inositides in response to light. In the dark,

all phosphoinositide levels were low, with the highest being PI(4)P at 0.16 mol%,

followed by PI(4,5)P2 at 0.02 mol% and PI(3)P at a barely detectable 0.001 mol%. Levels

of PI(3,4)P2, and PI(3,4,5)P3 were undetectable. After 12 hours of exposure to room light,

PI(3)P levels surged 35 fold, PI(4)P increased 1.7 fold, and PI(4,5)P2 levels increased 14

fold. The locations of these phosphoinositides were probed using the same binding

proteins fused to fluorescent tags, either expressed by plasmid electroporation of neonatal

retinas, or used to label fixed retinal sections. The PI(4,5)P2-binding PH-domain of PLCδ

localized to the cytoplasm of the inner segment, but not the outer segment. The PI(4)P-

binding domain from FAPP1 yielded bright punctate staining in the inner segment,

suggesting labeling of vesicles or organelles involved in membrane trafficking. The

PI(3)P-binding domains primarily localized to puncta in the distal inner segment, but

small dots suggesting vesicles were also observed in the outer segment. We have

generated mice with a rod-cell-specific knockout of the Type III PI-3 kinase Vps34, to

determine the function of the light induced increase in PI(3)P.

37

ALLORECOGNITION-BASED COOPERATION IS REQUIRED FOR MORPHOGENESIS IN SOCIAL

AMOEBA

Shigenori Hirose, Gad Shaulsky, Adam Kuspa

During morphogenesis, cells with specialized properties cooperate to establish tissues

with defined functions. It is difficult to analyze this process because morphogenesis

occurs over time and with considerable spatial complexity. Despite the biological

significance of the means by which a functional community of cells is established and

maintained, there are relatively few mechanistic studies of morphogenesis.

The social amoeba Dictyostelium discoideum has proven to be useful for studying the

cooperation of cells. Dictyostelium cells aggregate into a multicellular organism through

chemotaxis toward cAMP and progress a stereotyped developmental process. The system

is so durable that its development can be carried out even when constrained to two-

dimensional space. Since the growth and development of the organism are separate

processes, we can form chimeras to examine the behavior of single cells, or small groups

of cells, with different genotypes within the multicellular “body”.

A pair of transmembrane proteins with immunoglobulin domains, TgrB1 and TgrC1, are

expressed prior to multicellular development and function as allorecognition molecules.

Cells initiate cooperation and complete morphogenesis only with cells of the same

allotype (those that share identical tgrB1 and tgrC1 alleles).

When a few cells with one allotype are embedded in large majority of cells with different

allotypes, the minority cells fails to perform cooperative aggregation with the majority.

But once the minority cells physically contact to other same allotype, the migration defect

is restored and the group of the minority cells initiates cooperative migration. Cells that

establish cooperation also initiate the expression of genes required for multicellular

development. These results suggest that the allorecognition system is not only required to

protect against exploitation by unrelated cells, but is also required for morphogenesis

itself, thus ensuring cooperation among cells with similar genotypes.

38

VALIDATED NEAR-ATOMIC RESOLUTION STRUCTURE OF BACTERIOPHAGE EPSILON15

DERIVED FROM CRYO-EM AND MODELING

Corey Hryc, Matthew Baker, Qinfen Zhang, Weimin Wu, Joanita Jakana, Cameron

Hasse-Pettingell, Pavel Afonine, Paul Adams, Jonathan Kink, Wen Jiang, Wah Chiu

Bacteriophages infect nearly all bacteria, making them excellent potential alternatives to

antibiotics. In this work, we solved the structure of bacteriophage epsilon15 using cryo-

EM and computational modeling to a resolution at which an all-atomic model could be

constructed of the intact mature viral capsid. This structure allowed us to segment and

model the two primary structural proteins (GP7 and GP10), and describe the intricate

interactions required for capsid stability. Beyond the relevance to its own structure, the

use of a secondary staple protein in epsilon15 further illustrates the diverse mechanisms

of capsid assembly and stability found in tailed dsDNA bacteriophages.

Additionally, the technology development in this work represents the first time that a

complete validation of map and model has been used to assess the cryo-EM

reconstruction and associated models. Even at near-atomic resolution, the models in this

work were fully validated using best-practices from X-ray crystallography. Obtaining

near-atomic resolution allowed for a more extensive model building procedure to be

used. Previously our C-α models were focused on fit to density and proper bond lengths.

The modeling procedure for the all-atom structures now accounts for various rotamers,

side chain clashes, geometry and side chain fit to density. With this work we hope to have

established the standards for which all cryo-EM maps and models are to be evaluated.

Beyond the importance of establishing community wide standards, this work also reveals

for the first time that a virus, bacteriophage epsilon15, containing two canonical viral

structural proteins folds: the major capsid protein fold from HK97 bacteriophage fold and

the jellyroll fold found in a wide range of eukaryotic viruses. We propose that the

presence of such folds in epsilon15, in which a vertebrate host acted as a reservoir for

both bacteriophages and other viruses, evolved through genetic transfer and

recombination.

39

EVALUATING THE IMPACT OF CANCER MUTATIONS

Teng-Kuei Hsu, Panagiotis Katsonis, Olivier Lichtarge

Whole genome sequencing of cancer cells has uncovered a large number of genetic

alterations and estimating the functional impact and clinical consequences of these

alterations has become a major challenge. Toward this goal, we derived an “Evolutionary

Action” method, based on protein evolution information, to evaluate the impact of any

point mutation, such that a greater Evolutionary Action score corresponds to a greater

impact on protein function.

In cancer genes, high Action mutations disrupt or alter protein function, and therefore

they are positively selected during tumor development since they confer a growth

advantage to the cancer cells. In contrast, high Action mutations are not selected for in

the genes that control other cellular functions or are non-essential. As a result, the

distribution of Action scores for the somatic mutations of each gene is able to identify the

genes that are associated with cancer. Identifying cancer genes is crucial for

understanding the cancer mechanism, finding potential therapeutic targets and developing

novel strategies to treat cancer. The survival of patients can also be predicted from the

impact of genetic alterations in key cancer genes, such as TP53. Cancer patients with

high impact genetic alterations in cancer genes may have a lower survival rate than

patients with no or with low impact mutations. The integration of mutations occurring in

various genes is a major challenge that can improve the associations of mutations to

disease severity. Using protein networks can help in this integration, and lead to a better

understanding of the functional importance of genetic alterations.

40

STRUCTURAL BASIS FOR DIVERSE GLYCAN RECOGNITIONS BY ROTAVIRUS CAPSID

PROTEIN VP8*

Liya Hu, Sasirekha Ramani, Nicolas Cortes-Penfield, Sue Crawford, Rita Czako,

Gagandeep Kang, Jacques LePendu, Mary K. Estes, B.V.V. Prasad

As the leading cause of gastroenteritis in young children, rotavirus (RV) initially attaches

to host cells which are covered in a glycocalyx consisting of glycolipids, glycoproteins

and proteoglycans. Recognition of sialylated glycans by the VP8 domain of the spike

protein VP4 was thought to be the key event during rotavirus infection. Although

structurally well conserved with a galectin-like fold, VP8 is the least conserved RV

structural protein giving rise to 35 P genotypes. We discovered that subtle changes in the

glycan-binding site of VP8 alters glycan specificity from sialic acid, a key determinant of

infectivity for sialidase-sensitive (s-s) animal RVs, to non-sialylated histo-blood group

antigens (HBGAs) in the VP8 of a sialidase-insensitive (s-i) human rotavirus (hRV,

P[14]), provided a new paradigm in understanding hRV infectivity. To further study the

VP8 glycan specificity of other genotypes, we carried out glycan array analysis of VP8 of

a neonatal-specific hRVs (P[11]) and that of a globally prevalent P[4] hRV. We found

that the neonate-specific P[11] VP8 specifically binds to glycans containing a precursor

HBGA, whose modification is thought to be developmentally regulated, and the prevalent

P[4] VP8 binds to different types of HBGAs. Structural analysis of P[4] VP8/H-type

HBGA complex shows a novel glycan binding site on P[4] VP8; the crystal structure of

P[11] VP8 displays several significant conformational changes at the known glycan

binding sites. The in vivo relevance of these glycan binding specificities were validated

by hemagglutination and cell-based infectivity assays. Our studies suggest that RV strains

exhibit significant variations in glycan specificity that may explain tissue tropism and

host specificity.

These studies were supported by grants from NIAID and Robert Welch foundation.

