BACKGROUND DNA damage is one of the critical contributors to the occurrence and development of some cancers.APEX1 and APEX2 are the most important molecules in the DNA damage,and APEX1 has been identified as a diagnos...BACKGROUND DNA damage is one of the critical contributors to the occurrence and development of some cancers.APEX1 and APEX2 are the most important molecules in the DNA damage,and APEX1 has been identified as a diagnostic and prognostic biomarker in liver hepatocellular carcinoma(LIHC).However,the expression of APEX2 and its functional mechanisms in LIHC are still unclear.AIM To examine the expression of APEX2 and the potential mechanism network in LIHC.METHODS We conducted a pan-cancer analysis of the expression of APEX1 and APEX2 using the interactive TIMER tool.GEO datasets,including GSE14520,GSE22058,and GSE64041,were used to compare the APEX2 expression level in tumor tissues and adjacent non-tumor tissues.Then,we calculated the 5-year survival rate according to the web-based Kaplan-Meier analysis.We included the TCGA liver cancer database in GSEA analysis based on the high and low APEX2 expression,showing the potential mechanisms of APEX2 in LIHC.After that,we conducted Pearson correlation analysis using GEPIA2.Next,we performed quantitative polymerase chain reaction(qPCR)assay to examine the APEX2 levels in normal liver cell line LO2 and several liver cancer cell lines,including HepG2,Huh7,SMMC7721,and HCCLM3.APEX2 in HCCLM3 cells was knocked down using small interfering RNA.The role of APEX2 in cell viability was confirmed using CCK-8.Dualluciferase reporter assay was performed to examine the promoter activity of CCNB1 and MYC.RESULTS APEX1 and APEX2 are both highly expressed in the tumor tissues of BLCA,BRCA,CHOL,COAD,ESCA,HNSC,LIHC,LUAD,LUSC,READ,and STAD.APEX2 overexpression in LIHC was validated using GSE14520,GSE22058,and GSE64041 datasets.The survival analysis showed that LIHC patients with high expression of APEX2 had a lower overall survival rate,even in the AJCC T1 patients.High level of APEX2 could indicate a lower overall survival rate in patients with or without viral hepatitis.The GSEA analysis identified that kinetochore and spindle microtubules are the two main cellular components of APEX2 in GO Ontology.APEX2 was also positively associated with molecular function regulation of chromosome segregation and DNA replication.The results of KEGG analysis indicated that APEX2 expression was positively correlated with cell cycle pathway and pro-oncogenic MYC signaling.Pearson correlation analysis showed that APEX2 had a significant positive correlation with CCNB1 and MYC.APEX2 level was higher in liver cancer cell lines than in normal liver LO2 cells.Small interfering RNA could knock down the APEX2 expression in HCCLM3 cells.Knockdown of APEX2 resulted in a decrease in the viability of HCCLM3 cells as well as the expression and promoter activity of CCNB1 and MYC.CONCLUSION APEX2 is overexpressed in LIHC,and the higher APEX2 level is associated with a worse prognosis in overall survival.APEX2 is closely involved in the biological processes of chromosome segregation and DNA replication.APEX2 expression is positively correlated with the pro-oncogenic pathways.Knockdown of APEX2 could inhibit the cell viability and CCNB1 and MYC pathways,suggesting that APEX2 is an oncogene in LIHC,which could be a potential pharmaceutic target in the anti-tumor therapy.展开更多
The first part of this report describes the data reduction of non-merohedrally twinned crystals measured on Bruker and Agilent area-detector diffractometers. The image frames of methyl-2-aminopyrazine-3-carboxylate we...The first part of this report describes the data reduction of non-merohedrally twinned crystals measured on Bruker and Agilent area-detector diffractometers. The image frames of methyl-2-aminopyrazine-3-carboxylate were processed with APEX2 to furnish a set of overlapping diffraction indices that were used for solution and refinement. CrysAlisPRO was used for processing the frames of bis（diethyldicarbamato）nickel, which exists in monoclinic and tetragonal polymorphs, and in untwinned and twinned forms. In the second part, the crystal structure of [（3-formyl-4- hydroxyphenyl）methyl]triphenylphosphanium chloride was refined through the ‘HKLF 5＇（based on a combined set of diffraction