共查询到20条相似文献,搜索用时 31 毫秒
1.
Feng Hong Bei Liu Gabriela Chiosis Daniel T. Gewirth Zihai Li 《The Journal of biological chemistry》2013,288(25):18243-18248
Integrins play important roles in regulating a diverse array of cellular functions crucial to the initiation, progression, and metastasis of tumors. Previous studies have shown that a majority of integrins are folded by the endoplasmic reticulum chaperone gp96. Here, we demonstrate that the dimerization of integrin αL and β2 is highly dependent on gp96. The αI domain (AID), a ligand binding domain shared by seven integrin α-subunits, is a critical region for integrin binding to gp96. Deletion of AID significantly reduced the interaction between integrin αL and gp96. Overexpression of AID intracellularly decreased surface expression of gp96 clients (integrins and Toll-like receptors) and cancer cell invasion. The α7 helix region is crucial for AID binding to gp96. A cell-permeable α7 helix peptide competitively inhibited the interaction between gp96 and integrins and blocked cell invasion. Thus, targeting the binding site of α7 helix of AID on gp96 is potentially a new strategy for treatment of cancer metastasis. 相似文献
2.
Wu S Dole K Hong F Noman AS Issacs J Liu B Li Z 《The Journal of biological chemistry》2012,287(24):19896-19903
HSP90 chaperones a large number of proteins, and it plays essential roles in multiple signaling pathways to maintain protein homeostasis in the cytosol. In addition, HSP90 has been implicated in mediating recognition of lipopolysaccharide (LPS). However, no pharmacologic agents have been developed to interrogate this pathway. Herein we demonstrate that a peptide-based inhibitor that was previously reported to inhibit the master Toll-like receptor-chaperone gp96, an endoplasmic reticulum paralog of HSP90, in fact blocks HSP90-LPS interaction. It inhibited the binding of LPS to the cell surface of both wild type and gp96-null cells and thereby abrogated the cellular response to LPS but not to other Toll-like receptor ligands. We also generated a series of peptide derivatives (named peptide inhibitors of endotoxin responsiveness (PIERs)) from the N-terminal helix structure of HSP90 and demonstrated their effectiveness in blocking LPS activity. PIER inhibition of LPS signaling was partially reversed by CD14 expression. Moreover, we found that a cell-permeable PIER abrogated HSP90 function and caused degradation of multiple known HSP90 client proteins in cancer cells. Thus, targeting HSP90 is a promising modality for treatment of both LPS-mediated pathology and cancer. 相似文献
3.
Shin-ichi Yamada Toshio Ono Akio Mizuno Takayuki K Nemoto 《European journal of biochemistry》2003,270(1):146-154
The alpha isoform of human 90-kDa heat shock protein (HSP90alpha) is composed of three domains: the N-terminal (residues 1-400); middle (residues 401-615) and C-terminal (residues 621-732). The middle domain is simultaneously associated with the N- and C-terminal domains, and the interaction with the latter mediates the dimeric configuration of HSP90. Besides one in the N-terminal domain, an additional client-binding site exists in the C-terminal domain of HSP90. The aim of the present study is to elucidate the regions within the C-terminal domain responsible for the bindings to the middle domain and to a client protein, and to define the relationship between the two functions. A bacterial two-hybrid system revealed that residues 650-697 of HSP90alpha were essential for the binding to the middle domain. An almost identical region (residues 657-720) was required for the suppression of heat-induced aggregation of citrate synthase, a model client protein. Replacement of either Leu665-Leu666 or Leu671-Leu672 to Ser-Ser within the hydrophobic segment (residues 662-678) of the C-terminal domain caused the loss of bindings to both the middle domain and the client protein. The interaction between the middle and C-terminal domains was also found in human 94-kDa glucose-regulated protein. Moreover, Escherichia coli HtpG, a bacterial HSP90 homologue, formed heterodimeric complexes with HSP90alpha and the 94-kDa glucose-regulated protein through their middle-C-terminal domains. Taken together, it is concluded that the identical region including the hydrophobic segment of the C-terminal domain is essential for both the client binding and dimer formation of the HSP90-family molecular chaperone and that the dimeric configuration appears to be similar in the HSP90-family proteins. 相似文献
4.
