(Bag3) and Its P209L Mutant

Characterization and interaction studies of

Bcl-2-associated athanogene 3 (Bag3) and its P209L mutant

Jeffrey Li

Department of Biochemistry

McGill University, Montreal

August 2018

A thesis submitted to McGill University in partial fulfillment of the requirements of the degree

of Master of Science

Abstract:

A genetic form of myofibrillar myopathy (MFM) has been linked to a P209L mutation in

Bag3, with patients exhibiting childhood-onset progressive skeletal muscle weakness and associated cardiomyopathy. Microscopy studies on MFM patients show myofibril disintegration beginning at the Z-disk and formation of protein aggregates. Bag3 is a member of the BAG family of Hsp70 nucleotide exchange factors, and is associated with a number of pathways, such as selective autophagy, aggresome formation and Hippo pathway regulation. Previous work in zebrafish suggests Bag3-P209L is aggregation-prone and can sequester wildtype Bag3 into aggregates, leading to a loss of Bag3 function. How a loss in Bag3 function leads to the disease is unknown. We identified changes in Bag3 phosphorylation at residues linked to regulation of the heat shock response. BioID comparison of wildtype Bag3 and Bag3-P209L interactors in the soluble and insoluble fraction show potential changes in pathways such as autophagy, Hippo and

Wnt pathway regulation, signal transduction, and actin cytoskeleton maintenance. Interestingly, a

Bag3 function in Wnt signaling has never been reported. With these findings, we propose several hypotheses describing how the observed gain and loss of interaction caused by the P209L mutation may affect Bag3-associated pathways, and how these aberrancies could contribute to the MFM phenotype.

Résumé:

Une forme génétique de la myopathie myofibrillaire (MMF) a été liée à une mutation

P209L dans la protéine Bag3. Les patients avec cette version particulière de la myopathie ont démontré une faiblesse progressive débutant à l’enfance des muscles squelettiques avec une cardiomyopathie associée. Les études microscopiques sur les patients atteints du MMF démontrent une désintégration myofibrille commençante a la ligne Z et des formations d’agrégats de protéines. Bag3 est un membre de la famille BAG des facteurs d’échanges de nucléotides de Hsp70, et est associée avec un nombre de processus cellulaires tels que l’autophagie sélective, la formation des agrésomes (« aggresomes », en anglais) et la voie de signalisation Hippo. Les travaux antérieurs sur le poisson-zèbre suggèrent que Bag3-P209L est propice à former des agrégats et peut aussi pousser la version naturelle de Bag3 à former des agrégats. Cela conduit la perte de fonction de Bag3. Nous ne savons pas encore pourquoi la perte de fonction de Bag3 cause cette myopathie. Nous avons identifié des variations de phosphorylation de Bag3 sur des acides aminés liées au contrôle de la réponse au choc thermique. BioID de la version naturelle Bag3 et de Bag3-P209L a demontré des différences entre leurs interactions de protéines dans les fractions solubles et insolubles. Ces différences démontrent des changements potentiels dans les processus cellulaires tels que l’autophagie, le contrôle de Hippo et Wnt, transduction de signal, et l’entretien du cytosquelette d'actine. Bag3 n’a jamais été démontré auparavant à avoir une fonction dans la voie de signalisation Wnt. Avec ces résultats, nous proposons plusieurs hypothèses décrivant comment les gains ou pertes d’interactions observées causés par la mutation P209L peuvent affecter les processus biochimiques associés à Bag3, et comment ces changements en interactions peuvent contribuer au phénotype MMF.

Acknowledgements:

First and foremost, I would like to thank my supervisor Dr. Jason C. Young. His mentorship, insight, and patience in guiding me through this project has left me a more analytical and organized scientist and individual, and for that, I am deeply indebted. I also thank Jason for his time in editing this thesis. I would also like to thank all members of the Young Lab, past and present, for technical assistance and friendship over the years: Michael Wong, Dr. Imad Baaklini, Dr. Patrick Kim- Chiaw, Dr. Conrado Gonçalves, Sam Lee, Brittany Williamson, Kevin Guo, Eva Wang, and Yogita Patel. Special thanks to Yogita Patel, for the close friendship and support, as well as being an excellent laboratory mentor during my early years as a fledging scientist. Also, thank you to Kevin Simpson-Poirier for translating my abstract. My thanks to Dr. Kurt Dejgaard for providing help with my proteomic studies, as well as Dr. Simon Wing and Dr. Imed Gallouzi for their mentorship in my supervisory committee. Thank you to our collaborator Dr. Josée Lavoie for generously providing me with Bag3 plasmids and antibodies, as well as Dr. Gregor Jansen for BioID plasmids. Thank you to the Bellini Foundation for project funding. Special thanks to Derek Hall, Amr Omer, Dr. Sergio DiMarco and the rest of the Gallouzi lab for advice in microscopy and working with the C2C12 cell line. To my friends in the Schmeing lab, especially Frederik, thanks for the board games, roasting, and blessed beer hour, as well as the occasional ÄKTA assistance. To my skookum homies- Cynthia, Brittany, Colten, Shane, and Camille, thank you for the fantastic memories, support, and copious amounts of food and drink. I do apologize to and thank all friends and mentors that I have not mentioned. Most importantly, I thank my family- Mom, Dad, and Mark, for their undying love and believing in me throughout this project. I could have not made it through graduate school, or life for that matter, without their support.

Table of Contents:

Abstract: ...... 2 Résumé: ...... 3 Acknowledgements: ...... 4 Preface and Contribution of Authors: ...... 7 List of Figures and Tables:...... 8 List of Abbreviations: ...... 9 Chapter 1 : Introduction ...... 12 Myofibrillar myopathy: ...... 12 Skeletal muscle structure and mechanism: ...... 13 Skeletal muscle differentiation: ...... 15 Signaling pathways in myogenesis: ...... 16 Wnt signaling: ...... 16 Hippo signaling: ...... 18 Protein homeostasis: ...... 21 Chaperones: ...... 21 Hsp70 family: ...... 23 Hsp70 co-chaperones: ...... 25 Small heat shock proteins (sHsps): ...... 26 Protein degradation: ...... 28 Ubiquitin proteasome system (UPS): ...... 28 Autophagy: ...... 29 Bag3: ...... 31 Domains of Bag3: ...... 33 IPV motifs: ...... 33 WW domain: ...... 33 PXXP domains: ...... 33 Bag3 functions: ...... 34 Chaperone assisted selective autophagy (CASA): ...... 34 Hippo pathway regulation: ...... 37 Nutrient sensing and protein synthesis: ...... 37 Bag3 and stress granules: ...... 38 Bag3 and myofibrillar myopathy: ...... 39 Objectives and Rationale: ...... 42 Chapter 2 : Results...... 43 In vitro characterization of the Bag3 and HspB8 interaction: ...... 43 Bag3-P209L aggregates in C2C12 cells: ...... 47 JG-98 does not have an effect on Bag3-P209L aggregation: ...... 49 Bag3-P209L shows a reduction in phosphorylation: ...... 52 BioID studies with Bag3 show a diversity of potential interactors: ...... 55 Potential soluble fraction interactors: ...... 59 Potential insoluble fraction interactors: ...... 61 Microscopy and proteomics studies in differentiated C2C12 myotubes: ...... 63 Chapter 3 : Discussion ...... 75

Bag3 in Z-disk and actin cytoskeleton assembly and stability: ...... 76 Bag3-HSF1 dynamics and the heat shock response: ...... 78 Bag3 and Hippo pathway regulation: ...... 79 Bag3 and Wnt signaling: ...... 81 Sarcomere organization and regulation of clathrin: ...... 85 Desmosomal junctions: ...... 85 Conclusions and future perspectives: ...... 87 Chapter 4 : Methods ...... 88 Plasmids: ...... 88 Antibodies: ...... 88 Reagents: ...... 88 Protein purification: ...... 89 Anti-Bag3 purification from rabbit serum: ...... 89 Circular dichroism: ...... 90 Intrinsic fluorescence: ...... 90 Cell culture: ...... 91 Differentiation: ...... 91 Transfection: ...... 91 Western Blot: ...... 91 Fluorescence Microscopy: ...... 92 FLAG Immunoprecipitation: ...... 93 Trypsin on-bead digestion of FLAG peptides: ...... 94 Titanium dioxide (TiO2) enrichment of phosphopeptides: ...... 94 Phosphoproteomics Database Searching and Protein Identification: ...... 95 BioID: ...... 95 BioID Database Searching and Protein Identification: ...... 96 References:...... 98

Preface and Contribution of Authors:

The experiments shown in this thesis were performed and analyzed by myself, with supervision and guidance under Dr. Young. The phosphoproteomics and BioID experiments were performed and analyzed by myself with assistance from Dr. Kurt Dejgaard, who conducted the operation of the mass spectrometer as well as Mascot database searching of the raw mass spectrometry data. Further analysis of the processed mass spectrometry data was performed by myself.

List of Figures and Tables:

Figure 1.1: Sarcomere structure within myofibrils ...... 14

Figure 1.2: Crosstalk between the canonical Wnt and Hippo pathways...... 20

Figure 1.3: Proteostasis overview ...... 22

Figure 1.4: The Hsp70 cycle ...... 24

Figure 1.5: The major domains of Bag3 ...... 32

Figure 1.6: Chaperone-assisted selective autophagy ...... 36

Figure 2.1: Circular dichroism and intrinsic fluorescence spectra of Bag3 and HspB8 ...... 46

Figure 2.2: Bag3-P209L aggregates in C2C12 cells...... 48

Figure 2.3: JG-98 does not rescue Bag3-P209L solubility ...... 50

Figure 2.4: JG-98 does not significantly affect Bag3-P209L aggregation...... 51

Figure 2.5: Immunoprecipitation of Bag3-WT-3xFLAG and Bag3-P209L-3xFLAG ...... 53

Table 2.1: Phosphorylated peptide counts of Bag3-WT-3xFLAG and Bag3-P209L-3xFLAG ... 54

Figure 2.6: Expression of N- and C-terminal-BirA-tagged Bag3 ...... 55

Figure 2.7: Streptavidin pulldown of BirA-Bag3 ...... 58

Figure 2.8: Soluble BioID interactors of Bag3 in C2C12 myoblasts ...... 65

Figure 2.9: Insoluble BioID interactors of Bag3 in C2C12 myoblasts ...... 67

Table 2.2: Soluble BioID Hits ...... 69

Table 2.3: Insoluble BioID Hits ...... 71

Table 2.4: Excluded BioID background hits in the soluble fraction...... 72

Table 2.5: Excluded BioID background hits in the insoluble fraction...... 74

List of Abbreviations:

MFM: Myofibrillar myopathy MRF: Myogenic regulatory factor MYOG: Myogenin MyHC: Myosin heavy chain Fzd: Frizzled LRP5/6: LDL-receptor related proteins 5 and 6 APC: Adenomatous polyposis coli GSK3β: Serine-threonine kinase glycogen synthase kinase-3 DVL: Disheveled TCF: T cell factor LEF: Lymphoid enhancer-binding factor PCP: Planar cell polarity JNK: JUN-N-terminal kinase PLC: Phospholipase C PKC: Protein kinase C CamKII: Calcium-calmodulin-dependent kinase II NFAT: Nuclear factor associated with T cells NFκB: Nuclear factor kappa-light-chain-enhancer of activated B cells CREB: cAMP response element-binding protein YAP: Yes-associated protein TAZ/WWTR1: WW domain containing protein 1 MST1/2: Mammalian STE20-like protein kinases 1 and 2 SAV1: Salvador homolog 1 LATS1/2: Large tumor suppressor kinases 1 and 2 MOB1: Mob kinase activator 1 GPCR: G-protein coupled receptor PQC: Protein quality control Hsp: Heat shock protein sHsp: Small heat shock protein Hsp70: Heat shock protein 70 kDa ER: Endoplasmic reticulum NBD: Nucleotide-binding domain SBD: Substrate-binding domain Hsc70: Heat shock cognate protein 70 kDa CFTR: Cystic fibrosis transmembrane conductance regulator hERG: Human ether-a-go-go related gene Hsp110: Heat shock protein 110 kDa Ubl: ubiquitin-like domain ACD: α-crystallin domain UPS: Ubiquitin proteasome system CHIP: Carboxy-terminus of Hsp70 Interacting Protein TPR: Tetratricopeptide repeat CMA: Chaperone-mediated autophagy

LAMP2A: Lysosome-associated membrane protein type 2A Bag3: Bcl-2-associated athanogene 3 NEF: Nucleotide exchange factor SH3: Src-homology-3 CASA: Chaperone-assisted selective autophagy SYNPO2: Synaptopodin-2 TSC: Tuberous sclerosis proteins complex MTOC: Microtubule-organizing center Htt43Q: Huntingtin-exon 1 43-polyglutamine repeat Stv: Starvin ALS: Amyotrophic lateral sclerosis mTORC1: Mammalian target of rapamycin complex-1 SG: Stress granule LCD: Low-complexity domain DRiP: defective ribosome product ZASP: Z-band alternatively spliced PDZ motif-containing protein FHL1: Four and a half LIM domain 1 FLNC: Filamin C CapZβ1: F-actin capping protein subunit beta FRAP: Fluorescence recovery after photobleaching IPTG: Isopropyl-β-D-1-thiogalactopyranoside CV: Column volume DMEM: Dulbecco’s Modified Eagle Medium FBS: Fetal bovine serum TX-100: Triton X-100 TFA: Trifluoroacetic acid CD: Circular dichroism spectroscopy ITC: Isothermal titration calorimetry MKL2: Myocardin-like protein 2 LIMD1: LIM domain-containing protein 1 AFTIN: Aftiphilin CLH1: Clathrin heavy chain 1 WWOX: WW domain-containing oxidoreductase PPP2R3A: Serine/threonine-protein phosphatase 2A regulatory subunit B'' subunit alpha PP2A: Protein phosphatase 2A ABLM1: Actin-binding LIM protein 1 BI2L1: Brain-specific angiogenesis inhibitor 1 XRN1: 5’-3’ exoribonuclease 1 HAUS6: HAUS augmin like complex subunit 6 ASHWN: Ashwin CSKI2: Caskin-2 TCP: T-complex protein 1 subunit UBA1: Ubiquitin-like modifier-activating enzyme 1 UBP24: Ubiquitin carboxyl-terminal hydrolase 24 MLF2: Myeloid leukemia factor 2 CNN3: Calponin-3

DDB1: DNA damage-binding protein 1 CUL4: Cullin-4 LDHA: Lactate dehydrogenase SHMT2: Serine hydroxylmethyltransferase SERA: D-3-phosphoglycerate dehydrogenase PKM: Pyruvate kinase TCTP: Translationally-controlled tumor protein EIF3D: Eukaryotic initiation factor 3D PHLB2: Pleckstrin homology-like domain protein B KIF23: Kinesin-like protein PLAK: Plakoglobin DESP: Desmoplakin HSF1: Heat shock factor 1 NXN: Nucleoredoxin NKD: Naked cuticle

Chapter 1: Introduction

Myofibrillar myopathy:

The term myofibrillar myopathy (MFM) describes a varied group of neuromuscular disorders sharing morphologically distinct features of myofibril disintegration and progressive muscle weakness (1-5). Autosomal dominant inheritance, cardiomyopathy and peripheral neuropathy are also commonly associated with the disease (1,3,5). Electron microscopy studies indicate that MFM-associated myofibril disintegration begins at the Z-disk, a key structure in the fibril (described in detail below). In this process, proteins and filamentous material, mainly those associated with the Z-disk, accumulate or aggregate in abnormal localizations (1,2). This strongly implicates the aberrant behavior of Z-disk-related proteins as drivers of the disease phenotype, and multiple genetic studies of MFM patients have found a number of mutations in these proteins (1-8).

Of particular note, a P209L point mutation in the protein Bag3 has been linked to a severe childhood-onset form of myofibrillar myopathy (6). Patients exhibited progressive muscle weakness, cardiomyopathy, spine rigidity, and peripheral neuropathy, leading to respiratory failure in the second decade of life (6). However, the molecular mechanisms by which the mutation of Bag3 or other Z-disk proteins cause the disease phenotype remains unclear, though previous work suggests the loss of protein quality control as a potential driver of the phenotype (9,10).

Skeletal muscle structure and mechanism:

Skeletal muscle, also known as striated muscle, is comprised of structured arrangements of muscle cells called myofibers, which are multinucleated cells further composed of organized groups of multiple rod-like units called myofibrils (11). These myofibrils are made of repeated units of sarcomeres (Figure 1.1A), which are the basic contractile unit of muscle (11). The main components of sarcomeres are thick and thin myofilaments, predominantly composed of myosin and actin respectively, whose interactions form the basis for muscle contraction (12,13). Each sarcomere unit is separated by very “dark” bands called Z-disks or Z-lines, where the actin thin filaments are anchored (Figure 1.1B). These thin filaments extend from the Z-disk to the edges of the light band at the center of the sarcomere called the H-zone. Within the H-zone lies the M- line, where the thick filaments are anchored. The dark A-band of the sarcomere represents the length of the thick filaments. The lighter I-band near the Z-disk represents where only the thin filaments are found (12,14).

Muscle contraction involves the interaction between the myosin of the thick filaments and the actin of the thin filaments in a “cross-bridge cycle” (15). Myosin bound to ATP is not bound to actin. Upon ATP hydrolysis, the myosin head changes conformation and is in a “pre- powerstroke” state and binds to actin. When bound to actin, myosin releases its ADP-Pi, causing a “powerstroke” change in conformation, pulling the thin filament towards the M-line. The cycle then repeats, leading to the contraction of the sarcomeres as the thin filament “slides” between the thick filaments towards the M-line (12,14).

