Interferon-induced human MxA GTPase blocks nuclear import of Thogoto virus nucleocapsids.
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Interferon-induced human MxA protein belongs to the dynamin superfamily of large GTPases. It exhibits antiviral activity against a variety of RNA viruses, including Thogoto virus, an influenza virus-like orthomyxovirus transmitted by ticks. Here, we report that MxA blocks the transport of Thogoto virus nucleocapsids into the nucleus, thereby preventing transcription of the viral genome. This interaction can be abolished by a mAb that neutralizes the antiviral activity of MxA. Our results reveal an antiviral mechanism whereby an interferon-induced protein traps the incoming virus and interferes with proper transport of the viral genome to its ultimate target compartment within the infected cell. (+info)
Human deafness dystonia syndrome is a mitochondrial disease.
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The human deafness dystonia syndrome results from the mutation of a protein (DDP) of unknown function. We show now that DDP is a mitochondrial protein and similar to five small proteins (Tim8p, Tim9p, Tim10p, Tim12p, and Tim13p) of the yeast mitochondrial intermembrane space. Tim9p, Tim10p, and Tim12p mediate the import of metabolite transporters from the cytoplasm into the mitochondrial inner membrane and interact structurally and functionally with Tim8p and Tim13p. DDP is most similar to Tim8p. Tim8p exists as a soluble 70-kDa complex with Tim13p and Tim9p, and deletion of Tim8p is synthetically lethal with a conditional mutation in Tim10p. The deafness dystonia syndrome thus is a novel type of mitochondrial disease that probably is caused by a defective mitochondrial protein-import system. (+info)
Reversing adipocyte differentiation: implications for treatment of obesity.
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Conventional treatment of obesity reduces fat in mature adipocytes but leaves them with lipogenic enzymes capable of rapid resynthesis of fat, a likely factor in treatment failure. Adenovirus-induced hyperleptinemia in normal rats results in rapid nonketotic fat loss that persists after hyperleptinemia disappears, whereas pair-fed controls regain their weight in 2 weeks. We report here that the hyperleptinemia depletes adipocyte fat while profoundly down-regulating lipogenic enzymes and their transcription factor, peroxisome proliferator-activated receptor (PPAR)gamma in epididymal fat; enzymes of fatty acid oxidation and their transcription factor, PPARalpha, normally low in adipocytes, are up-regulated, as are uncoupling proteins 1 and 2. This transformation of adipocytes from cells that store triglycerides to fatty acid-oxidizing cells is accompanied by loss of the adipocyte markers, adipocyte fatty acid-binding protein 2, tumor necrosis factor alpha, and leptin, and by the appearance of the preadipocyte marker Pref-1. These findings suggest a strategy for the treatment of obesity by alteration of the adipocyte phenotype. (+info)
Human molybdopterin synthase gene: identification of a bicistronic transcript with overlapping reading frames.
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A universal molybdenum-containing cofactor (MoCo) is essential for the activity of all human molybdoenzymes, including sulphite oxidase. The free cofactor is highly unstable, and all organisms share a similar biosynthetic pathway. The involved enzymes exhibit homologies, even between bacteria and humans. We have exploited these homologies to isolate a cDNA for the heterodimeric molybdopterin (MPT)-synthase. This enzyme is necessary for the conversion of an unstable precursor into molybdopterin, the organic moiety of MoCo. The corresponding transcript shows a bicistronic structure, encoding the small and large subunits of the MPT-synthase in two different open reading frames (ORFs) that overlap by 77 nucleotides. In various human tissues, only one size of mRNA coinciding with the bicistronic transcript was detected. In vitro translation and mutagenesis experiments demonstrated that each ORF is translated independently, leading to the synthesis of a 10-kDa protein and a 21-kDa protein for the small and large subunits, respectively, and indicated that the 3'-proximal ORF of the bicistronic transcript is translated by leaky scanning. (+info)
Apolipoprotein B in the rough endoplasmic reticulum: translation, translocation and the initiation of lipoprotein assembly.
