skip to main content

Title: Bioorthogonal Reporters for Detecting and Profiling Protein Acetylation and Acylation

Protein acylation, exemplified by lysine acetylation, is a type of indispensable and widespread protein posttranslational modification in eukaryotes. Functional annotation of various lysine acetyltransferases (KATs) is critical to understanding their regulatory roles in abundant biological processes. Traditional radiometric and immunosorbent assays have found broad use in KAT study but have intrinsic limitations. Designing acyl–coenzyme A (CoA) reporter molecules bearing chemoselective chemical warhead groups as surrogates of the native cofactor acetyl-CoA for bioorthogonal labeling of KAT substrates has come into a technical innovation in recent years. This chemical biology platform equips molecular biologists with empowering tools in acyltransferase activity detection and substrate profiling. In the bioorthogonal labeling, protein substrates are first enzymatically modified with a functionalized acyl group. Subsequently, the chemical warhead on the acyl chain conjugates with either an imaging chromophore or an affinity handle or any other appropriate probes through an orthogonal chemical ligation. This bioorganic strategy reformats the chemically inert acetylation and acylation marks into a chemically maneuverable functionality and generates measurable signals without recourse to radioisotopes or antibodies. It offers ample opportunities for facile sensitive detection of KAT activity with temporal and spatial resolutions as well as allows for chemoproteomic profiling of protein acetylation pertaining to specific KATs of interest on the global scale. We reviewed here the past and current advances in bioorthogonal protein acylations and highlighted their wide-spectrum applications. We also discussed the design of other related acyl-CoA and CoA-based chemical probes and their deployment in illuminating protein acetylation and acylation biology.

more » « less
Author(s) / Creator(s):
Publisher / Repository:
SAGE Publications
Date Published:
Journal Name:
SLAS DISCOVERY: Advancing the Science of Drug Discovery
Page Range / eLocation ID:
p. 148-162
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. Abstract

    Histone acetyltransferases (HATs, also known as lysine acetyltransferases, KATs) catalyze acetylation of their cognate protein substrates using acetyl‐CoA (Ac‐CoA) as a cofactor and are involved in various physiological and pathological processes. Advances in mass spectrometry‐based proteomics have allowed the discovery of thousands of acetylated proteins and the specific acetylated lysine sites. However, due to the rapid dynamics and functional redundancy of HAT activities, and the limitation of using antibodies to capture acetylated lysines, it is challenging to systematically and precisely define both the substrates and sites directly acetylated by a given HAT. Here, we describe a chemoproteomic approach to identify and profile protein substrates of individual HAT enzymes on the proteomic scale. The approach involves protein engineering to enlarge the Ac‐CoA binding pocket of the HAT of interest, such that a mutant form is generated that can use functionalized acyl‐CoAs as a cofactor surrogate to bioorthogonally label its protein substrates. The acylated protein substrates can then be chemoselectively conjugated either with a fluorescent probe (for imaging detection) or with a biotin handle (for streptavidin pulldown and chemoproteomic identification). This modular chemical biology approach has been successfully implemented to identify protein substrates of p300, GCN5, and HAT1, and it is expected that this method can be applied to profile and identify the sub‐acetylomes of many other HAT enzymes. © 2022 Wiley Periodicals LLC.

    Basic Protocol 1: Labeling HAT protein substrates with azide/alkyne‐biotin

    Alternate Protocol: Labeling protein substrates of HATs with azide/alkyne‐TAMRA for in‐gel visualization

    Support Protocol 1: Expression and purification of HAT mutants

    Support Protocol 2: Synthesis of Ac‐CoA surrogates

    Basic Protocol 2: Streptavidin enrichment of biotinylated HAT substrates

    Basic Protocol 3: Chemoproteomic identification of HAT substrates

    Basic Protocol 4: Validation of specific HAT substrates with western blotting

    more » « less
  2. The family of lysine acetyltransferases (KATs) regulates epigenetics and signaling pathways in eukaryotic cells. So far, knowledge of different KAT members contributing to the cellular acetylome is limited, which limits our understanding of biological functions of KATs in physiology and disease. Here, we found that a clickable acyl-CoA reporter, 3-azidopropanoyl CoA (3AZ-CoA), presented remarkable cell permeability and effectively acylated proteins in cells. We rationally engineered the major KAT member, histone acetyltransferase 1 (HAT1), to generate its mutant forms that displayed excellent bio-orthogonal activity for 3AZ-CoA in substrate labeling. We were able to apply the bio-orthogonal enzyme–cofactor pair combined with SILAC proteomics to achieve HAT1 substrate targeting, enrichment, and proteomic profiling in living cells. A total of 123 protein substrates of HAT1 were disclosed, underlining the multifactorial functions of this important enzyme than hitherto known. This study demonstrates the first example of utilizing bio-orthogonal reporters as a chemoproteomic strategy for substrate mapping of individual KAT isoforms in the native biological contexts. 
    more » « less
  3. Abstract

