Interaction of 14-3-3 protein with regulator of G protein signaling 7 is dynamically regulated by tumor necrosis factor-alpha

Regulators of G protein signaling (RGS) constitute a family of proteins with a conserved RGS domain of approximately 120 amino acids that accelerate the intrinsic GTP hydrolysis of activated Galpha(i) and Galpha(q) subunits. The phosphorylation-dependent interaction of 14-3-3 proteins with a subset of RGS proteins inhibits their GTPase-accelerating activity in vitro. The inhibitory interaction between 14-3-3 and RGS7 requires phosphorylation of serine 434 of RGS7. We now show that phosphorylation of serine 434 is dynamically regulated by TNF-alpha. Cellular stimulation by TNF-alpha transiently decreased the phosphorylation of serine 434 of RGS7, abrogating the inhibitory interaction with 14-3-3. We examined the effect of 14-3-3 on RGS-mediated deactivation kinetics of G protein-coupled inwardly rectifying K(+) channels (GIRKs) in Xenopus oocytes. 14-3-3 inhibited the function of wild-type RGS7, but not that of either RSG7(P436R) or RGS4, two proteins that do not bind 14-3-3. Our findings are the first evidence that extracellular signals can modulate the activity of RGS proteins by regulating their interaction with 14-3-3.     

Myxoma virus leukemia-associated protein is responsible for major histocompatibility complex class I and Fas-CD95 down-regulation and defines scrapins, a new group of surface cellular receptor abductor proteins

Down-modulation of major histocompatibility class I (MHC-I) molecules is a viral strategy for survival in the host. Myxoma virus, a member of the Poxviridae family responsible for rabbit myxomatosis, can down-modulate the expression of MHC-I molecules, but the viral factor(s) has not been described. We cloned and characterized a gene coding for an endoplasmic reticulum (ER)-resident protein containing an atypical zinc finger and two transmembrane domains, which we called myxoma virus leukemia-associated protein (MV-LAP). MV-LAP down-regulated surface MHC-I and Fas-CD95 molecules upon transfection; the mechanism probably involves an exacerbation of endocytosis and was lost when the ER retention signal was removed. In addition, the lytic activity of MHC-I-restricted antigen-specific cytolytic T lymphocytes (CTL) against myxoma virus-infected antigen-presenting target cells was significantly reduced, revealing a strong correlation between MHC-I down-regulation by MV-LAP and CTL killing in vitro. In vivo experiments with a knockout virus showed that MV-LAP is a virulence factor, potentially involved in the immunosuppression characteristic of myxomatosis. Data bank analysis revealed that MV-LAP has homologs in herpesviruses and other poxviruses. We propose the name "scrapins" to define a new group of ER-resident surface cellular receptor abductor proteins. The down-regulation of cell surface molecules by scrapins probably helps protect infected cells during viral infections.     

Tat-vaccinated macaques do not control simian immunodeficiency virus SIVmac239 replication

The regulatory proteins of human immunodeficiency virus may represent important vaccine targets. Here we assessed the role of Tat-specific cytotoxic T lymphocytes (CTL) in controlling pathogenic simian immunodeficiency virus SIVmac239 replication after using a DNA-prime, vaccinia virus Ankara-boost vaccine regimen. Despite the induction of Tat-specific CTL, there was no significant reduction in either peak or viral set point compared to that of controls.     

Molecular structure of a novel cholesterol-responsive A subclass ABC transporter,ABCA9

We recently identified a novel ABC A subclass transporter, ABCA6, in human macrophages. Here, we report the molecular cloning of an additional ABC A subfamily transporter from macrophages denoted ABCA9. The identified coding sequence is 4.9 kb in size and codes for a 1624 amino acid protein product. In accordance with the proposed nomenclature, the novel transporter was designated ABCA9. The putative full-length ABC transporter polypeptide consists of two transmembrane domains and two nucleotide binding folds and thus conforms to the group of full-size ABC transporters. We identified alternative ABCA9 mRNA variants in human macrophages that predict the existence of three truncated forms of the novel transporter. Among the human ABC A subfamily transporters, ABCA9 exhibits the highest amino acid sequence homology with ABCA8 (72%) and ABCA6 (60%), respectively. The striking amino acid sequence similarity between these transporter molecules supports the notion that they represent an evolutionary more recently emerged subgroup within the family of ABC A transporters, which we refer to as "ABCA6-like transporters." ABCA9 mRNA is ubiquitously expressed with the highest mRNA levels in heart, brain, and fetal tissues. Analysis of the genomic structure revealed that the ABCA9 gene consists of 39 exons that are located within a genomic region of approximately 85 kb size on chromosome 17q24.2. In human macrophages, ABCA9 mRNA is induced during monocyte differentiation into macrophages and suppressed by cholesterol import indicating that ABCA9, like other known ABC A subfamily transporters, is a cholesterol-responsive gene. Based on this information, ABCA9 is likely involved in monocyte differentiation and macrophage lipid homeostasis.     