Matsumoto S Tanaka E Nemoto TK Ono T Takagi T Imai J Kimura Y Yahara I Kobayakawa T Ayuse T Oi K Mizuno A 《The Journal of biological chemistry》2002,277(38):34959-34966
At the primary structure level, the 90-kDa heat shock protein (HSP90) is composed of three regions: the N-terminal (Met(1)-Arg(400)), middle (Glu(401)-Lys(615)), and C-terminal (Asp(621)-Asp(732)) regions. In the present study, we investigated potential subregion structures of these three regions and their roles. Limited proteolysis revealed that the N-terminal region could be split into two fragments carrying residues Met(1) to Lys(281) (or Lys(283)) and Glu(282) (or Tyr(284)) to Arg(400). The former is known to carry the ATP-binding domain. The fragments carrying the N-terminal two-thirds (Glu(401)-Lys(546)) and C-terminal one-third of the middle region were sufficient for the interactions with the N- and C-terminal regions, respectively. Yeast HSC82 that carried point mutations in the middle region causing deficient binding to the N-terminal region could not support the growth of HSP82-depleted cells at an elevated temperature. Taken together, our data show that the N-terminal and middle regions of the HSP90 family protein are structurally divided into two respective subregions. Moreover, the interaction between the N-terminal and middle regions is essential for the in vivo function of HSP90 in yeast. 相似文献
5.
Roles of heat shock protein gp96 in the ER quality control: redundant or unique function? 总被引:2,自引:0,他引:2
Heat shock protein gp96 is an endoplasmic reticulum chaperone, belonging to the HSP90 family. The function of gp96 as a molecular chaperone was discovered more than 10 years ago, but its importance has been overshadowed by the brilliance of its role in immune responses. It is now clear that gp96 is instrumental in the initiation of both the innate and adaptive immunity. Recently, the roles of gp96 in protein homeostasis, as well as in cell differentiation and development, are beginning to draw more attention due to rapid development in the structural study of HSP90 and some surprising new discoveries from genetic studies of gp96. In this review, we focus on the aspect of gp96 as an ER molecular chaperone in protein maturation, peptide binding and the regulation of its activity. 相似文献
6.
《Bioorganic & medicinal chemistry》2019,27(21):115080
Cells constantly need to adopt to changing environmental conditions, maintaining homeostasis and proteostasis. Heat shock proteins are a diverse class of molecular chaperones that assist proteins in folding to prevent stress-induced misfolding and aggregation. The heat shock protein of 90 kDa (HSP90) is the most abundant heat shock protein. While basal expression of HSP90 is essential for cell survival, in many tumors elevated HSP90 levels are found, which is often associated with bad prognosis. Therefore, HSP90 has emerged as a major target in tumor therapy. The HSP90 machinery is very complex in that it involves large conformational changes during the chaperoning cycle and a variety of co-chaperones. At the same time, this complexity offers a plethora of possibilities to interfere with HSP90 function. The best characterized class of HSP90 modulators are competitive inhibitors targeting the N-terminal ATP-binding pocket. Nineteen compounds of this class entered clinical trials. However, due to severe adverse effects, including induction of the heat shock response, no N-terminal inhibitor has been approved by the FDA so far. As alternatives, compounds commonly referred to as “C-terminal inhibitors” have been developed, either as natural product-based analogues or by rational design, which employ multiple mechanisms to modulate HSP90 function, including modulation of the interaction with co-chaperones, induction of conformational changes that influence the chaperoning cycle, or inhibition of C-terminal dimerization. In this review, we summarize the current development state of characteristic C-terminal inhibitors, with an emphasis on their (proposed) molecular modes of action and binding sites. 相似文献
7.
