3-Hydroxy-3-methylglutaryl coenzyme A （HMG CoA） reductase produces mevalonate, an important intermediate in the synthesis of cholesterol and essential nonsterol isoprenoids. The reductase is subject to an exorbitant...3-Hydroxy-3-methylglutaryl coenzyme A （HMG CoA） reductase produces mevalonate, an important intermediate in the synthesis of cholesterol and essential nonsterol isoprenoids. The reductase is subject to an exorbitant amount of feedback control through multiple mechanisms that are mediated by sterol and nonsterol end-products of mevalonate metabolism. Here, I will discuss recent advances that shed light on one mechanism for control of reductase, which involves rapid degradation of the enzyme. Accumulation of certain sterols triggers binding of reductase to endoplasmic reticulum （ER） membrane proteins called Insig-1 and Insig-2. Reductase-Insig binding results in recruitment of a membrane-associated ubiquitin ligase called gp78, which initiates ubiquitination of reductase. This ubiquitination is an obligatory reaction for recognition and degradation of reductase from ER membranes by cytosolic 26S proteasomes. Thus, sterol-accelerated degradation of reductase represents an example of how a general cellular process （ER-associated degradation） is used to control an important metabolic pathway （cholesterol synthesis）.展开更多
The βC1 protein encoded by the Tomato yellow leaf curl China virus-associated betasatellite functions as a pathogenicity determinant. To better understand the molecular basis whereby βC1 functions in pathogenicity, ...The βC1 protein encoded by the Tomato yellow leaf curl China virus-associated betasatellite functions as a pathogenicity determinant. To better understand the molecular basis whereby βC1 functions in pathogenicity, a yeast two-hybrid screen of a tobacco cDNA library was carried out using βC1 as the bait. The screen revealed that βC1 interacts with a tobacco RING-finger protein designated NtRFP1, which was further confirmed by the bimolecular fluorescence complementation and co-immunoprecipitation assays in Nicotiana benthamiana cells. Expression of NtRFP1 was induced by βC1, and in vitro ubiquiti- nation assays showed that NtRFP1 is a functional E3 ubiquiUn ligase that mediates βC1 ubiquitination. In addition, βC1 was shown to be ubiquitinated in vivo and degraded by the plant 26S proteasome. After viral infection, plants overexpressing NtRFP1 developed attenuated symptoms, whereas plants with silenced expression of NtRFP1 showed severe symptoms. Other lines of evidence showed that NtRFP1 attenuates βC1-induced symptoms through promoting its degradation by the 26S proteasome. Taken together, our results suggest that tobacco RING E3 ligase NtRFP1 attenuates disease symptoms by interacting with βC1 to mediate its ubiquitination and degradation via the ubiquitin/26S proteasome system.展开更多
Transcription factor Oct4 plays critical roles in maintaining pluripotency and controlling lineage commitment of embryonic stem cells （ESCs）. Our previous study indicates that Wwp2, a mouse HECT-type E3 ubiquitin li...Transcription factor Oct4 plays critical roles in maintaining pluripotency and controlling lineage commitment of embryonic stem cells （ESCs）. Our previous study indicates that Wwp2, a mouse HECT-type E3 ubiquitin ligase, ubiquitinates Oct4 and promotes its degradation in a heterologous system. However, roles of Wwp2 in regulating en- dogenous Oct4 protein levels as well as molecular characteristics of the function of Wwp2 have not been determined. Here, we report that Wwp2 plays an important role in Oct4 ubiquitination and degradation during differentiation of embryonal carcinoma cells （ECCs）, although it does not appear to affect Oct4 protein levels in the undifferentiated ECCs and ESCs. Importantly, inhibition of Wwp2 expression by specific RNA interference elevates the Oct4 protein level, leading to attenuation in retinoid acid-induced activation of differentiation-related marker genes. Mechanisti- cally, Wwp2 catalyzes Oct4 poly-ubiquitination via the lysine 63 linkage in a dosage-dependent manner. Interest- ingly, Wwp2 also regulates its own ligase activity in a similar manner. Moreover, auto-ubiquitination of Wwp2 occurs through an intra-molecular mechanism. Taken together, these results demonstrate a crucial role of Wwp2 in con- trolling endogenous Oct4 protein levels during differentiation processes of ECCs and suggest an interesting dosage- dependent mechanism for regulating the catalytic activity of the E3 ubiquitin ligase, Wwp2.展开更多
Jasmonic acid(JA)is a crucial phytohormone that regulates plant immunity.The endogenous JA level is determined by the rates of its biosynthesis and catabolism in plants.The activation of JA biosynthesis has been well ...Jasmonic acid(JA)is a crucial phytohormone that regulates plant immunity.The endogenous JA level is determined by the rates of its biosynthesis and catabolism in plants.The activation of JA biosynthesis has been well documented;however,how plants repress JA catabolism upon pathogen infection remains elusive.In this study,we identified and characterized Botrytis cinerea-induced F-box protein 1(BFP1)in Arabidopsis.The expression of BFP1 was induced by B.cinerea in a JA signaling-dependent manner,and BFP1 protein was critical for plant defense against B.cinerea and plant response to JA.In addition,BFP1 overexpression increased plant defenses against broad-spectrum pathogens without fitness costs.Further experiments demonstrated that BFP1 interacts with and mediates the ubiquitination and degrada-tion of jasmonic acid oxidases(JAOs,also known as jasmonate-induced oxygenases,JoXs),the enzymes that hydroxylate JA to 12OH-JA.Consistent with this,BFP1 affects the accumulation of JA and 12OH-JA during B.cinerea infection.Moreover,mutation of JAo2 complemented the phenotypes of the bfp1 mutant.Collectively,our results unveil a new mechanism used by plants to activate immune responses upon path-ogeninfection:suppressing JA catabolism.展开更多
