Molecular Chaperone Machinery Research Articles

Two-colour single-molecule photoinduced electron transfer fluorescence imaging microscopy of chaperone dynamics

Many proteins are molecular machines, whose function is dependent on multiple conformational changes that are initiated and tightly controlled through biochemical stimuli. Their mechanistic understanding calls for spectroscopy that can probe simultaneously such structural coordinates. Here we present two-colour fluorescence microscopy in combination with photoinduced electron transfer (PET) probes as a method that simultaneously detects two structural coordinates in single protein molecules, one colour per coordinate. This contrasts with the commonly applied resonance energy transfer (FRET) technique that requires two colours per coordinate. We demonstrate the technique by directly and simultaneously observing three critical structural changes within the Hsp90 molecular chaperone machinery. Our results reveal synchronicity of conformational motions at remote sites during ATPase-driven closure of the Hsp90 molecular clamp, providing evidence for a cooperativity mechanism in the chaperone’s catalytic cycle. Single-molecule PET fluorescence microscopy opens up avenues in the multi-dimensional exploration of protein dynamics and allosteric mechanisms.

Structure and function of the co-chaperone protein phosphatase 5 in cancer.

Protein phosphatase 5 (PP5) is a serine/threonine protein phosphatase that regulates many cellular functions including steroid hormone signaling, stress response, proliferation, apoptosis, and DNA repair. PP5 is also a co-chaperone of the heat shock protein 90 molecular chaperone machinery that assists in regulation of cellular signaling pathways essential for cell survival and growth. PP5 plays a significant role in survival and propagation of multiple cancers, which makes it a promising target for cancer therapy. Though there are several naturally occurring PP5 inhibitors, none is specific for PP5. Here, we review the roles of PP5 in cancer progression and survival and discuss the unique features of the PP5 structure that differentiate it from other phosphoprotein phosphatase (PPP) family members and make it an attractive therapeutic target.

Dominant Steady State Proteome Changes in the Absence of CHIP Highlight a Role in Neuronal Cell Membrane Integrity Linked to the Actin Cytoskeleton

Functional integrity of the E3-ubiquitin ligase CHIP is essential for healthy aging in the brain. By virtue of its association with the core molecular chaperone machinery, studies into the physiological role(s) of CHIP have been dominated by its involvement in the quality control (QC) and degradation of unfolded, aggregated and/or mutated proteins. To elucidate the dominant changes in protein steady state levels in human neurons where CHIP function was ablated, we conducted an unbiased proteomic analysis in genetically engineered CHIP knockout compared to wild-type iPSC-derived cortical neurons. Rather than a broad effect on protein homeostasis, loss of CHIP function impacted on a focused cohort of proteins that analysis defined as actin cytoskeleton signalling and membrane integrity pathways. In support of the pathway analysis, CHIP KO cell-lines had enhanced sensitivity to mechanical and chemical membrane damage. Consistent with recent studies on the identification of a non-canonical HSP-independent E3-activity for CHIP, the lack of general QC defects in knock-out cells suggests that the major readout of CHIP function in cortical neurons is the regulation of native folded substrates involved in maintaining cellular ‘health’. Thus, CHIP-mediated regulation of the of cytoskeletal- and membrane-related proteins is likely to make a vital contribution to the neuroprotective activity of CHIP.

Hsp90 Chaperones Bluetongue Virus Proteins and Prevents Proteasomal Degradation.

