Novel modification of Human Myeloma proteasomes and development of non-active site directed inhibitors

Abstract

Multiple Myeloma (MM) is a plasma cell malignancy that is characterised by bone lesions and production of excessive amounts of monoclonal protein. Treatment involves the use of chemotherapy agents, immunomodulatory agents and proteasome inhibitors (PI). The 26S proteasome is a 2.5 MDa molecular machine that is integral to the viability of all eukaryote cell. Its main function is to hydrolyse proteins that are marked for degradation by a poly-ubiquitin chain. Although MM is treatable, it is as yet incurable with mean survival of ~6 years. In this thesis, I show that human proteasomes contain a charged polymeric posttranslational modification (PTM), one which has some similarity to poly-ADP-ribose. This modification is not normally resolvable via normal SDS-PAGE electrophoresis, but can be resolved by the lesser used CTAB-PAGE or after separation of the proteasome from other cellular components. This modification appears to be present predominantly in the nucleus of the cell, and may provide a mechanism for how nuclear proteasomes interact with chromatin, DNA and other nuclear components. The use of proteasome inhibitors as a valid therapy for MM has been hypothesised to be due to a high proteasome load in MM; therefore, a small amount of inhibition would suffice to perturb proteostasis. However, I show that myeloma cells experience severe proteasome inhibition upon treatment with compounds such as Bortezomib, to a degree that far exceeds the levels of inhibition observed with purified proteasomes. This suggests that, when PIs engage with proteasomal active-sites, they trigger a cellular mechanism which exacerbates this inhibition to a far greater degree than would otherwise be achieved. I excluded trivial explanations including additional binding of the inhibitor, caspase mediated proteasome inhibition or cell death initiation. Intriguingly, I found that early changes to CTAB-PAGE detectable PTMs coincided with PIs’ ability to achieve an excessive degree of cellular proteasome inhibition. In addition to this work I continue the development of an allosteric proteasome inhibitor identified by a phage display technique. Through a number of rounds of chemical optimisation, I show the ability of these compounds to inhibit proteasome degradation of an ubiquinated substrate and a lethality in myeloma cells at 25 nM.