SciCombinator

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Concept: Co-chaperone

27

Heat-shock protein 90 (Hsp90) is an ATP-dependent molecular chaperone that associates dynamically with various co-chaperones during its chaperone cycle. Here we analyzed the role of the activating co-chaperone Aha1 in the progression of the yeast Hsp90 chaperone cycle and identified a critical ternary Hsp90 complex containing the co-chaperones Aha1 and Cpr6. Aha1 accelerates the intrinsically slow conformational transitions of Hsp90 to an N-terminally associated state but does not fully close the nucleotide-binding pocket yet. Cpr6 increases the affinity between Aha1 and Hsp90 and further stimulates the Hsp90 ATPase activity. Synergistically, Aha1 and Cpr6 displace the inhibitory co-chaperone Sti1 from Hsp90. To complete the cycle, Aha1 is released by the co-chaperone p23. Thus, at distinct steps during the Hsp90 chaperone cycle, co-chaperones selectively trap statistically distributed Hsp90 conformers and thus turn Hsp90 into a deterministic machine.

Concepts: Proteins, Protein, Chaperone, Proteasome, Heat shock protein, Hsp90, Chaperonin, Co-chaperone

4

The Hsp90 chaperone is a central node of protein homeostasis, activating many diverse client proteins. Hsp90 functions as a molecular clamp that closes and opens in response to the binding and hydrolysis of ATP. Crystallographic studies have defined distinct conformational states of the mechanistic core, implying structural changes that have not yet been observed in solution. Here we engineered one-nanometer fluorescence probes based on photoinduced electron transfer into the yeast Hsp90 to observe these motions. We found that the ATPase activity of the chaperone was reflected in the kinetics of specific structural rearrangements at remote positions that acted cooperatively. Nanosecond single-molecule fluorescence fluctuation analysis uncovered that critical structural elements that undergo rearrangement were mobile on a sub-millisecond time scale. We identified a two-step mechanism for lid closure over the nucleotide-binding pocket. The activating co-chaperone Aha1 mobilized the lid of apo Hsp90, suggesting an early role in the catalytic cycle.

Concepts: Proteins, Protein, Electron, Metabolism, Protein folding, Chaperone, Heat shock protein, Co-chaperone

3

Small heat shock proteins (sHsps) are ubiquitous molecular chaperones that prevent the aggregation of unfolding proteins during proteotoxic stress. In Caenorhabditis elegans, Sip1 is the only sHsp exclusively expressed in oocytes and embryos. Here, we demonstrate that Sip1 is essential for heat shock survival of reproducing adults and embryos. X-ray crystallography and electron microscopy revealed that Sip1 exists in a range of well-defined globular assemblies consisting of two half-spheres, each made of dimeric “spokes.” Strikingly, the oligomeric distribution of Sip1 as well as its chaperone activity depend on pH, with a trend toward smaller species and higher activity at acidic conditions such as present in nematode eggs. The analysis of the interactome shows that Sip1 has a specific substrate spectrum including proteins that are essential for embryo development.

Concepts: DNA, Protein, Developmental biology, Chaperone, Heat shock protein, Hsp90, Chaperonin, Co-chaperone

3

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the selective loss of motor neurons in the spinal cord, brain stem, and motor cortex. Mutations in superoxide dismutase (SOD1) are associated with familial ALS and lead to SOD1 protein misfolding and aggregation. Here we show that the molecular chaperone, HSJ1 (DNAJB2), mutations in which cause distal hereditary motor neuropathy, can reduce mutant SOD1 aggregation and improve motor neuron survival in mutant SOD1 models of ALS. Overexpression of human HSJ1a (hHSJ1a) in vivo in motor neurons of SOD1(G93A) transgenic mice ameliorated disease. In particular, there was a significant improvement in muscle force, increased motor unit number and enhanced motor neuron survival. hHSJ1a was present in a complex with SOD1(G93A) and led to reduced SOD1 aggregation at late stages of disease progression. We also observed altered ubiquitin immunoreactivity in the double transgenic animals, suggesting that ubiquitin modification might be important for the observed improvements. In a cell model of SOD1(G93A) aggregation, HSJ1a preferentially bound to mutant SOD1, enhanced SOD1 ubiquitylation and reduced SOD1 aggregation in a J-domain and ubiquitin interaction motif (UIM) dependent manner. Collectively, the data suggest that HSJ1a acts on mutant SOD1 through a combination of chaperone, co-chaperone and pro-ubiquitylation activity. These results show that targeting SOD1 protein misfolding and aggregation in vivo can be neuroprotective and suggest that manipulation of DnaJ molecular chaperones might be useful in the treatment of ALS.

Concepts: Spinal cord, Gene expression, Superoxide dismutase, Protein folding, Amyotrophic lateral sclerosis, Chaperone, Protein targeting, Co-chaperone

2

Cellular protein homeostasis depends on heat shock proteins 70 kDa (Hsp70s), a class of ubiquitous and highly conserved molecular chaperone. Key to the chaperone activity is an ATP-induced allosteric regulation of polypeptide substrate binding and release. To illuminate the molecular mechanism of this allosteric coupling, here we present a novel crystal structure of an intact human BiP, an essential Hsp70 in ER, in an ATP-bound state. Strikingly, the polypeptide-binding pocket is completely closed, seemingly excluding any substrate binding. Our FRET, biochemical and EPR analysis suggests that this fully closed conformation is the major conformation for the ATP-bound state in solution, providing evidence for an active release of bound polypeptide substrates following ATP binding. The Hsp40 co-chaperone converts this fully closed conformation to an open conformation to initiate productive substrate binding. Taken together, this study provided a mechanistic understanding of the dynamic nature of the polypeptide-binding pocket in the Hsp70 chaperone cycle.

