Upload
lavonn
View
45
Download
2
Tags:
Embed Size (px)
DESCRIPTION
Identification of Novel HPV16 Binding Proteins using DNA Affinity Purification. Alton R. Johnson Jr. Barry University, Miami Shores, FL Summer Research Institution: University of Pennsylvania, Philadelphia, PA. History of Human Papillomavirus (HPV). At least 140 different strains - PowerPoint PPT Presentation
Citation preview
Identification of Novel HPV16 Binding Proteins using DNA Affinity
Purification
Alton R. Johnson Jr.
Barry University, Miami Shores, FL
Summer Research Institution: University of Pennsylvania, Philadelphia, PA
History of Human Papillomavirus (HPV)
• At least 140 different strains
• 40 of these strains affect the genital tract
• Low risk types such as HPV6 and HPV11
• High risk types such as HPV16 and HPV18
• Strain examined in this study: HPV16
History of HPV
• DNA from high risk strands (HPV16 and HPV18) is found in 98% of cervical cancer cases
• Suggesting HPV increases the production of these malignancies
• Cervical cancer is the 2nd most common cancer in women worldwide
E2 Protein• Plays critical role in viral transcription, DNA
replication, and genome maintenance
• Binds as a dimer to viral DNA
• Strong binding affinity with viral DNA and Brd4 protein
• Tethers to the episome to host chromosomes during mitosis
Brd4 Protein
• Contains a bromodomain and extraterminal domain (BET)
• A major E2- interacting protein
• Remains bound to condensed chromatin during mitosis
• Associates with histones H3 and H4
• Mediates Transcriptional Activation
Research Purpose
To identify additional cellular factors that may be involved in the episomal maintenance. We must first identify the other binding proteins that are bound to the HPV16 other than the
Brd4 protein.
Brd4/E2 interaction allows stable genomic maintenance
Parent Cell
HPV16 Episome
Nucleus
Mammalian CellDaughter Cell
HPV16 Episome
Nucleus
Daughter Cell
Proteins of Interest
??
?
HPV16 Episome
Nucleus
LacO
Mammalian Cell (NHOK)
+ -
NO HPV16
6xHis LacI HPV16H i s - L
6xHis
Hi s -L
Wash Wash
Elute
Proteins
of Interest
Elute
LacI
Background Proteins
Background Proteins
Cell Lysis to Extract Viral DNA
Identify Proteins of Interest Identify Background Proteins
HPV16
Cell Lysis
LacO
LacO/LacI Binding Mechanism
Mammalian Cell (NHOK)
Mammalian Cell (W12)
HPV16 Episome
Nucleus
Cell Lysis to Extract Viral DNA
Detect HPV16 by PCR
E2
-
R
ESIN
HPV16
Wash Resin
Run DNA Lysate through resin
containing E2 Protein
Elute Proteins
Identify Proteins of Interest
Cell Lysis
E2
-
R
ESIN
Wash Resin
Run DNA Lysate through resin
containing E2 Protein
Elute Proteins
+
Background
Proteins
Background
ProteinsProteins of Interest
Identify Background Proteins
Nucleus
Mammalian Cell (HFK)
-
E2/HPV16 Binding Mechanism
Background
Proteins
HPV episome from W12 cell lysate successfully binds to GSH-E2TA Resin
July 31, 2009M- 100bp LadderC- pUC19-HPV16-W12
250 µl of W12 cell lysate and 250 µl was added to the 10 µg of GSH-E2TA bead resin
WASH (PBS)
ELUTE (2M KCl)
PM
170 -
130 -
95 -
72 -
55 -
43 -
34 -
26 -
17 -
(kDa)PM GSH/GST
GSH/GST-E2TA
August 5, 2009
GST-E2TA
Silver Stain of Proteins of Interest
Proteins of Interests
Proteins of Interests
Proteins of Interests
Proteins of Interests
Conclusion
The proteins of interest have now been stained and molecular weights identified. The next
phase is to identify these proteins using mass spectrometry.
Significance
The identification of these novel proteins can provide better data to understand the
mechanisms involved in the tethering of the viral episome to chromosomes. If identified
there is a possibility of developing a drug that can inhibit this protein binding to reduce the
spread of the viral episome during mitotic division.
P.I. and Mentor
Laboratory Staff
Research in the Lab
Presenting Research
Culture/Sightseeing/Fun
The View
Carolina Pombo-Martinez and Me
Acknowledgements University of PennsylvaniaJianxin You, PhDJunpeng Yan, PhDQing Li, PhDSusan Ross, PhDEdward Marshall IIISusan Sheng
Barry UniversityFlona Redway, PhDPeter Lin, PhDTeresa Petrino,PhDChristoph Hengartner, PhD
Supported by NIH-NIGMS MBRS RISE grant R25 GM059244, Barry University