Caveolin-1 interacts with the chaperone complex TCP-1 and modulates its protein folding activity.

We report that caveolin-1, one of the major structural protein of caveolae, interacts with TCP-1, a hetero-oligomeric chaperone complex present in all eukaryotic cells that contributes mainly to the folding of actin and tubulin. The caveolin-TCP-1 interaction entails the first 32 amino acids of the N-terminal segment of caveolin. Our data show that caveolin-1 expression is needed for the induction of TCP-1 actin folding function in response to insulin stimulation. Caveolin-1 phosphorylation at tyrosine residue 14 induces the dissociation of caveolin-1 from TCP-1 and activates actin folding. We show that the mechanism by which caveolin-1 modulates TCP-1 activity is indirect and involves the cytoskeleton linker filamin. Filamin is known to bind caveolin-1 and to function as a negative regulator of insulin-mediated signaling. Our data support the notion that the caveolin-filamin interaction contributes to restore insulin-mediated phosphorylation of caveolin, thus allowing the release of active TCP-1.

The vacuolar transporter chaperone (VTC) complex is required for microautophagy.

Microautophagy involves direct invagination and fission of the vacuolar/lysosomal membrane under nutrient limitation. This occurs by an autophagic tube, a specialized vacuolar membrane invagination that pinches off vesicles into the vacuolar lumen. In this study we have identified the VTC (vacuolar transporter chaperone) complex as required for microautophagy. The VTC complex is present on the ER and vacuoles and at the cell periphery. On induction of autophagy by nutrient limitation the VTC complex is recruited to and concentrated on vacuoles. The VTC complex is inhomogeneously distributed within the vacuolar membranes, showing an enrichment on autophagic tubes. Deletion of the VTC complex blocks microautophagic uptake into vacuoles. The mutants still form autophagic tubes but the production of microautophagic vesicles from their tips is impaired. In line with this, affinity-purified antibodies to the Vtc proteins inhibit microautophagic uptake in a reconstituted system in vitro. Our data suggest that the VTC complex is an important constituent of autophagic tubes and that it is required for scission of microautophagic vesicles from these tubes.

A caveolin dominant negative mutant associates with lipid bodies and induces intracellular cholesterol imbalance

Recent studies have indicated a role for caveolin in regulating cholesterol-dependent signaling events. In the present study we have analyzed the role of caveolins in intracellular cholesterol cycling using a dominant negative caveolin mutant. The mutant caveolin protein, cav-3(DGV) specifically associates with the membrane surrounding large lipid droplets. These structures contain neutral lipids, and are accessed by caveolin 1-3 upon overexpression. Fluorescence, electron, and video microscopy observations are consistent with formation of the membrane-enclosed lipid rich structures by maturation of subdomains of the ER. The caveolin mutant causes the intracellular accumulation of free cholesterol (FC) in late endosomes, a decrease in surface cholesterol and a decrease in cholesterol efflux and synthesis. The amphiphile U18666A acts synergistically with cav(DGV) to increase intracellular accumulation of FC. Incubation of cells with oleic acid induces a significant accumulation of full-length caveolins in the enlarged lipid droplets. We conclude that caveolin can associate with the membrane surrounding lipid droplets and is a key component involved in intracellular cholesterol balance and lipid transport in fibroblasts.

Mutations in FKBP10 cause recessive osteogenesis imperfecta and Bruck syndrome.

