RESUMEN
Aging is a natural process that internal gene control and external stimuli mediate. Clinical data pointed out that homozygotic or heterozygotic mutation in the pyrroline-5-carboxylate reductase 1 (PYCR1) gene in humans caused cutis laxa (ARCL) disease, with progeroid appearance, lax and wrinkled skin, joint laxity, osteopenia, and mental retardation phenotypes. In this study, we aimed to generate pycr1 knockout (KO) zebrafish and carried out biochemical characterizations and behavior analyses. Marked apoptosis and senescence were detected in pycr1 KO zebrafish, which started from embryos/larvae stage. Biochemical assays showed that adult pycr1 KO fish have significantly reduced proline and extracellular matrix contents, lowered energy, and diminished superoxide dismutase (SOD) and telomerase activity when compared to the wild type fish, which suggested the pycr1 KO fish may have dysfunction in mitochondria. The pycr1 KO fish were viable; however, displayed progeria-like phenotype from the 4 months old and reach 50% mortality around six months old. In adult stage, we found that pycr1 KO fish showed reduced locomotion activity, aggression, predator avoidance, social interaction interest, as well as dysregulated color preference and circadian rhythm. In summary, we have identified multiple behavioral alterations in a novel fish model for aging with pycr1 gene loss-of-function by behavioral tests. This animal model may not only provide a unique vertebrate model to screen potential anti-aging drugs in the future, but also be an excellent in vivo model towards a better understanding of the corresponding behavioral alterations that accompany aging.
Asunto(s)
Envejecimiento/genética , Síntomas Conductuales/genética , Modelos Animales de Enfermedad , Modelos Animales , Progeria/genética , Pirrolina Carboxilato Reductasas/genética , Pez Cebra/genética , Envejecimiento/metabolismo , Animales , Metabolismo Energético , Matriz Extracelular/metabolismo , Técnicas de Inactivación de Genes , Locomoción , Mutación con Pérdida de Función , Mitocondrias/metabolismo , Prolina/metabolismo , Pirrolina Carboxilato Reductasas/fisiología , Superóxido Dismutasa/metabolismoRESUMEN
We have conducted biochemical studies with commercial available pyrroline-5-carboxylate (P5C) reductase (PYCR1) to investigate whether this enzyme plays a role in L-lysine degradation. Our recent studies with antiquitin/ALDH7A1 deficient fibroblasts revealed an alternative genesis of L-pipecolic acid, and we then hypothesized that PYCR1 was responsible for the conversion of Δ(1)-piperideine-6-carboxylate (P6C) into pipecolic acid. We here present evidence that PYCR1 is indeed able to produce L-pipecolic acid from P6C preparations, and the observed K m for this conversion is of the same magnitude as the K m described for the conversion of P5C to L-proline by PYCR1. Urine samples from antiquitin deficient individuals, who accumulate P6C, were also incubated with PYCR1 which resulted in a marked decrease of P6C and a huge increase of L-pipecolic acid as measured by LC-MS/MS, confirming that indeed PYCR1 generates L-pipecolic acid from P6C.
Asunto(s)
Ácidos Picolínicos/metabolismo , Ácidos Pipecólicos/metabolismo , Pirrolina Carboxilato Reductasas/fisiología , Humanos , delta-1-Pirrolina-5-Carboxilato ReductasaRESUMEN
The complete Bacillus subtilis genome contains four genes (proG, proH, proI, and comER) with the potential to encode Delta(1)-pyrroline-5-carboxylate reductase, a proline biosynthetic enzyme. Simultaneous defects in three of these genes (proG, proH, and proI) were required to confer proline auxotrophy, indicating that the products of these genes are mostly interchangeable with respect to the last step in proline biosynthesis.
Asunto(s)
Bacillus subtilis/enzimología , Genes Bacterianos , Prolina/biosíntesis , Pirrolina Carboxilato Reductasas/fisiología , Arginasa/metabolismo , Mutagénesis , Pirrolina Carboxilato Reductasas/genética , delta-1-Pirrolina-5-Carboxilato ReductasaRESUMEN
The Arabidopsis thaliana gene that encodes pyrroline-5-carboxylate reductase (At-P5R), the last enzyme in proline biosynthesis in A. thaliana, is developmentally regulated and is highly expressed in cells that divide rapidly or undergo changes in osmotic potential. A 69 bp region (P69; -120 to -51) has previously been identified in a 5' deletion analysis of the At-P5R promoter to be necessary for the basal expression. Here, the essential role of P69 for tissue-specific expression of At-P5R is demonstrated by loss- and gain-of-function experiments.
Asunto(s)
Arabidopsis/genética , Caulimovirus/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Fragmentos de Péptidos/genética , Regiones Promotoras Genéticas , Pirrolina Carboxilato Reductasas/genética , ARN Ribosómico/genética , Arabidopsis/enzimología , Secuencia de Bases , Regulación de la Expresión Génica/fisiología , Regulación Viral de la Expresión Génica/fisiología , Genes de Plantas , Genes Reporteros , Glucuronidasa/biosíntesis , Glucuronidasa/genética , Datos de Secuencia Molecular , Fragmentos de Péptidos/fisiología , Plantas Modificadas Genéticamente , Regiones Promotoras Genéticas/fisiología , Pirrolina Carboxilato Reductasas/fisiología , Semillas/genética , Eliminación de SecuenciaRESUMEN
Direct evidence is presented for a proline cycle using a cell-free experimental system which sequentially transfers 3H from [1-3H]glucose to NADP+ to delta 1-pyrroline-5-carboxylate and yields [3H]proline. The formation of [3H]proline depends on the presence of NADP, delta 1-pyrroline-5-carboxylate, and the enzymes glucose-6-phosphate dehydrogenase and delta 1-pyrroline-5-carboxylate reductase. The production of [3H]proline from unlabeled proline in the presence of mitochondria provides direct evidence for one complete turn of a proline cycle which transfers reducing equivalents produced by glucose oxidation in the pentose pathway into mitochondria. In this cycle, proline is oxidized to delta 1-pyrroline-5-carboxylate by mitochondrial proline oxidase. delta 1-pyrroline-5-carboxylate is released from mitochondria and is recycled back to proline by delta 1-pyrroline-5-carboxylate reductase with concomitant oxidation of NADPH. At the maximal rate observed, 60% of delta 1-pyrroline-5-carboxylate produced is recycled back to proline. This cycle provides a mechanism for transferring reducing equivalents from NADPH into mitochondria and is linked to glucose oxidation in the pentose pathway by NADPH turnover.
