Establishing the BioID2 System as a Tool to Study SHP2’s

i

Establishing the BioID2 System as a Tool to Study SHP2’s Interacting Proteins in

Osteoclasts

By

Kathleen Turajane

BS, Cornell University, 2017

Thesis

Submitted in partial fulfillment of the requirements for the Degree of Master of Science in the

Graduate Program of Biotechnology at Brown University

PROVIDENCE, RHODE ISLAND

MAY 2021

ii

AUTHORIZATION TO LEND AND REPRODUCE THE THESIS

As the sole author of this thesis, I authorize Brown University to lend it to other institutions

for the purpose of scholarly research.

Date

Kathleen Turajane, Author

I further authorize Brown University to reproduce this thesis by photocopying or other

means, in total or in part, at the request of other institutions or individuals for the purpose

of scholarly research.

Date

Kathleen Turajane, Author

iii

This thesis by Kathleen Thida Turajane is accepted in its present form by the Department of

Biotechnology as satisfying the thesis requirements for the degree of Master of Science

Date Signature:

Dr. Wentian Yang, Advisor

Date Signature:

Dr. Cynthia L. Jackson, Reader

Date Signature:

Douglas C. Moore, M.S., Reader

Approved by the Graduate Council

Date Signature:

Dr. Andrew G. Campbell, Dean of the Graduate School

iv

Acknowledgments

I would first like to express my deepest appreciation to my principal investigator, Dr.

Wentian Yang, for his constructive criticism, and unwavering support, especially during the

unprecedented times of COVID-19 pandemic.

I would also like to thank my thesis committee members, Dr. Cynthia L. Jackson and

Professor Douglas C. Moore, for their insightful feedback which greatly improved my

dissertation writing.

I would like to sincerely thank Jiayu Wei who initiated this project. Without her major

contributions, the completion of this project could not have been accomplished. In addition, I am

extremely grateful to my research colleagues, Lijun Wang, Huiliang Yang, Liang Wang, and

Jiahui Huang, for their time and support in training and helping me complete my dissertation.

Lastly, I cannot begin to express my thanks to my family for their love and continuous

support.

v

Table of Contents

Acknowledgements ....................................................................................................................... iv

Table of Figures ............................................................................................................................ vi

List of Abbreviations ................................................................................................................... vii

ABSTRACT ................................................................................................................................... 8

CHAPTER 1: INTRODUCTION ................................................................................................ 9

Reference .................................................................................................................................. 12

CHAPTER 2: MATERIALS AND METHODS ....................................................................... 14

2.1 BioID2 system .................................................................................................................... 14

2.2 Cell lines and plasmid constructs ..................................................................................... 15

2.3 Proximity biotinylation ..................................................................................................... 16

2.4 Immunoblot analysis ......................................................................................................... 17

Reference .................................................................................................................................. 18

CHAPTER 3: RESULTS ............................................................................................................ 20

3.1 Successful BioID2-SH2 and BioID2-SHP2 plasmid expressions in all cells ................. 20

3.2 Response to biotinylation detected in macrophage cells stably expressing BioID2

fusion protein ........................................................................................................................... 21

CHAPTER 4: CONCLUSIONS ................................................................................................. 22

REFERENCE .............................................................................................................................. 24

vi

Table of Figures

Figure 1. Protein structure and regulation of SHP2 tyrosine phosphatase

Figure 2. BioID2 system: A proximity-dependent biotin ligase labeling technique

Figure 3. Four combination plasmid constructs generated

Figure 4. Validation of BioID2 fusion protein functionality

Figure 5. Detection of biotinylated proteins of macrophage BAC1.2F5 cells stably expressing