Osteogenesis imperfecta (OI) is a genetic disorder of connective tissue characterized by bone fragility and alteration in synthesis and posttranslational modification of type I collagen. Autosomal dominant OI is caused by mutations in the genes (COL1A1 or COL1A2) encoding the chains of type I collagen. Bruck syndrome is a recessive disorder featuring congenital contractures in addition to bone fragility; Bruck syndrome type 2 is caused by mutations in PLOD2 encoding collagen lysyl hydroxylase, whereas Bruck syndrome type 1 has been mapped to chromosome 17, with evidence suggesting region 17p12, but the gene has remained elusive so far. Recently, the molecular spectrum of OI has been expanded with the description of the basis of a unique posttranslational modification of type I procollagen, that is, 3-prolyl-hydroxylation. Three proteins, cartilage-associated protein (CRTAP), prolyl-3-hydroxylase-1 (P3H1, encoded by the LEPRE1 gene), and the prolyl cis-trans isomerase cyclophilin-B (PPIB), form a complex that is required for fibrillar collagen 3-prolyl-hydroxylation, and mutations in each gene have been shown to cause recessive forms of OI. Since then, an additional putative collagen chaperone complex, composed of FKBP10 (also known as FKBP65) and SERPINH1 (also known as HSP47), also has been shown to be mutated in recessive OI. Here we describe five families with OI-like bone fragility in association with congenital contractures who all had FKBP10 mutations. Therefore, we conclude that FKBP10 mutations are a cause of recessive osteogenesis imperfecta and Bruck syndrome, possibly Bruck syndrome Type 1 since the location on chromosome 17 has not been definitely localized.

Stress-induced targeting of molecular chaperones in the yeast Saccharomyces cerevisiae

The eukaryotic stress response is an essential mechanism that helps protect cells from a variety of environmental stresses. Cell death can result if cells are not able to properly adapt and protect themselves against adverse stress conditions. Failure to properly deal with stress has implications in human diseases including neurodegenerative disorders and distinct cancers, emphasizing the importance of understanding the eukaryotic stress response in detail. As part of this response, expression of a battery of heat shock proteins (HSP) is induced, which act as molecular chaperones to assist in the repair or triage of unfolded proteins. The 90-kDa HSP (Hsp90) operates in the context of a multi-chaperone complex to promote the maturation of nuclear and cytoplasmic clients. I have discovered that Hsp90 and the co-chaperone Sba1 accumulate in the nucleus of quiescent Saccharomyces cerevisiae cells in a karyopherin-dependent manner. I isolated nuclear accumulation- defective HSP82 mutant alleles to probe the nature of this targeting event and identified a mutant with a single amino acid substitution (I578F) sufficient to prevent nuclear accumulation of Hsp90 in quiescent cells. Diploid hsp82-I578F cells exhibited pronounced defects in spore wall construction and maturation, resulting in catastrophic sporulation. The mislocalization and sporulation phenotypes were shared by another previously identified HSP82 mutant allele, further linking localization to Hsp90 functional status. Pharmacological inhibition of Hsp90 with macbecin in sporulating diploid cells also blocked spore formation, underscoring the importance of this chaperone in this developmental program. The yeast molecular chaperone Hsp104 is a member of the Hsp100 superfamily of AAA+ ATPases. Unlike the Hsp90 family of chaperones, Hsp104 is not restricted to a specific set of client proteins, but rather assists in reactivating stress-denatured proteins by solubilizing protein aggregates. I have discovered that Hsp104, along with the Hsp70 chaperone, Ssa1, and the sHSP Hsp26 accumulate into RNA processing bodies (P- bodies) and stress granules, sites of mRNA metabolism. I found that Hsp104 recruits both Ssa1 and Hsp26 to P-bodies and that these three chaperones are required for stress granule formation. These findings suggest a possible role for chaperones in mRNA metabolism by aiding in the assembly, disassembly or conversion of these enigmatic mRNP complexes. Taken together, the work presented in this dissertation serves to better understand the eukaryotic stress response by illustrating the importance of subcellular-chaperone localization in key biological processes.

