|Ph.D Student||Singh Sumeet Kumar|
|Subject||Expediting the Chemical Synthesis of Polyubiquitinated|
Proteins to Shed Light on Proteasomal Degradation
|Department||Department of Chemistry||Supervisor||Professor Ashraf Brik|
|Full Thesis text|
Recent advances in a chemical and semi-synthetic approach for protein synthesis offer unique opportunities to deliver the desired bio-conjugates in high homogeneity and workable quantities to support biochemical, biophysical, and functional studies. These tools become more critical in systems where biological methods are unable to afford the target in high homogeneity and good quantities. To understand how ubiquitin (Ub) chain length, linkage type, position on the substrate, and susceptibility to DUBs (especially RPN11) affect the processing of different substrates as well as Ub itself by proteasomes, we need efficient methods which can enable us to produce these conjugates.
To address this formidable challenge, we developed a strategy based on a selective modification of cysteine residue of the protein of interest with an aldehyde moiety to enable oxime conjugation with the desired Ub chain bearing hydroxylamine functionality at the C-terminus of Ub. The developed oxime chemistry yielded minimal side products, and it was highly selective and extremely rapid. Furthermore, we applied this strategy for the synthesis of polyubiquitinated proteins to generate a set of well-defined ubiquitinated proteins bearing an isopeptide mimic that is resistant to DUBs between the chain and the substrate. This approach enabled us to comprehend how Ub chain length governs protein degradation outcome. Our results support that (1) the cleavage of the proximal isopeptide bond is not a prerequisite for proteasomal degradation, (2) by overcoming trimming at the proteasome, tetraUb is a fundamentally different signal than shorter chains, and (3) the tetraUb chain can be degraded with the substrate. Together, these results highlight the usefulness of chemistry to dissect the contribution of proteasome-associated DUBs and the complexity of the degradation process.
To further investigate the unique aspects of proteasomal degradation, especially to comprehend the fate of different Ub during proteasomal degradation, we performed the chemical syntheses of a native ubiquitinated substrate with a Ub chain that is differentially labelled at the distal (Myc/Flag tag) and proximal Ub (Myc/Flag tag) as well as the substrate protein (HA tag). Using the specific antibodies against these different tags, we were able to follow and understand the fate of each part of the polyubiquitinated substrate. Our initial studies revealed that the distal Ub is more susceptible towards DUBs than proximal Ub, and interestingly, we found that proximal Ub seems to degrade along with substrate protein. These initial results are promising, and we are still working to confirm them and will soon finalise all the results related to this study.
Overall, controlling substrate features at the atomic level by applying innovative chemistry to lower degrees of freedom provides unprecedented opportunities to dissect a complicated enzymatic process, such as in the proteasome. During this thesis work, replacing the proximal isopeptide bond with an oxime bond provided new information about the contribution of the shaving step (attributed to Rpn11) to the overall degradation process. The finding that tetraUb can be degraded with the substrate triggers a fresh look at natural conjugates for which no known DUBs are identified for the shaving step.