Julia M. Eckl Matthias J. Scherr Lee Freiburger Marina A. Daake Michael Sattler Klaus Richter 《The Journal of biological chemistry》2015,290(52):30843-30854
Protein kinases are the most prominent group of heat shock protein 90 (Hsp90) clients and are recruited to the molecular chaperone by the kinase-specific cochaperone cell division cycle 37 (Cdc37). The interaction between Hsp90 and nematode Cdc37 is mediated by binding of the Hsp90 middle domain to an N-terminal region of Caenorhabditis elegans Cdc37 (CeCdc37). Here we map the binding site by NMR spectroscopy and define amino acids relevant for the interaction between CeCdc37 and the middle domain of Hsp90. Apart from these distinct Cdc37/Hsp90 interfaces, binding of the B-Raf protein kinase to the cochaperone is conserved between mammals and nematodes. In both cases, the C-terminal part of Cdc37 is relevant for kinase binding, whereas the N-terminal domain displaces the nucleotide from the kinase. This interaction leads to a cooperative formation of the ternary complex of Cdc37 and kinase with Hsp90. For the mitogen-activated protein kinase extracellular signal-regulated kinase 2 (Erk2), we observe that certain features of the interaction with Cdc37·Hsp90 are conserved, but the contribution of Cdc37 domains varies slightly, implying that different kinases may utilize distinct variations of this binding mode to interact with the Hsp90 chaperone machinery. 相似文献
8.
de la Fuente van Bentem S Vossen JH de Vries KJ van Wees S Tameling WI Dekker HL de Koster CG Haring MA Takken FL Cornelissen BJ 《The Plant journal : for cell and molecular biology》2005,43(2):284-298
Recent data suggest that plant disease resistance (R) proteins are present in multi-protein complexes. Tomato R protein I-2 confers resistance against the fungal pathogen Fusarium oxysporum. To identify components of the I-2 complex, we performed yeast two-hybrid screens using the I-2 leucine-rich repeat (LRR) domain as bait, and identified protein phosphatase 5 (PP5) as an I-2 interactor. Subsequent screens revealed two members of the cytosolic heat shock protein 90 (HSP90) family as interactors of PP5. By performing in vitro protein-protein interaction analysis using recombinant proteins, we were able to show a direct interaction between I-2 and PP5, and between I-2 and HSP90. The N-terminal part of the LRR domain was found to interact with HSP90, whereas the C-terminal part bound to PP5. The specific binding of HSP90 to the N-terminal region of the I-2 LRR domain was confirmed by co-purifying HSP90 from tomato lysate using recombinant proteins. Similarly, the interaction between PP5 and HSP90 was established. To investigate the role of PP5 and HSP90 for I-2 function, virus-induced gene silencing was performed in Nicotiana benthamiana. Silencing of HSP90 but not of PP5 completely blocked cell death triggered by I-2, showing that HSP90 is required for I-2 function. Together these data suggest that R proteins require, like steroid hormone receptors in animal systems, an HSP90/PP5 complex for their folding and functioning. 相似文献
9.
Man D He W Sze KH Gong K Smith DK Zhu G Ip NY 《The Journal of biological chemistry》2003,278(26):23285-23294
The functional receptor complex of ciliary neurotrophic factor (CNTF), a member of the gp130 family of cytokines, is composed of CNTF, the CNTF receptor alpha (CNTFR), gp130, and the leukemia inhibitory factor receptor (LIFR). However, the nature of the receptor-mediated interactions in this complex has not yet been resolved. To address this issue we have determined the solution structure of the C-terminal or BC domain of CNTFR and studied the interactions of CNTFR with LIFR and gp130. We reported previously that the membrane distal cytokine-binding domain (CBD1) of LIFR could interact in vitro with soluble CNTFR (sCNTFR) in the absence of CNTF. Here we show that the CBD of human gp130 can also bind in vitro to sCNTFR in the absence of CNTF. In addition, the gp130 CBD could compete with the LIFR CBD1 for the binding of sCNTFR. Substitution of residues in the gp130 CBD, the LIFR CBD1, and the CNTFR BC domain that are expected to be involved in receptor-receptor interactions significantly reduced their interactions. An NMR chemical shift perturbation study of the interaction between the BC domains of CNTFR and gp130 further mapped the interaction surface. These data suggest that both the gp130 CBD and the LIFR CBD1 interact with CNTFR in a similar way and provide insights into the nature of the CNTF receptor complex. 相似文献
10.
