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Concept: Hsp90

77

Nicotinamide mononucleotide adenylyl transferase 2 (NMNAT2) is neuroprotective in numerous preclinical models of neurodegeneration. Here, we show that brain nmnat2 mRNA levels correlate positively with global cognitive function and negatively with AD pathology. In AD brains, NMNAT2 mRNA and protein levels are reduced. NMNAT2 shifts its solubility and colocalizes with aggregated Tau in AD brains, similar to chaperones, which aid in the clearance or refolding of misfolded proteins. Investigating the mechanism of this observation, we discover a novel chaperone function of NMNAT2, independent from its enzymatic activity. NMNAT2 complexes with heat shock protein 90 (HSP90) to refold aggregated protein substrates. NMNAT2’s refoldase activity requires a unique C-terminal ATP site, activated in the presence of HSP90. Furthermore, deleting NMNAT2 function increases the vulnerability of cortical neurons to proteotoxic stress and excitotoxicity. Interestingly, NMNAT2 acts as a chaperone to reduce proteotoxic stress, while its enzymatic activity protects neurons from excitotoxicity. Taken together, our data indicate that NMNAT2 exerts its chaperone or enzymatic function in a context-dependent manner to maintain neuronal health.

Concepts: Proteins, Protein, Brain, Enzyme, Protein folding, Chaperone, Heat shock protein, Hsp90

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

25

Proteins participate in almost every cell physiological function, and to do so, they need to reach a state that allows its function by folding and/or exposing surfaces of interactions. Spontaneous folding in the cell is in general hindered by its crowded and viscous environment, which favors misfolding and nonspecific and deleterious self-interactions. To overcome this, cells have a system, in which Hsp70 and Hsp90 play a central role to aid protein folding and avoid misfolding. The topics of this review include the biophysical tools used for monitoring protein-ligand and protein-protein interactions and also some important results related to the study of molecular chaperones and heat shock proteins (Hsp), with a focus on the Hsp70/Hsp90 network. The biophysical tools and their use to probe the conformation and interaction of Hsp70 and Hsp90 are briefly reviewed.

Concepts: Protein, Enzyme, Endoplasmic reticulum, Protein folding, Chaperone, Proteasome, Heat shock protein, Hsp90

4

There is a pressing need for new technologies that improve the efficacy and efficiency of drug discovery. Structure-based methods have contributed towards this goal but they focus on predicting the binding affinity of protein-ligand complexes, which is notoriously difficult. We adopt an alternative approach that evaluates structural, rather than thermodynamic, stability. As bioactive molecules present a static binding mode, we devised dynamic undocking (DUck), a fast computational method to calculate the work necessary to reach a quasi-bound state at which the ligand has just broken the most important native contact with the receptor. This non-equilibrium property is surprisingly effective in virtual screening because true ligands form more-resilient interactions than decoys. Notably, DUck is orthogonal to docking and other ‘thermodynamic’ methods. We demonstrate the potential of the docking-undocking combination in a fragment screening against the molecular chaperone and oncology target Hsp90, for which we obtain novel chemotypes and a hit rate that approaches 40%.

Concepts: Protein, Atom, Thermodynamics, Chaperone, Drug discovery, Virtual screening, Heat shock protein, Hsp90

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

A potential cause of neurodegenerative diseases including Parkinson’s disease (PD) is protein misfolding and aggregation that in turn leads to neurotoxicity. Targeting Hsp90 is an attractive strategy to halt neurodegenerative diseases, and benzoquinone ansamycin (BQA) Hsp90 inhibitors such as geldanamycin (GA) and 17-allylamino-17-demethoxygeldanamycin (17-AAG) have been shown to be beneficial in mutant A53T α-synuclein PD models. However, current BQA inhibitors result in off-target toxicities via redox cycling and/or arylation of nucleophiles at the C19 position. We developed novel 19-substituted BQA (19BQA) as a means to prevent arylation. In this study, our data demonstrated that 19-phenyl-GA, a lead 19BQA in the GA series, was redox stable and exhibited little toxicity relative to its parent quinone GA in human dopaminergic SH-SY5Y cells as examined by oxygen consumption, trypan blue, MTT and apoptosis assays. Meanwhile, 19-phenyl-GA retained the ability to induce autophagy and potentially protective heat shock proteins (HSPs) such as Hsp70 and Hsp27. We found that transduction of A53T, but not wild type (WT) α-synuclein induced toxicity in SH-SY5Y cells. 19-phenyl-GA decreased oligomer formation and toxicity of A53T α-synuclein in transduced cells. Mechanistic studies indicated that mTOR/p70S6K signaling was activated by A53T but not WT α-synuclein and 19-phenyl-GA decreased mTOR activation that may be associated with A53T α-synuclein toxicity. In summary, our results indicate that 19BQAs such as 19-phenyl-GA may provide a means to modulate protein-handling systems including HSPs and autophagy thereby reducing the aggregation and toxicity of proteins such as mutant A53T α-synuclein.