Figure 1.1: Sarcomere structure within myofibrils (A): A skeletal muscle myofibril, highlighting its repeating sarcomeric structure. (B): electron microscopy image of a single sarcomere. The I-band and A-band are where only thin actin and thick myosin filaments are found, respectively. (C): A cartoon presentation of sarcomere structure, highlighting the myosin thick filaments (black), actin thin filaments (blue) and myosin molecules utilized in the cross-bridge cycle (red). The Z-line (Z-disk) marks the border between sarcomere and is the actin filament anchoring point. The M-line is the midpoint of the sarcomere where myosin thick filaments are anchored. Microscopy images (A and B) from (12).

Skeletal muscle differentiation:

In vertebrates, the entirety of skeletal muscle in the body originates from a region in the developing embryo called the paraxial mesoderm, which further subdivides into smaller segments called somites (16). These somites contain embryonic muscle progenitor cells, precursors that give rise to satellite cells and muscle tissue (discussed later) (17,18).

Several families of transcription factors are responsible for cellular myogenesis progression. The transcription factors PAX3 and PAX7 are critical upstream controllers of muscle tissue development (16) (17). Myogenic regulatory factors (MRFs) are a set of transcription factors essential for myogenic determination, where cells are committed to a myogenic cell fate, and terminal differentiation into myotubes (19). Comprised of the proteins

MYOD, MYF5, MRF4, and myogenin (MYOG), this family, together with co-factors called E- proteins, bind consensus sequences called E-boxes found in promoters of muscle-specific genes

(19). In embryonic muscle progenitors, PAX3 activates MYOD and MYF5, committing the cell for a myogenic cell fate (20,21). Myogenin and MRF4 act in later stages, serving roles in myoblast fusion and terminal differentiation (22). The importance of these genes in development is highlighted by the early lethality and loss of skeletal muscle phenotype in myogenin knockout mouse models (23,24).

As described previously, embryonic muscle progenitors are the origins of embryo muscle tissue and muscle tissue in adults. The term satellite cells describe a population of PAX7 and

MYF5-expressing mononuclear cells found in muscle tissue between the basal lamina and plasma membrane of myofibers (16,25). Normally, these satellite cells are quiescent, lying dormant and unproliferative. Under growth periods or muscle injury, these cells become activated and move towards the injury location, expressing MYOD and thus committing to the

15 myogenic program. These committed cells are called myoblasts, which after a period of proliferation, terminally differentiate into nonproliferative and mature myocytes. Myocytes are highlighted by their upregulated expression of myogenin and MRF4, and decreased PAX7 and

MYF5 levels compared to myoblasts. These mononuclear myocytes fuse with one another to form myotubes or with damaged myofibers for repair. Myotube formation is also associated with decreased MYOD expression, and increased expression of myosin heavy chain (MyHC) involved in myosin thick filament cross bridge cycling described previously (16,26).

Signaling pathways in myogenesis:

Wnt signaling:

The Wnt family of secreted glycoproteins is comprised of 19 members in most mammals, each member having a unique expression pattern and function (27). Secreted Wnt proteins generally interact with the transmembrane receptor Frizzled (Fzd) and its co-receptor LDL- receptor related proteins 5/6 (LRP5/6), initiating the Wnt signaling cascade. Wnt signaling can occur via its canonical and non-canonical pathways, both of which will be briefly described below (28).

The canonical Wnt pathway, also called the β-catenin dependent pathway, is initiated by the interaction of the Wnt ligand with Fzd/LRP5/6, as mentioned previously (Figure 1.2). A critical member of this pathway is the transcriptional coactivator β-catenin, which in the absence of Wnt-Fzd interaction, is phosphorylated by a “destruction complex” comprised of the proteins axin, adenomatous polyposis coli (APC), and serine-threonine kinase glycogen synthase kinase-3

(GSK3β). This phosphorylation event leads to β-catenin’s proteasomal degradation (27).

However, Wnt-Fzd interaction recruits the scaffold protein Dishevelled (DVL), inducing dissociation of the β-catenin destruction complex and subsequent accumulation of β-catenin in

16 the cytoplasm. β-catenin is then able to translocate to the nucleus, binding the transcription factors T cell factor (TCF) and lymphoid enhancer-binding factor (LEF) to promote the transcription of target genes involved in regulation of processes such as differentiation (Figure

1.2) (29,30).

Multiple non-canonical Wnt pathways exist, namely the planar cell polarity (PCP),

Wnt/Ca2+, and PI3K/AKT/mTOR pathways (31). In the PCP pathway, the Wnt-Fzd mediated recruitment of DVL leads to the eventual activation of JUN-N-terminal kinase (JNK), which is able to activate target transcription factors involved in cell polarity regulation (31,32). In contrast, the Wnt/Ca2+ pathway, which has roles in development and inflammation, triggers an increase in intracellular Ca2+ through a cascade involving phospholipase C (PLC), protein kinase

C (PKC), and calcium-calmodulin-dependent kinase II (CamKII) (31,33). This cascade ends with the activation of the transcription factors nuclear factor associated with T cells (NFAT), nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB), and cAMP response element- binding protein (CREB) (33). Studies performed in C2C12 mouse myotubes also elucidated a role of Wnt-Fzd in activating the Akt/mTOR growth pathway and subsequently causing muscle hypertrophy (34).

Both the canonical and non-canonical Wnt pathways maintain important roles in skeletal muscle development and regeneration. Several Wnt proteins were shown to activate MRFs critical for embryonic muscle development through both canonical and non-canonical Wnt pathways (35-37). When muscles are injured, the Wnt/PCP pathway is able to trigger expansion of uncommitted satellite cells residing in muscle tissue, increasing the number of available satellite cells to be entered into the myogenic repair program (38). On the other hand, the canonical Wnt cascade is able to induce the differentiation of satellite cells in C2C12 mouse

17 myoblasts and isolated mouse muscle cultures (31,39,40). These findings showcase the importance of Wnt signaling in muscle tissue growth and repair in both embryonic and adult muscle tissues.

Hippo signaling:

First discovered in Drosophila, the Hippo pathway modulates organ size through regulation of cell differentiation and proliferation (41). In mammals, Hippo signaling centers around the regulation of the transcriptional co-activators Yes-associated protein (YAP) and WW domain containing protein 1 (TAZ/WWTR1), which, when unphosphorylated, bind the TEAD and SMAD family of transcription factors to promote target gene transcription (42). The Hippo cascade begins with mammalian STE20-like protein kinases 1 and 2 (MST1/2) binding their cofactor Salvador homolog 1 (SAV1) and phosphorylating their targets, large tumor suppressor kinases 1 and 2 (LATS1/2) and Mob kinase activator 1 (MOB1) (Figure 1.2). Phosphorylation of

LATS1/2 permits formation of a MOB1-LATS1/2 complex, leading to the phosphorylation of

YAP and TAZ and their subsequent suppression in the cytoplasm. Consequently, the cytoplasmic

YAP and TAZ is unable to interact with their target transcription factors in the nucleus (41-43).

The Hippo pathway also has a number of overarching regulators (44-46). G-protein coupled receptors (GPCRs) are a family of transmembrane receptors that is able to trigger different cellular responses via signaling pathways dependent on the extracellular ligand bound to the GPCR. In line with this concept, binding of different extracellular ligands to GPCR have both inhibitory and activating effects on LATS1/2 activity, eliciting different cellular responses through the Hippo pathway (45). Cytoskeletal changes when under mechanical tension are also able to induce YAP/TAZ phosphorylation and hereby regulate its activity in a LATS- independent manner. This highlights a major role of YAP and TAZ in allowing cells to adapt to

18 outside forces, inducing cellular growth changes in response to their specific physical environment (42,44,46,47). Additionally, the Hippo pathway was also recently shown to have overlap with the Wnt pathway (Figure 1.2), where the Hippo effectors YAP/TAZ are an essential part of the β-catenin destruction complex involved in the canonical Wnt pathway. Activation of the Wnt pathway releases YAP/TAZ from this complex, allowing both YAP/TAZ and β-catenin to accumulate within the nucleus and induce expression of their respective target genes (48).

In skeletal muscle, the Hippo pathway has been shown to have important roles in myoblast differentiation. In mouse ex vivo satellite cells, YAP is expressed when in the proliferative state, and decreases significantly when differentiation is induced. Overexpression of constitutively active mutant YAP-S127A in satellite cells and satellite cell-derived myoblasts significantly increases their proliferation rate. Additionally, overexpression of YAP significantly inhibits differentiation of satellite cell-derived myoblasts, suggesting YAP is a major regulator of the switch between satellite cell proliferation and differentiation (49). This effect is also seen in

C2C12 myoblasts (50). In mice models, YAP overexpression is able to induce increases in protein synthesis and subsequent adult muscle fiber size through interaction with the TEAD transcription factors, a primary target of YAP/TAZ (51).

Figure 1.2: Crosstalk between the canonical Wnt and Hippo pathways The canonical Wnt pathway (yellow box) and Hippo pathway (pink box) share a common regulatory mechanism. The β-catenin “destruction complex” comprised of axin, APC, and

GSK3β (pathway highlighted in grey) also includes YAP and TAZ, preventing the accumulation of YAP, TAZ, and β-catenin and preventing Hippo and Wnt target genes from being expressed.

However, when Wnt ligand initiates the canonical Wnt pathway, the subsequent dissociation of the destruction complex frees YAP/TAZ, allowing their translocation to the nucleus for target gene expression (48).

Protein homeostasis:

Within the cell, protein homeostasis, also known as proteostasis, involves the maintenance of proper cellular protein levels through the balance of protein synthesis and degradation (52). Misfolding of these proteins can lead to their aggregation and subsequent disease states, such as Alzheimer’s and Parkinson’s. Though it is unknown how exactly these aggregates cause cellular toxicity, it has been proposed that aggregates may sequester proteins essential for viability. It is also possible that these aggregates induce further aggregation of protein monomers and overwhelm the protective proteostasis pathways in a positive feedback cycle, causing cell death. Thus, of particular importance is protein quality control (PQC), which prevents the accumulation of misfolded or unfolded proteins by assisting in their refolding or marking them for degradation (Figure 1.3) (52-54).

Chaperones:

An integral component of PQC are the molecular chaperones, proteins that assist in the folding or solubilization of non-native proteins or the assembly/disassembly of macromolecular structures. Many chaperones are heat shock proteins (Hsps) induced by stress conditions (53-58).

Categorized by molecular weight into structurally related families, these proteins can be either

ATP-dependent or ATP-independent (53). Generally, ATP-dependent chaperones, such as those in the Hsp70 family, maintain ATPase activity to allow them to actively refold aberrant proteins, or mark them for degradation (53,56). In contrast, the ATP-independent small Hsps (sHsps) comprise another family, and are thought to behave as “holdases”, binding to aberrant or unfolded proteins and preventing their aggregation. Additionally, these sHsps assist ATP- dependent chaperones in refolding or degradation of their substrates (53,59-61). When folding is

21 unproductive or otherwise unable to rescue native folding of aberrant proteins, they are primarily degraded through the activities of either the proteasome or the lysosome (Figure 1.3) (62,63).

Figure 1.3: Proteostasis overview Newly translated polypeptides must fold to reach their native conformations. If proteins unfold or misfold, they must be refolded through the assistance of chaperones, such as Hsp70.

Aggregate proteins must be degraded through autophagy, or targeted to the aggresome for

CASA. Monomeric unfolded proteins or unwanted native proteins can be targeted to the proteasome for degradation. These mechanisms will be described in detail in this review.

Hsp70 family:

One of the most well-characterized chaperone families is the heat shock protein 70 kDa family (Hsp70s), with the human genome encoding 13 different isoforms found in the nucleus, endoplasmic reticulum (ER), mitochondria, cytoplasm, and extracellular space (64-66).

Structurally, Hsp70 is comprised of an N-terminal nucleotide-binding domain (NBD) and a C- terminal substrate-binding domain (SBD) connected by an evolutionarily conserved linker

(67,68). Its major cytoplasmic members, the constitutively expressed Hsc70 (HspA8) and stress- inducible Hsp70 (HspA1A/A1B), share ~86% sequence identity, with their differences predominantly in their SBD (65,69). Additionally, they and most Hsp70s share the same general mechanism of action. As seen in Figure 1.4, ATP-bound Hsp70 maintains a very low substrate binding affinity, and cannot stably bind substrate proteins. Upon hydrolysis of ATP, the now

ADP-bound Hsp70 has a strong binding affinity for substrate. Exchange of ADP for ATP reverts

Hsp70 to its low binding-affinity form, releasing the substrate and allowing it to fold on its own.

If the substrate cannot fold correctly, this cycle can then repeat, promoting correct refolding or targeting these aberrant substrates to alternative pathways (56,70). However, the basal ATPase activity and substrate-binding cycle of Hsp70 is slow, and it cannot promote the efficient folding of substrate proteins without the assistance of other regulatory proteins, called co-chaperones

(71) This low basal rate of folding is reflected in studies using purified Hsc70 and denatured firefly luciferase, a model Hsp substrate. Incubation with Hsc70 alone is unable to rescue luciferase activity, whereas adding co-chaperones increases the rate of refolding and therefore significantly restores luciferase activity (72,73).

Figure 1.4: The Hsp70 cycle Hsp70 (yellow) has a low substrate-binding affinity in its ATP-bound state. A J-domain co- chaperone (red) brings substrate to Hsp70 and stimulates its ATPase activity, inducing a shift to a high substrate affinity form of Hsp70. Exchange of ADP to ATP on Hsp70 by a nucleotide exchange factor co-chaperone (blue) causes a decrease in substrate affinity, leading to substrate release and allowing the substrate to fold. Figure adapted from (70).

Hsp70 co-chaperones:

The two main co-chaperone types important for Hsp70 function include the J-domain proteins, and nucleotide exchange factors. J-domain proteins, also known as the Hsp40s or

DNAJs, are characterized by their conserved J-domain (homologous to Escherichia coli DnaJ), which stimulates ATP hydrolysis by Hsp70 and thereby promotes substrate binding. Many J- domain proteins can also bind to unfolded proteins and deliver them to Hsp70 for refolding or degradation (71). The ability of J-domain proteins to promote polypeptide refolding by Hsc70 has been shown both in vitro and in cultured cells, with different J-domain proteins promoting varying degrees of substrate folding. Previous work from our lab and others found that DNAJA2 was most effective at promoting luciferase refolding after stress and maintaining the activity of mutant cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, while

DNAJA1 could not (72-75). However DNAJA1 is important for the biosynthetic folding of

CFTR and the human ether-a-go-go related gene (hERG) potassium channel (76-78).

In contrast, nucleotide exchange factors facilitate the exchange of Hsp70-bound ADP to

ATP, permitting the release of substrate proteins from Hsp70. Three different families of NEFs exist, the Hsp110s, the Bag proteins, and the HspBP1 family (79-82).

The Hsp110 co-chaperones are evolutionarily divergent members of the Hsp70 family.

There are three cytoplasmic isoforms expressed in humans, Hsp110 (Hsp105), Apg1 and Apg2

(81,83,84). Structurally, Hsp110 contains an N-terminal nucleotide-binding domain (NBD), a beta-sandwich domain, and a three-alpha-helix bundle domain (85). Co-crystallization studies using Hsp110 from Saccharomyces cerevisiae and human or yeast Hsp70-NBD show that the

Hsp110 helix bundle and NBD contact the Hsp70 NBD, stabilizing it in an open conformation and permitting nucleotide to dissociate (85,86). While the putative Hsp110 SBD has been

25 reported to bind substrate, its role in biological function remains unclear (87-90). Additionally,

Hsp110, in cooperation with DNAJB1, DNAJA2 and Hsp70, has been shown to be involved in protein disaggregation (Figure 1.3) (91,92).

The five members of the Bag family of NEFs, numbered Bag1-5, are characterized by their conserved C-terminal Bag domain that lends them their regulatory activity towards the

ATPase domain of Hsp70 (79,93,94). Bag1 was identified first, in complexes with the anti- apoptotic protein Bcl-2 and preventing cell death under a variety of stresses (95). Bag1 was further characterized as the first eukaryotic NEF, with the identification of its four other family members shortly after (80,94). A crystallographic structure of Hsc70-NBD and the Bag domain of Bag1 indicated that the Bag domain forms a three-helix bundle that binds the Hsc70-NBD, forcing it into a conformation unsuitable for nucleotide binding and promoting nucleotide dissociation (93).

However, within the family, the BAG proteins have diverse domains and structures connecting them to various cellular processes (79,94). Bag1 contains an ubiquitin-like (Ubl) domain that has been shown to interact with the proteasome, and thus Bag1 in complex with

Hsp70 may target substrate proteins for degradation in this manner (described in detail below)

(96,97). Bag4 was found to bind tumor necrosis factor receptor 1 and death receptor 3, preventing apoptosis. Interestingly, its BAG domain is several helix turns shorter than that of

Bag1, suggesting it belongs to a separate BAG subfamily (98,99). Bag3, the subject of this thesis, will be described in more detail below.

