RESUMEN
Implementation of network systems biology principles offers unparalleled opportunity to synthesize and integrate inherently complex high-throughput data for meaningful interpretation. Nonstochastic network templates constructed from extant knowledge facilitate large data set prioritization and prognostication, identifying context-dependent candidates for follow-up functional validation and interpretation. In conjunction, enrichment and representation assessment refines comprehension of network-associated molecular functions and biological processes. Together, exploiting intrinsic network properties provides a value-added decision-support instrument for data deconvolution in systems biomedicine.
Asunto(s)
Ensayos Analíticos de Alto Rendimiento/métodos , Redes y Vías Metabólicas , Biología de Sistemas/métodos , Técnicas de Apoyo para la Decisión , HumanosRESUMEN
Systems biology provides a platform for integrating multiple components and interactions underlying cell, organ, and organism processes in health and disease. Beyond traditional approaches focused on individual molecules or pathways, bioinformatic network analysis of high-throughput data sets offers an opportunity for integration of biological complexity and multilevel connectivity. Emerging applications in rational drug discovery range from targeting and modeling disease-corrupted networks to screening chemical or ligand libraries to identification/validation of drug-target interactions for improved efficacy and safety.
Asunto(s)
Biología Computacional/tendencias , Descubrimiento de Drogas/tendencias , Redes Reguladoras de Genes , Biología de Sistemas/tendencias , Sistemas de Liberación de Medicamentos , Evaluación Preclínica de Medicamentos/estadística & datos numéricos , Modelos Estadísticos , Proteínas/fisiología , Receptores de Droga/efectos de los fármacos , Receptores de Droga/genéticaRESUMEN
Proteomic analyses encompass a suite of high-throughput technologies for large-scale separation and identification of proteins responsible for execution of physiological processes. As such, proteomics is ideally suited to dissecting developmental complexity and dynamics, an understanding of which is vital to the realization of regenerative therapeutic medicine. Pharmacoproteomics is increasingly targeting characterization of regenerative therapeutic strategies. A perspective on the application of proteomics to further our understanding of cardiac regenerative medicine, in concert with guided cardiogenic programming, is delineated herein.
Asunto(s)
Farmacogenética/tendencias , Medicina Regenerativa/tendencias , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Corazón/crecimiento & desarrollo , Cardiopatías/terapia , HumanosRESUMEN
Proteomic analysis of rabbit ventricular myocytes revealed a novel posttranslational modification to myosin light chain 1 (MLC1), consisting of phosphorylation at two sites. Subproteomic extraction to isolate myofilament-enriched fractions enabled determination of the extent of phosphorylation, which increased from 25.7+/-1.6% to 34.0+/-2.7% (mean+/-SE, n=4; P<0.05) after adenosine treatment at levels sufficient to pharmacologically precondition the myocytes (100 micromol/L). Mass spectrometry of MLC1 tryptic digests identified two peptide fragments modified by phosphorylation. These two phosphopeptides were characterized by peptide mass fingerprinting to determine the phosphorylation sites within rabbit ventricular MLC1, which correspond to Thr69 and Ser200 of rat MLC1, and to Thr64 and Ser194 or 195 of human MLC1. This proteomic analysis of preconditioned myocardium has revealed a previously unsuspected in vivo posttranslational modification to MLC1.
Asunto(s)
Ventrículos Cardíacos/metabolismo , Cadenas Ligeras de Miosina/metabolismo , Proteoma , Adenosina/farmacología , Animales , Western Blotting , Células Cultivadas , Electroforesis en Gel Bidimensional , Ventrículos Cardíacos/citología , Precondicionamiento Isquémico , Fosforilación/efectos de los fármacos , Procesamiento Proteico-Postraduccional , Conejos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
The development of proteomics is a timely one for cardiovascular research. Analyses at the organ, subcellular, and molecular levels have revealed dynamic, complex, and subtle intracellular processes associated with heart and vascular disease. The power and flexibility of proteomic analyses, which facilitate protein separation, identification, and characterization, should hasten our understanding of these processes at the protein level. Properly applied, proteomics provides researchers with cellular protein "inventories" at specific moments in time, making it ideal for documenting protein modification due to a particular disease, condition, or treatment. This is accomplished through the establishment of species- and tissue-specific protein databases, providing a foundation for subsequent proteomic studies. Evolution of proteomic techniques has permitted more thorough investigation into molecular mechanisms underlying cardiovascular disease, facilitating identification not only of modified proteins but also of the nature of their modification. Continued development should lead to functional proteomic studies, in which identification of protein modification, in conjunction with functional data from established biochemical and physiological methods, has the ability to further our understanding of the interplay between proteome change and cardiovascular disease.
