RESUMO
Proteolysis is an essential post-transcriptional process. The lysosome/vacuole is the central organelle for non-specific proteolysis in eukaryotes. Most proteases that work in the lysosome enter it via the secretory pathway. The bulk of proteins to be degraded enter this proteolytic compartment via endocytosis and autophagocytosis. Our understanding of the mechanisms involved in these processes has increased considerably, and the information obtained in these studies has enabled new, specific proteolytic pathways to be investigated in other cellular compartments, particularly in the cytoplasm.
Assuntos
Endopeptidases/metabolismo , Lisossomos/enzimologia , Processamento de Proteína Pós-Traducional , Vacúolos/enzimologia , Animais , Transporte Biológico , Catálise , Endocitose , Ativação Enzimática , Células Eucarióticas/metabolismo , Proteínas Fúngicas/metabolismo , Fagocitose , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/ultraestrutura , Especificidade por SubstratoRESUMO
Glutamate receptors of the kainate-preferring subtype have recently been shown to mediate synaptic transmission in the hippocampus. The low-affinity kainate receptor subunit GluR7 was found to be nonfunctional in previous studies. We report here that the GluR7 subunit and a novel carboxy-terminal splice variant, GluR7b, are functional glutamate receptors with unique pharmacological properties. In particular, glutamate exhibits a 10-fold lower potency for (non-desensitized) GluR7-mediated currents as compared to other non-NMDA receptor channels. These data will facilitate understanding of the distinct role played by GluR7 receptors in synaptic transmission.
Assuntos
DNA Recombinante , Variação Genética/fisiologia , Ácido Glutâmico/farmacologia , Receptores de Ácido Caínico/efeitos dos fármacos , Receptores de Ácido Caínico/genética , Receptores de Ácido Caínico/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Dados de Sequência Molecular , Ratos , Receptor de GluK3 CainatoRESUMO
The ionotropic glutamate receptor subunit GluR6 undergoes developmentally and regionally regulated Q/R site RNA editing that reduces the calcium permeability of GluR6-containing kainate receptors. To investigate the functional significance of this editing in vivo, we engineered mice deficient in GluR6 Q/R site editing. In these mutant mice but not in wild types, NMDA receptor-independent long-term potentiation (LTP) could be induced at the medial perforant path-dentate gyrus synapse. This indicates that kainate receptors with unedited GluR6 subunits can mediate LTP. Behavioral analyses revealed no differences from wild types, but mutant mice were more vulnerable to kainate-induced seizures. Together, these results suggest that GluR6 Q/R site RNA editing may modulate synaptic plasticity and seizure vulnerability.
Assuntos
Plasticidade Neuronal/fisiologia , Edição de RNA/fisiologia , Receptores de Ácido Caínico/metabolismo , Convulsões/metabolismo , Sinapses/metabolismo , Animais , Sítios de Ligação/genética , Cálcio/metabolismo , Células Cultivadas , Giro Denteado/citologia , Giro Denteado/metabolismo , Feminino , Técnicas In Vitro , Ácido Caínico , Potenciação de Longa Duração/fisiologia , Masculino , Camundongos , Camundongos Mutantes , Neurônios/metabolismo , Via Perfurante/citologia , Via Perfurante/metabolismo , Receptores de Ácido Caínico/genética , Convulsões/induzido quimicamente , Receptor de GluK2 CainatoRESUMO
We describe here the first example of an exonic polymorphism that affects the primary structure of a human ionotropic glutamate receptor. The human kainate receptor GluR7 gene contains a thymine (T)/guanine (G) nucleotide variation that determines a serine or alanine at position 310 in the extracellular region of GluR7 receptor subunits. Our finding contrasts with a previous report that suggested that GluR7 transcripts were RNA-edited at this site. Whole-cell patch-clamp recordings did not detect differences in receptor activation and desensitization between the human GluR7 receptor isoforms expressed in HEK-293 cells. Analysis of 41 tissue samples obtained from 30 human brains revealed expression level differences between GluR7 alleles expressed in the same brain. The expression level of the allelic GluR7 mRNAs differed in 27 samples from 1.2- to 12.7-fold. Unequal expression level of allelic mRNAs is characteristic for genes that are affected by genomic imprinting or that contain mutations. Genomic imprinting in most cases is conserved between human and mice. However, we did not detect unequal expression of allelic GluR7 mRNAs in mice. Our results are important for future studies that explore a potential role or roles for GluR7 receptors in the brain and for neurological disorders.
