L1CAM ubiquitination facilitates its lysosomal degradation

The cell adhesion molecule L1 is implicated in several processes in the developing and adult nervous system. Intracellular trafficking of L1 is important for cell migration, neurite growth and adhesion. We demonstrate here that L1 is ubiquitinated at the plasma membrane and in early endosomes. Mono-ubiquitination regulates L1 intracellular trafficking by enhancing its lysosomal degradation. We propose that L1’s ubiquitination might be an additional mechanism to control its re-appearance at the cell surface thereby influencing processes like neurite growth and cell adhesion.     

The role of synphilin-1 in synaptic function and protein degradation

The name synphilin-1 comes from its identification as an alpha-synuclein-interacting protein (SNCAIP) in yeast two-hybrid screens. Since alpha-synuclein (PARK1) was the first gene identified as causing inherited forms of Parkinsons disease (PD), synphilin-1 was quickly implicated in neurodegeneration in PD. Recently, the first genetic evidence for the direct contribution of synphilin-1 in the pathogenesis of PD has been defined with the identification of an R621C mutation as a susceptibility factor for PD in two German patients. Extensive in vitro studies have determined the physiological functions of synphilin-1, identified novel synphilin-1-interacting proteins, and linked synphilin-1 to ubiquitin-mediated protein degradation. The present article provides an overview of the current concepts of the role of synphilin-1 in synaptic function and protein degradation and in the molecular mechanisms leading to neurodegeneration in PD.The work of R.K. on synphilin-1 is supported by a grant from the Fritz Thyssen Foundation, a grant from the Federal Ministry of Education and Research (Fö 01KS9602), and the Interdisciplinary Center of Clinical Research Tübingen (IZKF)     

CHIP facilitates ubiquitination of inducible nitric oxide synthase and promotes its proteasomal degradation

Inducible nitric oxide synthase (iNOS) is responsible for nitric oxide (NO) synthesis from l-arginine in response to inflammatory mediators. It is reported that iNOS is degraded mainly by the ubiquitin-proteasome pathway in RAW264.7 cells and human embryonic kidney (HEK) 293 cells. In this study, we showed that iNOS was ubiquitinated and degraded dependent on CHIP (COOH terminus of heat shock protein 70-interacting protein), a chaperone-dependent ubiquitin ligase. The results from overexpression and RNAi experiments demonstrated that CHIP decreased the protein level of iNOS, shortened the half-life of iNOS and attenuated the production of NO. Furthermore, CHIP promoted ubiquitination and proteasomal degradation of iNOS by associating with iNOS. These results suggest that CHIP plays an important role in regulation iNOS activity.     

Fbxo45 facilitates pancreatic carcinoma progression by targeting USP49 for ubiquitination and degradation

Fbxo45, a conserved F-box protein, comprises of an atypical SKP1, CUL1, F-box protein (SCF) ubiquitin ligase complex that promotes tumorigenesis and development. However, the biological function and molecular mechanisms of Fbxo45 involved in pancreatic carcinogenesis are ambiguous. We conducted several approaches, including transfection, coIP, real-time polymerase chain reaction (RT-PCR), Western blotting, ubiquitin assays, and animal studies, to explore the role of Fbxo45 in pancreatic cancer. Here, we report that USP49 stability is governed by Fbxo45-mediated ubiquitination and is enhanced by the absence of Fbxo45. Moreover, Fbxo45 binds to a short consensus sequence of USP49 through its SPRY domain. Furthermore, Fbxo45-mediated USP49 ubiquitination and degradation are enhanced by NEK6 kinase. Functionally, Fbxo45 increases cell viability and motility capacity by targeting USP49 in pancreatic cancer cells. Xenograft mouse experiments demonstrated that ectopic expression of Fbxo45 enhanced tumor growth in mice and that USP49 overexpression inhibited tumor growth in vivo. Notably, Fbxo45 expression was negatively associated with USP49 expression in pancreatic cancer tissues. Fbxo45 serves as an oncoprotein to facilitate pancreatic oncogenesis by regulating the stability of the tumor suppressor USP49 in pancreatic cancer.Subject terms: Pancreatic cancer, Pancreatic cancer     

beta-arrestin-1 competitively inhibits insulin-induced ubiquitination and degradation of insulin receptor substrate 1

