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Selective Macrocyclic Peptide Modulators of Lys63-linked Ubiquitin Chains Disrupt DNA Damage Repair

ABSTRACT: Developing an effective binder for a specific ubiquitin (Ub) chain is a promising approach for modulating various biological processes with potential applications in drug discovery. In this study, we combined the Random Non-standard Peptides Integrated Discovery (RaPID) method and chemical protein synthesis to screen an extended library of macrocyclic peptides against synthetic Lys63-linked Di-Ub. This enabled us to discover a novel binder with low nanomolar affinity and specificity for this particular Ub chain. We further chemically modified the most effective binder to generate next-generation binders. We show that our potent cyclic peptide is cell-permeable and inhibits DNA damage repair, leading to cell cycle arrest and apoptotic cell death. Concordantly, a pulldown experiment with the biotinylated analog of our lead cyclic peptide identified various proteins involved in DNA synthesis and damage repair. Collectively, we established a novel and powerful strategy for selective inhibition of protein-protein interactions associated with Lys63-linked Di-Ub using cyclic peptides. This study offers an advancement in modulating central Ub pathways and provides new opportunities in drug discovery areas associated with Ub signaling.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Keren Bendalak

LAB HEAD: Ashraf Brik

PROVIDER: PXD035924 | Pride | 2022-09-19

REPOSITORIES: Pride

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Project description:The discovery of hepatitis C virus (HCV) in 1989 revealed the virus as the etiology of 40%-90% of the “essential” mixed cryoglobulinemia, where immune complex forms deposit called cryoprecipitate at temperatures below 37 °C. Cryoprecipitate constitutes monoclonal IgM and polyclonal IgG, some of which has reactivity against HCV core and NS3 epitopes. Resultant immune complex is considered entrapped on microvascular endothelium via C1q receptor, leading to complement activation and organ injury presenting predominantly as dermopathy, peripheral neuropathy and nephropathy. However, currently little is known on whether auto-reactive, cold-precipitating IgG components enriched in cryoprecipitate may play some role on the deposition of immune complex and subsequent complement-mediated injury of specific organs. Recently, with the advent of high-throughput immune repertoire sequencing and mass spectrometry, technical feasibility is growing to delineate antibodies of interest and their sequences directly from serum. To date, vast majority of studies actually utilized the antigen column for affinity purification of antibodies of interest, although this strategy is not applicable to disease entity with unknown antigen involvement. In such cases, disease-specific and organ-specific immune deposits may be a good alternative source of etiological antibodies. Herein, targeting HCV cryoglobulinemic vasculitis as a model, we conducted a proof-of-concept study aiming at characterizing the IgG components most prone to cryoprecipitation. To this end, we longitudinally studied one patient with cryoglobulinemic vasculitis with chronic HCV infection. After obtaining informed consent, cryoprecipitate and supernatant were separated from peripheral blood sample. Fab fragments from Protein G-purified IgG were recovered after papain digestion for isobaric tags for relative and absolute quantification (iTRAQ)-based quantitative proteomics. Simultaneously, total RNA was isolated from peripheral blood, and immunoglobulin heavy chain variable region (IGHV) was PCR-amplified with unique molecular identifier (UMI) strategy to construct a personal IGHV sequence library of immunoglobulin variable region. Sequencing output from MiSeq was bioinformatically converted into mass spectrometry database. Search was performed using MaxQuant software.

			Action	DRS
	74109-14_proteinGroups-genes.xlsx	Xlsx
	74109-14_proteinGroups-perseus.xlsx	Xlsx
	GlyGly__K_Sites.txt	Txt
	NAN.txt	Txt
	Oxidation__M_Sites.txt	Txt

Dataset Information

Selective Macrocyclic Peptide Modulators of Lys63-linked Ubiquitin Chains Disrupt DNA Damage Repair

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