Livrables
Aha1 binds to the middle domain of Hsp90, contributes to client protein activation and stimulates the ATPase activity of the molecular chaperone.
Hsp90 is a highly conserved, abundant and constitutively expressed homodimeric molecular chaperone of the eukaryotic cytosol. Together with Hsp70, it is specifically involved in the folding and conformational regulation of a limited subset of proteins. Almost all natural substrates of Hsp90 are medically relevant signal transduction molecules, including the nuclear receptors for steroid hormones, several protooncogenic kinases and disparate proteins such as nitric oxide synthase.
We have identified Aha1 (activator of Hsp90 ATPase as binding partner of Hsp90. Using genetic and biochemical approaches, the middle domain of Hsp90 (amino acids 272-617) was found to mediate the interaction with Aha1. In experiments with purified proteins, Aha1 stimulated the intrinsic ATPase activity of Hsp90 fivefold. To further establish its cellular role, we deleted the gene encoding Aha1 S. cerevisiae. In vivo experiments demonstrated that Aha1 contributes to efficient activation of the heterologous Hsp90 client protein v-src. Moreover, Aha1 became crucial for cell viability under non-optimal growth conditions when Hsp90 levels are limiting. Thus, our results identify a novel type of cofactor involved in the regulation of the molecular chaperone Hsp90. Aha1 might be especially relevant to the regulation of Hsp90/Hsp70 dependent signaling molecules like protooncogenic kinases that are relevant to the onset and propagation on cancer.
- Aha1 binds to the middle domain of Hsp90
- Aha1 is a stimulator of Hsp90 ATPase activity
- Aha1 increases the efficiency of Hsp90 to activate client proteins
- Aha1 seems to adjust ATP hydolysis rate of Hsp90 according to the demands of the client protein. In the activation of protooncogenic kinases, it might therefore enable Hsp90 to act as a cancer chaperone.
The human DnaJ (Hsp40) proteins HSJ1a and HSJ1b are type II DnaJ proteins with different C termini generated by alternate splicing. Both protein isoforms can regulate the ATPase activity and substrate binding of Hsp70. In this study, we have confirmed the neuronal expression of HSJ1a and HSJ1b proteins and localized their expression in human neural retina using isoform-specific antisera. HSJ1a and HSJ1b were enriched in photoreceptors, particularly the inner segments, but had different intracellular localization due to the prenylation of HSJ1b by a geranylgeranyl moiety. Because of their enrichment at the site of rhodopsin production, we investigated the effect of HSJ1 isoforms on the cellular processing of wild type and mutant rhodopsin apoprotein in SK-N-SH cells.
The expression of HSJ1b, but not HSJ1a, inhibited the normal cellular processing of wild-type rhodopsin-GFP, which co-localized with HSJ1b at the ER. HSJ1b expression also increased the incidence of inclusion formation by the wild-type protein. Both isoforms were recruited to mutant P23H rhodopsin inclusions, but only HSJ1b enhanced inclusion formation. Investigation of a prenylation-null mutant showed that the modulation of rhodopsin processing and inclusion formation was dependent on the correct subcellular targeting of HSJ1b to the cytosolic face of the ER. An Hsp70 interaction-null mutant of HSJ1b had the same effect as HSJ1b, suggesting that these phenomena were independent of Hsp70 and, furthermore, over-expression of Hsp70 with HSJ1b did not modulate the HSJ1b effect on inclusion formation, showing that Hsp70 was not limiting. The data provide evidence that cytoplasmic chaperones when targeted to the ER can influence the folding and processing of a GPCR and show that DnaJ protein function can be further specialized by alternative splicing and post-translational modification.
These results have contributed to workpackage 1
Immortalized human fibroblasts were used to investigate the putative interactions of the Hsp90 molecular chaperone with the wild-type p53 tumour suppressor protein. We show that geldanamycin or radicicol, specific inhibitors of Hsp90, diminish specific wild-type p53 binding to the p21 promoter sequence. Consequently, these inhibitors decrease p21 mRNA levels, which leads to a reduction in cellular p21/Waf1 protein, known to induce cell cycle arrest. In control experiments, we show that neither geldanamycin nor radicicol affect p53 mRNA levels.
