Chaperone Network Research Articles

Differentiation Drives Widespread Rewiring of the Neural Stem Cell Chaperone Network

Neural stem and progenitor cells (NSPCs) are critical for continued cellular replacement in the adult brain. Lifelong maintenance of a functional NSPC pool necessitates stringent mechanisms to preserve a pristine proteome. We find that the NSPC chaperone network robustly maintains misfolded protein solubility and stress resilience through high levels of the ATP-dependent chaperonin TRiC/CCT. Strikingly, NSPC differentiation rewires the cellular chaperone network, reducing TRiC/CCT levels and inducing those of the ATP-independent small heat shock proteins (sHSPs). This switches the proteostasis strategy in neural progeny cells to promote sequestration of misfolded proteins into protective inclusions. The chaperone network of NSPCs is more effective than that of differentiated cells, leading to improved management of proteotoxic stress and amyloidogenic proteins. However, NSPC proteostasis is impaired by brain aging. The less efficient chaperone network of differentiated neural progeny may contribute to their enhanced susceptibility to neurodegenerative diseases characterized by aberrant protein misfolding and aggregation.

DNAJB6, a Key Factor in Neuronal Sensitivity to Amyloidogenesis

CAG-repeat expansions in at least eight different genes cause neurodegeneration. The length of the extended polyglutamine stretches in the corresponding proteins is proportionally related to their aggregation propensity. Although these proteins are ubiquitously expressed, they predominantly cause toxicity to neurons. To understand this neuronal hypersensitivity, we generated induced pluripotent stem cell (iPSC) lines of spinocerebellar ataxia type 3 and Huntington's disease patients. iPSC generation and neuronal differentiation are unaffected by polyglutamine proteins and show no spontaneous aggregate formation. However, upon glutamate treatment, aggregates form in neurons but not in patient-derived neural progenitors. During differentiation, the chaperone network is drastically rewired, including loss of expression of the anti-amyloidogenic chaperone DNAJB6. Upregulation of DNAJB6 in neurons antagonizes glutamate-induced aggregation, while knockdown of DNAJB6 in progenitors results in spontaneous polyglutamine aggregation. Loss of DNAJB6 expression upon differentiation is confirmed invivo, explaining why stem cells areintrinsically protected against amyloidogenesis and protein aggregates are dominantly present in neurons.

Correction: Function of hTim8a in complex IV assembly in neuronal cells provides insight into pathomechanism underlying Mohr-Tranebjærg syndrome

Human Tim8a and Tim8b are members of an intermembrane space chaperone network, known as the small TIM family. Mutations in TIMM8A cause a neurodegenerative disease, Mohr-Tranebjaerg syndrome (MTS), which is characterised by sensorineural hearing loss, dystonia and blindness. Nothing is known about the function of hTim8a in neuronal cells or how mutation of this protein leads to a neurodegenerative disease. We show that hTim8a is required for the assembly of Complex IV in neurons, which is mediated through a transient interaction with Complex IV assembly factors, in particular the copper chaperone COX17. Complex IV assembly defects resulting from loss of hTim8a leads to oxidative stress and changes to key apoptotic regulators, including cytochrome c, which primes cells for death. Alleviation of oxidative stress with Vitamin E treatment rescues cells from apoptotic vulnerability. We hypothesise that enhanced sensitivity of neuronal cells to apoptosis is the underlying mechanism of MTS.

Structural and dynamic insights revealing how lipase binding domain MD1 of Pseudomonas aeruginosa foldase affects lipase activation

Folding and cellular localization of many proteins of Gram-negative bacteria rely on a network of chaperones and secretion systems. Among them is the lipase-specific foldase Lif, a membrane-bound steric chaperone that tightly binds (KD = 29 nM) and mediates folding of the lipase LipA, a virulence factor of the pathogenic bacterium P. aeruginosa. Lif consists of five-domains, including a mini domain MD1 essential for LipA folding. However, the molecular mechanism of Lif-assisted LipA folding remains elusive. Here, we show in in vitro experiments using a soluble form of Lif (sLif) that isolated MD1 inhibits sLif-assisted LipA activation. Furthermore, the ability to activate LipA is lost in the variant sLifY99A, in which the evolutionary conserved amino acid Y99 from helix α1 of MD1 is mutated to alanine. This coincides with an approximately three-fold reduced affinity of the variant to LipA together with increased flexibility of sLifY99A in the complex as determined by polarization-resolved fluorescence spectroscopy. We have solved the NMR solution structures of P. aeruginosa MD1 and variant MD1Y99A revealing a similar fold indicating that a structural modification is likely not the reason for the impaired activity of variant sLifY99A. Molecular dynamics simulations of the sLif:LipA complex in connection with rigidity analyses suggest a long-range network of interactions spanning from Y99 of sLif to the active site of LipA, which might be essential for LipA activation. These findings provide important details about the putative mechanism for LipA activation and point to a general mechanism of protein folding by multi-domain steric chaperones.

