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1.
Biochim Biophys Acta Mol Basis Dis ; 1863(6): 1422-1435, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28235644

RESUMO

The mitochondrial aspartate-glutamate carrier isoform 1 (AGC1) catalyzes a Ca2+-stimulated export of aspartate to the cytosol in exchange for glutamate, and is a key component of the malate-aspartate shuttle which transfers NADH reducing equivalents from the cytosol to mitochondria. By sustaining the complete glucose oxidation, AGC1 is thought to be important in providing energy for cells, in particular in the CNS and muscle where this protein is mainly expressed. Defects in the AGC1 gene cause AGC1 deficiency, an infantile encephalopathy with delayed myelination and reduced brain N-acetylaspartate (NAA) levels, the precursor of myelin synthesis in the CNS. Here, we show that undifferentiated Neuro2A cells with down-regulated AGC1 display a significant proliferation deficit associated with reduced mitochondrial respiration, and are unable to synthesize NAA properly. In the presence of high glutamine oxidation, cells with reduced AGC1 restore cell proliferation, although oxidative stress increases and NAA synthesis deficit persists. Our data suggest that the cellular energetic deficit due to AGC1 impairment is associated with inappropriate aspartate levels to support neuronal proliferation when glutamine is not used as metabolic substrate, and we propose that delayed myelination in AGC1 deficiency patients could be attributable, at least in part, to neuronal loss combined with lack of NAA synthesis occurring during the nervous system development.


Assuntos
Sistemas de Transporte de Aminoácidos/biossíntese , Ácido Aspártico/análogos & derivados , Proliferação de Células , Regulação para Baixo , Proteínas Mitocondriais/biossíntese , Neurônios/metabolismo , Sistemas de Transporte de Aminoácidos Acídicos/deficiência , Sistemas de Transporte de Aminoácidos Acídicos/genética , Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Antiporters/deficiência , Antiporters/genética , Antiporters/metabolismo , Ácido Aspártico/biossíntese , Linhagem Celular , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/genética , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/metabolismo , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/patologia , Humanos , Doenças Mitocondriais/genética , Doenças Mitocondriais/metabolismo , Doenças Mitocondriais/patologia , Neurônios/patologia , Transtornos Psicomotores/genética , Transtornos Psicomotores/metabolismo , Transtornos Psicomotores/patologia
2.
Eur J Med Chem ; 116: 36-45, 2016 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-27043269

RESUMO

An affinity capillary electrophoresis (ACE) method to estimate apparent dissociation constants between bovine brain calmodulin (CaM) and non-peptidic ligands was developed. The method was validated reproducing the dissociation constants of a number of well-known CaM ligands. In particular, the potent antagonist 125-C9 was ad hoc synthesized through an improved synthetic procedure. The ACE method was successfully applied to verify CaM affinity for lubeluzole, a well-known neuroprotective agent recently proved useful to potentiate the activity of anti-cancer drugs. Lubeluzole was slightly less potent than 125-C9 (Kd = 2.9 ± 0.7 and 0.47 ± 0.06 µM, respectively) and displayed Ca(2+)/calmodulin-dependent kinase II (CaMKII) inhibition (IC50 = 40 ± 1 µM). Possible binding modes of lubeluzole to CaM were explored by docking studies based on the X-ray crystal structures of several trifluoperazine-CaM complexes. An estimated dissociation constant in good agreement with the experimental one was found and the main aminoacidic residues and interactions contributing to complex formation were highlighted. The possibility that interference with Ca(2+) pathways may contribute to the previously observed chemosensitizing effects of lubeluzole on human ovarian adenocarcinoma and lung carcinoma cells are discussed.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Calmodulina/metabolismo , Piperidinas/metabolismo , Piperidinas/farmacologia , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Tiazóis/metabolismo , Tiazóis/farmacologia , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/química , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Bovinos , Humanos , Simulação de Acoplamento Molecular , Piperidinas/química , Conformação Proteica , Inibidores de Proteínas Quinases/química , Tiazóis/química
4.
JIMD Rep ; 14: 77-85, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24515575

RESUMO

BACKGROUND: Whole exome sequencing (WES) offers a powerful diagnostic tool to rapidly and efficiently sequence all coding genes in individuals presenting for consideration of phenotypically and genetically heterogeneous disorders such as suspected mitochondrial disease. Here, we report results of WES and functional validation in a consanguineous Indian kindred where two siblings presented with profound developmental delay, congenital hypotonia, refractory epilepsy, abnormal myelination, fluctuating basal ganglia changes, cerebral atrophy, and reduced N-acetylaspartate (NAA). METHODS: Whole blood DNA from one affected and one unaffected sibling was captured by Agilent SureSelect Human All Exon kit and sequenced on the Illumina HiSeq2000. Mutations were validated by Sanger sequencing in all family members. Protein from wild-type and mutant fibroblasts was isolated to assess mutation effects on protein expression and enzyme activity. RESULTS: A novel SLC25A12 homozygous missense mutation, c.1058G>A; p.Arg353Gln, segregated with disease in this kindred. SLC25A12 encodes the neuronal aspartate-glutamate carrier 1 (AGC1) protein, an essential component of the neuronal malate/aspartate shuttle that transfers NADH and H(+) reducing equivalents from the cytosol to mitochondria. AGC1 activity enables neuronal export of aspartate, the glial substrate necessary for proper neuronal myelination. Recombinant mutant p.Arg353Gln AGC1 activity was reduced to 15% of wild type. One prior reported SLC25A12 mutation caused complete loss of AGC1 activity in a child with epilepsy, hypotonia, hypomyelination, and reduced brain NAA. CONCLUSIONS: These data strongly suggest that SLC25A12 disease impairs neuronal AGC1 activity. SLC25A12 sequencing should be considered in children with infantile epilepsy, congenital hypotonia, global delay, abnormal myelination, and reduced brain NAA.

