Hsp90 and Hsp70 chaperones: Collaborators in protein remodeling.

Heat shock proteins 90 (Hsp90) and 70 (Hsp70) are two families of highly conserved ATP-dependent molecular chaperones that fold and remodel proteins. Both are important components of the cellular machinery involved in protein homeostasis and participate in nearly every cellular process. Although Hsp90 and Hsp70 each carry out some chaperone activities independently, they collaborate in other cellular remodeling reactions. In eukaryotes, both Hsp90 and Hsp70 function with numerous Hsp90 and Hsp70 co-chaperones. In contrast, bacterial Hsp90 and Hsp70 are less complex; Hsp90 acts independently of co-chaperones, and Hsp70 uses two co-chaperones. In this review, we focus on recent progress toward understanding the basic mechanisms of Hsp90-mediated protein remodeling and the collaboration between Hsp90 and Hsp70, with an emphasis on bacterial chaperones. We describe the structure and conformational dynamics of these chaperones and their interactions with each other and with client proteins. The physiological roles of Hsp90 in Escherichia coli and other bacteria are also discussed. We anticipate that the information gained from exploring the mechanism of the bacterial chaperone system will provide the groundwork for understanding the more complex eukaryotic Hsp90 system and its modulation by Hsp90 co-chaperones.

Modulation of hippocampal neuronal resilience during aging by the Hsp70/Hsp90 co-chaperone STI1.

Chaperone networks are dysregulated with aging, but whether compromised Hsp70/Hsp90 chaperone function disturbs neuronal resilience is unknown. Stress-inducible phosphoprotein 1 (STI1; STIP1; HOP) is a co-chaperone that simultaneously interacts with Hsp70 and Hsp90, but whose function in vivo remains poorly understood. We combined in-depth analysis of chaperone genes in human datasets, analysis of a neuronal cell line lacking STI1 and of a mouse line with a hypomorphic Stip1 allele to investigate the requirement for STI1 in aging. Our experiments revealed that dysfunctional STI1 activity compromised Hsp70/Hsp90 chaperone network and neuronal resilience. The levels of a set of Hsp90 co-chaperones and client proteins were selectively affected by reduced levels of STI1, suggesting that their stability depends on functional Hsp70/Hsp90 machinery. Analysis of human databases revealed a subset of co-chaperones, including STI1, whose loss of function is incompatible with life in mammals, albeit they are not essential in yeast. Importantly, mice expressing a hypomorphic STI1 allele presented spontaneous age-dependent hippocampal neurodegeneration and reduced hippocampal volume, with consequent spatial memory deficit. We suggest that impaired STI1 function compromises Hsp70/Hsp90 chaperone activity in mammals and can by itself cause age-dependent hippocampal neurodegeneration in mice. Cover Image for this issue: doi: 10.1111/jnc.14749.

Disrupting progression of the yeast Hsp90 folding pathway at different transition points results in client-specific maturation defects.

The protein molecular chaperone Hsp90 (Heat shock protein, 90 kilodalton) plays multiple roles in the biogenesis and regulation of client proteins impacting myriad aspects of cellular physiology. Amino acid alterations located throughout Saccharomyces cerevisiae Hsp90 have been shown to result in reduced client activity and temperature-sensitive growth defects. Although some Hsp90 mutants have been shown to affect activity of particular clients more than others, the mechanistic basis of client-specific effects is unknown. We found that Hsp90 mutants that disrupt the early step of Hsp70 and Sti1 interaction, or show reduced ability to adopt the ATP-bound closed conformation characterized by Sba1 and Cpr6 interaction, similarly disrupt activity of three diverse clients, Utp21, Ssl2, and v-src. In contrast, mutants that appear to alter other steps in the folding pathway had more limited effects on client activity. Protein expression profiling provided additional evidence that mutants that alter similar steps in the folding cycle cause similar in vivo consequences. Our characterization of these mutants provides new insight into how Hsp90 and cochaperones identify and interact with diverse clients, information essential for designing pharmaceutical approaches to selectively inhibit Hsp90 function.

Revealing the interaction mode of the highly flexible Sorghum bicolor Hsp70/Hsp90 organizing protein (Hop): A conserved carboxylate clamp confers high affinity binding to Hsp90.

