According to the clpX gene sequence of Vibrio alginolyticus HY9901,a pair of specific primers were designed,and the full length was cloned by PCR and subjected to bioinformatics analysis.The results showed that the cl...According to the clpX gene sequence of Vibrio alginolyticus HY9901,a pair of specific primers were designed,and the full length was cloned by PCR and subjected to bioinformatics analysis.The results showed that the clpX gene was 1281 bp in length and encoded 426 amino acids.Its molecular structure formula was C 3842 H 6405 N 1281 O 1598 S 260,with a theoretical protein molecular weight of approximately 1044473.4 kDa and a theoretical pI value of 5.04.The clpX gene was predominantly situated within the cytoplasm,exhibiting unstable and hydrophilic protein characteristics.It possessed a signal peptide cleavage site,lacked a transmembrane region,and was not associated with any KEGG metabolic pathway.Additionally,it possessed 2 glycine phosphorylation sites,a CAMP-dependent protein kinase phosphorylation site,a C-terminal amidation modification site,6 protein kinase C phosphorylation sites,7 microbody C-terminal target signal sites,and an ATP/GTP site.The clpX phylogenetic tree was constructed using the MEGA 5.0 software via the neighbor-joining method.The results demonstrated that the clpX of V.alginolyticus exhibited up to 100%affinity with the clpX of Vibrio spp.The single subunit 3D structure model of the ClpX protein was obtained using the SWISS-MODEL program.A structural and functional analysis of the protein revealed the presence of three distinct ClpX structural and functional domains.In the prediction of secondary structure,the proportions ofα-helix,random coil,β-sheet and extended strand were 40.38%,37.09%,5.40%and 17.14%,respectively.The analysis of the ClpX protein through the STRING database revealed that the proteins interacting with the ClpX protein were Tig,Atpd,Hflb,Msrb-2,Rpod,Clpp,Clpa,Lon-1,Hfq,and ANP63951.1.A computational analysis of the ClpX protein identified a number of post-translational modification sites,including phosphorylation,acetylation,ubiquitination,glycosylation,methylation,S-palmitoylation,and lactylation.The significance of this study is to analyze the function of the clpX gene and establish a robust foundation for subsequent investigations into the mechanism of the clpX gene in Vibrio alginolyticus.展开更多
Meiosis is the process of producing haploid gametes through a series of complex chromosomal events and the coordinated action of various proteins.The mitochondrial protease complex(ClpXP),which consists of caseinolyti...Meiosis is the process of producing haploid gametes through a series of complex chromosomal events and the coordinated action of various proteins.The mitochondrial protease complex(ClpXP),which consists of caseinolytic mitochondrial matrix peptidase X(ClpX)and caseinolytic protease P(ClpP)and mediates the degradation of misfolded,damaged,and oxidized proteins,is essential for maintaining mitochondrial homeostasis.ClpXP has been implicated in meiosis regulation,but its precise role is currently unknown.In this study,we engineered an inducible male germ cell-specific knockout caseinolytic mitochondrial matrix peptidase X(Clpx^(cKO))mouse model to investigate the function of ClpX in meiosis.We found that disrupting Clpx in male mice induced germ cell apoptosis and led to an absence of sperm in the epididymis.Specifically,it caused asynapsis of homologous chromosomes and impaired meiotic recombination,resulting in meiotic arrest in the zygotene-to-pachytene transition phase.The loss of ClpX compromised the double-strand break(DSB)repair machinery by markedly reducing the recruitment of DNA repair protein RAD51 homolog 1(RAD51)to DSB sites.This dysfunction may be due to an insufficient supply of energy from the aberrant mitochondria in Clpx^(cKO) spermatocytes,as discerned by electron microscopy.Furthermore,ubiquitination signals on chromosomes and the expression of oxidative phosphorylation subunits were both significantly attenuated in Clpx^(cKO) spermatocytes.Taken together,we propose that ClpX is essential for maintaining mitochondrial protein homeostasis and ensuring homologous chromosome pairing,synapsis,and recombination in spermatocytes during meiotic prophase I.展开更多
基金Supported by National Natural Science Foundation of China(32073015)Graduate Education Innovation Program of Guangdong Province(YJYH[2022]1)+1 种基金Undergraduate Innovation and Entrepreneurship Training Program of Guangdong Ocean University(CXXL2024007)Undergraduate Innovation Team of Guangdong Ocean University(CCTD201802).
