N-terminal domain of human Hsp90 triggers binding to the cochaperone p23

The molecular chaperone Hsp90 is a protein folding machine that is conserved from bacteria to man. Human, cytosolic Hsp90 is dedicated to folding of chiefly signal transduction components. The chaperoning mechanism of Hsp90 is controlled by ATP and various cochaperones, but is poorly understood and controversial. Here, we characterized the Apo and ATP states of the 170-kDa human Hsp90 full-length protein by NMR spectroscopy in solution, and we elucidated the mechanism of the inhibition of its ATPase by its cochaperone p23. We assigned isoleucine side chains of Hsp90 via specific isotope labeling of their δ-methyl groups, which allowed the NMR analysis of the full-length protein. We found that ATP caused exclusively local changes in Hsp90's N-terminal nucleotide-binding domain. Native mass spectrometry showed that Hsp90 and p23 form a 22 complex via a positively cooperative mechanism. Despite this stoichiometry, NMR data indicated that the complex was not fully symmetric. The p23-dependent NMR shifts mapped to both the lid and the adenine end of Hsp90's ATP binding pocket, but also to large parts of the middle domain. Shifts distant from the p23 binding site reflect p23-induced conformational changes in Hsp90. Together, we conclude that it is Hsp90's nucleotide-binding domain that triggers the formation of the Hsp90(2)p23(2) complex. We anticipate that our NMR approach has significant impact on future studies of full-length Hsp90 with cofactors and substrates, but also for the development of Hsp90 inhibiting anticancer drugs.     

CRINEPT-TROSY NMR reveals p53 core domain bound in an unfolded form to the chaperone Hsp90

The molecular chaperone Hsp90 sequesters oncogenic mutants of the tumor suppressor p53 that have unstable core domains. It is not known whether p53 is bound in an unfolded, partly folded, or distorted structure, as is unknown for the structure of any bound substrate of Hsp90. It is a particularly difficult problem to analyze in detail the structures of large complexes in which one component is (partly) unfolded. We have shown by transverse relaxation-optimized NMR spectroscopy combined with cross-correlated relaxation-enhanced polarization transfer (CRINEPT-TROSY) that p53 core domain bound in an approximately 200-kDa complex with Hsp90 was predominantly unfolded lacking helical or sheet secondary structure. This mode of binding might be a general feature of substrates of Hsp90.     

V600E B-Raf requires the Hsp90 chaperone for stability and is degraded in response to Hsp90 inhibitors

The Raf family includes three members, of which B-Raf is frequently mutated in melanoma and other tumors. We show that Raf-1 and A-Raf require Hsp90 for stability, whereas B-Raf does not. In contrast, mutated, activated B-Raf binds to an Hsp90-cdc37 complex, which is required for its stability and function. Exposure of melanoma cells and tumors to the Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin results in the degradation of mutant B-Raf, inhibition of mitogen-activated protein kinase activation and cell proliferation, induction of apoptosis, and antitumor activity. These data suggest that activated mutated B-Raf proteins are incompetent for folding in the absence of Hsp90, thus suggesting that the chaperone is required for the clonal evolution of melanomas and other tumors that depend on this mutation. Hsp90 inhibition represents a therapeutic strategy for the treatment of melanoma.     

Pbn1p: an essential endoplasmic reticulum membrane protein required for protein processing in the endoplasmic reticulum of budding yeast

PBN1 was identified as a gene required for production of protease B (PrB) activity in Saccharomyces cerevisiae. PBN1 encodes an endoplasmic reticulum (ER)-localized, type I membrane glycoprotein and is essential for cell viability. To study the essential function(s) of Pbn1p, we constructed a strain with PBN1 under control of the GAL promoter. Depletion of Pbn1p in this strain abrogates processing of the ER precursor forms of PrB, Gas1p, and Pho8p. Depletion of Pbn1p does not affect exit of proprotease A or procarboxypeptidase Y from the ER, indicating that Pbn1p is not required for global exit from the ER. Depleting Pbn1p leads to a significant increase in the unfolded protein response pathway, accompanied by an expansion of bulk ER membrane, indicating that there is a defect in protein folding in the ER. pbn1-1, a nonlethal allele of PBN1, displays synthetic lethality with the ero1-1 allele (ERO1 is required for oxidation in the ER) and synthetic growth defects with the cne1Delta allele (CNE1 encodes calnexin). ER-associated degradation of a lumenal substrate, CPY*, is blocked in the absence of Pbn1p. These results suggest that Pbn1p is required for proper folding and/or the stability of a subset of proteins in the ER. Thus, Pbn1p is an essential chaperone-like protein in the ER of yeast.     

