共查询到20条相似文献,搜索用时 15 毫秒
1.
Structural basis for inhibition of the Hsp90 molecular chaperone by the antitumor antibiotics radicicol and geldanamycin 总被引:2,自引:0,他引:2
SM Roe C Prodromou R O''Brien JE Ladbury PW Piper LH Pearl 《Canadian Metallurgical Quarterly》1999,42(2):260-266
OBJECTIVES: Prostate cancer recurrence, evidenced by rising prostate-specific antigen (PSA) levels after radical prostatectomy, is an increasingly prevalent clinical problem in need of new treatment options. Preclinical studies have suggested that for tumors in general, settings of minimal cancer volume may be uniquely suitable for recombinant vaccine therapy targeting tumor-associated antigens. A clinical study was undertaken to evaluate the safety and biologic effects of vaccinia-PSA (PROSTVAC) administered to subjects with postprostatectomy recurrence of prostate cancer and to assess the feasibility of interrupted androgen deprivation as a tool for modulating expression of the vaccine target antigen, as well as detecting vaccine bioactivity in vivo. METHODS: A limited Phase I clinical trial was conducted to evaluate the safety and biologic effects of vaccinia-PSA administered in 6 patients with androgen-modulated recurrence of prostate cancer after radical prostatectomy. End points included toxicity, serum PSA rise related to serum testosterone restoration, and immunologic effects measured by Western blot analysis for anti-PSA antibody induction. RESULTS: Toxicity was minimal, and dose-limiting toxicity was not observed. Noteworthy variability in time required for testosterone restoration (after interruption of androgen deprivation therapy) was observed. One subject showed continued undetectable serum PSA (less than 0.2 ng/mL) for over 8 months after testosterone restoration, an interval longer than those reported in previous androgen deprivation interruption studies. Primary anti-PSA IgG antibody activity was induced after vaccinia-PSA immunization in 1 subject, although such antibodies were detectable in several subjects at baseline. CONCLUSIONS: Interrupted androgen deprivation may be a useful tool for modulating prostate cancer bioactivity in clinical trials developing novel biologic therapies. Immune responses against PSA may be present among some patients with prostate cancer at baseline and may be induced in others through vaccinia-PSA immunization. 相似文献
2.
T Hoshino J Wang MP Devetten N Iwata S Kajigaya RJ Wise JM Liu H Youssoufian 《Canadian Metallurgical Quarterly》1998,91(11):4379-4386
The FAC protein encoded by the gene defective in Fanconi anemia (FA) complementation group C binds to at least three ubiquitous cytoplasmic proteins in vitro. We used here the complete coding sequence of FAC in a yeast two-hybrid screen to identify interacting proteins. The molecular chaperone GRP94 was isolated twice from a B-lymphocyte cDNA library. Binding was confirmed by coimmunoprecipitation of FAC and GRP94 from cytosolic, but not nuclear, lysates of transfected COS-1 cells, as well as from mouse liver cytoplasmic extracts. Deletion mutants of FAC showed that residues 103-308 were required for interaction with GRP94, and a natural splicing mutation within the IVS-4 of FAC that removes residues 111-148 failed to bind GRP94. Ribozyme-mediated inactivation of GRP94 in the rat NRK cell line led to significantly reduced levels of immunoreactive FAC and concomitant hypersensitivity to mitomycin C, similar to the cellular phenotype of FA. Our results demonstrate that GRP94 interacts with FAC both in vitro and in vivo and regulates its intracellular level in a cell culture model. In addition, the pathogenicity of the IVS-4 splicing mutation in the FAC gene may be mediated in part by its inability to bind to GRP94. 相似文献
3.