beta-arrestin-1 is an adaptor protein that mediates agonist-dependent internalization and desensitization of G-protein-coupled receptors (GPCRs) and also participates in the process of heterologous desensitization between receptor tyrosine kinases and GPCR signaling. In the present study, we determined whether beta-arrestin-1 is involved in insulin-induced insulin receptor substrate 1 (IRS-1) degradation. Overexpression of wild-type (WT) beta-arrestin-1 attenuated insulin-induced degradation of IRS-1, leading to increased insulin signaling downstream of IRS-1. When endogenous beta-arrestin-1 was knocked down by transfection of beta-arrestin-1 small interfering RNA, insulin-induced IRS-1 degradation was enhanced. Insulin stimulated the association of IRS-1 and Mdm2, an E3 ubiquitin ligase, and this association was inhibited to overexpression of WT beta-arrestin-1, which led by decreased ubiquitin content of IRS-1, suggesting that both beta-arrestin-1 and IRS-1 competitively bind to Mdm2. In summary, we have found the following: (i) beta-arrestin-1 can alter insulin signaling by inhibiting insulin-induced proteasomal degradation of IRS-1; (ii) beta-arrestin-1 decreases the rate of ubiquitination of IRS-1 by competitively binding to endogenous Mdm2, an E3 ligase that can ubiquitinate IRS-1; (iii) dephosphorylation of S412 on beta-arrestin and the amino terminus of beta-arrestin-1 are required for this effect of beta-arrestin on IRS-1 degradation; and (iv) inhibition of beta-arrestin-1 leads to enhanced IRS-1 degradation and accentuated cellular insulin resistance.     

Hrd1 facilitates tau degradation and promotes neuron survival

Intraneuronal accumulation of abnormal phosphorylated tau (p-tau) is a molecular pathology in many neurodegenerative tauopathies, including Alzheimer's disease (AD) and frontotemporal dementia with parkinsonism-linked to chromosome 17 (FTDP-17). However, the underlying mechanism remains unclear. Here, we showed an inverse relationship between endoplasmic reticulum membrane ubiquitin ligase (E3) Hrd1 expression and p-tau accumulation in the hippocampal neurons of AD, and proposed that Hrd1 may be a negative regulator of p-tau. This notion was further supported by in vitro study demonstrating that Hrd1 interacted with tau and promoted the degradation of total tau and p-tau as well. The degradation of tau depended on its Hrd1 E3 activity. Knockdown of endogenous Hrd1 with siRNA stabilized tau levels. In addition, inhibition of proteasome maintained tau level and increased Hrd1-mediated tau ubiquitination, suggesting the proteasome was involved in tau/p-tau degradation. Over-expression of Hrd1 significantly alleviated tau cytotoxicity and promoted cell survival. These results indicated that Hrd1 functions as an E3 targeting tau or abnormal p-tau for proteasome degradation. The study provides an important insight into the molecular mechanisms of human tauopathies.     

TRIAD1 inhibits MDM2-mediated p53 ubiquitination and degradation

Murine double minute (MDM2) is an E3 ligase that promotes ubiquitination and degradation of tumor suppressor protein 53 (p53). MDM2-mediated regulation of p53 has been investigated as a classical tumorigenesis pathway. Here, we describe TRIAD1 as a novel modulator of the p53-MDM2 axis that induces p53 activation by inhibiting its regulation by MDM2. Ablation of TRIAD1 attenuates p53 levels activity upon DNA damage, whereas ectopic expression of TRIAD1 promotes p53 stability by inhibiting MDM2-mediated ubiquitination/degradation. Moreover, TRIAD1 binds to the C-terminus of p53 to promote its dissociation from MDM2. These results implicate TRIAD1 as a novel regulatory factor of p53-MDM2.Structured summary of protein interactions:p53 physically interacts with Mdm2 and Triad1 by anti tag coimmunoprecipitation (View Interaction: 1, 2, 3)Mdm2physically interacts with Triad1 by anti tag coimmunoprecipitation (View interaction)p53physically interacts with Mdm2 by anti tag coimmunoprecipitation (View interaction)Triad1binds to p53 by pull down (View interaction)Mdm2physically interacts with p53 by anti tag coimmunoprecipitation (View interaction)p53physically interacts with Triad1 by anti tag coimmunoprecipitation (View interaction)     

Swf1-dependent palmitoylation of the SNARE Tlg1 prevents its ubiquitination and degradation

Protein palmitoylation is a post-translational modification that affects a great number of proteins. In most cases, the enzymes responsible for this modification have not been identified. Some proteins use palmitoylation to attach themselves to membranes; however, palmitoylation also occurs in transmembrane proteins, and the function of this palmitoylation is less clear. Here we identify Swf1, a member of the DHHC-CDR family of palmitoyltransferases, as the protein responsible for modifying the yeast SNAREs Snc1, Syn8 and Tlg1, at cysteine residues close to the cytoplasmic end of their single transmembrane domains (TMDs). In an swf1Delta mutant, Tlg1 is mis-sorted to the vacuole. This occurs because unpalmitoylated Tlg1 is recognised by the ubiquitin ligase Tul1, resulting in its targeting to the multivesicular body pathway. Our results suggest that one role of palmitoylation is to protect TMDs from the cellular quality control machinery, and that Swf1 may be the enzyme responsible for most, if not all, TMD-associated palmitoylation in yeast.     

