Valosin-containing protein （VCP） is a type-II adenosine triphosphatase （ATPase） wih extensive biological function in organisms. Silkworm is the second in- sect model for genetic studies and a bioreactor for protei...Valosin-containing protein （VCP） is a type-II adenosine triphosphatase （ATPase） wih extensive biological function in organisms. Silkworm is the second in- sect model for genetic studies and a bioreactor for proteinaceous drugs and biomaterials. In this paper, a new VCP-like gene was amplified from the fat body of silkworm follow- ing genome prediction and spliced expressed sequence tag sequences, using both reverse transcription polymerase chain reaction （RT-PCR） and 3＇-RACE （rapid amplification of complementary DNA ends） methods. Bioinformatical analysis showed that the translated amino acid sequence contained a highly conserved domain of VCPs similar to that of many insects. This domain consists of the conserved structure motifs of the ATP binding site and the catalytical center, which is closely related to the insect VCPs in a phylogenetic tree. The silkworm VCP-like gene was successfully inserted into the plasmid and transformed into Escherichia coli cells to express VCP-like protein with ATPase activity. The expression of silkworm VCP-like protein was also confirmed by Western blotting and mass spectromet- ric analyses. Distribution of the VCP-like gene in various tissues of the silkworm was also studied by real-time PCR. Results showed that the messenger RNA （mRNA） of VCP-like protein is widely expressed in fat body, reproductive organs （testis or ovary）, silk gland, head, Malpighian tubule, epidermis and midgut. Among them, fat body has the highest mRNA expression level of the VCP-like gene, while the midgut has the lowest expression level. This study provides groundwork for further study on the structure and function of the new VCP-like protein.展开更多
Background Hepatitis B virus （HBV） x protein （HBx） in HepG2 cells causes a moderate decrease in proteolysis activity of the proteasome. A highly conserved Kunitz-type serine protease inhibitor domain within 154 am...Background Hepatitis B virus （HBV） x protein （HBx） in HepG2 cells causes a moderate decrease in proteolysis activity of the proteasome. A highly conserved Kunitz-type serine protease inhibitor domain within 154 amino acid residues of HBx has been identified. In this study, a peptide chain derived from the Kunitz domain （PKD） was used to study its effect on the cell cycle and apoptosis of HepG2 cells, and investigated the function of PKD on the activities of proteasomes and AAA-ATPase p97, which involves in the ubiquitin-proteasome protein degradation pathway. Methods The PKD peptide （Phe-Val-Leu-Gly-Gly-Cys-Arg-His-Lys） was chemically synthesized. MTT assays were used to determine the effects of PKD on HepG2 cell growth. Mouse anti-p97 antibody was developed for Western blotting to detect the expression of p97. ATPase activity of proteasomes was measured using a colorimetric assay. Peptidase activities of proteasomes were analyzed with various peptidase-specific fluorogenic peptide substrates. Flow cytometry was used to determinate cell cycle phase and apoptosis. Results Viability of HepG2 cells decreased in a PKD-dose-dependent manner. Cells exhibited significant cytotoxicity in the presence of 15 mmol/L of PKD. Western blotting analysis showed that expression of p97 was suppressed in HepG2 cells treated with PKD compared to untreated cells. The ATPase activity of proteasomes from immunoprecipitates of HepG2 cells pretreated with PKD was apparently decreased. Chymotryptic activity of proteasomes in HepG2 cells was significantly inhibited by 10mmol/L PKD; tryptic activity and peptidylglutamyl peptide hydroiase activity of proteasomes were less inhibited by PKD than chymotryptic activity. The cell cycle phase of HepG2 cells treated with PKD for 36 hours was blocked largely at the G0-G1 phase, while untreated control cells were mainly in S phase. PKD also significantly induced apoptosis. Conclusions The peptide derived from Kunitz domain of HBx protein induces HepG2 cell growth arrest and apoptosis, which may result from down-regulation of p97 expression, and decrease of both the ATPase and chymotryptic activities of proteasomes.展开更多
