YWK-II protein/APLP2 is a member of an evolutionarily conserved protein family that includes amyloid precursor protein (APP) and amyloid precursor like protein-1 (APLP1). We have previously demonstrated that YWK-II/APLP2 functions as a novel G0-protein-coupled receptor for Müllerian inhibiting substance (MIS) in cell survival. However, factors regulating the stability and turnover of YWK-II/APLP2 have not been identified. Here we present evidence that human leukocyte antigen-B-associated transcript 3 (Bat3), an important regulator involved in apoptosis, can interact with YWK-II/APLP2 and enhance its stability by reducing its ubiquitination and degradation by the ubiquitin-proteasome system. Co-expression of different Bat3 domain deletion constructs with YWK-II/APLP2 reveals that the proline-rich domain of Bat3 is required for its binding to YWK-II/APLP2. In addition, we find that the protein levels of YWK-II/APLP2 could be enhanced by nuclear export of Bat3 under apoptotic stimulation. We also find elevated levels of Bat3 and YWK-II/APLP2 in human colorectal cancer with a positive correlation between the two. Taken together, these results have revealed a previously undefined mechanism regulating cell apoptosis and suggest that aberrant enhancement of YWK-II/APLP2 by nuclear export of Bat3 may play a role in cancer development by inhibiting cell apoptosis.
To analyze the functions of differentially expressed genes between abdominal aortic aneurysm and normal aortic tissue by cDNA microarray.Total RNAs were respectively isolated from the normal aorta and aortic aneurysm, purified into mRNAs by oligotex. Subsequently they were reverse-transcribed into cDNAs incorporated with fluorescent dUTP to make hybridization probes, which were hybridized as the cDNA microarray for scanning of fluorescent signals and differentially expressed genes between the normal aortic and aortic aneurysm by using GenePix Pro 3.0 software.A total of 18 differentially expressed genes were detected, accounting for 0.44% of total genes. Among these genes, 11 were related to cell cycle and the remaining 7 to cell apoptosis. The number of upregulated genes in the aortic aneurysm was 9 (mean ratio: 3.860) and that of the downregulated 9 (mean Ratio: 0.294). Bio-informative analysis showed that these 18 genes might influence the growth and apoptosis of smooth muscle cells in abdominal aortic aneurysms.During the development of abdominal aortic aneurysms, modulations of multi-gene expression would undergo various changes. Cell cycle and apoptosis-related genes were related to the growth and apoptosis of smooth muscle cells in abdominal aortic aneurysms. Further research into these genes will clarify the mechanisms of abdominal aortic aneurysms.
MicroRNAs (miRNAs) are posttranscriptional modulators of gene expression that play important roles in various biological processes. Spermatogenesis is a highly regulated process in which diploid spermatogonia eventually differentiate into haploid spermatozoa. In this study, we identified four differentially expressed miRNAs between two premeiotic male germ cells, made predictions about their putative targets, and confirmed cyclin T2 (Ccnt2) as a direct target of miR‐15a. We also report that miR‐15a inhibited muscle differentiation at least in part by targeting Ccnt2, which represents a novel interaction. Subsequently, miR‐15a and Ccnt2 were profiled in developing mice testes to observe their inverse correlations in the postnatal 3‐week period to understand their roles in spermatogenesis.
To develop a targeting protein for Xenopus kinesin-like protein 2 (TPX2) C' terminal SBP-3 x Flag-tagged HCT 116 cell model.Homologous arms were amplified by polymerase chain reaction (PCR), and then the adeno-associated virus (AAV) -targeting vector of TPX2 was constructed. HCT 116 cells were targeted after the viruses were packaged. Positive cell clones with neomycin resistance gene were obtained by G418 and PCR screening. Finally, the neomycin gene cassette was excised after the targeted clones were infected with adenovirus expressing Cre-recombinase, and the TPX2 C' terminal SBP and 3 x Flag endogenous double-tagged HCT 116 cells were obtained by PCR screening.Two positive cell clones with neomycin resistance gene were obtained by PCR screening. The positive clones with neomycin resistance gene excised were obtained by Cre adenovirus infection, and the knock-in of SBP-3 x Flag gene was verified by Western blot analysis.The TPX2 C' terminal SBP-3 x Flag tagged HCT 116 cell model was successfully established.
