Network Of Molecular Chaperones Research Articles

The Plasmodium falciparum exported J domain proteins fine-tune human and malarial Hsp70s: pathological exploitation of proteostasis machinery.

Cellular proteostasis requires a network of molecular chaperones and co-chaperones, which facilitate the correct folding and assembly of other proteins, or the degradation of proteins misfolded beyond repair. The function of the major chaperones, heat shock protein 70 (Hsp70) and heat shock protein 90 (Hsp90), is regulated by a cohort of co-chaperone proteins. The J domain protein (JDP) family is one of the most diverse co-chaperone families, playing an important role in functionalizing the Hsp70 chaperone system to form a powerful protein quality control network. The intracellular malaria parasite, Plasmodium falciparum, has evolved the capacity to invade and reboot mature human erythrocytes, turning them into a vehicles of pathology. This process appears to involve the harnessing of both the human and parasite chaperone machineries. It is well known that malaria parasite-infected erythrocytes are highly enriched in functional human Hsp70 (HsHsp70) and Hsp90 (HsHsp90), while recent proteomics studies have provided evidence that human JDPs (HsJDPs) may also be enriched, but at lower levels. Interestingly, P. falciparum JDPs (PfJDPs) are the most prominent and diverse family of proteins exported into the infected erythrocyte cytosol. We hypothesize that the exported PfJPDs may be an evolutionary consequence of the need to boost chaperone power for specific protein folding pathways that enable both survival and pathogenesis of the malaria parasite. The evidence suggests that there is an intricate network of PfJDP interactions with the exported malarial Hsp70 (PfHsp70-x) and HsHsp70, which appear to be important for the trafficking of key malarial virulence factors, and the proteostasis of protein complexes of human and parasite proteins associated with pathology. This review will critically evaluate the current understanding of the role of exported PfJDPs in pathological exploitation of the proteostasis machinery by fine-tuning the chaperone properties of both human and malarial Hsp70s.

Protein Misfolding and Aggregation in Proteinopathies: Causes, Mechanism and Cellular Response

Proteins are central to life functions. Alterations in the structure of proteins are reflected in their function. Misfolded proteins and their aggregates present a significant risk to the cell. Cells have a diverse but integrated network of protection mechanisms. Streams of misfolded proteins that cells are continuously exposed to must be continually monitored by an elaborated network of molecular chaperones and protein degradation factors to control and contain protein misfolding problems. Aggregation inhibition properties of small molecules such as polyphenols are important as they possess other beneficial properties such as antioxidative, anti-inflammatory, and pro-autophagic properties and help neuroprotection. A candidate with such desired features is important for any possible treatment development for protein aggregation diseases. There is a need to study the protein misfolding phenomenon so that we can treat some of the worst kinds of human ailments related to protein misfolding and aggregation.

HSP70-HSP90 Chaperone Networking in Protein-Misfolding Disease.

Molecular chaperones and their associated co-chaperones are essential in health and disease as they are key facilitators of protein-folding, quality control and function. In particular, the heat-shock protein (HSP) 70 and HSP90 molecular chaperone networks have been associated with neurodegenerative diseases caused by aberrant protein-folding. The pathogenesis of these disorders usually includes the formation of deposits of misfolded, aggregated protein. HSP70 and HSP90, plus their co-chaperones, have been recognised as potent modulators of misfolded protein toxicity, inclusion formation and cell survival in cellular and animal models of neurodegenerative disease. Moreover, these chaperone machines function not only in folding but also in proteasome-mediated degradation of neurodegenerative disease proteins. This chapter gives an overview of the HSP70 and HSP90 chaperones, and their respective regulatory co-chaperones, and explores how the HSP70 and HSP90 chaperone systems form a larger functional network and its relevance to counteracting neurodegenerative disease associated with misfolded proteins and disruption of proteostasis.

Molecular Effects of Elongation Factor Ts and Trigger Factor on the Unfolding and Aggregation of Elongation Factor Tu Induced by the Prokaryotic Molecular Chaperone Hsp33.