At least 25 genes,many involved in trafficking,localisation or shaping of membrane organelles,have been identified as causative genes for the neurodegenerative disorder hereditary spastic paraplegia（HSP）.One of the ...At least 25 genes,many involved in trafficking,localisation or shaping of membrane organelles,have been identified as causative genes for the neurodegenerative disorder hereditary spastic paraplegia（HSP）.One of the most commonly mutated HSP genes,atlastin-1, encodes a dynamin-like GTPase that mediates homotypic fusion of endoplasmic reticulum（ER） membranes.However,the molecular mechanisms of atlastin-1-related membrane fusion and axonopathy remain unclear.To better understand its mode of action,we used affinity purification coupled with mass spectrometry to identify protein interactors of atlastin in Drosophila.Analysis of 72 identified proteins revealed that the atlastin interactome contains many proteins involved in protein processing and transport,in addition to proteins with roles in mRNA binding,metabolism and mitochondrial proteins.The highest confidence interactor from mass spectrometry analysis, the ubiquitin-selective AAA-ATPase valosin-containing protein（VCP）,was validated as an atlastin-interacting protein,and VCP and atlastin showed overlapping subcellular distributions.Furthermore,VCP acted as a genetic modifier of atlastin：loss of VCP partially suppressed an eye phenotype caused by atlastin overexpression,whereas overexpression of VCP enhanced this phenotype.These interactions between atlastin and VCP suggest a functional relationship between these two proteins,and point to potential shared mechanisms between HSP and other forms of neurodegeneration.展开更多
Chikungunya virus(CHIKV)is a re-emerging mosquito-transmitted RNA virus causing joint and muscle pain.To better understand how CHIKV rewires the host cell and usurps host cell functions,we generated a systematic CHIKV...Chikungunya virus(CHIKV)is a re-emerging mosquito-transmitted RNA virus causing joint and muscle pain.To better understand how CHIKV rewires the host cell and usurps host cell functions,we generated a systematic CHIKV-human protein-protein interaction map and revealed several novel connections that will inform further mechanistic studies.One of these novel interactions,between the viral protein E1 and STIP1 homology and U-box containing protein 1(STUB1),was found to mediate ubiquitination of E1 and degrade E1 through the proteasome.Capsid associated with G3BP1,G3BP2 and AAAþATPase valosin-containing protein(VCP).Furthermore,VCP inhibitors blocked CHIKV infection,suggesting VCP could serve as a therapeutic target.Further work is required to fully understand the functional consequences of these interactions.Given that CHIKV proteins are conserved across alphaviruses,many virus-host protein-protein interactions identified in this study might also exist in other alphaviruses.Construction of interactome of CHIKV provides the basis for further studying the function of alphavirus biology.展开更多
文摘Valosin-containing protein （VCP） is a type-II adenosine triphosphatase （ATPase） wih extensive biological function in organisms. Silkworm is the second in- sect model for genetic studies and a bioreactor for proteinaceous drugs and biomaterials. In this paper, a new VCP-like gene was amplified from the fat body of silkworm follow- ing genome prediction and spliced expressed sequence tag sequences, using both reverse transcription polymerase chain reaction （RT-PCR） and 3＇-RACE （rapid amplification of complementary DNA ends） methods. Bioinformatical analysis showed that the translated amino acid sequence contained a highly conserved domain of VCPs similar to that of many insects. This domain consists of the conserved structure motifs of the ATP binding site and the catalytical center, which is closely related to the insect VCPs in a phylogenetic tree. The silkworm VCP-like gene was successfully inserted into the plasmid and transformed into Escherichia coli cells to express VCP-like protein with ATPase activity. The expression of silkworm VCP-like protein was also confirmed by Western blotting and mass spectromet- ric analyses. Distribution of the VCP-like gene in various tissues of the silkworm was also studied by real-time PCR. Results showed that the messenger RNA （mRNA） of VCP-like protein is widely expressed in fat body, reproductive organs （testis or ovary）, silk gland, head, Malpighian tubule, epidermis and midgut. Among them, fat body has the highest mRNA expression level of the VCP-like gene, while the midgut has the lowest expression level. This study provides groundwork for further study on the structure and function of the new VCP-like protein.
基金This study was supported by a grant from the National Natural Science Foundation of China （No. 30672433）.
