RESUMEN
Bartonellae are Gram-negative facultative-intracellular pathogens that use a type-IV-secretion system (T4SS) to translocate a cocktail of Bartonella effector proteins (Beps) into host cells to modulate diverse cellular functions. BepC was initially reported to act in concert with BepF in triggering major actin cytoskeletal rearrangements that result in the internalization of a large bacterial aggregate by the so-called 'invasome'. Later, infection studies with bepC deletion mutants and ectopic expression of BepC have implicated this effector in triggering an actin-dependent cell contractility phenotype characterized by fragmentation of migrating cells due to deficient rear detachment at the trailing edge, and BepE was shown to counterbalance this remarkable phenotype. However, the molecular mechanism of how BepC triggers cytoskeletal changes and the host factors involved remained elusive. Using infection assays, we show here that T4SS-mediated transfer of BepC is sufficient to trigger stress fiber formation in non-migrating epithelial cells and additionally cell fragmentation in migrating endothelial cells. Interactomic analysis revealed binding of BepC to a complex of the Rho guanine nucleotide exchange factor GEF-H1 and the serine/threonine-protein kinase MRCKα. Knock-out cell lines revealed that only GEF-H1 is required for mediating BepC-triggered stress fiber formation and inhibitor studies implicated activation of the RhoA/ROCK pathway downstream of GEF-H1. Ectopic co-expression of tagged versions of GEF-H1 and BepC truncations revealed that the C-terminal 'Bep intracellular delivery' (BID) domain facilitated anchorage of BepC to the plasma membrane, whereas the N-terminal 'filamentation induced by cAMP' (FIC) domain facilitated binding of GEF-H1. While FIC domains typically mediate post-translational modifications, most prominently AMPylation, a mutant with quadruple amino acid exchanges in the putative active site indicated that the BepC FIC domain acts in a non-catalytic manner to activate GEF-H1. Our data support a model in which BepC activates the RhoA/ROCK pathway by re-localization of GEF-H1 from microtubules to the plasma membrane.
Asunto(s)
Actinas/metabolismo , Proteínas Bacterianas/metabolismo , Bartonella/metabolismo , Membrana Celular/metabolismo , Proteína C/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Fibras de Estrés/fisiología , Proteínas Bacterianas/genética , Citoesqueleto/metabolismo , Células Endoteliales/citología , Células Endoteliales/metabolismo , Células HeLa , Humanos , Proteína C/genética , Factores de Intercambio de Guanina Nucleótido Rho/genéticaRESUMEN
The mammalian gene Nit1 (nitrilase-like protein 1) encodes a protein that is highly conserved in eukaryotes and is thought to act as a tumor suppressor. Despite being â¼35% sequence identical to ω-amidase (Nit2), the Nit1 protein does not hydrolyze efficiently α-ketoglutaramate (a known physiological substrate of Nit2), and its actual enzymatic function has so far remained a puzzle. In the present study, we demonstrate that both the mammalian Nit1 and its yeast ortholog are amidases highly active toward deaminated glutathione (dGSH; i.e., a form of glutathione in which the free amino group has been replaced by a carbonyl group). We further show that Nit1-KO mutants of both human and yeast cells accumulate dGSH and the same compound is excreted in large amounts in the urine of Nit1-KO mice. Finally, we show that several mammalian aminotransferases (transaminases), both cytosolic and mitochondrial, can form dGSH via a common (if slow) side-reaction and provide indirect evidence that transaminases are mainly responsible for dGSH formation in cultured mammalian cells. Altogether, these findings delineate a typical instance of metabolite repair, whereby the promiscuous activity of some abundant enzymes of primary metabolism leads to the formation of a useless and potentially harmful compound, which needs a suitable "repair enzyme" to be destroyed or reconverted into a useful metabolite. The need for a dGSH repair reaction does not appear to be limited to eukaryotes: We demonstrate that Nit1 homologs acting as excellent dGSH amidases also occur in Escherichia coli and other glutathione-producing bacteria.
Asunto(s)
Aminohidrolasas/metabolismo , Glutatión/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transaminasas/metabolismo , Aminohidrolasas/fisiología , Animales , Desaminación , Humanos , Hidrólisis , Ratones , Ratones Noqueados , Especificidad por SustratoRESUMEN
Glutarate, a side-product in the metabolism of tryptophan and lysine, is metabolized by conversion to glutaryl-CoA by a transferase using succinyl-CoA as a coenzyme donor. The enzyme catalyzing this conversion has not been formally identified. However, a benign form of glutaric aciduria (glutaric aciduria type III) is due to mutations in C7orf10, a putative member of the coenzyme A transferase class III family. In the present work, we show that recombinant human C7orf10 catalyzes the succinyl-CoA-dependent conversion of glutarate to glutaryl-CoA. C7orf10 could use many dicarboxylic acids as CoA acceptors, the best ones being glutarate, succinate, adipate, and 3-hydroxymethylglutarate. Confocal microscopy analysis of CHO cells transfected with a C7orf10-GFP fusion protein indicated that C7orf10 is a mitochondrial protein, in agreement with the presence of a predicted mitochondrial propeptide at its N-terminus. The effect of a missense mutation (p.Arg336Trp) found in the homozygous state in several patients with glutaric aciduria type III and present in the general population at a low frequency was also investigated. The p.Arg336Trp mutation led to the production of insoluble and inactive C7orf10 both in Escherichia coli and in HEK293T cells. These findings indicate that C7orf10 is implicated in the metabolism of glutarate, but possibly also of longer dicarboxylic acids. Homologues of this enzyme are found in numerous bacterial operons comprising also a putative glutaryl-CoA dehydrogenase, indicating that an enzyme with similar specificity exists in prokaryotes.