Small heat shock proteins (sHsps):

Another major component of the PQC network is the small heat shock protein family

(sHsps). Comprised of ten members in humans (HspB1-10), sHsps are believed to act as

“holdases”, binding to aberrant proteins and preventing their aggregation so they can be refolded or degraded by other pathways. Lacking an ATPase activity, sHsps are unable to actively promote refolding (100-104). Members of the sHsp family are characterized by their conserved central α-crystallin domain (ACD) and their relatively low monomeric molecular weight, between 16 and 25 kDa (101,103). They form homodimers through the ACD, and some of them also form larger oligomers. Their unstructured N- and C-terminal regions are less conserved, but are essential for sHsp oligomerization (101,103,105). Oligomeric sHsps contain an IxI/V motif in their C-terminal tails that interact with a hydrophobic groove formed by two β-strands in their

ACDs, allowing oligomerization. Additional interactions between N-terminal tails of sHsp subunits further stabilize their oligomerization (106,107). Within the cell, the structural states of sHsps can range from dimers to large oligomers of around 24 subunits (101,107). However, some sHsps, such as HspB8 and HspB6, lack the IxI/V motif, and cannot form large oligomers

(106).

Oligomeric state changes have been previously shown to be integral for sHsp chaperone function, with transitions to lower-molecular weight oligomers/dimers required for its activity

(108). It has also been shown that in cooperation with Hsp70, many sHsps are able to prevent aggregation of and refold model substrates, such as citrate synthase and luciferase (109-113).

Interestingly, the precise mechanism in which sHsps prevent aggregate-induced cytotoxicity varies within the family. Only HspB1 (Hsp27), HspB3, and HspB5 (α-B crystallin) were shown to assist in the active refolding of firefly luciferase after heat shock. However, despite their lack of luciferase refolding, HspB6, HspB7, HspB8, and HspB9 prevented aggregation and associated toxicity of Huntingtin polyQ aggregates, suggesting these two subgroups have separate mechanisms in which they prevent protein aggregation-induced toxicity (114,115). sHsps are

27 able to refold denatured luciferase Other than their holdase functions, several members of the sHsp family have been linked to modulation of cytoskeleton stability, assembly, and disassembly, with further implications in cell division and cytoskeletal maintenance under muscle cell tension (116-119). Particularly, the diversity of sHsp expression in skeletal and cardiac muscle is the highest among all tissues, including HspB1, B2, B3, B5, B6, B7, and B8

(115). HspB4 is only expressed in the eye lens, and HspB9 and B10 in the testis. Other than muscle, HspB1, B5, B6, and B8 are also expressed in a diverse set of tissues, such as the brain, colon, kidney, liver, lung, and stomach (115). Interestingly, mutations in four of these proteins are associated with neuromuscular-degenerative disorders, with mutations in HspB1, HspB3, and

HspB8 causing motor neuropathies, while HspB5 mutations have been linked to myopathy (120-

123).

Protein degradation:

Ubiquitin proteasome system (UPS):

One of the two major mechanisms of protein degradation in the cell, the ubiquitin proteasome system (UPS) involves the conjugation of ubiquitin polypeptide to lysine residues of target proteins and onto itself in long chains, hereby marking the proteins for degradation by the

26S proteasome (124,125). The proteasome unfolds these polyubiquitinated substrates, and channels them into its internal degradative chamber for proteolysis, yielding ubiquitin monomers and amino acids for recycling (62). The ubiquitination of protein targets involves three major enzyme types working in series, namely the E1, E2, and E3 enzymes. E1 enzymes activate ubiquitin into a high-energy intermediate state that is able to label targets. The charged ubiquitin is then transferred to E2-carrier proteins that deliver them to E3 ligases, which bind to specific targets and mark them with ubiquitin for proteasomal degradation (62).

A particularly important E3 ligase in the Hsp70 system is the Carboxy-terminus of Hsp70

Interacting Protein (CHIP) (126). CHIP contains a tetratricopeptide repeat (TPR) domain, allowing interaction with the C-terminal EEVD motifs of Hsp70 and Hsp90, another ATP- dependent chaperone family with different structures and mechanisms than Hsp70 (58,126,127).

Additionally, CHIP’s U-Box domain facilitates interaction with E2 ubiquitin-conjugating enzymes (128). As a result, CHIP is able to ubiquitinate chaperone substrates, targeting them for proteasomal degradation rather than refolding (127,129,130). This occurs through the involvement of the Hsp70 NEF Bag1, which is able to bind CHIP and transfer Hsp70 substrates to the CHIP ubiquitination machinery (96). In contrast, Bag2 has been shown to be an inhibitor of the E3 ubiquitin ligase activity of the Hsp70 cochaperone CHIP.

Autophagy:

The term “autophagy” encompasses a number of processes that involve the eventual degradation of proteins by the lysosome, such as macroautophagy, microautophagy, and chaperone-mediated autophagy (131,132). Macroautophagy, referred to as “autophagy” in this thesis, represents a major means of protein and organelle degradation, complementing the activities of the UPS (131). This process involves the concerted actions of ATG proteins in initial formation of a cytoplasmic double-membrane, called a phagophore, near the target proteins or organelle (133). A particularly important protein at this step is ATG8/LC3, which is initially cleaved to form a cytoplasmic LC3-I (134). This cleavage allows the conjugation of phosphatidylethanolamine to the LC3-I molecule, forming LC3-II and permitting the insertion of

LC3-II into the phagophore membrane (135,136). Due to its localization within the phagophore membrane, LC3-II is a classical marker of autophagy, and its generation can be used as a measure of autophagic flux (131). Additionally, the autophagic adaptor protein p62/SQSTM1,

29 which binds ubiquitinated material, is able to interact with LC3-II, and can hereby target ubiquitinated substrates to degradation via autophagy (137,138). The phagophore is then extended to encapsulate the entirety of the target cytoplasmic material, forming the autophagosome. This autophagosome is able to fuse with the lysosome, an organelle containing acidic proteases and hydrolases. This fusion event leads to the breakdown of the target cargo

(131-133,139). These degraded materials can be recycled for use in other cellular processes, which highlights why autophagy is induced during cellular starvation (139,140). Additionally, due to the ability of the phagophore to engulf large cargos, autophagy has major roles in the degradation of large aggregates and organelles that otherwise cannot be unfolded and degraded by the UPS (141-143). Interestingly, CHIP, in complex with Bag3, HspB8, and Hsp70, was also recently found to be involved in specific protein degradation through autophagy (144). This evidence presents CHIP as a master controller of Hsp70-substrate degradation, able to degrade substrates either through the proteasome or lysosome depending on its associations.

The two lesser-characterized forms of autophagy within the cell are microautophagy and chaperone-mediated autophagy, briefly described below. In microautophagy, the lysosome directly captures cytoplasmic regions for degradation by invagination of the lysosomal membrane, forming an “autophagic tube”, with the circular tip towards the center of the lysosomal lumen. Vesicles containing cytoplasmic material are formed at the tip of the autophagic tube, which are eventually budded and degraded fully in the lysosomal lumen. This process is generally non-selective, though alternative selective forms of microautophagy do exist

(145).

Chaperone-mediated autophagy (CMA) involves the translocation of specific Hsc70 substrates containing a KFERQ motif directly across the lysosomal membrane. The Hsc70-

30 bound substrate then interacts with the lysosomal transmembrane protein lysosome-associated membrane protein type 2A (LAMP2A), which oligomerizes to form a translocation complex to channel the substrate into the lysosome. Hsc70 assists in this process by unfolding the substrate, which is required for translocation. Additionally, a lysosomal form of Hsc70 assists in translocation, though its role remains unclear (146,147).

Bag3:

Bag3 (Bcl-2-associated athanogene 3) is a 76 kDa protein with a multitude of functions in multiple cellular pathways. Also known as Bis or CAIR-1, Bag3 was initially characterized as an interactor of Bcl-2, an inhibitor of apoptosis (148). Through this interaction, Bag3 was shown to synergistically assist Bcl-2 in resisting Bax and Fas-induced apoptosis (148). Further work identified Bag3 as a member of the BAG family of Hsp70 NEFs (93,94). Expressed highly in skeletal muscle, Bag3 has been shown to be required for the maintenance of muscle survival in mouse models and C2C12 mouse myotubes (149). In humans, various mutations in Bag3 have been linked to dilated cardiomyopathy and myofibrillar myopathy (6,150).

Bag3 contains numerous domains, leading to its role in a number of cellular pathways

(Figure 1.5). From N- to C-terminus, Bag3 contains a WW domain, two IPV motifs, a proline- rich (PXXP) region, and a Bag domain. As with other members of the Bag family, the conserved

Bag domain binds the N-terminal ATPase domain of Hsp70 and acts as a nucleotide exchange factor (NEF) as described previously (151). Via its IPV motifs, Bag3 maintains further connections to the chaperone system through interactions with sHsps, particularly HspB8, its strongest-binding sHsp interactor (106). Bag3’s WW domain and PXXP region allow interactions with proteins containing proline-rich sequences and Src-homology-3 (SH3) domains,

31 respectively (152). The diversity of these domains establishes Bag3 as a “scaffold” protein with involvement in a multitude of cellular pathways.

Figure 1.5: The major domains of Bag3 A brief overview of the various domains of Bag3 and their associated functions. Each domain is colored and point to their specific interactors. Each Bag3 interactor points towards their highlighted functions in the context of their Bag3 interaction. Numbers above each domain denote their starting and ending amino acid. These functions will be described in further detail below.

Domains of Bag3:

IPV motifs:

The two IPV motifs in Bag3 permit its interactions with sHsp chaperones and are found in the flexible region between its N-terminal WW domain and C-terminal BAG domain (106).

Dimeric HspB8 and HspB6 do not have IXI/V motifs, but still have the conserved groove in their

ACDs that can bind the motifs. Structurally, Bag3’s IPV motifs bind these sites in HspB8 and

HspB6 (106). Of all the sHsps, HspB8 is the strongest sHsp interactor, though other sHsps also interact with Bag3 weakly (106,153). Together with the Bag domain-mediated interaction with

Hsp70, Bag3’s interaction with HspB8 is an important component of the Bag3-mediated

“chaperone-assisted selective autophagy” (CASA) pathway, which will be discussed in detail further in this review. Interestingly, the Bag3-P209L mutation involved in myofibrillar myopathy targets the second IPV motif, though the mechanism in which the mutation causes the disease is unclear and will be discussed later in this review (6).

WW domain:

Bag3 contains an N-terminal WW domain, allowing interaction with proline-rich sequences and is required for autophagy initiation in glioma cells (152,154). Interactors of Bag3 via its WW domain include CASA component synaptopodin-2 (SYNPO2), the tuberous sclerosis proteins (TSC) complex involved in nutrient sensing, and Hippo pathway components LATS1 and AMOTL1, all of which are described later in this review.

PXXP domains:

In addition to its role in stimulating autophagy, Bag3 is involved in assisting in the transport of aberrant proteins to the aggresome (142). The term “aggresome” describes inclusion bodies of protein aggregates found at the microtubule-organizing center (MTOC) near the

33 nucleus. These structures are thought to prevent the accumulation of aggregates throughout the cell by concentrating aberrant proteins in a single location via retrograde transport along microtubules, and hereby allowing for their efficient sequestration and degradation (155,156).

Bag3, using its PXXP domain, is able to interact with the microtubule motor protein dynein, and thus connect Hsp70 and its bound substrates to the aggresome-formation pathway and subsequent autophagic degradation in the CASA pathway (described below) (142). Bag3’s

PXXP domain also interacts with the SH3 domain of PLC-γ, involved in signal transduction, suggesting Bag3 has a role in EGFR-PLC-γ pathway (151). However, further work on this function has yet to be published.

Bag3 functions:

Chaperone assisted selective autophagy (CASA):

One of Bag3’s most well-described functions is the role of the Hsp70-Bag3-HspB8 complex in the “chaperone-assisted selective autophagy” (CASA) of Hsp70 substrates (Figure

1.6). Bag3 links the Hsp70 chaperone network to autophagic machinery by interacting with p62/SQSTM1, which binds ubiquitinated substrates and recruits the autophagosome membrane to engulf these substrates (138,143). In addition to p62/SQSTM1, a required Bag3 interaction for proper CASA function is that with synaptopodin-2/myopodin (SYNPO2) (119). Originally known as an actin-bundling and cytoskeletal adapter protein at the Z-disk in skeletal muscle,

SYNPO2 forms an essential complex with VPS16 and VPS18, two proteins associated with membrane tethering and autophagsome-lysosome fusion (119,157-159). Knockdown of Bag3 or

SYNPO2 led to a reduction in autophagosome formation in A7r5 rat smooth muscle cells, suggesting SYNPO2 maintains a critical role in autophagy during the Bag3-mediated CASA pathway (119). Hence, in concert with its aggresome formation function, a CASA model

34 emerges where Bag3 mediates targeting of Hsp70/HspB8 substrates to the aggresome via transport by dynein motor proteins. Once at the aggresome, Bag3 is able to induce autophagy of these targets by recruiting p62/SQSTM1 and SYNPO2, leading to engulfment of aggresomal targets by the phagophore and their subsequent degradation by macroautophagy (Figure 1.6)

(142,156).

In line with the CASA model, overexpression of Bag3 or HspB8 led to increased degradation of the aggregation-prone model protein huntingtin-exon 1 with a 43-polyglutamine repeat (Htt43Q), along with an increase in LC3-II, indicating that Bag3 stimulates macroautophagy of aberrant proteins. In contrast, Bag3 knockdown caused increased accumulation of Htt43Q (160). The autophagic degradative mechanism of Bag3 complements that of Bag1, which is thought to stimulate degradation of ubiquitinated proteins via the proteasome (96,97). Interestingly, in human primary cells and mice brain models of aging, Bag1 is expressed at higher levels in young tissue compared to Bag3, but Bag3 expression transitions to be higher than that of Bag1 in aged tissues, suggesting a shift of ubiquitinated protein degradation from a proteasome-mediated mechanism to autophagy (143).

The importance of Bag3 and its roles in autophagy are particularly highlighted in the context of skeletal muscle, where Bag3 assists in maintenance of the Z-disk under stress during muscle contraction. The Drosophila melanogaster ortholog of Bag3, Starvin (Stv), was shown to be essential for muscle maintenance throughout the Drosophila lifespan, with reductions in Stv levels causing Z-disk disintegration, and overall progressive muscle defects. This phenotype is consistent with that seen in Bag3-knockout mice (described in detail later) (144,149). Hence, both in Drosophila and mammalian muscle cells under contraction, Stv/Bag3, in complex with

Hsp70, HspB8, and CHIP, associate with SYNPO2 and p62, and are able to target substrates,

35 such as the cytoskeletal protein filamin, to autophagic degradation via the muscle-essential

CASA pathway (119,144). Other targets of this pathway include Htt43Q and SOD1-G85R, linked to the neurodegenerative syndromes Huntington’s disease and amyotrophic lateral sclerosis (ALS), respectively (61,142,160).

Figure 1.6: Chaperone-assisted selective autophagy Certain ubiquitinated substrates can be targeted to the aggresome by a Hsp70-Bag3-HspB8 complex, which moves along microtubules through the action of dynein motor proteins. Bag3 can then recruit p62/SQSTM1 and SYNPO2 to the aggresome. p62, bound to the phagophore, will bind the ubiquitinated substrates and bridge them to the phagophore double-wall membrane.

This allows the engulfment of the aggresome to form an autophagosome, which can be fused with a lysosome and its contents subsequently degraded.

Hippo pathway regulation:

Bag3 modulates the activity of the Hippo pathway co-transcriptional activators YAP and

TAZ through its WW domain interactions with the YAP/TAZ inhibitors LATS1 and AMOTL1

(119). Amongst the many genes found to be upregulated by YAP/TAZ is filamin A, which cross- links actin filaments and is vital for cell adhesion and migration (119,161). Hence, through attenuation of LATS1 and AMOTL1, Bag3 is able to promote filamin A transcription through the YAP/TAZ pathway (119). Interestingly, Bag3 also regulates the autophagic degradation of filamins A and C via the CASA pathway, indicating a dual role of Bag3 in filamin regulation in mechanically stressed cells (119,144).

Nutrient sensing and protein synthesis:

Bag3 maintains the ability to spatially coordinate the activity of mammalian target of rapamycin complex-1 (mTORC1), a catabolic/anabolic master regulator in the cell that among other functions, regulates protein translation (162,163). Through its WW domain, Bag3 is able to interact with the tuberous sclerosis proteins (TSC) complex, a regulator of mTORC1. In muscle cells under mechanical stress, the Bag3-TSC complex is recruited to the cytoskeleton and locally promotes autophagy and inhibition of protein synthesis through inhibition of mTORC1. In contrast, mTORC1 in the cytoplasm separated from the Bag3-TSC complex remains active and is able to stimulate protein translation and repair in response to the damage induced by the muscle contraction (163). In addition to its role in CASA, Bag3’s role in nutrient regulation further highlights its importance in skeletal muscle maintenance and its associated diseases (6).

Bag3 and stress granules:

Other than its functions in aggresome targeting and CASA, it was recently discovered the

HspB8-Bag3-Hsp70 complex plays a role in stress granule regulation and disassembly (164).

Stress granules (SGs) are cytoplasmic membrane-less compartments containing mRNA and specific RNA-binding proteins, transiently formed during periods of acute stress (165).

Cytotoxic stresses can lead to translation inhibition and the suppression of mRNA into SGs

(166,167). This translational inhibition can occur through various pathways, such as mTOR inhibition or phosphorylation of initiation factor eIF2α (165,168). With this general translation inhibition, the cell is able to conserve its nutrients for cell survival and recovery after stress

(169). Interestingly, mRNAs encoding proteins related to the stress response are excluded from

SGs, and hence are able to be translated (166). When stress is relieved, SGs disassemble in an

Hsp70-dependent process, allowing normal translation to resume (170).

