NudC guides client transfer between the Hsp40/70 and Hsp90 chaperone systems.

In the eukaryotic cytosol, the Hsp70 and the Hsp90 chaperone machines work in tandem with the maturation of a diverse array of client proteins. The transfer of nonnative clients between these systems is essential to the chaperoning process, but how it is regulated is still not clear. We discovered that NudC is an essential transfer factor with an unprecedented mode of action: NudC interacts with Hsp40 in Hsp40-Hsp70-client complexes and displaces Hsp70. Then, the interaction of NudC with Hsp90 allows the direct transfer of Hsp40-bound clients to Hsp90 for further processing. Consistent with this mechanism, NudC increases client activation in vitro as well as in cells and is essential for cellular viability. Together, our results show the complexity of the cooperation between the major chaperone machineries in the eukaryotic cytosol.

Coordinated Conformational Processing of the Tumor Suppressor Protein p53 by the Hsp70 and Hsp90 Chaperone Machineries.

p53, the guardian of the genome, requires chaperoning by Hsp70 and Hsp90. However, how the two chaperone machineries affect p53 conformation and regulate its function remains elusive. We found that Hsp70, together with Hsp40, unfolds p53 in an ATP-dependent reaction. This unfolded state of p53 is susceptible to aggregation after release induced by the nucleotide exchange factor Bag-1. However, when Hsp90 and the adaptor protein Hop are present, p53 is transferred from Hsp70 to Hsp90, allowing restoration of the native state upon ATP hydrolysis. Our results suggest that the p53 conformation is constantly remodeled by the two major chaperone machineries. This connects p53 activity to stress, and the levels of free molecular chaperones are important factors regulating p53 activity. Together, our findings reveal an intricate interplay and cooperation of Hsp70 and Hsp90 in regulating the conformation of a client.

The Co-chaperone Cns1 and the Recruiter Protein Hgh1 Link Hsp90 to Translation Elongation via Chaperoning Elongation Factor 2.

The Hsp90 chaperone machinery in eukaryotes comprises a number of distinct accessory factors. Cns1 is one of the few essential co-chaperones in yeast, but its structure and function remained unknown. Here, we report the X-ray structure of the Cns1 fold and NMR studies on the partly disordered, essential segment of the protein. We demonstrate that Cns1 is important for maintaining translation elongation, specifically chaperoning the elongation factor eEF2. In this context, Cns1 interacts with the novel co-factor Hgh1 and forms a quaternary complex together with eEF2 and Hsp90. The in vivo folding and solubility of eEF2 depend on the presence of these proteins. Chaperoning of eEF2 by Cns1 is essential for yeast viability and requires a defined subset of the Hsp90 machinery as well as the identified eEF2 recruiting factor Hgh1.

Data-driven large-scale genomic analysis reveals an intricate phylogenetic and functional landscape in J-domain proteins.

The 70-kD heat shock protein (Hsp70) chaperone system is a central hub of the proteostasis network that helps maintain protein homeostasis in all organisms. The recruitment of Hsp70 to perform different and specific cellular functions is regulated by the J-domain protein (JDP) co-chaperone family carrying the small namesake J-domain, required to interact and drive the ATPase cycle of Hsp70s. Besides the J-domain, prokaryotic and eukaryotic JDPs display a staggering diversity in domain architecture, function, and cellular localization. Very little is known about the overall JDP family, despite their essential role in cellular proteostasis, development, and its link to a broad range of human diseases. In this work, we leverage the exponentially increasing number of JDP gene sequences identified across all kingdoms owing to the advancements in sequencing technology and provide a broad overview of the JDP repertoire. Using an automated classification scheme based on artificial neural networks (ANNs), we demonstrate that the sequences of J-domains carry sufficient discriminatory information to reliably recover the phylogeny, localization, and domain composition of the corresponding full-length JDP. By harnessing the interpretability of the ANNs, we find that many of the discriminatory sequence positions match residues that form the interaction interface between the J-domain and Hsp70. This reveals that key residues within the J-domains have coevolved with their obligatory Hsp70 partners to build chaperone circuits for specific functions in cells.

The Plasticity of the Hsp90 Co-chaperone System.

The Hsp90 system in the eukaryotic cytosol is characterized by a cohort of co-chaperones that bind to Hsp90 and affect its function. Although progress has been made regarding the underlying biochemical mechanisms, how co-chaperones influence Hsp90 client proteins in vivo has remained elusive. By investigating the effect of 12 Hsp90 co-chaperones on the activity of different client proteins in yeast, we find that deletion of co-chaperones can have a neutral or negative effect on client activity but can also lead to more active clients. Only a few co-chaperones are active on all clients studied. Closely related clients and even point mutants can depend on different co-chaperones. These effects are direct because differences in client-co-chaperone interactions can be reconstituted in vitro. Interestingly, some co-chaperones affect client conformation in vivo. Thus, co-chaperones adapt the Hsp90 cycle to the requirements of the client proteins, ensuring optimal activation.

