RESUMEN
Mitochondrial dysfunction increases oxidative stress and depletes ATP in a variety of disorders. Several antioxidant therapies and drugs affecting mitochondrial biogenesis are undergoing investigation, although not all of them have demonstrated favorable effects in the clinic. We recently reported a therapeutic mitochondrial drug mitochonic acid MA-5 (Tohoku J. Exp. Med., 2015). MA-5 increased ATP, rescued mitochondrial disease fibroblasts and prolonged the life span of the disease model "Mitomouse" (JASN, 2016). To investigate the potential of MA-5 on various mitochondrial diseases, we collected 25 cases of fibroblasts from various genetic mutations and cell protective effect of MA-5 and the ATP producing mechanism was examined. 24 out of the 25 patient fibroblasts (96%) were responded to MA-5. Under oxidative stress condition, the GDF-15 was increased and this increase was significantly abrogated by MA-5. The serum GDF-15 elevated in Mitomouse was likewise reduced by MA-5. MA-5 facilitates mitochondrial ATP production and reduces ROS independent of ETC by facilitating ATP synthase oligomerization and supercomplex formation with mitofilin/Mic60. MA-5 reduced mitochondria fragmentation, restores crista shape and dynamics. MA-5 has potential as a drug for the treatment of various mitochondrial diseases. The diagnostic use of GDF-15 will be also useful in a forthcoming MA-5 clinical trial.
Asunto(s)
Ácidos Indolacéticos/farmacología , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Enfermedades Mitocondriales/metabolismo , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Fenilbutiratos/farmacología , Multimerización de Proteína/efectos de los fármacos , Adenosina Trifosfato/metabolismo , Animales , Biomarcadores , Línea Celular , Supervivencia Celular/efectos de los fármacos , ADN Mitocondrial , Modelos Animales de Enfermedad , Factores de Crecimiento de Fibroblastos/metabolismo , Fibroblastos/metabolismo , Factor 15 de Diferenciación de Crecimiento/metabolismo , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Mitocondrias/genética , Enfermedades Mitocondriales/diagnóstico , Enfermedades Mitocondriales/tratamiento farmacológico , Enfermedades Mitocondriales/genética , Dinámicas Mitocondriales/efectos de los fármacos , ATPasas de Translocación de Protón Mitocondriales/química , Complejos Multiproteicos/metabolismo , Mutación , Biogénesis de Organelos , Pronóstico , Sustancias Protectoras , Unión ProteicaRESUMEN
Sarcopenia is associated with increased morbidity and mortality in chronic kidney disease (CKD). Pathogenic mechanism of skeletal muscle loss in CKD, which is defined as uremic sarcopenia, remains unclear. We found that causative pathological mechanism of uremic sarcopenia is metabolic alterations by uremic toxin indoxyl sulfate. Imaging mass spectrometry revealed indoxyl sulfate accumulated in muscle tissue of a mouse model of CKD. Comprehensive metabolomics revealed that indoxyl sulfate induces metabolic alterations such as upregulation of glycolysis, including pentose phosphate pathway acceleration as antioxidative stress response, via nuclear factor (erythroid-2-related factor)-2. The altered metabolic flow to excess antioxidative response resulted in downregulation of TCA cycle and its effected mitochondrial dysfunction and ATP shortage in muscle cells. In clinical research, a significant inverse association between plasma indoxyl sulfate and skeletal muscle mass in CKD patients was observed. Our results indicate that indoxyl sulfate is a pathogenic factor for sarcopenia in CKD.