High-level production and secretion of recombinant proteins by the dimorphic yeast Arxula adeninivorans

The non-conventional dimorphic thermo- and salt-resistant yeast Arxula adeninivorans has been developed as a host for heterologous gene expression. For assessment of the system two model genes have been selected: the GFP gene encoding the intracellular green fluorescent protein, and the HSA gene encoding the secreted human serum albumin. The expression system includes two host strains, namely A. adeninivorans LS3, which forms budding cells at 30 degrees C and mycelia at >42 degrees C, and the strain A. adeninivorans 135, which forms mycelia at temperatures as low as 30 degrees C. For expression control the constitutive A. adeninivorans-derived TEF1-promoter and S. cerevisiae-derived PHO5-terminator were selected. The basic A. adeninivorans transformation/expression vector pAL-HPH1 is further equipped with the Escherichia coli-derived hph gene conferring hygromycin B resistance and the 25S rDNA from A. adeninivorans for rDNA targeting. Transformants were obtained for both budding cells and mycelia. In both cell types similar expression levels were achieved and the GFP was localised in the cytoplasm while more than 95% of the HSA accumulated in the culture medium. In initial fermentation trials on a 200-ml shake flask scale maximal HSA product levels were observed after 96 h of cultivation.     

An european pupil project linked to the scientific aims of the experiment AQUARIUS-XENOPUS on the taxi Soyuz flight Andromede to ISS

The German-French biological experiment AQUARIUS-XENOPUS which flew on the Soyuz flight Andromede to the International Space Station ISS (launched October 21, 2001 in Baikonour/Kazakhstan) was extended by an outreach project. Pupils of class 10 to 12 from Ulm/D and Nancy-Tomblaine/F studied swimming behavior of Xenopus tadpoles on ground. They were instructed to perform all experimental steps following the protocol of similar video recordings on ISS. After the flight, they evaluated the kinetics of swimming of both ground controls and space animals. The pupil project included theoretical components to introduce them to the field of gravitational biology. One feature of the project was the exchange of ideas between pupils by meetings which took place in Ulm (June 2001), Nancy (February 2002) and Paris (May 2002). We consider our approach as a successful way to include young people in space experiments on a cheap cost level and to bring ideas of gravitational biology into the curricula of European schools.     

Reduced expression and increased CpG dinucleotide methylation of the rat APOBEC-1 promoter in transgenic rabbits

Editing of apolipoprotein (apo) B mRNA in liver limits the plasma LDL levels in horses, dogs, rats or mice. Species such as man or rabbit do not edit the hepatic apo B mRNA and are therefore susceptible to atherosclerosis and coronary artery disease due to elevated plasma LDL levels. The catalytic subunit APOBEC-1 is the only missing component of the apo B mRNA editing enzyme complex in the human or rabbit liver. Here we describe the generation of transgenic rabbits in which APOBEC-1 expression is mediated by the proximal promoter of the rat APOBEC-1 gene. These transgenic rabbits are healthy and fertile, and rat APOBEC-1 mRNA is expressed in liver, intestine, kidney, lung, brain and muscle. The transgenic APOBEC-1 expression is low and not sufficient to induce editing in rabbit liver. In rat, the proximal APOBEC-1 promoter demonstrates a progressive loss of CpG dinucleotide methylation towards the core promoter region that is entirely unmethylated. In the transgenic rabbits, this distinct pattern of CpG methylation is lost, and throughout the entire rat APOBEC-1 promoter, >90% of the CpGs are methylated. Thus, the weak proximal rat APOBEC-1 promoter appears to be down-regulated in the rabbit and may be species-specific.     

The ATP binding cassette transporter A1 contributes to the secretion of interleukin 1beta from macrophages but not from monocytes

Deficiency of ABCA1 causes high density lipoprotein deficiency and macrophage foam cell formation in Tangier disease. ABCA1 was also postulated to mediate the secretion of IL-1beta from monocytes and macrophages. We investigated the contribution of ABCA1 to IL-1beta secretion from human monocytes and macrophages of normal donors and Tangier disease patients. Neither an anti-ABCA1 antisense oligonucleotide nor ABCA1 deficiency interfered with LPS-induced secretion of IL-1beta from full blood or freshly isolated monocytes. By contrast, anti-ABCA1 antisense oligonucleotides decreased the LPS-induced secretion of IL-beta from macrophages by 30-50%. The secretion of the precursor pro-IL-1beta and TNFalpha was not inhibited. Compared to normal macrophages, LPS-stimulated Tangier disease macrophages secreted less IL-1beta relative to TNFalpha. Also the spontaneous secretion of IL-1beta by Tangier macrophages was lower than by control cells. We conclude that IL-1beta is secreted from monocytes by an ABCA1-independent pathway and from macrophages by ABCA1-dependent and -independent pathways.