41

CRYSTAL STRUCTURES OF A SINGLE DOMAIN IN PKGI REVEAL THE MOLECULAR

MECHANISM OF CGMP SELECTIVITY

Gilbert Huang, Jeong Joo Kim, Albert Reger, Robin Lorenz, Eui-Whan Moon, Chi

Zhao, Darren Casteel, Daniela Bertinetti, Choel Kim, Friedrich Herberg

Cyclic guanosine monophosphate (cGMP) is a key secondary messenger that is produced

in response to nitric oxide. One of the key mediators of cGMP signaling, cGMP-

dependent protein kinase (PKG), is activated upon binding to cGMP and phosphorylates

downstream substrates in a process required for important physiological processes such

as vasodilation, nociception, and memory formation. PKGs are also known to mediate

most effect of drugs that increase cellular cGMP levels, including nitric oxide-releasing

agents and phosphodiesterase inhibitors, which are used for the treatment of angina

pectoris and erectile dysfunction, respectively. We have investigated the mechanism of

cyclic nucleotide selectivity by PKG by determining crystal structures of the cGMP-

selective carboxyl-terminal cyclic nucleotide-binding domain (CNBD-B) of human PKG

I bound to cGMP and in the apo form. Our crystal structure of CNBD-B with bound

cGMP reveals that cGMP adopts the syn configuration in the binding pocket and is

coordinated by a previously unidentified arginine residue. Furthermore, comparison of

the cGMP-bound crystal structure of the apo structure suggests a role for a C-terminal

tyrosine residue in capping the nucleotide into the binding pocket. The interaction of this

tyrosine residue with cGMP stabilizes a conformational change in the C-terminal helix,

suggesting a mechanism for kinase activation by cGMP.

42

IDENTIFICATION OF SITES OF EVOLUTIONARY DIVERGENCE IN LIGAND SPECIFICITY OF

METABOTROPIC GLUTAMATE RECEPTORS

Hye Jin Kang, Angela D. Wilkins, Olivier Lichtarge, Theodore G. Wensel

Metabotropic glutamate receptors (mGluRs) are important for modulating signaling by

glutamate, the main excitatory neurotransmitter in the central nervous system. The

structural basis of ligand specificity differences among Group1 and Group2 (mGluR1, 2,

3, 5) and Group3 (mGluR4, 6, 7, 8) has not been examined in an evolutionary context.

By testing several amino acids known to exist in mammalian brain to mGluR1,-2,-4,-6

and mGluR7 expressing HEK-cells 293 cells that express endogenous G αq or transiently

co-expressing Gα15, both of which yield Ca2+

mobilization detectable by high-

throughput fluorescence assays, we confirm that L-Serine-O-Phosphate (L-SOP) seems to

be the only endogenous ligand that is specific to Group 3 mGluR and that is antagonist to

mGluR1. To determine important residues for L-SOP binding, the Lichtarge group and

ours have used the Evolutionary Trace to analyze sequences from the class C GPCR

family, and identified G319, Q170, L342, N415, S344 and S189 as candidates for

residues important for ligand binding and/or responses. Among mGluR4 mutants, D415N

and R344S decreased potency for L-SOP, a ligand of mGluR4. For reciprocal mGluR1

mutants, we tested if these mutants behave as better antagonist to L-SOP than wild type

mGluR1 through L-SOP/ L-Glu competitive assay. Interestingly, reciprocal mGluR1

mutant N415D enhanced L-SOP binding by 2.3 fold. Even though S344R showed

comparable L-SOP binding to WT mGluR1, it showed significant L-SOP selectivity over

L-Glu by 16 fold because it retained L-SOP binding potency but significant loss of L-Glu

binding potency. Therefore, these mGluR1 mutants N415D and S344R gained L-SOP

functions while the reciprocal mGluR4 mutants D415N and S344R showed loss of

function (potency) for L-SOP. Accordingly, through these reciprocal mutagenesis studies,

our data demonstrated that Evolutionary Trace has predictive power to identify L-SOP

recognition sites in Group 3 and Group 1 mGluRs.

43

METABOLOMIC PROFILING IDENTIFIES BIOCHEMICAL PATHWAYS ASSOCIATED WITH

CASTRATE RESISTANT PROSTATE CANCER

Akash Kaushik, Shaiju K. Vareed, Sumanta Basu, Vasanta Putluri, Nagireddy Putluri,

Katrin Panzitt, Ismael A. Vergara, Nicholas Erho, Nancy L. Weigel, Nicholas Mitsiades,

Ali Shojaie, Ganesh Palapattu, George Michailidis, Arun Sreekumar

Despite recent developments in treatment strategies, castrate resistant prostate cancer

(CRPC) is still the second leading cause of cancer associated mortality among American

men, the biological underpinnings of which are not well understood. To this end, we

measured levels of 150 metabolites and examined the rate of utilization of 184

metabolites in androgen dependent prostate cancer (AD) and CRPC cell lines using a

combination of targeted mass spectrometry and metabolic phenotyping. Metabolic data

were used to derive biochemical pathways that were enriched in CRPC, using Oncomine

Concept Maps (OCM). The enriched pathways were then examined in-silico for their

association with treatment failure (i.e., PSA recurrence or biochemical recurrence) using

published clinically annotated gene expression data sets. Our results indicate that a total

of 19 metabolites were altered in CRPC compared to AD cell lines. These altered

metabolites mapped to a highly interconnected network of biochemical pathways that

describe UDP glucuronosyltransferase (UGT) activity. We observed an association with

time to treatment failure in an analysis employing genes restricted to this pathway in

three independent gene expression data sets. In summary, our studies highlight the value

of employing metabolomic strategies in cell lines to derive potentially clinically useful

predictive tools.

44

THE BAG6 COMPLEX INTERACTION NETWORK AND ITS ROLE IN DNA DAMAGE RESPONSE

Giedre Krenciute, Shangfeng Liu, Yi Shi, Nur Yucer, Priscilla Ortiz, Beom-Jun Kim,

Ore Abiola Odejimi, Jun Qin, Yi Wang

BAG6 is a member of the BAG protein family, which is implicated in diverse cellular

processes including apoptosis, co-chaperone, and DNA damage response (DDR).

Recently, it has been shown that BAG6 forms a stable complex with UBL4A and GET4

and functions in membrane protein targeting and protein quality control. The BAG6

sequence contains a canonical nuclear localization signal and is localized predominantly

in the nucleus. However, GET4 and UBL4A are found mainly in the cytoplasm. This

raised the question whether GET4 and UBL4A are also involved in DDR in the context

of the BAG6 complex. Here, we provide evidence that nuclear BAG6-UBL4A-GET4

complex mediates DDR signaling and damage-induced cell death. BAG6 appears to be

the central component for the process, as depletion of BAG6 leads to the loss of both

UBL4A and GET4 proteins and resistance to cell killing by DNA-damaging agents. In

addition, nuclear localization of BAG6 and phosphorylation of BAG6 by ATM/ATR are

also required for cell killing. UBL4A and GET4 translocate to the nucleus upon DNA

damage and play redundant roles in cell killing, as depletion of either one has no effect

but co-depletion leads to resistance. All three components of the BAG6 complex are

required for optimal DDR signaling, as BAG6, and to a lesser extent, GET4 and UBL4A,

regulate the recruitment of BRCA1 to sites of DNA damage. Finally, we purified

endogenous BAG6, GET4 and UBL4A by affinity purification and analyzed precipitated

endogenous protein complexes by mass spectrometry to study the BAG6 complex

interactome upon DNA damage. Together our results suggest that the BAG6 complex is

an effector in the DNA damage response pathway and its phosphorylation and nuclear

localization are important determinants for its function. Mass-spectrometric analysis of

the complex precipitates revealed new components of the BAG6 complex interaction

network in the context of damaged DNA.

45

DYNAMIN CONTROLS SNARE DENSITY AT THE FUSION SITE

Aditya Kulkarni, Kannan Alpadi, Christopher Peters

The convergence of the antagonistic reactions of membrane fusion and fission at the

hemifusion/hemifission intermediate has generated a captivating enigma of whether

SNAREs and dynamin have unusual counter-functions in fission and fusion respectively.

SNARE-mediated fusion and dynamin-driven fission are fundamental membrane

remodeling reactions known to occur during ubiquitous cellular communication events

such as exocytosis, endocytosis and vesicle transport. Here we demonstrate the influence

of the dynamin homolog Vps1 on lipid mixing and content mixing properties of yeast

vacuoles, and on the incorporation of SNAREs into fusogenic complexes. We propose a

novel concept that Vps1, through its oligomerization and SNARE domain binding,

promotes the hemifusion-content mixing transition in yeast vacuole fusion by increasing

the number of trans-SNAREs.

46

NECROTIC CELLS SHARE A SIMILAR MECHANISM WITH APOPTOTIC CELLS IN BEING

RECOGNIZED BY ENGULFING CELLS IN C. ELEGANS

Zao Li, Victor Venegas, Prashant Raghavan, Yoshinobu Nakanishi, Zheng Zhou

Necrosis is the premature death of cells caused by external factors, such as acute cell

injury or trauma. In contrast to apoptosis, the programmed cell death, necrosis is caspase-

independent and necrotic cells are morphologically distinct from apoptotic cells.

Although these two categories of cell deaths are genetically different, it has been

suggested that necrotic cell corpses are actively removed by the same set of genes

required in apoptotic cell removal indicating they might share a similar clearance

mechanism. In the nematode Caenorhabditis elegans, gain-of-function mutations in

certain ion channel subunits result in necrotic-like cell death of six touch neurons.

Necrotic touch neurons are subsequently engulfed and degraded inside engulfing cells.

However, it is unclear how necrotic cells are recognized by phagocytes.