Weekes MP Antrobus R Talbot S Hör S Simecek N Smith DL Bloor S Randow F Lehner PJ 《Journal of proteome research》2012,11(3):1475-1484
The endoplasmic reticulum chaperone gp96 is required for the cell surface expression of a narrow range of proteins, including toll-like receptors (TLRs) and integrins. To identify a more comprehensive repertoire of proteins whose cell surface expression is dependent on gp96, we developed plasma membrane profiling (PMP), a technique that combines SILAC labeling with selective cell surface aminooxy-biotinylation. This approach allowed us to compare the relative abundance of plasma membrane (PM) proteins on gp96-deficient versus gp96-reconstituted murine pre-B cells. Analysis of unfractionated tryptic peptides initially identified 113 PM proteins, which extended to 706 PM proteins using peptide prefractionation. We confirmed a requirement for gp96 in the cell surface expression of certain TLRs and integrins and found a marked decrease in cell surface expression of four members of the extended LDL receptor family (LDLR, LRP6, Sorl1 and LRP8) in the absence of gp96. Other novel gp96 client proteins included CD180/Ly86, important in the B-cell response to lipopolysaccharide. We highlight common structural motifs in these client proteins that may be recognized by gp96, including the beta-propeller and leucine-rich repeat. This study therefore identifies the extended LDL receptor family as an important new family of proteins whose cell surface expression is regulated by gp96. 相似文献
11.
Activation of Stat proteins by cytokines is initiated by their Src homology 2 (SH2) domain-mediated association with the cytokine receptors. Previously, we identified an essential role of the coiled-coil domain of Stat3 in binding of the receptor peptides derived from the interleukin-6 receptor subunit, gp130. In this study, we further investigated the molecular basis of this regulation. We found that the C-terminal domain of Stat3 negatively regulates its receptor binding activity only in the absence of the first alpha-helix of the coiled-coil domain, which leads to a hypothesis of intramolecular interaction. Physical interactions between the coiled-coil domain and the C-terminal domain, as well as the SH2 domain, were indeed detected. Furthermore, a sub-region of the C-terminal domain (amino acids 720-740), which is also involved in the interaction with the coiled-coil domain, was demonstrated to be critical for the regulation of the receptor binding. Correspondingly, phosphorylation on Ser-727 within this region inhibits this interaction. In agreement with the peptide binding results, both the coiled-coil domain and the C-terminal sub-region are necessary for the functional recruitment of Stat3 to the cellular gp130 in response to interleukin-6, suggesting that the interdomain interaction is a prerequisite for the SH2-mediated receptor binding in interleukin-6 signaling. 相似文献
12.
Identification of the peptide-binding site in the heat shock chaperone/tumor rejection antigen gp96 (Grp94) 总被引:13,自引:0,他引:13
Heat shock protein (HSP)-peptide complexes from tumor cells elicit specific protective immunity when injected into inbred mice bearing the same specific type of tumor. The HSP-mediated specific immunogenicity also occurs with virus-infected cells. The immune response is solely due to endogenous peptides noncovalently bound to HSP. A vesicular stomatitis virus capsid-derived peptide ligand bearing a photoreactive azido group was specifically bound by and cross-linked to murine HSP glycoprotein (gp) 96. The peptide-binding site was mapped by specific proteolysis of the cross-links followed by analysis of the cross-linked peptides using a judicious combination of SDS-gel electrophoresis, mass spectrometry, and amino acid sequencing. The minimal peptide-binding site was mapped to amino acid residues 624-630 in a highly conserved region of gp96. A model of the peptide binding pocket of gp96 was constructed based on the known crystallographic structure of major histocompatibility complex class I molecule bound to a similar peptide. The gp96-peptide model predicts that the peptide ligand is held in a groove formed by alpha-helices and lies on a surface consisting of antiparallel beta-sheets. Interestingly, in this model, the peptide binding pocket abuts the dimerization domain of gp96, which may have implications for the extraordinary stability of peptide-gp96 complexes, and for the faithful relay of peptides to major histocompatibility complex class I molecule for antigen presentation. 相似文献
13.