Asunto(s)
Indicán/metabolismo , Músculo Esquelético/metabolismo , Insuficiencia Renal Crónica/metabolismo , Sarcopenia/metabolismo , Uremia/metabolismo , Animales , Modelos Animales de Enfermedad , Masculino , Ratones , Músculo Esquelético/patología , Insuficiencia Renal Crónica/patología , Sarcopenia/patología , Uremia/patologíaRESUMEN
Mitochondrial dysfunction causes increased oxidative stress and depletion of ATP, which are involved in the etiology of a variety of renal diseases, such as CKD, AKI, and steroid-resistant nephrotic syndrome. Antioxidant therapies are being investigated, but clinical outcomes have yet to be determined. Recently, we reported that a newly synthesized indole derivative, mitochonic acid 5 (MA-5), increases cellular ATP level and survival of fibroblasts from patients with mitochondrial disease. MA-5 modulates mitochondrial ATP synthesis independently of oxidative phosphorylation and the electron transport chain. Here, we further investigated the mechanism of action for MA-5. Administration of MA-5 to an ischemia-reperfusion injury model and a cisplatin-induced nephropathy model improved renal function. In in vitro bioenergetic studies, MA-5 facilitated ATP production and reduced the level of mitochondrial reactive oxygen species (ROS) without affecting activity of mitochondrial complexes I-IV. Additional assays revealed that MA-5 targets the mitochondrial protein mitofilin at the crista junction of the inner membrane. In Hep3B cells, overexpression of mitofilin increased the basal ATP level, and treatment with MA-5 amplified this effect. In a unique mitochondrial disease model (Mitomice with mitochondrial DNA deletion that mimics typical human mitochondrial disease phenotype), MA-5 improved the reduced cardiac and renal mitochondrial respiration and seemed to prolong survival, although statistical analysis of survival times could not be conducted. These results suggest that MA-5 functions in a manner differing from that of antioxidant therapy and could be a novel therapeutic drug for the treatment of cardiac and renal diseases associated with mitochondrial dysfunction.
Asunto(s)
Ácidos Indolacéticos/farmacología , Túbulos Renales/citología , Mitocondrias/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Fenilbutiratos/farmacología , Animales , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
The 26S proteasome is a 2.5-MDa multisubunit protease complex that degrades polyubiquitylated proteins. Although its functions and structure have been extensively characterized, little is known about its dynamics in living cells. Here, we investigate the absolute concentration, spatio-temporal dynamics and complex formation of the proteasome in living cells using fluorescence correlation spectroscopy. We find that the 26S proteasome complex is highly mobile, and that almost all proteasome subunits throughout the cell are stably incorporated into 26S proteasomes. The interaction between 19S and 20S particles is stable even in an importin-α mutant, suggesting that the 26S proteasome is assembled in the cytoplasm. Furthermore, a genetically stabilized 26S proteasome mutant is able to enter the nucleus. These results suggest that the 26S proteasome completes its assembly process in the cytoplasm and translocates into the nucleus through the nuclear pore complex as a holoenzyme.
Asunto(s)
Citoplasma/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Activo de Núcleo Celular , Núcleo Celular/metabolismo , Microscopía por Crioelectrón , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Cinética , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Microscopía Confocal , Microscopía Fluorescente , Mutación , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/ultraestructura , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/ultraestructura , Factores de Tiempo , Imagen de Lapso de Tiempo/métodos , Proteína Fluorescente RojaRESUMEN
The 26S proteasome is a highly conserved multisubunit protease that degrades ubiquitinated proteins in eukaryotic cells. It comprises a 20S core particle and two 19S regulatory particles that are further divided into the lid and base complexes. The lid is a nine subunits complex that is structurally related to the COP9 signalosome and the eukaryotic initiation factor 3. Although the assembly pathway of the 20S and the base are well described, that of the lid is still unclear. In this study, we dissected the lid assembly using yeast lid mutant cells, rpn7-3, Delta rpn9, and rpn12-1. Using mass spectrometry, we identified a number of lid subassemblies, such as Rpn3-Rpn7 pair and a lid-like complex lacking Rpn12, in the mutants. Our analysis suggests that the assembly of the lid is a highly ordered and multi-step process; first, Rpn5, 6, 8, 9, and 11 are assembled to form a core module, then a second module, consisting of Rpn3, 7, and Sem1, is attached, followed by the incorporation of Rpn12 to form the lid complex.