A minor decrease in p53 protein level following the treatment of human fibroblasts with the inhibitors suggests the potential involvement of Hsp90 in the stabilization of wild-type p53. To support our in vivo findings, we used a reconstituted system with highly purified recombinant proteins to examine the effects of Hsp90 on wild-type p53 binding to the p21 promoter sequence. The human recombinant Hsp90 alpha isoform as well as bovine brain Hsp90 were purified to homogeneity. Both of these molecular chaperones displayed ATPase activity and the ability to refold heat-inactivated luciferase in a geldanamycin and radicicol sensitive manner, suggesting that post-translational modifications are not involved in the modulation of Hsp90 alpha activity. We show that the incubation of recombinant p53 at 37?C decreases the level of its wild-type conformation and strongly inhibits the in vitro binding of p53 to the p21 promoter sequence. Interestingly, Hsp90 in an ATP-dependent manner can positively modulate p53 DNA binding after incubation at physiological temperature of 37?C.
Other recombinant human chaperones from Hsp70 and Hsp40 families were not able to efficiently substitute Hsp90 in this reaction. Consistent with our in vivo results, geldanamycin can suppress Hsp90 ability to regulate in vitro p53 DNA binding to the promoter sequence. In summary, the results presented in this paper state that chaperone activity of Hsp90 is important for the transcriptional activity of genotypically wild-type p53. These results have contributed to workpackage 1 and 2
Cofactor Tpr2 combines two TPR domains and a J domain to regulate the Hsp70/Hsp90 chaperone system.
Molecular chaperones aid in the folding of newly synthesized polypeptides, and the refolding of proteins after stress-induced denaturation. In the eukaryotic cytosol, Hsp70 and Hsp90 cooperate with various co-chaperone proteins in the folding of a growing set of substrates, including the glucocorticoid receptor (GR). By yeast two-hybrid screening, we identified the cytosolic protein Tpr2 as an interacting partner of both Hsp90 and Hsp70 and we analysed the function of the co-chaperone Tpr2, which contains two chaperone-binding TPR domains and a DnaJ-homologous J domain. In vivo, an increase or decrease in Tpr2 expression reduces GR activation, suggesting that Tpr2 is required at a narrowly defined expression level. As shown in vitro, Tpr2 recognizes both Hsp70 and Hsp90 through its TPR domains, and its J domain stimulates ATP hydrolysis and polypeptide binding by Hsp70. Furthermore, unlike other co-chaperones, Tpr2 induces ATP-independent dissociation of Hsp90 but not of Hsp70 from chaperone/substrate complexes. Excess Tpr2 inhibits the Hsp90-dependent folding of GR in cell lysates.
We propose a novel mechanism in which Tpr2 mediates the retrograde transfer of substrates from Hsp90 onto Hsp70. At normal levels substoichiometric to Hsp90 and Hsp70, this activity optimises the function of the multi-chaperone machinery. This activity provides a mechanism for recycling substrates through the multi-chaperone machinery and rescues polypeptides from premature release that would lead to no-productive or even harmful cellular aggregation.
- The in vitro role of the co-chaperone Tpr2 in protein folding and aggregation was established. The peptide domains relevant to these actions were identified.
- The role of Tpr2 within the Hsp70/Hsp90 chaperone machine was characterized in vivo.
- The mechanism of Tpr2 action on the Hsp70/Hsp90 chaperone machine could be deciphered on the molecular level. Whereas Tpr2 stimulates client protein binding and processing by Hsp70, the activity of Hsp90 on substrate proteins is mainly inhibited by Tpr2.