Simple SummaryProteins are versatile biological macromolecules involved in most biological processes. However, because of the highly labile nature of protein structures, protein quality control (PQC) to ensure proteostasis (i.e., protein homeostasis)is difficult. Therefore, proteins of a specialized class (i.e., molecular chaperones) are required that assist in proper folding and prevent aberrant folding of other proteins. Hsp33 was originally discovered as a holding chaperone that is overexpressed upon heat shock and activated by oxidation to prevent the misfolding of client proteins. Recently, an unfoldase/aggregase activity of Hsp33 was identified in its reduced state against a specific substrate, EF-Tu, which plays a key role in protein biosynthesis in cells. The present study demonstrates that EF-Tu unfolding/aggregation by Hsp33 can be accelerated by another molecular chaperone trigger factor. Given that the unfolded/aggregated EF-Tu is finally degraded by another chaperone, Lon protease, it is likely that a chaperone network dysregulating EF-Tu operates in heat shock to attenuate protein biosynthesis, which is harmful to cell survival under stressed conditions. Therefore, the apparently contradictory chaperone function (i.e., promotion of client misfolding) of Hsp33 can also be associated with the PQC processes to ensure proteostasis in cells.Hsp33, a prokaryotic redox-regulated holding chaperone, has been recently identified to be able to exhibit an unfoldase and aggregase activity against elongation factor Tu (EF-Tu) in its reduced state. In this study, we investigated the effect of elongation factor Ts (EF-Ts) and trigger factor (TF) on Hsp33-mediated EF-Tu unfolding and aggregation using gel filtration, light scattering, circular dichroism, and isothermal titration calorimetry. We found that EF-Tu unfolding and subsequent aggregation induced by Hsp33 were evident even in its complex state with EF-Ts, which enhanced EF-Tu stability. In addition, although TF alone had no substantial effect on the stability of EF-Tu, it markedly amplified the Hsp33-mediated EF-Tu unfolding and aggregation. Collectively, the present results constitute the first example of synergistic unfoldase/aggregase activity of molecular chaperones and suggest that the stability of EF-Tu is modulated by a sophisticated network of molecular chaperones to regulate protein biosynthesis in cells under stress conditions.

Deciphering Network Crosstalk: The Current Status and Potential of miRNA Regulatory Networks on the HSP40 Molecular Chaperone Network.

Molecular chaperone networks fulfill complex roles in protein homeostasis and are essential for maintaining cell health. Hsp40s (commonly referred to as J-proteins) have critical roles in development and are associated with a variety of human diseases, yet little is known regarding the J-proteins with respect to the post-transcriptional mechanisms that regulate their expression. With relatively small alterations in their abundance and stoichiometry altering their activity, post-transcriptional regulation potentially has significant impact on the functions of J-proteins. MicroRNAs (miRNAs) are a large group of non-coding RNAs that form a complex regulatory network impacting gene expression. Here we review and investigate the current knowledge and potential intersection of miRNA regulatory networks with the J-Protein chaperone network. Analysis of datasets from the current version of TargetScan revealed a great number of predicted microRNAs targeting J-proteins compared to the limited reports of interactions to date. There are likely unstudied regulatory interactions that influence chaperone biology contained within our analysis. We go on to present some criteria for prioritizing candidate interactions including potential cooperative targeting of J-Proteins by multiple miRNAs. In summary, we offer a view on the scope of regulation of J-Proteins through miRNAs with the aim of guiding future investigations by identifying key regulatory nodes within these two complex cellular networks.

The functions and regulation of heat shock proteins; key orchestrators of proteostasis and the heat shock response.