Phosphatidylserine (PS) is an important apoptotic cell surface signal that attracts

engulfing cells. Using ectopically expressed MFG-E8, a high-affinity PS-binding protein,

we observed that PS was actively present on the surface of necrotic touch neurons. In

addition, phagocytic receptor CED-1, whose function is needed for the efficient clearance

of apoptotic cells, also acts as a phagocytic receptor for necrotic cells. We demonstrate

that necrotic cells, like apoptotic cells, rely on cell-surface PS as an “eat me” signal to

attract CED-1. We further found CED-7, the worm homolog of mouse ABC1 transporter,

was necessary for PS-exposure on necrotic cell surfaces. Moreover, we discovered

ANOH-1, the worm homolog of mammalian scramblase TMEM16F could contribute to

the presentation of PS on necrotic cell surfaces as well and act in a parallel pathway to

CED-7. Our findings suggest between two distinct cell deaths, a conserved mechanism

may exist for the recognition of cell corpse.

47

PATHWAY-CENTRIC INTEGRATIVE ANALYSIS IDENTIFIES RRM2 AS A PROGNOSTIC

MARKER IN BREAST CANCER ASSOCIATED WITH POOR SURVIVAL AND TAMOXIFEN

RESISTANCE

Suman Maity, Nagireddy Putluri, Chad J. Creighton, Vasanta Putluri, Ramakrishna

Kommangani, Fengju Chen, Sarmishta Nanda, Salil Kumar Bhowmik, Atsushi

Terunuma, Tiffany Dorsey, Agostina Nardone, Xiaoyong Fu, Chad Shaw, Rachel Schiff,

Bert W. O’Malley, John P. Lydon, Stefan Ambs, George Michailidis, Arun Sreekumar

Abstract

Background: Breast cancer molecular subtypes include luminal A, B, normal-like, Her-2

enriched and basal-like tumors, among which luminal B and basal-like cancers are highly

aggressive. Biochemical pathways-associated with patient-survival or treatment response

in these more aggressive subtypes is not well understood. With the limited availability of

pathologically verified clinical specimens, cell line models are routinely used for

pathway-centric studies. To achieve translational relevance, bioinformatics methods to

identify clinically relevant pathways in these cell lines have to be developed.

Methods: We measured the metabolome of luminal and basal-like breast cancer cell lines

using mass spectrometry, linked metabolites to biochemical pathways using Gene Set

Analysis and developed a rank-based method to select pathways based on their

enrichment in patient-derived OMICs datasets and prognostic relevance. The selected

pathways were characterized for their role in breast cancer.

Results: Pyrimidine metabolism was altered in luminal vs basal breast cancer, while the

combined expression of its associated genes or expression of its key gene Ribonucleotide

Reductase M2 subunit (RRM2) alone, correlated well with decreased survival across all

breast cancer subtypes and in luminal node negative patients treated with tamoxifen.

Metabolic products of RRM2 were higher in tamoxifen-resistant cells and xenograft

tumors, while knockdown of the enzyme in tamoxifen-resistant cells significantly

decreased proliferation.

Conclusion: We developed a novel rank-based pathway-centric integromics method and

used it to identify a key role for pyrimidine metabolism in aggressive breast cancer and

tamoxifen resistance. Our findings also nominate RRM2 as a novel drug target for

tamoxifen resistant breast cancer patients.

48

TRPV2 AND TRPP2 STRUCTURE AND FUNCTION

Jennifer McGehee, Irina Serysheva, Vera Moiseenkova-Bell, Zhixian Zhang, Theodore

G. Wensel

TRP channel involvement in phototransduction signaling has been established in

invertebrates, however, most physiological roles for TRP channels in vertebrates have not

yet been identified. Recent studies from the Wensel lab investigated gene expression in

mouse retina, and several TRP channels were identified. TRP channels are important for

pain sensation, calcium homeostasis, and other important physiological functions; there

are numerous lines of evidence implicating TRP channels in diseases like congenital

stationary night blindness. The aims of this project are to establish a method for over-

expression, purification, and reconstitution of the purified channels into lipid vesicles to

test their functional properties by fluorometric methods and to obtain cryo-electron

micrographs for the reconstruction of three-dimensional models.

Both rat TRPV2-1D4 tag and human TRPP2-1D4 tag have been overexpressed in yeast

and affinity-purified using 1D4 monoclonal antibodies conjugated to sepharose beads.

Cryo-electron micrographs of TRPV2/amphipol complexes suspended in vitreous ice

were obtained. Reconstruction has been performed to obtain three-dimensional models.

TRPP2/amphipol complexes have been studied by negative stain electron microscopy. In

order to test the function of the purified channels in amphipol complexes or reconstituted

in vesicles, fluorometric and radiometric assays were performed. TRPV2, expressed in

HEK-293 cells, has been detected by Western blot and immunofluorescence. High

throughput fluorescence calcium flux technology for drug testing has been optimized, and

TRPV2 was shown to be activated by 2-APB and cannabidiol and blocked by Ruthenium

Red. Purified TRPP2 has been subjected to gel filtration, Blue Native-PAGE, and

chemical crosslinking studies to further characterize the stoichiometry of the purified

protein. Preliminary data suggests that TRPP2 exists as a trimer, unlike many of the other

tetrameric TRP channels. Understanding the structural requirements for the function of

these proteins could ultimately aid in designing targeted therapeutic drugs for diseases

linked to ion channel dysfunction.

49

APPROACHING THE PROBLEM OR ANTIBIOTIC RESISTANCE: MECHANISM OF β-LACTAM

HYDROLYSIS BY KPC-2 β-LACTAMASE

Shrenik Mehta, Moumita Samanta, Chone Chow-Dar, Timothy Palzkill

The KPC-2 β-lactamase poses a major threat to antibiotic therapy as it can hydrolyze the

carbapenem antibiotics which are considered a last line of defense. Despite several

biochemical and structural studies, the mechanism of carbapenem hydrolysis by KPC

remains elusive. In this study, we have performed alanine scanning mutagenesis of

several important active site residues of the KPC-2 enzyme. Each mutant was subjected

to detailed kinetic analysis, comparing their ability to hydrolyze carbapenem and

cephalosporin substrates with respect to wild-type. The general mechanism of class-A β-

lactamase involves the catalytic serine attacking the β-lactam carbonyl forming the acyl-

enzyme intermediate. Subsequently, the deacylation step involves an attack on the acyl-

enzyme intermediate by the catalytic water resulting in release of the active enzyme. The

study reveals that both E166A and N170A mutants are highly deacylation deficient. The

T237A mutant shows greater than 10-fold increase in catalytic efficiency for

cephalosporins as compared to wild-type. However, with both the carbapenem substrates

viz. imipenem and meropenem, this mutant deviates from Michaelis-Menten kinetics to

show a branched kinetic pathway. This indicates that on exposure to carbapenem

substrate, the T237A mutant changes conformation and exists as a mixture of kinetically

viable and non-viable isoforms. The R220A mutant displayed an 8-fold increase in

catalytic efficiency for cephalosporins while its catalytic efficiency was 2-fold lower than

wild-type for imipenem hydrolysis. Interestingly, meropenem hydrolysis followed a

branched kinetic pathway with R220A. Thus, this preliminary analysis of the KPC-2

active site indentified the importance of T237 for KPC-2 to function as a carbapenemase.

The loss of this residue is accompanied by loss in kinetic viability as a carbapenemase.

However since the cephalosporinase activity of this mutant seems unaffected, T237

possibly plays a role in substrate class discrimination. Similarly, the R220 residue is

essential for the hydrolysis of meropenem.

50

THE STRINGENT RESPONSE: A FOURTH, INDEPENDENT STRESS RESPONSE REQUIRED FOR

MUTAGENIC REPAIR OF DNA BREAKS IN E. COLI

Phillip J. Minnick, Ryan L. Frisch, Janet L. Gibson, Austen L. Terwilliger, Tyler J.

McCue, Michele C. Darrow, Christophe Herman, Susan M. Rosenberg,

Under stress, molecular mechanisms controlled by stress responses increase mutation

rates, potentially accelerating adaptation to stressors such as nutrient limitation,

antibiotics, and chemotherapies. One stress-induced mutagenesis (SIM) mechanism is the

mutagenic repair of DNA double-strand breaks (DSBs) in Escherichia coli. Under stress,

or if the general stress response is activated artificially, repair of DSBs switches from use

of the high fidelity DNA Pol III to use error-prone Pol IV. The resulting mutagenic repair

of DSBs localizes new mutations both in time (under stress), and in genomic space (near

DSBs). In the E. coli Lac assay these mutations can appear either in the form of point

mutations or gene amplifications. Three stress responses promote mutagenic DSB repair

in E. coli, the rpoE envelope-protein stress response promotes break formation; the SOS

DNA-damage stress response upregulates Pol IV, and the rpoS general stress response

licenses use of Pol IV in repair. Here we demonstrate that stringent-response-defective

∆relA or ∆dksA cells are defective for both point mutations and gene amplification. We

show that the stringent response role in SIM is not formation of DSBs (activation of rpoE

pathway), expression of dinB (activation of SOS pathway), nor expression of rpoS. We

conclude that the stringent response constitutes a fourth, independent, stress response

input into mutagenic break repair. Our data imply that the stringent response regulates the

expression of a currently unknown protein(s), critical to SIM.