Heat-induced oligomerization of the molecular chaperone Hsp90. Inhibition by ATP and geldanamycin and activation by transition metal oxyanions 总被引:1,自引:0,他引:1
It has been previously reported that heat shock protein 90 (Hsp90) oligomerizes at high temperatures and displays concomitantly a novel chaperone activity (Yonehara, M., Minami, Y., Kawata, Y., Nagai, J., and Yahara, I. (1996) J. Biol. Chem., 271, 2641-2645). In order to better define these oligomerization properties at high temperatures and to know whether they are influenced by modulators of Hsp90 function, heat-induced oligomerization of highly purified dimeric Hsp90 has been investigated over a wide range of temperature and protein concentrations by native polyacrylamide gel electrophoresis and size exclusion chromatography. Whereas below 50 degreesC, the dimeric form is maintained over a large range of concentrations, at the critical temperature of 50 degreesC, a sharp transition from dimeric to higher order oligomeric species takes place within minutes, in a highly ordered process, suggesting that a conformational change, leading to the appearance of a new oligomerization site, occurs in Hsp90 dimer. Moreover, at and above the critical temperature, the extent of oligomerization increases with Hsp90 concentration. Formation of high order oligomers at high temperatures is sensitive to modulators of Hsp90 function. ATP and geldanamycin, both known to bind to the same pocket of Hsp90, are inhibitors of this process, whereas molybdate, vanadate, and Nonidet P-40, which are thought to increase surface hydrophobicity of the protein, are activators. Thus, oligomerization of Hsp90 at high temperatures may be mediated through hydrophobic interactions that are hindered by ligands and favored by transition metal oxyanions. The fact that the heat-induced oligomerization of Hsp90 is affected by specific ligands that modulate its properties also suggests that this process may be involved in cell protection during heat shock. 相似文献
4.
KI Kang X Meng J Devin-Leclerc I Bouhouche A Chadli F Cadepond EE Baulieu MG Catelli 《Canadian Metallurgical Quarterly》1999,96(4):1439-1444
Hsp90, a molecular chaperone required for the functioning of glucocorticosteroid receptor (GR), ensures, by direct interaction, the conformational competence of the steroid-binding pocket. In addition to having this positive function, Hsp90 maintains steroid receptors in an inactive form in the absence of hormone. However, neither the participation of Hsp90 once the pathway has been activated by the ligand nor the importance of increased Hsp90 levels in determining the amplitude of the response has ever been assessed directly. Here, by increasing the Hsp90/GR ratio in the nuclear compartment, we found an attenuation of the response to glucocorticosteroids that was not due to a nonspecific or toxic effect of the Hsp90 modified by nuclear targeting. Since this negative effect was more pronounced at high levels of hormone, when receptor and Hsp90 are maximally dissociated, the possibility of an interaction between Hsp90 and GR, already activated to a DNA-binding form, was directly investigated. Indeed GR, after in vivo activation by ligand, was still able to reassociate with Hsp90, suggesting that this interaction plays a role in vivo, possibly in receptor recycling. Moreover, the GR binding to its DNA response element was inhibited by an excess of Hsp90, pointing to a function of Hsp90 in the nuclear compartment. It is thus proposed that an increased Hsp90/GR ratio influences the responsiveness to ligand at a step that is after receptor activation. This increased ratio may be of pathophysiological relevance in the different circumstances that lead to an elevated level of nuclear Hsp90. 相似文献
5.