BioID2 fusion protein

vii

List of Abbreviations

BCA Bicinchoninic acid

BioID Proximity-dependent biotin identification

BirA* Promiscuous biotin ligase enzyme BirA

BSA Bovine serum albumin

C-fms Macrophage colony-stimulating factor receptor 1

Ctsk Cathepsin k

FBS Fetal bovine serum

HRP Horseradish peroxidase

HSCs Hematopoietic stem cells

M-CSF Macrophage colony-stimulating factor

NF-κB Nuclear factor kappa B

OC Osteoclast

PBS Phosphate Buffered Saline

PTK Protein tyrosine kinase

PTP Protein tyrosine phosphatase

RANK Receptor activator of nuclear factor kappa B

RANKL Receptor activator of nuclear factor kappa B ligand

SHP2 Src-homology-2 domain containing protein tyrosine phosphatase 2

TBST Tris-buffered saline pH7.6, 0.1% Tween 20

8

ABSTRACT

Src-homology-2 domain containing protein tyrosine phosphatase 2 (SHP2), a widely

expressed protein tyrosine phosphatase, plays a critical role in osteoclast (OC) development and

skeletal remodeling. Conventional SHP2 knockout mice are embryonic lethal. To investigate the

role of SHP2 in OCs, our lab generated OC-specific SHP2 deficient mice using the “Cre-loxP”

system and Ctsk-Cre as a driver. Phenotypic characterization demonstrates that these SHP2

mutants are severely osteopetrotic, manifesting a marked increase in bone density. Additional

analyses revealed that SHP2 is required for OC development by regulating the fusion of pre-OCs

during osteoclastogenesis. However, the mechanism by which SHP2 regulates the

osteoclastogenic program is not fully understood. To identify the protein substrates of SHP2, we

adopted the BioID2 technology and have designed and built our unique plasmid constructs by

fusing SHP2 (full length) or its SH2 domains (N-SH2 + C-SH2) to a promiscuous biotin ligase

BirA (BirA*). Next, we validated the expression of these newly generated plasmids in 293T and

macrophage BAC1.2F5 cell lines. The expression of BioID2-SHP2 or BioID-SH2 fusion proteins

was detected with SHP2 antibodies. Finally, macrophage BAC1.2F5 lines that stably expressed

BioID2-SHP2 or BioID2-SH2 fusion protein were established and found to specifically respond

to biotinylation in the presence of biotin. Collectively, I have successfully constructed BioID2-

SHP2 or BioID2-SH2 plasmid constructs and validated their expression in 293T cells and OC

precursor BAC1.2F5 cells. These constructs and stably transfected cell lines will serve as a

promising tool to study SHP2 interacting proteins in osteoclasts and other types of cells.

9

CHAPTER 1: INTRODUCTION

As life expectancy continues to increase, the number of patients with osteoporosis is

expected to rise in the upcoming years. These patients develop weak and brittle bones that are

susceptible to fracture. Osteoporosis is associated with the enhanced activity of osteoclasts (OC),

a giant multinucleated cell that degrades bone matrix, resulting in bone mineral loss. The study of

OC development and functional regulation can provide insight into potential therapeutic targets

for osteoporosis [1]. Osteoclastogenesis refers to the multi-step development of OCs from

hematopoietic stem cells (HSCs). The first step is the commitment of HSCs into

monocyte/macrophage lineage in the bone marrow. Then, the binding of macrophage-colony-

stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL), produced by

osteoblasts, to their respective receptors (c-fms and RANK) on OC precursors stimulates pre-OC

proliferation, fusion, and OC differentiation. Finally, differentiated multinucleated OCs mature

through cell polarization, allowing them to perform their resorptive activity [2].

Protein phosphorylation at certain tyrosine residues regulates OC production and resorptive

activity [3]. In contrast to what we know about the role of protein tyrosine kinases (PTKs) in

osteoclastogenesis [4], little is known about the role of PTPs. SHP2, encoded by PTPN11, is a

ubiquitous non-receptor protein tyrosine phosphatase. The structure of SHP2 is comprised of two

10

SH2 domains (N-SH2 and C-SH2) and a

catalytic PTP domain (Figure 1) [5], [6].