Cells respond to protein-damaging (proteotoxic) stress by activation of the Heat Shock Response (HSR). The HSR provides cells with an enhanced ability to endure proteotoxic insults and plays a crucial role in determining subsequent cell death or survival. The HSR is, therefore, a critical factor that influences the toxicity of protein stress. While named for its vital role in the cellular response to heat stress, various components of the HSR system and the molecular chaperone network execute essential physiological functions as well as responses to other diverse toxic insults. The effector molecules of the HSR, the Heat Shock Factors (HSFs) and Heat Shock Proteins (HSPs), are also important regulatory targets in the progression of neurodegenerative diseases and cancers. Modulation of the HSR and/or its extended network have, therefore, become attractive treatment strategies for these diseases. Development of effective therapies will, however, require a detailed understanding of the HSR, important features of which continue to be uncovered and are yet to be completely understood. We review recently described and hallmark mechanistic principles of the HSR, the regulation and functions of HSPs, and contexts in which the HSR is activated and influences cell fate in response to various toxic conditions.

Violacein-Induced Chaperone System Collapse Underlies Multistage Antiplasmodial Activity.

Antimalarial drugs with novel modes of action and wide therapeutic potential are needed to pave the way for malaria eradication. Violacein is a natural compound known for its biological activity against cancer cells and several pathogens, including the malaria parasite, Plasmodium falciparum (Pf). Herein, using chemical genomic profiling (CGP), we found that violacein affects protein homeostasis. Mechanistically, violacein binds Pf chaperones, PfHsp90 and PfHsp70-1, compromising the latter’s ATPase and chaperone activities. Additionally, violacein-treated parasites exhibited increased protein unfolding and proteasomal degradation. The uncoupling of the parasite stress response reflects the multistage growth inhibitory effect promoted by violacein. Despite evidence of proteotoxic stress, violacein did not inhibit global protein synthesis via UPR activation—a process that is highly dependent on chaperones, in agreement with the notion of a violacein-induced proteostasis collapse. Our data highlight the importance of a functioning chaperone–proteasome system for parasite development and differentiation. Thus, a violacein-like small molecule might provide a good scaffold for development of a novel probe for examining the molecular chaperone network and/or antiplasmodial drug design.

Effect of molecular chaperone network modulators treatment on energy and tau metabolism in human cells

AbstractBackgroundAlzheimer disease (AD) is a complex multifactorial disease representing >60 % cases of dementia with no disease modifying therapies available in clinics largely due to its poorly understood pathology. One of the new proteins that recently emerged as a promising target in AD therapy is FKBP51, or FK506‐binding protein 51 kDa. FKBP51, together with its partner, a molecular chaperone heat shock protein 90 kDa (Hsp90), have been found to play an important role in metabolism of tau promoting its accumulation and aggregation. In vitro, in vivo and epidemiological studies also suggest an important role of FKBP51 in control of body weight and insulin resistance.MethodRecently, we have successfully obtained several active compounds able to inhibit FKBP51‐Hsp90 interactions both in in vitro assays and in human cell lines. SH‐SY5Y cells, differentiated human adipocytes were treated with FKBP51‐Hsp90 interaction inhibitors. Effect of compounds on energy metabolism, lipid metabolism as well on tau accumulation was analyzed. Using RNAseq we have analyzed transcriptome changes upon compound treatment.ResultAddition of compounds resulted in profound stimulation of energy metabolism both in SH‐SY5Y cells and differentiated adipocytes. Significant increase in mitochondrial membrane potential was also observed. Dramatic decrease in size and number of lipid droplets was observed in differentiated adipocytes upon compound treatment. RNAseq results show increased expression of genes involved in lipid catabolism, beta‐oxidation and thermogenesis. We have also observed decreased tau levels in the treated SH‐SY5Y cells.ConclusionOur data implicate FKBP51 as a possible target for AD treatment affecting cell energy metabolism and tau accumulation.

Characterization, expression profiling, and thermal tolerance analysis of heat shock protein 70 in pine sawyer beetle, Monochamus alternatus hope (Coleoptera: Cerambycidae).

