Objective:Glycogen synthase kinase-3β(GSK3β)has been recognized as a suppressor of Wnt/β-catenin signaling,which is critical for the stemness maintenance of breast cancer stem cells.However,the regulatory mechanism...Objective:Glycogen synthase kinase-3β(GSK3β)has been recognized as a suppressor of Wnt/β-catenin signaling,which is critical for the stemness maintenance of breast cancer stem cells.However,the regulatory mechanisms of GSK3βprotein expression remain elusive.Methods:Co-immunoprecipitation and mass spectral assays were performed to identify molecules binding to GSK3β,and to characterize the interactions of GSK3β,heat shock protein 90(Hsp90),and co-chaperones.The role of PGK1 in Hsp90 chaperoning GSK3βwas evaluated by constructing 293T cells stably expressing different domains/mutants of Hsp90α,and by performing a series of binding assays with bacterially purified proteins and clinical specimens.The influences of Hsp90 inhibitors on breast cancer stem cell stemness were investigated by Western blot and mammosphere formation assays.Results:We showed that GSK3βwas a client protein of Hsp90.Hsp90,which did not directly bind to GSK3β,interacted with phosphoglycerate kinase 1 via its C-terminal domain,thereby facilitating the binding of GSK3βto Hsp90.GSK3β-bound PGK1 interacted with Hsp90 in the“closed”conformation and stabilized GSK3βexpression in an Hsp90 activity-dependent manner.The Hsp90 inhibitor,17-AAG,rather than HDN-1,disrupted the interaction between Hsp90 and PGK1,and reduced GSK3βexpression,resulting in significantly reduced inhibition ofβ-catenin expression,to maintain the stemness of breast cancer stem cells.Conclusions:Our findings identified a novel regulatory mechanism of GSK3βexpression involving metabolic enzyme PGK1-coupled Hsp90,and highlighted the potential for more effective cancer treatment by selecting Hsp90 inhibitors that do not affect PGK1-regulated GSK3βexpression.展开更多
Severe fever with thrombocytopenia syndrome(SFTS)caused by the SFTS virus(SFTSV)is an emerging disease in East Asia with a fatality rate of up to 30%.However,the viral-host interaction of SFTSV remains largely unknown...Severe fever with thrombocytopenia syndrome(SFTS)caused by the SFTS virus(SFTSV)is an emerging disease in East Asia with a fatality rate of up to 30%.However,the viral-host interaction of SFTSV remains largely unknown.The heat-shock protein 90(Hsp90)family consists of highly conserved chaperones that fold and remodel proteins and has a broad impact on the infection of many viruses.Here,we showed that Hsp90 is an important host factor involved in SFTSV infection.Hsp90 inhibitors significantly reduced SFTSV replication,viral protein expression,and the formation of inclusion bodies consisting of nonstructural proteins(NSs).Among viral proteins,NSs appeared to be the most reduced when Hsp90 inhibitors were used,and further analysis showed that their translation was affected.Co-immunoprecipitation of NSs with four isomers of Hsp90 showed that Hsp90βspecifically interacted with them.Knockdown of Hsp90βexpression also inhibited replication of SFTSV.These results suggest that Hsp90βplays a critical role during SFTSV infection and could be a potential target for the development of drugs against SFTS.展开更多
Cytosolic chaperones are involved in the regulation of cellular protein homeostasis in general. Members of the families of heat stress proteins 70 （Hsp70） and 90 （Hsp90） assist the transport of preproteins to orga...Cytosolic chaperones are involved in the regulation of cellular protein homeostasis in general. Members of the families of heat stress proteins 70 （Hsp70） and 90 （Hsp90） assist the transport of preproteins to organelles such as chloroplasts or mitochondria. In addition, Hsp70 was described to be involved in the degradation of chloroplast preproteins that accumulate in the cytosol. Because a similar function has not been established for Hsp90, we analyzed the influences of Hsp90 and Hsp70 on the protein abundance in the cellular context using an in vivo system based on mesophyll protoplasts. We observed a differential behavior of preproteins with respect to the cytosolic chaperone-dependent regulation. Some preproteins such as pOE33 show a high dependence on Hsp90, whereas the abundance of preproteins such as pSSU is more strongly dependent on Hsp70. The E3 ligase, C-terminus of Hsp70-interacting protein （Chip）, appears to have a more general role in the control of cytosolic protein abundance. We discuss why the different reaction modes are comparable with the cytosolic unfolded protein response.展开更多