Li H Zhou M Han J Zhu X Dong T Gao GF Tien P 《Journal of immunology (Baltimore, Md. : 1950)》2005,174(1):195-204
Previously, we reported that a 7-mer HLA-A11-restricted peptide (YVNTNMG) of hepatitis B virus (HBV) core Ag (HBcAg(88-94)) was associated with heat shock protein (HSP) gp96 in liver tissues of patients with HBV-induced hepatocellular carcinoma (HCC). This peptide is highly homologous to a human HLA-A11-restricted 9-mer peptide (YVNVNMGLK) and to a mouse H-2-K(d)-restricted 9-mer peptide (SYVNTNMGL). To further characterize its immunogenicity, BALB/c mice were vaccinated with the HBV 7-mer peptide. It was found that a specific CTL response was induced by the 7-mer peptide, although the response was approximately 50% of that induced by the mouse H-2-K(d)-restricted 9-mer peptide, as detected by ELISPOT, tetramer, and (51)Cr release assays. To evaluate the adjuvant effect of HSP gp96, mice were coimmunized with gp96 and the 9-mer peptide, and a significant adjuvant effect was observed with gp96. To further determine whether the immune effect of gp96 was dependent on peptide binding, the N- and C-terminal fragments of gp96, which are believed to contain the putative peptide-binding domain, were cloned and expressed in Escherichia coli. CTL assays indicated that only the N-terminal fragment, but not the C-terminal fragment, was able to produce the adjuvant effect. These results clearly demonstrated the potential of using gp96 or its N-terminal fragment as a possible adjuvant to augment CTL response against HBV infection and HCC. 相似文献
14.
15.
16.
Calcineurin is composed of a catalytic subunit (CNA) and a regulatory subunit (CNB). CNA contains the catalytic domain and three regulatory domains: a CNB-binding domain (BBH), a C-terminal calmodulin-binding domain (CBD), and an autoinhibitory domain (AID). We constructed a series of mutants of CNA to explore the regulatory role of its C-terminal regulatory domain and CaM. We demonstrated a more precise mechanism of CNA regulation by C-terminal residues 389-511 in the presence of CNB. First, we showed that residues 389-413, which were identified in previous work as constituting a CaM binding domain (CBD), also have an autoinhibiting function. We also found that residues 389-413 were not sufficient for CaM binding and that the CBD comprises at least residues 389-456. In conclusion, two distinct segments of the C-terminal regulatory region (389-511) of CNA inhibit enzyme activity: residues 389-413 interact with the CNB binding helix (BBH), and residues 457-482 with the active center of CNA. 相似文献
17.
热休克蛋白90(HSP90)是一类ATPase依赖性蛋白,作为分子伴侣,可在辅分子伴侣协助下,通过自身构象改变,参与众多细胞的生物学事件,从而协助新合成蛋白的正确折叠、成功装配、功能稳定及异常蛋白的降解过程。HSP90功能的发挥依赖于辅分子伴侣及氨基末端结合的核苷酸。辅分子伴侣是一类可与分子伴侣(如,HSP90)结合并调节其功能的蛋白,通过参与ATPase循环从而调节HSP90分子伴侣的功能。近年来,辅分子伴侣的研究得到越来越多的关注,本文就辅分子伴侣调控HSP90功能的作用进行综述。 相似文献
18.
The tetratricopeptide repeat domain (TPR)-containing co-chaperone Hsp-organising protein (Hop) plays a critical role in mediating interactions between Heat Shock Protein (Hsp)70 and Hsp90 as part of the cellular assembly machine. It also modulates the ATPase activity of both Hsp70 and Hsp90, thus facilitating client protein transfer between the two. Despite structural work on the individual domains of Hop, no structure for the full-length protein exists, nor is it clear exactly how Hop interacts with Hsp90, although it is known that its primary binding site is the C-terminal MEEVD motif. Here, we have undertaken a biophysical analysis of the structure and binding of Hop to Hsp90 using a variety of truncation mutants of both Hop and Hsp90, in addition to mutants of Hsp90 that are thought to modulate the conformation, in particular the N-terminal dimerisation of the chaperone. The results establish that whilst the primary binding site of Hop is the C-terminal MEEVD peptide of Hsp90, binding also occurs at additional sites in the C-terminal and middle domain. In contrast, we show that another TPR-containing co-chaperone, CyP40, binds solely to the C-terminus of Hsp90.Truncation mutants of Hop were generated and used to investigate the dimerisation interface of the protein. In good agreement with recently published data, we find that the TPR2a domain that contains the Hsp90-binding site is also the primary site for dimerisation. However, our results suggest that residues within the TPR2b may play a role. Together, these data along with shape reconstruction analysis from small-angle X-ray scattering measurements are used to generate a solution structure for full-length Hop, which we show has an overall butterfly-like quaternary structure.Studies on the nucleotide dependence of Hop binding to Hsp90 establish that Hop binds to the nucleotide-free, ‘open’ state of Hsp90. However, the Hsp90-Hop complex is weakened by the conformational changes that occur in Hsp90 upon ATP binding. Together, the data are used to propose a detailed model of how Hop may help present the client protein to Hsp90 by aligning the bound client on Hsp70 with the middle domain of Hsp90. It is likely that Hop binds to both monomers of Hsp90 in the form of a clamp, interacting with residues in the middle domain of Hsp90, thus preventing ATP hydrolysis, possibly by the prevention of association of N-terminal and middle domains in individual Hsp90 monomers. 相似文献
19.
