The Deubiquitinating Enzyme USP8 Promotes Trafficking and Degradation of the Chemokine Receptor 4 at the Sorting Endosome

Reversible ubiquitination orchestrated by the opposition of ubiquitin ligases and deubiquitinating enzymes mediates endocytic trafficking of cell surface receptors for lysosomal degradation. Ubiquitin-specific protease 8 (USP8) has previously been implicated in endocytosis of several receptors by virtue of their deubiquitination. The present study explores an indirect role for USP8 in cargo trafficking through its regulation of the chemokine receptor 4 (CXCR4). Contrary to the effects of USP8 loss on enhanced green fluorescent protein, we find that USP8 depletion stabilizes CXCR4 on the cell surface and attenuates receptor degradation without affecting its ubiquitination status. In the presence of ligand, diminished CXCR4 turnover is accompanied by receptor accumulation on enlarged early endosomes and leads to enhancement of phospho-ERK signaling. Perturbation in CXCR4 trafficking, resulting from USP8 inactivation, occurs at the ESCRT-0 checkpoint, and catalytic mutation of USP8 specifically targeted to the ESCRT-0 complex impairs the spatial and temporal organization of the sorting endosome. USP8 functionally opposes the ubiquitin ligase AIP4 with respect to ESCRT-0 ubiquitination, thereby promoting trafficking of CXCR4. Collectively, our findings demonstrate a functional cooperation between USP8, AIP4, and the ESCRT-0 machinery at the early sorting phase of CXCR4 and underscore the versatility of USP8 in shaping trafficking events at the early-to-late endosome transition.     

The interaction of hemoglobin with phosphatidylserine vesicles

The interaction of hemoglobin with phosphatidylserine vesicles at low ionic strength and pH conditions was studied. The fluorescence intensity of a lipid embedded probe was quenched by bound Hb but could not be reversed by an elevation of ionic strength and pH. The irreversibility of the fluorescence quenching is a time-dependent process associated with changes in the heme Soret and visible spectra. The rate of these changes was much faster for methemoglobin than for either cyanomethemoglobin or oxyhemoglobin. Elevation of ionic strength released out of the bound hemoglobin into the water phase most of the globin but only a small fraction of the heme. The data are interpreted as demonstrating the ability of phosphatidylserine vesicles to compete with globin for the heme group. When Hb binds to the liposome, heme is being transferred into the lipid phase and the rate-limiting step is the dissociation of the heme-globin complex. The fact that binding of heme to the lipid vesicles is very strong was demonstrated by the failure of hemin to interact with globin when the two were rapidly mixed in the presence of phosphatidylserine vesicles. A multi-step process is suggested to explain the results of Hb phosphatidylserine interaction.     

Method for the quantitative assay of fatty acid-bile acid conjugates by tandem mass spectrometry

Fatty acid-bile acid conjugates and especially arachidyl amido cholic acid are synthetic molecules that were shown to prevent cholesterol gallstone formation in mice and hamsters as well as to dissolve pre-existing gallstones in mice. To measure these novel compounds we developed a liquid chromatography electrospray tandem mass spectrometry method based on the analysis of 100 microL of plasma with stearyl amido cholic acid (stamchol, 1.5 microM/L) added as internal standard. Repeatable calibrations between 0 and 50 microM/L exhibited consistent linearity and reproducibility. Inter- and intraassay C.V.s were 5.3-11.4% and 2.6-6.4%, respectively, at targeted concentrations of 0.1, 2.3 and 50 microM/L.     

Inhibition of Astrocyte Glutamine Production by α-Ketoisocaproic Acid

Abstract: We have evaluated the effect of α-ketoisocaproic acid (KIC), the ketoacid of leucine, on the production of glutamine by cultured astrocytes. We used ¹⁵NH₄Cl as a metabolic tracer to measure the production of both [5-¹⁵N]glutamine, reflecting amidation of glutamate via glutamine synthetase, and [2-¹⁵N]glutamine, representing the reductive amination of 2-oxoglutarate via glutamate dehydrogenase and subsequent conversion of [¹⁵N]-glutamate to [2-¹⁵N]glutamine. Addition of KIC (1 mM) to the medium diminished the production of [5-¹⁵N]glutamine and stimulated the formation of [2-¹⁵N]glutamine with the overall result being a significant inhibition of net glutamine synthesis. An external KIC concentration as low as 0.06 mM inhibited synthesis of [5-¹⁵N]glutamine and a level as low as 0.13 mM enhanced labeling (atom% excess) of [2-¹⁵N]glutamine. Higher concentrations of KIC in the medium had correspondingly larger effects. The presence of KIC in the medium did not affect flux through glutaminase, which was measured using [2-¹⁵N]glutamine as a tracer. Nor did KIC inhibit the activity of glutamine synthetase that was purified from sheep brain. Addition of KIC to the medium caused no increased release of lactate dehydrogenase from the astrocytes, suggesting that the ketoacid was not toxic to the cells. KIC treatment was associated with an approximately twofold increase in the formation of ¹⁴CO₂ from [U-¹⁴C]glutamate, indicating that transamination of glutamate with KIC increases intraastrocytic α-ketoglutarate, which is oxidized in the tricarboxylic acid cycle. KIC inhibited glutamine synthesis more than any other ketoacid tested, with the exception of hydroxypyruvate. The data indicate that KIC diminishes flux through glutamine synthetase by lowering the intraastrocytic glutamate concentration below the K_m of glutamine synthetase for glutamate, which we determined to be ～7 mM.     