The Role of HSF1 and the Chaperone Network in the Tumor Microenvironment.

Tumors are stressful environments. As tumors evolve from single mutated cancer cells into invasive malignancies they must overcome various constraints and barriers imposed by a hostile microenvironment. To achieve this, cancer cells recruit and rewire cells in their microenvironment to become pro-tumorigenic. We propose that chaperones are vital players in this process, and that activation of stress responses helps tumors adapt and evolve into aggressive malignancies, by enabling phenotypic plasticity in the tumor microenvironment (TME). In this chapter we will review evidence supporting non-cancer-cell-autonomous activity of chaperones in human patients and mouse models of cancer, discuss the mechanisms by which this non-cell-autonomous activity is mediated and provide an evolutionary perspective on the basis of this phenomenon.

Opposing effects of an F-box protein and the HSP90 chaperone network on microtubule stability and neurite growth in Caenorhabditis elegans.

Molecular chaperones often work collaboratively with the ubiquitylation-proteasome system (UPS) to facilitate the degradation of misfolded proteins, which typically safeguards cellular differentiation and protects cells from stress. In this study, however, we report that the Hsp70/Hsp90 chaperone machinery and an F-box protein, MEC-15, have opposing effects on neuronal differentiation, and that the chaperones negatively regulate neuronal morphogenesis and functions. Using the touch receptor neurons (TRNs) of Caenorhabditis elegans, we find that mec-15(-) mutants display defects in microtubule formation, neurite growth, synaptic development and neuronal functions, and that these defects can be rescued by the loss of Hsp70/Hsp90 chaperones and co-chaperones. MEC-15 probably functions in a Skp-, Cullin- and F-box- containing complex to degrade DLK-1, which is an Hsp90 client protein stabilized by the chaperones. The abundance of DLK-1, and likely other Hsp90 substrates, is fine-tuned by the antagonism between MEC-15 and the chaperones; this antagonism regulates TRN development, as well as synaptic functions of GABAergic motor neurons. Therefore, a balance between the UPS and the chaperones tightly controls neuronal differentiation.

Bacterial RF3 Senses Chaperone Function in Co-Translational Folding

Molecular chaperones assist in protein folding by interacting with nascent polypeptide chains (NCs) during translation. Whether the ribosome can sense chaperone defects and abort translation of misfolding NCs has not yet been explored. Here we used quantitative proteomics in E. coli to investigate the ribosome-associated chaperone network and the consequences of its dysfunction. Trigger factor and the DnaK (Hsp70) system are the major NC-binding chaperones. HtpG (Hsp90), GroEL and ClpB contribute increasingly, when DnaK is deficient. Surprisingly, misfolding of NCs due to defects in co-translational chaperone function or amino acid analog incorporation, results in recruitment of the non-canonical release factor RF3 to the ribosome. RF3 then cooperates with RF2 in mediating premature chain termination of a subset of abundant NCs, facilitating clearance by degradation. This function of RF3 efficiently reduces the accumulation of misfolded proteins. It is critical for proteostasis maintenance and cell survival under conditions of limited chaperone availability.

Challenging Proteostasis: Role of the Chaperone Network to Control Aggregation-Prone Proteins in Human Disease.

Protein homeostasis (Proteostasis) is essential for correct and efficient protein function within the living cell. Among the critical components of the Proteostasis Network (PN) are molecular chaperones that serve widely in protein biogenesis under physiological conditions, and prevent protein misfolding and aggregation enhanced by conditions of cellular stress. For Alzheimer's, Parkinson's, Huntington's diseases and ALS, multiple classes of molecular chaperones interact with the highly aggregation-prone proteins amyloid-β, tau, α-synuclein, huntingtin and SOD1 to influence the course of proteotoxicity associated with these neurodegenerative diseases. Accordingly, overexpression of molecular chaperones and induction of the heat shock response have been shown to be protective in a wide range of animal models of these diseases. In contrast, for cancer cells the upregulation of chaperones has the undesirable effect of promoting cellular survival and tumor growth by stabilizing mutant oncoproteins. In both situations, physiological levels of molecular chaperones eventually become functionally compromised by the persistence of misfolded substrates, leading to a decline in global protein homeostasis and the dysregulation of diverse cellular pathways. The phenomenon of chaperone competition may underlie the broad pathology observed in aging and neurodegenerative diseases, and restoration of physiological protein homeostasis may be a suitable therapeutic avenue for neurodegeneration as well as for cancer.