Background:Previous studies have revealed the critical role of transglutaminase 2(TGM2)as a potential therapeutic target in cancers,but the oncogenic roles and underlying mechanisms of TGM2 in gastric cancer(GC)are no...Background:Previous studies have revealed the critical role of transglutaminase 2(TGM2)as a potential therapeutic target in cancers,but the oncogenic roles and underlying mechanisms of TGM2 in gastric cancer(GC)are not fully understood.In this study,we examined the role and potential mechanism of TGM2 in GC.Methods:Western blotting,immunohistochemistry,CCK8,colony formation and transwell assays were used to measure TGM2 expression in the GC cells and tissues and to examine the in vitro role of TGM2 in GC.Xenograft and in vivo metastasis experiments were performed to examine the in vivo role of TGM2 in GC.Gene set enrichment analysis,quantitative PCR and western blotting were conducted to screen for potential TGM2 targets involved in GC.Gain/loss-offunction and rescue experiments were conducted to detect the biological roles of STAT1 in GC cells in the context of TGM2.Co-immunoprecipitation,mass spectrometry,quantitative PCR and western blotting were conducted to identify STAT1-interacting proteins and elucidate their regulatory mechanisms.Mutations in TGM2 and two molecules(ZM39923 and A23187)were used to identify the enzymatic activity of TGM2 involved in the malignant progression of GC and elucidate the underlying mechanism.Results:In this study,we demonstrated elevated TGM2 expression in the GC tissues,which closely related to pathological grade,and predicted poor survival in patients with GC.TGM2 overexpression or knockdown promoted(and inhibited)cell proliferation,migration,and invasion,which were reversed by STAT1 knockdown or overexpression.Further studies showed that TGM2 promoted GC progression by inhibiting STAT1 ubiquitination/degradation.Then,tripartite motif-containing protein 21(TRIM21)was identified as a ubiquitin E3 ligase of STAT1 in GC.TGM2 maintained STAT1 stability by facilitating the dissociation of TRIM21 and STAT1 with GTP-binding enzymatic activity.A23187 abolished the role of TGM2 in STAT1 and reversed the pro-tumor role of TGM2 in vitro and in vivo.Conclusions:This study revealed a criti展开更多
The precise control of receptor levels is crucial for initiating cellular signaling transduction in response to specific ligands;however,such mechanisms regulating nodulation factor(NF)receptor(NFR)-mediated perceptio...The precise control of receptor levels is crucial for initiating cellular signaling transduction in response to specific ligands;however,such mechanisms regulating nodulation factor(NF)receptor(NFR)-mediated perception of NFs to establish symbiosis remain unclear.In this study,we unveil the pivotal role of the NFR-interacting RING-type E3 ligase 1(NIRE1)in regulating NFR1/NFR5 homeostasis to optimize rhizobial infection and nodule development in Lotus japonicus.We demonstrated that NiRE1 has a dual function in this regulatory process.It associates with both NFR1 and NFR5,facilitating their degradation through K48-linked polyubiquitination before rhizobial inoculation.However,following rhizobial inoculation,NFR1 phosphorylates NIRE1ata conserved residue,Tyr-109,inducing a functional switch in NIRE1,which enables NIRE1tomediateK63-linkedpolyubiquitination,thereby stabilizing NFR1/NFR5 in infected root cells.The introduction of phospho-dead NIRE1Y1osF leads to delayed nodule development,underscoring the significance of phosphorylation at Tyr-1o9 in orchestrating symbiotic processes.Conversely,expression of the phospho-mimic NIRE1Y0E results in the formation of spontaneous nodules in L.japonicus,further emphasizing the critical role of the phosphorylation-dependent functional switch in NiRE1.In summary,these findings uncover a fine-tuned symbiotic mechanism that a single E3 ligase could undergo a phosphorylationdependent functional switch to dynamically and precisely regulate NF receptor protein levels.展开更多
目的探究脊髓性肌萎缩症(spinal muscular atrophy,SMA)中p53积累的分子机制。方法构建运动神经元生存基因(Survival motor neuron,Smn)敲低的稳转小鼠运动神经元细胞系NSC34,采用蛋白质印迹和荧光定量PCR检测蛋白和基因的变化;用蛋白...目的探究脊髓性肌萎缩症(spinal muscular atrophy,SMA)中p53积累的分子机制。方法构建运动神经元生存基因(Survival motor neuron,Smn)敲低的稳转小鼠运动神经元细胞系NSC34,采用蛋白质印迹和荧光定量PCR检测蛋白和基因的变化;用蛋白合成抑制剂放线菌酮(Cycloheximide,CHX)抑制蛋白合成,蛋白质印迹检测p53蛋白降解情况,采用免疫共沉淀检测p53与MDM2结合情况;采用免疫荧光检测小鼠脊髓组织运动神经元数量,运用流式细胞仪分析细胞周期的分布。结果在Smn敲低的NSC34细胞中,p53蛋白水平显著增加,但是其基因水平未发生明显变化;当蛋白合成被抑制后,SMN缺失会抑制p53蛋白降解,p53积累后会调控下游MDM2表达显著增加,但是其与p53结合却显著减少;p⁃p53(Ser18)和acetyl⁃p53(K382)水平显著升高,K382乙酰化位点会影响p53泛素化结合,导致其泛素化被抑制;最终p53积累导致NSC34细胞和SMA小鼠脊髓组织中细胞周期阻滞和凋亡增加。结论SMN蛋白缺失通过抑制p53泛素化降解途径导致其积累,进而引起细胞周期阻滞和凋亡。展开更多