Beyond Chaperoning: UCS Proteins Emerge as Regulators of Myosin-Mediated Cellular Processes.

The UCS (UNC-45/CRO1/She4p) family of proteins has emerged as chaperones specific for the folding, assembly, and function of myosin. UCS proteins participate in various myosin-dependent cellular processes including myofibril organization and muscle functions, cell differentiation, striated muscle development, cytokinesis, and endocytosis. Mutations in the genes that code for UCS proteins cause serious defects in myosin-dependent cellular processes. UCS proteins that contain an N-terminal tetratricopeptide repeat (TPR) domain are called UNC-45. Vertebrates usually possess two variants of UNC-45, the ubiquitous general-cell UNC-45 (UNC-45A) and the striated muscle UNC-45 (UNC-45B), which is exclusively expressed in skeletal and cardiac muscles. Except for the TPR domain in UNC-45, UCS proteins comprise of several irregular armadillo (ARM) repeats that are organized into a central domain, a neck region, and the canonical C-terminal UCS domain that functions as the chaperoning module. With or without TPR, UCS proteins form linear oligomers that serve as scaffolds that mediate myosin folding, organization into myofibrils, repair, and motility. This chapter reviews emerging functions of these proteins with a focus on UNC-45 as a dedicated chaperone for folding, assembly, and function of myosin at protein and potentially gene levels. Recent experimental evidences strongly support UNC-45 as an absolute regulator of myosin, with each domain of the chaperone playing different but complementary roles during the folding, assembly, and function of myosin, as well as recruiting Hsp90 as a co-chaperone to optimize key steps. It is becoming increasingly clear that UNC-45 also regulates the transcription of several genes involved in myosin-dependent cellular processes.

CHIP: A Co-chaperone for Degradation by the Proteasome and Lysosome.

Protein homeostasis relies on a balance between protein folding and protein degradation. Molecular chaperones like Hsp70 and Hsp90 fulfill well-defined roles in protein folding and conformational stability via ATP-dependent reaction cycles. These folding cycles are controlled by associations with a cohort of non-client protein co-chaperones, such as Hop, p23, and Aha1. Pro-folding co-chaperones facilitate the transit of the client protein through the chaperone-mediated folding process. However, chaperones are also involved in proteasomal and lysosomal degradation of client proteins. Like folding complexes, the ability of chaperones to mediate protein degradation is regulated by co-chaperones, such as the C-terminal Hsp70-binding protein (CHIP/STUB1). CHIP binds to Hsp70 and Hsp90 chaperones through its tetratricopeptide repeat (TPR) domain and functions as an E3 ubiquitin ligase using a modified RING finger domain (U-box). This unique combination of domains effectively allows CHIP to network chaperone complexes to the ubiquitin-proteasome and autophagosome-lysosome systems. This chapter reviews the current understanding of CHIP as a co-chaperone that switches Hsp70/Hsp90 chaperone complexes from protein folding to protein degradation.

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.

J-Domain Proteins Orchestrate the Multifunctionality of Hsp70s in Mitochondria: Insights from Mechanistic and Evolutionary Analyses.

Mitochondrial J-domain protein (JDP) co-chaperones orchestrate the function of their Hsp70 chaperone partner(s) in critical organellar processes that are essential for cell function. These include folding, refolding, and import of mitochondrial proteins, maintenance of mitochondrial DNA, and biogenesis of iron-sulfur cluster(s) (FeS), prosthetic groups needed for function of mitochondrial and cytosolic proteins. Consistent with the organelle's endosymbiotic origin, mitochondrial Hsp70 and the JDPs' functioning in protein folding and FeS biogenesis clearly descended from bacteria, while the origin of the JDP involved in protein import is less evident. Regardless of their origin, all mitochondrial JDP/Hsp70 systems evolved unique features that allowed them to perform mitochondria-specific functions. Their modes of functional diversification and specialization illustrate the versatility of JDP/Hsp70 systems and inform our understanding of system functioning in other cellular compartments.

Sequence-based features that are determinant for tail-anchored membrane protein sorting in eukaryotes.