Small Molecule Inhibitors Targeting the Heat Shock Protein System of Human Obligate Protozoan Parasites.

Obligate protozoan parasites of the kinetoplastids and apicomplexa infect human cells to complete their life cycles. Some of the members of these groups of parasites develop in at least two systems, the human host and the insect vector. Survival under the varied physiological conditions associated with the human host and in the arthropod vectors requires the parasites to modulate their metabolic complement in order to meet the prevailing conditions. One of the key features of these parasites essential for their survival and host infectivity is timely expression of various proteins. Even more importantly is the need to keep their proteome functional by maintaining its functional capabilities in the wake of physiological changes and host immune responses. For this reason, molecular chaperones (also called heat shock proteins)—whose role is to facilitate proteostasis—play an important role in the survival of these parasites. Heat shock protein 90 (Hsp90) and Hsp70 are prominent molecular chaperones that are generally induced in response to physiological stress. Both Hsp90 and Hsp70 members are functionally regulated by nucleotides. In addition, Hsp70 and Hsp90 cooperate to facilitate folding of some key proteins implicated in cellular development. In addition, Hsp90 and Hsp70 individually interact with other accessory proteins (co-chaperones) that regulate their functions. The dependency of these proteins on nucleotide for their chaperone function presents an Achille’s heel, as inhibitors that mimic ATP are amongst potential therapeutic agents targeting their function in obligate intracellular human parasites. Most of the promising small molecule inhibitors of parasitic heat shock proteins are either antibiotics or anticancer agents, whose repurposing against parasitic infections holds prospects. Both cancer cells and obligate human parasites depend upon a robust protein quality control system to ensure their survival, and hence, both employ a competent heat shock machinery to this end. Furthermore, some inhibitors that target chaperone and co-chaperone networks also offer promising prospects as antiparasitic agents. The current review highlights the progress made so far in design and application of small molecule inhibitors against obligate intracellular human parasites of the kinetoplastida and apicomplexan kingdoms.

Investigation of a dilated cardiomyopathy-associated variant in BAG3 using genome-edited iPSC-derived cardiomyocytes.

Mutations in B cell lymphoma 2-associated athanogene 3 (BAG3) are recurrently associated with dilated cardiomyopathy (DCM) and muscular dystrophy. Using isogenic genome-edited human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), we examined how a DCM-causing BAG3 mutation (R477H), as well as complete loss of BAG3 (KO), impacts myofibrillar organization and chaperone networks. Although unchanged at baseline, fiber length and alignment declined markedly in R477H and KO iPSC-CMs following proteasome inhibition. RNA sequencing revealed extensive baseline changes in chaperone- and stress response protein-encoding genes, and protein levels of key BAG3 binding partners were perturbed. Molecular dynamics simulations of the BAG3-HSC70 complex predicted a partial disengagement by the R477H mutation. In line with this, BAG3-R477H bound less HSC70 than BAG3-WT in coimmunoprecipitation assays. Finally, myofibrillar disarray triggered by proteasome inhibition in R477H cells was mitigated by overexpression of the stress response protein heat shock factor 1 (HSF1). These studies reveal the importance of BAG3 in coordinating protein quality control subsystem usage within the cardiomyocyte and suggest that augmenting HSF1 activity might be beneficial as a means to mitigate proteostatic stress in the context of BAG3-associated DCM.