Kelch样ECH关联蛋白1(Kelch-like ECH-associated protein 1,Keap1)是E3泛素连接酶的底物识别亚单位,在蛋白质的泛素化修饰中起重要作用。蛋白质的泛素化修饰作为一种重要且复杂的蛋白质翻译后修饰,在自噬和蛋白酶体系统中作为降解信号...Kelch样ECH关联蛋白1(Kelch-like ECH-associated protein 1,Keap1)是E3泛素连接酶的底物识别亚单位,在蛋白质的泛素化修饰中起重要作用。蛋白质的泛素化修饰作为一种重要且复杂的蛋白质翻译后修饰,在自噬和蛋白酶体系统中作为降解信号而被利用。野生型Keap1能够识别、结合多种底物蛋白质并通过Keap1-Cul3-Rbx1复合物泛素化。此外,Keap1还作为一种抑癌蛋白而被广泛研究,已发现诸多Keap1的体细胞突变或等位基因的异常缺失诱发人类多种疾病。当前的研究主要围绕在Keap1-Nrf2系统而很少涉及到其他下游底物。鉴于Keap1在细胞中的重要功能及广大的发展空间,这篇综述将对Keap1目前的研究现状进行总结。包括:泛素-蛋白酶体系统(ubiquitin-proteasome system,UPS),Keap1的结构、功能、突变,Keap1的泛素化底物及Keap1的相关疾病,探讨Keap1在疾病中的临床意义及研究中存在的机遇与挑战。展开更多
Protein abundance and localization at the plasma membrane(PM)shapes plant development and mediates adaptation to changing environmental conditions.It is regulated by ubiquitination,a post-translational modification cr...Protein abundance and localization at the plasma membrane(PM)shapes plant development and mediates adaptation to changing environmental conditions.It is regulated by ubiquitination,a post-translational modification crucial for the proper sorting of endocytosed PM proteins to the vacuole for subsequent degradation.To understand the significance and the variety of roles played by this reversible modification,the function of ubiquitin receptors,which translate the ubiquitin signature into a cellular response,needs to be elucidated.In this study,we show that TOL(TOM1-like)proteins function in plants as multivalent ubiquitin receptors,governing ubiquitinated cargo delivery to the vacuole via the conserved Endosomal Sorting Complex Required for Transport(ESCRT)pathway.TOL2 and TOL6 interact with components of the ESCRT machinery and bind to K63-linked ubiquitin via two tandemly arranged conserved ubiquitin-binding domains.Mutation of these domains results not only in a loss of ubiquitin binding but also altered localization,abolishing TOL6 ubiquitin receptor activity.Function and localization of TOL6 is itself regulated by ubiquitination,whereby TOL6 ubiquitination potentially modulates degradation of PM-localized cargoes,assisting in the fine-tuning of the delicate interplay between protein recycling and downregulation.Taken together,our findings demonstrate the function and regulation of a ubiquitin receptor that mediates vacuolar degradation of PM proteins in higher plants.展开更多
UBE2C(Ubiquitin conjugating enzyme E2 C), a key regulator of cell cycle progression, is a promising target for discovery of antitumor agents. However, it is challenging to develop inhibitors of UBE2C owing to its lack...UBE2C(Ubiquitin conjugating enzyme E2 C), a key regulator of cell cycle progression, is a promising target for discovery of antitumor agents. However, it is challenging to develop inhibitors of UBE2C owing to its lack of “druggable” pockets. Bio PROTACs(biological proteolysis targeting chimeras) are a kind of protein-based degraders by fusing an adaptor to a subunit of E3 ligase for ubiquitination and subsequent proteasome-dependent degradation of target protein. We report herein the design and biological evaluation of a UBE2C-targeting bio PROTAC based on the NEL(novel E3 ligase) domain of bacterial E3 ligase Ipa H9.8 and the UBE2C-binding WHB(winged-helix B) domain of APC_(2)(anaphase promoting complex subunit 2). The in vitro ubiquitination test and Mass Spectrometry analysis showed that the bio PROTAC could transfer ubiquitin to surface exposed lysines on UBE2C and catalyzed the formation of polyubiquitin chains. In addition, the transient co-expression experiment showed that the bio PROTAC could promote proteasomal degradation of heterologous UBE2C and rescue its downstream substrates in mammalian cells.展开更多
Background The mechanism of hepatitis B virus(HBV)-induced carcinogenesis remains an area of interest.The accumulation of hepatitis B surface antigen in the endoplasmic reticulum(ER)of hepatocytes stimulates persisten...Background The mechanism of hepatitis B virus(HBV)-induced carcinogenesis remains an area of interest.The accumulation of hepatitis B surface antigen in the endoplasmic reticulum(ER)of hepatocytes stimulates persistent ER stress.Activity of the unfolded protein response(UPR)pathway of ER stress may play an important role in inflammatory cancer transformation.How the protective UPR pathway is hijacked by cells as a tool for malignant transformation in HBV-related hepatocellular carcinoma(HCC)is still unclear.Here,we aimed to define the key molecule hyaluronan-mediated motility receptor(HMMR)in this process and explore its role under ER stress in HCC development.Methods An HBV-transgenic mouse model was used to characterize the pathological changes during the tumor progression.Proteomics and transcriptomics analyses were performed to identify the potential key molecule,screen the E3 ligase,and define the activation pathway.Quantitative real-time PCR and Western blotting were conducted to detect the expression of genes in tissues and cell lines.Luciferase reporter assay,chromatin immunoprecipitation,coimmunoprecipitation,immunoprecipitation,and immunofluorescence were employed to investigate the molecular mechanisms of HMMR under ER stress.Immunohistochemistry was used to clarify the expression patterns of HMMR and related molecules in human tissues.Results We found sustained activation of ER stress in the HBV-transgenic mouse model of hepatitis-fibrosis-HCC.HMMR was transcribed by c/EBP homologous protein(CHOP)and degraded by tripartite motif containing 29(TRIM29)after ubiquitination under ER stress,which caused the inconsistent expression of mRNA and protein.Dynamic expression of TRIM29 in the HCC progression regulated the dynamic expression of HMMR.HMMR could alleviate ER stress by increasing autophagic lysosome activity.The negative correlation between HMMR and ER stress,positive correlation between HMMR and autophagy,and negative correlation between ER stress and autophagy were verified in human tissues.Conclus展开更多