Proteostasis is dependent on the Hsp70/Hsp90 system (the two chaperones and their co-chaperones). Of these, Hop (Hsp70/Hsp90 organizing protein), also known as Sti1, forms an important scaffold to simultaneously binding to both Hsp70 and Hsp90. Hop/Sti1 has been implicated in several disease states, for instance cancer and transmissible spongiform encephalopathies. Therefore, human and yeast homologous have been better studied and information on plant homologous is still limited, even though plants are continuously exposed to environmental stress. Particularly important is the study of crops that are relevant for agriculture, such as Sorghum bicolor, a C4 grass that is among the five most important cereals and is considered as a bioenergy feedstock. To increase the knowledge on plant chaperones, the hop putative gene for Sorghum bicolor was cloned and the biophysical and structural characterization of the protein was done by cross-linking coupled to mass spectroscopy, small angle X-ray scattering and structural modeling. Additionally, the binding to a peptide EEVD motif, which is present in both Hsp70 and Hsp90, was studied by isothermal titration calorimetry and hydrogen/deuterium exchange and the interaction pattern structurally modeled. The results indicate SbHop as a highly flexible, mainly alpha-helical monomer consisting of nine tetratricopeptide repeat domains, of which one confers high affinity binding to Hsp90 through a conserved carboxylate clamp. Moreover, the present insights into the conserved interactions formed between Hop and Hsp90 can help to design strategies for potential therapeutic approaches for the diseases in which Hop has been implicated.

Evidence of a Cell Surface Role for Hsp90 Complex Proteins Mediating Neuroblast Migration in the Subventricular Zone.

In most mammalian brains, the subventricular zone (SVZ) is a germinative layer that maintains neurogenic activity throughout adulthood. Neuronal precursors arising from this region migrate through the rostral migratory stream (RMS) and reach the olfactory bulbs where they differentiate and integrate into the local circuitry. Recently, studies have shown that heat shock proteins have an important role in cancer cell migration and blocking Hsp90 function was shown to hinder cell migration in the developing cerebellum. In this work, we hypothesize that chaperone complexes may have an important function regulating migration of neuronal precursors from the subventricular zone. Proteins from the Hsp90 complex are present in the postnatal SVZ as well as in the RMS. Using an in vitro SVZ explant model, we have demonstrated the expression of Hsp90 and Hop/STI1 by migrating neuroblasts. Treatment with antibodies against Hsp90 and co-chaperone Hop/STI1, as well as Hsp90 and Hsp70 inhibitors hinder neuroblast chain migration. Time-lapse videomicroscopy analysis revealed that cell motility and average migratory speed was decreased after exposure to both antibodies and inhibitors. Antibodies recognizing Hsp90, Hsp70, and Hop/STI1 were found bound to the membranes of cells from primary SVZ cultures and biotinylation assays demonstrated that Hsp70 and Hop/STI1 could be found on the external leaflet of neuroblast membranes. The latter could also be detected in conditioned medium samples obtained from cultivated SVZ cells. Our results suggest that chaperones Hsp90, Hsp70, and co-chaperone Hop/STI1, components of the Hsp90 complex, regulate SVZ neuroblast migration in a concerted manner through an extracellular mechanism.

Hop/Sti1 - A Two-Faced Cochaperone Involved in Pattern Recognition Receptor Maturation and Viral Infection.

Perception of pathogens by host pattern recognition receptors (PRRs) or R proteins is a prerequisite to promote successful immune responses. The Hsp70/Hsp90 organizing protein Hop/Sti1, a multifunctional cochaperone, has been implicated in the maturation of a receptor-like kinase (RLK) necessary for chitin sensing. However, it remains unknown whether Hop/Sti1 is generally participating in PRR genesis. Using RNA-interference (RNAi), we silenced Hop/Sti1 expression in Nicotiana tabacum to gain further insight into the role of the cochaperone in plant defense responses. As expected, transgenic plants do not respond to chitin treatment anymore. In contrast to this, trafficking and functionality of the flagellin PRR FLS2 were unaltered, suggesting a selective involvement of Hop/Sti1 during PRR maturation. Furthermore, Hop/Sti1 was identified as a cellular determinant of Potato virus Y (PVY) symptom development in tobacco, since PVY was able to accumulate to near wild-type level without provoking the usual veinal necrosis phenotype. In addition, typical antiviral host defense responses were suppressed in the transgenic plants. These data suggest that perception of PVY is dependent on Hop/Sti1-mediated receptor maturation, while viral symptoms represent a failing attempt to restrict PVY spread. In addition, Hop/Sti1 colocalized with virus-induced membrane aggregates in wild-type plants. The retention of Hop/Sti1 in potential viral replication complexes suggests a role during viral translation/replication, explaining why RNAi-lines do not exhibit increased susceptibility to PVY. This study provides evidence for a dual role of Hop/Sti1 in PRR maturation and pathogen perception as well as in promoting viral proliferation.