Monochamus alternatus Hope (Coleoptera: Cerambycidae) warrants attention as a dominant transmission vector of the pinewood nematode, and it exhibits tolerance to high temperature. Heat shock protein 70 (HSP70) family members, including inducible HSP70 and heat shock cognate protein 70 (HSC70), are major contributors to the molecular chaperone networks of insects under heat stress. In this regard, we specifically cloned and characterized three MaltHSP70s and three MaltHSC70s. Bioinformatics analysis on the deduced amino acid sequences showed these genes, having close genetic relationships with HSP70s of Coleopteran species, collectively shared conserved signature structures and ATPase domains. Subcellular localization prediction revealed the HSP70s of M. alternatus were located not only in the cytoplasm and endoplasmic reticulum but also in the nucleus and mitochondria. The transcript levels of MaltHSP70s and MaltHSC70s in each state were significantly upregulated by exposure to 35-50°C for early 3 h, while MaltHSP70s reached a peak after exposure to 45°C for 2-3 h in contrast to less-upregulated MaltHSC70s. In terms of MaltHSP70s, the expression threshold in females was lower than that in males. Also, both fat bodies and Malpighian tubules were the tissues most sensitive to heat stress in M. alternatus larvae. Lastly, the ATPase activity of recombinant MaltHSP70-2 in vitro remained stable at 25-40°C, and this recombinant availably enhanced the thermotolerance of Escherichia coli. Overall, our findings unraveled HSP70s might be the intrinsic mediators of the strong heat tolerance of M. alternatus due to their stabilized structure and bioactivity.

Molecular and pharmacological chaperones for SOD1.

The efficacy of superoxide dismutase-1 (SOD1) folding impacts neuronal loss in motor system neurodegenerative diseases. Mutations can prevent SOD1 post-translational processing leading to misfolding and cytoplasmic aggregation in familial amyotrophic lateral sclerosis (ALS). Evidence of immature, wild-type SOD1 misfolding has also been observed in sporadic ALS, non-SOD1 familial ALS and Parkinson's disease. The copper chaperone for SOD1 (hCCS) is a dedicated and specific chaperone that assists SOD1 folding and maturation to produce the active enzyme. Misfolded or misfolding prone SOD1 also interacts with heat shock proteins and macrophage migration inhibitory factor to aid folding, refolding or degradation. Recognition of specific SOD1 structures by the molecular chaperone network and timely dissociation of SOD1-chaperone complexes are, therefore, important steps in SOD1 processing. Harnessing these interactions for therapeutic benefit is actively pursued as is the modulation of SOD1 behaviour with pharmacological and peptide chaperones. This review highlights the structural and mechanistic aspects of a selection of SOD1-chaperone interactions together with their impact on disease models.

Review: Protein misfolding diseases - the rare case of Marinesco-Sjögren syndrome.

Secretory and cell membrane proteins are synthesized in the endoplasmic reticulum (ER), where a network of molecular chaperones and folding factors ensure correct protein folding and export to post-ER compartments. Failure of this process leads to accumulation of unfolded/misfolded proteins, ER stress, and activation of the unfolded protein response (UPR), a complex signalling pathway aimed at restoring ER homeostasis, whose failure eventually leads to cell death. Suppressor of Ire1/Lhs1 double mutant (SIL1) is a nucleotide exchange factor for immunoglobulin binding protein, the main ER chaperone and primary sensor of ER stress. Loss of SIL1 function causes Marinesco-Sjögren syndrome (MSS), a rare multisystem disease of early infancy for which there is no cure. This review, examines the current understanding of SIL1 activities in the ER, and reviews experimental data describing the consequences of SIL1 deficiency in cell and animal models. We discuss the evidence supporting a role of the UPR - particularly the protein kinase RNA-like endoplasmic reticulum kinase branch - in the pathogenesis of MSS, and how this may be pharmacologically manipulated for treatment.

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.