The correct targeting and insertion of tail-anchored (TA) integral membrane proteins is critical for cellular homeostasis. TA proteins are defined by a hydrophobic transmembrane domain (TMD) at their C-terminus and are targeted to either the ER or mitochondria. Derived from experimental measurements of a few TA proteins, there has been little examination of the TMD features that determine localization. As a result, the localization of many TA proteins are misclassified by the simple heuristic of overall hydrophobicity. Because ER-directed TMDs favor arrangement of hydrophobic residues to one side, we sought to explore the role of geometric hydrophobic properties. By curating TA proteins with experimentally determined localizations and assessing hypotheses for recognition, we bioinformatically and experimentally verify that a hydrophobic face is the most accurate singular metric for separating ER and mitochondria-destined yeast TA proteins. A metric focusing on an 11 residue segment of the TMD performs well when classifying human TA proteins. The most inclusive predictor uses both hydrophobicity and C-terminal charge in tandem. This work provides context for previous observations and opens the door for more detailed mechanistic experiments to determine the molecular factors driving this recognition.

Protein interactions in the biological assembly of iron-sulfur clusters in Escherichia coli: Molecular and mechanistic aspects of the earliest assembly steps.

This contribution focuses on the earliest steps of the assembly of FeS clusters and their insertion into acceptor apoproteins, that call for transient formation of a 2Fe2S cluster on a scaffold protein from sulfide and iron salts. For the sake of simplicity, this report is essentially limited to the Escherichia coli isc-encoded proteins and does not take into account agents that modulate the enzymatic synthesis of sulfide by protein in the same operon or the redox events associated with both sulfide generation and conversion of 2Fe2S structures in clusters of higher nuclearity. Therefore, the results discussed here are based on chemical reconstitution systems using inorganic sulfide, ferric salts, and excess thiols. This simplification offers the possibility to address some mechanistic issues related to the role of protein/protein interaction as for modulating: (a) the rate of cluster assembly on scaffold proteins; (b) the stability of the cluster on the scaffold protein; and (c) the rate of transfer to acceptor apoproteins as also influenced by the acceptor concentration. The emerging picture highlights the mechanistic versatility of the systems, that is discussed in terms of the capability of such an apparently simple combination of proteins to cope with various physiological situation. The hypothetical mechanism presented here may represent an additional way of modulating the rate and outcome of the overall process while avoiding potential toxicity issues.

The co-chaperone HOP participates in TIR1 stabilisation and in auxin response in plants.

HOP (HSP70-HSP90 organising protein) is a conserved family of co-chaperones well known in mammals for its role in the folding of signalling proteins associated with development. In plants, HOP proteins have been involved in the response to multiple stresses, but their role in plant development remains elusive. Herein, we describe that the members of the HOP family participate in different aspects of plant development as well as in the response to warm temperatures through the regulation of auxin signalling. Arabidopsis hop1 hop2 hop3 triple mutant shows different auxin-related phenotypes and a reduced auxin sensitivity. HOP interacts with TIR1 auxin coreceptor in vivo. Furthermore, TIR1 accumulation and auxin transcriptional response are reduced in the hop1 hop2 hop3 triple mutant, suggesting that HOP's function in auxin signalling is related, at least, to TIR1 interaction and stabilisation. Interestingly, HOP proteins form part of the same complexes as SGT1b (a different HSP90 co-chaperone) and these co-chaperones synergistically cooperate in auxin signalling. This study provides relevant data about the role of HOP in auxin regulation in plants and uncovers that both co-chaperones, SGT1b and HOP, cooperate in the stabilisation of common targets involved in plant development.

Regulation of Translation, Translocation, and Degradation of Proteins at the Membrane of the Endoplasmic Reticulum.

The endoplasmic reticulum (ER) of mammalian cells is the central organelle for the maturation and folding of transmembrane proteins and for proteins destined to be secreted into the extracellular space. The proper folding of target proteins is achieved and supervised by a complex endogenous chaperone machinery. BiP, a member of the Hsp70 protein family, is the central chaperone in the ER. The chaperoning activity of BiP is assisted by ER-resident DnaJ (ERdj) proteins due to their ability to stimulate the low, intrinsic ATPase activity of BiP. Besides their co-chaperoning activity, ERdj proteins also regulate and tightly control the translation, translocation, and degradation of proteins. Disturbances in the luminal homeostasis result in the accumulation of unfolded proteins, thereby eliciting a stress response, the so-called unfolded protein response (UPR). Accumulated proteins are either deleterious due to the functional loss of the respective protein and/or due to their deposition as intra- or extracellular protein aggregates. A variety of metabolic diseases are known to date, which are associated with the dysfunction of components of the chaperone machinery. In this review, we will delineate the impact of ERdj proteins in controlling protein synthesis and translocation under physiological and under stress conditions. A second aspect of this review is dedicated to the role of ERdj proteins in the ER-associated degradation pathway, by which unfolded or misfolded proteins are discharged from the ER. We will refer to some of the most prominent diseases known to be based on the dysfunction of ERdj proteins.