The N-SH2 domain functions as a

conformational switch that is responsible

for SHP2 activation. Conversely, the C-

SH2 domain is not involved in SHP2

activation; however, it is important in

that it modulates binding energy. In the

basal state, the binding of the N-SH2

domain to the catalytic PTP domain

results in the autoinhibition of the PTP

activity. In the presence of ligand, the N-SH2 domain binds to a phosphopeptide on the ligand and

dissociates from the PTP domain. Consequently, the catalytic site of the PTP domain is exposed,

which results in SHP2 enzyme activation for substrate binding [5], [7].

To study the role of SHP2 in osteoclastogenesis and OC functional regulation, our lab

generated transgenic mice in which SHP2 was selectively knocked out in the cathepsin k (Ctsk)-

expressing cells, presumably osteoclasts, via “Ctsk-Cre-mediated Ptpn11 floxed allele deletion.

The inactivation of SHP2 expression in murine Ctsk-expressing cells resulted in an osteopetrotic

phenotype, a condition with abnormally increased cancellous bone mineral density and trabecular

number. Previous work in the lab found that SHP2 deletion has minimal effect on pre-OC

proliferation or survival in culture conditions with sufficient M-CSF and RANKL condition, and

that SHP2 specifically regulates and is required for the fusion of pre-OCs during

osteoclastogenesis [8]. The mechanism by which SHP2 regulates osteoclastogenic programs,

Figure 1. Protein structure and functional regulation of SHP2. The structure of tyrosine phosphatase Shp2 contains N-terminal (N) and C-terminal (C) Src homology 2 domain (SH2), and protein tyrosine phosphatase (PTP) catalytic domain (A). In the absence of phosphotyrosine proteins (pY), SHP2 remains in a closed conformation with the N-SH2 domain bound to the PTP domain, which blocks the catalytic site. When binding to the appropriate ligand, the closed confirmation is altered, allowing the substrates to bind to the active site (B). (Adapted from [4,5]).

A

B

11

however, is not fully understood. Based on the current knowledge, identifying SHP2’s substrate(s)

during osteoclastogenesis is essential to understanding the regulatory molecular mechanism(s) and

develop potential therapeutics for OC-mediated diseases.

To investigate protein-protein interactions, there are traditional methods such as yeast two-

hybrid and affinity purification [9]. However, there are major disadvantages to these classical

methods. These include the inability to detect transient or weak protein interactions and study

protein expression in non-native conditions. To overcome these challenges, proximity-dependent

labeling techniques have been developed [9]. Briefly, the protein of interest is expressed in a fused

construct with a modifying enzyme that covalently attaches a tag on proximal and interacting

proteins. These proteins are later identified via mass spectrometry [9]. Proximity-dependent biotin

identification (BioID) system is a unique proximity-dependent labeling technique to screen

candidate proteins that directly and indirectly interact with the protein of interest in a live condition

through biotinylation [10]. Later, the second generation of BioID, BioID2, was created to provide

more-selective targeting of the fusion protein, utilize less biotin, and improve labeling of

neighboring proteins [11]. Previously, this BioID proximity biotinylation technology has been

used to investigate proteins in unicellular eukaryotes and mammalian cells [12], such as interacting

proteins of invadopodia-specific protein Tks5a in breast cancer cells [13] and excitatory and

inhibitory postsynaptic proteins in cortical and hippocampal neurons [14].

In the study of osteoclastogenesis, BioID2 system has not been previously used to study

unique protein interactions with SHP2. Therefore, the aim of this study was to establish the system

and explore the application of BioID2 to study SHP2’s interacting proteins during the osteoclastic

differentiation of macrophage BAC1.2F5 cells induced by RANKL and/or M-CSF.

12

Reference [1] S. L. Teitelbaum, “Osteoclasts: What do they do and how do they do it?,” Am. J. Pathol.,

vol. 170, no. 2, pp. 427–435, 2007.

[2] N. Lampiasi, R. Russo, and F. Zito, “The Alternative Faces of Macrophage Generate

Osteoclasts,” Biomed Res. Int., vol. 2016, no. Figure 2, 2016.

