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A novel testicular RhoGAP-domain protein induces apoptosis.

ABSTRACT: The GTPase-activating proteins (GAPs) accelerate the hydrolysis of GTP to GDP by small GTPases. The GTPases play diverse roles in many cellular processes, including proliferation, cell motility, endocytosis, nuclear import/export, and nuclear membrane formation. Little is known about GAP-domain proteins in spermatogenesis. We isolated a novel RhoGAP domain-containing tGAP1 protein from male germ cells that exhibits unusual properties. The tGAP1 is expressed at low levels in early spermatogonia. Robust transcription initiates in midpachytene spermatocytes and continues after meiosis. The 175-kDa tGAP1 protein localizes to the cytoplasm of spermatocytes and to the cytoplasm and nucleus in spermatids. The protein contains four GAP domain-related sequences, in contrast to all other GAP proteins that harbor one such domain. No activity toward RhoA, Rac1, or Cdc42 could be detected. Results of transfection studies in various somatic cells indicated that low-level tGAP1 expression significantly slows down the cell cycle. Expression of higher levels of tGAP1 by infection of somatic cells with recombinant adenoviruses demonstrated that tGAP1 efficiently induces apoptosis, which to our knowledge is the first such demonstration for a RhoGAP protein. Based on its subcellular location in spermatids and its activity, tGAP1 may play a role in nuclear import/export.

SUBMITTER: Modarressi MH

PROVIDER: S-EPMC3158803 | biostudies-literature | 2004 Dec

REPOSITORIES: biostudies-literature

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A novel testicular RhoGAP-domain protein induces apoptosis.

Modarressi M Hossein MH Cheng Min M Tarnasky Heide A HA Lamarche-Vane Nathalie N de Rooij Dirk G DG Ruan Yibing Y van der Hoorn Frans A FA

Biology of reproduction 20040811 6

The GTPase-activating proteins (GAPs) accelerate the hydrolysis of GTP to GDP by small GTPases. The GTPases play diverse roles in many cellular processes, including proliferation, cell motility, endocytosis, nuclear import/export, and nuclear membrane formation. Little is known about GAP-domain proteins in spermatogenesis. We isolated a novel RhoGAP domain-containing tGAP1 protein from male germ cells that exhibits unusual properties. The tGAP1 is expressed at low levels in early spermatogonia. ...[more]

PMID: 15306557

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Project description:Mycoplasma bovis causes important diseases and great losses on feedlots and dairy farms. However, there are only a few measures to control M. bovis-related diseases. As in other mycoplasma species, this is predominantly because the virulence related factors of this pathogen are largely unknown. Therefore, in this study, we aimed to identify novel virulence-related factors among the secreted proteins of M. bovis. Using bioinformatic tools to analyze its secreted proteins, we preliminarily predicted 39 secreted lipoproteins, and then selected 11 of them for confirmation based on SignalP scores >0.6 or SceP scores >0.8 and conserved domains. These 11 genes were cloned after gene modification based on the codon bias of Escherichia coli and expressed. Mouse antiserum to each recombinant protein was developed. A western blotting assay with these antisera confirmed that MbovP280 and MbovP475 are strongly expressed and secreted proteins, but only MbovP280 significantly reduced the viability of bovine macrophages (BoMac). In further experiments, MbovP280 induced the apoptosis of BoMac treated with both live M. bovis and MbovP280 protein. The conserved coiled-coil domain of MbovP280 at amino acids 210-269 is essential for its induction of apoptosis. Further, immunoprecipitation, mass spectrometry, and coimmunoprecipitation assays identified the anti-apoptosis regulator αB-crystallin (CRYAB) as an MbovP280-binding ligand. An αβ-crystallin knockout cell line BoMac-cryab-, Mbov0280-knockout M. bovis strain T9.297, and its complemented M. bovis strain CT9.297 were constructed and the apoptosis of BoMac-cryab- induced by these strains was compared. The results confirmed that CRYAB is critical for MbovP280 function as an apoptosis inducer in BoMac. In conclusion, in this study, we identified MbovP280 as a novel secreted protein of M. bovis that induces the apoptosis of BoMac via its coiled-coil domain and cellular ligand CRYAB. These findings extend our understanding of the virulence mechanism of mycoplasmal species.

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A novel testicular RhoGAP-domain protein induces apoptosis.

Publications

A novel testicular RhoGAP-domain protein induces apoptosis.

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