Epichaperome Abundance Predicts Response to the Epichaperome Inhibitor, Puh-71, in Acute Myeloid Leukemia

The epichaperome, which was previously described by our team (Rodina et al. Nature 2016, Joshi et al. Nature Reviews Cancer 2018), is a complex hyper-connected network of chaperones and co-chaperones that are present in a subset of tumors, and its role is to facilitate the survival of malignant cells. We have previously shown abundant epichaperome networks in different tumor types, including AML. PU-H71 is a novel epichaperome inhibitor that selectively targets cells expressing the epichaperome. We hypothesize that personalizing PU-H71 in AML based on epichaperome abundance will significantly improve the clinical efficacy of PU-H71. To investigate this, we assessed the frequency of epichaperome abundance in primary AML samples and sought to develop a method with clinical application to predict response to PU-H71. We screened 55 de novo and relapsed/refractory primary AML bone marrow and peripheral blood samples for epichaperome abundance and cytotoxicity. Epichaperome abundance was assessed by incubating cells with FITC-bound PU-H71 (labeled F2) for 4 hours followed by analysis by multi-parameter flow cytometry. A chemically similar FITC-bound PU-H71, which does not bind the epichaperome, termed F9, was used as a control (Taldone et al. Bioorg Med Chem Lett 2011). MV411 cell lines were also used as positive controls (given known epichaperome abundance and high sensitivity to PU-H71), and intra-sample T-cells were used as negative controls. Furthermore, we assessed cell survival by incubating cells with 0.5uM PU-H71 for 48-hours followed by evaluation by multi-parameter flow cytometry. All analyses were performed in duplicates or triplicates. The ratio of blast F2 to F9 was combined with the F2-F9 blast to T cell ratio to calculate epichaperome abundance. Data on epichaperome abundance was available in 50 patient samples. Cytotoxicity data was available in 41 patients. 22/41 (54%) of patient samples underwent ≥60% cell death, respectively, and 7/41 (17%) were resistant to PU-H71, as defined by < 5% cytotoxicity. An ROC curve using epichaperome cut-offs of 1, 2.5, 4.5, 6, 8 and 15 provided an AUC of 0.97 (95% CI 0.889-1.056, p=0.006). Using a cut-off of 4.5 for epichaperome abundance, 22/50 (44%) of samples expressed high levels of the epichaperome, and sensitivity and specificity for predicting >60% in vitro cytotoxicity to PU-H71 were 81.8% (95% CI 59.7-94.8%) and 94.7% (95% CI 74-100%) (Figure 1). This method's diagnostic accuracy was 87.8%, with positive and negative likelihood ratios of 15.5 and 0.19. Collectively, our data suggests that this flow cytometry-based evaluation of epichaperome abundance is a promising tool for detecting epichaperome abundance and personalizing the treatment of PU-H71 in patients with AML. Disclosures Chiosis: Samus Therapeutics: Equity Ownership, Patents & Royalties: Intellectual rights to the PU-FITC assay. Roboz:Trovagene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sandoz: Consultancy, Membership on an entity's Board of Directors or advisory committees; Roche/Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Otsuka: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orsenix: Consultancy, Membership on an entity's Board of Directors or advisory committees; MEI Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celltrion: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Actinium: Consultancy, Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amphivena: Consultancy, Membership on an entity's Board of Directors or advisory committees; Argenx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astex: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bayer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Eisai: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees. Guzman:Samus Therapeutics: Patents & Royalties: intellectual rights to the PU-FITC assay; Cellectis: Research Funding; SeqRx: Consultancy.

PTPN11 Mutated Acute Myeloid Leukemia (AML) Features an Abundant Epichaperome Network and Is Sensitive to the Epichaperome Inhibitor PU-H71

PTPN11 Mutated Acute Myeloid Leukemia (AML) Features an Abundant Epichaperome Network and Is Sensitive to the Epichaperome Inhibitor PU-H71

Function of hTim8a in complex IV assembly in neuronal cells provides insight into pathomechanism underlying Mohr-Tranebjærg syndrome.

Human Tim8a and Tim8b are members of an intermembrane space chaperone network, known as the small TIM family. Mutations in TIMM8A cause a neurodegenerative disease, Mohr-Tranebjærg syndrome (MTS), which is characterised by sensorineural hearing loss, dystonia and blindness. Nothing is known about the function of hTim8a in neuronal cells or how mutation of this protein leads to a neurodegenerative disease. We show that hTim8a is required for the assembly of Complex IV in neurons, which is mediated through a transient interaction with Complex IV assembly factors, in particular the copper chaperone COX17. Complex IV assembly defects resulting from loss of hTim8a leads to oxidative stress and changes to key apoptotic regulators, including cytochrome c, which primes cells for death. Alleviation of oxidative stress with Vitamin E treatment rescues cells from apoptotic vulnerability. We hypothesise that enhanced sensitivity of neuronal cells to apoptosis is the underlying mechanism of MTS.