Mutations in the photopigment rhodopsin are the major cause of autosomal dominant retinitis pigmentosa. The majority of mutations in rhodopsin lead to misfolding of the protein. Through the detailed examination of P23H and K296E mutant opsin processing in COS-7 cells, we have shown that the mutant protein does not accumulate in the Golgi, as previously thought, instead it forms aggregates that have many of the characteristic features of an aggresome.
The aggregates form close to the centrosome and lead to the dispersal of the Golgi apparatus. Furthermore, these aggregates are ubiquitinated, recruit cellular chaperones and disrupt the intermediate filament network. Mutant opsin expression can disrupt the processing of normal opsin, as co-transfection revealed that the wild-type protein is recruited to mutant opsin aggregates. The degradation of mutant opsin is dependent on the proteasome machinery. Unlike the situation with DeltaF508-CFTR, proteasome inhibition does not lead to a marked increase in aggresome formation but increases the retention of the protein within the ER, suggesting that the proteasome is required for the efficient retrotranslocation of the mutant protein. Inhibition of N-linked glycosylation with tunicamycin leads to the selective retention of the mutant protein within the ER and increases the steady state level of mutant opsin. Glycosylation, however, has no influence on the biogenesis and targeting of wild-type opsin in cultured cells.
This demonstrates that N-linked glycosylation is required for ER-associated degradation of the mutant protein but is not essential for the quality control of opsin folding. The addition of 9-cis-retinal to the media increased the amount of P23H, but not K296E, that was soluble and reached the plasma membrane. These data show that rhodopsin autosomal dominant retinitis pigmentosa is similar to many other neurodegenerative diseases in which the formation of intracellular protein aggregates is central to disease pathogenesis, and they suggest a mechanism for disease dominance.
These results have contributed to workpackage 1 and 2
The heat-shock protein 70 chaperone machine is functionally connected to the ubiquitin-proteasome system by the co-chaperone CHIP. In this article, we discuss evidence that the neuronal DnaJ proteins HSJ1a and HSJ1b may represent a further link between the cellular protein folding and degradation machineries. We have demonstrated that HSJ1 proteins contain putative ubiquitin interaction motifs and can modulate the cellular processing of rhodopsin, a protein that is targeted for degradation by the proteasome when it is misfolded.
This result has contributed to workpackage 1
Dj2 is a member of the DnaJ family of proteins, which regulate the chaperoning function of the hsp70s. We isolated a monkey cDNA dj2 clone corresponding to the large mRNA species encoded by the gene. This mRNA differs from the small mRNA produced by the same gene in that it contains a long 3' untranslated region. Both messages were found to be equally stable and to produce the same protein, which is susceptible to farnesylation. Studies in mouse tissues and various cell lines revealed that these messages and their products are differentially expressed. Surprisingly, we found that only the non-farnesylated form of dj2 is capable of translocating to the cell nucleus, especially after heat shock. Finally, based on protein interaction studies, our results indicate that dj2 is a specific partner for hsc70 and not for hsp70.
GST fusion proteins of the wild type T antigen (Tag) and a mutant form unable to bind hsp70 were used in pull down assays in order to detect their interactions with the hsp70 chaperone machine members. Both hsc70 and hsp70 were found to bind Tag in vitro and in cellular extracts of CV1 and NIH3T3 cells (permissive and non permissive for SV40 cells) under control and heat shock conditions. Our efforts to delineate the composition of the hsp70 chaperone machine continue in order to detect the presence of other partners.
This work has contributed to workpackage 1 (chaperone regulation) and to workpackage 3 (role of hsps in transformation)
The invention relates to:
1. a method of obtaining a gene with an increased expression in mammalian cells;
2. a method of protein production in mammalian cells;
3. a method of obtaining a vector with modified gene allowing efficient expression in mammalian cells.