Upon recognition of dsRNA,toll-like receptor 3(TLR3)recruits the adaptor protein TRIF to activate IRF3 and NF-κB signaling,initiating innate immune responses.The ubiquitination of TLR3 downstream signaling molecules ...Upon recognition of dsRNA,toll-like receptor 3(TLR3)recruits the adaptor protein TRIF to activate IRF3 and NF-κB signaling,initiating innate immune responses.The ubiquitination of TLR3 downstream signaling molecules and their roles in the innate response have been discovered;however,whether TLR3 itself is ubiquitinated and then functionally involved remains to be elucidated.By immunoprecipitating TLR3-binding proteins in macrophages,we identified ring finger protein 170(RNF170)as a TLR3-binding E3 ligase.RNF170 mediated the K48-linked polyubiquitination of K766 in the TIR domain of TLR3 and promoted the degradation of TLR3 through the proteasome pathway.The genetic ablation of RNF170 selectively augmented TLR3-triggered innate immune responses both in vitro and in vivo.Our results reveal a novel role for RNF170 in selectively inhibiting TLR3-triggered innate immune responses by promoting TLR3 degradation.展开更多
Pregnane X receptor(PXR)is a ligand-activated nuclear receptor that transcriptionally upregulates drug-metabolizing enzymes[e.g.,cytochrome P4503A4(CYP3A4)]and transporters.Although the regulation of PXR target genes ...Pregnane X receptor(PXR)is a ligand-activated nuclear receptor that transcriptionally upregulates drug-metabolizing enzymes[e.g.,cytochrome P4503A4(CYP3A4)]and transporters.Although the regulation of PXR target genes is well-characterized,less is known about the regulation of PXR protein level.By screening an RNAi library,we identified the F-box-only protein 44(FBXO44)as a novel E3ligase for PXR.PXR abundance increases upon knockdown of FBXO44,and,inversely,decreases upon overexpression of FBXO44.Further analysis revealed that FBXO44 interacts with PXR,leading to its ubiquitination and proteasomal degradation,and we determined that the F-box associated domain of FBXO44 and the ligand binding domain of PXR are required for the functional interaction.In summary,FBXO44 regulates PXR protein abundance,which has downstream consequences for CYP3A4 levels and drug-drug interactions.The results of this study provide new insight into the molecular mechanisms that regulate PXR protein level and activity and suggest the importance of considering how modulating E3ubiquitin ligase activities will affect PXR-mediated drug metabolism.展开更多
CDK4 is crucial for Gl-to-S transition of cell cycle. It is well established that ubiquitin-mediated degradations of CDK inhibitors and cycUns are pivotal for the timely and unidirectional progression of cell cycle. H...CDK4 is crucial for Gl-to-S transition of cell cycle. It is well established that ubiquitin-mediated degradations of CDK inhibitors and cycUns are pivotal for the timely and unidirectional progression of cell cycle. However, how CDK4 itself is modulated by ubiquitin-mediated degradation has been elusive. Here we report that the steady-state level of CDK4 is controlled by PAQR4, a member of the progestin and adipoQ receptor family, and SKP2, an E3 ubiquitin ligase. Knockdown of PAQR4 leads to reduction of cell proliferation, accompanied by reduced protein level of CDK4. PAQR4 reduces polyubiquitination and degradation of CDK4. PAQR4 interacts with the C-terminal lobe of CDK4. On the other hand, SKP2 also interacts with the C-terminal lobe of CDK4 and enhances polyubiquitination and degradation of CDK4. importantly, PAQR4 and SKP2 bind to the same region in CDK4, and PAQR4 competes with SKP2 for the binding, thereby abrogating SKP2-mediated ubiquitination of CDK4. Using a two-stage DMBA/TPA-induced skin cancer model, we find that PAQR4-deleted mice are resistant to chemical carcinogen-induced tumor formation. Collectively, our findings reveal that the steady-state level of CDK4 is controlled by the antagonistic actions between PAQR4 and SKP2, contributing to modulation of cell proliferation and tumorigenesis.展开更多
文摘3-Hydroxy-3-methylglutaryl coenzyme A （HMG CoA） reductase produces mevalonate, an important intermediate in the synthesis of cholesterol and essential nonsterol isoprenoids. The reductase is subject to an exorbitant amount of feedback control through multiple mechanisms that are mediated by sterol and nonsterol end-products of mevalonate metabolism. Here, I will discuss recent advances that shed light on one mechanism for control of reductase, which involves rapid degradation of the enzyme. Accumulation of certain sterols triggers binding of reductase to endoplasmic reticulum （ER） membrane proteins called Insig-1 and Insig-2. Reductase-Insig binding results in recruitment of a membrane-associated ubiquitin ligase called gp78, which initiates ubiquitination of reductase. This ubiquitination is an obligatory reaction for recognition and degradation of reductase from ER membranes by cytosolic 26S proteasomes. Thus, sterol-accelerated degradation of reductase represents an example of how a general cellular process （ER-associated degradation） is used to control an important metabolic pathway （cholesterol synthesis）.