[3] M. Shalev and A. Elson, “The roles of protein tyrosine phosphatases in bone-resorbing

osteoclasts,” Biochim. Biophys. Acta - Mol. Cell Res., vol. 1866, no. 1, pp. 114–123, 2019.

[4] M. H. C. Sheng and K. H. W. Lau, “Role of protein-tyrosine phosphatases in regulation of

osteoclastic activity,” Cellular and Molecular Life Sciences. 2009.

[5] W. Qiu et al., “Structural insights into Noonan/LEOPARD syndrome-related mutants of

protein-tyrosine phosphatase SHP2 (PTPN11),” BMC Struct. Biol., 2014.

[6] M. Abbasi et al., “Regulation of brain-derived neurotrophic factor and growth factor

signaling pathways by tyrosine phosphatase Shp2 in the retina: A brief review,” Frontiers

in Cellular Neuroscience. 2018.

[7] Q. Liu, J. Qu, M. Zhao, Q. Xu, and Y. Sun, “Targeting SHP2 as a promising strategy for

cancer immunotherapy,” Pharmacol. Res., vol. 152, no. December 2019, 2020.

[8] Y. Zhou et al., “SHP2 regulates osteoclastogenesis by promoting preosteoclast fusion,”

FASEB J., vol. 29, no. 5, pp. 1635–1645, 2015.

[9] A. Bareja, C. P. Hodgkinson, E. Soderblom, G. Waitt, and V. J. Dzau, “The proximity-

labeling technique BioID identifies sorting nexin 6 as a member of the insulin-like growth

factor 1 (IGF1)–IGF1 receptor pathway,” J. Biol. Chem., vol. 293, no. 17, pp. 6449–6459,

2018.

[10] K. J. Roux, D. I. Kim, B. Burke, and D. G. May, “BioID: A Screen for Protein-Protein

13

Interactions,” Curr. Protoc. Protein Sci., 2018.

[11] D. I. Kim et al., “An improved smaller biotin ligase for BioID proximity labeling,” Mol.

Biol. Cell, vol. 27, no. 8, pp. 1188–1196, 2016.

[12] R. Varnaitė and S. A. MacNeill, “Meet the neighbors: Mapping local protein interactomes

by proximity-dependent labeling with BioID,” Proteomics, vol. 16, no. 19, pp. 2503–

2518, 2016.

[13] S. Thuault et al., “A proximity-labeling proteomic approach to investigate invadopodia

molecular landscape in breast cancer cells,” Sci. Rep., 2020.

[14] A. Uezu et al., “Identification of an elaborate complex mediating postsynaptic inhibition,”

Science (80-. )., 2016.

[15] K. J. Roux, D. I. Kim, M. Raida, and B. Burke, “A promiscuous biotin ligase fusion

protein identifies proximal and interacting proteins in mammalian cells,” J. Cell Biol.,

2012.

[16] S. Han, J. Li, and A. Y. Ting, “Proximity labeling: spatially resolved proteomic mapping

for neurobiology,” Current Opinion in Neurobiology. 2018.

[17] T. K. Kim and J. H. Eberwine, “Mammalian cell transfection: The present and the future,”

Anal. Bioanal. Chem., 2010.

[18] A. Dey and W. Li, “Cell cycle-independent induction of D1 and D2 cyclin expression, but

not cyclin-Cdk complex formation or Rb phosphorylation, by IFNγ in macrophages,”

Biochim. Biophys. Acta - Mol. Cell Res., 2000.

14

CHAPTER 2: MATERIALS AND METHODS

2.1 BioID2 system

BioID2 involves constructing a fusion protein of a promiscuous bacterial biotin ligase BirA

(BirA*) and the protein of interest, in our case SHP2. As depicted in Figure 2 [15], [16], BirA* is

a biotin ligase that biotinylates both interacting and neighboring protein targets. Using adenosine

triphosphate (ATP), BirA* converts free biotin into highly reactive biotinyl-5’-AMP (adenosine

monophosphate).