AT Bender AM Silverstein DR Demady KC Kanelakis S Noguchi WB Pratt Y Osawa 《Canadian Metallurgical Quarterly》1999,274(3):1472-1478
It is established that the multiprotein heat shock protein 90 (hsp90)-based chaperone system acts on the ligand binding domain of the glucocorticoid receptor (GR) to form a GR.hsp90 heterocomplex and to convert the receptor ligand binding domain to the steroid-binding state. Treatment of cells with the hsp90 inhibitor geldanamycin inactivates steroid binding activity and increases the rate of GR turnover. We show here that a portion of neuronal nitric-oxide synthase (nNOS) exists as a molybdate-stabilized nNOS. hsp90 heterocomplex in the cytosolic fraction of human embryonic kidney 293 cells stably transfected with rat nNOS. Treatment of human embryonic kidney 293 cells with geldanamycin both decreases nNOS catalytic activity and increases the rate of nNOS turnover. Similarly, geldanamycin treatment of nNOS-expressing Sf9 cells partially inhibits nNOS activation by exogenous heme. Like the GR, purified heme-free apo-nNOS is activated by the DE52-retained fraction of rabbit reticulocyte lysate, which also assembles nNOS. hsp90 heterocomplexes. However, in contrast to the GR, heterocomplex assembly with hsp90 is not required for increased heme binding and nNOS activation in this cell-free system. We propose that, in vivo, where access by free heme is limited, the complete hsp90-based chaperone machinery is required for sustained opening of the heme binding cleft and nNOS activation, but in the heme-containing cell-free nNOS-activating system transient opening of the heme binding cleft without hsp90 is sufficient to facilitate heme binding. 相似文献
6.
Role of the protein chaperone YDJ1 in establishing Hsp90-mediated signal transduction pathways 总被引:2,自引:0,他引:2
The substrate-specific protein chaperone Hsp90 (heat shock protein 90) from Saccharomyces cerevisiae functions in diverse signal transduction pathways. A mutation in YDJ1, a member of the DnaJ chaperone family, was recovered in a synthetic-lethal screen with Hsp90 mutants. In an otherwise wild-type background, the ydj1 mutation exerted strong and specific effects on three Hsp90 substrates, derepressing two (the estrogen and glucocorticoid receptors) and reducing the function of the third (the tyrosine kinase p60v-src). Analysis of one of these substrates, the glucocorticoid receptor, indicated that Ydj1 exerts its effects through physical interaction with Hsp90 substrates. 相似文献
7.
X Zhu X Zhao WF Burkholder A Gragerov CM Ogata ME Gottesman WA Hendrickson 《Canadian Metallurgical Quarterly》1996,272(5268):1606-1614
DnaK and other members of the 70-kilodalton heat-shock protein (hsp70) family promote protein folding, interaction, and translocation, both constitutively and in response to stress, by binding to unfolded polypeptide segments. These proteins have two functional units: a substrate-binding portion binds the polypeptide, and an adenosine triphosphatase portion facilitates substrate exchange. The crystal structure of a peptide complex with the substrate-binding unit of DnaK has now been determined at 2.0 angstroms resolution. The structure consists of a beta-sandwich subdomain followed by alpha-helical segments. The peptide is bound to DnaK in an extended conformation through a channel defined by loops from the beta sandwich. An alpha-helical domain stabilizes the complex, but does not contact the peptide directly. This domain is rotated in the molecules of a second crystal lattice, which suggests a model of conformation-dependent substrate binding that features a latch mechanism for maintaining long lifetime complexes. 相似文献
8.
9.
M Chevalier L King C Wang MJ Gething E Elguindi SY Blond 《Canadian Metallurgical Quarterly》1998,273(41):26827-26835
To investigate the role of each domain in BiP/GRP78 function, we have used a full-length recombinant BiP engineered to contain two enterokinase sites; one site is located after an N-terminal FLAG epitope, and a second site has been inserted at the junction between the N- and C-terminal domains (FLAG-BiP.ent). FLAG-BiP.ent oligomerizes into multiple species that interconvert with each other in a slow, concentration- and temperature-dependent equilibrium. Binding of ATP or AMP-PNP (adenosine 5'-(beta, gamma-imino)triphosphate), but not ADP, or of a peptidic substrate induces depolymerization of FLAG-BiP.ent and stabilization of monomeric species. Enterokinase cleavage of monomeric, nucleotide-free BiP.ent results in the physical dissociation of the 44-kDa N-terminal ATPase fragment (N44.ent) from the 30-kDa C-terminal substrate binding domain (C30.ent). Upon dissociation, the freed C-terminal substrate binding domain readily undergoes self-association while N44.ent remains monomeric. Enterokinase cleavage performed in the presence of a synthetic peptide prevents oligomerization of the freed C30.ent domain. Addition of ATP during enterokinase cleavage has no effect on C30.ent oligomerization. Our data clearly indicate that binding of a specific peptide onto the C-terminal domain, or ATP onto the N-terminal domain, induces internal conformational change(s) within the C30 domain that result(s) in BiP depolymerization. 相似文献
10.