文摘The βC1 protein encoded by the Tomato yellow leaf curl China virus-associated betasatellite functions as a pathogenicity determinant. To better understand the molecular basis whereby βC1 functions in pathogenicity, a yeast two-hybrid screen of a tobacco cDNA library was carried out using βC1 as the bait. The screen revealed that βC1 interacts with a tobacco RING-finger protein designated NtRFP1, which was further confirmed by the bimolecular fluorescence complementation and co-immunoprecipitation assays in Nicotiana benthamiana cells. Expression of NtRFP1 was induced by βC1, and in vitro ubiquiti- nation assays showed that NtRFP1 is a functional E3 ubiquiUn ligase that mediates βC1 ubiquitination. In addition, βC1 was shown to be ubiquitinated in vivo and degraded by the plant 26S proteasome. After viral infection, plants overexpressing NtRFP1 developed attenuated symptoms, whereas plants with silenced expression of NtRFP1 showed severe symptoms. Other lines of evidence showed that NtRFP1 attenuates βC1-induced symptoms through promoting its degradation by the 26S proteasome. Taken together, our results suggest that tobacco RING E3 ligase NtRFP1 attenuates disease symptoms by interacting with βC1 to mediate its ubiquitination and degradation via the ubiquitin/26S proteasome system.
基金We thank Dr Richard Baer （Pathology, Columbia University, New York, USA） for generously providing various Ub mutant plasmids. This study was supported by Grants from the National Natural Science Foundation of China （30871257, 30730051） and the National High Technology Research, Development Program of China （2006CB943901 and 2007CB947904）, the Shanghai Sci- ence and Technology Developmental Foundation （08JC1413100） and the Shanghai Leading Academic Discipline Project （$30201）.
文摘Transcription factor Oct4 plays critical roles in maintaining pluripotency and controlling lineage commitment of embryonic stem cells （ESCs）. Our previous study indicates that Wwp2, a mouse HECT-type E3 ubiquitin ligase, ubiquitinates Oct4 and promotes its degradation in a heterologous system. However, roles of Wwp2 in regulating en- dogenous Oct4 protein levels as well as molecular characteristics of the function of Wwp2 have not been determined. Here, we report that Wwp2 plays an important role in Oct4 ubiquitination and degradation during differentiation of embryonal carcinoma cells （ECCs）, although it does not appear to affect Oct4 protein levels in the undifferentiated ECCs and ESCs. Importantly, inhibition of Wwp2 expression by specific RNA interference elevates the Oct4 protein level, leading to attenuation in retinoid acid-induced activation of differentiation-related marker genes. Mechanisti- cally, Wwp2 catalyzes Oct4 poly-ubiquitination via the lysine 63 linkage in a dosage-dependent manner. Interest- ingly, Wwp2 also regulates its own ligase activity in a similar manner. Moreover, auto-ubiquitination of Wwp2 occurs through an intra-molecular mechanism. Taken together, these results demonstrate a crucial role of Wwp2 in con- trolling endogenous Oct4 protein levels during differentiation processes of ECCs and suggest an interesting dosage- dependent mechanism for regulating the catalytic activity of the E3 ubiquitin ligase, Wwp2.
基金This work was supported by the Natural Science Foundation of China(32000224,to L.L.)the Natural Science Foundation of Shandong Province(ZR2021QC142,to W.L.).
文摘Jasmonic acid(JA)is a crucial phytohormone that regulates plant immunity.The endogenous JA level is determined by the rates of its biosynthesis and catabolism in plants.The activation of JA biosynthesis has been well documented;however,how plants repress JA catabolism upon pathogen infection remains elusive.In this study,we identified and characterized Botrytis cinerea-induced F-box protein 1(BFP1)in Arabidopsis.The expression of BFP1 was induced by B.cinerea in a JA signaling-dependent manner,and BFP1 protein was critical for plant defense against B.cinerea and plant response to JA.In addition,BFP1 overexpression increased plant defenses against broad-spectrum pathogens without fitness costs.Further experiments demonstrated that BFP1 interacts with and mediates the ubiquitination and degrada-tion of jasmonic acid oxidases(JAOs,also known as jasmonate-induced oxygenases,JoXs),the enzymes that hydroxylate JA to 12OH-JA.Consistent with this,BFP1 affects the accumulation of JA and 12OH-JA during B.cinerea infection.Moreover,mutation of JAo2 complemented the phenotypes of the bfp1 mutant.Collectively,our results unveil a new mechanism used by plants to activate immune responses upon path-ogeninfection:suppressing JA catabolism.