Inhibition of Hsp90 via 17-DMAG induces apoptosis in a p53-dependent manner to prevent medulloblastoma

Elevated expression of HSP90 is observed in many tumor types and is associated with a limited clinical response. Targeting HSP90 using inhibitors such as 17-DMAG (17-desmethoxy-17-N,N-dimethylaminoethylaminogeldanamycin) has shown limited therapeutic success. HSP90 regulates the function of several proteins implicated in tumorigenesis although the precise mechanism through which 17-DMAG regulates tumor cell survival remains unclear. We observed a requirement for p53 in mediating 17-DMAG-induced cell death. The sensitivity of primary mouse embryonic fibroblasts and tumor cells to 17-DMAG-induced apoptosis depended on the p53 status. Wild-type MEFs underwent 17-DMAG-induced caspase-dependent cell death, whilst those lacking p53 failed to do so. Interestingly p53-dependent cell death occurred independently of Atm or Arf. Primary tumor cells derived from two models of murine medulloblastoma (Ptch1^+/−;Ink4c^−/− and p53^FL/FL;Nestin-Cre⁺; Ink4c^−/−) that retain and lack p53 function, respectively, displayed a dependence on functional p53 to engage 17-DMAG-induced apoptosis. Strikingly, 17-DMAG treatment in an allograft model of Ptch1^+/−;Ink4c^−/− but not p53^FL/FL;Nestin-Cre⁺; Ink4c^−/− tumor cells prevented tumor growth in vivo. Our data suggest that p53 status is a likely predictor of the sensitivity of tumors to 17-DMAG.Acquisition of inactivating p53 mutations or aberrant expression of signaling molecules that engage p53 are extremely common in tumors and can render them refractive to conventional therapies (1). Because the tumor suppressor activity of p53 is mediated largely by its ability to engage apoptosis, its inactivation provides tolerance to the tumor microenvironment (1) and is analogous to the survival promoting effects of heat shock proteins (HSPs) that, in response to stresses including hypoxia and nutrient deprivation and in collaboration with co-chaperone proteins, regulate the refolding and repair of damaged proteins (2). By doing so, they preserve protein function and maintain cellular survival in part by preventing apoptosis (3). The expression of several HSPs, including HSP90 is increased in tumors (4), suggesting that elevated HSP expression may contribute to aberrant tumor survival. New clinical strategies aim to exploit this weakness by targeting components of the stress pathway (5).HSP90 displays the unique ability to selectively associate with signaling molecules implicated in the aberrant survival of tumor cells (4). These include mutant (6, 7) and wild-type (wt) p53 (8), Raf-1 (9), and Akt (10). HSP90 is ubiquitously expressed in both normal and malignant tissues, but its altered ‘high-affinity’ conformation in tumor cells confers 100-fold selectivity for HSP90 inhibitors (11). Consequently, several HSP90 inhibitors derived from the ansamycin antibiotic geldanamycin (GA) are in clinical trials for the treatment of cancer (5, 12, 13). Ansamycin compounds bind tightly to the ATP-binding pocket of HSP90 to prevent its stable interaction with substrates and to target them for proteasomal degradation (14, 15). HSP90 inhibitors have shown promising but limited signs of clinical activity (5, 12, 13). It therefore remains important to understand how 17-DMAG acts as an effective anti-tumor agent and if its efficacy is likely to be challenged by features of tumor cells that confer resistance to conventional therapies.Elevated expression of HSP90 in human medulloblastoma (16) suggests that it may represent a candidate for therapeutic intervention in this disease. Here, we sought to preclinically evaluate if the HSP90 inhibitor, 17-DMAG, affects the growth of medulloblastoma, a form of pediatric cancer arising in the cerebellum that develops largely after birth due to the failure of granule neuron precursors (GNPs) to exit the cell cycle and differentiate (17). This aberrant process has been linked to human medulloblastomas involving TP53 inactivation (10% of human cases), defective Sonic Hedgehog/PATCHED (SHH/PTCH) signaling (27% of human cases), lesions in the WNT signaling pathway (15% of human cases), as well as the persistent expression of pro-proliferative genes (18).Several murine models for medulloblastoma that recapitulate causative genetic lesions identified in human medulloblastoma (19) are characterized by activation of the Shh/Ptch signaling pathway, two of which were used in our studies (20). The first (denoted throughout as Ptch1^+/−;Ink4c^−/−) was generated through a germline deletion of one copy of the Patched gene (Ptch1), the receptor for Shh (21), which, when combined with the deletion of Ink4c (22), induce medulloblastomas with an approximate 60% incidence (23). Importantly, all tumors retain functional p53 (20, 23) but lose expression of the wt Ptch1 allele (20). The second model (denoted throughout as p53^FL/FL;Ink4c^−/−) is generated by the conditional deletion of floxed p53 in the cerebellum using Cre recombinase under the control of the Nestin promoter (24) which, when combined with germ line deletion of Ink4c and irradiation of postnatal day 7 mice displays complete penetrance of medulloblastomas (20). Importantly, tumors arising in p53-deficient mice are characterized by the homozygous deletion of Ptch1 (20). Therefore, both the Ptch1^+/−;Ink4c^−/− and p53^FL/FL;Ink4c^−/− models of medulloblastoma are characterized by the constitutive activation of Shh/Ptch signaling, regardless of their founding mutations, with the only difference being the presence or absence of functional p53.Using in vitro assays in primary wt and p53-null mouse embryonic fibroblasts (MEFs) and purified GNP-like tumor cells, we show that 17-DMAG induced apoptosis in a p53- and caspase-dependent manner that required Puma or Bax/Bak, but was independent of p19^Arf and Atm signaling. Transfer of tumor cells derived from each of the murine models into immunocompromised recipients demonstrated that 17-DMAG efficiently prevented medulloblastoma tumor formation and growth in vivo but only when p53 was functional.Our studies establish a relationship between Hsp90 and p53 activity in vivo and provide evidence that the Hsp90 inhibitor, 17-DMAG requires an intact p53 response to exert its anti-tumorigenic effect. Although the relevance of these findings in a clinical setting remains to be examined, they predict that HSP90 inhibitors may be an effective treatment option for human medulloblastoma, a tumor type in which a significant percentage of tumors retain functional p53.     

