The coronavirus disease 2019(COVID-19)pandemic has led to worldwide efforts to understand the biological traits of the newly identified human coronavirus(HCoV-19)virus.In this mass spectrometry(MS)-based study,we reve...The coronavirus disease 2019(COVID-19)pandemic has led to worldwide efforts to understand the biological traits of the newly identified human coronavirus(HCoV-19)virus.In this mass spectrometry(MS)-based study,we reveal that out of 21 possible glycosites in the HCoV-19 spike protein(S protein),20 are completely occupied by N-glycans,predominantly of the oligomannose type.All seven glycosylation sites in human angiotensin I converting enzyme 2(hACE2)were found to be completely occupied,mainly by complex N-glycans.However,glycosylation did not directly contribute to the binding affinity between HCoV-19 S protein and hACE2.Additional post-translational modification(PTM)was identified,including multiple methylated sites in both proteins and multiple sites with hydroxylproline in hACE2.Refined structural models of HCoV-19 S protein and hACE2 were built by adding N-glycan and PTMs to recently published cryogenic electron microscopy structures.The PTM and glycan maps of HCoV-19 S protein and hACE2 provide additional structural details for studying the mechanisms underlying host attachment and the immune response of HCoV-19,as well as knowledge for developing desperately needed remedies and vaccines.展开更多
Many important crops(e.g.,tuber,root,and tree crops)are cross-pollinating.For these crops,no inbred lines are available for genetic study and breeding because they are self-incompatible,clonally propagated,or have a l...Many important crops(e.g.,tuber,root,and tree crops)are cross-pollinating.For these crops,no inbred lines are available for genetic study and breeding because they are self-incompatible,clonally propagated,or have a long generation time,making the identification of agronomically important genes difficult,particularly in crops with a complex autopolyploid genome.In this study,we developed a method,OutcrossSeq,for mapping agronomically important loci in outcrossing crops based on whole-genome low-coverage resequencing of a large genetic population,and designed three computation algorithms in OutcrossSeq for different types of outcrossing populations.We applied OutcrossSeq to a tuberous root crop(sweet potato,autopolyploid),a tree crop(walnut tree,highly heterozygous diploid),and hybrid crops(double-cross populations)to generate high-density genotype maps for the outcrossing populations,which enable precise identification of genomic loci underlying important agronomic traits.Candidate causative genes at these loci were detected based on functional clues.Taken together,our results indicate that OutcrossSeq is a robust and powerful method for identifying agronomically important genes in heterozygous species,including polyploids,in a cost-efficient way.The OutcrossSeq software and its instruction manual are available for downloading at www.xhhuanglab.cn/tool/OutcrossSeq.html.展开更多
Background:Tumor metastasis is a major factor for poor prognosis of hepatocellular carcinoma(HCC),but the relationship between ubiquitination and metastasis need to be studiedmore systematically.We analyzed the ubiqui...Background:Tumor metastasis is a major factor for poor prognosis of hepatocellular carcinoma(HCC),but the relationship between ubiquitination and metastasis need to be studiedmore systematically.We analyzed the ubiquitinome of HCC in this study to have a more comprehensive insight into human HCC metastasis.Methods:The protein ubiquitination levels in 15 HCC specimens with vascular invasion and 15 without vascular invasion were detected by ubiquitinome.Proteins with significantly different ubiquitination levels between HCCs with and without vascular invasion were used to predict E3 ubiquitin ligases associated with tumor metastasis.The topological network of protein substrates and corresponding E3 ubiquitin ligaseswas constructed to identify the key E3 ubiquitin ligase.Besides,the growth,migration and invasion ability of LM3 and HUH7 hepatoma cell lines with andwithout SYVN1 expression interferencewere measured by cell proliferation assay,subcutaneous tumor assay,umphal vein endothelium tube formation assay,transwell migration and invasion assays.Finally,the interacting proteins of SYVN1 were screened and verified by protein interaction omics,immunofluorescence,and immunoprecipitation.Ubiquitin levels of related protein substrates in LM3 and HUH7 cells were compared in negative control,SYVN1 knockdown,and SYVN1 overexpression groups.Results:In this study,our whole-cell proteomic dataset and ubiquitinomic dataset contained approximately 5600 proteins and 12,000 ubiquitinated sites.We discovered increased ubiquitinated sites with shorter ubiquitin chains during the progression ofHCC metastasis.In addition,proteomic and ubiquitinomic analyses revealed that high expression of E3 ubiquitin-protein ligase SYVN1 is related with tumor metastasis.Furthermore,we found that SYVN1 interacted with heat shock protein 90(HSP90)and impacted the ubiquitination of eukaryotic elongation factor 2 kinase(EEF2K).Conclusions:The ubiquitination profiles of HCC with and without vascular invasion were significantly different.SYVN1 was the most important E3 ubiquitin-protein ligase responsible for this phenomenon,and itwas related with tumormetastasis and growth.Therefore,SYVN1might be a potential therapeutic target for HCC.展开更多