基金the National Natural Science Foundation of China(81802996,81871946,and 82072708)Special Foundation for National Science and Technology Basic Research Program of China(2019FY101104)+2 种基金the Program for Development of Innovative Research Team in the First Affiliated Hospital of NJMUthe Priority Academic ProgramDevelopment of JiangsuHigher Education Institutions(JX10231801)Jiangsu Key Medical Discipline(General Surgery)(ZDXKA2016005),Jiangsu Key Lab of Cancer Biomarkers,Prevention and Treatment,Collaborative Innovation Center for Cancer Personalized Medicine,Nanjing Medical University.
文摘Background:Previous studies have revealed the critical role of transglutaminase 2(TGM2)as a potential therapeutic target in cancers,but the oncogenic roles and underlying mechanisms of TGM2 in gastric cancer(GC)are not fully understood.In this study,we examined the role and potential mechanism of TGM2 in GC.Methods:Western blotting,immunohistochemistry,CCK8,colony formation and transwell assays were used to measure TGM2 expression in the GC cells and tissues and to examine the in vitro role of TGM2 in GC.Xenograft and in vivo metastasis experiments were performed to examine the in vivo role of TGM2 in GC.Gene set enrichment analysis,quantitative PCR and western blotting were conducted to screen for potential TGM2 targets involved in GC.Gain/loss-offunction and rescue experiments were conducted to detect the biological roles of STAT1 in GC cells in the context of TGM2.Co-immunoprecipitation,mass spectrometry,quantitative PCR and western blotting were conducted to identify STAT1-interacting proteins and elucidate their regulatory mechanisms.Mutations in TGM2 and two molecules(ZM39923 and A23187)were used to identify the enzymatic activity of TGM2 involved in the malignant progression of GC and elucidate the underlying mechanism.Results:In this study,we demonstrated elevated TGM2 expression in the GC tissues,which closely related to pathological grade,and predicted poor survival in patients with GC.TGM2 overexpression or knockdown promoted(and inhibited)cell proliferation,migration,and invasion,which were reversed by STAT1 knockdown or overexpression.Further studies showed that TGM2 promoted GC progression by inhibiting STAT1 ubiquitination/degradation.Then,tripartite motif-containing protein 21(TRIM21)was identified as a ubiquitin E3 ligase of STAT1 in GC.TGM2 maintained STAT1 stability by facilitating the dissociation of TRIM21 and STAT1 with GTP-binding enzymatic activity.A23187 abolished the role of TGM2 in STAT1 and reversed the pro-tumor role of TGM2 in vitro and in vivo.Conclusions:This study revealed a criti
基金the National Key R&D Program of China(2019YFA0904700)the National Natural Science Foundation of China(32200207 and 32090063)+1 种基金the China Postdoctoral Science Foundation(2019M662652)a Self-lnnovation grant from National Laboratory(AML2023B01).
文摘The precise control of receptor levels is crucial for initiating cellular signaling transduction in response to specific ligands;however,such mechanisms regulating nodulation factor(NF)receptor(NFR)-mediated perception of NFs to establish symbiosis remain unclear.In this study,we unveil the pivotal role of the NFR-interacting RING-type E3 ligase 1(NIRE1)in regulating NFR1/NFR5 homeostasis to optimize rhizobial infection and nodule development in Lotus japonicus.We demonstrated that NiRE1 has a dual function in this regulatory process.It associates with both NFR1 and NFR5,facilitating their degradation through K48-linked polyubiquitination before rhizobial inoculation.However,following rhizobial inoculation,NFR1 phosphorylates NIRE1ata conserved residue,Tyr-109,inducing a functional switch in NIRE1,which enables NIRE1tomediateK63-linkedpolyubiquitination,thereby stabilizing NFR1/NFR5 in infected root cells.The introduction of phospho-dead NIRE1Y1osF leads to delayed nodule development,underscoring the significance of phosphorylation at Tyr-1o9 in orchestrating symbiotic processes.Conversely,expression of the phospho-mimic NIRE1Y0E results in the formation of spontaneous nodules in L.japonicus,further emphasizing the critical role of the phosphorylation-dependent functional switch in NiRE1.In summary,these findings uncover a fine-tuned symbiotic mechanism that a single E3 ligase could undergo a phosphorylationdependent functional switch to dynamically and precisely regulate NF receptor protein levels.