Biotinoyl‐5′‐AMP is then

released from the

enzyme’s active site,

allowing it to react with a

specific lysine side chain

on the nearby proteins.

This reaction leads to the

formation of an amide

bond between the biotin

and the lysine side chain

and the release of AMP.

Finally, the biotinylated

proteins are affinity-captured and identified via mass spectrometry [12].

Figure 1. BioID2 system: A proximity-dependent biotin ligase labeling technology. The protein of interest or bait protein (purple) is fused with a promiscuous form (BirA*, light blue) of the bacterial biotin ligase BirA and expressed in cells. BirA* converts exogenously added free biotin (red) to highly reactive biotinyl‐5′‐AMP (B-AMP) that is released from the enzyme's active site, allowing it to react with primary amines on proximal proteins (grey, pink, and yellow). These proteins interact either directly or indirectly with the fusion protein or remain only within the labeling radius. Non-proximal proteins (green and navy) are not biotinylated. After biotin labeling, cells are lysed, and proteins are extracted. Then, biotinylated proteins are captured and purified using streptavidin, and identified by mass spectrometry. (Adapted from [15, 16]).

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2.2 Cell lines and plasmid constructs

In this study we investigated creating BirA* fusion proteins with full-length SHP2, and a

truncated version containing only the SH2 domain. Two plasmids, mycBioID-pBABE-puro

(Addgene, plasmid #80901) and mycBioID2-13X Linker-MCS (Addgene, plasmid #92308) were

selected for stable and transient transfection, respectively. In stable transfection, the introduced

genetic materials are successfully integrated into the host genome and passed on to the next

generation. Conversely, transiently transfected genetic materials are not integrated into the host

genome so the genes are expressed for a limited period of time. Thus, stable and transient

transfections are great tools to study the long-term and short-term effects of gene expression,

respectively [17].

The coding regions of SHP2 and SH2 (Dr. Yang’s lab) were individually sub-cloned into

each of two plasmids (mycBioID-pBABE-puro and

mycBioID2-13X Linker-MCS) via standard cloning

techniques. Newly generated plasmids were

transformed into Escherichia coli DH5α cells for

amplification, then purified using a miniprep kit

(Qiagen, Valencia, CA). For simplicity, stable and

transient transfections are referred to as ‘pBABE-

myc’ and ‘myc’ tags respectively in this paper. The

chosen plasmids led to the expression of myc-tagged

proteins, which can be identified using an antibody

raised against the myc antigen. Overall, four plasmid

Figure 3. Diagrams depict the four plasmid constructs generated to express indicated fusion proteins. Blue: full-length of SHP2. Red: SH2 domain. Yellow: mycBioID-pBABE-puro plasmid or “pBABE-myc” (stable transfection). Light blue: mycBioID2-13X Linker-MCS plasmid or “myc” (transient transfection).

16

constructs were generated, and their nomenclatures are: (1) SHP2-pBABE-myc; (2) SH2-pBABE-

myc; (3) SHP2-myc; and (4) SH2-myc (Figure 3).

Then, HEK 293T cells were co-transfected with retroviral vectors expressing the generated

plasmid (SHP2-pBABE-myc and SH2-pBABE-myc for stable transfection; SHP2-myc and SH2-

myc for transient transfection) and a helper Ecopak plasmid, using FuGene transfection reagent

(Promega Corporation, Madison, WI). The culture media of the transfected 293T cells, containing

retroviral particles, was harvested and stored in the -80°C freezer for long-term usage.

To maintain the long-term expression of BioID2-SHP2 or BioID-SH2 fusion proteins and

study SHP2’s interacting proteins temporally in osteoclastic cells, we generated stably-transfected

mouse macrophage BAC1.2F5 cell line (ATCC, Manassas, VA). Briefly, macrophage BAC1.2F5

cell line was retrovirally transduced in culture medium with 8 ug/ml polybrene to generate cells

stably expressing SHP2-pBABE-myc and SH2-pBABE-myc. The transfected BAC1.2F5 cells

were selected in media containing 4 ug/ml of puromycin (Sigma-Aldrich, St. Louis, MO).