Stress granules are highly dynamic in nature, such that SG mRNAs and proteins can shuttle between the SG and the cytoplasm for translation (167,171). It has been recently suggested that SGs exist in a phase-separated liquid droplet state due to weak interactions involving low complexity domain (LCD) proteins and RNA (172,173). However, mutations in

SG-sequestered RNA-binding proteins, such as FUS and hnRNPA1, can induce the aggregation of SG-sequestered proteins over time into insoluble inclusions, possibly contributing to disease phenotypes like amyotrophic lateral sclerosis (ALS) (172,174). Interestingly, the Hsp70-Bag3-

HspB8 complex is involved in the clearance of incomplete polypeptides, coined “defective ribosome products” (DRiPs) that would otherwise accumulate in SGs and promote their aggregation when the cell’s protein quality control machinery is impaired (164,175). Knockdown of HspB8 or Bag3 slows SG disassembly during recovery after stress. Cells depleted of HspB8

38 or Bag3 also show an increase in SGs containing DRiPs as well as SGs containing mutant FUS and hnRNPA1 (164). However, the exact mechanism in which aberrant DRiPs are extracted from SGs or SGs disassembled remains unknown (164).

Bag3 and myofibrillar myopathy:

Mutations linked to myofibrillar myopathy (MFM) have been found in the Z-disk proteins desmin, α-B crystallin (CRYAB, HspB5), myotilin, Z-band alternatively spliced PDZ motif-containing protein (ZASP), four and a half LIM domain 1 (FHL1), and filamin C (FLNC) as well as Bag3 (4-6,8,120,176). Of these proteins, Bag3 is among the few that do not have a direct structural role at the Z-disk (60,61,160). Instead, Bag3 likely assists in the maintenance of the Z-disk through its links to the protein quality control pathway and CASA (142,149).

Blockages in Bag3-mediated CASA pathway have been linked to MFM caused by mutant

FLNC-W2710X (5,10). In zebrafish, expression of FLNC-W2710X leads to aberrant protein aggregation at the Z-disk. However, the FLNC mutant remains able to prevent fiber disintegration induced by FLNC knockout (10). Bag3 was also found to be sequestered within

FLNC-W2710X aggregates, implicating subsequent changes in Bag3 function as a mechanism for the disease. Surprisingly, knockdown of Bag3 promoted the clearance of mutant FLNC aggregates in an autophagy-dependent manner, suggesting a potential blockage of CASA as the main driver of the disease phenotype (10).

In mice, Bag3 was shown to be highly expressed in skeletal and cardiac muscle. Bag3-/- mice were significantly smaller than their heterozygous or wildtype littermates, with all Bag3-/- mice exhibiting early lethality by Day 25 (149). Tissue sections from Bag3-/- mice showed atrophic and varied-size myofibers. These myofibers showed increased apoptosis and Z- disk/myofibril disruption by Day 14, suggesting a critical role of Bag3 in Z-disk maintenance.

Consistent with this, knockdown of Bag3 in mouse C2C12 myoblasts caused increased apoptosis upon differentiation and decreased overall differentiation efficiency (149).

It has been proposed that the Bag3 mediates an interaction between Hsc70 and F-actin capping protein subunit beta (CapZβ1), thereby preserving Z-disk structure (177). CapZβ1 regulates the length of actin filaments at the Z-disk, a process vital for proper muscle function and development (178). Inhibition of CapZβ1 or Bag3 in rat neonatal cardiomyocytes led to myofibril disruption when the cells were subjected to mechanical strain. Knockdown of Bag3 destabilized CapZβ1 levels by promoting its degradation by the proteasome (177). Thus, Bag3 emerges as an important protector of Z-disk integrity through regulation of actin polymerization as well as protein quality control via the CASA pathway (144,177).

The P209L mutation in Bag3 causes an autosomal dominant and childhood-onset form of myofibrillar myopathy (6,9). Unlike other forms of MFM, such as those caused by ZASP or filamin mutations, Bag3-P209L myopathy appears in patients at an early age, with rapid development of symptoms and fatality by the second decade of life (5,6,179). As with a majority of myofibrillar myopathy patients, these patients exhibited progressive muscle weakness, cardiomyopathy, and respiratory insufficiency. Rigid spines and peripheral neuropathy were also present, but not among all patients. Analysis of skeletal muscle tissue showed Z-disk aberrations, debris accumulation, and myofibril disruption, with apoptotic nuclei present (6). However, the molecular mechanism by which the P209L mutation causes the disease phenotype is unknown.

Interestingly, the P209L mutation of Bag3 targets the proline residue within its second IPV motif, suggesting that Bag3 interaction with its sHsp interactors, such as HspB8, may be disrupted (106). Though deletion of one or both IPV motifs has been shown to reduce or prevent sHsp binding, it remains unclear if the P209L mutation causes the same effect (180).

Limited progress has been made elucidating the molecular mechanism behind Bag3-

P209L MFM. Zebrafish models expressing fluorescent Bag3-P209L exhibited significant Bag3-

P209L aggregates at the Z-disk in muscle tissue. Despite this, fluorescence recovery after photobleaching (FRAP) studies show that Bag3-P209L has no significant difference in recovery dynamics compared to Bag3-WT. Curiously, co-expression of Bag3-P209L with wildtype Bag3 or FLNC showed suppression of these proteins into Bag3-P209L aggregates (9). It is suggested that perhaps the P209L mutation, targeting Bag3’s second IPV motif, reduces its interactions with HspB8 and prevents proper CASA function (106). However, zebrafish models overexpressing Bag3-WT show no increase in aggregation when autophagy is inhibited or stimulated, indicating that autophagy inhibition alone is not responsible for the Bag3-P209L aggregation phenotype. Treatment of autophagy inhibitors and stimulators in Bag3-P209L models exhibit a slight increase and large decrease in aggregation respectively, indicating that

Bag3-P209L aggregates are actively degraded by autophagy and autophagy stimulation may be a possible path for therapy. Bag3-P209L overexpression is surprisingly able to rescue the myofibril destruction phenotype in Bag3-knockout zebrafish, showing that Bag3-P209L remains mostly functional. Knockdown of endogenous Bag3 in Bag3-WT or Bag3-P209L-overexpressing cells did not change the number of aggregate-containing cells, showing that the presence of

Bag3-P209L, rather than the loss of Bag3, is the driver of the aggregation phenotype. This evidence suggests a potential gain-of-function model in which Bag3-P209L acquires a susceptibility to aggregate, leading to a reduction in functional Bag3. This reduction is further exacerbated by sequestration of Bag3-WT within aggregates, leading to the disease phenotype

(9). Interestingly, this mechanism is similar to that proposed by the same authors for FLNC-

W2710X myofibrillar myopathy, highlighting the hypothesis that the progressive muscle

41 weakness seen in MFM patients is due to the gradual loss of Bag3 function due to suppression of

Bag3-WT in Bag3-P209L aggregates (9,10).

Objectives and Rationale:

Other than initial zebrafish work proposing a model of Bag3-P209L aggregate suppression of Bag3-WT, very little published work exists characterizing the mechanisms and effects of the P209L mutation on Bag3 function and interaction (9). The P209L mutation targets the second of Bag3’s two IPV motifs critical for sHsp binding, particularly HspB8. Additionally, immunoprecipitation data from our collaborator Josée Lavoie (Université Laval) showed that

Bag3-P209L does not completely prevent HspB8 binding (unpublished). We hypothesize that the

P209L mutation disrupts the normal interactions of Bag3 with its various skeletal muscle interactors by suppressing them into aggregates. To assess this, we utilized a proteomics approach using BioID and phosphoproteomics in C2C12 myoblasts. BioID involves tagging a protein of interest with a promiscuous biotin ligase, causing biotinylation of proteins in close proximity. Biotinylated proteins can then be captured using streptavidin resin and identified through mass spectrometry. Since the streptavidin-biotin interaction is incredibly strong, it permits the usage of harsh solubilization buffers during streptavidin pulldown, allowing the identification of weak or insoluble interactors missed in immunoprecipitations (181). In this case,

BioID allows identification of soluble and insoluble Bag3 interactors that may be disrupted or gained by the P209L mutation. Phosphoproteomics was used to evaluate if the P209L mutation affected Bag3 phosphorylation, which may further contribute to the disease. Overall, we wish to find insight into the molecular mechanisms in which Bag3-P209L causes myofibrillar myopathy, and identify potential avenues for therapeutic intervention.

Chapter 2: Results

In vitro characterization of the Bag3 and HspB8 interaction:

Previous literature showed the two IPV motifs of Bag3 mediate its interactions with

HspB8, with the Bag3-P209L mutation targeting the second IPV motif (106). We initially hypothesized that the P209L mutation disrupts HspB8 binding, destabilizing Bag3-P209L and subsequently leading to its aggregation. To answer this, we affinity purified wildtype Bag3

(Bag3-WT), Bag3-P209L, and wildtype HspB8 from bacteria. This work faced several major technical challenges, particularly poor protein stability and inconsistency between purifications

(data not shown). However, we were able to eventually purify both Bag3 and HspB8 with moderate yields and purity to begin our assays.

Our approach utilized circular dichroism spectroscopy (CD) and intrinsic fluorescence to assess the Bag3-HspB8 interaction. CD spectra of purified Bag3-WT, Bag3-P209L, and wildtype

HspB8 showed evidence of protein secondary structure, indicating the purified proteins were able to fold, as illustrated by the predicted secondary structure percentages given by deconvolution of the CD spectra (Figure 2.1A,B). The percentages in Figure 2.1B do not sum to

100%, highlighting their role as approximations, rather than precise measurements, of protein secondary structure. Nevertheless, when HspB8 and Bag3 were mixed together in a 2:1 stoichiometric ratio, the resulting spectrum showed no significant differences when compared to the additive sum of the individual Bag3 and HspB8 component spectra (Figure 2.1A). Thus, when mixed, neither Bag3 nor HspB8 have detectable secondary structure changes, suggesting these proteins do not interact or their interaction does not cause secondary structure changes.

This finding was also seen in CD experiments using HspB8:Bag3 ratios of 1:1 and 4:1 (data not shown).

Consistent with the CD experiments, intrinsic fluorescence assays also detected no significant changes in tryptophan fluorescence when stoichiometric amounts of Bag3 and HspB8 were mixed together (Figure 2.1C), corroborating the possibilities that these proteins do not interact or do not interact in a manner that affects intrinsic tryptophan fluorescence.

Due to the nature of CD spectroscopy and intrinsic fluorescence experiments as indirect assays of protein interaction, we could not conclude the absence of Bag3-HspB8 interaction or if their interaction did not cause any detectable structural change. To address this directly, we utilized isothermal titration calorimetry (ITC) and streptavidin pulldown assays using biotinylated Bag3 and synthetic Bag3 peptides. However, significant non-specific HspB8 binding rendered pulldowns with either full-length Bag3 or Bag3 peptide inconclusive (data not shown). Preliminary ITC trials showed no evidence of HspB8 binding to Bag3 (data not shown), contrary to published work at the time (106). Additionally, co-immunoprecipitations performed by our collaborator Dr. Josée Lavoie showed Bag3-WT interacts with HspB8, and the P209L mutation does not significantly affect HspB8 binding (recently published (182)). Compounded by emerging work showing Bag3 directly interacts with HspB8, we hypothesized that the Bag3-

P209L mutation affects its binding with skeletal muscle interactors other than HspB8, and this disruption leads to the MFM phenotype (180).

Figure 2.1: Circular dichroism and intrinsic fluorescence spectra of Bag3 and HspB8 (A). CD spectra of 3μM purified Bag3 (blue), 6μM purified HspB8 (red), or a mixture of 3μM Bag3 and 6μM HspB8 (green). If mixing Bag3 and HspB8 does not induce secondary structure changes in either protein, the spectrum of their mixture should match the theoretical CD spectrum (purple) representing the sum of the individual 3μM Bag3 and 6μM HspB8 spectra. (n=6) (B). Deconvolution of the CD spectra shown in (A) into approximate percentage estimates of secondary structure. Antiparallel and parallel refer to different classes of β-sheets. (C). Intrinsic fluorescence spectra of 2μM Bag3 (blue), 4μM HspB8 (red) or a mixture of 2μM Bag3 and 4μM HspB8 (green). The additive sum of the individual Bag3 and HspB8 spectra is plotted as a theoretical spectrum (purple). (n=3)

Bag3-P209L aggregates in C2C12 cells:

Previous published work in zebrafish show expression of Bag3-P209L-eGFP leads to its aggregation (9). To establish if this phenotype occurs in C2C12 cells, we overexpressed Bag3-

WT and Bag3-P209L C-terminally tagged with either a 3xFLAG epitope tag or a GFP tag. The

C2C12 mouse myoblast cell line was used in our study because it better represents muscle physiology compared to other common cell lines used to address chaperone function, such as

HEK293. Transient transfection of Bag3-WT-3xFLAG and Bag3-P209L-3xFLAG showed strong expression of exogeneous Bag3 able to be detected in Western blots by antibodies specific for the 3xFLAG epitope or Bag3 (Figure 2.2A). Overexpression of Bag3-WT showed a majority of expressed Bag3 is maintained in the soluble fraction with a small amount trapped in the insoluble pellet. Conversely, overexpression of Bag3-P209L exhibited major suppression of

Bag3-P209L into the insoluble fraction, consistent with previous findings in the literature (9)

(Figure 2.2A). Fluorescence microscopy of C2C12 myoblasts transfected with Bag3-WT-GFP shows few or no Bag3-GFP aggregates, whereas Bag3-P209L-GFP expression leads to cells containing numerous Bag3-GFP cytoplasmic aggregates (Figure 2.2B), confirming some degree of the Bag3-P209L seen in the insoluble pellet by Western blot are aggregates, and that overexpression of Bag3-WT and Bag3-P209L can serve as a model to study Bag3-P209L behavior in C2C12 cells.

Figure 2.2: Bag3-P209L aggregates in C2C12 cells. (A). C2C12 myoblasts were transfected with varying amounts of Bag3-WT-3xFLAG or Bag3- P209L-3xFLAG DNA and harvested 48 hours after transfection. Control cells were transfected with empty pcDNA 3.1 vector. Representative 2 μg pcDNA control lane shown. (B). C2C12 myoblasts were transfected with Bag3-WT-GFP or Bag3-P209L-GFP. 48 hours after transfection, cells were fixed and imaged under a 63X magnification.

JG-98 does not have an effect on Bag3-P209L aggregation:

Unpublished work presented by Dr. Jason Gestwicki (University of California, San

Francisco) showed deletion of the BAG domain in Bag3-P209L rescue its solubility in HEK293 cells. This effect was also seen with BAG domain point mutations preventing Hsp70 binding, as well as after treatment with the small molecule Hsp70 inhibitor JG-98. JG-98 is an allosteric inhibitor of Hsp70, stabilizing its ADP-bound form and thereby inhibiting its substrate cycling.

Additionally, it has been shown that treatment with JG-98 is able to prevent the Hsp70-Bag3 interaction (183,184). To assess if the rescue effect is seen in our model, we treated C2C12 myoblasts transiently transfected with empty vector, FLAG-Bag3-WT or FLAG-Bag3-P209L with 1 μM JG-98 and 3 μM JG-98 (Figure 2.3). Consistent with our previous data (Figure 2.2), overexpression of Bag3-WT does not lead to significant accumulation in the insoluble fraction, whereas Bag3-P209L overexpression led to a majority of Bag3-P209L being trapped in the insoluble fraction (Figure 2.3A). However, treatment with 1 μM or 3 μM JG-98 did not lead to a significant change in the amount of Bag3 in either fraction (Figure 2.3), suggesting that contrary to previous findings in HEK293 cells, JG-98 treatment does not rescue the solubility of Bag3-

P209L in C2C12 myoblasts. Fluorescence microscopy of Bag3-GFP-transfected C2C12 cells showed cytoplasmic distribution of Bag3-WT and significant Bag3-P209L aggregation consistent with Figure 2.2 (Figure 2.4). Treatment with JG-98 either did not change Bag3-P209L aggregation, or caused Bag3-P209L to localize around the nucleus (Figure 2.4). However, this experiment remained inconclusive due to few fluorescent cells detected, most likely caused by cell toxicity induced by the combined stresses of DNA transfection and JG-98 treatment.

Figure 2.3: JG-98 does not rescue Bag3-P209L solubility (A). Representative Western blot of the experiments quantified in Figure 2.3B/C. C2C12 myoblasts were transfected with empty pcDNA3.1 vector, Bag3-WT-FLAG, or Bag3-P209L- FLAG plasmids. 24 hours after transfection, cells were treated with DMSO or JG-98 for 24 hours prior to harvest and Western blot. (B) and (C). Quantification of (A), relative to the amount of Bag3 in the non-treated control lane (n=5). NT = non-treated control. Error bars represent one standard deviation.

Figure 2.4: JG-98 does not significantly affect Bag3-P209L aggregation. C2C12 myoblasts were transfected with plasmids encoding GFP-tagged Bag3-WT, Bag3-P209L, or empty GFP. 24 hours after transfection, cells were treated with 3 μM JG-98 or DMSO for 24 hours prior to fixing and mounting. Cells were imaged under a 63X magnification.

Bag3-P209L shows a reduction in phosphorylation:

Phosphorylation of specific protein residues are widely known to induce changes in protein behavior (185). Specific phosphorylation of Bag3 at Ser-187 in Chinese hamster ovary cells has been linked to regulating Bag3’s role in epithelial-mesenchymal transitions (186). A number of other phosphorylation sites were found in large scale analyses, but have not been validated (187-190). To assess if the P209L mutation of Bag3 changes its phosphorylation state, we utilized a proteomics approach studying Bag3-WT-3xFLAG and Bag3–P209L-3xFLAG after

FLAG immunoprecipitation and titanium dioxide (TiO2) enrichment.

