Dataset Information

The SGK3-triggered ubiquitin-proteasome degradation of podocalyxin (PC) and ezrin in podocytes was associated with the stability of the PC/ezrin complex.

ABSTRACT: Podocyte damage is commonly accompanied by destabilization of the podocalyxin (PC)/ezrin complex. Serum- and glucocorticoid-inducible kinase 3 (SGK3) plays a role in the maintenance of podocyte function, but the details of this role are poorly understood. Herein we demonstrated that SGK3 and its downstream target protein neural precursor cell expressed developmentally downregulated protein 4 subtype 2 (Nedd4-2) triggered PC and ezrin interaction. In adriamycin (ADR)-induced nephritic mice, and after puromycin aminonucleoside (PAN)-induced podocyte damage in vitro, PC and ezrin protein expression levels decreased significantly, while Nedd4-2 activity increased. Moreover, PAN treatment increased PC and ezrin ubiquitination and decreased PC/ezrin interaction in cultured mouse podocytes. The downregulation of SGK3 activity in mouse podocytes resulted in decreased PC and ezrin protein expression and increased the ubiquitin-proteasome degradation of PC and ezrin. Furthermore, upregulation of SGK3 activity mostly reversed the PAN-induced decrease in PC and ezrin protein expression. Overexpression of Nedd4-2 led to decreased ezrin protein expression via the upregulation of ezrin ubiquitination. In contrast, Nedd4-2 knockdown resulted in increased ezrin protein expression but decreased ezrin ubiquitination. In PC-transfected human embryonic kidney (HEK293T) cells, SGK3 activity downregulation and Nedd4-2 overexpression resulted in decreased PC/ezrin interaction. These results suggested that SGK3 triggers the ubiquitin-proteasome degradation of PC and ezrin, while the SGK3/Nedd4-2 signaling pathway regulates ezrin, but not PC, ubiquitination. Thus SGK3 helps to regulate podocyte function by maintaining the stability of the PC/ezrin complex.

SUBMITTER: Yuan YP

PROVIDER: S-EPMC6212497 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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The SGK3-triggered ubiquitin-proteasome degradation of podocalyxin (PC) and ezrin in podocytes was associated with the stability of the PC/ezrin complex.

Yuan Ya-Pei YP Zhao Hong H Peng Li-Qin LQ Li Zi-Fang ZF Liu Song S Yuan Cheng-Yan CY Mwamunyi Mercy-Julian MJ Pearce David D Yao Li-Jun LJ

Cell death & disease 20181101 11

Podocyte damage is commonly accompanied by destabilization of the podocalyxin (PC)/ezrin complex. Serum- and glucocorticoid-inducible kinase 3 (SGK3) plays a role in the maintenance of podocyte function, but the details of this role are poorly understood. Herein we demonstrated that SGK3 and its downstream target protein neural precursor cell expressed developmentally downregulated protein 4 subtype 2 (Nedd4-2) triggered PC and ezrin interaction. In adriamycin (ADR)-induced nephritic mice, and a ...[more]

PMID: 30385740

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Project description:BackgroundUbiquitination is a post-translational modification where ubiquitin is covalently attached to lysine residues on substrate proteins to signal their degradation by the 26S proteasome or initiate other non-degradation functions such as cellular trafficking. The diversity of ubiquitin modifications can be attributed to the variable number of ubiquitin molecules attached to a lysine residue (mono- vs. poly-ubiquitin chains), the type of covalent linkages within poly-ubiquitin chains and the number of lysine residues on a substrate that are occupied by ubiquitin at any given time. The integral role ubiquitination plays in cell homeostasis is reflected by the multitude of diseases associated with impaired ubiquitin modification, rendering it the focus of extensive research initiatives and proteomic discovery studies. However, determining the functional role of distinct ubiquitin modifications directly from proteomic data remains challenging and represents a bottleneck in the process of deciphering how ubiquitination at specific substrate sites impacts cell signaling.MethodsIn this study SILAC coupled with LC-MS/MS is used to identify ubiquitinated proteins in SKOV3 ovarian cancer cells, with the implementation of a computational approach that measures relative ubiquitin occupancy at distinct modification sites upon 26S proteasome inhibition and uses that data to infer functional significance.ResultsIn addition to identifying and quantifying relative ubiquitin occupancy at distinct post-translational modification sites to distinguish degradation from non-degradation signaling, this research led to the discovery of nine ubiquitination sites in the oncoprotein HER2 that have not been previously reported in ovarian cancer. Subsequently the computational approach applied in this study was utilized to infer the functional role of individual HER2 ubiquitin-modified residues.ConclusionsIn summary, the computational method, previously described for glycosylation analysis, was used in this study for the assessment of ubiquitin stoichiometries and applied directly to proteomic data to distinguish degradation from non-degradation ubiquitin functions.

Project description:Although the neurobiological basis of autism spectrum disorder (ASD) is not fully understood, recent studies have indicated the potential role of GABAA receptors in the pathophysiology of ASD. GABAA receptors play a crucial role in various neurodevelopmental processes and adult neuroplasticity. However, the mechanism(s) of regulation of GABAA receptors in ASD remains poorly understood.Postmortem middle frontal gyrus tissues (13 ASD and 13 control subjects) were used. In vitro studies were performed in primary cortical neurons at days in vitro (DIV) 14. The protein levels were examined by western blotting. Immunofluorescence studies were employed for cellular localization. The gene expression was determined by RT-PCR array and qRT-PCR.A significant decrease in GABAA?1 protein, but not mRNA levels was found in the middle frontal gyrus of ASD subjects indicating a post-translational regulation of GABAA receptors in ASD. At the cellular level, treatment with proteasomal inhibitor, MG132, or lactacystin significantly increased GABAA?1 protein levels and Lys48-linked polyubiquitination of GABAA?1, but reduced proteasome activity in mouse primary cortical neurons (DIV 14 from E16 embryos). Moreover, treatment with betulinic acid, a proteasome activator significantly decreased GABAA?1 protein levels in cortical neurons indicating the role of polyubiquitination of GABAA?1 proteins with their subsequent proteasomal degradation in cortical neurons. Ubiquitination specific RT-PCR array followed by western blot analysis revealed a significant increase in SYVN1, an endoplasmic reticulum (ER)-associated degradation (ERAD) E3 ubiquitin ligase in the middle frontal gyrus of ASD subjects. In addition, the inhibition of proteasomal activity by MG132 increased the expression of GABAA?1 in the ER. The siRNA knockdown of SYVN1 significantly increased GABAA?1 protein levels in cortical neurons. Moreover, reduced association between SYVN1 and GABAA?1 was found in the middle frontal gyrus of ASD subjects.SYVN1 plays a critical role as an E3 ligase in the ubiquitin proteasome system (UPS)-mediated GABAA?1 degradation. Thus, inhibition of the ubiquitin-proteasome-mediated GABAA?1 degradation may be an important mechanism for preventing GABAA?1 turnover to maintain GABAA?1 levels and GABA signaling in ASD.

Dataset Information

The SGK3-triggered ubiquitin-proteasome degradation of podocalyxin (PC) and ezrin in podocytes was associated with the stability of the PC/ezrin complex.

Publications

The SGK3-triggered ubiquitin-proteasome degradation of podocalyxin (PC) and ezrin in podocytes was associated with the stability of the PC/ezrin complex.

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