It has been demonstrated that genes containing a high proportion of G and C nucleotides in their coding sequence are expressed more efficiently that genes containing a low proportion of G and C nucleotides. Therefore, a method according to the present invention consists in changing the sequence of a gene encoding a protein in such a way as to obtain a high proportion of G and C nucleotides in the coding sequence, without changing the amino acid sequence of the protein. We discovered this phenomenon by comparing the sequence of hsp70 and hsc70 human genes. This invention could be used in biotechnology in the production of therapeutic proteins like enzymes, hormones, cytokines, growth factors, receptors and antagonists, and in gene therapy by increasing the expression of transferred gene in human cells. These results have contributed to workpackage 4.
Heat shock protein 70 (Hsp70) is a potent survival protein whose depletion triggers massive caspase-independent tumour cell death. Here, we show that Hsp70 exerts its pro-survival function by inhibiting lysosomal membrane permebilization. The cell death induced by Hsp70 depletion was preceded by the release of lysosomal enzymes into the cytosol and inhibited by pharmacological inhibitors of lysosomal cysteine proteases. Accordingly, the Hsp70-mediated protection against various death stimuli in Hsp70 expressing human tumour cells as well as in immortalized Hsp70 transgenic murine fibroblasts occurred at the level of the lysosomal permeabilization.
On the contrary, Hsp70 failed to inhibit the cytochrome c-induced apoptosome-dependent caspase activation in vitro and Fas ligand-induced caspase-dependent apoptosis in immortalized fibroblasts. Immuno-electron microscopy revealed that endosomal and lysosomal membranes of tumour cells contained Hsp70. Permeabilization of purified endo/lysosomes by digitonin failed to release Hsp70 suggesting that it is physically associated with the membranes. Finally, Hsp70 positive lysosomes displayed increased size and resistance against chemical and physical membrane destabilization. These data identify Hsp70 as the first survival protein that functions by inhibiting the death-associated permeabilization of lysosomes.
Heat shock protein 70 (Hsp70) chaperone family comprises six highly homologous cytosolic proteins. Here we show that the "testis-associated" Hsp70-2 is expressed in cancer cells of various origins and is crucial for their growth and survival. Surprisingly, Hsp70-2 promotes cell growth by a mechanism clearly distinct from those used by the stress-inducible (Hsp70) or the cognate (Hsc70) members of the family. Knockdown of Hsp70-2 by RNA interference induces G1 arrest and dramatic senescence-like morphology mediated by macrophage inhibitory cytokine-1, a divergent member of the transforming growth factor-? family. Importantly, Hsp70-2 knockdown induces senescence exclusively in cancer cells and its expression is strongly elevated in a subset of human breast cancer tissues demonstrating a new chaperone-dependent survival mechanism in human cancer.
CHIP is a co-chaperone of Hsp70 that inhibits Hsp70-dependent refolding in vitro. However, the effect of altered expression of CHIP on the fate of unfolded proteins in mammalian cells has not been determined. Surprisingly, we found that over-expression of CHIP in fibroblasts increased the refolding of proteins after thermal denaturation. This effect was insensitive to geldanamycin, an Hsp90 inhibitor, and required the TPR motifs but not the U-box domain of CHIP. Inhibition of Hsp70 chaperone activity abolished the effects of CHIP on protein folding, indicating that the CHIP-mediated events were Hsp70-dependent.
Hsp40 competitively inhibited the CHIP-dependent refolding, which is consistent with in vitro data indicating that these co-factors act on Hsp70 in the ATP-bound state and have opposing effects on Hsp70 ATPase activity. Consistent with these observations, CHIP over-expression did not alter protein folding in the setting of ATP depletion, when Hsp70 is in the ADP-bound state. Concomitant with its effects on refolding heat-denatured substrates, CHIP increased the fraction of nascent chains co-immuno-precipitating with Hsc70, but only when sufficient ATP was present to allow Hsp70 to cycle rapidly. Our data suggest that, consistent with in vitro studies, CHIP attenuates the Hsp70 cycle in living cells. The impact of this effect on the fate of unfolded proteins in cells however, is different from what might be expected from the in vitro data. Rather than resulting in inhibited refolding, CHIP increases the folding capacity of Hsp70 in eukaryotic cells.
These results have contributed to workpackage 1
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