Heat Shock Proteins as Targets for Novel Antimalarial Drug Discovery.

Plasmodium falciparum, the parasitic agent that is responsible for a severe and dangerous form of human malaria, has a history of long years of cohabitation with human beings with attendant negative consequences. While there have been some gains in the fight against malaria through the application of various control measures and the use of chemotherapeutic agents, and despite the global decline in malaria cases and associated deaths, the continual search for new and effective therapeutic agents is key to achieving sustainable development goals. An important parasite survival strategy, which is also of serious concern to the scientific community, is the rate at which the parasites continually develop resistance to drugs. Among the key players in the parasite's ability to develop resistance, maintain cellular integrity, and survives within an unusual environment of the red blood cells are the molecular chaperones of the heat shock proteins (HSP) family. HSPs constitute a novel avenue for antimalarial drug discovery and by exploring their ubiquitous nature and multifunctional activities, they may be suitable targets for the discovery of multi-targets antimalarial drugs, needed to fight incessant drug resistance. In this chapter, features of selected families of plasmodial HSPs that can be exploited in drug discovery are presented. Also, known applications of HSPs in small molecule screening, their potential usefulness in high throughput drug screening, as well as possible challenges are highlighted.

Heat Shock Proteins of Malaria: Highlights and Future Prospects.

The deadliest malaria parasite of humans, Plasmodium falciparum, is an obligate parasite that has had to develop mechanisms for survival under the unfavourable conditions it confronts within host cells. The chapters in the book "Heat Shock Proteins of Malaria" provide a critique of the evidence that heat shock proteins (Hsps) play a key role in the survival of P. falciparum in host cells. The role of the plasmodial Hsp arsenal is not limited to the protection of the parasite cell (largely through their role as molecular chaperones), as some of these proteins also promote the pathological development of malaria. This is largely due to the export of a large number of these proteins into the infected erythrocyte cytosol. Although P. falciparum erythrocyte membrane protein 1 (PfEMP1) is the main virulence factor for the malaria parasite, some of the exported plasmodial Hsps appear to augment parasite virulence. While this book largely delves into experimentally validated information on the role of Hsps in the development and pathogenicity of malaria, some of the information is based on hypotheses yet to be fully tested. Therefore, here we highlight what we know to be definite roles of plasmodial Hsps. Furthermore, we distill information that could provide practical insights on the options available for future research directions, including interventions against malaria that may target the role of Hsps in the development of the disease.

Introductory Chapter: The Importance of Heat Shock Proteins in Survival and Pathogenesis of the Malaria Parasite Plasmodium falciparum.

Malaria did not die with the end of the age of western colonization but is still a major public health issue in large parts of the world. Despite repeated and concerted efforts to eradicate this disease, it has proved remarkably resilient, and constant vigilance and continuous research are required to discover new chinks in the parasite's armor and alleviate the suffering at both the individual and societal levels. A deeper understanding of the fundamental processes underlying parasite survival, propagation, virulence, and ability to cause disease is the key to the development of desperately needed new therapies and prophylactic drugs. Malaria parasites, by the nature of their lifecycle, are subject to a number of environmental and cellular stresses which they must overcome to survive. To this end, they express a number of heat shock proteins (HSPs), molecules specialized on buffering the effects of external stimuli, but which are also essential for normal cellular biochemistry. In this introductory chapter, I give a brief overview of the diversity of structure, function, and importance of these HSPs, and highlight some of the current and future research questions in this field. Additionally, this chapter acts as a bridge to the other chapters in this book. These chapters, I think you will agree, demonstrate that with regard to HSPs malaria parasites, as in so many things, obey the adage "Same same, but different."

Aha-type co-chaperones: the alpha or the omega of the Hsp90 ATPase cycle?

Heat shock protein 90 (Hsp90) is a dimeric molecular chaperone that plays an essential role in cellular homeostasis. It functions in the context of a structurally dynamic ATP-dependent cycle to promote conformational changes in its clientele to aid stability, maturation, and activation. The client activation cycle is tightly regulated by a cohort of co-chaperone proteins that display specific binding preferences for certain conformations of Hsp90, guiding Hsp90 through its functional ATPase cycle. Aha-type co-chaperones are well-known to robustly stimulate the ATPase activity of Hsp90 but other roles in regulating the functional cycle are being revealed. In this review, we summarize the work done on the Aha-type co-chaperones since the 1990s and highlight recent discoveries with respect to the complexity of Hsp90 cycle regulation.