BAC1.2F5 macrophages were maintained in growth medium consisting α-MEM with 10% fetal

bovine serum (FBS), glutamine, antibiotics (penicillin and streptomycin sulfate) and L cell-

conditioned medium as the source of murine M-CSF [18]. These cells can differentiate into

osteoclasts in the presence of M-CSF and RANKL.

2.3 Proximity biotinylation

To perform biotin labeling, we used methods developed by Roux et al. [10]. Briefly, after

cell confluency was reached, cells stably expressing BioID2 fusion protein were treated with media

containing 50 µM biotin overnight. Then, lysis buffer, containing 8 M urea in 50 mM Tris·Cl, pH

7.4, protease inhibitor, and 1 mM dithiothreitol (DTT), was added and cell debris was removed by

centrifugation at 14,000 rpm for 10 minutes. Next, biotinylated proteins were captured by

17

incubating the cell lysate with Streptavidin Sepharose High Performance Beads (GE Healthcare,

17511301) for 4 hours at 4°C. After bead washing, the protein concentration of each replicate was

measured by bicinchoninic acid (BCA) protein assay kit (Thermo Scientific/Pierce Products,

Rockland, ME, USA) with a Nanodrop Spectrophotometer (Thermo Scientific).

2.4 Immunoblot analysis

To detect the presence of the protein, we used previously described methods [8], [10].

Approximately 50 µg of cell lysates were resolved on a 10% SDS-PAGE, transferred to PVDF

membrane, and incubated with anti-SHP2 (Santa Cruz), anti-SH2 (Santa Cruz), and anti-Myc

(Invitrogen) antibodies at a dilution of 1:1000 and streptavidin-horseradish peroxidase (HRP)

antibody at a dilution of 1:40000 overnight at 4°C. After washing in TBS-T buffer for 1 hour, the

membranes were incubated with HRP–conjugated secondary anti-Rabbit or anti-Mouse antibodies

(Bio-Rad) at the dilution of 1:2000 for 40-60 minutes at room temperature. After washing with

TBST for 4 x 15 minutes again, detection of the proteins of interest was performed by enhanced

chemiluminescence (Thermo Scientific).

18

Reference

[1] S. L. Teitelbaum, “Osteoclasts: What do they do and how do they do it?,” Am. J. Pathol.,

vol. 170, no. 2, pp. 427–435, 2007.















FASEB J., vol. 29, no. 5, pp. 1635–1645, 2015.




2018.


19






2518, 2016.




Science (80-. )., 2016.



2012.








20

CHAPTER 3: RESULTS

3.1 Successful BioID2-SH2 and BioID2-SHP2 plasmid expressions in all cells

After various construct plasmids were designed and generated (Figure 4, left), we

performed western blots to evaluate the expression of BioID2 fusion proteins. First, we validated

our protocols for transient and stable transfection of 293T cells, a packaging cell line for producing

retroviral particles. The endogenous SHP2 protein, which serves as an endogenous/positive

control, was expressed by the 293T cells as at the expected 65-kD protein mass (Figure 4A, lane

1). An anti-myc antibody was used to distinguish between endogenous protein expression and

myc-tagged proteins generated by the chosen plasmids. The immunoblot analysis revealed the

expression of BioID2-SH2 and BioID2-SHP2 (Figure 4A, lane 2-4). Second, retroviral

transduction of macrophage BAC1.2F5 cell line was performed and we stably expressed SH2 and

SHP2 fused to BirA* in macrophage BAC1.2F5 cell line (Figure 4B). The data suggested that

Figure 4. Validation of BioID2-SHP2 and BioID2-SH2 fusion protein expression in 293T and Bac1.2F5 cells. The table represents endogenous SHP2 (lane 1) and plasmid constructs and their corresponding lane number on the immunoblots (lanes 2-5). Western blots demonstrated the expression of four plasmids in 293T cells (A) and the expression of myc-tagged full-length SHP2 and SH2 domain in Bac1.2F5 cells via stable transfection (B).