    The side‐chain acetylation of lysine residues in histones and non‐histone proteins catalyzed by lysine acetyltransferases (KATs) represents a widespread posttranslational modification (PTM) in the eukaryotic cells. Lysine acetylation plays regulatory roles in major cellular pathways inside and outside the nucleus. In particular, KAT‐mediated histone acetylation has an effect on all DNA‐templated epigenetic processes. Aberrant expression and activation of KATs are commonly observed in human diseases, especially cancer. In recent years, the study of KAT functions in biology and disease has greatly benefited from chemical biology tools and strategies. In this Review, we present the past and current accomplishments in the design of chemical biology approaches for the interrogation of KAT activity and function. These methods and probes are classified according to their mechanisms of action and respective applications, with both strengths and limitations discussed.

    more » « less
  4. Protein lipidation plays critical roles in regulating protein function and localization. However, the chemical diversity and specificity of fatty acyl group utilization have not been investigated using untargeted approaches, and it is unclear to what extent structures and biosynthetic origins ofS-acyl moieties differ fromN- andO-fatty acylation. Here, we show that fatty acylation patterns inCaenorhabditis elegansdiffer markedly between different amino acid residues. Hydroxylamine capture revealed predominant cysteineS-acylation with 15-methylhexadecanoic acid (isoC17:0), a monomethyl branched-chain fatty acid (mmBCFA) derived from endogenous leucine catabolism. In contrast, enzymatic protein hydrolysis showed that N-terminal glycine was acylated almost exclusively with straight-chain myristic acid, whereas lysine was acylated preferentially with two different mmBCFAs and serine was acylated promiscuously with a broad range of fatty acids, including eicosapentaenoic acid. Global profiling of fatty acylated proteins using a set of click chemistry–capable alkyne probes for branched- and straight-chain fatty acids uncovered 1,013S-acylated proteins and 510 hydroxylamine-resistantN- orO-acylated proteins. Subsets ofS-acylated proteins were labeled almost exclusively by either a branched-chain or a straight-chain probe, demonstrating acylation specificity at the protein level. Acylation specificity was confirmed for selected examples, including theS-acyltransferase DHHC-10. Last, homology searches for the identified acylated proteins revealed a high degree of conservation of acylation site patterns across metazoa. Our results show that protein fatty acylation patterns integrate distinct branches of lipid metabolism in a residue- and protein-specific manner, providing a basis for mechanistic studies at both the amino acid and protein levels.

    more » « less
  5. Syntrophomonas wolfei is an anaerobic syntrophic microbe that degrades short-chain fatty acids to acetate, hydrogen, and/or formate. This thermodynamically unfavorable process proceeds through a series of reactive acyl-Coenzyme A species (RACS). In other prokaryotic and eukaryotic systems, the production of intrinsically reactive metabolites correlates with acyl-lysine modifications, which have been shown to play a significant role in metabolic processes. Analogous studies with syntrophic bacteria, however, are relatively unexplored and we hypothesized that highly abundant acylations could exist in S. wolfei proteins, corresponding to the RACS derived from degrading fatty acids. Here, by mass spectrometry-based proteomics (LC–MS/MS), we characterize and compare acylome profiles of two S. wolfei subspecies grown on different carbon substrates. Because modified S. wolfei proteins are sufficiently abundant to analyze post-translational modifications (PTMs) without antibody enrichment, we could identify types of acylations comprehensively, observing six types (acetyl-, butyryl-, 3- hydroxybutyryl-, crotonyl-, valeryl-, and hexanyl-lysine), two of which have not been reported in any system previously. All of the acyl-PTMs identified correspond directly to RACS in fatty acid degradation pathways. A total of 369 sites of modification were identified on 237 proteins. Structural studies and in vitro acylation assays of a heavily modified enzyme, acetyl-CoA transferase, provided insight on the potential impact of these acyl-protein modifications. The extensive changes in acylation-type, abundance, and modification sites with carbon substrate suggest that protein acylation by RACS may be an important regulator of syntrophy. 
    more » « less