Concepts: Photosynthesis, Protein, Gene, Nitrogen, Neurology, Chaperone, Heat shock protein, Hsp90

3

Efficient elimination of misfolded proteins by the proteasome system is critical for proteostasis. Inadequate proteasome capacity can lead to aberrant aggregation of misfolded proteins and inclusion body formation, a hallmark of neurodegenerative disease. The proteasome system cannot degrade aggregated proteins; however, it stimulates autophagy-dependent aggregate clearance by producing unanchored lysine (K)63-linked ubiquitin chains via the proteasomal deubiquitinating enzyme Poh1. The canonical function of Poh1, which removes ubiquitin chains en bloc from proteasomal substrates prior to their degradation, requires intact 26S proteasomes. Here we present evidence that during aggresome clearance, 20S proteasomes dissociate from protein aggregates, while Poh1 and selective subunits of 19S proteasomes are retained. The dissociation of 20S proteasome components requires the molecular chaperone Hsp90. Hsp90 inhibition suppresses 26S proteasome remodeling, unanchored ubiquitin chain production, and aggresome clearance. Our results suggest that 26S proteasomes undergo active remodeling to generate a Poh1-dependent K63-deubiquitinating enzyme to facilitate protein aggregate clearance.

Concepts: Proteins, Protein, Protein folding, Aggregate, Chaperone, Proteasome, Heat shock protein, Hsp90

3

One function of the glucocorticoid receptor (GR) in corticotroph cells is to suppress the transcription of the gene encoding proopiomelanocortin (POMC), the precursor of the stress hormone adrenocorticotropin (ACTH). Cushing disease is a neuroendocrine condition caused by partially glucocorticoid-resistant corticotroph adenomas that excessively secrete ACTH, which leads to hypercortisolism. Mutations that impair GR function explain glucocorticoid resistance only in sporadic cases. However, the proper folding of GR depends on direct interactions with the chaperone heat shock protein 90 (HSP90, refs. 7,8). We show here that corticotroph adenomas overexpress HSP90 compared to the normal pituitary. N- and C-terminal HSP90 inhibitors act at different steps of the HSP90 catalytic cycle to regulate corticotroph cell proliferation and GR transcriptional activity. C-terminal inhibitors cause the release of mature GR from HSP90, which promotes its exit from the chaperone cycle and potentiates its transcriptional activity in a corticotroph cell line and in primary cultures of human corticotroph adenomas. In an allograft mouse model, the C-terminal HSP90 inhibitor silibinin showed anti-tumorigenic effects, partially reverted hormonal alterations, and alleviated symptoms of Cushing disease. These results suggest that the pathogenesis of Cushing disease caused by overexpression of heat shock proteins and consequently misregulated GR sensitivity may be overcome pharmacologically with an appropriate HSP90 inhibitor.

Concepts: Protein, Gene expression, Chaperone, Cortisol, Heat shock protein, Cushing's syndrome, Adrenocorticotropic hormone, Hsp90

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 Hsp90 molecular chaperone and its Cdc37 cochaperone help stabilize and activate more than half of the human kinome. However, both the mechanism by which these chaperones assist their “client” kinases and the reason why some kinases are addicted to Hsp90 while closely related family members are independent are unknown. Our structural understanding of these interactions is lacking, as no full-length structures of human Hsp90, Cdc37, or either of these proteins with a kinase have been elucidated. Here we report a 3.9 angstrom cryo-electron microscopy structure of the Hsp90-Cdc37-Cdk4 kinase complex. Surprisingly, the two lobes of Cdk4 are completely separated with the β4-β5 sheet unfolded. Cdc37 mimics part of the kinase N lobe, stabilizing an open kinase conformation by wedging itself between the two lobes. Finally, Hsp90 clamps around the unfolded kinase β5 strand and interacts with exposed N- and C-lobe interfaces, protecting the kinase in a trapped unfolded state. On the basis of this structure and an extensive amount of previously collected data, we propose unifying conceptual and mechanistic models of chaperone-kinase interactions.

Concepts: Proteins, Protein, Protein folding, Chaperone, Proteasome, Heat shock protein, Protein targeting, Hsp90