Phosphorylation at Bag3’s Thr-406 in the PXXP region was the most abundant phosphorylation detected in both wildtype and P209L-mutated Bag3 (Table 2.1). Other phosphorylation sites were between the WW domain and PXXP (residues 55-302), or in the

PXXP region (residues 302-412). Phosphorylation at Ser-289, which was readily detected in

Bag3-WT-3xFLAG, was not found in Bag3-P209L (Table 2.1). Significantly fewer total phosphopeptides were detected in Bag3-P209L-3xFLAG samples, as particularly reflected in the counts of T406-phosphorylated peptides and S194/195 (S194 or S195) phosphorylated peptides

(Table 2.1). This reduction in Bag3-P209L phosphopeptides was also seen in those phosphorylated at T285 and S289, S291, S399/T400/T406, and S289/S291. However, Bag3-WT was much more abundant in the soluble fraction compared to Bag3-P209L, and subsequently, approximately two-fold more Bag3-WT was able to be immunoprecipitated as seen by Western blot (Figure 2.5D). To correct for this difference, the phosphopeptide counts of each replicate in

Table 2.1 were normalized to the relative amount of Bag3-WT immunoprecipitated compared to

Bag3-P209L and then summed (Table 2.1). These normalized peptide counts highlight that the significant differences in Bag3 T406-phosphopeptides and S289/S291-phosphopeptides are due

52 to differences in total Bag3-WT and Bag3 P209L precipitated. Nevertheless, Bag3 phosphorylation at S289 was significantly enriched in Bag3-WT samples, whereas it was not detected in Bag3-P209L samples (Table 2.1).

Figure 2.5: Immunoprecipitation of Bag3-WT-3xFLAG and Bag3-P209L-3xFLAG (A). C2C12 myoblasts transfected with Bag3-WT-3xFLAG or Bag3-P209L-3xFLAG were immunoprecipitated 48 hours after transfection. Cell lysates are shown as inputs, while flowthrough refers to the lysate after incubation with FLAG beads. (B) and (C). FLAG immunoprecipitations of the two replicates used in the phosphoproteomics study shown in Table 2.1. Protocol performed is identical to (A), as described in the Methods section. (D). The quantified Bag3-WT IP band in Figure 2.5B and C was divided by its corresponding Bag3- P209L IP band to measure the fold-difference in total Bag3 immunoprecipitated between Bag3- WT and Bag3-P209L in the given replicate. MT = Bag3-P209L.

Total Total Normalized Normalized Phosphorylated peptide peptide peptide peptide Residue: counts counts counts counts Bag3 WT Bag3 P209L Bag3 WT Bag3 P209L T406 66 38 31.2 38 S289 64 0 29.7 0 T285 and S289 6 0 2.7 0 S291 5 0 2.3 0 T285 4 8 2.0 8 S194 or S195 28 10 12.5 10 S289 or S291 3 0 1.3 0 S399 or T400 or 2 0 1.0 0 T406

Table 2.1: Phosphorylated peptide counts of Bag3-WT-3xFLAG and Bag3-P209L-3xFLAG C2C12 myoblasts were transfected with Bag3-WT-3xFLAG or Bag3-P209L-3xFLAG. 48 hours after transfection, the 3xFLAG-tagged proteins were immunoprecipitated from the soluble fractions of the cell lysates and digested with trypsin. Phosphopeptides in the tryptic digests were enriched using TiO2 enrichment prior to mass spectrometry. Total counts shown are summed counts of two replicates. Normalized counts are summed counts of two replicates normalized to the amount of Bag3-WT/Bag3-P209L immunoprecipitated as visualized by Western blot.

BioID studies with Bag3 show a diversity of potential interactors:

To determine Bag3 interactors, including normally insoluble proteins, we utilized a proximal biotinylation (BioID) approach to identify potential Bag3 interactors in the insoluble and soluble fraction that would be otherwise undetectable by traditional co-immunoprecipitation.

Importantly, the biotin interaction with streptavidin is resistant to the harsh conditions required to extract proteins from the insoluble fraction of lysates.

Figure 2.6: Expression of N- and C-terminal-BirA-tagged Bag3 (A). C2C12 myoblasts were transiently transfected with pcDNA5-pDEST-BirA-FLAG plasmid encoding for Bag3-WT and –P209L tagged at either the N- or C-terminus. 48 hours after transfection, cells were harvested and visualized by Western blot. (B). Quantification of (A). Expression of Bag3-BirA is shown as percentage of endogenous Bag3 in the same lane. MT= Bag3-P209L

Bag3-WT and Bag3-P209L were subcloned into two different pcDNA5-pDEST-BirA-

FLAG Gateway plasmids encoding for either an N- or C-terminal FLAG-BirA tag (gift from Dr.

David Y Thomas). To assess the expression and solubility of BirA-tagged Bag3, the plasmids were transiently transfected into C2C12 myoblasts and the resulting lysates analyzed by Western blotting (Figure 2.6). Consistent with the Bag3-P209L aggregation seen in Figures 2.2 and 2.3,

Bag3-P209L BirA-tagged at either terminus significantly aggregates and is mostly found in the insoluble fraction (Figure 2.6A). C-terminally-tagged Bag3 appears to have slightly higher levels of insoluble Bag3-BirA compared to the N-terminal variant. However, in the soluble fraction, N- terminally-tagged Bag3 is seen at higher levels than C-terminally Bag3. When quantified, N- terminally-tagged Bag3-WT expression is approximately twice that of endogenous Bag3, whereas C-terminally-tagged Bag3-WT expression is only half of endogenous Bag3 (Figure

2.6B). When comparing the effect of tag location on soluble Bag3-P209L, this difference becomes more marked. The level of soluble N-terminally tagged Bag3-P209L is approximately the same as endogenous Bag3, while expression of soluble C-terminally-tagged Bag3 is only

25% of endogenous Bag3 levels (Figure 2.6B). Because the levels of soluble C-terminally tagged

Bag3 is significantly lower than endogenous Bag3, we decided to utilize the N-terminally-tagged forms in our BioID study, as their expression levels would be more similar to those of endogenous Bag3. In addition, consistent with previous studies in zebrafish, we observed that expression of Bag3-P209L caused endogenous Bag3 to be suppressed into the insoluble fraction, which was not seen as clearly in Figure 2.2 due to lack of separation between the 3xFLAG epitope-tagged Bag3 and the non-tagged endogenous Bag3 (Figure 2.6) (9).

Having successfully created a BirA-Bag3 fusion for expression in C2C12, we began our

BioID experiment. C2C12 myoblasts were transiently transfected with N-terminally tagged

BirA-Bag3-WT, BirA-Bag3–P209L, or BirA alone. After 24 hours, the transfected cells were supplied with 50 μM biotin for 24 hours. Cells were lysed in phosphate-buffered saline (PBS) and 1% Triton X-100 (TX-100) to extract soluble proteins. The lysate was centrifuged at

20,000xg to pellet insoluble material. The insoluble fraction was resuspended in 8M urea and

0.5% SDS, and subjected to sonication. The samples were then normalized, precleared, and biotinylated proteins pulled down with streptavidin resin. Prior to on-bead tryptic digestion and

LC-MS/MS analysis, a portion of this resin was analyzed by Western blot to confirm that BirA-

Bag3 was successfully precipitated from the lysate (Figure 2.7). Though no Bag3 was seen in the pulldown elution because the biotin-streptavidin bond is resistant to SDS, there was a clear reduction in BirA-Bag3, especially in the soluble fraction, when comparing the precleared input to the post-binding flowthrough (Figure 2.7). This suggests that BirA-Bag3 is functional, as it is able to bind to the streptavidin resin. Consistent with this, in both the soluble and insoluble fractions, significantly more Bag3-WT and Bag3-P209L peptides were detected by mass spectrometry compared to the empty BirA control, indicating that our BioID method is able to recover biotinylated proteins proximal to BirA-Bag3, including BirA-Bag3 itself (Tables 2.2 and

2.3).

LC-MS/MS analysis of the BioID replicates revealed 27 and 26 “significant hits” in the soluble and insoluble fractions, respectively (Figure 2.8 and 2.9, Tables 2.2 and 2.3). Hits were deemed significant if their Bag3-WT or Bag3-P209L spectral counts for each replicate were equal or greater to 3, at least 2-fold greater than the untagged BirA control, and the protein appeared in at least 2 replicates of the 3 replicates. A complete list of soluble and insoluble

57 significant hits is shown in Tables 2.2 and 2.3. Nuclear splicing and ribosomal proteins were removed as they are known background contaminants in BioID and are highly abundant in the insoluble fraction (shown in Tables 2.4 and 2.5).

Figure 2.7: Streptavidin pulldown of BirA-Bag3 (A) and (B). C2C12 myoblasts were transiently transfected with pcDNA5-pDEST-FLAG-BirA- Bag3-WT and –Bag3-P209L, and treated with 50 μM biotin 24 hours later. After 24 hours of biotin treatment, cells were harvested and biotinylated proteins precipitated by streptavidin pulldown prior to visualization by Western blot. EV = empty BirA vector, WT = BirA-Bag3- WT, MT = BirA-Bag3-P209L, FT = flowthrough

Potential soluble fraction interactors:

BioID analysis of soluble protein revealed that all but one significant hit were enriched in the Bag3-WT samples compared to Bag3-P209L samples, which is expected due to the tendency of the mutant to aggregate (Figure 2.8, Table 2.2). This is reflected by the higher soluble peptide counts of Bag3 compared to Bag3-P209L (Table 2.2). Additionally, the identified hits in the soluble fraction were of a variety of functions. Of the soluble hits, 9 have chaperone-related functions. The hit most Bag3-WT enriched relative to Bag3-P209L found, UBP24, belongs to the ubiquitin-specific peptidase family and cleaves ubiquitin from tagged proteins (191). The Hsp70- interacting E3 ligase CHIP was the second-most Bag3-WT enriched protein identified. Other chaperone proteins enriched in Bag3-WT samples include Hsp70 and Hsc70, the co-chaperones

Bag2, and DNAJB12, and the small heat shock proteins HspB1, HspB8, and HspB5 (CRYAB).

Additionally, myeloid leukemia factor 2 (MLF2), slightly enriched in Bag3-WT samples, interacts with Huntington polyQ aggregates and assists in their suppression. MLF2 was shown to interact with Bag2 and Hsc70 to regulate gene transcription (192,193).

MKL2, a cytoplasmic nucleus-shuttling transcription factor co-activator required for skeletal muscle differentiation, was also detected as a potential Bag3 interactor, but was only slightly enriched in Bag3-WT samples compared to Bag3-P209L (194). LIMD1, which inhibits the Hippo pathway effectors LATS1/2, particularly in mechanically strained cells, was detected at higher levels in Bag3-WT samples compared to Bag3-P209L (195) (196). However, LATS1, which is known to interact with the Bag3-Hsp70 complex, was not detected in our assay (119).

Aftiphilin (AFTIN) and clathrin heavy chain (CLH1), major components in clathrin coated vesicle transport, were significantly enriched in soluble Bag3-WT samples (197,198).

CLH1 is also required for maintenance of sarcomere organization in skeletal muscle (199).

Interestingly, our BioID assay detected 5 different proteins involved in Wnt signaling in the soluble fraction, including pathway members Dvl1, Dvl2, and negatively regulatory proteins nucleoredoxin, WWOX, and PPP2R3A. Interestingly, these proteins, all enriched in Bag3-WT samples, inhibit Wnt signaling by affecting Dvl; WWOX and nucleoredoxin inhibits the β- catenin/Wnt pathway via direct interaction with Dvl, whereas PPP2R3A indirectly downregulates Dvl protein levels (200-202). PPP2R3A is also a regulatory subunit of protein phosphatase 2A (PP2A) (203). PP2A is a component of protein complexes called STRIPAK involved in the Wnt and Hippo pathways and also have roles in differentiation and the cell cycle

(204).

We also observed Bag3-WT sample enrichment (relative to Bag3-P209L) of multiple proteins with cytoskeleton-related functions. Actin-binding LIM protein 1 (ABLM1) and brain- specific angiogenesis inhibitor-1 (BI2L1, also called IRTKS) are adaptor proteins that mediate actin cytoskeleton interactions with LIM-domain proteins and GTPases, respectively. In doing so, these proteins have roles in cell proliferation and migration (205,206). Interestingly, in skeletal muscle, ABLIM1 is localized to the Z-disk and abnormal splicing of its mRNA has been linked to muscular dystrophy (207).

5’-3’ exoribonuclease 1 (XRN1), significantly more abundant in Bag3-WT samples, is involved in mRNA decay and negative regulation of autophagy (208,209). HAUS6 is involved in microtubule and mitotic spindle assembly (210). Ashwin (ASHWN) and Caskin-2 (CSKI2) are relatively uncharacterized in terms of their function, but were detected in our assay (211-213).

Ashwin was the only soluble protein found that was enriched in Bag3-P209L samples compared to Bag3-WT.

Potential insoluble fraction interactors:

Much like the potential interactors of the soluble fraction, those found in the insoluble fraction also were of many functions (Figure 2.9, Table 2.3). In addition, a number of hits were found with higher enrichment in Bag3-P209L samples compared to Bag3-WT, suggesting that the insoluble mutant Bag3 has gained interactions which are possibly abnormal. In line with

Bag3’s role as an Hsp70 co-chaperone and sHsp interactor, both Hsp70 and Hsc70, as well as

HspB1, HspB5, and HspB8 were detected. However, unlike the small heat shock proteins, Hsp70 and Hsc70 were enriched in Bag3-P209L samples, relative to Bag3-WT. Additionally, four subunits of the TRiC/CCT chaperonin (TCP-B/E/G/Z) were enriched in Bag3-WT samples relative to Bag3-P209L, with TCPG being the second most differentially enriched Bag3-WT protein identified. The TRiC/CCT chaperonin is a large 1-megadalton cylindrical complex that has a central cylindrical “chamber” in which proteins can be folded in a separated environment.

TRiC has been shown to be required for folding of tubulin, actin, and other cytoskeletal proteins, and interacts with Bag3 to influence actin dynamics (214-216). The most differentially enriched protein found in Bag3-WT samples compared to Bag3-P209L was the ubiquitin-like modifier- activating enzyme 1 (UBA1), an E1-activating enzyme involved in proteasomal degradation. The loss of UBA1 has been linked to X-linked spinal muscular atrophy, and its disregulation leading to the accumulation of β-catenin and other UPS targets (217,218). CSKI2, previously detected in the soluble fraction, was also found to be enriched in Bag3-WT samples. Calponin-3 (CNN3), enriched in Bag3-WT samples, is an actin binding protein that stabilizes the actin cytoskeleton, and has functions in myogenesis and myoblast fusion (219,220).

DNA damage-binding protein 1 (DDB1), involved in DNA repair, was enriched in Bag3-

WT samples. DDB1 can interact with the CUL4 E3 ligase to form an ubiquitin-ligase complex

61 targeting substrates for degradation. Interestingly, the CUL4/DDB1 complex targets MyoD in

C2C12 myoblasts, preventing differentiation (221). Myosin light-chain-12A, a subunit of the actin-binding protein myosin-II, is involved in cell adhesion and migration and was enriched in

Bag3-WT samples (222).

Several nutrient-sensing and biosynthesis proteins were also isolated, comprised of lactate dehydrogenase (LDHA), serine hydroxylmethyltransferase (SHMT2), D-3- phosphoglycerate dehydrogenase (SERA), pyruvate kinase (PKM), and translationally-controlled tumor protein (TCTP). LDHA and PKM play important roles in the glycolysis pathway, while

SERA and SHMT2 are involved in serine and glycine biosynthesis, respectively (223-226).

TCTP is a highly regulated protein with cytoprotective, stress response, and cell growth/proliferation functions (227). Of these proteins, SERA was the most enriched in Bag3-

WT samples, with TCTP and LDHA also enriched in Bag3-WT samples. SHMT2 and PKM were enriched in Bag3-P209L samples, relative to Bag3-WT.

The eukaryotic initiation factor-3D, a subunit of the EIF3 translation complex, was detected at similar levels between Bag3-WT and Bag3-P209L samples. The abundances of

Pleckstrin homology-like domain protein B (PHLB2) and kinesin-like protein (KIF23) were also not significantly different between Bag3-WT and P209L samples. PHLB2 is involved in microtubule stabilization, whereas KIF23 is a microtubule motor protein involved in mitosis and cytokinesis (228-230).

The abundance of cell junction proteins plakoglobin (PLAK) and desmoplakin (DESP) was significantly higher in Bag3-P209L samples compared to Bag3-WT, suggesting a gain of interaction. These proteins are major components of desmosomes, a type of cell junction permitting strong inter-cell adhesion (231). PLAK, also known as γ-catenin, can modulate the

62 canonical Wnt/β-catenin pathway, and can compete for binding to the Wnt pathway TCF/LEF transcription factors. Consequently, DESP can indirectly influence the canonical Wnt pathway through controlling PLAK (232-235). Another hit enriched in Bag3-P209L samples, CRK, is also involved in cell adhesion and migration along with its other roles in signal transduction

(236-238).

Microscopy and proteomics studies in differentiated C2C12 myotubes:

The work presented in this thesis was conducted in undifferentiated C2C12 myoblasts.

