RESUMEN
The central nervous system plays a critical role in the normal control of arterial blood pressure and in its elevation in virtually all forms of hypertension. Mitochondrial dysfunction has been increasingly associated with the development of hypertension. Therefore, we examined whether mitochondrial dysfunction occurs in the brain in hypertension and characterized it at the molecular scale. Mitochondria from whole brain and brain stem from 12-week-old spontaneously hypertensive rats with elevated blood pressure (190+/-5 mm Hg) were compared against those from age-matched normotensive (134+/-7 mm Hg) Wistar Kyoto rats (n=4 in each group). Global differential analysis using 2D electrophoresis followed by tandem mass spectrometry-based protein identification suggested a downregulation of enzymes involved in cellular energetics in hypertension. Targeted differential analysis of mitochondrial respiratory complexes using the classical blue-native SDS-PAGE/Western method and a complementary combination of sucrose-gradient ultracentrifugation/tandem mass spectrometry revealed previously unknown assembly defects in complexes I, III, IV, and V in hypertension. Interestingly, targeted examination of the brain stem, a regulator of cardiovascular homeostasis and systemic blood pressure, further showed the occurrence of mitochondrial complex I dysfunction, elevated reactive oxygen species production, decreased ATP synthesis, and impaired respiration in hypertension. Our findings suggest that in already-hypertensive spontaneously hypertensive rats, the brain respiratory complexes exhibit previously unknown assembly defects. These defects impair the function of the mitochondrial respiratory chain. This mitochondrial dysfunction localizes to the brain stem and is, therefore, likely to contribute to the development, as well as to pathophysiological complications, of hypertension.
Asunto(s)
Encéfalo/enzimología , Hipertensión/complicaciones , Hipertensión/fisiopatología , Enfermedades Mitocondriales/etiología , Complejos Multienzimáticos/genética , Procesamiento Proteico-Postraduccional , Animales , Sistema Cardiovascular/fisiopatología , Doxiciclina/farmacología , Electroforesis en Gel Bidimensional , Inhibidores Enzimáticos/farmacología , Homeostasis , Hipertensión/enzimología , Metaloendopeptidasas/antagonistas & inhibidores , Enfermedades Mitocondriales/enzimología , Complejos Multienzimáticos/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteómica , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Espectrometría de Masas en TándemRESUMEN
The initiation of DNA replication in Saccharomyces cerevisiae depends upon the destruction of the Clb-Cdc28 inhibitor Sic1. In proliferating cells Cln-Cdc28 complexes phosphorylate Sic1, which stimulates binding of Sic1 to SCF(Cdc4) and triggers its proteosome mediated destruction. During sporulation cyclins are not expressed, yet Sic1 is still destroyed at the G1-/S-phase boundary. The Cdk (cyclin dependent kinase) sites are also required for Sic1 destruction during sporulation. Sic1 that is devoid of Cdk phosphorylation sites displays increased stability and decreased phosphorylation in vivo. In addition, we found that Sic1 was modified by ubiquitin in sporulating cells and that SCF(Cdc4) was required for this modification. The meiosis-specific kinase Ime2 has been proposed to promote Sic1 destruction by phosphorylating Sic1 in sporulating cells. We found that Ime2 phosphorylates Sic1 at multiple sites in vitro. However, only a subset of these sites corresponds to Cdk sites. The identification of multiple sites phosphorylated by Ime2 has allowed us to propose a motif for phosphorylation by Ime2 (PXS/T) where serine or threonine acts as a phospho-acceptor. Although Ime2 phosphorylates Sic1 at multiple sites in vitro, the modified Sic1 fails to bind to SCF(Cdc4). In addition, the expression of Ime2 in G1 arrested haploid cells does not promote the destruction of Sic1. These data support a model where Ime2 is necessary but not sufficient to promote Sic1 destruction during sporulation.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sitios de Unión , Proteínas de Ciclo Celular/genética , Proteínas Inhibidoras de las Quinasas Dependientes de la Ciclina , Estabilidad de Enzimas , Fase G1/fisiología , Regulación Fúngica de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular , Fosforilación , Proteínas Quinasas/genética , Procesamiento Proteico-Postraduccional , Proteínas Serina-Treonina Quinasas , Proteínas de Saccharomyces cerevisiae/genética , Esporas Fúngicas/metabolismoRESUMEN
Aziridine analogues of diaminopimelic acid (DAP) have been prepared stereoselectively for the first time and evaluated as inhibitors of DAP epimerase. (2R,3S,3'S)-3-(3'-Aminopropane)aziridine-2,3'-dicarboxylate was synthesised and shown to be a reversible inhibitor of DAP epimerase with an IC(50) value of 2.88 mM. (2S,4S)- and (2S,4R)-2-(4-Amino-4-carboxybutyl)aziridine-2-carboxylic acid (ll-azi-DAP and dl-azi-DAP ) were made as pure diastereomers, and both were shown to be irreversible inhibitors of DAP epimerase. ll-Azi-DAP selectively binds to Cys-73 of the enzyme active site whereas dl-azi-DAP binds to Cys-217 via attack of sulfhydryl on the methylene of the inhibitor aziridine ring. These observations are consistent with the two base mechanism proposed for the epimerization of ll-DAP and meso-DAP by DAP epimerase.
Asunto(s)
Isomerasas de Aminoácido/metabolismo , Aziridinas/química , Ácido Diaminopimélico/química , Ácido Diaminopimélico/metabolismo , Isomerasas de Aminoácido/antagonistas & inhibidores , Aziridinas/síntesis química , Aziridinas/farmacología , Cisteína/química , Cisteína/metabolismo , Isomerismo , Espectrometría de Masas , Datos de Secuencia Molecular , EstereoisomerismoRESUMEN
Here we describe a proteomic analysis of Escherichia coli in which 3,199 protein forms were detected, and of those 2,160 were annotated and assigned to the cytosol, periplasm, inner membrane, and outer membrane by biochemical fractionation followed by two-dimensional gel electrophoresis and tandem mass spectrometry. Represented within this inventory were unique and modified forms corresponding to 575 different ORFs that included 151 proteins whose existence had been predicted from hypothetical ORFs, 76 proteins of completely unknown function, and 222 proteins currently without location assignments in the Swiss-Prot Database. Of the 575 unique proteins identified, 42% were found to exist in multiple forms. Using DIGE, we also examined the relative changes in protein expression when cells were grown in the presence and absence of amino acids. A total of 23 different proteins were identified whose abundance changed significantly between the two conditions. Most of these changes were found to be associated with proteins involved in carbon and amino acid metabolism, transport, and chemotaxis. Detailed information related to all 2,160 protein forms (protein and gene names, accession numbers, subcellular locations, relative abundances, sequence coverage, molecular masses, and isoelectric points) can be obtained upon request in either tabular form or as interactive gel images.