indices） and PLATON（based on one set of diffraction indices） routes to give identical outcomes because the amount of overlap of the twin domains is small. For the third part, in a proof-of-concept investigation, the diffraction pattern of untwinned and twinned 4-{（E）-（4-aminophenyl）diazenyl]phenylamine was recorded simultaneously in one run; the three domains could be indexed and the crystal structure satisfactorily refined. The refinement was identical to those derived from independent measurements; the crystal structure features two independent centrosymmetric molecules, one of which is ordered and the other whole-molecule-disordered. This two-in-one run opens up the possibility that two or more crystals having different atomic compositions can be measured simultaneously if their reciprocal lattices do not overlap significantly.展开更多
The innate immune sensor NLRP3 inflammasome overactivation is involved in the pathogenesis of ulcerative colitis.PGAM5 is a mitochondrial phosphatase involved in NLRP3 inflammasome activation in macrophages.However,th...The innate immune sensor NLRP3 inflammasome overactivation is involved in the pathogenesis of ulcerative colitis.PGAM5 is a mitochondrial phosphatase involved in NLRP3 inflammasome activation in macrophages.However,the role of PGAM5 in ulcerative colitis and the mechanisms underlying PGAM5 regulating NLRP3 activity remain unknown.Here,we show that PGAM5 deficiency ameliorates dextran sodium sulfate(DSS)-induced colitis in mice via suppressing NLRP3 inflammasome activation.By combining APEX2-based proximity labeling focused on PGAM5 with quantitative proteomics,we identify NEK7 as the new binding partner of PGAM5 to promote NLRP3 inflammasome assembly and activation in a PGAM5 phosphatase activity-independent manner upon inflammasome induction.Interfering with PGAM5eNEK7 interaction by punicalagin inhibits the activation of the NLRP3 inflammasome in macrophages and ameliorates DSS-induced colitis in mice.Altogether,our data demonstrate the PGAM5eNEK7 interaction in macrophages for NLRP3 inflammasome activation and further provide a promising therapeutic strategy for ulcerative colitis by blocking the PGAM5eNEK7 interaction.展开更多
Chronic hepatitis B virus(HBV)infection is a leading cause of liver cirrhosis and he-patocellular carcinoma,representing a global health problem for which a functional cure is difficult to achieve.The HBV core protein...Chronic hepatitis B virus(HBV)infection is a leading cause of liver cirrhosis and he-patocellular carcinoma,representing a global health problem for which a functional cure is difficult to achieve.The HBV core protein(HBc)is essential for multiple steps in the viral life cycle.It is the building block of the nucleocapsid in which viral DNA reverse transcription oc-curs,and its mediation role in viral-host cell interactions is critical to HBV infection persis-tence.However,systematic studies targeting HBc-interacting proteins remain lacking.Here,we combined HBc with the APEX2 to systematically identify HBc-related host proteins in living cells.Using functional screening,we confirmed that proteasome activator subunit 1(PSME1)is a potent HBV-associated host factor.PSME1 expression was up-regulated upon HBV infection,and the protein level of HBc decreased after PSME1 knockdown.Mechanistically,the interac-tion between PSME1 and HBc inhibited the degradation of HBc by the 26S proteasome,thereby improving the stability of the HBc protein.Furthermore,PSME1 silencing inhibits HBV tran-scription in the HBV infection system.Our findings reveal an important mechanism by which PSME1 regulates HBc proteins and may facilitate the development of new antiviral therapies targeting PSME1 function.展开更多
As a common feature of tumors,chromosomal instability(CIN)not only forces carcinomatous evolution,but also loads cancer cells with extra pressure through a robust imbalance of genome patterning that may be used for ca...As a common feature of tumors,chromosomal instability(CIN)not only forces carcinomatous evolution,but also loads cancer cells with extra pressure through a robust imbalance of genome patterning that may be used for cancer treatment.Errors in cytokinesis increase CIN,so cytokinesis components are valuable targets for treating cancer.However,due to the short time span and confined space of cytokinesis bridges,profiling cytokinesis fac-tors is challenging.Taking advantage of engineered ascorbate peroxidase(APEX2),we established a cytokinesis bridge-APEX reaction in living cells.A total of 218 cytokinesis bridge proteins were identified with high relia-bility.Knockdown of cytokinesis bridge genes generated micronuclei that activate the cGAS-pathway and cause apoptosis in cancer cells bearing high CIN rather than low CIN.Thus,our study proposes a strategy for killing high-CIN tumors regardless of tumor type,and provides a proteome resource of cytokinetic bridges for future research.展开更多