We hypothesized that the expression of Bag3-P209L would inhibit differentiation of these cells into myotubes, thereby providing a possible mechanism in which the myofibrillar myopathy phenotype arises. Additionally, both the phosphoproteomics and BioID screens were to be performed in differentiated C2C12 myotubes to observe how Bag3-P209L interactions or phosphorylation may change in terminally differentiated myotubes. However, our initial approach of transfecting differentiated C2C12 myotubes, or transiently transfecting myoblasts just prior to differentiation were hampered by poor differentiation and transfection efficiency when analyzed by Western blot or fluorescence microscopy (data not shown). To overcome these obstacles, we adopted a lentiviral approach to improve the efficiency of Bag3 expression in

C2C12 myotubes. Due to time constraints, we were unable to optimize our lentiviral protocol to permit our proposed microscopy and proteomics studies in C2C12 myotubes (data not shown).

Log(Average Soluble WT/P209L) -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6

UBP24 CHIP AFTIN XRN1 CLH1 LIMD1 DJB12 BI2L1 ABLM1 HAUS6 ARGL1 NXN BAG2 MLF2 HS71A MKL2 CSKI2 BAG3 HSP7C DVL1 PPP2R3A WWOX DVL2 HSPB1 HSPB8 CRYAB ASHWN

Figure 2.8: Soluble BioID interactors of Bag3 in C2C12 myoblasts C2C12 myoblasts were transfected with N-terminally tagged BirA-FLAG-Bag3-WT and Bag3- P209L, or empty BirA-FLAG vector. Biotinylated proteins in the soluble fraction were precipitated using streptavidin-conjugated resin and digested with trypsin prior to mass spectrometry analysis (n=3). Peptide spectral counts of each significant hit were averaged and the log ratio of average hits in the Bag3-WT samples compared to the Bag3-P209L sample were calculated and tabulated in the figure above (peptide counts shown in Table 2.2). Protein hits in Bag3-WT or –P209L samples were deemed significant if their spectral counts were equal or greater to 3, at least 2-fold greater than the empty vector control counts, and appeared in at least 2 replicates. Nuclear splicing proteins, ribosomal proteins, and other common BirA contaminants were excluded as background hits (shown in Table 2.4)

Log(Average Insoluble WT/P209L) -1.5 -1 -0.5 0 0.5 1

UBA1 TCPG SERA CRYAB CSKI2 TCPE TCPZ HSPB1 MYL12A TCPB HSPB8 TCTP CNN3 LDHA DDB1 PHLB2 EIF3D KIF23 BAG3 SHMT2 HS71A PYM CRK HSP7C PLAK DESP

Figure 2.9: Insoluble BioID interactors of Bag3 in C2C12 myoblasts C2C12 myoblasts were transfected with N-terminally tagged BirA-FLAG-Bag3-WT and Bag3- P209L, or empty BirA-FLAG vector. Biotinylated proteins in the insoluble fraction were isolated by resuspension in an 8M urea and 0.5% SDS solution followed by sonication. After dilution, the fraction was precipitated using streptavidin-conjugated resin and digested with trypsin prior to mass spectrometry analysis (n=3). Peptide spectral counts of each significant hit were averaged and the log ratio of average hits in the Bag3-WT samples compared to the Bag3-P209L sample were calculated and tabulated in the figure above (peptide counts shown in Table 2.3). Protein hits in Bag3-WT or –P209L samples were deemed significant if their spectral counts were equal or greater to 3, at least 2-fold greater than the empty vector control counts, and appeared in at least 2 replicates. Nuclear splicing proteins, ribosomal proteins, and other common BirA contaminants were excluded as background hits (shown in Table 2.5)

Average BirA WT P209L BirA WT P209L BirA WT P209L Rep. Protein WT/P209L R1 R1 R1 R2 R2 R2 R3 R3 R3

3 UBP24 22.00 1 8 0 0 9 0 0 5 1

2 CHIP 10.00 0 6 0 0 4 1 0 0 0

3 AFTIN 5.50 3 4 2 0 4 0 1 3 0

3 XRN1 5.00 7 3 0 3 7 0 1 5 0

3 CLH1 4.71 0 4 2 2 15 2 0 14 3

3 LIMD1 4.60 7 10 5 1 7 0 3 6 0

2 DJB12 4.50 0 4 0 0 0 0 0 5 0

3 BI2L1 4.06 10 31 12 3 22 2 4 16 3

2 ABLM1 4.00 3 3 0 0 5 0 0 0 0

3 HAUS6 3.33 6 2 2 1 5 0 1 3 1

3 ARGL1 3.00 0 0 2 0 3 0 1 3 0

3 NXN 2.80 1 4 3 0 6 0 0 4 2

3 BAG2 2.67 0 1 0 0 4 0 0 3 0

3 MLF2 2.50 0 3 4 0 9 1 0 8 3

3 HS71A 2.39 23 94 57 16 129 35 21 121 52

2 MKL2 2.27 2 10 9 0 0 0 2 15 2

2 CSKI2 2.17 0 3 2 0 0 0 0 10 4

3 BAG3 1.85 13 127 91 7 97 65 6 201 74

3 HSP7C 1.83 104 331 200 107 390 168 88 432 261

3 DVL1 1.80 9 8 7 2 10 3 5 9 5

PPP2R3 3 1.57 3 10 12 1 9 0 1 3 2 A

2 WWOX 1.38 5 7 15 4 15 1 0 0 0

3 DVL2 1.30 20 25 40 13 44 13 16 31 24

3 HSPB1 1.29 7 10 13 6 15 9 7 20 13

3 HSPB8 1.21 4 17 32 2 64 24 2 45 48

3 CRYAB 1.21 5 11 11 2 8 4 4 16 14

2 ASHWN 0.36 0 0 7 0 0 0 0 4 4

Table 2.2: Soluble BioID Hits Peptide counts for each BioID replicate (R1, R2, R3) are shown in the table above. The replicate column (Rep.) indicates how many replicates does the given protein appear in. Average WT/P209L ratio was calculated by averaging the peptide counts of Bag3-WT and Bag3-P209L across all replicates, and dividing the results to find the WT/P209L ratio.

Average BirA WT P209L BirA WT P209L BirA WT P209L Rep. Protein WT/P209L R1 R1 R1 R2 R2 R2 R3 R3 R3

3 UBA1 5.33 0 0 0 0 13 3 0 3 0

3 TCPG 5.00 0 0 0 0 5 1 0 5 1

3 SERA 3.25 1 0 0 0 9 3 1 4 1

3 CRYAB 3.00 0 0 0 0 5 2 0 4 1

3 TCPE 2.00 0 0 0 1 7 4 3 5 2

3 TCPZ 2.00 0 0 0 1 4 4 0 4 0

2 CSKI2 2.00 0 1 3 0 0 0 0 5 0

3 HSPB1 1.75 0 0 1 0 15 8 3 13 7

3 MYL12A 1.50 0 0 0 0 5 0 1 1 4

3 TCPB 1.40 0 0 0 0 5 4 0 2 1

3 HSPB8 1.38 0 0 1 0 6 4 0 5 3

2 TCTP 1.33 0 0 0 0 5 3 1 3 3

3 CNN3 1.29 0 0 0 0 6 6 1 3 1

3 LDHA 1.25 1 0 0 2 7 6 1 3 2

3 DDB1 1.13 0 0 0 0 11 13 1 7 3

3 PHLB2 1.13 0 0 0 1 7 0 2 2 8

3 EIF3D 1.11 1 0 0 2 8 2 4 12 16

2 KIF23 0.90 0 0 0 6 10 0 2 8 20

3 BAG3 0.82 0 63 106 0 98 156 0 95 52

2 SHMT2 0.78 0 0 0 0 4 4 0 3 5

3 HS71A 0.75 9 12 38 5 35 46 3 29 17

2 PKM 0.73 2 0 6 7 22 24 0 0 0

2 CRK 0.67 1 0 8 2 14 13 0 0 0

3 HSP7C 0.67 17 27 131 17 159 154 34 93 134

3 PLAK 0.14 34 0 10 2 0 25 0 5 1

3 DESP 0.05 29 0 22 0 0 57 0 4 0

Table 2.3: Insoluble BioID Hits Peptide counts for each BioID replicate (R1, R2, R3) are shown in the table above. The replicate column indicates how many replicates does the given protein appear in. Average WT/P209L ratio was calculated by averaging the peptide counts of Bag3-WT and Bag3-P209L across all replicates, and dividing the results to find the WT/P209L ratio.

BirA WT P209L BirA WT P209L BirA WT P209L Rep. Protein R1 R1 R1 R2 R2 R2 R3 R3 R3

3 ACACA 362 466 959 772 710 1390 369 644 1696

2 ATPO 4 5 9 0 0 0 5 7 17

2 LC7L2 3 3 9 0 0 0 0 4 2

2 MCCA 90 100 177 0 0 0 92 155 278

2 MCCB 23 36 51 0 0 0 16 35 73

2 PCCA 88 92 255 0 0 0 82 116 401

2 PCCB 39 63 127 0 0 0 55 77 173

2 PGCB 1 2 0 0 0 0 2 4 0

3 PRC2C 19 9 9 4 9 0 2 11 0

2 PYC 62 74 174 0 0 0 75 109 218

3 Q9CPN9 2 13 28 22 8 160 10 40 109

2 RBM39 0 6 6 0 0 0 2 2 6

2 RL36 0 0 0 0 1 4 2 1 1

2 SCMC1 6 11 36 0 0 0 10 14 25

2 SRSF2 0 0 4 0 0 0 1 0 0

2 SRSF5 0 0 4 0 0 0 1 0 0

2 TIM16 0 0 2 0 0 0 0 2 3

2 TSPO 3 1 2 0 0 0 1 0 6

Table 2.4: Excluded BioID background hits in the soluble fraction. Peptide counts for each BioID replicate (R1, R2, R3) are shown in the table above sorted alphabetically by protein name. The replicate column indicates how many replicates does the given protein appear in. These proteins appeared in at least two replicates, but were excluded because they are ribosomal proteins, splicing proteins, or other BirA contaminants.

BirA WT P209L BirA WT P209L BirA WT P209L Rep. Protein R1 R1 R1 R2 R2 R2 R3 R3 R3

2 E9QAZ2 7 0 3 0 0 0 13 36 57

2 EF2 8 0 3 8 33 26 0 0 0

2 EIF3A 0 1 0 1 3 1 0 0 0

2 FBRL 10 0 1 0 0 0 11 31 87

2 H11 5 0 2 0 0 0 16 311 54

2 H14 40 0 45 0 0 0 91 902 138

2 H2AV 22 14 33 0 0 0 41 59 535

2 H4 56 13 45 0 0 0 136 272 762

2 NOP2 2 0 0 0 0 0 9 12 40

3 PYR1 0 0 0 0 21 2 2 9 1

2 Q6PGF5 0 0 0 2 3 0 2 2 21

3 Q9CPN9 107 211 181 27 6 177 4 2 0

3 RL10 2 0 7 7 12 5 7 21 48

2 RL13 6 0 13 0 0 0 7 29 82

3 RL13A 6 0 6 5 8 2 4 16 22

2 RL14 1 0 2 0 0 0 8 14 20

3 RL17 2 0 6 5 19 3 7 24 55

2 RL18 10 0 7 0 0 0 9 34 84

2 RL18A 5 0 6 0 0 0 5 10 35

3 RL21 2 0 1 3 10 0 6 12 38

3 RL22 1 0 2 1 3 0 1 5 3

3 RL22L 0 0 1 2 3 1 0 1 5

2 RL26 4 0 0 0 0 0 5 12 47

2 RL27 8 0 5 0 0 0 12 22 117

2 RL27A 2 0 2 0 0 0 3 4 21

3 RL28 4 0 5 6 9 0 3 15 40

2 RL3 7 0 8 0 0 0 16 39 96

3 RL30 2 0 2 3 7 2 1 7 7

3 RL32 0 0 0 1 11 0 2 9 34

2 RL34 2 0 7 0 0 0 4 24 10

3 RL35A 7 0 6 9 15 4 6 16 46

2 RL37A 1 0 0 0 0 0 2 3 11

2 RL4 5 1 3 0 0 0 10 44 73

2 RL6 10 0 16 0 0 0 16 24 127

2 RL7 5 0 8 0 0 0 17 29 106

2 RL7A 6 0 16 0 0 0 10 44 91

3 RL9 1 0 4 6 12 5 6 11 18

2 RRP5 0 2 0 0 0 0 4 6 29

2 RS13 8 0 5 0 0 0 16 22 129

2 RS14 11 0 12 0 0 0 6 16 36

2 RS15 3 0 0 0 0 0 3 4 33

2 RS16 5 0 2 0 0 0 11 16 52

2 RS19 8 0 6 2 11 14 0 0 0

3 RS20 7 0 0 1 6 1 4 8 7

2 RS24 5 0 4 0 0 0 10 18 63

3 RS25 2 0 0 9 66 4 4 12 22

2 RS26 1 0 3 0 0 0 7 14 20

2 RS27 0 0 0 3 5 3 2 5 16

2 RS5 3 1 0 0 5 0 0 0 0

3 RS6 6 0 10 20 18 5 10 20 76

2 RS8 6 0 7 0 0 0 17 32 104

2 RSSA 3 0 7 3 11 12 0 0 0

2 SMD1 1 0 1 0 0 0 6 10 70

2 SMD3 3 0 0 0 0 0 2 8 13

2 SRSF1 3 5 6 0 0 0 1 7 11

2 SYG 0 0 0 0 5 3 0 4 1

3 SYNC 0 0 0 0 6 2 1 3 2

2 SYRC 1 0 0 0 5 2 0 0 0

Table 2.5: Excluded BioID background hits in the insoluble fraction. Peptide counts for each BioID replicate (R1, R2, R3) are shown in the table above sorted alphabetically by protein name. The replicate column indicates how many replicates does the given protein appear in. These proteins appeared in at least two replicates, but were excluded because they are ribosomal proteins, splicing proteins, or other BirA contaminants.

Chapter 3: Discussion

The physiological phenotype of Bag3-P209L myofibrillar myopathy has been extensively described, but insight into the molecular mechanism behind its pathology is limited

(3,6,9,239,240). We show that disruption of the Hsp70-Bag3 interaction with JG-98 does not affect Bag3-P209L aggregation. We thus hypothesized that Bag3 interactions other than Hsp70 are impacted by the P209L mutation, leading to the disease phenotype. To address this, we used proteomics methodology to show Bag3-P209L exhibits changes in phosphorylation as well as potential changes in interactions in C2C12 myoblasts. We observed significant loss of phosphorylation at S289 and other, albeit less dramatic, changes in phosphorylation in Bag3-

P209L. Through BioID, we establish that Bag3 has previously-unknown potential interactions that also may be perturbed by the P209L mutation. Many of the identified interactions have already been characterized, such as Hsp70 and the small heat shock proteins HspB8, HspB1, and

HspB5 (Tables 2.2 and 2.3). However, we report novel potential interactors, such as LIMD1,

NXN, and desmoplakin, implicated in a number of pathways and functions, such as Hippo or

Wnt signaling (Tables 2.2 and 2.3).

Based on these data, several hypotheses are presented below, about the effects of changes in phosphorylation and potential interactions due to the Bag3-P209L mutation. However, regardless of the hypothesis, foundational experiments must be performed to assess the validity of Bag3 interaction, due to limitations of the BioID method. Namely, because BioID uses proximal biotinylation rather than direct interaction to pull down identified proteins, a BioID

“hit” may be a true interactor of Bag3 or a non-interactor that is in close proximity to the BirA tag of Bag3. Hence, before their relationship to Bag3 can be assessed, the identified BioID hits

75 of interest must be shown to interact with Bag3 either directly or in complex. This can be addressed by either co-immunoprecipitation (co-IP) or in vitro interaction assays.

Bag3 in Z-disk and actin cytoskeleton assembly and stability:

It has already been shown that the Bag3-mediated CASA pathway is required for the degradation of damaged filamin to ensure proper maintenance of the actin cytoskeleton in mechanically strained muscle cells (119). In the proposed model, the Hsp70-Bag3-HspB8 complex, in cooperation with the CHIP E3 ligase, degrades filamin damaged by mechanical strain through the CASA pathway while simultaneously upregulating filamin transcription by the

Hippo pathway (119,144). The CASA pathway mediated by the CHIP-Hsp70-Bag3-HspB8 complex was also found to be essential for Z-disk integrity (144). Remarkably, in our BioID screen, CHIP peptides were found to be 10-fold more enriched in Bag3-WT samples compared to Bag3-P209L, while Hsc70/Hsp70 and HspB8 peptide counts were not significantly different

(Table 2.2). This suggests a P209L-driven loss of interaction between CHIP and the Hsp70-

Bag3-HspB8 complex, which would lead to CASA aberrations and resulting Z-disk instability

(144).

Moreover, Bag2, a member of the same family as Bag3, was also enriched in Bag3-WT

BioID samples (Table 2.2). Other than its classical roles as a Hsp70 co-chaperone, Bag2 has been shown to be an inhibitor of CHIP, suggesting it may also play a role in the interplay between Bag3 and CHIP and thereby affect CASA indirectly (241,242). To address these hypothesized changes in CASA, assessment of LC3-II/LC3-I ratios and p62 turnover by Western blot in control and lysosome-inhibited cells would allow measurement of autophagic flux and assessment of CASA changes induced by Bag3-P209L. Immunofluorescence studies of LC3 and

76 p62 in cells treated with and without lysosomal inhibitors could also be used to assess autophagy flux and punctae formation, as well as the localization of Bag3-P209L in autophagic punctae.

Based on this idea of Bag3-P209L inducing defects in CASA, it is interesting that

UBP24, the most differentially Bag3-WT enriched soluble protein identified in our BioID screen, is a ubiquitin-specific protease that cleaves ubiquitin from tagged proteins (243). Though a role of UBP24 and autophagy has not been shown, our data imply wildtype Bag3 and UBP24 interact in a P209L-sensitive manner. Thus, it is possible that this interaction is critical for modulation of

CASA or its ubiquitinated targets, and loss of Bag3-UBP24 interaction could lead to aberrant degradation or aggresomal suppression of proteins otherwise essential for cellular health.

