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Title:Investigation and application of substrate tolerant thioether forming enzymes
Author(s):He, Chang
Director of Research:van der Donk, Wilfred A
Doctoral Committee Chair(s):van der Donk, Wilfred A
Doctoral Committee Member(s):Chen, Jie; Sweedler, Jonathan V; Hergenrother, Paul J
Department / Program:Chemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Lanthipeptide engineering
Lanthipeptide library
LanC-like protein
Abstract:Thioether-containing compounds are involved in different biological functions in both prokaryotic and eukaryotic systems. Nature has evolved a treasure box of intriguing thioether containing natural products. Lanthipeptides contain thioether rings and have demonstrated diverse biological activities. This group of natural products are of great interest due to their diverse ring topology and high substrate tolerance of the biosynthetic enzymes. Amongst them lanthipeptides are one of the most studied ribosomally synthesized and post-translationally modified peptides (RiPPs). In general, a precursor peptide with an N-terminal leader peptide and a C-terminal core peptide is ribosomally synthesized. The core peptides undergoes a series of post-translational modification including dehydration of serines and threonines to the α,β-unsaturated residues 2,3-didehydroalanine (Dha) and (Z)-2,3-didehydrobutyrine (Dhb), respectively. Thioether rings are formed through Michael addition of thiol groups on cysteine residues to theses dehydroamino acids. The removal of the leader peptide yields the mature product, which is exported from the producing cells. The enzymes involved in lanthipeptides biosynthesis are well studied and several platforms have been employed for lanthipeptide engineering using the highly substrate tolerant lanthipeptides synthetases. The potential of lanthipeptide engineering is further demonstrated in chapter 2 using a substrate-tolerant synthetase (ProcM) from Prochlorococcus that dehydrates and cyclizes up to 30 different linear precursor peptides encoded in its genome. A bicyclic lanthipeptide library was constructed, characterized and coupled to a selection system to screen for a protein-protein interaction (PPI) inhibitor. XY3-3 was identified as an inhibitor for the PPI between the HIV p6 protein and the ubiquitin E2 variant (UEV) domain of the human TSG101 protein, which is essential for HIV virus budding from infected cells. A detailed in vitro characterization was performed to identify the binding site of XY3-3 using fluorescence polarization. XY3-3 competes with full length p6 protein for binding to UEV. To optimize XY3-3, two yeast display systems are investigated. Unfortunately, initial data suggested that the displayed peptide formed a disulfide bond which inhibited ProcM modification. Further optimization of yeast display systems or adaption of other display systems may allow the selection of a lanthipeptide scaffold with better binding affinity. While my work illustrated applications for bacterial lanthipeptide synthetases, their mammalian homologs remain mysterious. LanCL proteins, which are homologs of the lanthipeptide cyclase LanC, share similar a structure and conserved zinc binding residues with their bacterial counterparts. Moreover, LanCL proteins bind to glutathione at the putative active site. Recent studies suggested diverse functions of LanCL proteins, yet their enzymatic activity is not well understood. One hypothesis is that LanCL proteins function in a similar way as LanC, by catalyzing glutathionylation of dehydro amino acids to afford the small molecule lanthionine in mammalian systems. To test this hypothesis, in chapter 3, I developed two LC/MS/MS methods to quantify endogenous lanthionine concentrations in both wild type and LanCL knockout mouse brains, where LanCL proteins are highly expressed. Similar levels of about 0.5-2.5 nmol/g tissue were detected for both WT and TKO mouse, suggesting LanCL proteins are not involved in lanthionine synthesis. However, it is still possible that LanCL proteins use glutathione as one of the substrates. Indeed, recent findings in our lab showed LanCL proteins interact with a wide range of kinases and catalyze glutathionylation of Dha/Dhb containing kinase proteins and peptides. In chapter 4, I expanded the substrate scope with non-kinase lanthipeptides, demonstrating LanCL proteins are highly substrate tolerant. To identify LanCL targets, I developed a chemoproteomics method to enrich proteins with LanCL-catalyzed glutathionylation. The initial proteomics data suggest that LanCL protein targets are not limited to kinases and LanCL-catalyzed glutathionylation may function as a general mechanism to eliminate functions of electrophilic Dha/Dhb containing proteins.
Issue Date:2020-01-24
Rights Information:Copyright 2020 Chang He
Date Available in IDEALS:2020-08-27
Date Deposited:2020-05

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