UCS proteins: managing the myosin motor

Recent studies indicate that myosin molecular motors interact inside cells with proteins containing a conserved 'UCS' domain. This appears to ensure proper folding of myosin heads so that they can perform their ATP-dependent actin-based motor functions.     

Geranylgeranyl switching regulates GDI-Rab GTPase recycling

Rab GTPases, key regulators of membrane targeting and fusion, require the covalent attachment of geranylgeranyl lipids to their C terminus for function. To elucidate the role of lipid in Rab recycling, we have determined the crystal structure of Rab guanine nucleotide dissociation inhibitor (alphaGDI) in complex with a geranylgeranyl (GG) ligand (H(2)N-Cys-(S-GG)-OMe). The lipid is bound beneath the Rab binding platform in a shallow hydrophobic groove. Mutation of the binding pocket in the brain-specific alphaGDI leads to mental retardation. Strikingly, lipid binding acts through a conserved allosteric switching mechanism to promote release of the GDI-Rab[GDP] complex from the membrane.     

Influence of Genotype on Endoreduplication of Giant Chromosomes and Expressivity of Character eyelessin Drosophila melanogaster

In Drosophila melanogaster lines LA (low activity), HA (high activity), andOregon-R (wild type), the effect of genetic background on endoreplication in giant chromosomes of salivary glands, fecundity, and expression of mutation eywas studied. The degree of chromosome polyteny and the number of adult flies in saturated lines ey _L, ey _H, and ey _Or were significantly higher than in the original lines. The degree of chromosome polyteny was correlated with fecundity. The expressivity of the ey character was shown to be far lower in lines ey _L, ey _H, and ey _Or than in the original eyelessline. In the saturated lines, the eye facets were reduced to a similar degree. All the lines studied displayed clear-cut sexual distinctions in this parameter. In the ey _L line, the coefficients of variation for the degree of chromosome polyteny, fecundity, and expressivity of the ey mutation were much lower than in lines eyeless, ey _LAandey _Or.     

The chemokine receptor CCR5 is not a necessary inflammatory mediator in kainic acid-induced hippocampal injury: evidence for a compensatory effect by increased CCR2 and CCR3

Chemokines and their receptors have been strongly implicated in the inflammatory process. However, their roles in excitotoxic brain injury are largely unknown. In this study we used C-C chemokine receptor 5 (CCR5) knockout (KO) mice to investigate the role of CCR5 in neurodegeneration induced by intranasal administration of the excitotoxin kainic acid (KA). Although KA treatment resulted in an increased CCR5 mRNA level in the hippocampi of wild-type mice, a CCR5 deficiency in KO mice did not affect either the clinical and pathological changes in vivo or the neuronal susceptibilities to KA insult in vitro. KA treatment stimulated mRNA expression of the monocyte chemoattractant protein-2 (MCP-2) in both the wild-type and KO mice. KA treatment did not affect mRNA levels for the macrophage inflammatory protein-1alpha (MIP-1alpha) or the regulated upon activation normal T cells expressed and secreted protein (RANTES) in either wild-type or CCR5 KO mice. CCR2 mRNA expression was undetectable in the hippocampi of wild-type mice regardless of KA treatment. In contrast, CCR5 KO mice showed CCR2 mRNA expression that was remarkably increased after KA treatment. KA treatment did not affect CCR3 mRNA expression in the wild-type mice, whereas KO mice showed both a higher basal level of CCR3 mRNA expression as well as a strong upregulation following KA treatment. These results indicate that CCR5 is not a necessary inflammatory mediator in KA induced neurodegeneration. The roles of CCR5 in excitotoxic injury in CCR5 deficient mice are compensated by increased CCR2 and CCR3 expression, which share the common MCP-2 ligand with CCR5.     

