Signal transduction gRABs attention

Rab proteins are small GTPases involved in the regulation of vesicular membrane traffic. Research done in the past years has demonstrated that some of these proteins are under the control of signal transduction pathways. Still, several recent papers point out to a new unexpected role for this family of Ras-related proteins, as potential regulators of intracellular signaling pathways. In particular, several evidence indicate that members of the Rab family of small GTPases, through their effectors, are key molecules participating to the regulation of numerous signal transduction pathways profoundly influencing cell proliferation, cell nutrition, innate immune response, fragmentation of compartments during mitosis and apoptosis. Even more surprisingly, direct involvement of Rab proteins in signaling to the nucleus has been demonstrated. This review will focus on aspects of Rab proteins function connected to signal transduction and, in particular, connections between membrane traffic and other cell pathways will be examined

Reverse transcriptase-PCR differential display analysis of meningococcal transcripts during infection of human cells: Up-regulation of priA and its role in intracellular replication

<p>Abstract</p> <p>Background</p> <p><it>In vitro </it>studies with cell line infection models are beginning to disclose the strategies that <it>Neisseria meningitidis </it>uses to survive and multiply inside the environment of the infected host cell. The goal of this study was to identify novel virulence determinants that are involved in this process using an <it>in vitro </it>infection system.</p> <p>Results</p> <p>By using reverse transcriptase-PCR differential display we have identified a set of meningococcal genes significantly up-regulated during residence of the bacteria in infected HeLa cells including genes involved in L-glutamate transport (<it>gltT </it>operon), citrate metabolism (<it>gltA</it>), disulfide bond formation (<it>dsbC</it>), two-partner secretion (<it>hrpA-hrpB</it>), capsulation (<it>lipA</it>), and DNA replication/repair (<it>priA</it>). The role of PriA, a protein that in <it>Escherichia coli </it>plays a central role in replication restart of collapsed or arrested DNA replication forks, has been investigated. <it>priA </it>inactivation resulted in a number of growth phenotypes that were fully complemented by supplying a functional copy of <it>priA</it>. The <it>priA</it>-defective mutant exhibited reduced viability during late logarithmic growth phase. This defect was more severe when it was incubated under oxygen-limiting conditions using nitrite as terminal electron acceptors in anaerobic respiration. When compared to wild type it was more sensitive to hydrogen peroxide and the nitric oxide generator sodium nitroprusside. The <it>priA</it>-defective strain was not affected in its ability to invade HeLa cells, but, noticeably, exhibited severely impaired intracellular replication and, at variance with wild type and complemented strains, it co-localized with lysosomal associated membrane protein 1.</p> <p>Conclusion</p> <p>In conclusion, our study i.) demonstrates the efficacy of the experimental strategy that we describe for discovering novel virulence determinants of <it>N. meningitidis </it>and ii.) provides evidence for a role of <it>priA </it>in preventing both oxidative and nitrosative injury, and in intracellular meningococcal replication.</p

Mycobacterial Nucleoside Diphosphate Kinase Blocks Phagosome Maturation in Murine Raw 264.7 Macrophages

bacille Calmette-Guérin (BCG), disrupt the normal function of host Rab5 and Rab7, two small GTPases that are instrumental in the control of phagosome fusion with early endosomes and late endosomes/lysosomes respectively. nucleoside diphosphate kinase (Ndk) exhibits GTPase activating protein (GAP) activity towards Rab5 and Rab7. Then, using a model of latex bead phagosomes, we demonstrated that Ndk inhibits phagosome maturation and fusion with lysosomes in murine RAW 264.7 macrophages. Maturation arrest of phagosomes containing Ndk-beads was associated with the inactivation of both Rab5 and Rab7 as evidenced by the lack of recruitment of their respective effectors EEA1 (early endosome antigen 1) and RILP (Rab7-interacting lysosomal protein). Consistent with these findings, macrophage infection with an Ndk knocked-down BCG strain resulted in increased fusion of its phagosome with lysosomes along with decreased survival of the mutant.Our findings provide evidence in support of the hypothesis that mycobacterial Ndk is a putative virulence factor that inhibits phagosome maturation and promotes survival of mycobacteria within the macrophage

Role of the small GTPase Rab7 in the late endocytic pathway.

Rab7 is a small GTPase localized to the late endosomal compartment. Its function was investigated by overexpressing dominant negative or constitutively active mutants in BHK-21 cells. The effects of such overexpression on the internalization and/or degradation of different endocytic markers and on the morphology of the late endosomal compartment were analyzed. We observed a marked inhibition of the degradation of 125I-low density lipoproteins in cells transfected with the Rab7 dominant negative mutants while the rate of internalization was not affected. Moreover in these cells there was an accumulation of many small vesicles scattered throughout the cytoplasm. In contrast, overexpression of the activating mutants led to the appearance of atypically large endocytic structures and caused a dramatic change in the distribution of the cation-independent mannose 6-phosphate receptor. Our data indicate that the Rab7 protein in mammalian cells is present on a late endosomal compartment much larger than the compartment labeled by the cation-independent mannose 6-phosphate receptor. Rab7 also appears to play a fundamental role in controlling late endocytic membrane traffic

Proyecto Micaela para las Empresas

Este escrito tiene como propósito ofrecer un breve racconto de un proyecto de extensión y transferencia de conocimiento desarrollado en el marco del Centro Interdisciplinario de Estudios sobre Historia de Mujeres y Género: trabajo y política (CIEHMGE), ubicado en la Facultad de Ciencia Política y Relaciones Internacionales de la Universidad Nacional de Rosario, Argentina. Las tareas desarrolladas en el período 2020-2021 tuvieron como misión fundamental trabajar con el sector productivo en la conformación de espacios de capacitación y facilitación de introducción de la perspectiva de género en empresas nacionales e internacionales radicadas en el país. Este camino que recorrimos implicó un trabajo interdisciplinario y el involucramiento de docentes, no docentes, graduados/as y estudiantes en la construcción de saberes y espacios democráticos en su carácter de miembros/as del CIEHMGE