51

ROLES OF NUCLEOID-ASSOCIATED PROTEINS IN STRESS-INDUCED MUTAGENESIS IN

STARVING ESCHERICHIA COLI

Jessica Moore, PC Thornton, Susan M. Rosenberg, PJ Hastings

Nucleoid-Associated Proteins (NAPs) are a super-family of Escherichia coli proteins

required for compaction of the bacterial chromosome or “nucleoid”. NAPs control access

to the genome and thereby regulate DNA transactions such as DNA damage, repair, and

transcription. E. coli under stress such as starvation increase mutation rate transiently

under the control of stress responses, until a mutation occurs that allows adaptation. A

well characterized mechanism is the stress response-controlled switch to mutagenic repair

of DNA breaks. lac frameshift-bearing cells starved on lactose medium acquire

compensating frameshift (“point”) mutations, or amplifications of the leaky lac allele to

20-50 copies, which confers sufficient enzyme activity for growth. The mutagenesis

requires (spontaneous) double-stranded DNA breaks and their repair, which requires

access to the DNA. We report that several NAPs are either partly or wholly required for

point mutation and amplification. We focus on four NAPs: H-NS, Fis, Dps, and CbpA.

H-NS and Fis are two major NAPs used during growth. Dps is the major stationary-phase

NAP with CbpA, a late stationary-phase NAP. Deletions of H-NS, Fis, and CbpA cause

strong reductions in SIM, whereas deletion of Dps increases SIM. We report that the H-

NS role in SIM may be promotion of induction of the SOS response and/or homologous

recombination, and that Dps inhibits SIM via protection against reactive oxygen species.

Despite extensive previous gene-hunts in this assay, the roles of NAPs and reactive

oxygen species are newly discovered here. Models for their functions are considered.

52

STRUCTURAL BASIS OF SUBSTRATE SPECIFICITY AND PROTEASE INHIBITION IN NORWALK

VIRUS PROTEASE

Zana Muhaxhiri, Lisheng Deng, Sreejesh Shanker, Timothy G. Palzkill, Mary K. Estes,

Yongcheng Song, B.V.V. Prasad

Noroviruses (NoVs) are the leading cause of non-bacterial acute gastroenteritis. Norwalk

Virus (NV) is the prototype human Calicivirus and the NV protease cleaves the

polyprotein, encoded by the open reading frame 1 of the viral genome, at five non-

homologous sites (P1-P1ʹ) to release six nonstructural proteins that are essential for viral

replication. The structural details of how NV protease recognizes multiple substrates are

unclear. In our X-ray structure of NV protease construct, we observed that the C-terminal

tail, representing a native substrate P5-P1, is inserted into the active site cleft of the

neighboring protease molecule providing atomic details of how NV protease recognizes a

substrate. The crystallographic structure of NV protease with the C-terminal tail

redesigned to mimic P4-P1 of another substrate site provided further structural details of

how the active site accommodates sequence variations in the substrates. Based on these

structural analyses, substrate-based aldehyde inhibitors were synthesized and screened for

their inhibition potency. Crystallographic structures of the protease in complex with each

of the three most potent inhibitors were determined. These structures showed concerted

conformational changes in the S4-S2 pockets of the protease to accommodate variations

in the P4-P2 residues of the substrate/inhibitor, which could be a mechanism for how the

NV protease recognizes multiple sites in the polyprotein with differential affinities during

virus replication. These structures further indicate that the mechanism of inhibition by

these inhibitors involves covalent bond formation with the side-chain of the conserved

cysteine in the active site by nucleophilic addition, and such substrate-based aldehydes

could be effective protease inhibitors.

53

STRUCTURAL BASIS OF MEMBRANE FUSION INDUCED BY INFLUENZA B VIRUS

HEMAGGLUTININ

Fengyun Ni, Xiaorui Chen, Jun Shen, Jianpeng Ma, Qinghua Wang

Influenza virus remains one of the major threats to human health. The virus infects the

host cell through the binding between its surface glycoprotein hemagglutinin (HA) and

sialic acid-containing receptors on the cell surface. HA undergoes a dramatic

conformational change in response to low pH in the late endosome. This conformational

change is associated with the fusion of viral and host endosomal membranes, thereby

releasing the viral genome into the host cells. We inspect the available HA structures at

neutral pH (pre-fusion state) in terms of the structural characteristics that are related to

the fusion ability of HA, and compare the structural differences between influenza A

virus HA (AHA) and influenza B virus HA (BHA). We also determine the crystal

structure of BHA at low pH (post-fusion state), and reveal specific strategies employed

by influenza B virus to stabilize its post-fusion state of BHA. In summary, our structural

and functional studies have provided important new insights into the mechanism of

membrane fusion induced by BHA. These results lead to a better understanding of the

replication of influenza B virus and design of small molecules to inhibit the

conformational change of BHA, thus the infection of influenza B virus.

54

FUNCTIONALLY-IMPORTANT NUCLEOTIDES IN NON-CODING RNAS EVOLVE IN HIGHLY

COMPACT CLUSTERS: SEQUENCE-STRUCTURE ANALYSIS OF RNA-BASED MACHINES

Ilya Novikov, Angela Wilkins, Olivier Lichtarge

Functional non-coding RNAs play a vital role in a variety of subcellular processes

including RNA and DNA modification, maintenance of genome stability, and gene

regulation. In this work we aim to gain better insight into the evolutionary relationship

between sequence, structure, and function of these molecules. We hypothesize that in

structured RNAs, evolutionarily-important nucleotides form three-dimensional clusters

that define the functional sites of the molecule. In particular, we focus on several classes

of RNAs that have solved three-dimensional structures in the Protein Data Bank. For

each of these molecules, we identify nucleotides of evolutionary importance with the help

of Evolutionary Trace (ET). ET quantifies patterns of sequence and phylogenetic

divergence in a molecule to provide a measure of relative evolutionary importance for

each nucleotide in the sequence. Combining this information with structural analysis

shows that the more evolutionarily-important nucleotides form well-defined, non-random

clusters on the structure of the molecule and often constitute known functionally-relevant

regions, such as metabolite and ion binding sites, protein interfaces, and catalytic pockets.

This suggests that evolutionarily-important nucleotides in structured ncRNA molecules

evolve in a manner that is detectable, and that functional regions in these ncRNA

molecules can be predicted based on sequence and phylogenetic information.

55

PATHWAY-BASED INTEGRATIVE ANALYSIS REVEALS A KEY ROLE FOR THE HEXOSAMINE

BIOSYNTHETIC PATHWAY IN PROSTATE CANCER PROGRESSION

Nagireddy Putluri, Katrin Panzitt, Ali Shojaie, Akash K. Kaushik, Vasanta Putluri,

Vadiraja Bhat, Rajni Sonavane, Yiqing Zhang, Xuhong Cao, Harene Venghatakrishnan,

Hangwen Li, Sajna Vithayathil, Shaiju K. Vareed, Alexander Zaslavsky, Benny Abraham

Kaipparettu, Nicholas Erho, Ismael Vergara, Elai Davicioni, Robert B. Jenkins, Nicholas

Mitsiades, Nancy L. Weigel, Michael M. Ittmann, Arul M. Chinnaiyan, Ganesh S.

Palapattu, George Michailidis, Arun Sreekumar

Castrate resistant prostate cancer (CRPC) is a lethal disease thought to be associated with

specific metabolic alterations. To obtain better insights into these biochemical changes,

we developed a pathway-based integrated analytical method to examine prostate cancer

(PCa) gene expression and metabolomic datasets, and found distinct alterations in the

Hexosamine Biosynthetic Pathway (HBP) between androgen dependent (AD) PCa and

CRPC, with elevated expression of HBP genes associated with poor clinical outcome.

Expression of the HBP enzyme glucosamine-phosphate N-acetyltransferase 1

(GNPNAT1) was regulated by androgens and elevated in PCa, but diminished in CRPC.

Genetic loss of function experiments for GNPNAT1 in CRPC-like cells led to increased

aggressiveness, which was accompanied by alterations in bio-energetic processes.

Addition of an HBP downstream metabolite, UDP-N-acetylglucosamine, significantly

enhanced efficacy of the clinically employed anti-androgen MDV3100 in CRPC-like

cells. Our study demonstrates the potential therapeutic value of targeting altered

biochemical pathways in PCa.

56

INVESTIGATING THE FUNCTIONAL IMPACT OF AMINO ACID SUBSTITUTIONS ON THE HIGHLY

PROFICIENT OROTIDINE DECARBOXYLASE BY RANDOMIZATION MUTAGENESIS

Sam Regenbogen, Benu Atri, Panagiotis Katsonis, Olivier Lichtarge, Timothy Palzkill

Discerning the relationship between protein structure and function is one of the major

goals of modern biology, and many methods have been devised to both predict and

observe this relationship. We are using two methods to investigate the structure-function

relationship of the E. coli enzyme orotidine-5’-monophosphate decarboxylase (ODCase):

a computational method to predict the impact of specific mutations on protein function,

and large-scale randomization to experimentally assay the functional effects of mutations.

ODCase is an essential enzyme in the pyrimidine biosynthesis pathway and is found in all

domains of life. It is one of the most proficient enzymes known, increasing reaction rate

1017

-fold. This has made ODCase an attractive target for structure-function studies, and

yet, despite catalytic residues and overall structure that are virtually invariant among

species, its mechanism is still not fully understood.

We have used a computational method of predicting the functional effects of amino acid

substitutions in ODCase and are currently undertaking an experimental method of

investigating the real outcomes of the same mutations. We are designing randomized

primers for each of the 245 codons in the pyrF gene. Using site-directed mutagenes we

will produce, for each codon, a library containing all 20 residues. These libraries will

then be transformed into cells lacking pyrF, and selected on minimal media. These

selected libraries will then be pooled and submitted for deep sequencing, allowing us to

identify all single-residue substitutions that still maintain ODCase function.