Concepts: Enzyme kinetics, Proteins, Protein, Endoplasmic reticulum, Protein folding, Chaperone, Heat shock protein, Co-chaperone

2

The aggregation of α-synuclein (α-syn) into amyloid fibrils is associated with neurodegenerative diseases, collectively referred to as the α-synucleinopathies. In vivo, molecular chaperones, such as the small heat-shock proteins (sHsps), normally act to prevent protein aggregation; however, it remains to be determined how aggregation-prone α-syn evades sHsp chaperone action leading to its disease-associated deposition. This work examines the molecular mechanism by which two canonical sHsps, αB-crystallin (αB-c) and Hsp27, interact with aggregation-prone α-syn to prevent its aggregation in vitro. Both sHsps are very effective inhibitors of α-syn aggregation, but no stable complex between the sHsps and α-syn was detected, indicating that the sHsps inhibit α-syn aggregation via transient interactions. Moreover, the ability of these sHsps to prevent α-syn aggregation was dependent on the kinetics of aggregation; the faster the rate of aggregation (shorter the lag phase), the less effective the sHsps were at inhibiting fibril formation of α-syn. Thus, these findings indicate that the rate at which α-syn aggregates in cells may be a significant factor in how it evades sHsp chaperone action in the α-synucleinopathies.

Concepts: Protein, Protein folding, Chaperone, Proteasome, Heat shock protein, Hsp90, Chaperonin, Co-chaperone

1

Hsp90 belongs to a family of some of the most highly expressed heat shock proteins that function as molecular chaperones to protect the proteome not only from the heat shock but also from other misfolding events. As many client proteins of Hsp90 are involved in oncogenesis, this chaperone has been the focus of intense research efforts. Yet, we lack structural information for how Hsp90 interacts with co-chaperones and client proteins. Here, we developed a mass-spectrometry-based approach that allowed quantitative measurements of in vitro and in vivo effects of small-molecule inhibitors on Hsp90 conformation, and interaction with co-chaperones and client proteins. From this analysis, we were able to derive structural models for how Hsp90 engages its interaction partners in vivo, and how different drugs affect these structures. In addition, the methodology described here offers a new approach to probe the effects of virtually any inhibitor treatment on the proteome level.

Concepts: Proteins, Protein, Protein folding, Chaperone, Proteasome, Heat shock protein, Hsp90, Co-chaperone

1

The mechanisms underlying tau-related synaptic and cognitive deficits and the interrelationships between tau species, their clearance pathways and synaptic impairments remain poorly understood. To gain insight into these mechanisms, we examined these interrelationships in aged non-mutant genomic human tau (htau) mice, with established tau pathology and neuron loss. We also examined how these interrelationships changed with an intervention by feeding mice either a control diet or one containing the brain permeable amyloid- and tau-binding molecule curcumin. Transgene dependent elevations in soluble and insoluble phospho-tau monomer and soluble tau dimers accompanied deficits in behavior, hippocampal excitatory synaptic markers and molecular chaperones (heat shock proteins, HSPs) involved in tau degradation and microtubule stability. In htau mice but not control mice, HSP70, HSP70/HSP72 and HSP90 were selectively reduced in membrane- enriched fractions but not in cytosolic fractions. The synaptic proteins PSD95 and NR2B were reduced in dendritic fields and redistributed into perikarya, corresponding to changes observed by immunoblot. Curcumin selectively suppressed levels of soluble tau dimers, but not of insoluble and monomeric phospho-tau, while correcting behavioral, synaptic and HSP deficits. Treatment increased PSD95 co-immunoprecipitating with NR2B and increased transgene-independent HSPs implicated in tau clearance. It elevated HSP90 and HSC70 without increasing HSP mRNAs, that is without induction of the heat shock response. Instead curcumin differentially impacted HSP90 client kinases, selectively reducing fyn, without reducing Akt. In summary curcumin reduced soluble tau and elevated HSPs involved in tau clearance, showing that even after tangles have formed, tau-dependent behavioral and synaptic deficits can be corrected.

Concepts: Proteins, Protein, Chaperone, Proteasome, Heat shock protein, Hsp90, Co-chaperone

0

Stress-Inducible Protein-1 (STIP1) is a co-chaperone that associates directly with heat shock proteins, and regulates motility of various types of cancer. In the present study, we investigated the role of STIP1 on metastasis of gastric cancer (GC).

Concepts: Present, Proteins, Metastasis, Chaperone, Heat shock protein, Stomach cancer, Co-chaperone

0

Small heat shock proteins are chaperones with variable mechanisms of action. The function of cardiac family member Hspb7 is unknown, despite being identified through GWAS as a potential cardiomyopathy risk gene. We discovered that zebrafish hspb7 mutants display mild focal cardiac fibrosis and sarcomeric abnormalities. Significant mortality was observed in adult hspb7 mutants subjected to exercise stress, demonstrating a genetic and environmental interaction that determines disease outcome. We identified large sarcomeric proteins FilaminC and Titin as Hspb7 binding partners in cardiac cells. Damaged FilaminC undergoes autophagic processing to maintain sarcomeric homeostasis. Loss of Hspb7 in zebrafish or human cardiomyocytes stimulated autophagic pathways and expression of the sister gene encoding Hspb5. Inhibiting autophagy caused FilaminC aggregation in HSPB7 mutant human cardiomyocytes and developmental cardiomyopathy in hspb7 mutant zebrafish embryos. These studies highlight the importance of damage-processing networks in cardiomyocytes, and a previously unrecognized role in this context for Hspb7.

Concepts: Protein, Gene, Genetics, Gene expression, Cell, Chaperone, Heat shock protein, Co-chaperone