Charcot-Marie-Tooth (CMT) type 2 neuropathies are a group of autosomal-dominant axonal disorders genetically and clinically heterogeneous. In particular, CMT type 2B (CMT2B) neuropathies are characterized by severe sensory loss, often complicated by infections, arthropathy, and amputations. Recently, four missense mutations in the small GTPase Rab7 associated with the Charcot-Marie Tooth type 2B phenotype have been identified. These mutations target highly conserved amino acid residues. However, nothing is known about whether and how these mutations affect Rab7 function. We investigated the biochemical and functional properties of three of the mutant proteins. Interestingly, all three proteins exhibited higher nucleotide exchange rates and hydrolyzed GTP slower than the wild-type protein. In addition, whereas 23% of overexpressed wild-type Rab7 was GTP bound in HeLa cells, the large majority of the mutant proteins (82-89%) were in the GTP-bound form, consistent with the data on GTP hydrolysis and exchange rates. The CMT2B-associated Rab7 proteins were also able to bind the Rab7 effector RILP (Rab-interacting lysosomal protein) and to rescue Rab7 function after silencing. Altogether, these data demonstrate that all tested CMT2B-associated Rab7 mutations are mechanistically similar, suggesting that activated forms of the Rab7 are responsible for CMT2B disease.

Abstract-Stress-dependent regulation of cardiac action potential duration is mediated by the sympathetic nervous system and the hypothalamic-pituitary-adrenal axis. It is accompanied by an increased magnitude of the slow outward potassium ion current, I Ks . KCNQ1 and KCNE1 subunits coassemble to form the I Ks channel. Mutations in either subunit cause long QT syndrome, an inherited cardiac arrhythmia associated with an increased risk of sudden cardiac death.Here we demonstrate that exocytosis of KCNQ1 proteins to the plasma membrane requires the small GTPase RAB11, whereas endocytosis is dependent on RAB5. We further demonstrate that RAB-dependent KCNQ1/KCNE1 exocytosis is enhanced by the serum-and glucocorticoid-inducible kinase 1, and requires phosphorylation and activation of phosphoinositide 3-phosphate 5-kinase and the generation of PI(3,5)P 2 . Identification of KCNQ1/KCNE1 recycling and its modulation by serum-and glucocorticoid-inducible kinase 1-phosphoinositide 3-phosphate 5-kinase -PI(3,5)P 2 provides a mechanistic insight into stress-induced acceleration of cardiac repolarization. (Circ Res. 2007;100:686-692.)Key Words: kinase Ⅲ PIP2 Ⅲ RAB Ⅲ trafficking Ⅲ PIKfyve E motional stress activates the sympathetic nervous system 1 and the release of stress hormones such as cortisol via the hypothalamic-pituitary-adrenal (HPA) axis 2 and is a common trigger of sudden cardiac death. 3,4 One of the many genes regulated by cortisol is the serum-and glucocorticoidinducible kinase 1 (SGK1). 5,6 In vitro experiments have shown that SGK1 stimulates I Ks 7 , a repolarizing potassium current conducted by channels composed of KCNQ1 ␣-subunits and KCNE1 ␤-subunits. 8,9 Moreover, a gain-offunction variant of SGK1 is associated with shortening of the QT interval. 10 SGK1-mediated regulation of I Ks might be particularly important in patients with KCNQ1 (Kv7.1, Kv-LQT1) or KCNE1 (minK) mutations that are prone to fatal cardiac arrhythmias triggered by physical and psychological stress. 4 The mechanism responsible for regulation of I Ks channels by SGK1 have remained elusive. SGK1 enhances the abundance of other types of channel protein in the plasma membrane by inhibiting the ubiquitin ligase Nedd4 -2 11 in addition to other mechanisms (summarized by Lang et al 2006 12 ).Other candidate signaling molecules that may affect channel trafficking include RAB family proteins, GTPases involved in vesicle cycling. [13][14][15][16][17][18] RAB5, a monomeric GTPase of the Ras superfamily, has been implicated in the regulation of early steps in the endocytic pathway, whereas the RAB11 GTPase is localized at the trans-Golgi network, post-Golgi vesicles and the recycling endosome. 19 Both RAB5 and RAB11 are expressed in cardiac tissue. 17 Mammalian cells and Xenopus laevis oocytes have been shown to possess and use highly conserved RAB-dependent trafficking pathways. 20,21 Endocytosis by RAB5 and exocytosis by RAB11 have been reported to participate in the regulation of CFTR chloride channels 22 and the glucose transporter GluT4. 15...

While much data exist in the literature about how Neisseria meningitidis adheres to and invades human cells, its behavior inside the host cell is largely unknown. One of the essential meningococcal attributes for pathogenesis is the polysaccharide capsule, which has been shown to be important for bacterial survival in extracellular fluids. To investigate the role of the meningococcal capsule in intracellular survival, we used B1940, a serogroup B strain, and its isogenic derivatives, which lack either the capsule or both the capsule and the lipooligosaccharide outer core, to infect human phagocytic and nonphagocytic cells and monitor invasion and intracellular growth. Our data indicate that the capsule, which negatively affects bacterial adhesion and, consequently, entry, is, in contrast, fundamental for the intracellular survival of this microorganism. The results of in vitro assays suggest that an increased resistance to cationic antimicrobial peptides (CAMPs), important components of the host innate defense system against microbial infections, is a possible mechanism by which the capsule protects the meningococci in the intracellular environment. Indeed, unencapsulated bacteria were more susceptible than encapsulated bacteria to defensins, cathelicidins, protegrins, and polymyxin B, which has long been used as a model compound to define the mechanism of action of CAMPs. We also demonstrate that both the capsular genes (siaD and lipA) and those encoding an efflux pump involved in resistance to CAMPs (mtrCDE) were up-regulated during the intracellular phase of the infectious cycle.

GdhR is a meningococcal transcriptional regulator that was previously shown to positively control the expression of gdhA, encoding the NADP-specific L-glutamate dehydrogenase (NADP-GDH), in response to the growth phase and/or to the carbon source. In this study we used reverse transcriptase-PCR-differential display (to identify additional GdhR-regulated genes. The results indicated that GdhR, in addition to NADP-GDH, controls the expression of a number of genes involved in glucose catabolism by the Entner-Doudoroff pathway and in L-glutamate import by an unknown ABC transport system. The genes encoding the putative periplasmic substrate-binding protein (NMB1963) and the permease (NMB1965) of the ABC transporter were genetically inactivated. Uptake experiments demonstrated an impairment of L-glutamate import in the NMB1965-defective mutant in the absence or in the presence of a low sodium ion concentration. In contrast, at a sodium ion concentration above 60 mM, the uptake defect disappeared, possibly because the activity of a sodium-driven secondary transporter became predominant. Indeed, the NMB1965-defective mutant was unable to grow at a low sodium ion concentration (<20 mM) in a chemically defined medium containing L-glutamate and four other amino acids that supported meningococcal growth, but it grew when the sodium ion concentration was raised to higher values (>60 mM). The same growth phenotype was observed in the NMB1963-defective mutant. Cell invasion and intracellular persistence assays and expression data during cell invasion provided evidence that the L-glutamate ABC transporter, tentatively named GltT, was critical for meningococcal adaptation in the low-sodium intracellular environment.

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