For each position in the ODCase protein sequence, we will be able to determine the

relative effect on ODCase function of all 20 possible amino acids using their relative

frequencies in the pooled sequence data. This information will provide a strong test for

the predictions made computationally, and will provide further insight to the mechanistic

requirements of this highly proficient enzyme.

57

CONTRIBUTION OF EUKARYOTIC CHAPERONIN TRIC/CCT TO BIOGENESIS AND FOLDING OF

A LEUKEMIC ONCOGENIC TRANSCRIPTION FACTOR, AML1-ETO

Soung-Hun Roh, Wah Chiu, David Tweardy

The t(8;21) is one of the most frequent chromosomal translocations associated with acute

myeloid leukemia (AML). This translocation creates a fusion protein consisting of the

acute myeloid leukemia-1 transcription factor (AML1) and the ETO corepressor (AML1-

ETO), which represses transcription through AML1 (RUNX1) DNA binding sites and

immortalizes hematopoietic progenitor cells.

Although AML1-ETO is a important therapeutic target to treat acute leukemia, it is hard

to be managed by conventional drug development strategies since it does not mediate its

function by ligand-binding or enzyme activity, where are eligible drug-targeting sites by

small molecules. In order to control this undruggable oncoprotein, a novel approach of

altering the oncoprotein proteostasis network through type II eukaryotic chaperonin,

TCP-1 Ring Complex (TRiC/CCT), has been suggested. Here, we have shown AML1-

ETO is a TRiC client and its folding intermediate directly associates to TRiC through the

DNA binding domain (AML_1~175). Chemical cross-linking and cryo-EM study also

revealed that multiple TRiC subunits (CCT4, 6) bind cooperatively to AML1-ETO. More

interestingly, total expression level of AML1-ETO is dramatically decreased in vitro

translational system when endogenous TRiC was immune-depleted. Our study suggests

not only TRiC is required for AML1-ETO’s biogenesis but also translational AML1-

ETO-TRiC interaction is a potential drug target to control Acute Myeloid Leukemia.

58

RETINITIS PIGMENTOSA AND NONSENSE-MEDIATED DECAY IN MOUSE PHOTORECEPTORS

Ramon Roman-Sanchez, John Wilson

Retinitis pigmentosa (RP) is an inherited degenerative disease of the retina that results in

rod and cone photoreceptor death. Initially as rods degenerate, subjects lose peripheral

and night vision; this is followed by cone photoreceptor degeneration leaving the patients

completely blind in some instances. RP affects 1 in every 4000 individuals worldwide

and around 30-40% of these are the result of autosomal-dominant (ADRP) mutations.

Rhodopsin (Rho) is a G protein-coupled receptor of the rods and it initiates the

phototransduction cascade upon photon capture. Mutations in Rho, which account for

about 25% of all ADRP cases, affect many processes, including Rho post-translational

modifications, transport, folding and signaling capabilities.

More than 150 mutations that result in RP have been identified within Rho. Seven of

these are nonsense mutations of which five cause dominant RP (dRP) and the two cause

recessive RP (rRP). One possible explanation for this difference is that rRP mutants

undergo nonsense-mediated mRNA decay (NMD) whereas dRP do not, thereby giving

life to a toxic Rho truncation. The NMD pathway is the cell’s quality control mechanism

for the detection and degradation of aberrant mRNA transcripts. If an mRNA molecule

contains a premature termination codon (PTC), defined as a stop codon ~50nt upstream

of an exon-exon junction, then degradation will ensue by NMD.

Our lab has found that the transcript of Rho-Q64X, which causes dRP in humans, is

present a similar levels to wild type Rho in a mouse model. The same observation was

made in a mouse model with a duplicated exon 2 that results in a PTC at the exon-exon

junction. One study in cells detected degradation by NMD of the Rho Q249X nonsense

mutant that causes rRP, but in vivo studies are absent. These observations raise the

question of whether Rho is insensitive to NMD in photoreceptors. I am studying Rho

nonsense transcripts and NMD in the context of the retina so as to have a better

understanding of RP, its mechanisms and aid in the development of gene therapy

strategies based on suppression and replacement of the mutant Rho.

59

MYC-DRIVEN 2-HYDROXYGLUTARATE ASSOCIATES WITH POOR PROGNOSIS IN BREAST

CANCER

Susmita Samanta, Atsushi Terunuma, Nagireddy Putluri, Prachi Mishra, Ewy A. Mathé,

Tiffany H. Dorsey, Ming Yi, Tiffany A. Wallace, Haleem J. Issaq, Ming Zhou, J. Keith

Killian, Holly S. Stevenson, Edward D. Karoly, King Chan, DaRue Prieto, Tiffany Y.T.

Hsu, Sarah J. Kurley, Vasanta Putluri, Rajni Sonavane, Daniel C. Edelman, Jacob Wulff,

Adrienne M. Starks, Yinmeng Yang, Rick A. Kittles, Harry G. Yfantis, Dong H. Lee,

Olga B. Ioffe, Rachel Schiff, Robert M. Stephens, Paul S. Meltzer, Timothy D. Veenstra,

Thomas F. Westbrook, Stefan Ambs, Arun Sreekumar

Using an untargeted discovery approach and validation of key metabolites, we

characterized the metabolomic profile of human breast tumors and uncovered intrinsic

metabolite signatures in these tumors. Importantly, the oncometabolite, 2-

hydroxyglutarate (2HG), accumulated in a subset of tumors and human breast cancer cell

lines. 2HG reached mmolar concentrations comparable to those in isocitrate

dehydrogenase (IDH)-mutant gliomas, despite the absence of IDH mutations. Instead, we

discovered a significant association between increased 2HG levels and MYC pathway

activation in breast cancer, which was corroborated in human mammary epithelial cells

with inducible MYC. Further analyses showed a global increase of DNA methylation in

2HG-high tumors and identified a poor survival tumor subtype with distinct DNA

methylation, high tissue 2HG, and heightened occurrence in African-American patients.

Tumors of this subtype had a stem cell-like transcriptional signature with WNT and

MYC pathway activation. These tumors over-expressed glutaminase, suggesting a

functional relationship between glutamine and 2HG metabolism in breast cancer.

Accordingly, 13C-labeled glutamine was metabolized into 2HG in cells with aberrant

2HG accumulation, whereas pharmacologic and siRNA-mediated inhibition of

glutaminase markedly reduced 2HG. Our findings highlight 2HG as a candidate breast

cancer oncometabolite associated with MYC activation and poor prognosis.

60

A GFP-BASED ASSAY FOR CAG REPEAT INSTABILITY

Beatriz A. Santillan, John Wilson

Myotonic dystrophy, Huntington disease, and several spinocerebellar ataxias are

members of a group of disorders that correlate with CAG trinucleotide repeats (TNRs)

that increase in length (expand) in specific genes. Despite their long-term study and their

severe effects on patients, these diseases still lack effective treatment strategies. Our lab

is trying to define the mechanisms that underlie CAG repeat instability, with the ultimate

therapeutic goal of devising ways to prevent expansion or promote contraction.

In order to efficiently search for modifiers of instability, we have generated a human cell

assay carrying an unstable CAG89 repeat within a GFP reporter. A large contraction

within the CAG tract will yield a functional GFP transcript, giving rise to a green cell

detectable by flow cytometry. Through cell sorting experiments we have shown that the

intensity of green fluorescence is dependent on the size of the repeat tract. This allows us

to detect a range of contraction sizes and therefore instability events. Furthermore, the

ability to sort and subsequently isolate cells enables us to rapidly characterize the entire

spectrum of length changes at the TNR locus, including expansions and contractions.

Finally the frequency of events is high enough that the system is amenable to large-scale

screens.

This system gives us the ability to uncover the major genetic and environmental effectors

of TNR instability using small interfering RNAs or drug treatments.

61

STRUCTURAL ANALYSIS OF DETERMINANTS OF HBGA BINDING AMONG GI NOROVIRUSES

Sreejesh Shanker, Rita Czako, Banumathi Sankaran, Robert L. Atmar, Mary K. Estes,

B.V.V. Prasad

Noroviruses (NoVs) cause acute gastroenteritis worldwide. They use histo-blood group

antigens (HBGAs) as susceptibility and cell attachment factors. Observed periodic

emergence of new NoVs strains is attributed to altered HBGA binding specificities and

antigenic drift. Human NoVs are classified into two major genogroups (GI and GII) with

each genogroup further divided into several genotypes. GIIs are more prevalent and well-

studied but recent epidemiology studies show increased activity levels among GI NoVs

with some members having gained the ability to bind non-secretor HBGAs in contrast to

the prototype Norwalk virus (GI.1). NoVs bind HBGAs through the protruding (P)

domain of the major capsid protein VP1. Previous structural studies of both GI and GII

genogroup have provided insights into P domain -HBGA interactions. To further

understand the determinants of strain-specific HBGA binding among GI NoVs we

determined the structure of the P domain of a novel GI.7 genotype strain and compared it

to the P domain structures of previously determined GI.1 and GI.2 strains. Our

crystallographic studies revealed significant structural differences in the loop regions of

the GI.7 P domain. The GI.7 strain bound to ABH, Lewis secretor and Lewis non-

secretor family of HBGAs allowing us to further elucidate the determinants of non-

secretor Lewis HBGA binding among GI NoVs. Our GI.7-VLP glycan array binding

studies reveal sialoglycans as novel ligands to GI.7 NoVs. Overall our studies provide a

structural basis for strain-dependent alterations in HBGA-binding and in the possible

antigenic variation among GI NoVs.

