Development and application of ubiquitin-based chemical probes

Sui, Xin; Wang, Yu; Du, Yunxiang; Liang, Lujun; Zheng, Qin; Li, Yiming; Liu, Lei

doi:10.1039/d0sc03295f

Cited by 50 publications

(33 citation statements)

References 130 publications

(158 reference statements)

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“…In eukaryotic cells, proteins can be post-translationally modified by Ub or Ubl proteins to undergo proteasome degradation or serve as function regulators for a large variety of cellular processes [ 35 , 36 , 37 , 38 , 39 ]. To study pathways involving Ub and Ubls, diverse Ub and Ubl probes have been synthesized and used for various research purposes [ 3 , 40 , 41 , 42 ]. Dha probes which contain an α, β-unsaturated carbonyl structure at the C -terminus of Ub and Ubl proteins are ideal in the formation of covalent adducts with a catalytic cysteine in many enzymes in Ub and Ubl pathways.…”

Section: Resultsmentioning

confidence: 99%

“…In cells, proteins are usually post-translationally modified via enzymatic reactions. Due to the fact that the enzymes which install PTMs are either unknown or promiscuous, chemical biologists have long been focusing on the development of synthetic and semi-synthetic methods for the synthesis of post-translationally modified proteins for their functional investigations [ 1 , 2 , 3 , 4 ]. Notable methods include native chemical ligation [ 5 ], expressed protein ligation [ 6 ], and the genetic code expansion technique that relies on the suppression of amber codon for the incorporation of noncanonical amino acids that are post-translationally modified proteinogenic amino acids themselves, or can be chemically modified to form amino acids with PTMs [ 7 , 8 , 9 , 10 , 11 ].…”

Section: Introductionmentioning

confidence: 99%

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Site-Specific Conversion of Cysteine in a Protein to Dehydroalanine Using 2-Nitro-5-thiocyanatobenzoic Acid

Qiao

Leeuwon

et al. 2021

Molecules

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Dehydroalanine exists natively in certain proteins and can also be chemically made from the protein cysteine. As a strong Michael acceptor, dehydroalanine in proteins has been explored to undergo reactions with different thiolate reagents for making close analogues of post-translational modifications (PTMs), including a variety of lysine PTMs. The chemical reagent 2-nitro-5-thiocyanatobenzoic acid (NTCB) selectively modifies cysteine to form S-cyano-cysteine, in which the S–C? bond is highly polarized. We explored the labile nature of this bond for triggering E2 elimination to generate dehydroalanine. Our results indicated that when cysteine is at the flexible C-terminal end of a protein, the dehydroalanine formation is highly effective. We produced ubiquitin and ubiquitin-like proteins with a C-terminal dehydroalanine residue with high yields. When cysteine is located at an internal region of a protein, the efficiency of the reaction varies with mainly hydrolysis products observed. Dehydroalanine in proteins such as ubiquitin and ubiquitin-like proteins can serve as probes for studying pathways involving ubiquitin and ubiquitin-like proteins and it is also a starting point to generate proteins with many PTM analogues; therefore, we believe that this NTCB-triggered dehydroalanine formation method will find broad applications in studying ubiquitin and ubiquitin-like protein pathways and the functional annotation of many PTMs in proteins such as histones.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Site-Specific Conversion of Cysteine in a Protein to Dehydroalanine Using 2-Nitro-5-thiocyanatobenzoic Acid

Qiao

Leeuwon

et al. 2021

Molecules

View full text Add to dashboard Cite

show abstract

“…The design of a robust reconstituted in vitro assay to monitor the ubiquitylation of substrates, suitable for HTS would enable both mechanistic studies of Ub transfer as well as small‐molecule modulator screens for TRIM proteins. To this extent, a variety of synthetic and semisynthetic approaches to obtain Ub and UBL‐based reagents have been developed [209] allowing for different assay readouts. Fluorescence polarization (FP)‐based screening methods have been successfully used to monitor DUBs activity, mainly employing Ub‐AMC or Ub‐rhodamine, which fluoresce only after cleavage [210] .…”

Section: Recent Advances To Explore the Ubiquitylation Machinerymentioning

confidence: 99%

Targeting TRIM Proteins: A Quest towards Drugging an Emerging Protein Class

et al. 2021

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The ubiquitylation machinery regulates several fundamental biological processes from protein homeostasis to a wide variety of cellular signaling pathways. As a consequence, its dysregulation is linked to diseases including cancer, neurodegeneration, and autoimmunity. With this review, we aim to highlight the therapeutic potential of targeting E3 ligases, with a special focus on an emerging class of RING ligases, named tri‐partite motif (TRIM) proteins, whose role as targets for drug development is currently gaining pharmaceutical attention. TRIM proteins exert their catalytic activity as scaffolds involved in many protein–protein interactions, whose multidomains and adapter‐like nature make their druggability very challenging. Herein, we give an overview of the current understanding of this class of single polypeptide RING E3 ligases and discuss potential targeting options.

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“…Answers to these questions will provide greater insights into our understanding of ESCs and iPSCs, and further their applications in basic and clinical research of human diseases. One of the technical challenges is to develop antibodies or Ub-based chemical probes to capture any Ub linkage of interest with high specificity and sensitivity [ 188 ].…”

Section: Concluding Remarks and Perspectivesmentioning

confidence: 99%

The Role of E3s in Regulating Pluripotency of Embryonic Stem Cells and Induced Pluripotent Stem Cells

Zhang

2021

IJMS

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Pluripotent embryonic stem cells (ESCs) are derived from early embryos and can differentiate into any type of cells in living organisms. Induced pluripotent stem cells (iPSCs) resemble ESCs, both of which serve as excellent sources to study early embryonic development and realize cell replacement therapies for age-related degenerative diseases and other cell dysfunction-related illnesses. To achieve these valuable applications, comprehensively understanding of the mechanisms underlying pluripotency maintenance and acquisition is critical. Ubiquitination modifies proteins with Ubiquitin (Ub) at the post-translational level to monitor protein stability and activity. It is extensively involved in pluripotency-specific regulatory networks in ESCs and iPSCs. Ubiquitination is achieved by sequential actions of the Ub-activating enzyme E1, Ub-conjugating enzyme E2, and Ub ligase E3. Compared with E1s and E2s, E3s are most abundant, responsible for substrate selectivity and functional diversity. In this review, we focus on E3 ligases to discuss recent progresses in understanding how they regulate pluripotency and somatic cell reprogramming through ubiquitinating core ESC regulators.

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Development and application of ubiquitin-based chemical probes

Abstract: This article reviews the design, synthesis, and application of different classes of Ub-based chemical probes.

Cited by 50 publications

References 130 publications

Site-Specific Conversion of Cysteine in a Protein to Dehydroalanine Using 2-Nitro-5-thiocyanatobenzoic Acid

Site-Specific Conversion of Cysteine in a Protein to Dehydroalanine Using 2-Nitro-5-thiocyanatobenzoic Acid

Targeting TRIM Proteins: A Quest towards Drugging an Emerging Protein Class

The Role of E3s in Regulating Pluripotency of Embryonic Stem Cells and Induced Pluripotent Stem Cells

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