skip to main content


Title: Identification and Profiling of Histone Acetyltransferase Substrates by Bioorthogonal Labeling
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
Award ID(s):
1808087
NSF-PAR ID:
10381136
Author(s) / Creator(s):
 ;  ;  
Publisher / Repository:
Wiley Blackwell (John Wiley & Sons)
Date Published:
Journal Name:
Current Protocols
Volume:
2
Issue:
7
ISSN:
2691-1299
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. 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
  2. Abstract

    Protein S‐acylation, predominately in the form of palmitoylation, is a reversible lipid post‐translational modification on cysteines that plays important roles in protein localization, trafficking, activity, and complex assembly. The functions and regulatory mechanisms of S‐acylation have been extensively studied in mammals owing to remarkable development of high‐resolution proteomics and the discovery of the S‐acylation‐related enzymes. However, the advancement of S‐acylation studies in plants lags behind that in mammals, mainly due to the lack of knowledge about proteins responsible for this process, such as protein acyltransferases and their substrates. In this article, a set of systematic protocols to study global S‐acylation inArabidopsisseedlings is described. The procedures are presented in detail, including preparation ofArabidopsisseedlings, enrichment of plasma membrane (PM) proteins, ensuing enrichment of S‐acylated proteins/peptides based on the acyl‐biotin exchange method, and large‐scale identification of S‐acylated proteins/peptides via mass spectrometry. This approach enables researchers to study S‐acylation of PM proteins in plants in a systematic and straightforward way. © 2020 Wiley Periodicals LLC.

    Basic Protocol 1: Preparation ofArabidopsisseedling materials

    Basic Protocol 2: Isolation and enrichment of plasma membrane proteins

    Support Protocol 1: Determination of protein concentration using BCA assay

    Basic Protocol 3: Enrichment of S‐acylated proteins by acyl‐biotin exchange method

    Support Protocol 2: Protein precipitation by methanol/chloroform method

    Basic Protocol 4: Trypsin digestion and proteomic analysis

    Alternate Protocol: Pre‐resin digestion and peptide‐level enrichment

     
    more » « less
  3. 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
  4. Abstract

    Reversible addition‐fragmentation chain‐transfer (RAFT) polymerization is a commonly used polymerization methodology to generate synthetic polymers. The products of RAFT polymerization, i.e., RAFT polymers, have been widely employed in several biologically relevant areas, including drug delivery, biomedical imaging, and tissue engineering. In this article, we summarize a synthetic methodology to display an azide group at the chain end of a RAFT polymer, thus presenting a reactive site on the polymer terminus. This platform enables a click reaction between azide‐terminated polymers and alkyne‐containing molecules, providing a broadly applicable scaffold for chemical and bioconjugation reactions on RAFT polymers. We also highlight applications of these azide‐terminated RAFT polymers in fluorophore labeling and for promoting organelle targeting capability. © 2020 Wiley Periodicals LLC.

    Basic Protocol 1: Synthesis of the azide derivatives of chain transfer agent and radical initiator

    Basic Protocol 2: Installation of an azide group on the α‐end of RAFT polymers

    Alternate Protocol: Installation of an azide group on the ω‐end of RAFT polymers

    Basic Protocol 3: Click reaction between azide‐terminated RAFT polymers and alkyne derivatives

     
    more » « less
  5. Abstract

    Immobilization of proteins and enzymes on solid supports has been utilized in a variety of applications, from improved protein stability on supported catalysts in industrial processes to fabrication of biosensors, biochips, and microdevices. A critical requirement for these applications is facile yet stable covalent conjugation between the immobilized and fully active protein and the solid support to produce stable, highly bio-active conjugates. Here, we report functionalization of solid surfaces (gold nanoparticles and magnetic beads) with bio-active proteins using site-specific and biorthogonal labeling and azide-alkyne cycloaddition, a click chemistry. Specifically, we recombinantly express and selectively label calcium-dependent proteins, calmodulin and calcineurin, and cAMP-dependent protein kinase A (PKA) with N-terminal azide-tags for efficient conjugation to nanoparticles and magnetic beads. We successfully immobilized the proteins on to the solid supports directly from the cell lysate with click chemistry, forgoing the step of purification. This approach is optimized to yield low particle aggregation and high levels of protein activity post-conjugation. The entire process enables streamlined workflows for bioconjugation and highly active conjugated proteins.

    Graphical Abstract

     
    more » « less