62

PHAGOCYTIC RECEPTOR SIGNALING REGULATES CLATHRIN AND EPSIN-MEDIATED

CYTOSKELETAL REMODELING DURING APOPTOTIC CELL ENGULFMENT IN C. ELEGANS

Qian Shen, Bin He, Nan Lu, Barbara Conradt, Barth D. Grant, Zheng Zhou

The engulfment and subsequent degradation of apoptotic cells by phagocytes is an

evolutionarily conserved process that efficiently removes dying cells from animal bodies

during development. Here we reported that clathrin heavy chain (CHC-1), key

component of a vesicle coating protein clathrin, and its adaptor protein epsin (EPN-1),

play crucial roles in removing apoptotic cells in C. elegans. Clathrin is a coat protein well

known for its function in receptor-mediated endocytosis, but unknown for acting in

phagocytosis, neither are epsins or other clathrin adaptors. Our study has identified the

novel roles of clathrin and epsin in phagocytosis. Inactivating epn-1 or chc-1 specifically

reduces the speed of engulfment through impairing actin polymerization, the driving

force for engulfment. Clathrin-actin crosstalk not only induces membrane curvature, but

also directs actin polymerization and drives pseudopod extension around apoptotic cells.

Epistasis analysis places epn-1 and chc-1 in the same genetic pathway as ced-1, ced-6,

ced-7, and dyn-1 for cell-corpse engulfment. The CED-1 signaling pathway is necessary

for the pseudopod enrichment of EPN-1 and CHC-1. As a result, CED-1, CED-6, and

DYN-1, like EPN-1 and CHC-1, are essential for the assembly and stable maintenance of

actin fibers along pseudopods, indicating that in addition to driving ‘focal exocytosis’ for

membrane expansion, the CED-1 signaling pathway also regulates the remodeling of the

cytoskeleton for cell-corpse engulfment. Our work identified a novel mechanism

employed by clathrin and its adaptor to promote pseudopod extension and the engulfment

of apoptotic cells, and ties the CED-1 signaling pathway to the actin cytoskeleton.

63

MULTIDISCIPLINARY APPROACHES CONVERGE TO REVEAL THE ACTIVE STRUCTURES OF

DNA

Lynn Zechiedrich, JM Fogg, RN Irobalieva, DJ Catanese Jr, W Chiu, M Schmid

The double-helical structure of DNA imparts incredible stability, protecting the encoded

genetic information from chemical and mechanical stress. Hydrophobic bases, the

molecular readout of the genetic code, are buried within the interior of the helix. In

contrast, the monotonous, hydrophilic, highly negatively charged sugar-phosphate

backbone that contains no genetic information forms the outside, thus accessible, part of

DNA. The same stability that makes relaxed B-form DNA the safe repository of the

genetic code prevents access to the information encoded by the bases. We hypothesized

that the seemingly contradictory requirements of genetic stability and DNA activity are

accomplished via a tightly-regulated switch whereby torsional strain causes localized

structural alterations, including base-flipping, denaturation and other non-canonical, non-

B form DNA structures. We used gel electrophoresis, cryo-electron microscopy, and

human topoisomerase IIalpha binding to demonstrate that our hypothesis was correct.

The structural alterations brought about by torsional stress facilitate access to the genetic

code to initiate DNA activity and recruit DNA-acting enzymes.

64

COMPUTATIONAL DE NOVO DESIGN OF TRANSMEMBRANE PEPTIDE INHIBITORS TARGETING

ONCOGENIC RECEPTORS

Jiaming Sun, Patrick Barth

Interaction specificity ensures proper cellular functions mediated by biomolecules and

selective action of therapeutics. How this property is achieved by proteins interacting

within lipid membranes remains poorly understood. Designing molecules targeting

selectively any membrane protein would greatly improve our knowledge of their action

and abilities to regulate their disease-associated dysfunctions. However, in absence of

precise structural information on membrane protein associations, engineering interaction

specificity using atom-level predictions and selection of physical interactions for many

competing targets is a daunting computational and experimental challenge. We describe a

general integrated computational and experimental approach using de novo structure

modeling and design from sequence only, and apply it to engineer highly-specific

transmembrane peptide inhibitors of self-association of receptors bearing an oncogenic

mutation. We have modeled the structures of oncogenic, constitutively active single-point

mutant variants of the Fibroblast Growth Factor Receptor 3 (FGFR3) and thrombopoietin

receptor (TpoR) from sequence, then computationally designed, and experimentally

optimized peptides that strongly inhibit their self-association, but do not bind the wild

type receptors. When compared to peptides selected in vivo for target binding from

random mutagenesis libraries, the highest-ranked computationally designed peptides

exhibit complex networks of non-polar and polar residue interaction motifs that are close

to optimal for binding affinity and specificity to their target. Our results indicate that

precise atomic-level tertiary interactions can be designed de novo to discriminate

between membrane protein variants that differ by a few atoms only. More generally, our

approach paves the road for designing molecules that target a large diversity of

uncharacterized membrane receptors with unprecedented selectivity.

65

NANOTECHNOLOGY ENABLED GENE DELIVERY IN VIVO

Xianzhou Song, Fude Feng, Ruogu Qi, Jin Wang

A novel biodegradable star-shaped poly-asparamide was developed for gene delivery. We

discovered that the star-shaped polymer showed higher gene transfection efficiency in

cell culture than the linear counterparts. What is more intriguing is that the plasmid DNA

nanoparticles formed with the star polymer can specifically deliver the gene to the lung in

mice and completely avoid the liver accumulation, where most of the nanoparticle

delivery systems end up with. In contrast, the plasmid DNA nanoparticles formed with

the linear polymer can only accumulate in the liver and express the gene with much lower

efficiency.

Furthermore, the low toxicity and biodegradable characters make it a privileged

alternative for virus based or liposome mediated gene delivery system, in which biosafety

is always a concern. In future studies, we will explore the usage of poly-asparamides in

genetic mutation caused diseases.

66

SUBSTRATE-INDUCED REVERSIBLE INACTIVATION OF TEM-1 β-LACTAMASE VARIANTS BY

CEPHALOSPORIN ANTIBIOTICS

Vlatko Stojanoski, Timothy Palzkill

Serine β-lactamases are bacterial enzymes that hydrolyze β-lactam antibiotics.

Mechanistically, serine β-lactamases are very similar to serine proteases such as

chymotrypsin. They both utilize acylation and deacylation of an active site serine in their

mechanism of catalysis. Also, they have strategically positioned a residue that acts as a

general base to activate the catalytic serine. His57 acts as the general base in the

hydrolysis reaction of chymotrypsin and is part of the catalytic triad essential for the

function of the enzyme. TEM-1, a common plasmid-encoded serine β lactamase,

catalyzes the hydrolysis of early penicillins and cephalosporins. Here we examine a

previously identified triple mutant of TEM-1 165-TyrTyrGly-167 (wild type165-

TrpGluPro-167) with switched substrate specificity from ampicillin to ceftazidime. When

compared to chymotrypsin, the Glu166Tyr substitution in the TEM-1 triple mutant is

analogous to a substitution of His57 in chymotrypsin that results in an enzyme that

maintains function. Our findings agree with previous observations of altered substrate

specificity of the β-lactamase triple mutant, which displays increased hydrolysis of

ceftazidime. Additionally, enzyme kinetic analysis of the triple mutant shows that the

hydrolysis of ceftazidime follows branched pathway characteristic of substrate-induced

reversible inactivation. These results, together with an ongoing crystallography studies,

will help elucidate the mechanism of hydrolysis utilized by the triple mutant to switch in

the specificity of the enzyme from ampicillin to ceftazidime when the critical Glu166

residue is substituted with tyrosine. This will provide insights into alternate pathways of

β-lactam catalysis and, more generally, alternate mechanisms for hydrolysis reactions

catalyzed by enzymes.

67

SPECIFICITY DETERMINANTS OF DOPAMINE RECEPTOR LIGAND RECOGNITION AND

DOWNSTREAM COUPLING

Yun-Min Sung, Angela Dawn Wilkins, Gustavo J. Rodriguez, Olivier Lichtarge,

Theodore G. Wensel

The D2 dopamine receptors (D2Rs) and 5-HT2A serotonin receptors (5-HT2ARs) both

belong to the class A subfamily of G protein-coupled receptors. Both receptors are

expressed in the central nervous system and serve as potential targets for antipsychotic

drugs. Although their binding sites are predicted to be structurally similar, they are able

to discriminate between the neurotransmitters dopamine and serotonin and mediate

distinct physiological processes. Previous studies using Evolutionary Trace (ET)

identified residues important for functional specificity of D2R and 5-HT2AR. Replacing

the ET-residues in D2R with the corresponding ET-residues from 5-HT2AR (ET-residue

swapping) in some cases led to a significant enhancement of serotonin-stimulated Gα16

protein activation or reduced dopamine responsiveness. However, some swaps showed no

effect at all. One possible explanation for these results is that some ET-residues may

work in pairs or larger groups of residues to perform specific functions. Therefore, in this

study, we investigated the effects of combined ET-residue swaps, which are predicted to

be covariant during evolution to acquire specific functions by ET analysis. The level of

Gαi activation induced by agonist-stimulated D2Rs was determined by the membrane

potential assay in which activated Gαi leads to opening of the TRPC4β channel and

cation influx across the plasma membrane in HEK293 cells. Some combined ET-residue

swaps showed more enhanced serotonin or diminished dopamine response compared to

the individual swaps, suggesting the functional coupling between the chosen ET-residues

in terms of the specificity of G protein activation induced by agonist-stimulated D2Rs.