The molecular chaperone machinery is important for the maintenance of protein homeostasis within the cells. The principle activities of the chaperone machinery are to facilitate protein folding and organize conformationally dynamic client proteins. Prominent among the members of the chaperone family are heat shock protein 70 (Hsp70) and 90 (Hsp90). Like cellular proteins, viral proteins depend upon molecular chaperones to mediate their stabilization and folding. Bluetongue virus (BTV), which is a model system for the Reoviridae family, is a nonenveloped arbovirus that causes hemorrhagic disease in ruminants. This constitutes a significant burden upon animals of commercial significance, such as sheep and cattle. Here, for the first time, we examined the role of chaperone proteins in the viral lifecycle of BTV. Using a combination of molecular, biochemical, and microscopic techniques, we examined the function of Hsp90 and its relevance to BTV replication. We demonstrate that Hsp70, the chaperone that is commonly usurped by viral proteins, does not influence virus replication, while Hsp90 activity is important for virus replication by stabilizing BTV proteins and preventing their degradation via the ubiquitin-proteasome pathway. To our knowledge this is the first report showing the involvement of Hsp90 as a modulator of BTV infection.IMPORTANCE Protein chaperones are instrumental for maintaining protein homeostasis, enabling correct protein folding and organization; prominent members include heat shock proteins 70 and 90. Virus infections place a large burden on this homeostasis. Identifying and understanding the underlying mechanisms that facilitate Bluetongue virus replication and spread through the usurpation of host factors is of primary importance for the development of intervention strategies. Our data identify and show that heat shock protein 90, but not heat shock protein 70, stabilizes bluetongue virus proteins, safeguarding them from proteasomal degradation.

Natural compounds as potential Hsp90 inhibitors for breast cancer-Pharmacophore guided molecular modelling studies

Breast cancer is one of the major impediments affecting women globally. The ATP-dependant heat shock protein 90 (Hsp90) forms the central component of molecular chaperone machinery that predominantly governs the folding of newly synthesized peptides and their conformational maturation. It regulates the stability and function of numerous client proteins that are frequently upregulated and/or mutated in cancer cells, therefore, making Hsp90 inhibition a promising therapeutic strategy for the development of new efficacious drugs to treat breast cancer. In the present in silico investigation, a structure-based pharmacophore model was generated with hydrogen bond donor, hydrogen bond acceptor and hydrophobic features complementary to crucial residues Ala55, Lys58, Asp93, Ile96, Met98 and Thr184 directed at inhibiting the ATP-binding activity of Hsp90. Subsequently, the phytochemical dataset of 3210 natural compounds was screened to retrieve the prospective inhibitors after rigorous validation of the model pharmacophore. The retrieved 135 phytocompounds were further filtered by drug-likeness parameters including Lipinski’s rule of five and ADMET properties, then investigated via molecular docking-based scoring. Molecular interactions were assessed using Genetic Optimisation for Ligand Docking program for 95 drug-like natural compounds against Hsp90 along with two clinical drugs as reference compounds – Geldanamycin and Radicicol. Docking studies revealed three phytochemicals are better than the investigated clinical drugs. The reference and hit compounds with dock scores of 48.27 (Geldanamycin), 40.90 (Radicicol), 73.04 (Hit1), 72.92 (Hit2) and 68.12 (Hit3) were further validated for their binding stability through molecular dynamics simulations. We propose that the non-macrocyclic scaffolds of three identified phytochemicals might aid in the development of novel therapeutic candidates against Hsp90-driven cancers.

The amino-terminal domain of Mycobacteriumtuberculosis ClpB protein plays a crucial role in its substrate disaggregation activity.

Mycobacterium tuberculosis (Mtb) is known to persist in extremely hostile environments within host macrophages. The ability to withstand such proteotoxic stress comes from its highly conserved molecular chaperone machinery. ClpB, a unique member of the AAA+ family of chaperones, is responsible for resolving aggregates in Mtb and many other bacterial pathogens. Mtb produces two isoforms of ClpB, a full length and an N‐terminally truncated form (ClpB∆N), with the latter arising from an internal translation initiation site. It is not clear why this internal start site is conserved and what role the N‐terminal domain (NTD) of Mtb ClpB plays in its function. In the current study, we functionally characterized and compared the two isoforms of Mtb ClpB. We found the NTD to be dispensable for oligomerization, ATPase activity and prevention of aggregation activity of ClpB. Both ClpB and ClpB∆N were found to be capable of resolubilizing protein aggregates. However, the efficiency of ClpB∆N at resolubilizing higher order aggregates was significantly lower than that of ClpB. Further, ClpB∆N exhibited reduced affinity for substrates as compared to ClpB. We also demonstrated that the surface of the NTD of Mtb ClpB has a hydrophobic groove that contains four hydrophobic residues: L97, L101, F140 and V141. These residues act as initial contacts for the substrate and are crucial for stable interaction between ClpB and highly aggregated substrates.