Objectives:Resistance to apoptosis in esophageal squamous cell carcinoma(ESCC)constitutes a significant impediment to treatment efficacy.Exploring alternative cell death pathways and their regulatory factors beyond ap...Objectives:Resistance to apoptosis in esophageal squamous cell carcinoma(ESCC)constitutes a significant impediment to treatment efficacy.Exploring alternative cell death pathways and their regulatory factors beyond apoptosis is crucial for overcoming drug resistance and enhancing therapeutic outcomes in ESCC.Methods:Mammalian Ste 20-like kinase 1(MST1)is implicated in regulating various cell deaths,including apoptosis,autophagy,and pyroptosis.Employing enhanced ascorbate peroxidase 2(APEX2)proximity labeling coupled with immunoprecipitation-mass spectrometry(IP-MS),we elucidated the interactomes of MST1 across these three cell death paradigms.Results:Proteomic profiling unveiled the functional roles and subcellular localization of MST1 and its interacting proteins during normal proliferation and various cell death processes.Notably,MST1 exhibited an expanded interactome during cell death compared to normal proliferation and chromosome remodeling functions consistently.In apoptosis,there was a notable increase of mitosis-associated proteins such as INCENP,ANLN,KIF23,SHCBP1 and SUPT16H,which interacted with MST1,alongside decreased expression of the pre-apoptotic protein STK3.During autophagy,the bindings of DNA repair-related proteins CBX8 and m6A reader YTHDC1 to MST1 were enhanced.In pyroptosis,LRRFIP2 and FLII which can inhibit pyroptosis increasingly binding to MST1.Conclusions:Our findings delineate potential mechanisms through which MST1 and its interactomes regulate cell death,paving the way for further investigation to validate and consolidate these observations.展开更多
基金Wenzhou Science and Technology Bureau,No.Y20180147.
文摘BACKGROUND DNA damage is one of the critical contributors to the occurrence and development of some cancers.APEX1 and APEX2 are the most important molecules in the DNA damage,and APEX1 has been identified as a diagnostic and prognostic biomarker in liver hepatocellular carcinoma(LIHC).However,the expression of APEX2 and its functional mechanisms in LIHC are still unclear.AIM To examine the expression of APEX2 and the potential mechanism network in LIHC.METHODS We conducted a pan-cancer analysis of the expression of APEX1 and APEX2 using the interactive TIMER tool.GEO datasets,including GSE14520,GSE22058,and GSE64041,were used to compare the APEX2 expression level in tumor tissues and adjacent non-tumor tissues.Then,we calculated the 5-year survival rate according to the web-based Kaplan-Meier analysis.We included the TCGA liver cancer database in GSEA analysis based on the high and low APEX2 expression,showing the potential mechanisms of APEX2 in LIHC.After that,we conducted Pearson correlation analysis using GEPIA2.Next,we performed quantitative polymerase chain reaction(qPCR)assay to examine the APEX2 levels in normal liver cell line LO2 and several liver cancer cell lines,including HepG2,Huh7,SMMC7721,and HCCLM3.APEX2 in HCCLM3 cells was knocked down using small interfering RNA.The role of APEX2 in cell viability was confirmed using CCK-8.Dualluciferase reporter assay was performed to examine the promoter activity of CCNB1 and MYC.RESULTS APEX1 and APEX2 are both highly expressed in the tumor tissues of BLCA,BRCA,CHOL,COAD,ESCA,HNSC,LIHC,LUAD,LUSC,READ,and STAD.APEX2 overexpression in LIHC was validated using GSE14520,GSE22058,and GSE64041 datasets.The survival analysis showed that LIHC patients with high expression of APEX2 had a lower overall survival rate,even in the AJCC T1 patients.High level of APEX2 could indicate a lower overall survival rate in patients with