5.
Mol Biotechnol ; 56(2): 157-65, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23949658

RESUMO

The Lpp2981 gene from Legionella pneumophila, the causative agent of Legionnaire's disease, was cloned into the pMWT7 plasmid. The construct was used to express this gene in Escherichia coli. Five different bacterial strains were tested to overexpress the gene but without success. Sequence analysis revealed a cluster of four rare codons near the 5'-end of the gene. These codons were replaced with those commonly used in E. coli. The mutated Lpp2981 gene was successfully expressed in all the E. coli strains tested. The expressed protein (with an apparent molecular mass of 30 kDa) was collected in the insoluble fraction of the cell lysate, purified as inclusion bodies and functionally reconstituted into liposomes. The highest level of overexpression was obtained in E. coli C0214 after 6 h of induction with isopropyl-ß-D-thiogalactopyranoside at 37 °C, yielding 74 mg of purified protein per liter of culture. We conclude that the clustering of rare codons at the 5'-end of the open-reading frame is a critical factor for the heterologous expression of Lpp2981 in E. coli.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Escherichia coli/metabolismo , Legionella pneumophila/metabolismo , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Códon , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Isopropiltiogalactosídeo/farmacologia , Legionella pneumophila/genética , Lipossomos/metabolismo , Mutação , Fases de Leitura Aberta , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
6.
PLoS Pathog ; 8(1): e1002459, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22241989

RESUMO

The Mitochondrial Carrier Family (MCF) is a signature group of integral membrane proteins that transport metabolites across the mitochondrial inner membrane in eukaryotes. MCF proteins are characterized by six transmembrane segments that assemble to form a highly-selective channel for metabolite transport. We discovered a novel MCF member, termed Legionellanucleotide carrier Protein (LncP), encoded in the genome of Legionella pneumophila, the causative agent of Legionnaire's disease. LncP was secreted via the bacterial Dot/Icm type IV secretion system into macrophages and assembled in the mitochondrial inner membrane. In a yeast cellular system, LncP induced a dominant-negative phenotype that was rescued by deleting an endogenous ATP carrier. Substrate transport studies on purified LncP reconstituted in liposomes revealed that it catalyzes unidirectional transport and exchange of ATP transport across membranes, thereby supporting a role for LncP as an ATP transporter. A hidden Markov model revealed further MCF proteins in the intracellular pathogens, Legionella longbeachae and Neorickettsia sennetsu, thereby challenging the notion that MCF proteins exist exclusively in eukaryotic organisms.


Assuntos
Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos/fisiologia , Proteínas de Transporte/metabolismo , Legionella pneumophila/metabolismo , Doença dos Legionários/metabolismo , Proteínas de Membrana/metabolismo , Trifosfato de Adenosina , Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Teste de Complementação Genética , Células HeLa , Humanos , Legionella pneumophila/genética , Legionella pneumophila/patogenicidade , Doença dos Legionários/genética , Proteínas de Membrana/genética , Neorickettsia sennetsu/genética , Neorickettsia sennetsu/metabolismo , Neorickettsia sennetsu/patogenicidade , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
7.
J Biol Chem ; 283(42): 28445-53, 2008 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-18682385

RESUMO

In Saccharomyces cerevisiae, alpha-isopropylmalate (alpha-IPM), which is produced in mitochondria, must be exported to the cytosol where it is required for leucine biosynthesis. Recombinant and reconstituted mitochondrial oxalacetate carrier (Oac1p) efficiently transported alpha-IPM in addition to its known substrates oxalacetate, sulfate, and malonate and in contrast to other di- and tricarboxylate transporters as well as the previously proposed alpha-IPM transporter. Transport was saturable with a half-saturation constant of 75 +/- 4 microm for alpha-IPM and 0.31 +/- 0.04 mm for beta-IPM and was inhibited by the substrates of Oac1p. Though not transported, alpha-ketoisocaproate, the immediate precursor of leucine in the biosynthetic pathway, inhibited Oac1p activity competitively. In contrast, leucine, alpha-ketoisovalerate, valine, and isoleucine neither inhibited nor were transported by Oac1p. Consistent with the function of Oac1p as an alpha-IPM transporter, cells lacking the gene for this carrier required leucine for optimal growth on fermentable carbon sources. Single deletions of other mitochondrial carrier genes or of LEU4, which is the only other enzyme that can provide the cytosol with alpha-IPM (in addition to Oac1p) exhibited no growth defect, whereas the double mutant DeltaOAC1DeltaLEU4 did not grow at all on fermentable substrates in the absence of leucine. The lack of growth of DeltaOAC1DeltaLEU4 cells was partially restored by adding the leucine biosynthetic cytosolic intermediates alpha-ketoisocaproate and alpha-IPM to these cells as well as by complementing them with one of the two unknown human mitochondrial carriers SLC25A34 and SLC25A35. Oac1p is important for leucine biosynthesis on fermentable carbon sources catalyzing the export of alpha-IPM, probably in exchange for oxalacetate.


Assuntos
Acetatos/metabolismo , Transporte Biológico , Leucina/metabolismo , Malatos/metabolismo , Mitocôndrias/metabolismo , Saccharomyces cerevisiae/metabolismo , Acetatos/química , Catálise , Citosol/metabolismo , Hidrolases/metabolismo , Isoleucina/química , Lipossomos/metabolismo , Modelos Biológicos , Modelos Moleculares , Conformação Molecular , Fatores de Tempo
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