Unfolding the chaperone story.

Protein folding in the cell was originally assumed to be a spontaneous process, based on Anfinsen’s discovery that purified proteins can fold on their own after removal from denaturant. Consequently cell biologists showed little interest in the protein folding process. This changed only in the mid and late 1980s, when the chaperone story began to unfold. As a result, we now know that in vivo, protein folding requires assistance by a complex machinery of molecular chaperones. To ensure efficient folding, members of different chaperone classes receive the nascent protein chain emerging from the ribosome and guide it along an ordered pathway toward the native state. I was fortunate to contribute to these developments early on. In this short essay, I will describe some of the critical steps leading to the current concept of protein folding as a highly organized cellular process.

From chaperonins to Rubisco assembly and metabolic repair.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) mediates the fixation of atmospheric CO2 in photosynthesis by catalyzing the carboxylation of the 5-carbon sugar ribulose-1,5-bisphosphate (RuBP). Despite its pivotal role, Rubisco is an inefficient enzyme and thus has been a key target for bioengineering. However, efforts to increase crop yields by Rubisco engineering remain unsuccessful, due in part to the complex machinery of molecular chaperones required for Rubisco biogenesis and metabolic repair. While the large subunit of Rubisco generally requires the chaperonin system for folding, the evolution of the hexadecameric Rubisco from its dimeric precursor resulted in the dependence on an array of additional factors required for assembly. Moreover, Rubisco function can be inhibited by a range of sugar-phosphate ligands. Metabolic repair of Rubisco depends on remodeling by the ATP-dependent Rubisco activase and hydrolysis of inhibitors by specific phosphatases. This review highlights our work toward understanding the structure and mechanism of these auxiliary machineries.

Cellular Regulation of Amyloid Formation in Aging and Disease.

As the population is aging, the incidence of age-related neurodegenerative diseases, such as Alzheimer and Parkinson disease, is growing. The pathology of neurodegenerative diseases is characterized by the presence of protein aggregates of disease specific proteins in the brain of patients. Under certain conditions these disease proteins can undergo structural rearrangements resulting in misfolded proteins that can lead to the formation of aggregates with a fibrillar amyloid-like structure. Cells have different mechanisms to deal with this protein aggregation, where the molecular chaperone machinery constitutes the first line of defense against misfolded proteins. Proteins that cannot be refolded are subjected to degradation and compartmentalization processes. Amyloid formation has traditionally been described as responsible for the proteotoxicity associated with different neurodegenerative disorders. Several mechanisms have been suggested to explain such toxicity, including the sequestration of key proteins and the overload of the protein quality control system. Here, we review different aspects of the involvement of amyloid-forming proteins in disease, mechanisms of toxicity, structural features, and biological functions of amyloids, as well as the cellular mechanisms that modulate and regulate protein aggregation, including the presence of enhancers and suppressors of aggregation, and how aging impacts the functioning of these mechanisms, with special attention to the molecular chaperones.

Regulation of the mammalian heat shock factor 1.

Living organisms are endowed with the capability to tackle various forms of cellular stress due to the presence of molecular chaperone machinery complexes that are ubiquitous throughout the cell. During conditions of proteotoxic stress, the transcription factor heat shock factor 1 (HSF1) mediates the elevation of heat shock proteins, which are crucial components of the chaperone complex machinery and function to ameliorate protein misfolding and aggregation and restore protein homeostasis. In addition, HSF1 orchestrates a versatile transcriptional programme that includes genes involved in repair and clearance of damaged macromolecules and maintenance of cell structure and metabolism, and provides protection against a broad range of cellular stress mediators, beyond heat shock. Here, we discuss the structure and function of the mammalian HSF1 and its regulation by post-translational modifications (phosphorylation, sumoylation and acetylation), proteasomal degradation, and small-molecule activators and inhibitors.

