Research Projects

Ubiquitin (Ub) signaling is a central keystone in regulating cellular processes like protein degradation. Deubiquitinating enzymes (DUBs) are essential participants in Ub signaling and work at different phases of the Ub life cycle, from producing mature Ub to modifying the Ub chain and regulating the fate and function of ubiquitinated proteins. Synthetic strategies have supplied novel Ub chains in their free or anchored form with large quantities and high purity, which allowed probing the specificity of certain DUBs and the molecular basis of their recognition, giving an insight into their functions in a native environment. Despite various improvements, studying Ub signaling in live cells remains challenging due to the inability to deliver large functional Ub probes across cellular membranes. Currently, activity-based probes (ABPs) for deubiquitinating enzymes (DUBs) are limited to mono- and di-Ub systems due to challenges in synthesizing stable, lengthier probes for more complex Ub chains and their live-cell delivery due to the existing platform's inability. This project addresses the critical need to chemically synthesize a stable, functional, and non-hydrolyzable Lys48-linked tetra-ubiquitin probes (~35 kDa) as a model system and develop an efficient cell-penetrating peptide (CPP) based delivery platform to facilitate the intracellular delivery of such probes to enable DUB activity in live cells. CPPs will be designed by incorporating histidine and 2-amino histidine substitutions in cyclic deca-arginine (cR10), optimizing membrane compatibility and reducing cytotoxicity. Stable tetra-ubiquitin probes with triazole linkages will be chemically synthesized by coupling between Ub units via well-established azide-alkyne click chemistry and conjugated to CPPs via cleavable disulfide bonds for cytosolic access to the probe in native form. Confocal microscopy and proteomics will assess intracellular delivery efficiency and DUB activity in live-cell environments. Combining the chemical synthesis of stable tetra-Ub probes and unique CPPs offers a unique approach to cellular studies in Ub signaling. This addresses a potential research gap by improving our understanding of the biological roles of DUBs in a physiologically suitable setting, applicable to answer fundamental biological questions on live-cell tetra-Ub delivery. Moreover, delivering large and complex stable Ub ABPs and studying complex Ub signaling in live cells will set the ground for future studies with even more complicated and giant protein targets (e.g., heterotypic branched or mixed Ub-chains) to understand their Ub signaling and extended application in protein therapeutics. Broadly, the research will benefit scientists in chemistry, chemical biology, drug delivery, and drug discovery, together with more comprehensive applications in the biotechnology and pharmaceutical industries, contributing to advancements in therapeutic interventions.