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Apolipoprotein (apo) B and the microsomal triglyceride transfer protein are essential for the hepatic assembly and secretion of triglyceride-rich VLDL. To understand how apoB initiates the process of lipoprotein formation, interest has focused on the biogenesis of its amino terminal globular domain (alpha1 domain). When only this domain is expressed in hepatoma cells, no lipoprotein particle will form. However, proper folding of the alpha1 domain is essential for the internal lipophilic regions of apoB to engage in cotranslational lipid recruitment. The essential function of this domain may be related to its capacity to promote a specific physical interaction with the microsomal triglyceride transfer protein, necessary for apoB's proper folding and lipidation. Alternatively, this domain may promote an autonomous lipid recruitment step that nucleates microsomal triglyceride transfer protein-dependent lipid sequestration by apoB. Forms of apoB that fail to initiate particle assembly or forms associated with aberrant underlipidated particles are targeted for intracellular turnover. Two sites of apoB degradation have been identified. In hepatocarcinoma-derived cells, misassembled apoB may undergo progressive reverse translocation from the endoplasmic reticulum lumen to the cytosol, a process that is mechanistically coupled to polyubiquitination and proteasome-mediated degradation on the cytosolic side of the membrane. Alternatively, studies in primary hepatocytes reveal that apoB may undergo sorting to a post-endoplasmic reticulum compartment for presecretory degradation. In either case, the balance between assembly and presecretory degradation of apoB may represent a control point for the production of hepatic VLDL. (+info)
The yeast non-Mendelian factor [ETA+] is a variant of [PSI+], a prion-like form of release factor eRF3.
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The yeast non-Mendelian factor [ETA+] is lethal in the presence of certain mutations in the SUP35 and SUP45 genes, which code for the translational release factors eRF3 and eRF1, respectively. One such mutation, sup35-2, is now shown to contain a UAG stop codon prior to the essential region of the gene. The non-Mendelian inheritance of [ETA+] is reminiscent of the yeast [PSI+] element, which is due to a self-propagating conformation of Sup35p. Here we show that [ETA+] and [PSI+] share many characteristics. Indeed, like [PSI+], the maintenance of [ETA+] requires the N-terminal region of Sup35p and depends on an appropriate level of the chaperone protein Hsp104. Moreover, [ETA+] can be induced de novo by excess Sup35p, and [ETA+] cells have a weak nonsense suppressor phenotype characteristic of weak [PSI+]. We conclude that [ETA+] is actually a weak, unstable variant of [PSI+]. We find that although some Sup35p aggregates in [ETA+] cells, more Sup35p remains soluble in [ETA+] cells than in isogenic strong [PSI+] cells. Our data suggest that the amount of soluble Sup35p determines the strength of translational nonsense suppression associated with different [PSI+] variants. (+info)
RNA-cytoskeletal associations.
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It has become evident over the past years that a large fraction of messenger RNAs is tightly associated with the cytoskeleton. Whereas microtubules are involved in RNA-cytoskeletal association in large cells like oocytes, neurons, or oligodendrocytes, microfilaments play the major role in smaller somatic cell types. Association of RNA with cytoskeletal filaments clearly is required for mRNA transport, but also appears to be crucial for efficient protein synthesis. Recent data now shed light on how mRNAs attach to the cytoskeleton. Messenger RNA seems to interact with microtubules or microfilaments in the form of large ribonucleoprotein particles, which in some cases also contain components of the protein synthesis apparatus. Recently, a number of RNA binding proteins have been identified in flies, amphibians, and mammals that are essential for the interaction of mRNA with cytoskeletal filaments or with microtubule- or actin-associated proteins. Such proteins include heterologous ribonucleoproteins, which are also involved in nuclear export of RNA. (+info)
RNA determinants for translational editing. Mischarging a minihelix substrate by a tRNA synthetase.
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The fidelity of protein synthesis requires efficient discrimination of amino acid substrates by aminoacyl-tRNA synthetases. Accurate discrimination of the structurally similar amino acids, valine and isoleucine, by isoleucyl-tRNA synthetase (IleRS) results, in part, from a hydrolytic editing reaction, which prevents misactivated valine from being stably joined to tRNAIle. The editing reaction is dependent on the presence of tRNAIle, which contains discrete D-loop nucleotides that are necessary to promote editing of misactivated valine. RNA minihelices comprised of just the acceptor-TPsiC helix of tRNAIle are substrates for specific aminoacylation by IleRS. These substrates lack the aforementioned D-loop nucleotides. Because minihelices contain determinants for aminoacylation, we thought that they might also play a role in editing that has not previously been recognized. Here we show that, in contrast to tRNAIle, minihelixIle is unable to trigger the hydrolysis of misactivated valine and, in fact, is mischarged with valine. In addition, mutations in minihelixIle that enhance or suppress charging with isoleucine do the same with valine. Thus, minihelixIle contains signals for charging (by IleRS) that are independent of the amino acid and, by itself, minihelixIle provides no determinants for editing. An RNA hairpin that mimics the D-stem/loop of tRNAIle is also unable to induce the hydrolysis of misactivated valine, both by itself and in combination with minihelixIle. Thus, the native tertiary fold of tRNAIle is required to promote efficient editing. Considering that the minihelix is thought to be the more ancestral part of the tRNA structure, these results are consistent with the idea that, during the development of the genetic code, RNA determinants for editing were added after the establishment of an aminoacylation system. (+info)