K Nagata 《Canadian Metallurgical Quarterly》1998,16(7):379-386
Heat shock protein (HSP) 47 is a collagen-binding stress protein localized in the endoplasmic reticulum (ER). In addition to stress-inducibility through heat shock element-heat shock factor interaction, the expression of HSP47 under normal conditions always correlates with that of collagens in various cell types and tissues. Both HSP47 and types I and III collagens are also dramatically induced under pathophysiological conditions such as liver fibrosis. HSP47 transiently associates with procollagen in the ER and dissociates from it in the cis-Golgi compartment. Possible functions of HSP47 as a molecular chaperone specific for procollagen are discussed: prevention of nascent procollagen chains from forming aggregates, effect on the modification of procollagen, inhibition of intracellular degradation of procollagen, quality control mechanisms under stress conditions, and effect on the secretion from the ER to the Golgi compartment. 相似文献
11.
Starvation of mouse hepatoma cells for essential amino acids or glucose results in the ADP-ribosylation of the molecular chaperone BiP/GRP78. Addition of the missing nutrient to the medium reverses the reaction. The signal mediating the response to environmental nutrients involves the translational efficiency. An inhibitor of proteins synthesis, cycloheximide, or reduced temperature, both of which reduce translational efficiency, stimulate the ADP-ribosylation of BiP/GRP78. Inhibition of N-linked glycosylation of proteins results in the overproduction of BiP/GRP78. The over produced protein is not ADP-ribosylated suggesting that this is the functional form of BiP/GRP78. The over produced BiP/GRP78 can, however, be ADP-ribosylated if the cells are starved for an essential amino acid. BiP/GRP78 resides in the lumen of the endoplasmic reticulum where it participates in the assembly of secretory and integral membrane proteins. ADP-ribosylation of BiP/GRP78 during starvation is probably part of a nutritional stress response which conserves limited nutrients by slowing flow through the secretory pathway. 相似文献
12.
The endoplasmic reticulum (ER) of live starfish oocytes was observed during meiotic maturation and fertilization. The ER was visualized by injection into the cytoplasm of an oil drop saturated with the fluorescent lipophilic dye DiI; DiI spread throughout the oocyte endoplasmic reticulum and the pattern was imaged by confocal microscopy. The ER in the immature (germinal vesicle stage) oocyte was composed of interconnected membrane sheets. In response to 1-methyladenine, the sheets of ER appeared to become associated with the yolk platelets, forming spherical shells. A few of these spherical shells could sometimes be seen in immature oocytes, but their number was much greater in the egg at the first meiotic spindle stage. At about the time that the first polar body formed, the spherical shells disappeared, and the ER returned to a form like that of the immature oocyte. The spherical shells did not reappear during the second meiotic cycle. During maturation, the ER also began to move; the movement was apparent by the time of germinal vesicle breakdown and continued throughout both meiotic cycles and in eggs with second polar bodies. When eggs at the first meiotic spindle stage were fertilized, the form of the ER changed. Within 1 min after sperm addition to the observation chamber, the circular cross sections of the spherical shells of the unfertilized egg ER were no longer distinct. At this point, the form of the ER could not be discerned with the resolution of the light microscope; however, the rate of spreading of DiI from an injected oil drop decreased, providing strong evidence that the ER had become fragmented. The ER remained in this form for several minutes and then gradually, the appearance of the ER and the rate of DiI spreading returned to be like those of the unfertilized egg. Injection of inositol trisphosphate caused a similar change in the ER structure. These results indicate that the ER is a dynamic structure, the form of which changes during oocyte maturation and fertilization. 相似文献
13.