or without viral hepatitis.The GSEA analysis identified that kinetochore and spindle microtubules are the two main cellular components of APEX2 in GO Ontology.APEX2 was also positively associated with molecular function regulation of chromosome segregation and DNA replication.The results of KEGG analysis indicated that APEX2 expression was positively correlated with cell cycle pathway and pro-oncogenic MYC signaling.Pearson correlation analysis showed that APEX2 had a significant positive correlation with CCNB1 and MYC.APEX2 level was higher in liver cancer cell lines than in normal liver LO2 cells.Small interfering RNA could knock down the APEX2 expression in HCCLM3 cells.Knockdown of APEX2 resulted in a decrease in the viability of HCCLM3 cells as well as the expression and promoter activity of CCNB1 and MYC.CONCLUSION APEX2 is overexpressed in LIHC,and the higher APEX2 level is associated with a worse prognosis in overall survival.APEX2 is closely involved in the biological processes of chromosome segregation and DNA replication.APEX2 expression is positively correlated with the pro-oncogenic pathways.Knockdown of APEX2 could inhibit the cell viability and CCNB1 and MYC pathways,suggesting that APEX2 is an oncogene in LIHC,which could be a potential pharmaceutic target in the anti-tumor therapy.
文摘The first part of this report describes the data reduction of non-merohedrally twinned crystals measured on Bruker and Agilent area-detector diffractometers. The image frames of methyl-2-aminopyrazine-3-carboxylate were processed with APEX2 to furnish a set of overlapping diffraction indices that were used for solution and refinement. CrysAlisPRO was used for processing the frames of bis（diethyldicarbamato）nickel, which exists in monoclinic and tetragonal polymorphs, and in untwinned and twinned forms. In the second part, the crystal structure of [（3-formyl-4- hydroxyphenyl）methyl]triphenylphosphanium chloride was refined through the ‘HKLF 5＇（based on a combined set of diffraction indices） and PLATON（based on one set of diffraction indices） routes to give identical outcomes because the amount of overlap of the twin domains is small. For the third part, in a proof-of-concept investigation, the diffraction pattern of untwinned and twinned 4-{（E）-（4-aminophenyl）diazenyl]phenylamine was recorded simultaneously in one run; the three domains could be indexed and the crystal structure satisfactorily refined. The refinement was identical to those derived from independent measurements; the crystal structure features two independent centrosymmetric molecules, one of which is ordered and the other whole-molecule-disordered. This two-in-one run opens up the possibility that two or more crystals having different atomic compositions can be measured simultaneously if their reciprocal lattices do not overlap significantly.
基金supported by the National Natural Science Foundation of China(82174010 and 81973512)the Open Fund of the State Key Laboratory of Pharmaceutical Biotechnology,Nanjing University(Grant No.KF-202206,China)the Double First-Class Project of China Pharmaceutical University(CPUQNJC22_02,China)。
文摘The innate immune sensor NLRP3 inflammasome overactivation is involved in the pathogenesis of ulcerative colitis.PGAM5 is a mitochondrial phosphatase involved in NLRP3 inflammasome activation in macrophages.However,the role of PGAM5 in ulcerative colitis and the mechanisms underlying PGAM5 regulating NLRP3 activity remain unknown.Here,we show that PGAM5 deficiency ameliorates dextran sodium sulfate(DSS)-induced colitis in mice via suppressing NLRP3 inflammasome activation.By combining APEX2-based proximity labeling focused on PGAM5 with quantitative proteomics,we identify NEK7 as the new binding partner of PGAM5 to promote NLRP3 inflammasome assembly and activation in a PGAM5 phosphatase activity-independent manner upon inflammasome induction.Interfering with PGAM5eNEK7 interaction by punicalagin inhibits the activation of the NLRP3 inflammasome in macrophages and ameliorates DSS-induced colitis in mice.Altogether,our data demonstrate the PGAM5eNEK7 interaction in macrophages for NLRP3 inflammasome activation and further provide a promising therapeutic strategy for ulcerative colitis by blocking the PGAM5eNEK7 interaction.
基金supported by the National Key R&D Program of China (No.2022YFA1303600).