Regulation of the heat shock response by thiol-reactive compounds in the yeast Saccharomyces cerevisiae

Cells govern their activities and modulate their interactions with the environment to achieve homeostasis. The heat shock response (HSR) is one of the most well studied fundamental cellular responses to environmental and physiological challenges, resulting in rapid synthesis of heat shock proteins (HSPs), which serve to protect cellular constituents from the deleterious effects of stress. In addition to its role in cytoprotection, the HSR also influences lifespan and is associated with a variety of human diseases including cancer, aging and neurodegenerative disorders. In most eukaryotes, the HSR is primarily mediated by the highly conserved transcription factor HSF1, which recognizes target hsp genes by binding to heat shock elements (HSEs) in their promoters. In recent years, significant efforts have been made to identify small molecules as potential pharmacological activators of HSF1 that could be used for therapeutic benefit in the treatment of human diseases relevant to protein conformation. However, the detailed mechanisms through which these molecules drive HSR activation remain unclear. In this work, I utilized the baker's yeast Saccharomyces cerevisiae as a model system to identify a group of thiol-reactive molecules including oxidants, transition metals and metalloids, and electrophiles, as potent activators of yeast Hsf1. Using an artificial HSE-lacZ reporter and the glucocorticoid receptor system (GR), these diverse thiol-reactive compounds are shown to activate Hsf1 and inhibit Hsp90 chaperone complex activity in a reciprocal, dose-dependent manner. To further understand whether cells sense these reactive compounds through accumulation of unfolded proteins, the proline analog azetidine-2-carboxylic acid (AZC) and protein cross-linker dithiobis(succinimidyl propionate) (DSP) were used to force misfolding of nascent polypeptides and existing cytosolic proteins, respectively. Both unfolding reagents display kinetic HSP induction profiles dissimilar to those generated by thiol-reactive compounds. Moreover, AZC treatment leads to significant cytotoxicity, which is not observed in the presence of the thiol-reactive compounds at the concentrations sufficient to induce Hsf1. Additionally, DSP treatment has little to no effect on Hsp90 functions. Together with the ultracentrifugation analysis of cell lysates that detected no insoluble protein aggregates, my data suggest that at concentrations sufficient to induce Hsf1, thiol-reactive compounds do not induce the HSR via a mechanism based on accumulation of unfolded cytosolic proteins. Another possibility is that thiol-reactive compounds may influence aspects of the protein quality control system such as the ubiquitin-proteasome system (UPS). To address this hypothesis, β-galactosidase reporter fusions were used as model substrates to demonstrate that thiol-reactive compounds do not inhibit ubiquitin activating enzymes (E1) or proteasome activity. Therefore, thiol-reactive compounds do not activate the HSR by inhibiting UPS-dependent protein degradation. I therefore hypothesized that these molecules may directly inactivate protein chaperones, known as repressors of Hsf1. To address this possibility, a thiol-reactive biotin probe was used to demonstrate in vitro that the yeast cytosolic Hsp70 Ssa1, which partners with Hsp90 to repress Hsf1, is specifically modified. Strikingly, mutation of conserved cysteine residues in Ssa1 renders cells insensitive to Hsf1 activation by cadmium and celastrol but not by heat shock. Conversely, substitution with the sulfinic acid and steric bulk mimic aspartic acid led to constitutive activation of Hsf1. Cysteine 303, located in the nucleotide-binding/ATPase domain of Ssa1, was shown to be modified in vivo by a model organic electrophile using Click chemistry technology, verifying that Ssa1 is a direct target for thiol-reactive compounds through adduct formation. Consistently, cadmium pretreatment promoted cells thermotolerance, which is abolished in cells carrying SSA1 cysteine mutant alleles. Taken together, these findings demonstrate that Hsp70 acts as a sensor to induce the cytoprotective heat shock response in response to environmental or endogenously produced thiol-reactive molecules and can discriminate between two distinct environmental stressors.