Proteins are targeted to the membrane and matrix of peroxisomes by distinct pathways. Recent observations suggest a further route: a subset of peroxisomal membrane proteins might be targeted first to the endoplasmic reticulum, and from there to peroxisomes by vesicle-mediated transport. 相似文献
14.
CE Stebbins AA Russo C Schneider N Rosen FU Hartl NP Pavletich 《Canadian Metallurgical Quarterly》1997,89(2):239-250
The Hsp90 chaperone is required for the activation of several families of eukaryotic protein kinases and nuclear hormone receptors, many of which are protooncogenic and play a prominent role in cancer. The geldanamycin antibiotic has antiproliferative and antitumor effects, as it binds to Hsp90, inhibits the Hsp90-mediated conformational maturation/refolding reaction, and results in the degradation of Hsp90 substrates. The structure of the geldanamycin-binding domain of Hsp90 (residues 9-232) reveals a pronounced pocket, 15 A deep, that is highly conserved across species. Geldanamycin binds inside this pocket, adopting a compact structure similar to that of a polypeptide chain in a turn conformation. This, and the pocket's similarity to substrate-binding sites, suggest that the pocket binds a portion of the polypeptide substrate and participates in the conformational maturation/refolding reaction. 相似文献
15.
Hop, an abundant and conserved protein of unresolved function, binds concomitantly with heat shock protein 70 (Hsp70) and Hsp90, participates with heat shock proteins at an intermediate stage of progesterone receptor assembly, and is required for efficient assembly of mature receptor complexes in vitro. A largely untested hypothesis is that Hop functions as an adaptor that targets Hsp90- to Hsp70-substrate complexes; if true, then loss of either Hsp70 binding or Hsp90 binding by Hop should equally disrupt its ability to promote assembly of mature receptor complexes. To generate Hop mutants that selectively disrupt heat shock protein interactions, highly conserved amino acids in the previously mapped Hsp70 and Hsp90 binding domains of Hop and in a conserved C-terminal domain were targeted for small substitutions and deletions. In co-precipitation assays, these mutants displayed selective loss of association with heat shock proteins. In assays using Hop-depleted rabbit reticulocyte lysate for the cell-free assembly of receptor complexes, none of the Hop mutants inhibited Hsp70 binding to receptor, but all mutants were defective in supporting Hsp90-receptor interactions. Thus, Hop has a novel role in the chaperone machinery as an adaptor that can integrate Hsp70 and Hsp90 interactions. 相似文献
16.
C Berasain D Patil E Perara SM Huang H Mouly C Bréchot 《Canadian Metallurgical Quarterly》1998,16(10):1277-1288
Cyclins are major cell cycle regulators which role in malignant transformation remains controversial. In this report we describe a new mechanism of cyclin oncogenic activation. We demonstrate that an altered form of cyclin A2 (S2A) which N-terminal part is replaced by the hepatitis B virus envelope protein transforms normal rat kidney cells and cooperates with ras to transform rat embryo fibroblasts. In contrast, neither the viral moiety, nor a full length or N-terminally deleted cyclin A2 show these oncogenic properties. S2A oncogenicity arises from its binding to cyclin dependent kinases, since mutation in the MRAIL sequence abolishes transformation and correlates with an abnormal cellular localization in the endoplasmic reticulum membrane. Together, these results implicate modification in the cellular distribution of a cell cycle regulator as a mechanism of virally-induced transformation. 相似文献
17.