Emiliene Berinyuy 《Journal of receptor and signal transduction research》2016,36(2):119-129
Inhibition of human immunodeficiency virus (HIV) entry into target human cells is considered as a critical strategy for preventing HIV infection. Conformational shifts of the HIV-1 envelope glycoprotein (gp120) facilitates the attachment of the virus to target cells, therefore gp120 remains an attractive target for antiretroviral therapy development. Compound 18A has been recently identified as a broad-spectrum anti-HIV inhibitor. It was proposed that 18A disrupts rearrangements of V1/V2 region in gp120; however, the precise mechanism by which 18A interferes with the inherent motion of V1/V2 domain remains obscure. In this report, we elaborate on the binding mode of compound 18A to the closed conformation of a soluble cleaved gp120 and further examine the dynamic motion of V1/V2 region in both gp120 and the gp120–18A complex via all-atom molecular dynamics simulations. In this work, comparative molecular dynamic analyses revealed that 18A makes contact with Leu179, Ile194, Ile424, Met426 W427, E370 and Met475 in the main hydrophobic cavity of the unliganded gp120 and disrupts the restructuring of V1/V2 domain observed in apo gp120. The unwinding of α1 and slight inversion of β2 in gp120 leads to the shift of VI/V2 domain away from the V3 N-terminal regions and toward the outer domain. Stronger contacts between Trp425 and Trp112 rings may contribute to the reduced flexibility of α1 observed upon 18A binding thereby inhibiting the shifts of the V1/V2 region. Binding of 18A to gp120: (1) decreases the overall flexibility of the protein and (2) inhibits the formation a gp120 conformation that closely ressembles a CD4-bound-like conformation. Information gained from this report not only elaborates on important dynamic features of gp120, but will also assist with the future designs of potent gp120 inhibitors as anti-HIV. 相似文献
20.
T K Nemoto T Ono T Kobayakawa E Tanaka T T Baba K Tanaka T Takagi T Gotoh 《European journal of biochemistry》2001,268(20):5258-5269
In the present study, we investigated the domain structure and domain-domain interactions of HtpG, an Escherichia coli homologue of eukaryotic HSP90. Limited proteolysis of recombinant HtpG, revealed three major tryptic sites, i.e. Arg7-Gly8, Arg336-Glu337 and Lys552-Leu553, of which the latter two were located at the positions equivalent to the major cleavage sites of human HSP90alpha. A similar pattern was obtained by papain treatment under nondenaturing conditions but not under denaturing conditions. Thus, HtpG consists of three domains, i.e. Domain A, Met1-Arg336; domain B, Glu337-Lys552; and domain C, Leu553-Ser624, as does HSP90. The domains of HtpG were expressed and their interactions were estimated on polyacrylamide gel electrophoresis under nondenaturing conditions. As a result, two kinds of domain-domain interactions were revealed: domain B interaction with domain A of the same polypeptide and domain C of one partner with domain B of the other in the dimer. Domain B could be structurally and functionally divided into two subdomains, the N-terminal two-thirds (subdomain BI) that interacted with domain A and the C-terminal one-third (subdomain BII) that interacted with domain C. The C-terminal two-thirds of domain A, i.e. Asp116-Arg336, were sufficient for the binding to domain B. We finally propose the domain organization of an HtpG dimer. 相似文献