To explore the protein factors that could interact with the testis-specific protein encoded by HSD-3.8 gene (GenBank Accession Number AF311312) related with female fertilization.Yeast two-hybrid system was used to screen the human ovary MATCHMAKER cDNA library with constructed "bait plasmid" containing the 0.7 kb fragment (HSD-0.7) of HSD-3.8. The interaction with the positive fragments using a series of truncated bait plasmids was investigated.One positive gene fragment was obtained, which coded for 144 amino acids of the C-terminus of human G protein beta subunit 1. Truncated bait plasmids couldn't interact with the fish protein fragment in yeast.The protein encoded by HSD-3.8 gene may function through G protein signal transduction pathway and the interaction depends on the integration of the bait protein.
rsb-66 is a novel gene from a suppression subtracted hybridization (SSH) library of round spermatid-specific cDNAs against those of primary spermatocytes. It was found to be specifically expressed in round spermatids. To explore the function of RSB-66, a yeast two-hybrid system was used to screen for potential interacting partners in a human testis cDNA library. HSD45, also known as INCA1 (inhibitor of Cdk interacting with cyclin A1), was identified as one of the positive clones. The interaction between RSB-66 and INCA1 was demonstrated to occur by GST pull down and coimmunoprecipitation. Using immunofluorescence, RSB-66 was found to be specifically expressed in round spermatids, mainly in the cytoplasm. When being transfected into HeLa cells, RSB-66 and INCA1 were found to be co-localized principally in the cytoplasm. The α helix in the RSB-66 C terminal and two amino acid residues (tyr117 and his119) appear to be crucial for its function.
Apoptosis is a regulated cellular suicide program that is critical for the development and maintenance of healthy tissues. Previous studies have shown that small kinetochore associated protein (SKAP) cooperates with kinetochore and mitotic spindle proteins to regulate mitosis. However, the role of SKAP in apoptosis has not been investigated. We have identified a new interaction involving SKAP, and we propose a mechanism through which SKAP regulates cell apoptosis. Our experiments demonstrate that both overexpression and knockdown of SKAP sensitize cells to UV-induced apoptosis. Further study has revealed that SKAP interacts with Pre-mRNA processing Factor 19 (Prp19). We find that UV-induced apoptosis can be inhibited by ectopic expression of Prp19, whereas silencing Prp19 has the opposite effect. Additionally, SKAP negatively regulates the protein levels of Prp19, whereas Prp19 does not alter SKAP expression. Finally, rescue experiments demonstrate that the pro-apoptotic role of SKAP is executed through Prp19. Taken together, these findings suggest that SKAP promotes UV-induced cell apoptosis by negatively regulating the anti-apoptotic protein Prp19.
A general mammalian expression vector designated pSV2-EP was reconstructed by inserting an oligonucleotide fragment into pSV2-dhfr. This vector allowed insertion of cDNAs with EcoRI cohesive ends. The pSV2-EP contains a simian virus 40 (SV40) early promoter, origin for DNA replication, SV40 poly-A site, splicing site, an initiator ATG downstream from the promoter and an EcoRI site for the insertion of cDNA fragment screened from lambda gt11 expression libraries. A recombinant plasmid (pS-VRS-1) was constructed by inserting RSD-1, a cDNA encoding a rabbit sperm tail protein, into the EcoRI site of the pSV2-EP vector. Chinese hamster ovarian (CHO) dhfr-negative cells were cotransformed with pSV2-dhfr and pSVRS-1 by the calcium phosphate method. In selective culture medium without thymidine and hypoxanthine, several cell lines were obtained containing mRNA and DNA that hybridized with RSD-1. One of these transformed cell lines stained intensely with anti-rSMP-B antibodies, demonstrating that the RSD-1 was expressed in the transformed CHO cells.
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