Forced periodic expression of G1 cyclins phase-locks the budding yeast cell cycle

Phase-locking (frequency entrainment) of an oscillator, in which a periodic extrinsic signal drives oscillations at a frequency different from the unperturbed frequency, is a useful property for study of oscillator stability and structure. The cell cycle is frequently described as a biochemical oscillator; however, because this oscillator is tied to key biological events such as DNA replication and segregation, and to cell growth (cell mass increase), it is unclear whether phase locking is possible for the cell cycle oscillator. We found that forced periodic expression of the G₁ cyclin CLN2 phase locks the cell cycle of budding yeast over a range of extrinsic periods in an exponentially growing monolayer culture. We characterize the behavior of cells in a pedigree using a return map to determine the efficiency of entrainment to the externally controlled pulse. We quantify differences between mothers and daughters and how synchronization of an expanding population differs from synchronization of a single oscillator. Mothers only lock intermittently whereas daughters lock completely and in a different period range than mothers. We can explain quantitative features of phase locking in both cell types with an analytically solvable model based on cell size control and how mass is partitioned between mother and daughter cells. A key prediction of this model is that size control can occur not only in G₁, but also later in the cell cycle under the appropriate conditions; this prediction is confirmed in our experimental data. Our results provide quantitative insight into how cell size is integrated with the cell cycle oscillator.     

Mutant glucocerebrosidase in Gaucher disease recruits Hsp27 to the Hsp90 chaperone complex for proteasomal degradation