Genetic Interactions between KAR7/SEC71, KAR8/JEM1, KAR5, and KAR2 during Nuclear Fusion in Saccharomyces cerevisiae

During mating of Saccharomyces cerevisiae, two nuclei fuse to produce a single diploid nucleus. Two genes, KAR7 and KAR8, were previously identified by mutations that cause defects in nuclear membrane fusion. KAR7 is allelic to SEC71, a gene involved in protein translocation into the endoplasmic reticulum. Two other translocation mutants, sec63-1 and sec72Δ, also exhibited moderate karyogamy defects. Membranes from kar7/sec71Δ and sec72Δ, but not sec63-1, exhibited reduced membrane fusion in vitro, but only at elevated temperatures. Genetic interactions between kar7 and kar5 mutations were suggestive of protein–protein interactions. Moreover, in sec71 mutants, Kar5p was absent from the SPB and was not detected by Western blot or immunoprecipitation of pulse-labeled protein. KAR8 is allelic to JEMI, encoding an endoplasmic reticulum resident DnaJ protein required for nuclear fusion. Overexpression of KAR8/JEM1 (but not SEC63) strongly suppressed the mating defect of kar2-1, suggesting that Kar2p interacts with Kar8/Jem1p for nuclear fusion. Electron microscopy analysis of kar8 mutant zygotes revealed a nuclear fusion defect different from kar2, kar5, and kar7/sec71 mutants. Analysis of double mutants suggested that Kar5p acts before Kar8/Jem1p. We propose the existence of a nuclear envelope fusion chaperone complex in which Kar2p, Kar5p, and Kar8/Jem1p are key components and Sec71p and Sec72p play auxiliary roles.

Cholesterol-induced caveolin targeting to lipid droplets in adipocytes: A role for caveolar endocytosis

We have investigated the targeting of caveolin to lipid bodies in adipocytes that express high levels of caveolins and contain well-developed lipid droplets. We observed that the lipid droplets isolated from adipocytes of caveolin-1 knock out mice contained dramatically reduced levels of cholesterol, indicating that caveolin is required for maintaining the cholesterol content of this organelle. Analysis of caveolin distribution by cell fractionation and fluorescent light microscopy in 3T3-L1 adipocytes indicated that addition of cholesterol rapidly stimulated translocation of caveolin to lipid droplets. The cholesterol-induced trafficking of caveolins to lipid droplets was shown to be dynamin- and protein kinase C (PKC)-dependent and modulated by src tyrosine kinase activation, suggesting a role for caveolar endocytosis in this novel trafficking pathway. Consistent with this, caveolae budding was stimulated by cholesterol addition. The present data identify lipid droplets as potential target organelles for caveolar endocytosis and demonstrate a role for caveolin-1 in the maintenance of free cholesterol levels in adipocyte lipid droplets.

Solution structure of DFF40 and DFF45 N-terminal domain complex and mutual chaperone activity of DFF40 and DFF45

Apoptotic DNA fragmentation is mediated by a caspase-activated DNA fragmentation factor (DFF)40. Expression and folding of DFF40 require the presence of DFF45, which also acts as a nuclease inhibitor before DFF40 activation by execution caspases. The N-terminal domains (NTDs) of both proteins are homologous, and their interaction plays a key role in the proper functioning of this two-component system. Here we report that the NTD of DFF45 alone is unstructured in solution, and its folding is induced upon binding to DFF40 NTD. Therefore, folding of both proteins regulates the formation of the DFF40/DFF45 complex. The solution structure of the heterodimeric complex between NTDs of DFF40 and DFF45 reported here shows that the mutual chaperoning includes the formation of an extensive network of intermolecular interactions that bury a hydrophobic cluster inside the interface, surrounded by intermolecular salt bridges.