Although most combined ET-residue swaps had effects intermediate between those of the

individual swaps for ligand binding affinity, we did observe that one combined ET-

residue swap showed higher binding affinity for serotonin compared to the individual

swaps. This synergistic effect on serotonin binding affinity suggests the functional

coupling for specificity of ligand binding. Taken together, these findings indicate that the

ET-residue pairs in some cases revealed correlated evolution towards obtaining

functional specificity of D2R, implying co-evolutionary interactions between the

predicted covariant ET-residues during the evolution of GPCR function and signaling.

68

THE ROLE OF ADAPTIVE IMMUNE SYSTEM IN THE ESCAPE OF BREAST CANCER CELLS FROM

PRIMARY TUMORS

Lin Tian, Thomas Welte, Xiang Zhang

Metastasis is one of the hallmarks of cancer, and is the direct cause of more than 90% of

cancer-related deaths. Intravasation into the circulation is a critical step for tumor cells to

reach distant organs. We have recently observed a dramatic effect of the adaptive

immune system on vascular structures and metastasis. In the project, we hypothesize that

adaptive immune cells may prevent the cancer cells from entering into the circulation

through normalizing the tumor-associated vasculatures.

We utilized a p53-null murine mammary tumor model to test our hypothesis. To

determine the role of adaptive immune cells in intravasation, we transplanted tumor cells

into mammary gland of Balb/c mice (immunocompetent) and nude mice

(immunodeficient). Through quantifying circulating tumor cells (CTCs) by qPCR, we

found that the number of CTCs in Balb/c mice was around 100 times lower than that in

nude mice. To further investigate the relation between adaptive immune cells and

vascular normalization, we compared pericyte coverage of tumor-associated vasculatures

in Balb/c mice and nude mice, and found that pericyte coverage in tumors of Balb/c mice

was higher than that of nude mice. To directly test the function of T lymphocytes in

vascular normalization, we reconstituted T lymphocytes in tumor bearing nude mice, and

found that pericyte coverage of tumor-associated vasculatures increased significantly in

nude mice with T cell reconstituted.

This study indicates that adaptive immune cells may decrease vasculature permeability

through increasing pericyte coverage. As a consequence, could inhibit intravasation and

decrease metastasis frequency.

69

GENETIC MANIPULATION, GENOME ENGINEERING, AND HUMAN DISEASE MODELING IN

DROSOPHILA MELANOGASTER

Koen Venken

Previously, I developed the novel transgenesis platform, phiC31 artificial chromosome

for manipulation or P[acman] —a system that allows the selective retrieval of large DNA

fragments, subsequent gene tagging, and the site-specific integration of transgenes up to

146 kb for the first time in vivo. We also made two genomic P[acman] DNA libraries (20

kb and 80 kb) to perform large scale gene tagging and generate a full “TransgeneOmic”

complement of the entire 20 Mbp X chromosome.

Besides, I engineered a multi-functional transposon-based platform, Minos Mediated

Integration Cassette or MiMIC. MiMIC allows limitless in vivo modification of genes

using recombinase-mediated cassette exchange of genetic elements for gene expression

and protein tagging.

Within the immediate future, my lab will utilize both resources in as many innovative

possibilities as feasible. Currently, we are finalizing the P[acman] endeavor by

integrating extreme-sized retrofitted P[acman] plasmids of more than 200kb into the fly

genome. We are also gearing towards high-throughput protein tagging of transgenes in a

96 well format allowing for large scale expression analysis, primarily focused on genes

involved in the synaptic vesicle cycle. In addition, we are combining both platforms into

a versatile gene-targeting paradigm, based on site-specific recombination, resulting in a

localized duplication, followed by nuclease driven tandem repeat reduction. This will

form the foundation for the analysis of fly genes orthologous to human neurological

disease causing genes using three engineering venues: binary factor tagging to

manipulate corresponding cells including visualization, protein tagging to analyze protein

expression patterns and protein complexes, and precise engineering of disease causing

mutations followed by phenotyping.

Other projects that are actively being explored are: endogenous sparse protein labeling to

reveal restricted protein expression patterns, engineering of transposon arrays to perform

targeted transposition for disease gene discovery including neuronal cancers, and

synthetic assembly of presumable disease pathways.

70

PROBING THE SITES OF INTERACTIONS OF ROTAVIRAL PROTEINS INVOLVED IN

REPLICATION

Maria Viskovska, Anish Ramakrishnan, Liya Hu, Dar-Chone Chow, Timothy Palzkill,

Mary Estes, B.V.V. Prasad

Replication and packaging of rotavirus genome occur in cytoplasmic compartments

called viroplasms that form during virus infection. These processes are orchestrated by

yet to be understood complex networks of interactions involving non-structural proteins

(NSPs) 2,5,6 and structural proteins (VPs) 1,2,3,6. The multifunctional enzyme NSP2, an

octamer with RNA binding activity, is critical for both coordinating viroplasm formation

with its binding partner NSP5, and in genome replication/packaging through its

interactions with replicating RNA, VP1 and VP2. In the current study, using various

techniques including phage display, peptide-array screening, isothermal calorimetry and

bio-layer interferometry, we examined the interactions between NSP2, VP1, VP2, NSP5

and NSP6. These studies provided first evidence for NSP2 interacting directly with VP1,

VP2, and NSP6. Our findings lead us to propose that NSP2’s interaction with VP2 may

serve two functions, first – in preventing premature self-assembly of the VP2 cores, and

second, inside viroplasms in anchoring the VP1/VP3/RNA complex onto the assembling

VP2 pentamers. Interaction between NSP2 and NSP6 has not been reported previously

and the role NSP6 may serve during infection is unknown. Our studies indicate that

NSP6-binding sites on NSP2 overlap with the VP1-binding regions suggesting that VP1

and NSP6 interact with NSP2 at different stages during the life cycle. It is likely that

NSP6 functions early in the infection, before viral replication is initiated. Our finding that

NSP5 binding sites overlap with VP2- and VP1-binding sites on NSP2 suggest that

interaction of these proteins with NSP2 is spatially and/or temporally regulated.

71

BIODEGRADABLE NANOPARTICLES FOR IN VIVO NON-VIRAL GENE DELIVERY

Jin Wang, Fude Feng, Xianzhou Song, Jin Wang

We successfully developed a novel biodegradable material based on polyaspartic acid for

gene therapy. In this project, we discovered that branched polymers showed higher gene

transfection efficiency in cell culture than the linear counterparts. What is more intriguing

is that intravenous injection of the plasmid DNA nanoparticles formed with the branched

polymer can specifically deliver the gene to the lung in mice and completely avoid the

liver accumulation. In contrast, the plasmid DNA nanoparticles formed with the linear

polymer can only accumulate in the liver and also express the gene with much lower

efficiency. Other routes of injections were also explored, including intra-peritoneal,

intramuscular and footpad. High efficiency of gene expression were achieved in all the

injection routes. Our study will provide a platform for future gene therapy.

72

DETERMINING THE ROLE OF REGULATOR OF G PROTEIN SIGNALING (RGS) PROTEIN,

RGS7, IN NEURONAL SIGNALING

Sara Wright, Theodore G. Wensel

Heterotrimeric G protein signaling is essential for the ability of eukaryotic cells to sense

stimuli such as chemicals, peptides, and light. The heterotrimer consists of a G alpha

subunit and a G beta/G gamma dimer, and is coupled intracellularly to G protein coupled

receptors (GPCRs). In the retina, G protein signaling is crucial for detection and

processing of light. There are two types of light-sensing cells in the retina – rod

photoreceptor cells, which detect low levels of light, and cone photoreceptors, which are

active in bright light conditions and can differentiate color. Defects in the rod

photoreceptor pathway are responsible for many diseases which affect night vision. For

example, congenital stationary night blindness (CSNB) is caused by mutations in genes

expressed in both rod photoreceptor cells and downstream ON-bipolar cells. Photons of

light are sensed by the GPCR rhodopsin in rod photoreceptors, which leads to a halt in

the release of glutamate at the photoreceptor/ON-bipolar cell synapse. ON-bipolar cells

sense the presense or absense of glutamate through another GPCR, mGluR6. In the dark,

photoreceptor cells release glutamate, which binds to mGluR6 on the surface of bipolar

cells and activates G protein signaling through G alpha o. This pathway is negatively

regulated by RGS7 and RGS11, which are members of the R7 family of regulator of G

protein signaling (RGS) proteins. R7 family proteins bind to the atypical G beta-like

protein, G beta 5. It has been previously determined that RGS7 and RGS11 co-localize

with G beta 5 and mGluR6 in the dendritic tips of ON-bipolar cells. The localization of

RGS11 is dependent on interaction with the membrane bound protein R9AP. An

analogous membrane localizing protein has yet to be identified for RGS7 in ON-bipolar

cells. This project involves identification of novel RGS7 interacting proteins by immuno-

precipitation and mass spectrometry of RGS7-containing complexes from retina tissue.