The expanding world of plant J-domain proteins

Plants maintain cellular proteostasis during different phases of growth and development despite a barrage of biotic and abiotic stressors in an ever-changing environment. This requires a collaborative effort of a cadre of molecular chaperones. Hsp70s and their obligate co-chaperones, J-domain proteins (JDPs), are arguably the most ubiquitous and formidable components of the cellular chaperone network, facilitating numerous and diverse cellular processes and allowing survival under a plethora of stressful conditions. JDPs are also among the most versatile chaperones. Compared to Hsp70s, the number of JDP-encoding genes has proliferated, suggesting the emergence of highly complex Hsp70-JDP networks, particularly in plants. Recent studies indicate that besides the increase in the number of JDP encoding genes; regulatory differences, neo- and sub-functionalization, and inter- and intra-class combinatorial interactions, is rapidly expanding the repertoire of Hsp70-JDP systems. This results in highly robust and functionally diverse chaperone networks in plants. Here, we review the current status of plant JDP research and discuss how the paradigm shift in the field can be exploited toward a better understanding of JDP function and evolution.

Different Heat Shock Proteins Bind α-Synuclein With Distinct Mechanisms and Synergistically Prevent Its Amyloid Aggregation

α-Synuclein (α-Syn) forms pathological amyloid aggregates deposited in Lewy bodies and Lewy neurites in the brain of Parkinson’s disease (PD) patients. Heat shock proteins (Hsps) are the major components of the cellular chaperone network, which are responsible for preventing proteins from amyloid aggregation. Different Hsps were reported to interact with α-syn. However, the underlying mechanism of the interplay between α-syn and different Hsps remains unclear. Here, by combing NMR spectroscopy, electron microscope and other biochemical approaches, we systemically investigated the interaction between α-syn and three Hsps from different families including Hsp27, HDJ1, and Hsp104. We found that all three Hsps can weakly bind to α-syn and inhibit it from amyloid aggregation. Intriguingly, different Hsps recognize distinct regions of α-syn monomer, and act synergistically in chaperoning α-syn from fibril formation in sub-stoichiometry. Our results revealed the diverse binding mechanisms employed by different Hsps to tackle α-syn, and suggested that different Hsps form a network for cooperatively chaperoning α-syn from pathological aggregation.

Modulation of hippocampal neuronal resilience during aging by the Hsp70/Hsp90 co-chaperone STI1.

Chaperone networks are dysregulated with aging, but whether compromised Hsp70/Hsp90 chaperone function disturbs neuronal resilience is unknown. Stress-inducible phosphoprotein 1 (STI1; STIP1; HOP) is a co-chaperone that simultaneously interacts with Hsp70 and Hsp90, but whose function in vivo remains poorly understood. We combined in-depth analysis of chaperone genes in human datasets, analysis of a neuronal cell line lacking STI1 and of a mouse line with a hypomorphic Stip1 allele to investigate the requirement for STI1 in aging. Our experiments revealed that dysfunctional STI1 activity compromised Hsp70/Hsp90 chaperone network and neuronal resilience. The levels of a set of Hsp90 co-chaperones and client proteins were selectively affected by reduced levels of STI1, suggesting that their stability depends on functional Hsp70/Hsp90 machinery. Analysis of human databases revealed a subset of co-chaperones, including STI1, whose loss of function is incompatible with life in mammals, albeit they are not essential in yeast. Importantly, mice expressing a hypomorphic STI1 allele presented spontaneous age-dependent hippocampal neurodegeneration and reduced hippocampal volume, with consequent spatial memory deficit. We suggest that impaired STI1 function compromises Hsp70/Hsp90 chaperone activity in mammals and can by itself cause age-dependent hippocampal neurodegeneration in mice. Cover Image for this issue: doi: 10.1111/jnc.14749.

Protein quality control in the secretory pathway.

Protein folding is inherently error prone, especially in the endoplasmic reticulum (ER). Even with an elaborate network of molecular chaperones and protein folding facilitators, misfolding can occur quite frequently. To maintain protein homeostasis, eukaryotes have evolved a series of protein quality-control checkpoints. When secretory pathway quality-control pathways fail, stress response pathways, such as the unfolded protein response (UPR), are induced. In addition, the ER, which is the initial hub of protein biogenesis in the secretory pathway, triages misfolded proteins by delivering substrates to the proteasome or to the lysosome/vacuole through ER-associated degradation (ERAD) or ER-phagy. Some misfolded proteins escape the ER and are instead selected for Golgi quality control. These substrates are targeted for degradation after retrieval to the ER or delivery to the lysosome/vacuole. Here, we discuss how these guardian pathways function, how their activities intersect upon induction of the UPR, and how decisions are made to dispose of misfolded proteins in the secretory pathway.

