Abstract
ITCH (aka Atrophin-1-interacting protein 4) is a prominent member of the NEDD4 HECT (Homologous to E6AP C-Terminus) E3 ubiquitin ligase family that regulates numerous cellular functions including inflammatory responses through T-cell activation, cell differentiation, and apoptosis. Known intracellular targets of ITCH-dependent ubiquitylation include receptor proteins, signaling molecules, and transcription factors. The HECT C-terminal lobe of ITCH contains the conserved catalytic cysteine required for the covalent attachment of ubiquitin onto a substrate and polyubiquitin chain assembly. We report here the complete experimentally determined 1H, 13C, and 15N backbone and sidechain resonance assignments for the HECT C-terminal lobe of ITCH (residues 784–903) using heteronuclear, multidimensional NMR spectroscopy. These resonance assignments will be used in future NMR-based studies to examine the role of dynamics and conformational flexibility in HECT-dependent ubiquitylation as well as deciphering the structural and biochemical basis for polyubiquitin chain synthesis and specificity by ITCH.
Highlights
Ubiquitylation is an important posttranslational modification that maintains cellular health and homeostasis by targeting proteins for proteosomal or autophagic degradation (Cohen-Kaplan et al 2016)
Interesting New Gene) E3 ubiquitin ligases that primarily act as scaffolds by orienting the E2 ~ ubiquitin thiolester complex and target protein for ubiquitin transfer, the HECT (Homologous to E6AP C-Terminus) E3 ubiquitin ligases play a catalytic role in the final attachment of ubiquitin by forming a thiolester intermediate with ubiquitin before transferring it to a substrate protein (Lorenz 2018; Metzger et al 2012)
Each HECT domain is comprised of two lobes—a larger N-terminal lobe responsible for recruiting an E2 ~ ubiquitin complex, and a smaller C-terminal lobe that contains the catalytic cysteine required to covalently attach ubiquitin to its substrates (Lorenz 2018)
Summary
Ubiquitylation is an important posttranslational modification that maintains cellular health and homeostasis by targeting proteins for proteosomal or autophagic degradation (Cohen-Kaplan et al 2016). The 3D structures of several NEDD4 HECT domains family members have been solved by X-ray crystallography, either alone or complexed with E2 and/or ubiquitin (Lorenz 2018), there are many unanswered questions regarding how these enzymes assemble into multi-protein complexes to synthesize polyubiquitin chains. ITCH is an intriguing HECT E3 ubiquitin ligase due to its ability to synthesize different polyubiquitin lysine linkages (i.e. K29, K48, and/or K63-linked) depending on the substrate it modifies, and this ubiquitin linkage specificity is dependent on the last 60 amino acids of the C-terminal lobe of ITCH (Kim and Huibregtse 2009). We report the complete backbone and sidechain 1H, 13C, and 15N resonance assignments for the catalytic C-terminal lobe of ITCH (residues 784–903) using 3D heteronuclear NMR spectroscopy These resonance assignments will enable future structural and mechanistic studies to better understand ITCH-dependent ubiquitylation
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