Development of cell-permeable ubiquitin probes for investigation of deubiquitinases
Date
2020
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
University of Delaware
Abstract
Ubiquitination is a reversible enzymatic post-translational modification system regulates a broad range of cellular processes in eukaryotes. Abnormal function and dysregulation of the ubiquitination system is known to cause a variety of human diseases including neurological disorders, cancer. During last decades, huge success has been achieved in targeting the proteasome system with several FDA approved drugs which tremendously encouraged the development of better and less toxic therapeutics against the human ubiquitination system in treating cancer and neurological disorders. The human genome encodes close to 100 deubiquitinating enzymes (DUBs), which are responsible for removing mono- and poly-ubiquitin from over 5,000 human proteins in cells in order to maintain the normal function and regulation of cells. Moreover, a large number of proteins can interact with DUBs and act as regulators and adaptors of DUBs by regulating the activity and function of DUBs, which further increase the diversity and specificity of DUBs. During the past decades, human DUBs emerging as novel targets for drug development, attract tremendous attention from academic research as well as industrial investment, due to their better-defined active and allosteric sites in comparison to many Ub ligases. ☐ To gain a deeper understanding of DUBs and their biological functions and activities requires biochemical tools permitting interrogation of DUB activities under physiologically relevant conditions with temporal precision. Activity-based probes for DUBs have been widely used in investigating the structure, function and activity of DUBs and helped to figure out the reaction mechanism of how DUBs remove ubiquitin chain from substrate proteins. However, most ubiquitin-based probes developed previously are not cell-permeable, limiting their utility to purified proteins and cell lysates. Lysis of cells usually leads to massive dilution of the cytoplasm and disruption of the normal cellular organization, which may alter the activity of many DUBs especially DUBs in complex with binding partners. To generate a new class of Ub-based cell-permeable probes can help to gain a deeper understanding of the intracellular activities of DUBs. In the second chapter, I described the generation and utility of cell-permeable ubiquitin-based probes containing a reactive warhead at the C-terminal for covalent trapping of DUBs with a catalytic cysteine. I employed cellpenetrating peptides (CPPs), particularly cyclic polyarginine (cR10), linked to the Nterminus of Ub in order to deliver Ub-based probe into cells, as confirmed using livecell fluorescence microscopy. In comparison to TAT containing cell-permeable Ubbased probe, enhanced intracellular delivery was observed through conjugation of a cyclic polyarginine (cR10) to the N-terminus of ubiquitin via a disulfide linkage. Using the new cR10 containing cell-permeable probes, I carried out intracellular DUB profiling of HeLa cells, and identified active DUBs been captured using immunocapture and label-free quantitative mass spectrometry. Additionally, I demonstrated that the cell-permeable probe can be used in assessing the inhibition efficiency of DUBs inhibitors in cells. My results indicate that cell-permeable Ubbased probe hold great promise in providing a better understanding of the cellular functions and activity of DUBs and can facilitate the discovery of potent therapeutics against human DUBs. ☐ The current developed Ub-based DUBs probes, including cell-permeable DUB probes, are constantly active. As we know, DUBs function and activity are highly regulated in cells. Photocaged cell-permeable ubiquitin probes hold the promise to profile the activity of DUBs with tempral resolution. In the third chapter, I described my initial attempt to photocage the reaction wardhead including Michael acceptor and aldehyde included at the Ub C-terminus probe by o-nitrobenzyl photo-removable group. However, the result was not optimal, as photocaged Michael acceptor warhead showed background labeling of USP2-CD even without UV irradiation and photocaged aldehyde probe showed weak labeling activity upon UV treatment. In the fourth chapter, I described the generaton of a photocaged cell-permeable Ub probe using tetrazole as the photocaged warhead. Upon 365 nm UV irradiation for 15 min, the tetrazole probe undergoes photoactivation and generates nitrilimine in situ, which covalently traps the active site cysteine of DUBs. I used a semisynthetic approach to generate ubiquitin-based probe containing a tetrazole warhead at the C-terminus of ubiquitin and employed a cyclic polyarginine (cR10) cell-penetrating peptide conjugated to the N-terminus of ubiquitin via a disulfide linkage to deliver the probe into live HeLa cells. The photocaged cell-permeable probe can label purified DUBs, including USP2, UCHL1 and UCHL3 through the trapping of the catalytic cysteine in the DUB active site only opon UV irradiation. Moreover, the cell-permeable Ubtetrazole probe showed robust labelling activity in HeLa cell lysate and live HeLa cells only with 365 nm UV irradiation. Using label-free quantitative mass spectrometry, I carried out protein profiling in intact HeLa cells by the new photocaged cellpermeable ubiquitin probe and identified 15 DUBs that were specifically captured by the probe upon 365 nm UV irradiation. Moreover, as a proof of concept, I did cell cycle dependent profiling of DUBs. The result showed the new photocaged cell-permeable ubiquitin probe can capture DUBs in G1/S and G2/M phases. Moreover, the probe allowed intracellular temporal profiling of DUBs at different time points (0 h, 1 h and 4 h) following the release of HeLa cells from G1/S phase. My results indicate that photocaged cell-permeable probe holds great promise in providing a better understanding of the cellular functions of DUBs and other ubiquitin binding proteins.
Description
Keywords
Cell-permeable, Cancer, Catalytic cysteine, UV treatment, Ubtetrazole