Cytosolic Prostaglandin E Synthase Is Involved in c-Fos Expression in Rat Fibroblastic 3Y1 Cells.

Cytosolic prostaglandin (PG) E synthase (cPGES/p23) plays a role in the biosynthesis of PGE2 and in the molecular chaperone machinery. Studies of knockout mice lacking cPGES/p23 have demonstrated that cPGES/p23 is essential in fetal mouse development. A cDNA microarray analysis revealed that a lack of cPGES/p23 decreases the expression of some immediate early genes, such as c-fos and activating transcription factor 3 (ATF3). Here we report the involvement of cPGES/p23 in c-Fos expression. A stable knockdown of cPGES/p23 in cultured fibroblasts not only reduced serum-induced c-Fos expression, but also decreased the phosphorylation of extracellular signal regulated kinase (ERK). These results suggest that cPGES/p23 is involved in the activation of ERK to promote c-Fos expression.

The human HSP70 family of chaperones: where do we stand?

The 70-kDa heat shock protein (HSP70) family of molecular chaperones represents one of the most ubiquitous classes of chaperones and is highly conserved in all organisms. Members of the HSP70 family control all aspects of cellular proteostasis such as nascent protein chain folding, protein import into organelles, recovering of proteins from aggregation, and assembly of multi-protein complexes. These chaperones augment organismal survival and longevity in the face of proteotoxic stress by enhancing cell viability and facilitating protein damage repair. Extracellular HSP70s have a number of cytoprotective and immunomodulatory functions, the latter either in the context of facilitating the cross-presentation of immunogenic peptides via major histocompatibility complex (MHC) antigens or in the context of acting as "chaperokines" or stimulators of innate immune responses. Studies have linked the expression of HSP70s to several types of carcinoma, with Hsp70 expression being associated with therapeutic resistance, metastasis, and poor clinical outcome. In malignantly transformed cells, HSP70s protect cells from the proteotoxic stress associated with abnormally rapid proliferation, suppress cellular senescence, and confer resistance to stress-induced apoptosis including protection against cytostatic drugs and radiation therapy. All of the cellular activities of HSP70s depend on their adenosine-5'-triphosphate (ATP)-regulated ability to interact with exposed hydrophobic surfaces of proteins. ATP hydrolysis and adenosine diphosphate (ADP)/ATP exchange are key events for substrate binding and Hsp70 release during folding of nascent polypeptides. Several proteins that bind to distinct subdomains of Hsp70 and consequently modulate the activity of the chaperone have been identified as HSP70 co-chaperones. This review focuses on the regulation, function, and relevance of the molecular Hsp70 chaperone machinery to disease and its potential as a therapeutic target.