Molecular cloning, expression, and functional characterization of 70-kDa heat shock protein, DnaK, from Bacillus halodurans

In the present study, we report cloning, sequencing, and functional characterization dnaK gene of B. halodurans that is the central component in cellular network of molecular chaperones. The 3D structures of DnaK obtained by I-TASSER server showed that the overall structures of DnaK from B. halodurans and human HSP70 chaperone BiP are very similar with a homology of 88.8%. The purified recombinant DnaK consists of a His-tag at C-terminus and show a band on approximately 70-kDa region in SDS-PAGE. The resultant refolding assay revealed that the refolding rate was considerably improved by the addition of the novel DnaK chaperone for the refolding of heat-denatured carbonic anhydrase. Also, salt resistance experiments indicated that E. coli + DnaK survival had enhanced by 4.4-fold as compared with control cells in 0.4 M NaCl. The number of E. coli + DnaK colonies was 2.5-fold higher than control colonies in pH 9.5. We showed that DnaK refolding functions were decreased by increasing Cd2+ in nanomolar concentrations. Hg2+ had a biphasic effect on recombinant DnaK refolding function: inhibition at low and stimulation at high concentrations. It was concluded that the DnaK from B. halodurans can potentially be employed for improving functional properties of proteins in various applications.

Interplay between the Hsp90 Chaperone and the HslVU Protease To Regulate the Level of an Essential Protein in Shewanella oneidensis.

Protein synthesis, folding, and degradation are an accurately regulated process occurring in every organism and called proteostasis. This process is essential to maintain a healthy proteome since proteostasis dysregulation is responsible for devastating cellular issues. Proteostasis is controlled by a complex network of molecular chaperones and proteases. Among them, eukaryotic Hsp90, assisted by many cochaperones and the Hsp70 chaperone system, plays a major role in activating hundreds of client proteins, and Hsp90 inhibition usually leads to proteasomal degradation of these clients. In bacteria, however, the precise function of Hsp90 remains quite unclear, and only a few clients are known. Recently, we have shown that Hsp90 is essential at elevated temperature in the aquatic model bacterium Shewanella oneidensis, and we have identified a client of Hsp90, TilS, involved in tRNA modification. Here we found that two members of the proteostasis network with antagonist activities, the Hsp90 chaperone and the HslVU protease, which is considered the proteasome ancestor, together regulate the level of TilS. In particular, we show that deletion of the genes coding for the HslVU protease suppresses the growth defect of an S. oneidensis strain with hsp90 deleted, by increasing the cellular level of the essential TilS protein. These results open up new avenues for understanding how proteostasis is controlled in bacteria, and new Hsp90 clients are much needed now to confirm the interplay between Hsp90 and proteases.IMPORTANCE Maintaining a healthy proteome is essential in every living cell from bacteria to humans. For example, proteostasis (protein homeostasis) imbalance in humans leads to devastating diseases, including neurodegenerative diseases and cancers. Therefore, proteins need to be assisted from their synthesis to their native folding and ultimately to their degradation. To ensure efficient protein turnover, cells possess an intricate network of molecular chaperones and proteases for protein folding and degradation. However, these networks need to be better defined and understood. Here, using the aquatic bacterium Shewanella oneidensis as a model organism, we demonstrate interplay between two proteins with antagonist activities, the Hsp90 chaperone and the HslVU protease, to finely regulate the level of an essential client of Hsp90. Therefore, this work provides a new bacterial model to better study protein regulation and turnover, and it sheds light on how proteostasis by Hsp90 and proteases could be controlled in bacteria.

Chaperoning Endoplasmic Reticulum-Associated Degradation (ERAD) and Protein Conformational Diseases.

Misfolded proteins compromise cellular homeostasis. This is especially problematic in the endoplasmic reticulum (ER), which is a high-capacity protein-folding compartment and whose function requires stringent protein quality-control systems. Multiprotein complexes in the ER are able to identify, remove, ubiquitinate, and deliver misfolded proteins to the 26S proteasome for degradation in the cytosol, and these events are collectively termed ER-associated degradation, or ERAD. Several steps in the ERAD pathway are facilitated by molecular chaperone networks, and the importance of ERAD is highlighted by the fact that this pathway is linked to numerous protein conformational diseases. In this review, we discuss the factors that constitute the ERAD machinery and detail how each step in the pathway occurs. We then highlight the underlying pathophysiology of protein conformational diseases associated with ERAD.