Vps45p stabilizes the syntaxin homologue Tlg2p and positively regulates SNARE complex formation

Sec1p-like/Munc-18 (SM) proteins bind to t-SNAREs and inhibit ternary complex formation. Paradoxically, the absence of SM proteins does not result in constitutive membrane fusion, Here, we show that in yeast cells lacking the SM protein Vps45p, the t-SNARE Tlg2p is down-regulated, to undetectable levels, by rapid proteasomal degradation. In the absence of Vps45p, Tlg2p can be stabilized through abolition of proteasome activity. Surprisingly, the stabilized Tlg2p was targeted to the correct intracellular location. However, the stabilized Tlg2p is non-functional and unable to bind its cognate SNARE binding partners, Tlg1p and Vti1p, in the absence of Vps45p, A truncation mutant lacking the first 230 residues of Tlg2p no longer bound Vps45p but was able to form complexes with Tlg1p and Vti1p in the absence of the SM protein. These data provide us with two valuable insights into the function of SM proteins. First, SM proteins act as chaperone-like molecules for their cognate t-SNAREs, Secondly, SM proteins play an essential role in the activation process allowing their cognate t-SNARE to participate in ternary complex formation.

Transthyretin Amyloidosis: Chaperone Concentration Changes and Increased Proteolysis in the Pathway to Disease

Transthyretin amyloidosis is a conformational pathology characterized by the extracellular formation of amyloid deposits and the progressive impairment of the peripheral nervous system. Point mutations in this tetrameric plasma protein decrease its stability and are linked to disease onset and progression. Since non-mutated transthyretin also forms amyloid in systemic senile amyloidosis and some mutation bearers are asymptomatic throughout their lives, non-genetic factors must also be involved in transthyretin amyloidosis. We discovered, using a differential proteomics approach, that extracellular chaperones such as fibrinogen, clusterin, haptoglobin, alpha-1-anti-trypsin and 2-macroglobulin are overrepresented in transthyretin amyloidosis. Our data shows that a complex network of extracellular chaperones are over represented in human plasma and we speculate that they act synergistically to cope with amyloid prone proteins. Proteostasis may thus be as important as point mutations in transthyretin amyloidosis.

Regulation of PIDD auto-proteolysis and activity by the molecular chaperone Hsp90.

In response to DNA damage, p53-induced protein with a death domain (PIDD) forms a complex called the PIDDosome, which either consists of PIDD, RIP-associated protein with a death domain and caspase-2, forming a platform for the activation of caspase-2, or contains PIDD, RIP1 and NEMO, important for NF-κB activation. PIDDosome activation is dependent on auto-processing of PIDD at two different sites, generating the fragments PIDD-C and PIDD-CC. Despite constitutive cleavage, endogenous PIDD remains inactive. In this study, we screened for novel PIDD regulators and identified heat shock protein 90 (Hsp90) as a major effector in both PIDD protein maturation and activation. Hsp90, together with p23, binds PIDD and inhibition of Hsp90 activity with geldanamycin efficiently disrupts this association and impairs PIDD auto-processing. Consequently, both PIDD-mediated NF-κB and caspase-2 activation are abrogated. Interestingly, PIDDosome formation itself is associated with Hsp90 release. Characterisation of cytoplasmic and nuclear pools of PIDD showed that active PIDD accumulates in the nucleus and that only cytoplasmic PIDD is bound to Hsp90. Finally, heat shock induces Hsp90 release from PIDD and PIDD nuclear translocation. Thus, Hsp90 has a major role in controlling PIDD functional activity.

Dynamic impacts of the inhibition of the molecular chaperone hsp90 on the T-cell proteome have implications for anti-cancer therapy.

