|Ph.D Student||Mansour Wissam|
|Subject||Modifying the Modifier: Recognition and Processing of|
Ubiquitin at the Proteasome
|Department||Department of Biology||Supervisors||Professor Michael Glickman|
|Dr. Akram Alian|
|Full Thesis text|
Attachment of ubiquitin (Ub) to cellular proteins is among the most common post translation modifications (PTMs) in eukaryotic cells. Once a single Ub molecule is covalently attached to a substrate protein, additional Ub units can be added to the previously attached Ub at any of Ub seven lysine’s, allowing the resulting polyUb signal to initiate a variety of cellular pathways. Substrates modified with Lys48-linked polyUb are targeted to degradation at the 26S proteasome. Numerous deubiquitinases (DUBs) - special isopeptidases - reverse ubiquitination by hydrolyzing polyUb chains back to monomeric Ub units. At the proteasome, Ubp6 and Rpn11 are the DUBs responsible for deconjugating polyUb from substrates.
The aims of this research were to 1) determine the Ub linkage preferences and enzymatic properties of proteasomal DUBs, 2) determine effect of phosphorylation on recognition of the Ub signal, and 3) uncover additional polyUb receptors on the proteasome. These aims were achieved by designing a panel of polyUb conjugates and by devising assays for study of proteasomal DUBs uncoupled form the proteasome. Through comparative studies, it was uncovered that Rpn11 and Ubp6 processed Lys11- and Lys63-linkages with a similar efficiency, which increased accordingly with Ub chain length. However, increasing chain length with Lys48 linkages produced the opposite effect, as most DUBs failed to process these chains. The highly compact nature of Lys48-linked polyUb may provide a structural explanation for this effect. Proteasomal DUBs were activated by roughly two orders of magnitude upon incorporation to proteasome compared to their enzymatic efficiency in isolation as standalone enzymes. Furthermore, the proteasome is a highly competent DUB capable of processing all Ub-Ub linkage types as demonstrated by the Ubch5b-Ubn substrate.
Ser65-phospho monoUb was chemically synthesized as well as Lys63-Ub2 that was phosphorylated either at the proximal unit or the distal unit, or both. Serving as a control, unmodified WT Ub and WT Lys63-Ub2 were similarity synthesized. Phosphorylated monoUb revealed that modified Ub can be activated by E1, while only selected E2s were able to utilize the activated phosphorylated Ub. Selective phosphorylation at different positions differentially effected several DUBs, suggesting that DUBs form unique interactions with polyUb chains. In particular, the proteasome exhibited sensitivity to the position of the phospho modification in Lys63-Ub2.
Photo-leucine cross-linker polyUb (pLeu-Ub) chains uncovered new polyUb receptors on the proteasome. Covalent cross-linking of pLeu-Ub chains to known Ub receptors, as well as linkage-selective receptors provided strong validation of this reagent. By applying pLeu-Ub to proteasome, the Rpn1 subunit was uncovered as a new polyUb receptor. Bioinformatic analysis provided a structural model for Rpn1 and predicted an interaction between the PC domain of Rpn1 and Ub hydrophobic patch. This was confirmed by applying pLeu-Ub to full length and truncated forms of recombinant Rpn1.
Collectively this work represents a major advance in the synthesis and application of well-defined Ub conjugates of diverse lengths, linkage types, and modifications to proteasome function. These results establish substrate preferences of proteasomal DUBs, how phospho-Ub is processed by DUBs, and characterization of Rpn1 as a novel polyUb receptor.