Defining Hsp33's Redox-regulated Chaperone Activity and Mapping Conformational Changes on Hsp33 Using Hydrogen-deuterium Exchange Mass Spectrometry.

Living organisms regularly need to cope with fluctuating environments during their life cycle, including changes in temperature, pH, the accumulation of reactive oxygen species, and more. These fluctuations can lead to a widespread protein unfolding, aggregation, and cell death. Therefore, cells have evolved a dynamic and stress-specific network of molecular chaperones, which maintain a "healthy" proteome during stress conditions. ATP-independent chaperones constitute one major class of molecular chaperones, which serve as first-line defense molecules, protecting against protein aggregation in a stress-dependent manner. One feature these chaperones have in common is their ability to utilize structural plasticity for their stress-specific activation, recognition, and release of the misfolded client. In this paper, we focus on the functional and structural analysis of one such intrinsically disordered chaperone, the bacterial redox-regulated Hsp33, which protects proteins against aggregation during oxidative stress. Here, we present a toolbox of diverse techniques for studying redox-regulated chaperone activity, as well as for mapping conformational changes of the chaperone, underlying its activity. Specifically, we describe a workflow which includes the preparation of fully reduced and fully oxidized proteins, followed by an analysis of the chaperone anti-aggregation activity in vitro using light-scattering, focusing on the degree of the anti-aggregation activity and its kinetics. To overcome frequent outliers accumulated during aggregation assays, we describe the usage of Kfits, a novel graphical tool which allows easy processing of kinetic measurements. This tool can be easily applied to other types of kinetic measurements for removing outliers and fitting kinetic parameters. To correlate the function with the protein structure, we describe the setup and workflow of a structural mass spectrometry technique, hydrogen-deuterium exchange mass spectrometry, that allows the mapping of conformational changes on the chaperone and substrate during different stages of Hsp33 activity. The same methodology can be applied to other protein-protein and protein-ligand interactions.

Mapping interactions with the chaperone network reveals factors that protect against tau aggregation.

A network of molecular chaperones is known to bind proteins (“clients”) and balance their folding, function and turnover. However, it is often not clear which chaperones are critical for selective recognition of individual clients. It is also not clear why these key chaperones might fail in protein aggregation diseases. In this study, we utilized human microtubule-associated protein tau (MAPT or tau) as a model client to survey interactions between ~30 purified chaperones and ~20 disease-associated tau variants (~600 combinations). From this large-scale analysis, we identified human DnaJA2 as an unexpected, but potent, inhibitor of tau aggregation. DnaJA2 levels were correlated with tau pathology in human brains, supporting the idea that it is an important regulator of tau homeostasis. Of significance, we found that some disease-associated tau variants were relatively immune to interactions with chaperones, suggesting a model in which avoiding physical recognition by chaperone networks may contribute to disease.Impact StatementLarge-scale screening of chaperone interactions with tau and its variants identified DnaJA2 as a key protective factor in tauopathy.

Development of GMP-1 a molecular chaperone network modulator protecting mitochondrial function and its assessment in fly and mice models of Alzheimer's disease.

Mitochondrial dysfunction is an early feature of Alzheimer's disease (AD) and may play an important role in the pathogenesis of disease. It has been shown that amyloid beta peptide (Aβ) and amyloid precursor protein (APP) interact with mitochondria contributing to the mitochondrial dysfunction in AD. Prevention of abnormal protein targeting to mitochondria can protect normal mitochondrial function, increase neuronal survival and at the end, ameliorate symptoms of AD and other neurodegenerative disorders. First steps of mitochondrial protein import are coordinated by molecular chaperones Hsp70 and Hsp90 that bind to the newly synthesized mitochondria‐destined proteins and deliver them to the protein import receptors on the surface of organelle. Here, we have described the development of a novel compound named GMP‐1 that disrupts interactions between Hsp70/Hsp90 molecular chaperones and protein import receptor Tom70. GMP‐1 treatment of SH‐SY5Y cells results in decrease in mitochondria‐associated APP and protects SH‐SY5Y cells from toxic effect of Aβ1‐42 exposure. Experiments in drosophila and mice models of AD demonstrated neuroprotective effect of GMP‐1 treatment, improvement in memory and behaviour tests as well as restoration of mitochondrial function.