文摘Chronic hepatitis B virus(HBV)infection is a leading cause of liver cirrhosis and he-patocellular carcinoma,representing a global health problem for which a functional cure is difficult to achieve.The HBV core protein(HBc)is essential for multiple steps in the viral life cycle.It is the building block of the nucleocapsid in which viral DNA reverse transcription oc-curs,and its mediation role in viral-host cell interactions is critical to HBV infection persis-tence.However,systematic studies targeting HBc-interacting proteins remain lacking.Here,we combined HBc with the APEX2 to systematically identify HBc-related host proteins in living cells.Using functional screening,we confirmed that proteasome activator subunit 1(PSME1)is a potent HBV-associated host factor.PSME1 expression was up-regulated upon HBV infection,and the protein level of HBc decreased after PSME1 knockdown.Mechanistically,the interac-tion between PSME1 and HBc inhibited the degradation of HBc by the 26S proteasome,thereby improving the stability of the HBc protein.Furthermore,PSME1 silencing inhibits HBV tran-scription in the HBV infection system.Our findings reveal an important mechanism by which PSME1 regulates HBc proteins and may facilitate the development of new antiviral therapies targeting PSME1 function.
基金supported by the National Natural Science Foundation of China(NSFC)(Grants No.81672610,81521002,81871160)to ML,and by the“Clinic+X”program(to ML)of Peking University.
文摘As a common feature of tumors,chromosomal instability(CIN)not only forces carcinomatous evolution,but also loads cancer cells with extra pressure through a robust imbalance of genome patterning that may be used for cancer treatment.Errors in cytokinesis increase CIN,so cytokinesis components are valuable targets for treating cancer.However,due to the short time span and confined space of cytokinesis bridges,profiling cytokinesis fac-tors is challenging.Taking advantage of engineered ascorbate peroxidase(APEX2),we established a cytokinesis bridge-APEX reaction in living cells.A total of 218 cytokinesis bridge proteins were identified with high relia-bility.Knockdown of cytokinesis bridge genes generated micronuclei that activate the cGAS-pathway and cause apoptosis in cancer cells bearing high CIN rather than low CIN.Thus,our study proposes a strategy for killing high-CIN tumors regardless of tumor type,and provides a proteome resource of cytokinetic bridges for future research.
基金supported by the funds of Guangdong Basic and Applied Basic Research Foundation(2019B030302012)the National Key R&D Program of China(2021YFC2501001,2022YFC3401002)+3 种基金Shenzhen Medical Research Funds(C2303002)the National Natural Science Foundation of China(82341024,82172930,82203286)the Major Program of Shenzhen Bay Laboratory(S201101004)China Postdoctoral Science Foundation(BX20220214).
文摘Objectives:Resistance to apoptosis in esophageal squamous cell carcinoma(ESCC)constitutes a significant impediment to treatment efficacy.Exploring alternative cell death pathways and their regulatory factors beyond apoptosis is crucial for overcoming drug resistance and enhancing therapeutic outcomes in ESCC.Methods:Mammalian Ste 20-like kinase 1(MST1)is implicated in regulating various cell deaths,including apoptosis,autophagy,and pyroptosis.Employing enhanced ascorbate peroxidase 2(APEX2)proximity labeling coupled with immunoprecipitation-mass spectrometry(IP-MS),we elucidated the interactomes of MST1 across these three cell death paradigms.Results:Proteomic profiling unveiled the functional roles and subcellular localization of MST1 and its interacting proteins during normal proliferation and various cell death processes.Notably,MST1 exhibited an expanded interactome during cell death compared to normal proliferation and chromosome remodeling functions consistently.In apoptosis,there was a notable increase of mitosis-associated proteins such as INCENP,ANLN,KIF23,SHCBP1 and SUPT16H,which interacted with MST1,alongside decreased expression of the pre-apoptotic protein STK3.During autophagy,the bindings of DNA repair-related proteins CBX8 and m6A reader YTHDC1 to MST1 were enhanced.In pyroptosis,LRRFIP2 and FLII which can inhibit pyroptosis increasingly binding to MST1.Conclusions:Our findings delineate potential mechanisms through which MST1 and its interactomes regulate cell death,paving the way for further investigation to validate and consolidate these observations.
