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Producción Científica, The Fas-activated serine/threonine kinase (FASTK) family of proteins has recently emerged as a central regulator of mitochondrial gene expression through the function of an unusual RNA-binding domain named RAP (for RNA-binding domain abundant in Apicomplexans), shared by all six members of the family. Here we describe the role of one of the less characterized members, FASTKD3, in mitochondrial RNA metabolism. First, we show that, in contrast to FASTK, FASTKD2, and FASTKD5, FASTKD3 does not localize in mitochondrial RNA granules, which are sites of processing and maturation of mtRNAs and ribosome biogenesis. Second, we generated FASTKD3 homozygous knock-out cell lines by homologous recombination and observed that the absence of FASTKD3 resulted in increased steady-state levels and half-lives of a subset of mature mitochondrial mRNAs: ND2, ND3, CYTB, COX2, and ATP8/6. No aberrant processing of RNA precursors was observed. Rescue experiments demonstrated that RAP domain is required for FASTKD3 function in mRNA stability. Besides, we describe that FASTKD3 is required for efficient COX1 mRNA translation without altering mRNA levels, which results in a decrease in the steady-state levels of COX1 protein. This finding is associated with reduced mitochondrial complex IV assembly and activity. Our observations suggest that the function of this family of proteins goes beyond RNA processing and ribosome assembly and includes RNA stability and translation regulation within mitochondria., Junta de Castilla y León (grants BIO/ VA20/15 and BIO/VA21/15), Swiss National Science Foundation (grant 310030B_160257/1)

Producción Científica, Abnormal vascular smooth muscle cell (VSMC) proliferation contributes to occlusive and proliferative disorders of the vessel wall. Salicylate and other nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit VSMC proliferation by an unknown mechanism unrelated to anti-inflammatory activity. In search for this mechanism, we have studied the effects of salicylate and other NSAIDs on subcellular Ca2+ homeostasis and Ca2+-dependent cell proliferation in rat aortic A10 cells, a model of neointimal VSMCs. We found that A10 cells displayed both store-operated Ca2+ entry (SOCE) and voltage-operated Ca2+ entry (VOCE), the former being more important quantitatively than the latter. Inhibition of SOCE by specific Ca2+ released-activated Ca2+ (CRAC/Orai) channels antagonists prevented A10 cell proliferation. Salicylate and other NSAIDs, including ibuprofen, indomethacin, and sulindac, inhibited SOCE and thereby Ca2+-dependent, A10 cell proliferation. SOCE, but not VOCE, induced mitochondrial Ca2+ uptake in A10 cells, and mitochondrial depolarization prevented SOCE, thus suggesting that mitochondrial Ca2+ uptake controls SOCE (but not VOCE) in A10 cells. NSAIDs depolarized mitochondria and prevented mitochondrial Ca2+ uptake, suggesting that they favor the Ca2+-dependent inactivation of CRAC/Orai channels. NSAIDs also inhibited SOCE in rat basophilic leukemia cells where mitochondrial control of CRAC/Orai is well established. NSAIDs accelerate slow inactivation of CRAC currents in rat basophilic leukemia cells under weak Ca2+ buffering conditions but not in strong Ca2+ buffer, thus excluding that NSAIDs inhibit SOCE directly. Taken together, our results indicate that NSAIDs inhibit VSMC proliferation by facilitating the Ca2+-dependent inactivation of CRAC/Orai channels which normally is prevented by mitochondria clearing of entering Ca2+., Junta de Castilla y León (grants SAN191/VA1806 and CSI12A08), Ministerio de Ciencia, Innovación y Universidades (grant BFU2009-0867), Deutsche Forschungsgemeinschaft (grant A3)

CPT1c is a carnitine palmitoyltransferase 1 (CPT1) isoform that is expressed only in the brain. The enzyme has recently been localized in neuron mitochondria. Although it has high sequence identity with the other two CPT1 isoenzymes (a and b), no CPT activity has been detected to date. Our results indicate that CPT1c is expressed in neurons but not in astrocytes of mouse brain sections. Overexpression of CPT1c fused to the green fluorescent protein in cultured cells demonstrates that CPT1c is localized in the endoplasmic reticulum rather than mitochondria and that the N-terminal region of CPT1c is responsible for endoplasmic reticulum protein localization. Western blot experiments with cell fractions from adult mouse brain corroborate these results. In addition, overexpression studies demonstrate that CPT1c does not participate in mitochondrial fatty acid oxidation, as would be expected from its subcellular localization. To identify the substrate of CPT1c enzyme, rat cDNA was overexpressed in neuronal PC-12 cells, and the levels of acylcarnitines were measured by high-performance liquid chromatography-mass spectrometry. Palmitoylcarnitine was the only acylcarnitine to increase in transfected cells, which indicates that palmitoyl-CoA is the enzyme substrate and that CPT1c has CPT1 activity. Microsomal fractions of PC-12 and HEK293T cells overexpressing CPT1c protein showed a significant increase in CPT1 activity of 0.57 and 0.13 nmol·mg-1·min-1, respectively, which is ∼50% higher than endogenous CPT1 activity. Kinetic studies demonstrate that CPT1c has similar affinity to CPT1a for both substrates but 20–300 times lower catalytic efficiency. info:eu-repo/semantics/acceptedVersion

Carnitine palmitoyltransferase (CPT) I, which catalyzes the conversion of palmitoyl-CoA to palmitoylcarnitine facilitating its transport through the mitochondrial membranes, is inhibited by malonyl-CoA. By using the SequenceSpace algorithm program to identify amino acids that participate in malonyl-CoA inhibition in all carnitine acyltransferases, we found 5 conserved amino acids (Thr314, Asn464, Ala478, Met593, and Cys608, rat liver CPT I coordinates) common to inhibitable malonyl-CoA acyltransferases (carnitine octanoyltransferase and CPT I), and absent in noninhibitable malonyl-CoA acyltransferases (CPT II, carnitine acetyltransferase (CAT) and choline acetyltransferase (ChAT)). To determine the role of these amino acid residues in malonyl-CoA inhibition, we prepared the quintuple mutant CPT I T314S/N464D/A478G/M593S/C608A as well as five single mutants CPT I T314S, N464D, A478G, M593S, and C608A. In each case the CPT I amino acid selected was mutated to that present in the same homologous position in CPT II, CAT, and ChAT. Because mutant M593S nearly abolished the sensitivity to malonyl-CoA, two other Met593mutants were prepared: M593A and M593E. The catalytic efficiency (V max/K m) of CPT I in mutants A478G and C608A and all Met593 mutants toward carnitine as substrate was clearly increased. In those CPT I proteins in which Met593 had been mutated, the malonyl-CoA sensitivity was nearly abolished. Mutations in Ala478, Cys608, and Thr314 to their homologous amino acid residues in CPT II, CAT, and ChAT caused various decreases in malonyl-CoA sensitivity. Ala478 is located in the structural model of CPT I near the catalytic site and participates in the binding of malonyl-CoA in the low affinity site (Morillas, M., Gómez-Puertas, P., Rubı́, B., Clotet, J., Ariño, J., Valencia, A., Hegardt, F. G., Serra, D., and Asins, G. (2002) J. Biol. Chem. 277, 11473–11480). Met593 may participate in the interaction of malonyl-CoA in the second affinity site, whose location has not been reported. info:eu-repo/semantics/publishedVersion