Asunto(s)
Enfermedades Cardiovasculares/metabolismo , Sistema Cardiovascular/metabolismo , Proteínas/metabolismo , Proteoma/metabolismo , Proyectos de Investigación/tendencias , Animales , Enfermedades Cardiovasculares/etiología , Sistema Cardiovascular/química , Cromatografía de Afinidad , Cromatografía Líquida de Alta Presión , Electroforesis en Gel Bidimensional , Humanos , Focalización Isoeléctrica , Espectrometría de Masas , Procesamiento Proteico-Postraduccional , Proteínas/análisis , Proteoma/química , Proteoma/genética , Manejo de Especímenes/métodosRESUMEN
Selective troponin I (TnI) modification has been demonstrated to be in part responsible for the contractile dysfunction observed with myocardial ischemia/reperfusion injury. We have isolated and characterized modified TnI products in isolated rat hearts after 0, 15, or 60 minutes of ischemia followed by 45 minutes of reperfusion using affinity chromatography with cardiac troponin C (TnC) and an anti-TnI antibody, immunological mapping, reversed-phase high-performance liquid chromatography, and mass spectrometry. Rat cardiac TnI becomes progressively degraded from 210 amino acid residues to residues 1-193, 63-193, and 73-193 with increased severity of injury. Degradation is accompanied by formation of covalent complexes between TnI 1-193 and, respectively, TnC residues 1-94 and troponin T (TnT) residues 191-298. The covalent complexes are likely a result of isopeptide bond formation between lysine 193 of TnI and glutamine 191 of TnT by the cross-linking enzyme transglutaminase. With severe ischemia, cellular necrosis results in specific release of TnI 1-193 into the reperfusion effluent and TnT degradation in the myocardium (25-, 27-, and 33-kDa products). Two-dimensional electrophoresis demonstrated that phosphorylation of TnI prevents ischemia-induced degradation. This study characterized the modified TnI products in isolated rat hearts reperfused after a brief or severe period of ischemia, revealing the progressive nature of TnI degradation, changes in phosphorylation, and covalent complexes with ischemia/reperfusion injury. Finally, we propose a model for ischemia/reperfusion injury in which the extent of proteolytic and transglutaminase activities ultimately determines whether apoptosis or necrosis is achieved.
Asunto(s)
Isquemia Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/metabolismo , Miocardio/metabolismo , Troponina I/metabolismo , Animales , Western Blotting , Cromatografía de Afinidad , Electroforesis en Gel de Poliacrilamida , Cinética , Masculino , Ratas , Ratas Sprague-Dawley , Troponina I/aislamiento & purificaciónRESUMEN
Abnormal smooth muscle contraction may contribute to diseases such as asthma and hypertension. Alterations to myosin light chain kinase or phosphatase change the phosphorylation level of the 20-kDa myosin regulatory light chain (MRLC), increasing Ca2+ sensitivity and basal tone. One Rho family GTPase-dependent kinase, Rho-associated kinase (ROK or p160(ROCK)) can induce Ca2+-independent contraction of Triton-skinned smooth muscle by phosphorylating MRLC and/or myosin light chain phosphatase. We show that another Rho family GTPase-dependent kinase, p21-activated protein kinase (PAK), induces Triton-skinned smooth muscle contracts independently of calcium to 62 +/- 12% (n = 10) of the value observed in presence of calcium. Remarkably, PAK and ROK use different molecular mechanisms to achieve the Ca2+-independent contraction. Like ROK and myosin light chain kinase, PAK phosphorylates MRLC at serine 19 in vitro. However, PAK-induced contraction correlates with enhanced phosphorylation of caldesmon and desmin but not MRLC. The level of MRLC phosphorylation remains similar to that in relaxed muscle fibers (absence of GST-mPAK3 and calcium) even as the force induced by GST-mPAK3 increases from 26 to 70%. Thus, PAK uncouples force generation from MRLC phosphorylation. These data support a model of PAK-induced contraction in which myosin phosphorylation is at least complemented through regulation of thin filament proteins. Because ROK and PAK homologues are present in smooth muscle, they may work in parallel to regulate smooth muscle contraction.