APC/Cdh1 targets PECAM-1 for ubiquitination and degradation in endothelial cells

Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a member of the immunoglobulin superfamily and is expressed by hematopoietic and endothelial cells (ECs). Recent studies have shown that PECAM-1 plays a crucial role in promoting the development of the EC inflammatory response in the context of disturbed flow. However, the mechanistic pathways that control PECAM-1 protein stability remain largely unclear. Here, we identified PECAM-1 as a novel substrate of the APC/Cdh1 E3 ubiquitin ligase. Specifically, lentivirus-mediated Cdh1 depletion stabilized PECAM-1 in ECs. Conversely, overexpression of Cdh1 destabilized PECAM-1. The proteasome inhibitor MG132 blocked Cdh1-mediated PECAM-1 degradation. In addition, Cdh1 promoted K48-linked polyubiquitination of PECAM-1 in a destruction box-dependent manner. Furthermore, we demonstrated that compared with pulsatile shear stress (PS), oscillatory shear stress decreased the expression of Cdh1 and the ubiquitination of PECAM-1, therefore stabilizing PECAM-1 to promote inflammation in ECs. Hence, our study revealed a novel mechanism by which fluid flow patterns regulate EC homeostasis via Cdh1-dependent ubiquitination and subsequent degradation of PECAM-1.     

Ubiquitin ligase Smurf1 targets TRAF family proteins for ubiquitination and degradation

The HECT-type E3 Smad ubiquitination regulation factor 1 (Smurf1) functions in regulation of cell polarity and bone homeostasis by targeting Smads, Runx2, RhoA and MEKK2 for ubiquitination and degradation. In a yeast two-hybrid screening, we identified TNF receptor-associated factor 4 (TRAF4) as a candidate substrate and was further validated. The PY motifs of TRAF4 mediated the interaction with the second WW domain of Smurf1. Overexpression of Smurf1 reduced the protein levels of TRAF4 dependent of its E3 activity and the proteasome. Further, we showed that all six members of TRAF family could be ubiquitinated by Smurf1. Consequently, Smurf1 interfered with the functions of TRAFs in NF-κB signaling under stimulation or not. These results suggested a new role of Smurf1 in inflammation and immunity through controlling the degradation of TRAFs.     

Anaphase-promoting complex/cyclosome-cdh1 mediates the ubiquitination and degradation of TRB3

We have recently demonstrated that TRB3, a novel endoplasmic reticulum (ER) stress-inducible protein, is induced by CHOP and ATF4 to regulate their function and ER stress-induced cell death; however, the regulation of TRB3 function has not been well characterized. Here we demonstrate that TRB3 is an unstable protein regulated by the ubiquitin-proteasome system. The carboxyl-terminal domain of TRB3 is necessary for protein degradation, and in this region, we found the typical D-box motif, which is a critical sequence for the anaphase-promoting complex/cyclosome (APC/C) dependent proteolysis. TRB3 proteins were stabilized by deletion of its D-box motif and interacted with APC/C coactivator proteins, Cdc20 and Cdh1. The expression level of TRB3 protein is down-regulated by over-expression of Cdh1 but not by that of Cdc20. In addition, knockdown of Cdh1 enhanced the endogenous TRB3 expression level and suppressed its ubiquitination level. These results suggest that APC/C^Cdh1 is involved in ubiquitination and down-regulating the stability of TRB3 protein.     

E3 ubiquitin ligase SIAH1 mediates ubiquitination and degradation of TRB3

Tribbles 3 homolog (TRB3) is recently identified as a scaffold-like regulator of various signal transducers and has been implicated in several processes including insulin signaling, NF-kappaB signaling, lipid metabolism and BMP signaling. To further understand cellular mechanisms of TRB3 regulation, we performed a yeast two-hybrid screen to identify novel TRB3 interacting proteins and totally obtained ten in-frame fused preys. Candidate interactions were validated by co-immunoprecipitation assays in mammalian cells. We further characterized the identified proteins sorted by Gene Ontology Annotation. Its interaction with the E3 ubiquitin ligase SIAH1 was further investigated. SIAH1 could interact with TRB3 both in vitro and in vivo. Importantly, SIAH1 targeted TRB3 for proteasome-dependent degradation. Cotransfection of SIAH1 could withdraw up-regulation of TGF-beta signaling by TRB3, suggesting SIAH1-induced degradation of TRB3 represents a potential regulatory mechanism for TGF-beta signaling.