21

plasmid constructs generated are functional and macrophage

BAC1.2F5 cells were stably infected to express BioID2-SH2

and BiolD2-SHP2 fusion proteins.

3.2 Response to biotinylation detected in macrophage cells

stably expressing BioID2 fusion protein

Here, we investigated the biotinylation activity in the

transfected macrophage BAC1.2F5 cells. Biotinylated proteins

were pulled down by streptavidin beads and assessed by western

blot using streptavidin-HRP. We observed that biotinylated

proteins were detected in BAC1.2F5 cells stably expressing

SHP2-pBABE-myc and SH2-pBABE-myc, but not in cells

expressing endogenous SHP2 (Figure 5). These findings

demonstrated that our BioID2 fusion proteins are functional.

Figure 5. Detection of biotinylated proteins of cells stably expressing BioID2 fusion protein. Compared to the control conditions, multiple proteins stably expressed by Bac 1.2F5 macrophages were biotinylated and detected by HRP-Conjugated Streptavidin.

22

CHAPTER 4: CONCLUSIONS

To further the long-term objective of identifying SHP2’s substrate(s) in osteoclastogenesis,

we established a system and explored the use of BioID2 to study protein-protein interaction. The

preliminary data confirmed the functionality of our unique BioID2 fusion proteins and indicated

the feasible application of this proximity-dependent labeling technology in macrophage cell lines.

We successfully performed transient and stable transfections of HEK 293 cells using the

four generated plasmid constructs. Results obtained through immunoblot analysis indicated that

HEK 293 cells expressed proteins corresponding to the SHP2-pBABE-myc, SH2-pBABE-myc,

SHP2-myc, and SH2-myc plasmids. In addition, retroviral transduction of macrophage BAC1.2F5

was achieved because SHP2-pBABE-myc and SH2-pBABE-myc proteins were expressed.

Roux et al. (2018) reported that one of the anticipated results of using BioID2 method is

that the generated cells stably express BioID2 fusion protein at the expected molecular weight and

the endogenous proteins are biotinylated [10]. Based on our data, when stable macrophage

BAC1.2F cells were treated with biotin, multiple protein bands were detected, indicating that (1)

proteins were successfully biotinylated and (2) that various proteins interacted with SH2 and SHP2

expressed by macrophage BAC1.2F cells.

BioID2 was developed to overcome the limitations of the traditional methods for studying

protein-protein interaction. Nevertheless, one limitation of BioID2 is that the technique identifies

proximal and interacting proteins of the bait protein but cannot discriminate between direct and

indirect protein-protein interaction. In addition, false-negative findings may occur if the primary

amine side chain, which is required for biotinylation, of the candidate proteins becomes

inaccessible [10]. The present study has only investigated and validated the functionality of our

novel BioID2 fusion proteins. Consequently, additional experiments are necessary to identify

23

protein substrate(s) of SHP2 through which SHP2 modulates RANKL and/or M-CSF signaling

and osteoclastogenesis. Future studies should stimulate osteoclast differentiation by treating

macrophage BAC1.2F5 cells stably expressing BioID2-SH2 and BioID2-SHP2 proteins with

RANKL and/or M-CFS, evaluate the response to biotinylation, and identify candidate proteins via

mass spectrometry.

In summary, my data have validated that our plasmid constructs are functional and could

serve as a promising tool to study SHP2 interacting proteins in osteoclasts and potentially other

types of cells as well.

24

REFERENCE

[1] S. L. Teitelbaum, “Osteoclasts: What do they do and how do they do it?,” Am. J. Pathol.,

vol. 170, no. 2, pp. 427–435, 2007.















FASEB J., vol. 29, no. 5, pp. 1635–1645, 2015.




2018.


25






2518, 2016.




Science (80-. )., 2016.



2012.








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Establishing the BioID2 System as a Tool to Study SHP2’s