文摘According to the clpX gene sequence of Vibrio alginolyticus HY9901,a pair of specific primers were designed,and the full length was cloned by PCR and subjected to bioinformatics analysis.The results showed that the clpX gene was 1281 bp in length and encoded 426 amino acids.Its molecular structure formula was C 3842 H 6405 N 1281 O 1598 S 260,with a theoretical protein molecular weight of approximately 1044473.4 kDa and a theoretical pI value of 5.04.The clpX gene was predominantly situated within the cytoplasm,exhibiting unstable and hydrophilic protein characteristics.It possessed a signal peptide cleavage site,lacked a transmembrane region,and was not associated with any KEGG metabolic pathway.Additionally,it possessed 2 glycine phosphorylation sites,a CAMP-dependent protein kinase phosphorylation site,a C-terminal amidation modification site,6 protein kinase C phosphorylation sites,7 microbody C-terminal target signal sites,and an ATP/GTP site.The clpX phylogenetic tree was constructed using the MEGA 5.0 software via the neighbor-joining method.The results demonstrated that the clpX of V.alginolyticus exhibited up to 100%affinity with the clpX of Vibrio spp.The single subunit 3D structure model of the ClpX protein was obtained using the SWISS-MODEL program.A structural and functional analysis of the protein revealed the presence of three distinct ClpX structural and functional domains.In the prediction of secondary structure,the proportions ofα-helix,random coil,β-sheet and extended strand were 40.38%,37.09%,5.40%and 17.14%,respectively.The analysis of the ClpX protein through the STRING database revealed that the proteins interacting with the ClpX protein were Tig,Atpd,Hflb,Msrb-2,Rpod,Clpp,Clpa,Lon-1,Hfq,and ANP63951.1.A computational analysis of the ClpX protein identified a number of post-translational modification sites,including phosphorylation,acetylation,ubiquitination,glycosylation,methylation,S-palmitoylation,and lactylation.The significance of this study is to analyze the function of the clpX gene and establish a robust foundation for subsequent investigations into the mechanism of the clpX gene in Vibrio alginolyticus.
基金supported by the Shenzhen Science and Technology Program,China(No.KQTD20190929172749226).
文摘Meiosis is the process of producing haploid gametes through a series of complex chromosomal events and the coordinated action of various proteins.The mitochondrial protease complex(ClpXP),which consists of caseinolytic mitochondrial matrix peptidase X(ClpX)and caseinolytic protease P(ClpP)and mediates the degradation of misfolded,damaged,and oxidized proteins,is essential for maintaining mitochondrial homeostasis.ClpXP has been implicated in meiosis regulation,but its precise role is currently unknown.In this study,we engineered an inducible male germ cell-specific knockout caseinolytic mitochondrial matrix peptidase X(Clpx^(cKO))mouse model to investigate the function of ClpX in meiosis.We found that disrupting Clpx in male mice induced germ cell apoptosis and led to an absence of sperm in the epididymis.Specifically,it caused asynapsis of homologous chromosomes and impaired meiotic recombination,resulting in meiotic arrest in the zygotene-to-pachytene transition phase.The loss of ClpX compromised the double-strand break(DSB)repair machinery by markedly reducing the recruitment of DNA repair protein RAD51 homolog 1(RAD51)to DSB sites.This dysfunction may be due to an insufficient supply of energy from the aberrant mitochondria in Clpx^(cKO) spermatocytes,as discerned by electron microscopy.Furthermore,ubiquitination signals on chromosomes and the expression of oxidative phosphorylation subunits were both significantly attenuated in Clpx^(cKO) spermatocytes.Taken together,we propose that ClpX is essential for maintaining mitochondrial protein homeostasis and ensuring homologous chromosome pairing,synapsis,and recombination in spermatocytes during meiotic prophase I.