Assuntos
Encéfalo/metabolismo , RNA Mensageiro/biossíntese , Receptores de Ácido Caínico/biossíntese , Alelos , Substituição de Aminoácidos , Animais , Linhagem Celular , Éxons/genética , Frequência do Gene , Impressão Genômica/genética , Humanos , Camundongos , Técnicas de Patch-Clamp , Mutação Puntual , Polimorfismo Genético/genética , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Ratos , Receptores de Ácido Caínico/genética , Transfecção , População Branca/genética , Receptor de GluK3 CainatoRESUMO
The physiological significance of RNA editing of transcripts that code for kainate-preferring glutamate receptor subunits is unknown, despite the fact that the functional consequences of this molecular modification have been well characterized in cloned receptor subunits. RNA editing of the codon that encodes the glutamine/arginine (Q/R) site in the second membrane domain (MD2) of glutamate receptor 5 (GluR5) and GluR6 kainate receptor subunits produces receptors with reduced calcium permeabilities and single-channel conductances. Approximately 50% of the GluR5 subunit transcripts from adult rat brain are edited at the Q/R site in MD2. To address the role of glutamate receptor mRNA editing in the brain, we have made two strains of mice with mutations at amino acid 636, the Q/R-editing site in GluR5, using embryonic stem cell-mediated transgenesis. GluR5(RloxP/RloxP) mice encode an arginine at the Q/R site of the GluR5 subunit, whereas GluR5(wt(loxP)/wt(loxP)) mice encode a glutamine at this site, similar to wild-type mice. Mutant animals do not exhibit developmental abnormalities, nor do they show deficits in the behavioral paradigms tested in this study. Kainate receptor current densities were reduced by a factor of six in acutely isolated sensory neurons of dorsal root ganglia from GluR5(RloxP/RloxP) mice compared with neurons from wild-type mice. However, the editing mutant mice did not exhibit altered responses to thermal and chemical pain stimuli. Our investigations with the GluR5-editing mutant mice have therefore defined a set of physiological processes in which editing of the GluR5 subunit is unlikely to play an important role.
Assuntos
Camundongos Mutantes/genética , Receptores de Ácido Caínico/metabolismo , Animais , Comportamento Animal , Eletrofisiologia , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Ácido Caínico , Camundongos , Camundongos Mutantes/fisiologia , Camundongos Mutantes/psicologia , Neurônios/metabolismo , Dor/psicologia , Recombinação Genética , Valores de Referência , Convulsões/induzido quimicamenteRESUMO
The etiology of mood disorders remains elusive, despite our increasing understanding of the neurotransmitter systems and brain regions that are involved. We performed a large family-based association study to test if the human kainate receptor GluR7 gene (GRIK3) is associated with bipolar disorder (BP) or recurrent major depressive disorder (R-MDD). One hundred fifty-three multiplex BP families from the National Institute of Mental Health (NIMH) Genetics Initiative on Bipolar Disorder were analyzed with the transmission disequilibrium test (TDT). We detected a significant linkage disequilibrium (LD) indicated by preferential maternal transmission of the GluR7 S310 allele to R-MDD patients (P = 0.012), but not to bipolar I disorder (BPI) patients (P = 1.00). We performed a second independent study by applying the TDT in 81 parent-offspring triads from families that inherit recurrent early-onset major depressive disorder (RE-MDD). The results from this second study showed only a suggestive maternal association (P = 0.068). Our findings imply that the GluR7 gene is a susceptibility factor in R-MDD and that the glutamatergic receptor system plays a critical role in the disease etiology.
Assuntos
Transtorno Depressivo Maior/genética , Receptores de Ácido Caínico/genética , Alelos , Sequência de Bases , Transtorno Bipolar/genética , DNA/genética , Transtorno Depressivo Maior/metabolismo , Feminino , Humanos , Desequilíbrio de Ligação , Masculino , Mães , Recidiva , Receptor de GluK3 CainatoRESUMO
The vacuolar proteinase yscB (PrB) has been implicated in the final maturation of procarboxypeptidase yscY (pro-CpY) to the mature wild-type form CpYb of 61 kDa. In PrB-deficient mutants, only the proteinase yscA processed form CpYa of 62 kDa is found [Mechler, B., Müller, H. & Wolf, D. H. (1987) EMBO J. 6, 2157-2163]. We report now that, akin to CpY, two forms of mature proteinase yscA (PrA) can be distinguished. In PrB-deficient mutant cells, PrAa, migrating at about 43 kDa in SDS/PAGE, is found, whereas PrAb, found in wild-type cells, had the known molecular mass of 42 kDa. In the PrB-deficient strain, pro-PrA and pro-CpY matured only to the higher-molecular-mass forms, PrAa and CpYa, and the maturation of both precursors was slower than in the isogenic wild-type strain. Pulse-labeling experiments indicated that the mature forms, PrAb or CpYb, are generated directly in the PrB-containing wild-type strain in vivo. In vitro experiments showed that PrB is able to trigger maturation of its 42-kDa pro-PrB precursor to mature PrB in the absence of PrA. Mature PrB and its proteolytic activity, however, shows a higher stability in the presence of mature PrA. The data indicate a molecular and kinetic participation of proteinase yscB in vacuolar hydrolase precursor maturation.