Posttranslational modifications(PTMs)of proteins,particularly acetylation,phosphorylation,and ubiquitination,play critical roles in the host innate immune response.PTMs’dynamic changes and the crosstalk among them ar...Posttranslational modifications(PTMs)of proteins,particularly acetylation,phosphorylation,and ubiquitination,play critical roles in the host innate immune response.PTMs’dynamic changes and the crosstalk among them are complicated.To build a comprehensive dynamic network of inflammation-related proteins,we integrated data from the whole-cell proteome(WCP),acetylome,phosphoproteome,and ubiquitinome of human and mouse macrophages.Our datasets of acetylation,phosphorylation,and ubiquitination sites helped identify PTM crosstalk within and across proteins involved in the inflammatory response.Stimulation of macrophages by lipopolysaccharide(LPS)resulted in both degradative and non-degradative ubiquitination.Moreover,this study contributes to the interpretation of the roles of known inflammatory molecules and the discovery of novel inflammatory proteins.展开更多
The devastating coronavirus disease 2019(COVID-19)pandemic has prompted worldwide efforts to study structural biological traits of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)and its viral components.Co...The devastating coronavirus disease 2019(COVID-19)pandemic has prompted worldwide efforts to study structural biological traits of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)and its viral components.Compared to the Spike protein,which is the primary target for currently available vaccines or antibodies,knowledge about other virion structural components is incomplete.Using high-resolution mass spectrometry,we report a comprehensive post-translational modification(PTM)analysis of nucleocapsid phosphoprotein(NCP),the most abundant structural component of the SARS-CoV-2 virion.In addition to phosphoryl groups,we show that the SARS-CoV-2 NCP is decorated with a variety of PTMs,including N-glycans and ubiquitin.Based on newly identified PTMs,refined protein structural models of SARS-CoV-2 NCP were proposed and potential immune recognition epitopes of NCP were aligned with PTMs.These data can facilitate the design of novel vaccines or therapeutics targeting NCP,as valuable alternatives to the current vaccination and treatment paradigm that is under threat of the ever-mutating SARS-CoV-2 Spike protein.展开更多
Autoimmune hepatitis(AIH)is a severe globally distributed liver disease that could occur at any age.Human menstrual blood-derived stem cells(MenSCs)have shown therapeutic effect in acute lung injury and liver failure....Autoimmune hepatitis(AIH)is a severe globally distributed liver disease that could occur at any age.Human menstrual blood-derived stem cells(MenSCs)have shown therapeutic effect in acute lung injury and liver failure.However,their role in the curative effect of AIH remains unclear.Here,a classic AIH mouse model was constructed through intravenous injection with concanavalin A(Con A).MenSCs were intravenously injected while Con A injection in the treatment groups.The results showed that the mortality by Con A injection was significantly decreased by MenSCs treatment and liver function tests and histological analysis were also ameliorated.The results of phosphoproteomic analysis and RNA-seq revealed that MenSCs improved AIH,mainly by apoptosis and c-Jun N-terminal kinase/mitogen-activated protein signaling pathways.Apoptosis analysis demonstrated that the protein expression of cleaved caspase 3 was increased by Con A injection and reduced by MenSCs transplantation,consistent with the TUNEL staining results.An AML12 co-culture system and JNK inhibitor(SP600125)were used to verify the JNK/MAPK and apoptosis signaling pathways.These findings suggested that MenSCs could be a promising strategy for AIH.展开更多
基金This work was supported by the National Key Research and Development Program(2017YFC1200204,2017YFA0504803,and 2018YFA0507700)Emergency Project of Zhejiang Provincial Department of Science and Technology(2020C03123-1)+1 种基金Fundamental Research Funds for the Central Universities(2018XZZX001-13)Independent Project Fund of the State Key Laboratory for Diagnosis and Treatment of Infectious Disease.