Gaucher disease is caused by mutations of the GBA1 gene, which encodes the lysosomal anchored gluococerebrosidase (GCase). GBA1 mutations commonly result in protein misfolding, abnormal chaperone recognition, and premature degradation, but are less likely to affect catalytic activity. In the present study, we demonstrate that the Hsp90/HOP/Cdc37 complex recruits Hsp27 after recognition of GCase mutants with subsequent targeting of GCase mutant peptides to degradation mechanisms such as VCP and the 26S proteasome. Inhibition of Hsp27 not only increased the quantity of enzyme but also enhanced GCase activity in fibroblasts derived from patients with Gaucher disease. These findings provide insight into a possible therapeutic strategy for protein misfolding diseases by correcting chaperone binding and altering subsequent downstream patterns of protein degradation.Gaucher disease (GD) is an autosomal recessive disorder caused by mutations in the GBA1 gene encoding the lysosomal enzyme, glucocerebrosidase (GCase). Genetic alterations in GBA1 lead to quantitative losses of GCase and accumulation of toxic amounts of glucocerebroside. As a consequence, patients suffer widespread organ and metabolic dysfunction. Among the 360 mutations that have been identified in GD, the majority of nucleotide changes are missense mutations, in which a single amino acid is substituted by another (1, 2). However, instead of catastrophic loss of intrinsic enzymatic catalytic function, most GBA1 mutations lead to changes in the natural conformation of the peptide during protein folding, followed by retention within the endoplasmic reticulum (ER) (3–6). Subsequently, the unnatural peptide conformation affects chaperone recognition, which then stimulates premature protein degradation via mechanisms, involving c-cbl associated proteasomal complexes and the E3 ligases Parkin and Itch (4, 7, 8). Identifying key chaperone proteins that determine GCase proteostasis is a plausible approach for developing targeted therapies in GD especially in cases of disease manifestations in the central nervous system where standard enzyme replacement treatment is ineffective.Heat shock protein 27 (Hsp27, also known as heat shock protein beta-1, HSPB1) is a chaperone protein of the small heat shock protein group that also includes ubiquitin, α-crystallin, and Hsp20 among others. In addition to its chaperone activities, Hsp27 is involved in thermotolerance, inhibition of apoptosis, and regulation of cell development and differentiation (9). Hsp27 acts as an ATP-independent chaperone by inhibiting protein aggregation and stabilizing partially denatured proteins ensuring proper refolding by the Hsp70 complex. In addition, Hsp27 may be a key player in proteasomal activation. For example, Hsp27 is integral to degradation of phosphorylated I-κBα in the proteasome (10). In addition, Hsp27 has been shown to interact with p27^Kip1 and to guide its degradation (11). These findings imply that Hsp27 plays a significant role in the proteasomal degradation of misfolded proteins in mammalian cells.We explored potential binding partners to mutant GCases using an unbiased mass spectrometric approach (20535800). We found that Hsp27 was a previously unidentified binding partner that specifically recognized two common mutations for type I (N370S/N370S) and type II/III (L444P/L444P) GD, but not its wild-type counterpart. When presented with the mutant GCase, the Hsp90 complex recruited Hsp27 before induction of the VCP/proteasomal degradation pathway. Targeting Hsp27 not only rescued quantitative enzymatic levels but also increased the catalytic activity of GCase in patient derived fibroblasts. These findings suggest that Hsp27 may be a useful target for the treatment of GD.     

Reassignment of the human CSF1 gene to chromosome 1p13-p21

Human macrophage colony-stimulating factor (CSF-1 or M-CSF) is encoded by a single gene that was previously assigned to the long arm of chromosome 5, band q33.1, in a region adjacent to the gene encoding its receptor (Pettenati MJ, et al, Proc Natl Acad Sci USA 84:2970, 1987). Using fluorescence in situ hybridization with genomic probes to examine normal metaphase chromosomes, we reassigned the human CSF1 gene to the short arm of chromosome 1, bands p13-p21. We confirmed this result by hybridizing a CSF1 cDNA probe to filters containing flow-sorted chromosomes and by identifying CSF1 sequences in DNAs extracted from human x rodent somatic cell hybrids that contained human chromosome 1 but not human chromosome 5. Our findings are consistent with studies that have shown tight linkage between the murine CSF1 and amylase genes, as part of a conserved linkage group between mouse chromosome 3 and the short arm of human chromosome 1, which also includes the genes encoding the beta subunits of thyrotropin and nerve growth factor. Assignment of the CSF1 gene to chromosome 1 at bands p13-p21 raises the possibility that it may be altered by certain nonrandom chromosomal abnormalities arising in human hematopoietic malignancies and solid tumors.     

Yeast Dam1p has a role at the kinetochore in assembly of the mitotic spindle

During mitosis, replicated chromosomes are separated to daughter cells by the microtubule-based mitotic spindle. Chromosomes attach to the mitotic spindle through specialized DNA/protein structures called kinetochores, but the mechanism of attachment is not well understood. We show here that the yeast microtubule-binding protein, Dam1p, associates physically and functionally with kinetochores, suggesting a role in kinetochore attachment to the spindle. An epitope-tagged version of Dam1p colocalizes with the integral kinetochore component Ndc10p/Cbf2p in immunofluorescence analysis of chromosome spreads. In addition, Dam1p is associated preferentially with centromeric DNA as shown by chromatin immunoprecipitation experiments, and this association depends on Ndc10p/Cbf2p. We also demonstrate genetic interactions between DAM1 and CTF19 or SLK19 genes encoding kinetochore proteins. Although the defect caused by the dam1-1 mutation leads to activation of the spindle checkpoint surveillance system and consequent persistence of sister chromatid cohesion, the metaphase arrest spindle abnormally elongates, resulting in virtually complete chromosome missegregation. Execution point experiments indicate that Dam1p has a role in formation of a metaphase spindle and in anaphase spindle elongation. Finally, we have observed that the protein encoded by the dam1-1 allele becomes delocalized at the nonpermissive temperature, correlating with the subsequent onset of the mutant phenotype. Our studies are consistent with a role for Dam1p in attachment of sister chromatids through the kinetochore to the mitotic spindle before chromosome segregation.     