文摘目的探究脊髓性肌萎缩症(spinal muscular atrophy,SMA)中p53积累的分子机制。方法构建运动神经元生存基因(Survival motor neuron,Smn)敲低的稳转小鼠运动神经元细胞系NSC34,采用蛋白质印迹和荧光定量PCR检测蛋白和基因的变化;用蛋白合成抑制剂放线菌酮(Cycloheximide,CHX)抑制蛋白合成,蛋白质印迹检测p53蛋白降解情况,采用免疫共沉淀检测p53与MDM2结合情况;采用免疫荧光检测小鼠脊髓组织运动神经元数量,运用流式细胞仪分析细胞周期的分布。结果在Smn敲低的NSC34细胞中,p53蛋白水平显著增加,但是其基因水平未发生明显变化;当蛋白合成被抑制后,SMN缺失会抑制p53蛋白降解,p53积累后会调控下游MDM2表达显著增加,但是其与p53结合却显著减少;p⁃p53(Ser18)和acetyl⁃p53(K382)水平显著升高,K382乙酰化位点会影响p53泛素化结合,导致其泛素化被抑制;最终p53积累导致NSC34细胞和SMA小鼠脊髓组织中细胞周期阻滞和凋亡增加。结论SMN蛋白缺失通过抑制p53泛素化降解途径导致其积累,进而引起细胞周期阻滞和凋亡。
文摘Kelch样ECH关联蛋白1(Kelch-like ECH-associated protein 1,Keap1)是E3泛素连接酶的底物识别亚单位,在蛋白质的泛素化修饰中起重要作用。蛋白质的泛素化修饰作为一种重要且复杂的蛋白质翻译后修饰,在自噬和蛋白酶体系统中作为降解信号而被利用。野生型Keap1能够识别、结合多种底物蛋白质并通过Keap1-Cul3-Rbx1复合物泛素化。此外,Keap1还作为一种抑癌蛋白而被广泛研究,已发现诸多Keap1的体细胞突变或等位基因的异常缺失诱发人类多种疾病。当前的研究主要围绕在Keap1-Nrf2系统而很少涉及到其他下游底物。鉴于Keap1在细胞中的重要功能及广大的发展空间,这篇综述将对Keap1目前的研究现状进行总结。包括:泛素-蛋白酶体系统(ubiquitin-proteasome system,UPS),Keap1的结构、功能、突变,Keap1的泛素化底物及Keap1的相关疾病,探讨Keap1在疾病中的临床意义及研究中存在的机遇与挑战。
基金This work has been supported by grants fromthe Austrian Science Fund(FWF P30850,V382 Richter-Programm to B.kFWF P31493 to C.L)by a Doc fellowship from the Austrian Academy of Sciences to L.D.-A.
文摘Protein abundance and localization at the plasma membrane(PM)shapes plant development and mediates adaptation to changing environmental conditions.It is regulated by ubiquitination,a post-translational modification crucial for the proper sorting of endocytosed PM proteins to the vacuole for subsequent degradation.To understand the significance and the variety of roles played by this reversible modification,the function of ubiquitin receptors,which translate the ubiquitin signature into a cellular response,needs to be elucidated.In this study,we show that TOL(TOM1-like)proteins function in plants as multivalent ubiquitin receptors,governing ubiquitinated cargo delivery to the vacuole via the conserved Endosomal Sorting Complex Required for Transport(ESCRT)pathway.TOL2 and TOL6 interact with components of the ESCRT machinery and bind to K63-linked ubiquitin via two tandemly arranged conserved ubiquitin-binding domains.Mutation of these domains results not only in a loss of ubiquitin binding but also altered localization,abolishing TOL6 ubiquitin receptor activity.Function and localization of TOL6 is itself regulated by ubiquitination,whereby TOL6 ubiquitination potentially modulates degradation of PM-localized cargoes,assisting in the fine-tuning of the delicate interplay between protein recycling and downregulation.Taken together,our findings demonstrate the function and regulation of a ubiquitin receptor that mediates vacuolar degradation of PM proteins in higher plants.
基金supported by the National Natural Science Foundation of China (No. 21907006 to Yuxin Ye)the Natural Science Foundation of Guangdong Province (No. 2020A1515011544to Yuxin Ye)the Shenzhen Science and Technology Innovation Committee (No. JCYJ20180302150357309 to Yuxin Ye, and No.JCYJ20200109140401752 to Hao Huang)。
文摘UBE2C(Ubiquitin conjugating enzyme E2 C), a key regulator of cell cycle progression, is a promising target for discovery of antitumor agents. However, it is challenging to develop inhibitors of UBE2C owing to its lack of “druggable” pockets. Bio PROTACs(biological proteolysis targeting chimeras) are a kind of protein-based degraders by fusing an adaptor to a subunit of E3 ligase for ubiquitination and subsequent proteasome-dependent degradation of target protein. We report herein the design and biological evaluation of a UBE2C-targeting bio PROTAC based on the NEL(novel E3 ligase) domain of bacterial E3 ligase Ipa H9.8 and the UBE2C-binding WHB(winged-helix B) domain of APC_(2)(anaphase promoting complex subunit 2). The in vitro ubiquitination test and Mass Spectrometry analysis showed that the bio PROTAC could transfer ubiquitin to surface exposed lysines on UBE2C and catalyzed the formation of polyubiquitin chains. In addition, the transient co-expression experiment showed that the bio PROTAC could promote proteasomal degradation of heterologous UBE2C and rescue its downstream substrates in mammalian cells.