Heat shock protein 90(Hsp90)is a highly conserved molecular chaperone that plays a vital role in the signal transduction of cancers.Hsp90 inhibitors are able to inhibit Hsp90 or the complex of Hsp90 and co-chaperones ...Heat shock protein 90(Hsp90)is a highly conserved molecular chaperone that plays a vital role in the signal transduction of cancers.Hsp90 inhibitors are able to inhibit Hsp90 or the complex of Hsp90 and co-chaperones resulting in the degradation of Hsp90-dependent client proteins through the ubiqui tina tion-proteasome pathway,thereby leading to the growth inhibition of tumor cells.This review will briefy discuss the molecular structure and biological function of Hsp90,and focus on a summary of recent progress in the development and testing of natural Hsp90 inhibitors and their different means by which they interact with Hsp90.展开更多
Heat shock proteins (Hsps) or molecular chaperones, are highly conserved protein families present in allstudied organisms. Following cellular stress, the intracellular concentration of Hsps generally increases several...Heat shock proteins (Hsps) or molecular chaperones, are highly conserved protein families present in allstudied organisms. Following cellular stress, the intracellular concentration of Hsps generally increases several folds.Hsps undergo ATP-driven conformational changes to stabilize unfolded proteins or unfold them for translocationacross membranes or mark them for degradation. They are broadly classified in several families according to theirmolecular weights and functional properties. Extensive studies during the past few decades suggest that Hsps play avital role in both normal cellular homeostasis and stress response. Hsps have been reported to interact with numeroussubstrates and are involved in many biological functions such as cellular communication, immune response, proteintransport, apoptosis, cell cycle regulation, gametogenesis and aging. The present review attempts to provide a briefoverview of various Hsps and summarizes their involvement in diverse biological activities.展开更多
The SMYD(SET and MYND domain)family of lysine methyltransferases(KMTs)plays pivotal roles in various cellular processes,including gene expression regulation and DNA damage response.Initially identified as genuine hist...The SMYD(SET and MYND domain)family of lysine methyltransferases(KMTs)plays pivotal roles in various cellular processes,including gene expression regulation and DNA damage response.Initially identified as genuine histone methyltransferases,specific members of this family have recently been shown to methylate non-histone proteins such as p53,VEGFR,and the retinoblastoma tumor suppressor(pRb).To gain further functional insights into this family of KMTs,we generated the protein interaction network for three different human SMYD proteins(SMYD2,SMYD3,and SMYD5).Characterization of each SMYD protein network revealed that they associate with both shared and unique sets of proteins.Among those,we found that HSP90 and several of its co-chaperones interact specifically with the tetratrico peptide repeat(TPR)-containing SMYD2 and SMYD3.Moreover,using proteomic and biochemical techniques,we provide evidence that SMYD2 methylates K209 and K615 on HSP90 nucleotide-binding and dimerization domains,respectively.In addition,we found that each methylation site displays unique reactivity in regard to the presence of HSP90 co-chaperones,pH,and demethylation by the lysine amine oxidase LSD1,suggesting that alternative mechanisms control HSP90 methylation by SMYD2.Altogether,this study highlights the ability of SMYD proteins to form unique protein complexes that may underlie their various biological functions and the SMYD2-mediated methylation of the key molecular chaperone HSP90.展开更多
基金This work was supported by grants from the NSFC Shandong Joint Fund(Grant No.U1606403)the National Natural Science Foundation of China(Grant No.81673450)+4 种基金the State Key Program of the National Natural Science Foundation of China(Grant No.82030074)the NSFC-Shandong Joint Fund(Grant No.U1906212)the Qingdao National Laboratory for Marine Science and Technology(Grant No.2015ASKJ02)the National Science and Technology Major Project for Significant New Drugs Development(Grant No.2018ZX09735-004)the Shandong Provincial Natural Science Foundation(major basic research projects,Grant No.ZR2019ZD18).
