establishing the bioid2 system as a tool to study shp2’s
TRANSCRIPT
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]).
15
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.
[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
19
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.
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.
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