文摘The coronavirus disease 2019(COVID-19)pandemic has led to worldwide efforts to understand the biological traits of the newly identified human coronavirus(HCoV-19)virus.In this mass spectrometry(MS)-based study,we reveal that out of 21 possible glycosites in the HCoV-19 spike protein(S protein),20 are completely occupied by N-glycans,predominantly of the oligomannose type.All seven glycosylation sites in human angiotensin I converting enzyme 2(hACE2)were found to be completely occupied,mainly by complex N-glycans.However,glycosylation did not directly contribute to the binding affinity between HCoV-19 S protein and hACE2.Additional post-translational modification(PTM)was identified,including multiple methylated sites in both proteins and multiple sites with hydroxylproline in hACE2.Refined structural models of HCoV-19 S protein and hACE2 were built by adding N-glycan and PTMs to recently published cryogenic electron microscopy structures.The PTM and glycan maps of HCoV-19 S protein and hACE2 provide additional structural details for studying the mechanisms underlying host attachment and the immune response of HCoV-19,as well as knowledge for developing desperately needed remedies and vaccines.
基金We are grateful to Prof.Lars M.Steinmetz for the Tn5 plasmid.This work was funded by the Ministry of Science and Technology of China(2016YFD0100902 to X.H.,2018YFD1000701 and 2019YFD1000703 to J.Y.)the National Natural Science Foundation of China(31825015 to X.H.)+4 种基金the Program of Shanghai Academic Research Leader(18XD1402900 to X.H.)the Innovation Program of the Shanghai Municipal Education Commission(2017-01-07-00-02-E00039 to X.H.)the Shanghai Municipal Afforestation&City Appearance and Environmental Sanitation Administration(G182402,G192413,G192414,and G202402 to J.Y.)the Youth Innovation Promotion Association CAS(to J.Y.)the State Key Laboratory of Tree Genetics and Breeding support Program(CAFYBB2019ZY001 to D.P.).
文摘Many important crops(e.g.,tuber,root,and tree crops)are cross-pollinating.For these crops,no inbred lines are available for genetic study and breeding because they are self-incompatible,clonally propagated,or have a long generation time,making the identification of agronomically important genes difficult,particularly in crops with a complex autopolyploid genome.In this study,we developed a method,OutcrossSeq,for mapping agronomically important loci in outcrossing crops based on whole-genome low-coverage resequencing of a large genetic population,and designed three computation algorithms in OutcrossSeq for different types of outcrossing populations.We applied OutcrossSeq to a tuberous root crop(sweet potato,autopolyploid),a tree crop(walnut tree,highly heterozygous diploid),and hybrid crops(double-cross populations)to generate high-density genotype maps for the outcrossing populations,which enable precise identification of genomic loci underlying important agronomic traits.Candidate causative genes at these loci were detected based on functional clues.Taken together,our results indicate that OutcrossSeq is a robust and powerful method for identifying agronomically important genes in heterozygous species,including polyploids,in a cost-efficient way.The OutcrossSeq software and its instruction manual are available for downloading at www.xhhuanglab.cn/tool/OutcrossSeq.html.
基金National Key Research and Development Program,Grant/Award Number:2017YFC1200100National Natural Science Foundation of China,Grant/Award Number:81400589Chinese National Science and Technology Major Project of the 13th Five-year plan,Grant/Award Number:2017ZX10202202-001-008。
文摘Background:Tumor metastasis is a major factor for poor prognosis of hepatocellular carcinoma(HCC),but the relationship between ubiquitination and metastasis need to be studiedmore systematically.We analyzed the ubiquitinome of HCC in this study to have a more comprehensive insight into human HCC metastasis.Methods:The protein ubiquitination levels in 15 HCC specimens with vascular invasion and 15 without vascular invasion were detected by ubiquitinome.Proteins with significantly different ubiquitination levels between HCCs with and without vascular invasion were used to predict E3 ubiquitin ligases associated with tumor metastasis.The topological network of protein substrates and corresponding E3 ubiquitin ligaseswas constructed to identify the key E3 ubiquitin ligase.Besides,the growth,migration and invasion ability of LM3 and HUH7 hepatoma cell lines with andwithout SYVN1 expression interferencewere measured by cell proliferation assay,subcutaneous tumor assay,umphal vein endothelium tube formation assay,transwell migration and invasion assays.Finally,the interacting proteins of SYVN1 were screened and verified by protein interaction omics,immunofluorescence,and immunoprecipitation.Ubiquitin levels of related protein substrates in LM3 and HUH7 cells were compared in negative control,SYVN1 knockdown,and SYVN1 overexpression groups.Results:In this study,our whole-cell proteomic dataset and ubiquitinomic dataset contained approximately 5600 proteins and 12,000 ubiquitinated sites.We discovered increased ubiquitinated sites with shorter ubiquitin chains during the progression ofHCC metastasis.In addition,proteomic and ubiquitinomic analyses revealed that high expression of E3 ubiquitin-protein ligase SYVN1 is related with tumor metastasis.Furthermore,we found that SYVN1 interacted with heat shock protein 90(HSP90)and impacted the ubiquitination of eukaryotic elongation factor 2 kinase(EEF2K).Conclusions:The ubiquitination profiles of HCC with and without vascular invasion were significantly different.SYVN1 was the most important E3 ubiquitin-protein ligase responsible for this phenomenon,and itwas related with tumormetastasis and growth.Therefore,SYVN1might be a potential therapeutic target for HCC.