4D Imaging of Protein Aggregation in Live Cells

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental conditions and an intracellular environment that is tightly packed, sticky, and hazardous to protein stability1. The ability to dynamically balance protein production, folding and degradation demands highly-specialized quality control machinery, whose absolute necessity is observed best when it malfunctions. Diseases such as ALS, Alzheimer's, Parkinson's, and certain forms of Cystic Fibrosis have a direct link to protein folding quality control components2, and therefore future therapeutic development requires a basic understanding of underlying processes. Our experimental challenge is to understand how cells integrate damage signals and mount responses that are tailored to diverse circumstances. The primary reason why protein misfolding represents an existential threat to the cell is the propensity of incorrectly folded proteins to aggregate, thus causing a global perturbation of the crowded and delicate intracellular folding environment1. The folding health, or "proteostasis," of the cellular proteome is maintained, even under the duress of aging, stress and oxidative damage, by the coordinated action of different mechanistic units in an elaborate quality control system3,4. A specialized machinery of molecular chaperones can bind non-native polypeptides and promote their folding into the native state1, target them for degradation by the ubiquitin-proteasome system5, or direct them to protective aggregation inclusions6-9. In eukaryotes, the cytosolic aggregation quality control load is partitioned between two compartments8-10: the juxtanuclear quality control compartment (JUNQ) and the insoluble protein deposit (IPOD) (Figure 1 - model). Proteins that are ubiquitinated by the protein folding quality control machinery are delivered to the JUNQ, where they are processed for degradation by the proteasome. Misfolded proteins that are not ubiquitinated are diverted to the IPOD, where they are actively aggregated in a protective compartment. Up until this point, the methodological paradigm of live-cell fluorescence microscopy has largely been to label proteins and track their locations in the cell at specific time-points and usually in two dimensions. As new technologies have begun to grant experimenters unprecedented access to the submicron scale in living cells, the dynamic architecture of the cytosol has come into view as a challenging new frontier for experimental characterization. We present a method for rapidly monitoring the 3D spatial distributions of multiple fluorescently labeled proteins in the yeast cytosol over time. 3D timelapse (4D imaging) is not merely a technical challenge; rather, it also facilitates a dramatic shift in the conceptual framework used to analyze cellular structure. We utilize a cytosolic folding sensor protein in live yeast to visualize distinct fates for misfolded proteins in cellular aggregation quality control, using rapid 4D fluorescent imaging. The temperature sensitive mutant of the Ubc9 protein10-12 (Ubc9ts) is extremely effective both as a sensor of cellular proteostasis, and a physiological model for tracking aggregation quality control. As with most ts proteins, Ubc9ts is fully folded and functional at permissive temperatures due to active cellular chaperones. Above 30 °C, or when the cell faces misfolding stress, Ubc9ts misfolds and follows the fate of a native globular protein that has been misfolded due to mutation, heat denaturation, or oxidative damage. By fusing it to GFP or other fluorophores, it can be tracked in 3D as it forms Stress Foci, or is directed to JUNQ or IPOD.

4D Imaging of Protein Aggregation in Live Cells

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental conditions and an intracellular environment that is tightly packed, sticky, and hazardous to protein stability1. The ability to dynamically balance protein production, folding and degradation demands highly-specialized quality control machinery, whose absolute necessity is observed best when it malfunctions. Diseases such as ALS, Alzheimer's, Parkinson's, and certain forms of Cystic Fibrosis have a direct link to protein folding quality control components2, and therefore future therapeutic development requires a basic understanding of underlying processes. Our experimental challenge is to understand how cells integrate damage signals and mount responses that are tailored to diverse circumstances.The primary reason why protein misfolding represents an existential threat to the cell is the propensity of incorrectly folded proteins to aggregate, thus causing a global perturbation of the crowded and delicate intracellular folding environment1. The folding health, or "proteostasis," of the cellular proteome is maintained, even under the duress of aging, stress and oxidative damage, by the coordinated action of different mechanistic units in an elaborate quality control system3,4. A specialized machinery of molecular chaperones can bind non-native polypeptides and promote their folding into the native state1, target them for degradation by the ubiquitin-proteasome system5, or direct them to protective aggregation inclusions6-9.In eukaryotes, the cytosolic aggregation quality control load is partitioned between two compartments8-10: the juxtanuclear quality control compartment (JUNQ) and the insoluble protein deposit (IPOD) (Figure 1 - model). Proteins that are ubiquitinated by the protein folding quality control machinery are delivered to the JUNQ, where they are processed for degradation by the proteasome. Misfolded proteins that are not ubiquitinated are diverted to the IPOD, where they are actively aggregated in a protective compartment.Up until this point, the methodological paradigm of live-cell fluorescence microscopy has largely been to label proteins and track their locations in the cell at specific time-points and usually in two dimensions. As new technologies have begun to grant experimenters unprecedented access to the submicron scale in living cells, the dynamic architecture of the cytosol has come into view as a challenging new frontier for experimental characterization. We present a method for rapidly monitoring the 3D spatial distributions of multiple fluorescently labeled proteins in the yeast cytosol over time. 3D timelapse (4D imaging) is not merely a technical challenge; rather, it also facilitates a dramatic shift in the conceptual framework used to analyze cellular structure.We utilize a cytosolic folding sensor protein in live yeast to visualize distinct fates for misfolded proteins in cellular aggregation quality control, using rapid 4D fluorescent imaging. The temperature sensitive mutant of the Ubc9 protein10-12 (Ubc9ts) is extremely effective both as a sensor of cellular proteostasis, and a physiological model for tracking aggregation quality control. As with most ts proteins, Ubc9ts is fully folded and functional at permissive temperatures due to active cellular chaperones. Above 30 °C, or when the cell faces misfolding stress, Ubc9ts misfolds and follows the fate of a native globular protein that has been misfolded due to mutation, heat denaturation, or oxidative damage. By fusing it to GFP or other fluorophores, it can be tracked in 3D as it forms Stress Foci, or is directed to JUNQ or IPOD.