Palmitoylation of the Autographa californica multicapsid nucleopolyhedrovirus envelope glycoprotein GP64: mapping,functional studies,and lipid rafts

Budded virions (BV) of the baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) contain a major envelope glycoprotein known as GP64, which was previously shown to be palmitoylated. In the present study, we used truncation and amino acid substitution mutations to map the palmitoylation site to cysteine residue 503. Palmitoylation of GP64 was not detected when Cys503 was replaced with alanine or serine. Palmitoylation-minus forms of GP64 were used to replace wild-type GP64 in AcMNPV, and these viruses were used to examine potential functions of GP64 palmitoylation in the context of the infection cycle. Analysis by immunoprecipitation and cell surface studies revealed that palmitoylation of GP64 did not affect GP64 synthesis or its transport to the cell surface in Sf9 cells. GP64 proteins lacking palmitoylation also mediated low-pH-triggered membrane fusion in a manner indistinguishable from that of wild-type GP64. Cells infected with viruses expressing palmitoylation-minus forms of GP64 produced infectious virions at levels similar to those from cells infected with wild-type AcMNPV. In combination, these data suggest that virus entry and exit in Sf9 cells were not significantly affected by GP64 palmitoylation. To determine whether GP64 palmitoylation affected the association of GP64 with membrane microdomains, the potential association of GP64 with lipid raft microdomains was examined. These experiments showed that: (i) AcMNPV-infected Sf9 cell membranes contain lipid raft microdomains, (ii) GP64 association with lipid rafts was not detected in infected Sf9 cells, and (iii) GP64 palmitoylation did not affect the apparent exclusion of GP64 from lipid raft microdomains.     

Human immunodeficiency virus type 1 Vif is efficiently packaged into virions during productive but not chronic infection

Packaging of the human immunodeficiency virus type 1 Vif protein into virus particles is mediated through an interaction with viral genomic RNA and results in the association of Vif with the nucleoprotein complex. Despite the specificity of this process, calculations of the amount of Vif packaged have produced vastly different results. Here, we compared the efficiency of packaging of Vif into virions derived from acutely and chronically infected H9 cells. We found that Vif was efficiently packaged into virions from acutely infected cells (60 to 100 copies per virion), while packaging into virions from chronically infected H9 cells was near the limit of detection (four to six copies of Vif per virion). Superinfection by an exogenous Vif-defective virus did not rescue packaging of endogenous Vif expressed in the chronically infected culture. In contrast, exogenous Vif expressed by superinfection of wild-type virus was readily packaged (30 to 40 copies per virion). Biochemical analyses suggest that the differences in the relative packaging efficiencies were not due to gross differences in the steady-state distribution of Vif in chronically or acutely infected cells but are likely due to differences in the relative rates of de novo synthesis of Vif. Despite its low packaging efficiency, endogenously expressed Vif was sufficient to direct the production of viruses with almost wild-type infectivity. The results from our study provide novel insights into the biochemical properties of Vif and offer an explanation for the reported differences regarding Vif packaging.     

Mu-opioid receptor desensitization: role of receptor phosphorylation,internalization, and representation

It is generally accepted that the internalization and desensitization of mu-opioid receptor (MOR) involves receptor phosphorylation and beta-arrestin recruitment. However, a mutant MOR, which is truncated after the amino acid residue Ser363 (MOR363D), was found to undergo phosphorylation-independent internalization and desensitization. As expected, MOR363D, missing the putative agonist-induced phosphorylation sites, did not exhibit detectable agonist-induced phosphorylation. MOR363D underwent slower internalization as reflected in the attenuation of membrane translocation of beta-arrestin 2 when compared with wild type MOR, but the level of receptor being internalized was similar to that of wild type MOR after 4 h of etorphine treatment. Furthermore, MOR363D was observed to desensitize faster than that of wild type MOR upon agonist activation. Surface biotinylation assay demonstrated that the wild type receptors recycled back to membrane after agonist-induced internalization, which contributed to the receptor resensitization and thus partially reversed the receptor desensitization. On the contrary, MOR363D did not recycle after internalization. Hence, MOR desensitization is controlled by the receptor internalization and the recycling of internalized receptor to cell surface in an active state. Taken together, our data indicated that receptor phosphorylation is not absolutely required in the internalization, but receptor phosphorylation and subsequent beta-arrestin recruitment play important roles in the resensitization of internalized receptors.