Chemical Perturbation of Oncogenic Protein Folding: from the Prediction of Locally Unstable Structures to the Design of Disruptors of Hsp90-Client Interactions.

Protein folding quality control in cells requires the activity of a class of proteins known as molecular chaperones. Heat shock protein-90 (Hsp90), a multidomain ATP driven molecular machine, is a prime representative of this family of proteins. Interactions between Hsp90, its co-chaperones, and client proteins have been shown to be important in facilitating the correct folding and activation of clients. Hsp90 levels and functions are elevated in tumor cells. Here, we computationally predict the regions on the native structures of clients c-Abl, c-Src, Cdk4, B-Raf and Glucocorticoid Receptor, that have the highest probability of undergoing local unfolding, despite being ordered in their native structures. Such regions represent potential ideal interaction points with the Hsp90-system. We synthesize mimics spanning these regions and confirm their interaction with partners of the Hsp90 complex (Hsp90, Cdc37 and Aha1) by Nuclear Magnetic Resonance (NMR). Designed mimics selectively disrupt the association of their respective clients with the Hsp90 machinery, leaving unrelated clients unperturbed and causing apoptosis in cancer cells. Overall, selective targeting of Hsp90 protein-protein interactions is achieved without causing indiscriminate degradation of all clients, setting the stage for the development of therapeutics based on specific chaperone:client perturbation.

Origin and Evolution of the Human Bcl2-Associated Athanogene-1 (BAG-1).

Molecular chaperones, particularly the 70-kDa heat shock proteins (Hsp70s), are key orchestrators of the cellular stress response. To perform their critical functions, Hsp70s require the presence of specific co-chaperones, which include nucleotide exchange factors containing the BCL2-associated athanogene (BAG) domain. BAG-1 is one of these proteins that function in a wide range of cellular processes, including apoptosis, protein refolding, and degradation, as well as tumorigenesis. However, the origin of BAG-1 proteins and their evolution between and within species are mostly uncharacterized. This report investigated the macro- and micro-evolution of BAG-1 using orthologous sequences and single nucleotide polymorphisms (SNPs) to elucidate the evolution and understand how natural variation affects the cellular stress response. We first collected and analyzed several BAG-1 sequences across animals, plants, and fungi; mapped intron positions and phases; reconstructed phylogeny; and analyzed protein characteristics. These data indicated that BAG-1 originated before the animals, plants, and fungi split, yet most extant fungal species have lost BAG-1. Furthermore, although BAG-1's structure has remained relatively conserved, kingdom-specific conserved differences exist at sites of known function, suggesting functional specialization within each kingdom. We then analyzed SNPs from the 1000 genomes database to determine the evolutionary patterns within humans. These analyses revealed that the SNP density is unequally distributed within the BAG1 gene, and the ratio of non-synonymous/synonymous SNPs is significantly higher than 1 in the BAG domain region, which is an indication of positive selection. To further explore this notion, we performed several biochemical assays and found that only one out of five mutations tested altered the major co-chaperone properties of BAG-1. These data collectively suggest that although the co-chaperone functions of BAG-1 are highly conserved and can probably tolerate several radical mutations, BAG-1 might have acquired specialized and potentially unexplored functions during the evolutionary process.

Phenylalanine hydroxylase variants interact with the co-chaperone DNAJC12.

DNAJC12, a type III member of the HSP40/DNAJ family, has been identified as the specific co-chaperone of phenylalanine hydroxylase (PAH) and the other aromatic amino acid hydroxylases. DNAJ proteins work together with molecular chaperones of the HSP70 family to assist in proper folding and maintenance of intracellular stability of their clients. Autosomal recessive mutations in DNAJC12 were found to reduce PAH levels, leading to hyperphenylalaninemia (HPA) in patients without mutations in PAH. In this work, we investigated the interaction of normal wild-type DNAJC12 with mutant PAH in cells expressing several PAH variants associated with HPA in humans, as well as in the Enu1/1 mouse model, homozygous for the V106A-Pah variant, which leads to severe protein instability, accelerated PAH degradation and mild HPA. We found that mutant PAH exhibits increased ubiquitination, instability, and aggregation compared with normal PAH. In mouse liver lysates, we showed that DNAJC12 interacts with monoubiquitin-tagged PAH. This form represented a major fraction of PAH in the Enu1/1 but was also present in liver of wild-type PAH mice. Our results support a role of DNAJC12 in the processing of misfolded ubiquitinated PAH by the ubiquitin-dependent proteasome/autophagy systems and add to the evidence that the DNAJ proteins are important players both for proper folding and degradation of their clients.