Hsp70s and J Proteins of Plasmodium Parasites Infecting Rodents and Primates: Structure, Function, Clinical Relevance, and Drug Targets

Human malaria is an economically important disease caused by single-celled parasites of the Plasmodium genus whose biology displays great evolutionary adaptation to both its mammalian host and transmitting vectors. While the parasite has multiple life cycle stages, it is in the blood stage where clinical symptoms of the disease are manifested. Following erythrocyte entry, the parasite resides in the parasitophorous vacuole and actively transports its own proteins to the erythrocyte cytosol. This host-parasite "cross-talk" results in tremendous modifications of the infected erythrocyte imparting properties that allow it to adhere to the endothelium preventing splenic clearance. The Hsp70-J protein (DnaJ/Hsp40) molecular chaperone machinery, involved in cellular protein homeostasis, is being investigated as a novel drug target in various cellular systems including malaria. In Plasmodium the diverse chaperone complement is intimately involved in infected erythrocyte remodelling associated with the development and pathogenesis of malaria. In this review, we provide an overview of the Hsp70-J protein chaperone complement in Plasmodium falciparum and compare it with other Plasmodium species including the ones that serve as experimental study models for malaria. We propose that the unique traits possessed by this machinery not only provide avenues for drug targeting but also inform the evolutionary fitness of this parasite to its environment.

Hsp70s and J Proteins of Plasmodium Parasites Infecting Rodents and Primates: Structure, Function, Clinical Relevance, and Drug Targets

Human malaria is an economically important disease caused by single-celled parasites of the Plasmodium genus whose biology displays great evolutionary adaptation to both its mammalian host and transmitting vectors. While the parasite has multiple life cycle stages, it is in the blood stage where clinical symptoms of the disease are manifested. Following erythrocyte entry, the parasite resides in the parasitophorous vacuole and actively transports its own proteins to the erythrocyte cytosol. This host-parasite “cross-talk” results in tremendous modifications of the infected erythrocyte imparting properties that allow it to adhere to the endothelium preventing splenic clearance. The Hsp70-J protein (DnaJ/Hsp40) molecular chaperone machinery, involved in cellular protein homeostasis, is being investigated as a novel drug target in various cellular systems including malaria. In Plasmodium the diverse chaperone complement is intimately involved in infected erythrocyte remodelling associated with the development and pathogenesis of malaria. In this review, we provide an overview of the Hsp70-J protein chaperone complement in Plasmodium falciparum and compare it with other Plasmodium species including the ones that serve as experimental study models for malaria. We propose that the unique traits possessed by this machinery not only provide avenues for drug targeting but also inform the evolutionary fitness of this parasite to its environment. Keywords: Cell stress, drug targets, heat shock proteins, J proteins, Hsp70, malaria, molecular chaperones, Plasmodium, transmitting vectors, erythrocyte.

Probing Molecular Mechanisms of the Hsp90 Chaperone: Biophysical Modeling Identifies Key Regulators of Functional Dynamics