Pervasive convergent evolution and extreme phenotypes define chaperone requirements of protein homeostasis

Maintaining protein homeostasis is an essential requirement for cell and organismal viability. An elaborate regulatory system within cells, the protein homeostasis network, safeguards that proteins are correctly folded and functional. At the heart of this regulatory system lies a class of specialized protein quality control enzymes called chaperones that are tasked with assisting proteins in their folding, avoiding aggregation and degradation. Failure and decline of protein homeostasis are directly associated with conditions of aging and aging-related neurodegeneration. However, it is not clear what tips the balance of protein homeostasis and leads to onset of aging and diseases. Here, using a comparative genomics approach we report general principles of maintaining protein homeostasis across the eukaryotic tree of life. Expanding a previous study of 16 eukaryotes to the quantitative analysis of 216 eukaryotic genomes, we find a strong correlation between the composition of eukaryotic chaperone networks and genome complexity that is distinct for different species kingdoms. Organisms with pronounced phenotypes clearly buck this trend. Northobranchius furzeri, the shortest-lived vertebrate and a widely used model for fragile protein homeostasis, is found to be chaperone limited while Heterocephalus glaber as the longest-lived rodent and thus an especially robust organism is characterized by above-average numbers of chaperones. Strikingly, the relative size of chaperone networks is found to generally correlate with longevity in Metazoa. Our results thus indicate that the balance in protein homeostasis may be a key variable in explaining organismal robustness.

Nascent Polypeptide Domain Topology and Elongation Rate Direct the Cotranslational Hierarchy of Hsp70 and TRiC/CCT

Cotranslational protein folding requires assistance from elaborate ribosome-associated chaperone networks. It remains unclear how the changing information in a growing nascent polypeptide dictates the recruitment of functionally distinct chaperones. Here, we used ribosome profiling to define the principles governing the cotranslational action of the chaperones TRiC/CCT and Hsp70/Ssb. We show that these chaperones are sequentially recruited to specific sites within domain-encoding regions of select nascent polypeptides. Hsp70 associates first, binding select sites throughout domains, whereas TRiC associates later, upon the emergence of nearly complete domains that expose an unprotected hydrophobic surface. This suggests that transient topological properties of nascent folding intermediates drive sequential chaperone association. Moreover, cotranslational recruitment of both TRiC and Hsp70 correlated with translation elongation slowdowns. We propose that the temporal modulation of the nascent chain structural landscape is coordinated with local elongation rates to regulate the hierarchical action of Hsp70 and TRiC for cotranslational folding.

Abstract 934: Physical and Genetic Interaction Analyses in Human Pluripotent Stem Cell-Derived Cardiomyocytes to Study Protein Quality Control Disease in the Heart

Cardiomyocytes must maintain constant contractile function of the heart throughout a human lifetime. To achieve this, a complex network of chaperones and other proteins maintains the homeostasis of the proteins in the cell. Protein quality control pathways are increasingly recognized for their importance in both inherited and sporadic cardiac disease, and as potential therapeutic targets. The co-chaperone BAG3 is particularly interesting, because genetic evidence suggests that variants of BAG3 can be both pathologic and protective. However, understanding the molecular mechanism underlying most of these disease-genetic variant associations remains a major challenge. Unbiased analysis of physical and genetic interaction networks holds the potential to provide critical information to fill in this knowledge gap. Using genome engineering tools, we have generated a series of isogenic human induced pluripotent (iPS) cell lines bearing different variants on the BAG3 gene, along with an epitope tag fusion. Using mass spectrometry methods, we have been able to identify interacting partners for the BAG3 protein expressed at endogenous levels in iPS-derived cardiomyocytes. Our results show a surprisingly high number of cardiac-specific interactions. In addition, some BAG3 protein variants present distinct gain or loss of specific interactions, allowing us to narrow down the list of candidate hits. Using microscopy image analysis, we performed a targeted knockdown screening on BAG3 interaction partners to dissect pathways of BAG3 interactors that are involved in the development disease phenotype.We hope the information obtained from this study will improve our understanding of the heart proteostasis network, enable the identification of potential therapeutic targets, and provide clues towards a broader understanding of the role of genetic variation in complex disease.