RESUMO
The stringent expression of the hypoxia inducible factor-1α (HIF-1α) is critical to a variety of pathophysiological conditions. We reveal that, in normoxia, enzymatic action of cystathionine ß-synthase (CBS) produces H2S, which persulfidates prolyl hydroxylase 2 (PHD2) at residues Cys21 and Cys33 (zinc finger motif), augmenting prolyl hydroxylase activity. Depleting endogenous H2S either by hypoxia or by inhibiting CBS via chemical or genetic means reduces persulfidation of PHD2 and inhibits activity, preventing hydroxylation of HIF-1α, resulting in stabilization. Our in vitro findings are further supported by the depletion of CBS in the zebrafish model that exhibits axis defects and abnormal intersegmental vessels. Exogenous H2S supplementation rescues both in vitro and in vivo phenotypes. We have identified the persulfidated residues and defined their functional significance in regulating the activity of PHD2 via point mutations. Thus, the CBS/H2S/PHD2 axis may provide therapeutic opportunities for pathologies associated with HIF-1α dysregulation in chronic diseases.
Assuntos
Cistationina beta-Sintase , Subunidade alfa do Fator 1 Induzível por Hipóxia , Prolina Dioxigenases do Fator Induzível por Hipóxia , Animais , Cistationina beta-Sintase/metabolismo , Sulfeto de Hidrogênio , Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Peixe-Zebra/metabolismoRESUMO
Although mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common cause of genetic Parkinson's disease, their function is largely unknown. LRRK2 is pleiotropic in nature, shown to be involved in neurodegeneration and in more peripheral processes, including kidney functions, in rats and mice. Recent studies in zebrafish have shown conflicting evidence that removal of the LRRK2 WD40 domain may or may not affect dopaminergic neurons and/or locomotion. This study shows that â¼50% LRRK2 knockdown in zebrafish causes not only neuronal loss but also developmental perturbations such as axis curvature defects, ocular abnormalities, and edema in the eyes, lens, and otic vesicles. We further show that LRRK2 knockdown results in significant neuronal loss, including a reduction of dopaminergic neurons. Immunofluorescence demonstrates that endogenous LRRK2 is expressed in the lens, brain, heart, spinal cord, and kidney (pronephros), which mirror the LRRK2 morphant phenotypes observed. LRRK2 knockdown results further in the concomitant upregulation of ß-synuclein, PARK13, and SOD1 and causes ß-synuclein aggregation in the diencephalon, midbrain, hindbrain, and postoptic commissure. LRRK2 knockdown causes mislocalization of the Na(+) /K(+) ATPase protein in the pronephric ducts, suggesting that the edema might be linked to renal malfunction and that LRRK2 might be associated with pronephric duct epithelial cell differentiation. Combined, our study shows that LRRK2 has multifaceted roles in zebrafish and that zebrafish represent a complementary model to further our understanding of this central protein. © 2016 Wiley Periodicals, Inc.