In addition, BioID analysis of the insoluble fraction detected enrichment of TRiC/CCT chaperonin subunits in Bag3-WT samples from 1.5-fold to 5-fold above that of Bag3-P209L samples (Table 2.3). The TRiC chaperonin is a molecular chaperone particularly known for its importance in folding monomeric actin, and has been identified in zebrafish skeletal muscle as an essential part of sarcomere assembly at the Z-disk (244,245). Bag3 has been shown to be a

TRiC interactor, and also has roles in Z-disk actin filament assembly in muscle through the

Bag3-Hsp70 interaction with the actin-capping protein CapZβ1 (177,244). Hence, a loss or reduction in TRiC interaction with Bag3-P209L could disrupt actin filament assembly at the Z- disk, contributing to the progressive muscle weakness seen in Bag3-P209L-expressing zebrafish and human patients (6,9). Consistent with this idea, disruptions of Z-disk actin assembly induced by TRiC subunit mutations have been shown to cause muscle weakness in zebrafish (244).

Hence, further experiments, such as co-IPs, are needed to address the effect of the P209L mutation on CHIP and TRiC interaction with Bag3.

In line with the idea of Bag3-P209L disrupting actin assembly, the actin cytoskeleton adaptor protein BI2L1 (IRTKS) was found to have an average 4-fold enrichment in soluble

Bag3-WT BioID samples compared to Bag3-P209L, with this enrichment consistently seen across all 3 replicates. IRTKS is a known regulator of actin filaments, able to induce remodeling of the actin cytoskeleton in cell migration (246). Overregulated in some cancers, IRTKS has also been shown to activate EGFR/ERK signaling and promote cell proliferation, as well as have roles in development (205,247). Interestingly, Bag3 was also found to regulate EGFR and ERK signaling in breast cancer cells, which allows the possibility of a combinatorial role of Bag3 and

IRTKS in signal transduction (248,249). However, the interaction between Bag3 and IRTKS has not been shown, but IRTKS does contain a SH3 domain, which is known to generally bind the

PXXP region of Bag3 (246,250). Hence, it is possible that Bag3 and IRTKS interact and have functions in either actin cytoskeleton remodeling or signal transduction that are somehow impacted by the Bag3-P209L mutation.

Bag3-HSF1 dynamics and the heat shock response:

We show Bag3-P209L displays a loss of phosphorylation at residue S289, and increased phosphorylation at T285 compared to Bag3-WT (Table 2.1). Interestingly, Bag3 has been shown to be phosphorylated at these residues under oxidative stress conditions in an ERK-dependent manner (251). In normal conditions, Bag3 shows strong interaction with HSF1 by co-IP, whereas this interaction is significantly reduced under stress conditions. However, the binding of Bag3 to

HSF1 is not changed if a short sequence containing the S289 and T285 residues is deleted, and this mutant inhibits the nuclear import of the transcription factor heat shock factor 1 (HSF1), leading to downregulation of Hsp70 expression and increased cell death in H2O2 treated-A172 cells (251). In conjunction with these findings, our data shows the loss of S289-phosphorylation

78 in Bag3-P209L, suggesting the mutant may aberrantly regulate HSF1 localization and activity during cellular stress. Hence, the cell would exhibit reduced expression of HSF1-induced chaperones needed to overcome stresses and prevent cell death. Additionally, expression of Bag3 is known to be stress-inducible by HSF1 (252,253). Therefore, during stress conditions in heterozygous cells, Bag3-P209L-mediated suppression of HSF1 activity may inhibit the expression of wild-type Bag3. This reduction in expression, combined with suppression of Bag3 into amorphous aggregates, would reduce the pool of Bag3 available to function in anti-apoptosis and protein quality control and subsequently leading to cell death, in line with the mechanism initially proposed by Ruparelia et al. (9).

HSF1 was not detected in the BioID experiments, possibly because it is in relatively low abundance, and an interaction with Bag3 is plausible in the C2C12 cells. Since Bag3-P209L has a tendency to aggregate, HSF1 may also be suppressed in Bag3 aggregates, which can be assessed by immunofluorescence microscopy or Western blot of HSF1 in the insoluble fraction in a Bag3-P209L overexpression context. Microscopy may also show that HSF1 escapes aggregation but is otherwise trapped in the cytoplasm. Additionally, because wildtype Bag3 has been shown to interact with HSF1 in co-IPs, we can use the same methodology to address the effect of the P209L mutation on its association with HSF1 in normal and oxidative stress conditions, where we expect the loss of S289-phosphorylation in Bag3-P209L would prevent the loss of Bag3-HSF1 interaction during stress (251). Detection of Hsp70 mRNA and protein levels before and after stress would serve as possible reporters for HSF1 activity.

Bag3 and Hippo pathway regulation:

In the context of the Hippo pathway, Bag3 has been shown to interact with the YAP and

TAZ inhibitors LATS1/2 under mechanical tension, hereby leading to YAP and TAZ activation

79 and transcription of target genes. This regulation was shown to be particularly important for the upregulation of the cytoskeletal protein filamin in response to its unfolding and degradation from mechanical strain (119). The Hippo pathway protein identified in our BioID screen, LIMD1, is a member of the Ajuba family proteins that was shown to also bind LATS1/2 and inhibit their activity in cells under mechanical stress, leading to increased YAP and TAZ-mediated transcription (195,254). Interestingly, in our screen, LIMD1 was also approximately 4.5-fold higher in soluble Bag3-WT samples compared to Bag3-P209L, suggesting a possible P209L- driven loss of Bag3-LIMD1 interaction (Table 2.2). It is unexpected that our BioID screen did not detected LATS1/2, a known Bag3 and LIMD1 interactor, though a recently published co-

IP/LC-MS/MS study of Bag3 also did not detect LATS1/2 binding and could only detect bound

LATS1/2 through Western blot (255). However, it is possible that either the abundance of

LATS1/2 is too low to be detected by BioID, or it ionizes poorly, preventing identification by mass spectrometry.

Nevertheless, the reduction of detected LIMD1 in Bag3-P209L samples suggests the mutant may be less able to sequester LATS1/2, leading to lower YAP and TAZ activity.

Mechanically strained tissues, such as skeletal muscle, may then be less able to respond to tension and subsequent accumulation of cytoskeletal damage (119). In addition, YAP has been shown to be a major regulator of satellite cell proliferation and differentiation, where YAP activity increases proliferation and inhibits differentiation of satellite cell-derived myoblasts

(49,50). Thus, the progressive muscle weakness phenotype seen in myofibrillar myopathy patients could be due to disruptions of Bag3 or LIMD1 Hippo pathway regulation leading to irregular satellite cell differentiation and a general inability to repair damaged muscle tissue (6).

Since YAP is downstream of LIMD1 or Bag3 in the Hippo pathway, measuring the dephosphorylation of YAP by Western blot, which indicates YAP activation, can be used as a

Hippo pathway reporter as previously published (255). To induce YAP activation, we propose using proteasome inhibition, which has been shown to lead to YAP dephosphorylation (255).

Tension-based stresses have also been shown to be involved in Bag3-mediated YAP activation

(119). Using these methods, the effect of Bag3-P209L overexpression on YAP activation can then be assessed, which based on our hypothesis, is expected to prevent YAP dephosphorylation presumably acting through LATS1/2 suppression. Since our BioID screen failed to detect

LATS1/2, co-IPs of Bag3-P209L and LATS1/2 are also needed to assess how the P209L mutation affects their interaction, since changes in Bag3 or LIMD1-mediated Hippo pathway regulation likely involve LATS1/2 to some degree. Additionally, since Bag3 and LIMD1 may have some interaction or synergetic role, assessing YAP activation in Bag3-WT and Bag3-

P209L overexpressing cells depleted of LIMD1 may give insight into the potential interplay between Bag3 and LIMD1 in Hippo signaling.

Bag3 and Wnt signaling:

While Bag3 is involved in Hippo pathway regulation, there has been no previous study linking Bag3 to Wnt signaling. However, our BioID assay identified five soluble and one insoluble proteins involved in Wnt signaling or its regulation (Tables 2.2 and 2.3). In the soluble fraction, only one protein, nucleoredoxin (NXN), was found with an above 2-fold peptide enrichment in Bag3-WT samples compared to Bag3-P209L, suggesting the mutation interferes with the interaction. The peptide abundances of the other four soluble Wnt proteins, DVL1,

DVL2, WWOX, and PPP2R3A, were not significantly different (less than 2-fold change) between Bag3 samples. NXN has been shown to directly interact with DVL proteins and prevent

81 their ubiquitination and degradation, allowing them to act downstream of Wnt in the canonical pathway. Under normal conditions in NIH3T3 mouse fibroblasts and HEK293, NXN is involved in inhibition of Wnt signaling. Conversely, when exposed to H2O2 stress, the canonical Wnt pathway is activated in a NXN-dependent manner (202,256). Thus, NXN emerges as an important Dvl regulator sensitive to varying cell challenges, with NXN-knockout mice exhibiting abnormal bone and heart development (256). Our observation of a reduced number of NXN peptides in Bag3-P209L expressing samples suggests a potential interaction or co-localization between Bag3 and NXN that is impacted by the Bag3-P209L mutation. Because the canonical and non-canonical Wnt pathways have been linked to expansion and differentiation of myogenic satellite cells, the idea that Bag3 may be involved in the regulation of DVL or NXN through an unknown mechanism also permits the possibility that the P209L mutation could affect this, disrupting normal Wnt signaling and hereby contribute to the myofibrillar myopathy phenotype

(31,38-40).

BioID analysis of the insoluble fraction revealed UBA1 was the most differentially enriched protein in Bag3-WT samples with peptide counts approximately 5-fold higher than

Bag3-P209L. UBA1 is an E1 ubiquitin-activating enzyme whose dysregulation has been linked to spinal muscular atrophy through disruption of the ubiquitin-proteasome degradation system.

Particularly, it was shown that β-catenin, the main component of the canonical Wnt pathway, is a target of UBA1, and the loss of UBA1 leads to accumulation of β-catenin (218). The differential enrichment of UBA1 peptides in our Bag3-WT BioID samples reveals the possibility of a Bag3-

UBA1 interaction that may have functions in protein degradation. If Bag3-P209L disrupts this interaction or its localization, it is possible that UBA1 targets, including β-catenin, will no longer be regulated, leading to dysregulation of processes such as the canonical Wnt pathway. One first-

82 glance approach could be assessment of β-catenin levels and localization in Bag3-WT and Bag3-

P209L expressing cells, which would provide insight into how the P209L mutation influences

Wnt signaling and how such changes may lead to myopathy. However, because mammalian cells only express two E1 enzymes, dysregulation of UBA1 would likely have far-reaching effects in the cell due to its importance in protein degradation (257). Hence, aside from UBA1’s specific effects on β-catenin, Bag3-P209L may lead to the disease phenotype partly through general dysregulation of UBA1-mediated protein ubiquitination and degradation by both proteasomes and autophagy. It is also possible that aberrancies in UBA1 could lead to toxicity or phenotype changes by affecting another signaling pathway other than Wnt, It is clear that care must be given when addressing the many possible mechanisms in which the interaction between Bag3-

P209L and UBA1 may contribute to the disease.

As mentioned previously, WWOX and PPP2R3A, two Wnt-related proteins, were identified by our BioID screen, but neither protein was significantly differentially enriched in

Bag3-WT or Bag3-P209L samples. WWOX (WW domain-containing oxidoreductase) is a proapoptotic protein, typically associated with cancer due to its tumor suppressor function (258).

However, WWOX has been shown to inhibit the canonical Wnt/β-catenin pathway, partly through cytoplasmic suppression of DVL proteins and subsequent destabilization of β-catenin

(201). Additionally, WWOX inhibits the activity of the protein BCL9-2, a transcription coactivator of β-catenin, further inhibiting the canonical Wnt pathway (259). WWOX was also shown to bind GSK3β, a member of the β-catenin degradation complex, though how this interaction is involved in Wnt pathway regulation is unknown (260). These findings present

WWOX as a negative regulator of the canonical Wnt pathway through multiple possible

83 mechanisms. Interaction with Bag3 could be either essential for WWOX function, or Bag3 may regulate WWOX activity.

PPP2R3A is a regulatory subunit of the serine/threonine protein phosphatase (PP2A) involved in the modulation of signaling pathways such as Wnt and MAPK. Specifically,

PPP2R3A has two isoforms produced by alternative splicing, PR72 and PR130 that bind the Wnt protein Naked cuticle (NKD) (261). NKD inhibits the activity of DVL, preventing Wnt signaling

(262,263). Interestingly, PR72 and PR130 have contrasting functions in NKD regulation, with

PR72 being required for NKD function whereas PR130 promotes Wnt signaling by inhibiting

NKD (200,264). Because of this, Bag3 interaction with PPP2R3A may influence Wnt signaling, but whether this would be stimulatory or inhibitory is unclear due to the possibility of either

PPP2R3A isoform interacting. Additionally, the phosphatase activity of PP2A has been shown to be regulated in part by nucleoredoxin, suggesting the possibility that a Bag3-nucleoredoxin interaction (discussed earlier) may exercise its regulatory effects on the Wnt pathway through modulation of PP2A activity (265). Alternatively, PP2A has been shown to stabilize β-catenin and prevent its degradation by the proteasome during development, and perhaps Bag3, by binding subunits of PP2A, plays some role in this function (266). However, if this is true, it is unusual that only the PPP2R3A subunit was detected in our BioID screen. Hence, further work is required to elucidate how Bag3 is involved in the functions of WWOX and PP2A. An initial approach, aside from co-IP to validate interaction, could be assessing the effect of Bag3- knockdown on WWOX, PP2A, and β-catenin expression levels and localization by Western blot and microscopy.

Sarcomere organization and regulation of clathrin:

Clathrin, a protein made of trimers of heavy chains and light chains, is primarily known for its roles as a membrane coat in endocytosis and intracellular trafficking (267). However, it has been also proposed that in C2C12 and mouse skeletal muscle, clathrin heavy chains are able to associate with actin filaments and are required for the formation and maintenance of costameres, structures linking the Z-disk to the plasma membrane (199). In our BioID screen, we observed significantly higher peptide counts of clathrin heavy chain in soluble Bag3-WT samples compared to Bag3-P209L, suggesting a potential loss of interaction (Table 2.2). Though interaction between clathrin and Bag3 needs to be validated, our data suggests the possibility of

Bag3 having an unknown P209L-sensitive role that involves clathrin in some way. Perhaps, if

Bag3-P209L somehow affects actin filament behavior at the Z-disk due to mechanisms such as disruptions in TRiC/CCT interaction, it is possible that these changes will also influence the interaction between clathrin and actin in costameres and thereby destabilize sarcomeric integrity.

Aside from co-IPs to validate the Bag3-clathrin interaction, immunofluorescence experiments in undifferentiated and differentiated muscle could be one approach to assess how the P209L mutation may affect Bag3 and clathrin localization, and how these changes, if significant, could lead to negative muscle phenotypes.

Desmosomal junctions:

In cardiac muscle, muscle cells are connected through Z-disk localized cell junctions called intercalated disks (268). One component of these junctions are desmosomes, a form of cell structure providing strong intercellular adhesion (269). Desmoplakin, a primary constituent of desmosomes, bind to intermediate filaments of the cytoskeleton, while the protein plakoglobin anchors desmoplakin to the transmembrane proteins at the desmosome. Hence, these proteins are

85 able to connect the cellular cytoskeleton to the plasma membrane, granting mechanical stability when under muscle contraction (269,270). We observe that both desmoplakin and plakoglobin are generally enriched in the insoluble fraction of Bag3-P209L BioID samples, though there is inconsistency in peptide counts between replicates (Table 2.3). Because desmosomes may be partially insoluble in our conditions, they may be biased to have BioID interactions with insoluble Bag3-P209L, though Bag3-WT peptides were also readily found in the insoluble fraction (9) (Table 2.3). It has been previously shown that both desmoplakin and plakoglobin primarily localize to the Triton-insoluble fraction using lysis conditions similar to the methods used in this thesis (271,272). Nevertheless, from these data emerges the possibility of two possible gain-of-function models; Bag3-P209L mislocalizes to the desmosome and disrupts its functions, or alternatively, Bag3-P209L captures desmosome components and suppresses them into its aggregates. In either case, desmosome aberrations induced by Bag3-P209L could cause disruptions in cell adhesion between cardiomyocytes, leading to loss of structural integrity under mechanical tension and the overall cardiomyopathy phenotype seen in Bag3-P209L patients.

Immunofluorescence microscopy may then be the best way to assess if Bag3-P209L induces a shift in localization of desmoplakin or plakoglobin by suppressing them into Bag3-P209L aggregates or if Bag3-P209L localizes at the desmosome unlike Bag3-WT. Additionally, a possible experiment would be to use harsher solubilization conditions to assess differences in soluble and insoluble desmoplakin and plakoglobin in the context of untransfected, Bag3-WT transfected, and Bag3-P209L transfected cells.

Conclusions and future perspectives:

Numerous published works have already established Bag3 as a multifunctional scaffold protein, with roles in autophagy, Hippo pathway regulation, protein quality control, and aggresome formation (119,160). Despite this, the way in which the P209L mutation causes myofibrillar myopathy remains unknown (6). Through a proteomics approach, we identify changes in Bag3 phosphorylation and potential Bag3 interactors impacted by the P209L mutation, hereby laying the groundwork for future experiments assessing how such changes may lead to the disease phenotype. We propose several different hypotheses addressing how Bag3-

P209L exhibits aberrant behavior in pathways such as the heat shock response, Hippo and Wnt signaling, and actin filament assembly.