Protein-Protein Interactions in the Molecular Chaperone Network.

Molecular chaperones play a central role in protein homeostasis (a.k.a. proteostasis) by balancing protein folding, quality control, and turnover. To perform these diverse tasks, chaperones need the malleability to bind nearly any "client" protein and the fidelity to detect when it is misfolded. Remarkably, these activities are carried out by only ∼180 dedicated chaperones in humans. How do a relatively small number of chaperones maintain cellular and organismal proteostasis for an entire proteome? Furthermore, once a chaperone binds a client, how does it "decide" what to do with it? One clue comes from observations that individual chaperones engage in protein-protein interactions (PPIs)-both with each other and with their clients. These physical links coordinate multiple chaperones into organized, functional complexes and facilitate the "handoff" of clients between them. PPIs also link chaperones and their clients to other cellular pathways, such as those that mediate trafficking (e.g., cytoskeleton) and degradation (e.g., proteasome). The PPIs of the chaperone network have a wide range of affinity values (nanomolar to micromolar) and involve many distinct types of domain modules, such as J domains, zinc fingers, and tetratricopeptide repeats. Many of these motifs have the same binding surfaces on shared partners, such that members of one chaperone class often compete for the same interactions. Somehow, this collection of PPIs draws together chaperone families and creates multiprotein subnetworks that are able to make the "decisions" of protein quality control. The key to understanding chaperone-mediated proteostasis might be to understand how PPIs are regulated. This Account will discuss the efforts of our group and others to map, measure, and chemically perturb the PPIs within the molecular chaperone network. Structural biology methods, including X-ray crystallography, NMR spectroscopy, and electron microscopy, have all played important roles in visualizing the chaperone PPIs. Guided by these efforts and -omics approaches to measure PPIs, new advances in high-throughput chemical screening that are specially designed to account for the challenges of this system have emerged. Indeed, chemical biology has played a particularly important role in this effort, as molecules that either promote or inhibit specific PPIs have proven to be invaluable research probes in cells and animals. In addition, these molecules have provided leads for the potential treatment of protein misfolding diseases. One of the major products of this research field has been the identification of putative PPI drug targets within the chaperone network, which might be used to change chaperone "decisions" and rebalance proteostasis.

Systems analysis of the genetic interaction network of yeast molecular chaperones.

Molecular chaperones are typically promiscuous interacting proteins that function globally in the cell to maintain protein homeostasis. Recently, we had carried out experiments that elucidated a comprehensive interaction network for the core 67 chaperones and 15 cochaperones in the budding yeast Saccharomyces cerevisiae [Rizzolo et al., Cell Rep., 2017, 20, 2735-2748]. Here, the genetic (i.e. epistatic) interaction network obtained for chaperones was further analyzed, revealing that the global topological parameters of the resulting network have a more central role in mediating interactions in comparison to the rest of the proteins in the cell. Most notably, we observed Hsp10, Hsp70 Ssz1 chaperone, and Hsp90 cochaperone Cdc37 to be the main drivers of the network architecture. Systematic analysis on the physicochemical properties for all chaperone interactors further revealed the presence of preferential domains and folds that are highly interactive with chaperones such as the WD40 repeat domain. Further analysis with established cellular complexes revealed the involvement of R2TP chaperone in quaternary structure formation. Our results thus provide a global overview of the chaperone network properties in yeast, expanding our understanding of their functional diversity and their role in protein homeostasis.