Mining the TRAF6/P62 Interactome for Preferred Substrates and Target Ubiquitination Sites: Developing a “Code Hypothesis”

Abstract

Ubiquitination is the second most common protein modification studied in terms of biochemistry and cell physiology. It plays a central regulatory role in number of eukaryotic cellular and molecular processes. This three step process of concerted action of the E1-E2-E3 enzymes produces an ubiquitinated protein. How E3 ligases select substrates and achieve selectivity at a Lysine residue remains unsolved. I undertook studies to identify both ubiquitin and SUMO (small ubiquitin-related modifier) substrates with the goal of understanding how Lysine selectivity is achieved in these two processes. Although distinct from ubiquitination, SUMOylation pathway draws many parallels with it. Based upon recent findings, I present a model that explains how an individual ubiquitin ligase may target specific Lysine residue(s) with the co-operation from a scaffold protein, p62. Tumor necrosis factor receptor-associated factor 6 (TRAF6) is an ubiquitin ligase that regulates a diverse array of physiological processes via forming Lys-63 linked polyubiquitin chains. Described here is a new approach to predict ubiquitinated substrates of TRAF6/p62 complex. Interactome knowledge was used to predict potential TRAF6 substrates. Observations showed that there was low linear conservation of a single consensus motif at predicted ubiquitinated sites. However, a substantial structural and sequence conservation was observed across mammalian species for a novel ubiquitination defined as [–(hydrophobic)–k–(hydrophobic)–x–x–(hydrophobic)–(polar)–(hydrophobic) – (polar)–(hydrophobic)]. These findings revealed that the identified target sites have structural preferences and depend on accessibility within the protein molecule.