文摘Objective:Glycogen synthase kinase-3β(GSK3β)has been recognized as a suppressor of Wnt/β-catenin signaling,which is critical for the stemness maintenance of breast cancer stem cells.However,the regulatory mechanisms of GSK3βprotein expression remain elusive.Methods:Co-immunoprecipitation and mass spectral assays were performed to identify molecules binding to GSK3β,and to characterize the interactions of GSK3β,heat shock protein 90(Hsp90),and co-chaperones.The role of PGK1 in Hsp90 chaperoning GSK3βwas evaluated by constructing 293T cells stably expressing different domains/mutants of Hsp90α,and by performing a series of binding assays with bacterially purified proteins and clinical specimens.The influences of Hsp90 inhibitors on breast cancer stem cell stemness were investigated by Western blot and mammosphere formation assays.Results:We showed that GSK3βwas a client protein of Hsp90.Hsp90,which did not directly bind to GSK3β,interacted with phosphoglycerate kinase 1 via its C-terminal domain,thereby facilitating the binding of GSK3βto Hsp90.GSK3β-bound PGK1 interacted with Hsp90 in the“closed”conformation and stabilized GSK3βexpression in an Hsp90 activity-dependent manner.The Hsp90 inhibitor,17-AAG,rather than HDN-1,disrupted the interaction between Hsp90 and PGK1,and reduced GSK3βexpression,resulting in significantly reduced inhibition ofβ-catenin expression,to maintain the stemness of breast cancer stem cells.Conclusions:Our findings identified a novel regulatory mechanism of GSK3βexpression involving metabolic enzyme PGK1-coupled Hsp90,and highlighted the potential for more effective cancer treatment by selecting Hsp90 inhibitors that do not affect PGK1-regulated GSK3βexpression.
基金supported by grants from the National Natural Science Foundation of China(31900146)the key Biosafety Science and Technology Program of Hubei Jiangxia Laboratory(JXBS001)+1 种基金the Hubei Natural Science Foundation for Distinguished Young Scholars(2021CFA050)the Creative Research Group Program of Natural Science Foundation of Hubei Province(2022CFA021).
文摘Severe fever with thrombocytopenia syndrome(SFTS)caused by the SFTS virus(SFTSV)is an emerging disease in East Asia with a fatality rate of up to 30%.However,the viral-host interaction of SFTSV remains largely unknown.The heat-shock protein 90(Hsp90)family consists of highly conserved chaperones that fold and remodel proteins and has a broad impact on the infection of many viruses.Here,we showed that Hsp90 is an important host factor involved in SFTSV infection.Hsp90 inhibitors significantly reduced SFTSV replication,viral protein expression,and the formation of inclusion bodies consisting of nonstructural proteins(NSs).Among viral proteins,NSs appeared to be the most reduced when Hsp90 inhibitors were used,and further analysis showed that their translation was affected.Co-immunoprecipitation of NSs with four isomers of Hsp90 showed that Hsp90βspecifically interacted with them.Knockdown of Hsp90βexpression also inhibited replication of SFTSV.These results suggest that Hsp90βplays a critical role during SFTSV infection and could be a potential target for the development of drugs against SFTS.
