Huoh, Yu-San
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Publication The Molecular Basis of Substrate Recognition by the Family of Pellino E3 Ubiquitin Ligases(2013-01-01) Huoh, Yu-SanThe four mammalian Pellinos (Pellinos 1, 2, 3a, and 3b) are E3 ubiquitin ligases that have emerging roles in the regulation of Toll-like receptors, interleukin-1 receptor, T-cell receptor, Nod2, and TNF receptor signaling pathways. While each Pellino has a distinct role in facilitating various cellular processes, the underlying mechanisms by which these highly homologous proteins act selectively in these signaling pathways are not clear. In this dissertation, we elucidate the molecular basis of Pellino substrate specificity in order to gain a better understanding of the roles that individual Pellinos play in orchestrating inflammation and cell death. Pellino substrate recognition is mediated by a non-canonical FHA domain, a well-characterized phosphothreonine binding module. We identify a high-affinity Pellino-FHA domain binding motif in the Pellino substrate, interleukin 1 receptor associated kinase 1 (IRAK1). Binding analysis of the different Pellinos to a panel of pT-peptides reveals that each Pellino has a distinct binding specificity. This specificity also manifests in the interaction of Pellinos with a number of known Pellino substrates. These results argue that the non-redundant roles that Pellinos play in immune signaling are in part due to their divergent substrate specificities. In addition to elucidating Pellino substrate binding preferences, we also sought to determine how Pellinos interact with substrates. Through mutational analyses, we have found that the Pellino2 FHA domain β9/β10 loop mediates interactions with IRAK1, but is dispensable for pT-peptide binding. These results show that Pellino substrate binding determinants may require more than a short pT-peptide motif.