Assuntos
Precursores Enzimáticos/metabolismo , Hidrolases/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Serina Endopeptidases/metabolismo , Vacúolos/enzimologia , Western Blotting , Eletroforese em Gel de Campo Pulsado , Estabilidade Enzimática , Cinética , Processamento de Proteína Pós-Traducional , Serina Endopeptidases/genéticaRESUMO
The codon of the catalytic serine in the active site of the vacuolar serine proteinase yscB (PrB) was changed to alanine, yielding the mutant gene prb1-Ala519. Following replacement of the wild-type PRB1 allele with prb1-Ala519, only a 73-kDa molecule was detected by immunoprecipitation with PrB-specific antiserum. The size of the mutant molecule corresponds to the unprocessed cytoplasmic precursor (pre-super-pro-PrB), as detected in sec61 mutants, when translocation into the endoplasmic reticulum is blocked. However, the mutant molecule is completely translocated into the secretory pathway, as indicated by protection from proteinase K digestion in spheroplast lysates in the absence of detergent. When N-glycosylation was inhibited in prb1-Ala519 mutant cells by tunicamycin, a smaller molecule of about 71 kDa appeared consistent with single N-glycosylation and signal-sequence cleavage of the translocated mutant PrB molecule in the endoplasmic reticulum. Thus, the active-site mutation prevents the wild-type processing of the N-glycosylated 73-kDa precursor of PrB to the 41.5 kDa pro-PrB in the endoplasmic reticulum. In order to characterize the processing of wild-type super-pro-PrB in more detail, we generated antibodies against the non-enzymatic superpeptide domain of the 73-kDa precursor expressed in Escherichia coli. We find that, in addition to pro-PrB, a distinct protein (superpeptide) with a mobility of about 41 kDa in SDS/PAGE is generated in the endoplasmic reticulum. Pulse-chase experiments indicate rapid degradation of the 41-kDa superpeptide in wild-type cells. Correspondingly, the superpeptide was virtually undetectable by immunoblotting wild-type cell extracts. In contrast, no degradation of radioactively labeled 41-kDa superpeptide was observed within 60 min in mutant strains deficient in the vacuolar proteinase yscA (PrA), in which maturation of vacuolar pro-PrB to active PrB is blocked. Accordingly, superpeptide antigenic material was readily detected by immunoblotting cell extracts and enriched in vacuolar preparations of PrA deficient mutant cells. These results indicate that the superpeptide and pro-PrB travel to the vacuole, where the superpeptide is rapidly degraded upon pro-PrB activation to PrB. Using purified vacuoles, rapid degradation of the superpeptide was reconstituted in vitro by addition of either mature PrA or mature PrB. However, the PrA-triggered in vitro degradation of the superpeptide required PrB activity, as this process was inhibited in the presence of the PrB inhibitor chymostatin.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Precursores Enzimáticos/metabolismo , Lisossomos/enzimologia , Saccharomyces cerevisiae/enzimologia , Serina Endopeptidases/metabolismo , Sítios de Ligação , Catálise , Eletroforese em Gel de Poliacrilamida , Retículo Endoplasmático/enzimologia , Precursores Enzimáticos/genética , Escherichia coli/enzimologia , Glicosilação , Hidrólise , Mutagênese Sítio-Dirigida , Plasmídeos , Processamento de Proteína Pós-Traducional , Saccharomyces cerevisiae/efeitos dos fármacos , Serina Endopeptidases/biossíntese , Serina Endopeptidases/genética , Tunicamicina/farmacologiaRESUMO
The G-protein-coupled receptor (GPCR) superfamily is one of the largest classes of proteins in mammalian genomes. GPCRs mediate diverse physiological functions and are the targets of >50% of all clinical drugs. The sequencing of the human genome and large-scale polymorphism discovery efforts have established an abundant source of single nucleotide polymorphisms (SNPs), particularly those that result in a change in the encoded amino acids (cSNPs), many are of which in GPCRs. Although the majority of these cSNPs are assumed not to be disease-causing (nDCs), experimental data on their functional impact are lacking. Here, we have computationally analyzed the distribution of 454 cSNPs within the GPCR gene family and have found that disease-causing cSNPs (DCs) are overrepresented, whereas nDCs are underrepresented or neutral in transmembrane and extracellular loop domains, respectively. This finding reflects the relative importance of these domains to GPCR function and implies different biological characteristics for the two sets of human polymorphisms.