RESUMEN
BACKGROUND: Meiotic prophase is a critical stage in sexual reproduction. Aberrant chromosome recombination during this stage is a leading cause of human miscarriages and birth defects. However, due to the experimental intractability of mammalian gonads, only a very limited number of meiotic genes have been characterized. Here we aim to identify novel meiotic genes important in human reproduction through computational mining of cross-species and cross-sex time-series expression data from budding yeast, mouse postnatal testis, mouse embryonic ovary, and human fetal ovary. RESULTS: Orthologous gene pairs were ranked by order statistics according to their co-expression profiles across species, allowing us to infer conserved meiotic genes despite obvious differences in cellular synchronicity and composition in organisms. We demonstrated that conserved co-expression networks could successfully recover known meiotic genes, including homologous recombination genes, chromatin cohesion genes, and genes regulating meiotic entry. We also showed that conserved co-expression pairs exhibit functional connections, as evidenced by the annotation similarity in Gene Ontology and overlap with physical interactions. More importantly, we predicted six new meiotic genes through their co-expression linkages with known meiotic genes, and subsequently used the genetically more amenable yeast system for experimental validation. The deletion mutants of all six genes showed sporulation defects, equivalent to a 100% validation rate. CONCLUSIONS: We identified evolutionarily conserved gene modules in meiotic prophase by integrating cross-species and cross-sex expression profiles from budding yeast, mouse, and human. Our co-expression linkage analyses confirmed known meiotic genes and identified several novel genes that might be critical players in meiosis in multiple species. These results demonstrate that our approach is highly efficient to discover evolutionarily conserved novel meiotic genes, and yeast can serve as a valuable model system for investigating mammalian meiotic prophase.
Asunto(s)
Regulación de la Expresión Génica , Profase/genética , Reproducción/genética , Saccharomycetales/citología , Saccharomycetales/genética , Animales , Secuencia Conservada , Redes Reguladoras de Genes , Humanos , Ratones , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reproducibilidad de los Resultados , Especificidad de la EspecieRESUMEN
Calsequestrin (CASQ) is a major Ca(2+) storage protein within the sarcoplasmic reticulum (SR) of both cardiac and skeletal muscles. CASQ reportedly acts as a Ca(2+) buffer and Ca(2+)-channel regulator through its unique Ca(2+)-dependent oligomerization, maintaining the free Ca(2+) concentration at a low level (0.5-1mM) and the stability of SR Ca(2+) releases. Our approach, employing isothermal titration calorimetry and light scattering in parallel, has provided valuable information about the affinity of human cardiac CASQ (hCASQ2) for a variety of drugs, which have been associated with heart- or muscle-related side effects. Those strongly binding drugs included phenothiazines, anthracyclines and Ca(2+) channel blockers, such as trifluoperazine, thioridazine, doxorubicin, daunorubicin, amlodipine and verapamil, having an average affinity of ~18 µM. They exhibit an inhibitory effect on in vitro Ca(2+)-dependent polymerization of hCASQ2 in a manner proportional to their binding affinity. Therefore accumulation of such drugs in the SR could significantly hinder the Ca(2+)-buffering capacity of the SR and/or the regulation of the Ca(2+) channel, RyR2. These effects could result in serious cardiac problems in people who have genetically impaired hCASQ2, defects in other E-C coupling components or problems with metabolism and clearance of those drugs.
Asunto(s)
Calsecuestrina/efectos adversos , Calsecuestrina/metabolismo , Miocardio/metabolismo , Preparaciones Farmacéuticas/metabolismo , Calcio , Calorimetría , Calsecuestrina/química , Humanos , Miocardio/citología , Unión Proteica , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Retículo Sarcoplasmático/metabolismoRESUMEN
Burkholderia cepacia AC1100 completely degrades 2,4,5-trichlorophenol, in which an FADH(2)-dependent monooxygenase (TftD) and an NADH:FAD oxidoreductase (TftC) catalyze the initial steps. TftD oxidizes 2,4,5-trichlorophenol (2,4,5-TCP) to 2,5-dichloro-p-benzoquinone, which is chemically reduced to 2,5-dichloro-p-hydroquinone (2,5-DiCHQ). Then, TftD oxidizes the latter to 5-chloro-2-hydroxy-p-benzoquinone. In those processes, TftC provides all the required FADH(2). We have determined the crystal structures of dimeric TftC and tetrameric TftD at 2.0 and 2.5 A resolution, respectively. The structure of TftC was similar to those of related flavin reductases. The stacked nicotinamide:isoalloxazine rings in TftC and sequential reaction kinetics suggest that the reduced FAD leaves TftC after NADH oxidation. The structure of TftD was also similar to the known structures of FADH(2)-dependent monooxygenases. Its His-289 residue in the re-side of the isoalloxazine ring is within hydrogen bonding distance with a hydroxyl group of 2,5-DiCHQ. An H289A mutation resulted in the complete loss of activity toward 2,5-DiCHQ and a significant decrease in catalytic efficiency toward 2,4,5-TCP. Thus, His-289 plays different roles in the catalysis of 2,4,5-TCP and 2,5-DiCHQ. The results support that free FADH(2) is generated by TftC, and TftD uses FADH(2) to separately transform 2,4,5-TCP and 2,5-DiCHQ. Additional experimental data also support the diffusion of FADH(2) between TftC and TftD without direct physical interaction between the two enzymes.
Asunto(s)
Burkholderia cepacia/enzimología , FMN Reductasa/química , FMN Reductasa/metabolismo , Flavina-Adenina Dinucleótido/metabolismo , Oxigenasas de Función Mixta/química , Oxigenasas de Función Mixta/metabolismo , NAD/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Biodegradación Ambiental , Calorimetría , Clorofenoles/metabolismo , Cristalografía por Rayos X , FMN Reductasa/genética , Cinética , Luz , Oxigenasas de Función Mixta/genética , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oxidación-Reducción , Bifenilos Policlorados/aislamiento & purificación , Bifenilos Policlorados/metabolismo , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Dispersión de Radiación , TermodinámicaRESUMEN
Potato (Solanum tuberosum) multicystatin (PMC) is a crystalline Cys protease inhibitor present in the subphellogen layer of potato tubers. It consists of eight tandem domains of similar size and sequence. Our in vitro results showed that the pH/PO(4)(-)-dependent oligomeric behavior of PMC was due to its multidomain nature and was not a characteristic of the individual domains. Using a single domain of PMC, which still maintains inhibitor activity, we identified a target protein of PMC, a putative Cys protease. In addition, our crystal structure of a representative repeating unit of PMC, PMC-2, showed structural similarity to both type I and type II cystatins. The N-terminal trunk, alpha-helix, and L2 region of PMC-2 were most similar to those of type I cystatins, while the conformation of L1 more closely resembled that of type II cystatins. The structure of PMC-2 was most similar to the intensely sweet protein monellin from Dioscorephyllum cumminisii (serendipity berry), despite a low level of sequence similarity. We present a model for the possible molecular organization of the eight inhibitory domains in crystalline PMC. The unique molecular properties of the oligomeric PMC crystal are discussed in relation to its potential function in regulating the activity of proteases in potato tubers.