Chapter 4: Methods

Plasmids:

DNA constructs encoding Bag3-WT, Bag3-P209L, and HspB8 were subcloned from GST-Bag3 plasmids (gift from Dr. Josée Lavoie, Université Laval) into pPROEX-HTa plasmids

(Invitrogen) for bacterial protein purification. For usage in cell culture, constructs encoding

Bag3-WT and Bag3-P209L were inserted into pcDNA 3.1 (Invitrogen) with an engineering N- terminal FLAG tag and p3xFLAG-CMV-14 (gift from Dr. Kalle Gehring, McGill University) encoding a C-terminal 3xFLAG tag. pCAGGS-GFP plasmid was a gift from an now-unknown source. pEGFP-N1 plasmids encoding Bag3-WT-GFP and Bag3-P209L-GFP were gifts from Dr.

Josée Lavoie. N-terminal and C-terminally tagged pcDNA5-pDEST-BirA-FLAG Gateway vectors (originally from Dr. Anne-Claude Gingras, University of Toronto) and pDONR221-Bag3 were gifts from Dr. David Y. Thomas, McGill University. Gateway cloning using pDONR221-

Bag3 was used to insert the Bag3-WT coding sequence into N-terminal and C-terminal tagged pcDNA5-pDEST-BirA-FLAG plasmids. Site-directed mutagenesis was used to introduce the

P209L mutation in these plasmids.

Antibodies:

Anti-FLAG M2 monoclonal antibody (used at 1:1000 dilution) was purchased from Sigma

Aldrich. Anti-Bag3 polyclonal rabbit serum was a gift from Dr. Josée Lavoie and was purified using the method described below (used at 1:5000 dilution).

Reagents:

Chemical reagents are from Bioshop unless otherwise stated. The Hsp70 small molecule inhibitor JG-98 is a gift from Dr. Jason Gestwicki (University of California, San Francisco).

Protein purification: pPROEX-HTa plasmids encoding His-tagged Bag3-WT, Bag3-P209L, and wildtype HspB8 were grown in BL21 Escherichia coli cells. Protein expression was induced with 1mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) at 18°C overnight. After harvesting, the cells were resuspended in equilibration buffer (500mM NaCl, 20mM imidazole, 20mM KH2PO4 pH

7.5) and lysed by cavitation in an EmulsiFlex homogenizer (AVESTIN). The lysate was clarified by ultracentrifugation. The lysate was loaded onto a 5 mL Ni-sepharose Nuvia IMAC resin column (Bio-Rad) and eluted with elution buffer (300mM imidazole, 20mM KH2PO4 pH 7.5).

Fractions containing HspB8 and Bag3 were determined by SDS-PAGE and pooled. The pooled

HspB8 was stored at -80°C. The pooled Bag3 fractions were diluted 8-fold with 20mM KH2PO4 pH 7.5, and loaded onto a 2 mL HiTrap Q Sepharose HP column (GE Healthcare). Proteins were eluted using a shallow 20 column volume (CV) gradient from 50mM NaCl to 600mM NaCl in

20mM KH2PO4 pH 7.5, and a subsequent 5 CV gradient from 600mM NaCl to 1M NaCl in the same buffer. Fractions containing Bag3 were determined by SDS-PAGE, pooled, and dialyzed into working buffer (100mM KOAc, 20mM HEPES-KOH pH 7.5, 5mM MgOAc2) prior to storage at -80°C.

Anti-Bag3 purification from rabbit serum:

Cyanogen bromide activated sepharose resin (Sigma Aldrich) was hydrated in 1mM HCl. The hydrated resin was activated by washing in 0.1M KH2PO4 pH 7.5 immediately before adding

1mg of purified Bag3. After overnight incubation at 4°C, the reaction was quenched with 1M monoethanolamine pH 7.5, incubating overnight at 4°C. Rabbit serum containing anti-Bag3 antibody (gift from Dr. Josée Lavoie) was clarified by centrifugation at 10,000×g for 15 minutes prior to loading onto the Bag3-sepharose resin for 4 hours. The antibody-bound resin was

89 washed with PBS 5 times before antibody elution with 0.1M glycine-HCl pH 3 and subsequent quenching into 1M Tris-HCl pH 8.

Circular dichroism:

Purified Bag3-WT, Bag3-P209L, and HspB8 were buffer exchanged using Bio-Rad Micro Bio- spin 6 desalting columns into 20mM KH2PO4 pH 7.5. Samples of each individual protein and mixtures of Bag3 and HspB8 were diluted to experimental concentrations with 20mM KH2PO4 pH 7.5 buffer prior to circular dichroism measurement using an Applied Photophysics Chirascan spectrometer. Measurement temperature was set at 25°C controlled by a Quantum Northwest

TC125 temperature controller. Measurements were taken at 180 nm to 260 nm, sampling 1 second-per-point and repeating 3 times per sample. 20mM KH2PO4 pH 7.5 buffer was used as a background blank. Measurements were averaged and smoothed using the Pro-Data Chirascan

“Smooth Trace” function. Smoothed CD spectra were deconvoluted into percentage secondary structures using CDNN (Circular Dichroism analysis using Neural Networks) 2.1 (273).

Intrinsic fluorescence:

Purified Bag3-WT, Bag3-P209L, and HspB8 were buffer exchanged using Bio-Rad Micro Bio- spin 6 desalting columns into 20mM KH2PO4 pH 7.5. The maximum excitation wavelength for

Bag3-WT, Bag3-P209L, and HspB8 was found to be 283nm. Using this excitation wavelength, emission spectra of each individual protein and mixtures of Bag3 and HspB8 were measured from 300nm to 400nm using a Horiba Fluorolog spectrofluorometer.

Cell culture:

Cells were grown on polystyrene dishes (Corning) kept in a humid 37°C incubator in 5% CO2.

C2C12 mouse myoblasts were maintained in Dulbecco’s Modified Eagle Medium (DMEM) containing 4.5 g/L glucose, L-Glutamine, and sodium pyruvate (Wisent), and supplemented with

20% fetal bovine serum (FBS) (Wisent), 100 units/mL penicillin (Wisent), and 100 μg/mL streptomycin (Wisent). C2C12 myoblasts were maintained below 70% confluency at all times to prevent unwanted differentiation.

Differentiation:

C2C12 myoblasts were grown to 100% confluency using the growth medium described previously. Upon reaching confluency, the medium was switched to differentiation medium, comprised of DMEM supplemented with 2% horse serum (Sigma), 100 units/mL penicillin, and

100 μg/mL streptomycin. Differentiation of C2C12 myoblasts into myotubes was monitored over the course of four to five days, with differentiation medium refreshed every 2 days.

Transfection:

C2C12 mouse myoblasts were grown to approximately 40-50% confluency prior to transfection.

A 1:2 ratio of plasmid to Jetprime reagent (Polyplus-transfection) was used for transfection.

Growth medium was refreshed just prior to and 4 hours after transfection.

Western Blot:

48 hours after transient plasmid transfection, cells were washed with PBS, scraped into PBS, and centrifuged at 1000×g for 10 minutes. The cell pellet was resuspended in PBS containing 1%

Triton X-100 (TX-100) and lysed for 10 minutes on ice. The lysate was centrifuged at 20,000×g for 10 minutes to separate insoluble material. The supernatant (soluble lysate) was aliquoted to a pre-cooled sample tube. The insoluble pellet was resuspended in 8M urea and 0.5% SDS and

91 sonicated using a Fisher Scientific Model 505 Sonic Dismembrator to break up insoluble material. Protein concentrations of the soluble and insoluble samples were measured using

PierceTM BCA Protein Assay Kit (Thermo Fisher). After the lysate samples were adjusted to the same protein concentraion with PBS containing 1% TX-100, Laemmli loading buffer (100 mM

Tris-HCl pH 6.8, 50 mM DTT, 10% glycerol, 10% SDS, bromophenol blue) was added to all samples and heated at 65°C for 10 minutes. Protein samples were separated with 10% SDS-

PAGE. Following protein separation, the proteins were transferred to a 0.45 μm nitrocellulose membrane (GE Healthcare) using a Bio-Rad Trans-blot SD Semi-Dry Transfer Cell in Towbin transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol) at constant 100 mA per gel for 30 minutes. Membranes were blocked with PBS containing 5% skim milk for 1 hour at room temperature. Membranes were incubated with primary antibody in PBS containing 5% skim milk overnight at 4°C. Membranes were washed three times with 10 mL PBS containing 0.1% TX-

100. A 1:10,000 dilution of secondary antibody (Jackson Immunoresearch) was added for 1 hour at room temperature. Membranes were washed three times with 10 mL PBS containing 0.1%

TX-100. The membranes were treated with Amersham ECL Western blotting reagent (GE

Healthcare) for 1 minute. Membranes were developed using film or a FluorChem HD2 digital camera (Alpha Innotech). Blots were quantified using Image Studio Lite (Licor)

Fluorescence Microscopy:

C2C12 myoblasts were seeded onto autoclaved coverslips (Fisher Scientific) coated with poly-

D-lysine (Corning). Cells were transfected with pEGFP-N1-Bag3WT, pEGFP-N1-Bag3P209L, or empty pCAGGS-GFP according to the protocol described previously. 48 hours after transfection, the cells were washed with PBS prior to fixation with a solution of PBS containing

4% formaldehyde (Electron Microscopy Sciences) for 20 minutes at room temperature. The

92 fixed cells were washed 3 times with PBS for 5 minutes each wash before permeabilization with

PBS containing 0.1% TX-100 for 10 minutes at room temperature. After washing 3 times with

PBS, 0.3 μM DAPI stain (Invitrogen) was added and incubated in the dark for 10 minutes at room temperature before washing 3 times with PBS. The coverslips were mounted onto microscopy slides (Fisher Scientific) with ProLong Gold Antifade reagent (Thermo Fisher) for at least 24 hours at room temperature in the dark prior to imaging with a Zeiss Observer.Z1 fluorescence microscope with an AxioCam MRm camera.

FLAG Immunoprecipitation:

C2C12 mouse myoblasts were seeded on 100mm dishes and transfected with empty pcDNA 3.1,

Bag3-p3xFLAG-CMV-14 and Bag3-P209L-p3xFLAG-CMV-14 as described previously. 48 hours after transfection, cells were washed with PBS, scraped in PBS, and centrifuged at 1000×g for 10 minutes at 4°C. The cell pellet was resuspended in lysis buffer, PBS containing 1% Triton

X-100 (TX-100) and Complete EDTA-free protease inhibitor cocktail (Roche). After 10 minutes of incubation on ice, the lysate was centrifuged at 20,000×g for 10 minutes at 4°C. The supernatant (lysate) was taken and its protein concentration measured using PierceTM BCA

Protein Assay Kit (Thermo Fisher). Samples were adjusted to the same protein concentration with lysis buffer. For FLAG immunoprecipitations to be used in mass spectrometry, the lysate was then pre-cleared through incubation with Sephadex G-25 Coarse beads (Amersham

Pharmacia) for 30 minutes, rotating at 4°C prior to centrifugation at 20,000×g for 10 minutes. 10

μL of packed anti-FLAG M2 magnetic beads (Sigma) in lysis buffer were added to each pre- cleared lysate sample and left rotating for 2.5 hours at 4°C. The beads were washed three times with pre-chilled PBS containing 0.1% TX-100 (wash buffer). For mass spectrometry studies, the beads were subsequently washed with 100mM ammonium bicarbonate (Sigma) buffer prior to

93 on-bead digestion. 20% of the packed beads were resuspended in Laemmli loading buffer for

Western blotting. For IP-Western blotting, the washed beads were eluted with 100 μg/mL

3xFLAG peptide (ApeXBio) in PBS for 60 minutes on ice.

Trypsin on-bead digestion of FLAG peptides:

FLAG immunoprecipitations were performed as previously described. After the 100mM ammonium bicarbonate wash, the FLAG magnetic beads were incubated at room temperature in

10mM DTT (Fisher Scientific) diluted in 100mM ammonium bicarbonate for 30 minutes.

Subsequently, 55 mM iodoacetamide was added and the samples incubated in the dark for 45 minutes. Mass spectrometry-grade trypsin (Promega) was added to the alkylated samples, which were incubated at 30°C overnight. 50% acetonitrile (Fisher Scientific) was used to wash the digested peptides from the beads. The supernatant was collected and trifluoroacetic acid (TFA)

(Sigma Aldrich) was added to a final concentration of 6%.

Titanium dioxide (TiO2) enrichment of phosphopeptides:

After tryptic digestion of FLAG peptides, the samples were incubated with TiO2 beads (GL

Sciences) for 30 minutes at 4°C based on previously published protocols (274-276). The supernatant was aspirated and the beads washed twice with pre-chilled solution of 0.5% TFA and

200 mM NaCl in 50% acetonitrile. The beads were then washed once with a 0.1% TFA in 50% acetonitrile. Phosphopeptides were eluted from the beads with two 5 minute incubations with

NH4OH (Sigma Aldrich) in 50% acetonitrile, pooling the two elutions together. The elution was evaporated in a centrifugal evaporator and resuspended prior to analysis by an Orbitrap Q-

Exactive HF nano-LC-MS/MS mass spectrometer.

Phosphoproteomics Database Searching and Protein Identification:

All MS/MS samples were analyzed using Mascot 2.5.1 (Matrix Science) and X! Tandem (Global

Proteome Machine) against the UniProt annotated human protein database assuming sample digestion by trypsin. Mascot and X! Tandem were searched with a fragment ion mass tolerance of 0.050 Da and a parent ion tolerance of 5.0 PPM. Glu->pyro-Glu of the N-terminus, ammonia- loss of the N-terminus, Gln->pyro-Glu of the N-terminus, oxidation of methionine and phosphorylation of serine, threonine and tyrosine were specified in X! Tandem as variable modifications. Oxidation of methionine and phosphorylation of serine, threonine and tyrosine were specified in Mascot as variable modifications. Scaffold 4.8.4 (Proteome Software Inc.) was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 95.0% probability. Protein identifications were accepted if they could be established at greater than 99.0% probability and contained at least 1 identified peptide. Phosphosites were counted and validated by hand.

BioID:

BioID experiments were designed based on published procedures (181,277,278). C2C12 mouse myoblasts were seeded and transfected with empty pcDNA5-BirA-FLAG expressing

BirA, pcDNA5-Bag3-BirA-FLAG and pcDNA5-Bag3-P209L-BirA-FLAG as described previously. 24 hours after transfection, the growth medium was replaced with growth medium supplemented with 50 μM biotin (Bioshop). After 24 hours of biotin incubation, the dishes were washed with 5 mL PBS, scraped in 5 mL pre-chilled PBS, and centrifuged at 1000×g for 10 minutes at 4°C. The cell pellet was lysed in lysis buffer (PBS containing 1% TX-100 and

Complete EDTA-free protease inhibitor cocktail) for 10 minutes at 4°C. The lysate was centrifuged at 20,000×g for 10 minutes at 4°C. The supernatant containing the soluble cellular

95 material was collected, and the insoluble pellet was resuspended in 8M urea and 0.5% sodium dodecyl sulfate (SDS). The insoluble pellet was sonicated with a Fisher Scientific Model 505

Sonic Dismembrator. The protein concentrations of the soluble and insoluble material were measured with PierceTM BCA Protein Assay Kit. The soluble samples were normalized with lysis buffer. The insoluble samples were normalized with lysis buffer, but were diluted at least 8-fold to reduce the urea concentration at or below 1M. The normalized samples were pre-cleared with

Sephadex G-25 Coarse beads for 30 minutes at 4°C. The pre-cleared samples were centrifuged at

20,000×g for 10 minutes prior to incubation with 15 μL packed Pierce streptavidin agarose resin

(Thermo Fisher) for 2 hours at 4°C. The resin was washed three times with pre-chilled PBS containing 0.1% TX-100, followed by a wash with 100 mM ammonium bicarbonate buffer.

Mass-spectrometry grade trypsin (Promega) was then added to the washed resin and incubated at

30°C overnight. 20% of the packed resin was kept for Western blot analysis. The digested peptides were washed from the resin with 50% acetonitrile, evaporated in a centrifugal evaporator, and resuspended in 0.1% formic acid (Fluka Analytical) prior to analysis by an

Orbitrap Q-Exactive HF nano-LC-MS/MS mass spectrometer.

BioID Database Searching and Protein Identification:

All MS/MS samples were analyzed using Mascot 2.5.1 (Matrix Science) and X! Tandem (Global

Proteome Machine) against the UniProt annotated mouse protein database assuming sample digestion by trypsin. Mascot and X! Tandem were searched with a fragment ion mass tolerance of 0.050 Da and a parent ion tolerance of 5.0 PPM. Glu->pyro-Glu of the N-terminus, ammonia- loss of the N-terminus, Gln->pyro-Glu of the N-terminus and oxidation of methionine were specified in X! Tandem as variable modifications. Oxidation of methionine was specified in

Mascot as a variable modification. Scaffold 4.8.4 (Proteome Software Inc.) was used to validate

MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at an FDR less than 5.0%. Protein identifications were accepted if they could be established at an FDR less than 5.0% and contained at least 1 identified peptide. The identified peptides were filtered to identify “hits”, which were defined as having peptide counts greater than 3 in either Bag3-WT or Bag3-P209L samples and counts at least two-fold higher than the BirA control.

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