Deciphering functional mechanisms of the Hsp90 chaperone machinery is an important objective in cancer biology aiming to facilitate discovery of targeted anti-cancer therapies. Despite significant advances in understanding structure and function of molecular chaperones, organizing molecular principles that control the relationship between conformational diversity and functional mechanisms of the Hsp90 activity lack a sufficient quantitative characterization. We combined molecular dynamics simulations, principal component analysis, the energy landscape model and structure-functional analysis of Hsp90 regulatory interactions to systematically investigate functional dynamics of the molecular chaperone. This approach has identified a network of conserved regions common to the Hsp90 chaperones that could play a universal role in coordinating functional dynamics, principal collective motions and allosteric signaling of Hsp90. We have found that these functional motifs may be utilized by the molecular chaperone machinery to act collectively as central regulators of Hsp90 dynamics and activity, including the inter-domain communications, control of ATP hydrolysis, and protein client binding. These findings have provided support to a long-standing assertion that allosteric regulation and catalysis may have emerged via common evolutionary routes. The interaction networks regulating functional motions of Hsp90 may be determined by the inherent structural architecture of the molecular chaperone. At the same time, the thermodynamics-based “conformational selection” of functional states is likely to be activated based on the nature of the binding partner. This mechanistic model of Hsp90 dynamics and function is consistent with the notion that allosteric networks orchestrating cooperative protein motions can be formed by evolutionary conserved and sparsely connected residue clusters. Hence, allosteric signaling through a small network of distantly connected residue clusters may be a rather general functional requirement encoded across molecular chaperones. The obtained insights may be useful in guiding discovery of allosteric Hsp90 inhibitors targeting protein interfaces with co-chaperones and protein binding clients.

The Role of Ubiquitin in Autophagy-Dependent Protein Aggregate Processing

The efficient management of misfolded protein aggregates is essential for cell viability and requires three interconnected pathways: the molecular chaperone machinery that assists protein folding, the proteasome pathway that degrades misfolded proteins, and the aggresomal pathway that sequesters and delivers toxic proteins aggregates to autophagy for clearance. Although autophagy is generally considered as non-selective degradative machinery, growing evidence supports the existence of a selective autophagy that specifically targets protein aggregates for clearance. This so-called "quality control autophagy" is established by specific ubiquitin E3 ligases, autophagic substrate ubiquitination, and specific ubiquitin binding proteins p62 and HDAC6. In this context, quality control autophagy is similar to the proteasome system and utilizes ubiquitin tags for substrate recognition and processing. Here I will discuss the recent progress towards understanding the molecular basis of this unique form of ubiquitin-dependent autophagy in protein aggregate clearance and its relevance to disease.

Cooperative Regulation of the Interferon Regulatory Factor-1 Tumor Suppressor Protein by Core Components of the Molecular Chaperone Machinery

Our understanding of the post-translational processes involved in regulating the interferon regulatory factor-1 (IRF-1) tumor suppressor protein is limited. The introduction of mutations within the C-terminal Mf1 domain (amino acids 301-325) impacts on IRF-1-mediated gene repression and growth suppression as well as the rate of IRF-1 degradation. However, nothing is known about the proteins that interact with this region to modulate IRF-1 function. A biochemical screen for Mf1-interacting proteins has identified an LXXLL motif that is required for binding of Hsp70 family members and cooperation with Hsp90 to regulate IRF-1 turnover and activity. These conclusions are supported by the finding that Hsp90 inhibitors suppress IRF-1-dependent transcription shortly after treatment, although at later time points inhibition of Hsp90 leads to an Hsp70-dependent depletion of nuclear IRF-1. Conversely, the half-life of IRF-1 is increased by Hsp90 in an ATPase-dependent manner leading to the accumulation of nuclear but not cytoplasmic IRF-1. This study begins to elucidate the role of the Mf1 domain of IRF-1 in orchestrating the recruitment of regulatory factors that can impact on both its turnover and transcriptional activity.

From hatching to dispatching: the multiple cellular roles of the Hsp70 molecular chaperone machinery

Molecular chaperones are best recognized for their roles in de novo protein folding and the cellular response to stress. However, many molecular chaperones, and in particular the Hsp70 chaperone machinery, have multiple diverse cellular functions. At the molecular level, chaperones are mediators of protein conformational change. To facilitate conformational change of client/substrate proteins, in manifold contexts, chaperone power must be closely regulated and harnessed to specific cellular locales--this is controlled by cochaperones. This review considers specialized functions of the Hsp70 chaperone machinery mediated by its cochaperones. We focus on vesicular trafficking, protein degradation and a potential role in G protein-coupled receptor processing.