Freeze-fracturing and surface labelling of embryonic neural retina cells

Freeze-fracturing of dissociated and aggregating neural retina cells from 7-day chick embryos revealed on the inner faces (PF) of the cell membrane numerous particles 6–20 nm in size. In contrast, the PF faces of blebs and some of the lobopodia that project from the cell surface were practically devoid of such particles. However, the elongated filopodia that abound on these cells showed numerous particles on their PF faces. These regional differences in the distribution of particles on PF faces of these cells are interpreted as reflecting membrane activity that leads to the formation of blebs and lobopodia. The frequent presence of “pits” at the basis of blebs and lobopodia is described. It is suggested that the “pits” are associated with the formation of these membrane projections; they may represent anchoring sites for microfilaments and for microtubules involved in the dynamic structure of the cell surface. ConA-binding sites on these cells were studied by scanning electron microscopy, using labeling with hemocyanin. The distribution of these sites on different regions of the cell surface coincided with the regional differences in the distribution of the inner membrane particles.     

Leishmania infection in humans, dogs and sandflies in a visceral leishmaniasis endemic area in Maranhão, Brazil

Leishmania infection in humans, dogs and sandflies was examined in the endemic visceral leishmaniasis (VL) municipality of Raposa, state of Maranh?o, Brazil. In this study, we examined Leishmania chagasi infection in the blood serum of both humans and Canis familiaris and the natural Leishmania sp. infection rate in the sandfly vector, Lutzomyia longipalpis. Enzyme-linked immunosorbent assay, indirect immunofluorescence reaction and polymerase chain reaction were performed to detect Leishmania infections in humans, dogs and sandflies, respectively. Overall, 186 out of 986 studied human beings were infected with L. chagasi parasites, representing an infection prevalence of 18.9%. An even higher infection rate was detected in dogs, where 66 (47.8%) out of 138 were infected. Among all Lu. longipalpis captured (n = 1,881), only 26.7% were females. The Leishmania infection frequency for the vector Lu. longipalpis was 1.56%. Remarkably, all infected sandflies were found in the peridomiciliary area. Furthermore, a high incidence of asymptomatic forms of VL in the human and canine populations was observed. The results of this study suggest autochthonous transmission of L. chagasi in this endemic area for visceral leishmaniasis because infection by Leishmania sp. was identified in all important elements of the transmission chain.     

Photosynthetic circadian rhythmicity patterns of Symbiodium,the coral endosymbiotic algae

Biological clocks are self-sustained endogenous timers that enable organisms (from cyanobacteria to humans) to anticipate daily environmental rhythms, and adjust their physiology and behaviour accordingly. Symbiotic corals play a central role in the creation of biologically rich ecosystems based on mutualistic symbioses between the invertebrate coral and dinoflagellate protists from the genus Symbiodinium. In this study, we experimentally establish that Symbiodinium photosynthesis, both as a free-living unicellular algae and as part of the symbiotic association with the coral Stylophora pistillata, is ‘wired’ to the circadian clock mechanism with a ‘free-run’ cycle close to 24 h. Associated photosynthetic pigments also showed rhythmicity under light/dark conditions and under constant light conditions, while the expression of the oxygen-evolving enhancer 1 gene (within photosystem II) coincided with photosynthetically evolved oxygen in Symbiodinium cultures. Thus, circadian regulation of the Symbiodinium photosynthesis is, however, complicated as being linked to the coral/host that have probably profound physiochemical influence on the intracellular environment. The temporal patterns of photosynthesis demonstrated here highlight the physiological complexity and interdependence of the algae circadian clock associated in this symbiosis and the plasticity of algae regulatory mechanisms downstream of the circadian clock.     

A multifaceted cellular damage repair and prevention pathway promotes high‐level tolerance to β‐lactam antibiotics

Bactericidal antibiotics are powerful agents due to their ability to convert essential bacterial functions into lethal processes. However, many important bacterial pathogens are remarkably tolerant against bactericidal antibiotics due to inducible damage repair responses. The cell wall damage response two‐component system VxrAB of the gastrointestinal pathogen Vibrio cholerae promotes high‐level β‐lactam tolerance and controls a gene network encoding highly diverse functions, including negative control over multiple iron uptake systems. How this system contributes to tolerance is poorly understood. Here, we show that β‐lactam antibiotics cause an increase in intracellular free iron levels and collateral oxidative damage, which is exacerbated in the ∆vxrAB mutant. Mutating major iron uptake systems dramatically increases ∆vxrAB tolerance to β‐lactams. We propose that VxrAB reduces antibiotic‐induced toxic iron and concomitant metabolic perturbations by downregulating iron uptake transporters and show that iron sequestration enhances tolerance against β‐lactam therapy in a mouse model of cholera infection. Our results suggest that a microorganism''s ability to counteract diverse antibiotic‐induced stresses promotes high‐level antibiotic tolerance and highlights the complex secondary responses elicited by antibiotics.     

A mutation in SNAP29, coding for a SNARE protein involved in intracellular trafficking, causes a novel neurocutaneous syndrome characterized by cerebral dysgenesis, neuropathy, ichthyosis, and palmoplantar keratoderma

Neurocutaneous syndromes represent a vast, largely heterogeneous group of disorders characterized by neurological and dermatological manifestations, reflecting the common embryonic origin of epidermal and neural tissues. In the present report, we describe a novel neurocutaneous syndrome characterized by cerebral dysgenesis, neuropathy, ichthyosis, and keratoderma (CEDNIK syndrome). Using homozygosity mapping in two large families, we localized the disease gene to 22q11.2 and identified, in all patients, a 1-bp deletion in SNAP29, which codes for a SNARE protein involved in vesicle fusion. SNAP29 expression was decreased in the skin of the patients, resulting in abnormal maturation of lamellar granules and, as a consequence, in mislocation of epidermal lipids and proteases. These data underscore the importance of vesicle trafficking regulatory mechanisms for proper neuroectodermal differentiation.