We have previously shown that the molecular chaperone HSC70 self-associates in solution into dimers, trimers, and probably high order oligomers, according to a slow temperature- and concentration-dependent equilibrium that is shifted toward the monomer upon binding of ATP peptides or unfolded proteins. To determine the structural basis of HSC70 self-association, the oligomerization properties of the isolated amino- and carboxyl-terminal domains of this protein have been analyzed by gel electrophoresis, size exclusion chromatography, and analytical ultracentrifugation. Whereas the amino-terminal ATPase domain (residues 1-384) was found to be monomeric in solution even at high concentrations, the carboxyl-terminal peptide binding domain (residues 385-646) exists as a slow temperature- and concentration-dependent equilibrium involving monomers, dimers, and trimers. The association equilibrium constant obtained for this domain alone is on the order of 10(5) M-1, very close to that determined previously for the entire protein, suggesting that self-association of HSC70 is determined solely by its carboxyl-terminal domain. Furthermore, oligomerization of the isolated carboxyl-terminal peptide binding domain is, like that of the entire protein, reversed by peptide binding, indicating that self-association of the protein may be mediated by the peptide binding site and, as such, should play a role in the regulation of HSC70 chaperone function. A general model for self-association of HSP70 is proposed in which the protein is in equilibrium between two states differing by the conformation of their carboxyl-terminal domain and their self-association properties. 相似文献
18.
The endoplasmic reticulum (ER) and the closely connected, single dictyosomal Golgi apparatus of Tetrahymena pyriformis cells showed random distribution in the cytoplasm. Ribosomes were evident, and coated vesicles pinched off from the ER were seen. The membranes of the endoplasmic reticulum generally formed a tube-like structure, although after histamine treatment multiple, folded and circular structures were observed. The number of coated vesicles detaching from the endoplasmic reticulum increased as a result of histamine treatment. 相似文献
19.
Several lines of evidence suggest that soluble peptide:N-glycanase (PNGase) is involved in the quality control system for newly synthesized glycoproteins in mammalian cells. Here we report the occurrence of a soluble PNGase activity in Saccharomyces cerevisiae. The enzyme, which was recovered in the cytosolic fraction, has a neutral pH optimum, and dithiothreitol is required for activity. All of these properties were similar to those of earlier described for mammalian PNGases. Interestingly, the yeast enzyme activity was found to be present almost exclusively in cells in stationary phase; little activity was detected in logarithmic growth phase cells. Upon incubation of a glycosylatable peptide R-Asn-X-Thr-R' with permeabilized yeast spheroplasts, we detected formation of both glycosylated peptide and the peptide product expected from PNGase-mediated deglycosylation of this glycopeptide, namely, R-Asp-X-Thr-R'. Recent findings that yeast have an active system for the retrograde transport of unfolded (glyco)proteins and glycopeptides out of the endoplasmic reticulum (ER) into the cytosol raise the possibility that this PNGase may participate in an early step in degradation of these molecules following their export from the ER. 相似文献
20.
The evolutionarily conserved Sec61 protein complex mediates the translocation of secretory proteins into the endoplasmic reticulum. To investigate the role of Sec61p, which is the main subunit of this complex, we generated recessive, cold-sensitive alleles of sec61 that encode stably expressed proteins with strong defects in translocation. The stage at which posttranslational translocation was blocked was probed by chemical crosslinking of radiolabeled secretory precursors added to membranes isolated from wild-type and mutant strains. Two classes of sec61 mutants were distinguished. The first class of mutants was defective in preprotein docking onto a receptor site of the translocon that included Sec61p itself. The second class of mutants allowed docking of precursors onto the translocon but was defective in the ATP-dependent release of precursors from this site that in wild-type membranes leads to pore insertion and full translocation. Only mutants of the second class were partially suppressed by overexpression of SEC63, which encodes a subunit of the Sec61 holoenzyme complex responsible for positioning Kar2p (yeast BiP) at the translocation channel. These mutants thus define two early stages of translocation that require SEC61 function before precursor protein transfer across the endoplasmic reticulum membrane. 相似文献