RESUMEN
N-glycanase 1(NGLY1) catalyzes the removal of N-linked glycans from newly synthesized or misfolded protein. NGLY1 deficiency is a recently diagnosed rare genetic disorder. The affected individuals present a broad spectrum of clinical features. Recent studies explored several possible molecular mechanisms of NGLY1 deficiency including defects in proteostasis, mitochondrial homeostasis, innate immunity, and water/ion transport. We demonstrate abnormal accumulation of endoplasmic reticulum-associated degradation (ERAD) substrates in NGLY1-deficient cells. Global quantitative proteomics discovered elevated levels of endogenous proteins in NGLY1-defective human and mouse cells. Further biological validation assays confirmed the altered abundance of several key candidates that were subjected to isobarically labeled proteomic analysis. CCN2 was selected for further analysis due to its significant increase in different cell models of NGLY1 deficiency. Functional assays show elevated CCN2 and over-stimulated TGF-ß signaling in NGLY1-deficient cells. Given the important role of CCN2 and TGF-ß pathway in mediating systemic fibrosis, we propose a potential link of increased CCN2 and TGF-ß signaling to microscopic liver fibrosis in NGLY1 patients.
Asunto(s)
Trastornos Congénitos de Glicosilación , Factor de Crecimiento del Tejido Conjuntivo , Degradación Asociada con el Retículo Endoplásmico , Animales , Humanos , Ratones , Trastornos Congénitos de Glicosilación/genética , Trastornos Congénitos de Glicosilación/metabolismo , Degradación Asociada con el Retículo Endoplásmico/genética , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/genética , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/metabolismo , Polisacáridos/metabolismo , Proteómica , Factor de Crecimiento Transformador beta/metabolismo , Agua/metabolismo , Factor de Crecimiento del Tejido Conjuntivo/genética , Factor de Crecimiento del Tejido Conjuntivo/metabolismoRESUMEN
Inorganic phosphate (Pi) is an essential nutrient for the human body which exerts adverse health effects in excess and deficit. High Pi-mediated cytotoxicity has been shown to induce systemic organ damage, though the underlying molecular mechanisms are poorly understood. In this study, we employed proteomics and phosphoproteomics to analyze Pi-mediated changes in protein abundance and phosphorylation. Bioinformatic analyses and literature review revealed that the altered proteins and phosphorylation were enriched in signaling pathways and diverse biological processes. Western blot analysis confirms the extensive change in protein level and phosphorylation in key effectors that modulate pre-mRNA alternative splicing. Global proteome and phospho-profiling provide a bird-eye view of excessive Pi-rewired cell signaling networks, which deepens our understanding of the molecular mechanisms of phosphate toxicity.
RESUMEN
Inorganic phosphate (Pi) is an essential nutrient for human health. Due to the changes in our dietary pattern, dietary Pi overload engenders systemic phosphotoxicity, including excessive Pi-related vascular calcification and chronic tissue injury. The molecular mechanisms of the seemingly distinct phenotypes remain elusive. In this study, we investigated Pi-mediated cellular response in HEK293 and HeLa cells. We found that abnormally high Pi directly mediates diverse cellular toxicity in a dose-dependent manner. Up to 10 mM extracellular Pi promotes cell proliferation by activating AKT signaling cascades and augmenting cell cycle progression. By introducing additional Pi, higher than the concentration of 40 mM, we observed significant cell damage caused by the interwoven Pi-related biological processes. Elevated Pi activates mitogen-activated protein kinase (MAPK) signaling, encompassing extracellular signal-regulated kinase 1/2 (ERK1/2), p38 and Jun amino-terminal kinase (JNK), which consequently potentiates Pi triggered lethal epithelial-mesenchymal transition (EMT). Synergistically, high Pi-caused endoplasmic reticulum (ER) stress also contributes to apparent apoptosis. To counteract, Pi-activated AKT signaling promotes cell survival by activating the mammalian target of rapamycin (mTOR) signaling and blocking ER stress. Pharmacologically or genetically abrogating Pi transport, the impact of high Pi-induced cytotoxicity could be reduced. Taken together, abnormally high extracellular Pi results in a broad spectrum of toxicity by rewiring complicated signaling networks that control cell growth, cell death, and homeostasis.
Asunto(s)
Apoptosis/efectos de los fármacos , Citotoxinas/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Fosfatos/farmacología , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Células HEK293 , Células HeLa , Humanos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Women with polycystic ovary syndrome (PCOS) are reported to have greater lean mass and insulin resistance. To examine muscular changes in a prenatally androgenized (PNA) rat model for PCOS, Sprague-Dawley rats were exposed to 5 mg testosterone or vehicle daily on gestational days 16-19. At 15 weeks of age, endurance on a rota-rod treadmill was measured. At 16 weeks of age, fasting blood glucose and insulin, hindlimb skeletal muscle mass, muscle fiber cross-sectional area (CSA) and composition, and intra- and peri-muscular lipid droplets were examined. Expression of mitochondrial marker ATP synthase and insulin signaling proteins were also investigated. Compared with controls, PNA female rats demonstrated greater total body and hindlimb muscle weights, greater muscle fiber CSA, and trending reduced time on the rota-rod. An increase in fibers co-expressing the slow and fast isoforms of myosin (90 vs. 86%, p < 0.05) and greater expression of ATP synthase (6-fold, p < 0.005) were observed in the gastrocnemius (GN) muscle. More lipid content was observed in GN and tibialis anterior (TA) muscles. PNA rats had elevated fasting serum insulin (1.9 vs. 1.2 ng/mL, p < 0.005) but comparable fasting glucose. Expression of total and Ser636/9-phosphorylated IRS1 were altered in PNA rat hindlimb muscles. Together, skeletal muscle alterations in hindlimb muscles of a PNA rat model for PCOS may represent consequences of, or adaptations to, insulin resistance in this model.