Structure of the Sec23p/24p and Sec13p/31p complexes of COPII

COPII-coated vesicles carry proteins from the endoplasmic reticulum to the Golgi complex. This vesicular transport can be reconstituted by using three cytosolic components containing five proteins: the small GTPase Sar1p, the Sec23p/24p complex, and the Sec13p/Sec31p complex. We have used a combination of biochemistry and electron microscopy to investigate the molecular organization and structure of Sec23p/24p and Sec13p/31p complexes. The three-dimensional reconstruction of Sec23p/24p reveals that it has a bone-shaped structure, (17 nm in length), composed of two similar globular domains, one corresponding to Sec23p and the other to Sec24p. Sec13p/31p is a heterotetramer composed of two copies of Sec13p and two copies of Sec31p. It has an elongated shape, is 28-30 nm in length, and contains five consecutive globular domains linked by relatively flexible joints. Putting together the architecture of these Sec complexes with the interactions between their subunits and the appearance of the coat in COPII-coated vesicles, we present a model for COPII coat organization.     

Single methyl group determines prion propagation and protein degradation activities of yeast heat shock protein (Hsp)-70 chaperones Ssa1p and Ssa2p

Organisms encode multiple homologous heat shock protein (Hsp)-70s, which are essential protein chaperones that play the major role in cellular protein "quality control." Although Hsp70s are functionally redundant and highly homologous, many possess distinct functions. A regulatory motif underlying such distinctions, however, is unknown. The 98% identical cytoplasmic Hsp70s Ssa1p and Ssa2p function differently with regard to propagation of yeast [URE3] prions and in the vacuolar-mediated degradation of gluconeogenesis enzymes, such as FBPase. Here, we show that the Hsp70 nucleotide binding domain (NBD) regulates these functional specificities. We find little difference in ATPase, protein refolding, and amyloid inhibiting activities of purified Ssa1p and Ssa2p, but show that interchanging NBD residue alanine 83 (Ssa1p) and glycine 83 (Ssa2p) switched functions of Ssa1p and Ssa2p in [URE3] propagation and FBPase degradation. Disrupting the degradation pathway did not affect prion propagation, however, indicating these are two distinct processes where Ssa1/2p chaperones function differently. Our results suggest that the primary evolutionary pressure for Hsp70 functional distinctions is not to specify interactions of Hsp70 with substrate, but to specify the regulation of this activity. Our data suggest a rationale for maintaining multiple Hsp70s and suggest that subtle differences among Hsp70s evolved to provide functional specificity without affecting overall enzymatic activity.     

p115 is a general vesicular transport factor related to the yeast endoplasmic reticulum to Golgi transport factor Uso1p.

A recently discovered vesicular transport factor, termed p115, is required along with N-ethylmaleimide-sensitive fusion protein (NSF) and soluble NSF attachment proteins for in vitro Golgi transport. p115 is a peripheral membrane protein found predominantly on the Golgi. Biochemical and electron microscopic analyses indicate that p115 is an elongated homodimer with two globular "heads" and an extended "tail" reminiscent of myosin II. We have cloned and sequenced cDNAs for bovine and rat p115. The predicted translation products are 90% identical, and each can be divided into three domains. The predicted 108-kDa bovine protein consists of an N-terminal 73-kDa globular domain followed by a 29-kDa coiled-coil dimerization domain, a linker segment of 4 kDa, and a highly acidic domain of 3 kDa. p115 is related to Uso1p, a protein required for endoplasmic reticulum to Golgi vesicular transport in Saccharomyces cerevisiae, which has a similar "head-coil-acid" domain structure. The p115 and Uso1p heads are similar in size, have approximately 25% sequence identity, and possess two highly homologous regions (62% and 60% identity over 34 and 53 residues, respectively). There is a third region of homology (50% identity over 28 residues) between the coiled-coil and acidic domains. Although the acidic nature of the p115 and Uso1p C termini is conserved, the primary sequence is not. We discuss these results in light of the proposed function of p115 in membrane targeting and/or fusion.