文摘Background Hepatitis B virus （HBV） x protein （HBx） in HepG2 cells causes a moderate decrease in proteolysis activity of the proteasome. A highly conserved Kunitz-type serine protease inhibitor domain within 154 amino acid residues of HBx has been identified. In this study, a peptide chain derived from the Kunitz domain （PKD） was used to study its effect on the cell cycle and apoptosis of HepG2 cells, and investigated the function of PKD on the activities of proteasomes and AAA-ATPase p97, which involves in the ubiquitin-proteasome protein degradation pathway. Methods The PKD peptide （Phe-Val-Leu-Gly-Gly-Cys-Arg-His-Lys） was chemically synthesized. MTT assays were used to determine the effects of PKD on HepG2 cell growth. Mouse anti-p97 antibody was developed for Western blotting to detect the expression of p97. ATPase activity of proteasomes was measured using a colorimetric assay. Peptidase activities of proteasomes were analyzed with various peptidase-specific fluorogenic peptide substrates. Flow cytometry was used to determinate cell cycle phase and apoptosis. Results Viability of HepG2 cells decreased in a PKD-dose-dependent manner. Cells exhibited significant cytotoxicity in the presence of 15 mmol/L of PKD. Western blotting analysis showed that expression of p97 was suppressed in HepG2 cells treated with PKD compared to untreated cells. The ATPase activity of proteasomes from immunoprecipitates of HepG2 cells pretreated with PKD was apparently decreased. Chymotryptic activity of proteasomes in HepG2 cells was significantly inhibited by 10mmol/L PKD; tryptic activity and peptidylglutamyl peptide hydroiase activity of proteasomes were less inhibited by PKD than chymotryptic activity. The cell cycle phase of HepG2 cells treated with PKD for 36 hours was blocked largely at the G0-G1 phase, while untreated control cells were mainly in S phase. PKD also significantly induced apoptosis. Conclusions The peptide derived from Kunitz domain of HBx protein induces HepG2 cell growth arrest and apoptosis, which may result from down-regulation of p97 expression, and decrease of both the ATPase and chymotryptic activities of proteasomes.
基金supported by Marie Curie Individual Fellowship 236777
文摘At least 25 genes,many involved in trafficking,localisation or shaping of membrane organelles,have been identified as causative genes for the neurodegenerative disorder hereditary spastic paraplegia（HSP）.One of the most commonly mutated HSP genes,atlastin-1, encodes a dynamin-like GTPase that mediates homotypic fusion of endoplasmic reticulum（ER） membranes.However,the molecular mechanisms of atlastin-1-related membrane fusion and axonopathy remain unclear.To better understand its mode of action,we used affinity purification coupled with mass spectrometry to identify protein interactors of atlastin in Drosophila.Analysis of 72 identified proteins revealed that the atlastin interactome contains many proteins involved in protein processing and transport,in addition to proteins with roles in mRNA binding,metabolism and mitochondrial proteins.The highest confidence interactor from mass spectrometry analysis, the ubiquitin-selective AAA-ATPase valosin-containing protein（VCP）,was validated as an atlastin-interacting protein,and VCP and atlastin showed overlapping subcellular distributions.Furthermore,VCP acted as a genetic modifier of atlastin：loss of VCP partially suppressed an eye phenotype caused by atlastin overexpression,whereas overexpression of VCP enhanced this phenotype.These interactions between atlastin and VCP suggest a functional relationship between these two proteins,and point to potential shared mechanisms between HSP and other forms of neurodegeneration.
基金supported by National Natural Science Foundation of China (82072270 and 82272306)Taishan Scholars Program (tstp20221142)+1 种基金Shandong Provincial Natural Science Foundation (ZR2021QC095)Academic Promotion Programme of Shandong First Medical University (2019LJ001).
文摘Chikungunya virus(CHIKV)is a re-emerging mosquito-transmitted RNA virus causing joint and muscle pain.To better understand how CHIKV rewires the host cell and usurps host cell functions,we generated a systematic CHIKV-human protein-protein interaction map and revealed several novel connections that will inform further mechanistic studies.One of these novel interactions,between the viral protein E1 and STIP1 homology and U-box containing protein 1(STUB1),was found to mediate ubiquitination of E1 and degrade E1 through the proteasome.Capsid associated with G3BP1,G3BP2 and AAAþATPase valosin-containing protein(VCP).Furthermore,VCP inhibitors blocked CHIKV infection,suggesting VCP could serve as a therapeutic target.Further work is required to fully understand the functional consequences of these interactions.Given that CHIKV proteins are conserved across alphaviruses,many virus-host protein-protein interactions identified in this study might also exist in other alphaviruses.Construction of interactome of CHIKV provides the basis for further studying the function of alphavirus biology.