文摘Cytosolic chaperones are involved in the regulation of cellular protein homeostasis in general. Members of the families of heat stress proteins 70 （Hsp70） and 90 （Hsp90） assist the transport of preproteins to organelles such as chloroplasts or mitochondria. In addition, Hsp70 was described to be involved in the degradation of chloroplast preproteins that accumulate in the cytosol. Because a similar function has not been established for Hsp90, we analyzed the influences of Hsp90 and Hsp70 on the protein abundance in the cellular context using an in vivo system based on mesophyll protoplasts. We observed a differential behavior of preproteins with respect to the cytosolic chaperone-dependent regulation. Some preproteins such as pOE33 show a high dependence on Hsp90, whereas the abundance of preproteins such as pSSU is more strongly dependent on Hsp70. The E3 ligase, C-terminus of Hsp70-interacting protein （Chip）, appears to have a more general role in the control of cytosolic protein abundance. We discuss why the different reaction modes are comparable with the cytosolic unfolded protein response.
基金This work was supported by National Science Foundation of China(90913020 and 30901847)Science and Technology Program of China(2012ZX09103-101-053)Science and Technology Star of Zhujiang of Guangzhou City(Dongmei Zhang).
文摘Heat shock protein 90(Hsp90)is a highly conserved molecular chaperone that plays a vital role in the signal transduction of cancers.Hsp90 inhibitors are able to inhibit Hsp90 or the complex of Hsp90 and co-chaperones resulting in the degradation of Hsp90-dependent client proteins through the ubiqui tina tion-proteasome pathway,thereby leading to the growth inhibition of tumor cells.This review will briefy discuss the molecular structure and biological function of Hsp90,and focus on a summary of recent progress in the development and testing of natural Hsp90 inhibitors and their different means by which they interact with Hsp90.
基金Department of Biotechnology(DBT),Department of Science and Technology(DST),New Delhi and Delhi University Research and Development Grant to SS.DST-BOYSCAST fellowship to SS is also gratefully acknowledged.
文摘Heat shock proteins (Hsps) or molecular chaperones, are highly conserved protein families present in allstudied organisms. Following cellular stress, the intracellular concentration of Hsps generally increases several folds.Hsps undergo ATP-driven conformational changes to stabilize unfolded proteins or unfold them for translocationacross membranes or mark them for degradation. They are broadly classified in several families according to theirmolecular weights and functional properties. Extensive studies during the past few decades suggest that Hsps play avital role in both normal cellular homeostasis and stress response. Hsps have been reported to interact with numeroussubstrates and are involved in many biological functions such as cellular communication, immune response, proteintransport, apoptosis, cell cycle regulation, gametogenesis and aging. The present review attempts to provide a briefoverview of various Hsps and summarizes their involvement in diverse biological activities.
基金supported by a Canadian Institutes for Health Research grant (GMX-209406 to J.-F.C.and D.F.).
文摘The SMYD(SET and MYND domain)family of lysine methyltransferases(KMTs)plays pivotal roles in various cellular processes,including gene expression regulation and DNA damage response.Initially identified as genuine histone methyltransferases,specific members of this family have recently been shown to methylate non-histone proteins such as p53,VEGFR,and the retinoblastoma tumor suppressor(pRb).To gain further functional insights into this family of KMTs,we generated the protein interaction network for three different human SMYD proteins(SMYD2,SMYD3,and SMYD5).Characterization of each SMYD protein network revealed that they associate with both shared and unique sets of proteins.Among those,we found that HSP90 and several of its co-chaperones interact specifically with the tetratrico peptide repeat(TPR)-containing SMYD2 and SMYD3.Moreover,using proteomic and biochemical techniques,we provide evidence that SMYD2 methylates K209 and K615 on HSP90 nucleotide-binding and dimerization domains,respectively.In addition,we found that each methylation site displays unique reactivity in regard to the presence of HSP90 co-chaperones,pH,and demethylation by the lysine amine oxidase LSD1,suggesting that alternative mechanisms control HSP90 methylation by SMYD2.Altogether,this study highlights the ability of SMYD proteins to form unique protein complexes that may underlie their various biological functions and the SMYD2-mediated methylation of the key molecular chaperone HSP90.
