E3 Ubiquitin Ligase E6AP Negatively Regulates Adipogenesis by Downregulating Proadipogenic Factor C/EBPalpha

Abstract

CCAAT/Enhancer Binding Protein Alpha (C/EBPα) is a key transcription factor involved in the adipocyte differentiation. Here for the first time we demonstrate that E6AP, an E3 ubiquitin ligase inhibits adipocyte differentiation in 3T3-L1 cells as revealed by reduced lipid staining with oil red. Knock down of E6AP in mouse 3T3L1 preadipocytes is sufficient to convert them to adipocytes independent of external hormonal induction. C/EBPα protein level is drastically increased in E6AP deficient 3T3L1 preadipocytes while inverse is observed when wild type E6AP is over expressed. We show that transient transfection of wild type E6AP downregulates C/EBPα protein expression in a dose dependent manner while catalytically inactive E6AP-C843A rather stabilizes it. In addition, wild type E6AP inhibits expression of proadipogenic genes while E6AP-C843A enhances them. More importantly, overexpression of E6AP-C843A in mesenchymal progenitor cells promotes accumulation of lipid droplets while there is drastically reduced lipid droplet formation when E6AP is over expressed. Taken together, our finding suggests that E6AP may negatively control adipogenesis by inhibiting C/EBPα expression by targeting it to ubiquitin-proteasome pathway for degradation.

Progranulin Controls Sepsis via C/EBPα-RegulatedIl10Transcription and Ubiquitin Ligase/Proteasome-Mediated Protein Degradation

“…C/EBPα has been reported to be degraded through the ubiquitin-proteasome pathway (47, 48), and GRN mutations cause the up-regulation of Ube2z, an E2 ubiquitin conjugating enzyme, in the periphery of frontotemporal dementia patients with GRN mutations (79). Although we did not find evidence of Ube2z’s involvement in PGRN-regulated C/EBPα level (data not shown), we did identify a clear role of E6AP in this process, i.e., it causes the degradation of C/EBPα by physically binding to the target protein, thus corroborating two previous studies in different contexts (49, 50). This activity is antagonized by the presence of PGRN.…”

“…It had been reported that E6AP, an E3 ubiquitin ligase negatively regulates granulopoiesis and adipogenesis by targeting C/EBPα for ubiquitin-mediated proteasome degradation (49, 50). To investigate the role of this enzyme in PGRN-regulated C/EBPα protein level, we first overexpressed an HA-tagged E6AP and C/EBPα in 293T cells, and found that the former decreased the latter’s protein level dose-dependently (Fig 7A).…”

Subcellular organization of UBE3A in neurons

“…At the cellular level, AS mice exhibit abnormalities in synaptic transmission and plasticity (Weeber et al, 2003;van Woerden et al, 2007;Yashiro et al, 2009;Greer et al, 2010;Margolis et al, 2010;Wallace et al, 2012), Golgi acidification (Condon et al, 2013), and mitochondrial function Llewellyn et al, 2015;Santini et al, 2015) . This phenotypic diversity suggests a multiplicity of roles for UBE3A in neural function.Previous immunohistochemical studies found UBE3A mainly in the nuclei of mature neurons, consistent with a proposed role in the co-regulation of transcription (Nawaz et al, 1999;Bernassola et al, 2008;Pal et al, 2013). However, the majority of putative UBE3A substrates are cytoplasmic proteins, and most cellular phenotypes in AS mice implicate loss of UBE3A function in non-nuclear compartments.…”

E3 Ubiquitin Ligase Fbw7 Negatively Regulates Osteoblast Differentiation by Targeting Runx2 for Degradation

“…Co-precipitates were immunoblotted with anti-HA and anti-Runx2 antibodies. A Western blotting and cycloheximide half-life experiment was performed as described previously (32,33). Protein samples from femur bone of different experimental groups were isolated as described by Wejheden et al (34).…”