BackgroundSpartin protein is involved in degradation of epidermal growth factor receptor and turnover of lipid droplets and a lack of expression of this protein is responsible for hereditary spastic paraplegia type 20 (SPG20). Spartin is a multifunctional protein that associates with many cellular organelles, including lipid droplets. Recent studies showed that spartin interacts with E3 ubiquitin ligases that belong to the neural precursor cell-expressed developmentally downregulated gene (Nedd4) family, including atrophin-1-interacting protein 4 (AIP4/ITCH). However, the biological importance of the spartin-AIP4 interaction remains unknown.ResultsIn this study, we show that spartin is not a substrate for AIP4 activity and that spartin's binding to AIP4 significantly increases self-ubiquitination of this E3 ligase, indicating that spartin disrupts the AIP4 autoinhibitory intramolecular interaction. Correspondingly, spartin has a seven times higher binding affinity to the WW region of AIP4 than the binding of the WW region has to the catalytic homologues of the E6-associated protein C-terminus (HECT) domain, as measured by enzyme-linked immunosorbent assay. We also show that spartin recruits AIP4 to lipid droplets and promotes ubiquitination of lipid droplet-associated protein, adipophilin, which regulates turnover of lipid droplets.ConclusionsOur findings demonstrate that spartin acts as an adaptor protein that activates and recruits AIP4 E3 ubiquitin ligase to lipid droplets and by this means regulates the level of ubiquitination of adipophilin and potentially other lipid-associated proteins. We propose that this is one of the mechanisms by which spartin regulates lipid droplet turnover and might contribute to the pathology of SPG20.

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The nuclear receptor (NR) superfamily is a group of transcriptional regulators that control multiple aspects of both physiology and pathology and are broadly recognized as viable therapeutic targets. While receptor-modulating drugs have been successful in many cases, the discovery of new drug targets is still an active area of research, because resistance to NR-targeting therapies remains a significant clinical challenge. Many successful targeted therapies have harnessed the control of receptor activity by targeting events within the NR signaling pathway. In this review, we explore the role of NR ubiquitylation and discuss how the expanding roles of ubiquitin could be leveraged to identify additional entry points to control receptor function for future therapeutic development. Key WordsJournal of Molecular Endocrinology (2015) 54, R151-R167

Background: NEMO is a known ubiquitin-binding protein that functions as a key regulator of NF-B activation. Results: WA is able to covalently modify NEMO to induce a gain-of-function binding to long Lys-48-linked polyubiquitin chains. Conclusion: WA can change the NEMO specificity for polyubiquitin chain interaction. Significance: This study reveals a novel drug class to target the UBD:ubiquitin interaction.

MEKK1 is a mitogen activated protein kinase kinase kinase (MAP3K) that regulates MAPK activation, and is the only known mammalian kinase that is also a ubiquitin ligase. MEKK1 contains a RING domain within its amino-terminal regulatory region, and MEKK1 has been shown to ubiquitylate the AP-1 transcription factor protein c-Jun, but the mechanism by which MEKK1 interacts with c-Jun to induce ubuiquitylation has not been defined. Proximal to RING domain is a SWI2/SNF2 and MuDR (SWIM) domain of undetermined function. In this report, we demonstrate that the MEKK1 SWIM domain, but not the RING domain directly associates with the c-Jun DNA binding domain, and that the SWIM domain is required for MEKK1-dependent c-Jun ubiquitylation. We further show that this MEKK1 SWIM/Jun interaction is specific, as SWIM domains from other proteins failed to bind c-Jun. We reveal that although the Jun and Fos DNA-binding domains are highly conserved, the MEKK1 SWIM domain does not bind Fos. Finally, we identify the sequence unique to Jun proteins required for specific interaction with the MEKK1 SWIM domain. Therefore we propose that the MEKK1 SWIM domain represents a novel substrate-binding domain necessary for direct interaction between c-Jun and MEKK1 that promotes MEKK1-dependent c-Jun ubiquitylation.

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Spartin contributes to the formation of dendritic aggresome-like induced structures (DALIS) through a unique ubiquitin-binding region (UBR). Using NMR and in vitro binding, the authors characterize spartin's UBR and show that DALIS formation depends on key residues within its UBR.

The NF-κB family of transcription factors regulates numerous cellular processes, including cell proliferation and survival responses. The constitutive activation of NF-κB has also emerged as an important oncogenic driver in many malignancies, such as activated B-cell like diffuse large B cell lymphoma, among others. In this study, we investigated the impact and mechanisms of action of Withaferin A, a naturally produced steroidal lactone, against both signal-inducible as well as constitutive NF-κB activities. We found that Withaferin A is a robust inhibitor of canonical and constitutive NF-κB activities, leading to apoptosis of certain lymphoma lines. In the canonical pathway induced by TNF, Withaferin A did not disrupt RIP1 polyubiquitination or NEMO-IKKβ interaction and was a poor direct IKKβ inhibitor, but prevented the formation of TNF induced NEMO foci which colocalized with TNF ligand. While GFP-NEMO efficiently formed TNF-induced foci, a GFP-NEMOY308S mutant that is defective in binding to polyubiquitin chains did not form foci. Our study reveals that Withaferin A is a novel type of IKK inhibitor which acts by disrupting NEMO reorganization into ubiquitin-based signaling structures in vivo.