RILP regulates vacuolar ATPase through interaction with the V1G1 subunit

Rab-interacting lysosomal protein (RILP) is a downstream effector of the Rab7 GTPase. GTP-bound Rab7 recruits RILP to endosomal membranes and, together, they control late endocytic traffic, phagosome and autophagosome maturation and are responsible for signaling receptor degradation. We have identified, using different approaches, the V1G1 (officially known as ATP6V1G1) subunit of the vacuolar ATPase (V-ATPase) as a RILP-interacting protein. V1G1 is a component of the peripheral stalk and is fundamental for correct V-ATPase assembly. We show here that RILP regulates the recruitment of V1G1 to late endosomal and lysosomal membranes but also controls V1G1 stability. Indeed, we demonstrate that V1G1 can be ubiquitylated and that RILP is responsible for proteasomal degradation of V1G1. Furthermore, we demonstrate that alterations in V1G1 expression levels impair V-ATPase activity. Thus, our data demonstrate for the first time that RILP regulates the activity of the V-ATPase through its interaction with V1G1. Given the importance of V-ATPase in several cellular processes and human diseases, these data suggest that modulation of RILP activity could be used to control V-ATPase function

Allele-specific silencing as therapy for familial amyotrophic lateral sclerosis caused by the p.G376D TARDBP mutation

Amyotrophic lateral sclerosis is a neurodegenerative disease characterized by the degeneration of motor neurons. There is no treatment for this disease that affects the ability to move, eat, speak and finally breathe, causing death. In an Italian family, a heterozygous pathogenic missense variant has been previously discovered in Exon 6 of the gene TARDBP encoding the TAR DNA-binding protein 43 protein. Here, we developed a potential therapeutic tool based on allele-specific small interfering RNAs for familial amyotrophic lateral sclerosis with the heterozygous missense mutation c.1127G &gt; A. We designed a small interfering RNA that was able to diminish specifically the expression of the exogenous Green Fluorescent Protein (TAR DNA-binding protein 43(G376D) mutant protein) in HEK-293T cells but not that of the Green Fluorescent Protein (TAR DNA-binding protein 43 wild-type). Similarly, this small interfering RNA silenced the mutated allele in fibroblasts derived from patients with amyotrophic lateral sclerosis but did not silence the wild-type gene in control fibroblasts. In addition, we established that silencing the mutated allele was able to strongly reduce the pathological cellular phenotypes induced by TAR DNA-binding protein 43(G376D) expression, such as the presence of cytoplasmic aggregates. Thus, we have identified a small interfering RNA that could be used to silence specifically the mutated allele to try a targeted therapy for patients carrying the p.G376D TAR DNA-binding protein 43 mutation

Alterations of autophagy in the peripheral neuropathy Charcot-Marie-Tooth type 2B

Charcot-Marie-Tooth type 2B (CMT2B) disease is a dominant axonal peripheral neuropathy caused by 5 mutations in the RAB7A gene, a ubiquitously expressed GTPase controlling late endocytic trafficking. In neurons, RAB7A also controls neuronal-specific processes such as NTF (neurotrophin) trafficking and signaling, neurite outgrowth and neuronal migration. Given the involvement of macroautophagy/autophagy in several neurodegenerative diseases and considering that RAB7A is fundamental for autophagosome maturation, we investigated whether CMT2B-causing mutants affect the ability of this gene to regulate autophagy. In HeLa cells, we observed a reduced localization of all CMT2B-causing RAB7A mutants on autophagic compartments. Furthermore, compared to expression of RAB7AWT, expression of these mutants caused a reduced autophagic flux, similar to what happens in cells expressing the dominant negative RAB7AT22N mutant. Consistently, both basal and starvation-induced autophagy were strongly inhibited in skin fibroblasts from a CMT2B patient carrying the RAB7AV162M mutation, suggesting that alteration of the autophagic flux could be responsible for neurodegeneration.Peer reviewe

An altered lipid metabolism characterizes Charcot-Marie-Tooth type 2B peripheral neuropathy.

Charcot-Marie Tooth type 2B (CMT2B) is a rare inherited peripheral neuropathy caused by five missense mutations in the RAB7A gene, which encodes a small GTPase of the RAB family. Currently, no cure is available for this disease. In this study, we approached the disease by comparing the lipid metabolism of CMT2B-derived fibroblasts to that of healthy controls. We found that CMT2B cells showed increased monounsaturated fatty acid level and increased expression of key enzymes of monounsaturated and polyunsaturated fatty acid synthesis. Moreover, in CMT2B cells a higher expression of acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS), key enzymes of de novo fatty acid synthesis, with a concomitantly increased [1-14C]acetate incorporation into fatty acids, was observed. The expression of diacylglycerol acyltransferase 2, a rate-limiting enzyme in triacylglycerol synthesis, as well as triacylglycerol levels were increased in CMT2B compared to control cells. In addition, as RAB7A controls lipid droplet breakdown and lipid droplet dynamics have been linked to diseases, we analyzed these organelles and showed that in CMT2B cells there is a strong accumulation of lipid droplets compared to control cells, thus reinforcing our data on abnormal lipid metabolism in CMT2B. Furthermore, we demonstrated that ACC and FAS expression levels changed upon RAB7 silencing or overexpression in HeLa cells, thus suggesting that metabolic modifications observed in CMT2B-derived fibroblasts can be, at least in part, related to RAB7 mutations