Asunto(s)
Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Complejo de la Endopetidasa Proteasomal/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
The dynamic and reversible process of ubiquitin modification controls various cellular activities. Ubiquitin exists as monomers, unanchored chains, or protein-conjugated forms, but the regulation of these interconversions remains largely unknown. Here, we identified a protein designated Rfu1 (regulator of free ubiquitin chains 1), which regulates intracellular concentrations of monomeric ubiquitins and free ubiquitin chains in Saccharomyces cerevisiae. Rfu1 functions as an inhibitor of Doa4, a deubiquitinating enzyme. Rapid loss of free ubiquitin chains upon heat shock, a condition in which more proteins require ubiquitin conjugation, was mediated in part by Doa4 and Rfu1. Thus, regulation of ubiquitin homeostasis is controlled by a balance between a deubiquitinating enzyme and its inhibitor. We propose that free ubiquitin chains function as a ubiquitin reservoir that allows maintenance of monomeric ubiquitins at adequate levels under normal conditions and rapid supply for substrate conjugation under stress conditions.
Asunto(s)
Endopeptidasas/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismo , Regulación Alostérica , Endopeptidasas/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte , Endosomas/metabolismo , Humanos , Mutación , Complejo de la Endopetidasa Proteasomal/genética , Unión Proteica , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/antagonistas & inhibidores , Transducción de Señal , Estrés Fisiológico , Ubiquitina Tiolesterasa , Complejos de Ubiquitina-Proteína Ligasa/metabolismoRESUMEN
The 26S proteasome is a highly conserved multisubunit protease that degrades ubiquitinated proteins in eukaryotic cells. The 26S proteasome consists of the proteolytic core particle (CP) and one or two 19S regulatory particles (RPs). Although the mechanisms of CP assembly are well described, the mechanism of RP assembly is largely unknown. Here, we show that four proteasome-interacting proteins (PIPs), Nas2/p27, Nas6/gankyrin, Rpn14/PAAF1, and Hsm3/S5b, bind specific Rpt subunits of the RP and interact each other genetically. Lack of these PIPs resulted in defective assembly of the 26S proteasome at an early stage, suggesting that these proteins are bona fide RP chaperones. Each of the RP chaperones formed distinct specific subassemblies of the base components and escorted them to mature RPs. Our results indicate that the RP assembly is a highly organized and elaborate process orchestrated by multiple proteasome-dedicated chaperones.
Asunto(s)
Complejo de la Endopetidasa Proteasomal/metabolismo , Humanos , Chaperonas Moleculares/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Recruitment of substrates to the 26S proteasome usually requires covalent attachment of the Lys48-linked polyubiquitin chain. In contrast, modifications with the Lys63-linked polyubiquitin chain and/or monomeric ubiquitin are generally thought to function in proteasome-independent cellular processes. Nevertheless, the ubiquitin chain-type specificity for the proteasomal targeting is still poorly understood, especially in vivo. Using mass spectrometry, we found that Rsp5, a ubiquitin-ligase in budding yeast, catalyzes the formation of Lys63-linked ubiquitin chains in vitro. Interestingly, the 26S proteasome degraded well the Lys63-linked ubiquitinated substrate in vitro. To examine whether Lys63-linked ubiquitination serves in degradation in vivo, we investigated the ubiquitination of Mga2-p120, a substrate of Rsp5. The polyubiquitinated p120 contained relatively high levels of Lys63-linkages, and the Lys63-linked chains were sufficient for the proteasome-binding and subsequent p120-processing. In addition, Lys63-linked chains as well as Lys48-linked chains were detected in the 26S proteasome-bound polyubiquitinated proteins. These results raise the possibility that Lys63-linked ubiquitin chain also serves as a targeting signal for the 26S proteaseome in vivo.