基金supported by the National Key R&D Program of China(Grant Nos.2016YFC1101304/3 and 2017YFC1200100)the National Natural Science Foundation of China(Grant No.81400589)+1 种基金the National S&T Major Project,China(Grant No.2017ZX10202202-001-008)the Science Fund for Creative Research Groups of the National Natural Science Foundation,China(Grant No.81721091)。
文摘Posttranslational modifications(PTMs)of proteins,particularly acetylation,phosphorylation,and ubiquitination,play critical roles in the host innate immune response.PTMs’dynamic changes and the crosstalk among them are complicated.To build a comprehensive dynamic network of inflammation-related proteins,we integrated data from the whole-cell proteome(WCP),acetylome,phosphoproteome,and ubiquitinome of human and mouse macrophages.Our datasets of acetylation,phosphorylation,and ubiquitination sites helped identify PTM crosstalk within and across proteins involved in the inflammatory response.Stimulation of macrophages by lipopolysaccharide(LPS)resulted in both degradative and non-degradative ubiquitination.Moreover,this study contributes to the interpretation of the roles of known inflammatory molecules and the discovery of novel inflammatory proteins.
基金supported by the National Natural Science Foundation of China(U20A20343)the National Key Research and Development Program(2017YFC1200204)+1 种基金Emergency Project of Zhejiang Provincial Department of Science and Technology(2020C03123-1)Fundamental Research Funds for the Central Universities(2018XZZX001-13).
文摘The devastating coronavirus disease 2019(COVID-19)pandemic has prompted worldwide efforts to study structural biological traits of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)and its viral components.Compared to the Spike protein,which is the primary target for currently available vaccines or antibodies,knowledge about other virion structural components is incomplete.Using high-resolution mass spectrometry,we report a comprehensive post-translational modification(PTM)analysis of nucleocapsid phosphoprotein(NCP),the most abundant structural component of the SARS-CoV-2 virion.In addition to phosphoryl groups,we show that the SARS-CoV-2 NCP is decorated with a variety of PTMs,including N-glycans and ubiquitin.Based on newly identified PTMs,refined protein structural models of SARS-CoV-2 NCP were proposed and potential immune recognition epitopes of NCP were aligned with PTMs.These data can facilitate the design of novel vaccines or therapeutics targeting NCP,as valuable alternatives to the current vaccination and treatment paradigm that is under threat of the ever-mutating SARS-CoV-2 Spike protein.
基金supported by Science Fund for Creative Research Groups of the National Natural Science Foundation of China(No.81721091)The Independent Task of State Key Laboratory for Diagnosis and Treatment of Infectious Diseases,The First Affiliated Hospital,Zhejiang University School of Medicine。
文摘Autoimmune hepatitis(AIH)is a severe globally distributed liver disease that could occur at any age.Human menstrual blood-derived stem cells(MenSCs)have shown therapeutic effect in acute lung injury and liver failure.However,their role in the curative effect of AIH remains unclear.Here,a classic AIH mouse model was constructed through intravenous injection with concanavalin A(Con A).MenSCs were intravenously injected while Con A injection in the treatment groups.The results showed that the mortality by Con A injection was significantly decreased by MenSCs treatment and liver function tests and histological analysis were also ameliorated.The results of phosphoproteomic analysis and RNA-seq revealed that MenSCs improved AIH,mainly by apoptosis and c-Jun N-terminal kinase/mitogen-activated protein signaling pathways.Apoptosis analysis demonstrated that the protein expression of cleaved caspase 3 was increased by Con A injection and reduced by MenSCs transplantation,consistent with the TUNEL staining results.An AML12 co-culture system and JNK inhibitor(SP600125)were used to verify the JNK/MAPK and apoptosis signaling pathways.These findings suggested that MenSCs could be a promising strategy for AIH.