73

CHARACTERIZING THE ROLE OF THE HER2 L755S MUTATION IN LAPATINIB RESISTANCE

OF HER2+ BREAST CANCER

Xiaowei Xu, Huizhong Hu, Agostina Nardone, Sarmistha Nanda, C. Kent Osborne,

Rachel Schiff

HER2-targeting therapies, including the HER2 monoclonal antibody trastuzumab (T) and

the HER1/2 tyrosine kinase inhibitor lapatinib (L), have shown great efficacy in HER2+

breast cancer (BC) patients. Yet resistance commonly exists. To investigate resistance

mechanisms, our lab has developed a large panel of HER2+ BC cell lines made resistant

to L, T, or L+T(LT), and has molecularly profiled them on various platforms.

Interestingly, whole-exome sequencing results revealed two HER2 mutations (G572V,

L755S) unique to BT474AZ late-phase-L-resistant (LLR) cells. The L755S mutation has

been reported to confer LR by a random site-mutagenesis screen in Ba/F3 cells.

Therefore, we hypothesize that in BT474AZ LLR cells, L755S mutation is the driver of

resistance by inducing an active conformation of HER2 which prevents L binding. Our

hypothesis also states that this mutation originates by clonal selection from parental cells

by long term L treatment.

Applying a self-developed nested-Q-PCR assay that specifically amplifies mutant DNA, I

have detected high HER2 L755S mutation levels in BT474AZ LLR cells, low L755S

levels in BT474AZ parental cells, and high L755S levels in LT-resistant cells of

BT474ATCC model as well. This finding supports the scenario of two independent

clonal selection processes of this mutation in BT474AZ/ATCC models and also excluded

the possibility that resistance was related to the specific AZ subclone of the BT474 cell

line. Importantly, we have found that the irreversible inhibitor afatinib (Afa), which binds

both active and inactive conformations of HER2 kinase domain, showed robust efficacy

in inhibiting LLR cell growth (Afa IC50 0.02µM vs. L IC50 3.3 µM). The potent

inhibitory effect of Afa in LLR supports the hypothesis that the L755S mutation is the

driver of LR in LLR cells. Afatinib might serve as a more effective alternative to

lapatinib in HER2+ BC patients with the HER2 L755S somatic mutation in tumors.

74

ACTOMYOSIN RING CONTRACTION OCCURS IN TWO MECHANISTICALLY DISTINCT PHASES

DURING DROSOPHILA CELLULARIZATION

Zenghui Xue, Anna Marie Sokac

While we have a cartoon picture suggesting that non-muscle actin/Myosin-2

(actomyosin) arrays contract like muscle sarcomeres, many molecular details of this

contraction remain unclear. For example, we do not know why the contractile rings in

cells undergoing cytokinesis do not get thicker or accumulate increasing actin density as

they contract. In addition, recent studies suggest that Myosin-2 motor activity is not

required for successful cytokinesis, leaving the mechanism of contraction in question.

To better understand the mechanism of actomyosin ring contraction, I am studying

Drosophila cellularization, which is a modified cytokinetic event. I find that the process

of actomyosin ring contraction during cellularization can be separated into two

morphologically and mechanistically distinct phases, called Phase 1 and 2. Using a

genetic mutant of the regulatory light chain of Myosin-2 (Spaghetti-squash; Sqh), I find

that Phase 1 of actomyosin ring contraction requires Myosin-2 motor activity, but Phase 2

does not. Quantitative live and fixed cell imaging shows that Phase 1 of contraction is

accompanied by an increase in actin levels, while Phase 2 is accompanied by a decrease

in actin levels. Based on these results, I propose a bi-phasic model for actomyosin ring

contraction during Drosophila cellularization. I hypothesize that Phase 1 proceeds like

contraction in the muscle sarcomere, but Phase 2 contraction is driven by actin

depolymerization.

75

THREE-DIMENSIONAL STRUCTURE OF THE ROD SENSORY CILIUM AND BASAL BODY

COMPLEX IN NORMAL AND DISEASED RETINA BY CRYO-ELECTRON TOMOGRAPHY

Zhixian Zhang, Feng He, Aiden Eblimit, RuiChen, Michael F. Schmid, Theodore G.

Wensel

The light-sensing portion of rod and cone photoreceptor cells, known as the outer

segment, is a modified primary cilium. Specialized cellular machinery is needed to

assemble and maintain its structure, and to carry out highly specific and active transport

and sorting of molecular components between the inner segment, where most

biosynthesis occurs, and the outer segment, where those components are utilized for

phototransduction. A narrow sub-cellular highway connecting the inner and outer

segment, known as the connecting cilium, is the site of action of numerous proteins

encoded by genes implicated in inherited diseases known as ciliopathies. At the heart of

the connecting cilium is a nine + zero bundle of microtubules (axoneme) that grows out

of the mother centriole of a mother-daughter pair, or basal body complex. We are using

cryo-electron tomography of isolated rods to determine the structures of the connecting

cilium and associated machinery in wildtype mice and in mouse models of human

disease. In recent work we have identified structural defects consisting of unraveled and

flattened axonemes in mouse knockouts of Spata7, a homologue of a gene associated

with severe blinding neurodegeneration in humans. We have also used computational

sub-tomogram averaging to determine the three-dimensional structure of a daughter

centriole in greater detail than seen previously. This procedure reveals a striking re-

organization of the geometric arrangement of the microtubule bundle along the axis

progressing from the minus (triplet) end to the plus (doublet) end of the centriole. A

better definition of the cilium-associated structures in wildtype rods will help to

understand their normal function as well as the pathogenesis of retinal neurodegenerative

conditions arising from structural defects.

76

DKSA GUARDS ELONGATING RNA POLYMERASE AGAINST RIBOSOME-STALLING-INDUCED

ARREST

Yan Zhang, Rachel A. Mooney, Jeffrey A. Grass, Priya Sivaramakrishnan, Christophe

Herman, Robert Landick, Jue Wang

RNAP elongation is coupled to translation in bacteria, and translation-transcription

coupling inhibits RNAP stalling. Here we present genome-wide evidence to suggest that,

upon amino acid starvation, ribosomes may promote rather than inhibit RNAP stalling.

We developed an algorithm to evaluate genome-wide polymerase progression

independently of local noise, to reveal that the transcription factor DksA modestly

prevents promoter-proximal pausing and dramatically increases RNAP elongation upon

uncoupling of transcription from translation by depletion of charged tRNA. DksA has

minimal effect on elongation in vitro and on noncoding RNA, in both cases transcript can

form RNA secondary structure, which could prevent transcript backtracking. Thus we

conclude that the effect of DksA on RNAP elongation is primarily on transcripts with a

strong tendency to backtrack, which can be induced by ribosome slowing/stalling or

occur promoter-proximally. We propose a model in which ribosome binding prevents

formation of backtrack-blocking RNA secondary structure in mRNAs, and show that,

under this circumstance, DksA prevents backtracking, thus acting as a transcription

elongation factor in vivo.

77

THE MATERNAL-TO-ZYGOTIC TRANSITION TARGETS ACTIN TO PROMOTE ROBUSTNESS

DURING MORPHOGENESIS

Liuliu Zheng, Leonardo A. Sepúlveda, Rhonald C. Lua, Olivier Lichtarge, Ido Golding,

Anna Marie Sokac

Robustness is a property built into biological systems to ensure stereotypical outcomes

despite fluctuating inputs from gene dosage, biochemical noise, and the environment.

During development, robustness safeguards embryos against structural and functional

defects. Yet, our understanding of how robustness is achieved in embryos is limited.

While much attention has been paid to the role of gene and signaling networks in

promoting robust cell fate determination, little has been done to rigorously assay how

mechanical processes like morphogenesis are designed to buffer against variable

conditions. Here we show that the cell shape changes that drive morphogenesis can be

made robust by mechanisms targeting the actin cytoskeleton. We identified two novel

members of the Vinculin/α-Catenin Superfamily that work together to promote

robustness during Drosophila cellularization, the dramatic tissue-building event that

generates the primary epithelium of the embryo. We find that zygotically-expressed

Serendipity-α (Sry-α) and maternally-loaded Spitting Image (Spt) share a redundant,

actin-regulating activity during cellularization. Spt alone is sufficient for cellularization at

an optimal temperature, but both Spt plus Sry-α are required at high temperature and

when actin assembly is compromised by genetic perturbation. Our results offer a clear

example of how the maternal and zygotic genomes interact to promote the robustness of

early developmental events. Specifically, the Spt and Sry-α collaboration is informative

when it comes to genes that show both a maternal and zygotic requirement during a given

morphogenetic process. For Drosophilid cellularization, Sry-α and its expression profile

may represent a genetic adaptive trait whose sole purpose is to make this extreme event

more reliable. Since all morphogenesis depends on cytoskeletal remodeling, both in

embryos and adults, we suggest that robustness-promoting mechanisms aimed at actin

could be effective at all life stages.