The molecular chaperone Hsp90-dependent proteome represents a complex protein network of critical biological and medical relevance. Known to associate with proteins with a broad variety of functions termed clients, Hsp90 maintains key essential and oncogenic signalling pathways. Consequently, Hsp90 inhibitors are being tested as anti-cancer drugs. Using an integrated systematic approach to analyse the effects of Hsp90 inhibition in T-cells, we quantified differential changes in the Hsp90-dependent proteome, Hsp90 interactome, and a selection of the transcriptome. Kinetic behaviours in the Hsp90-dependent proteome were assessed using a novel pulse-chase strategy (Fierro-Monti et al., accompanying article), detecting effects on both protein stability and synthesis. Global and specific dynamic impacts, including proteostatic responses, are due to direct inhibition of Hsp90 as well as indirect effects. As a result, a decrease was detected in most proteins that changed their levels, including known Hsp90 clients. Most likely, consequences of the role of Hsp90 in gene expression determined a global reduction in net de novo protein synthesis. This decrease appeared to be greater in magnitude than a concomitantly observed global increase in protein decay rates. Several novel putative Hsp90 clients were validated, and interestingly, protein families with critical functions, particularly the Hsp90 family and cofactors themselves as well as protein kinases, displayed strongly increased decay rates due to Hsp90 inhibitor treatment. Remarkably, an upsurge in survival pathways, involving molecular chaperones and several oncoproteins, and decreased levels of some tumour suppressors, have implications for anti-cancer therapy with Hsp90 inhibitors. The diversity of global effects may represent a paradigm of mechanisms that are operating to shield cells from proteotoxic stress, by promoting pro-survival and anti-proliferative functions. Data are available via ProteomeXchange with identifier PXD000537.

Facilitating folding of outer membrane proteins, roles of the periplasmic chaperone Skp and the outer membrane lipoprotein BamD

This thesis describes several important advancements in the understanding of the assembly of outer membrane proteins of Gram-negative bacteria like Escherichia coli. A first study was performed to identify binding regions in the trimeric chaperone Skp for outer membrane proteins. Skp is known to facilitate the passage of unfolded outer membrane proteins (OMPs) through the periplasm to the outer membrane (OM). A gene construct named “synthetic chaperone protein (scp)” gene was used to express a fusion protein (Scp) into the cytoplasm of E. coli. The scp gene was used as a template to design mutants of Scp suitable for structural and functional studies using site-directed spectroscopy. Fluorescence resonance energy transfer (FRET) was used to identify distances in Skp-OmpA complexes that separate regions in Scp and in outer membrane protein A (OmpA) from E. coli. For this study, single cysteine (Cys) mutants and single Cys - single tryptophan (Trp) double mutants of Scp were prepared. For FRET experiments, the cysteines were labeled with the tryptophan fluorescence energy acceptor IAEDANS. Single Trp mutants of OmpA were used as fluorescence energy donors. In the second part of this thesis, the function of BamD and the structure of BamD-Scp complexes were examined. BamD is an essential component of the β-barrel assembly machinery (BAM) complex of the OM of Gram-negative bacteria. Fluorescence spectroscopy was used to probe the interactions of BamD with lipid membranes and to investigate the interactions of BamD with possible partner proteins from the periplasm and from the OM. A range of single cysteine (Cys) and single tryptophan (Trp) mutants of BamD were prepared. A very important conclusion from the extensive FRET study is that the essential lipoprotein BamD interacts and binds to the periplasmic chaperone Skp. BamD contains tetratrico peptide repeat (TPR) motifs that are suggested to serve as docking sites for periplasmic chaperones such as Skp.

Resolving the energy paradox of chaperone/usher-mediated fibre assembly

Periplasmic chaperone/usher machineries are used for assembly of filamentous adhesion organelles of Gram-negative pathogens in a process that has been suggested to be driven by folding energy. Structures of mutant chaperone-subunit complexes revealed a final folding transition (condensation of the subunit hydrophobic core) on the release of organelle subunit from the chaperone-subunit pre-assembly complex and incorporation into the final fibre structure. However, in view of the large interface between chaperone and subunit in the pre-assembly complex and the reported stability of this complex, it is difficult to understand how final folding could release sufficient energy to drive assembly. In the present paper, we show the X-ray structure for a native chaperone-fibre complex that, together with thermodynamic data, shows that the final folding step is indeed an essential component of the assembly process. We show that completion of the hydrophobic core and incorporation into the fibre results in an exceptionally stable module, whereas the chaperone-subunit preassembly complex is greatly destabilized by the high-energy conformation of the bound subunit. This difference in stabilities creates a free energy potential that drives fibre formation.