基金National Natural Science Foundation of China,Grant/Award Number:82130084Shaanxi Provincial Key R&D Program,Grant/Award Number:2021SF-110。
文摘Background The mechanism of hepatitis B virus(HBV)-induced carcinogenesis remains an area of interest.The accumulation of hepatitis B surface antigen in the endoplasmic reticulum(ER)of hepatocytes stimulates persistent ER stress.Activity of the unfolded protein response(UPR)pathway of ER stress may play an important role in inflammatory cancer transformation.How the protective UPR pathway is hijacked by cells as a tool for malignant transformation in HBV-related hepatocellular carcinoma(HCC)is still unclear.Here,we aimed to define the key molecule hyaluronan-mediated motility receptor(HMMR)in this process and explore its role under ER stress in HCC development.Methods An HBV-transgenic mouse model was used to characterize the pathological changes during the tumor progression.Proteomics and transcriptomics analyses were performed to identify the potential key molecule,screen the E3 ligase,and define the activation pathway.Quantitative real-time PCR and Western blotting were conducted to detect the expression of genes in tissues and cell lines.Luciferase reporter assay,chromatin immunoprecipitation,coimmunoprecipitation,immunoprecipitation,and immunofluorescence were employed to investigate the molecular mechanisms of HMMR under ER stress.Immunohistochemistry was used to clarify the expression patterns of HMMR and related molecules in human tissues.Results We found sustained activation of ER stress in the HBV-transgenic mouse model of hepatitis-fibrosis-HCC.HMMR was transcribed by c/EBP homologous protein(CHOP)and degraded by tripartite motif containing 29(TRIM29)after ubiquitination under ER stress,which caused the inconsistent expression of mRNA and protein.Dynamic expression of TRIM29 in the HCC progression regulated the dynamic expression of HMMR.HMMR could alleviate ER stress by increasing autophagic lysosome activity.The negative correlation between HMMR and ER stress,positive correlation between HMMR and autophagy,and negative correlation between ER stress and autophagy were verified in human tissues.Conclus
基金This work was supported by grants from the National Natural Science Foundation of China(grant 81788101 to X.C.grant 81871236 to M.J.)the National 135 Mega Program of China(grant 2017ZX10202203-002 to M.J.grant 2017ZX10203206-001 to X.C.)+1 种基金the CAMS Innovation Fund for Medical Sciences(grant 2016-12M-1-003 to X.C.)the Medical Epigenetics Research Center,Chinese Academy of Medical Sciences(2018PT31015).
文摘Upon recognition of dsRNA,toll-like receptor 3(TLR3)recruits the adaptor protein TRIF to activate IRF3 and NF-κB signaling,initiating innate immune responses.The ubiquitination of TLR3 downstream signaling molecules and their roles in the innate response have been discovered;however,whether TLR3 itself is ubiquitinated and then functionally involved remains to be elucidated.By immunoprecipitating TLR3-binding proteins in macrophages,we identified ring finger protein 170(RNF170)as a TLR3-binding E3 ligase.RNF170 mediated the K48-linked polyubiquitination of K766 in the TIR domain of TLR3 and promoted the degradation of TLR3 through the proteasome pathway.The genetic ablation of RNF170 selectively augmented TLR3-triggered innate immune responses both in vitro and in vivo.Our results reveal a novel role for RNF170 in selectively inhibiting TLR3-triggered innate immune responses by promoting TLR3 degradation.
基金supported by the National Institutes of Health National Institute of General Medical Sciences[Grant R35GM118041]supported in part by the National Cancer Institute of the National Institutes of Health under Award Number P30 CA021765。
文摘Pregnane X receptor(PXR)is a ligand-activated nuclear receptor that transcriptionally upregulates drug-metabolizing enzymes[e.g.,cytochrome P4503A4(CYP3A4)]and transporters.Although the regulation of PXR target genes is well-characterized,less is known about the regulation of PXR protein level.By screening an RNAi library,we identified the F-box-only protein 44(FBXO44)as a novel E3ligase for PXR.PXR abundance increases upon knockdown of FBXO44,and,inversely,decreases upon overexpression of FBXO44.Further analysis revealed that FBXO44 interacts with PXR,leading to its ubiquitination and proteasomal degradation,and we determined that the F-box associated domain of FBXO44 and the ligand binding domain of PXR are required for the functional interaction.In summary,FBXO44 regulates PXR protein abundance,which has downstream consequences for CYP3A4 levels and drug-drug interactions.The results of this study provide new insight into the molecular mechanisms that regulate PXR protein level and activity and suggest the importance of considering how modulating E3ubiquitin ligase activities will affect PXR-mediated drug metabolism.
文摘CDK4 is crucial for Gl-to-S transition of cell cycle. It is well established that ubiquitin-mediated degradations of CDK inhibitors and cycUns are pivotal for the timely and unidirectional progression of cell cycle. However, how CDK4 itself is modulated by ubiquitin-mediated degradation has been elusive. Here we report that the steady-state level of CDK4 is controlled by PAQR4, a member of the progestin and adipoQ receptor family, and SKP2, an E3 ubiquitin ligase. Knockdown of PAQR4 leads to reduction of cell proliferation, accompanied by reduced protein level of CDK4. PAQR4 reduces polyubiquitination and degradation of CDK4. PAQR4 interacts with the C-terminal lobe of CDK4. On the other hand, SKP2 also interacts with the C-terminal lobe of CDK4 and enhances polyubiquitination and degradation of CDK4. importantly, PAQR4 and SKP2 bind to the same region in CDK4, and PAQR4 competes with SKP2 for the binding, thereby abrogating SKP2-mediated ubiquitination of CDK4. Using a two-stage DMBA/TPA-induced skin cancer model, we find that PAQR4-deleted mice are resistant to chemical carcinogen-induced tumor formation. Collectively, our findings reveal that the steady-state level of CDK4 is controlled by the antagonistic actions between PAQR4 and SKP2, contributing to modulation of cell proliferation and tumorigenesis.
