Development of A Continuous Fluorescence-Based Assay for N-Terminal Acetyltransferase D.

Yi-Hsun Ho,Lan Chen,Rong Huang

doi:10.3390/ijms22020594

Abstract

N-terminal acetylation catalyzed by N-terminal acetyltransferases (NATs) has various biological functions in protein regulation. N-terminal acetyltransferase D (NatD) is one of the most specific NAT with only histone H4 and H2A proteins as the known substrates. Dysregulation of NatD has been implicated in colorectal and lung cancer progression, implying its therapeutic potential in cancers. However, there is no reported inhibitor for NatD yet. To facilitate the discovery of small-molecule NatD inhibitors, we report the development of a fluorescence-based acetyltransferase assay in 384-well high-throughput screening (HTS) format through monitoring the formation of coenzyme A. The fluorescent signal is generated from the adduct in the reaction between coenzyme A and fluorescent probe ThioGlo4. The assay exhibited a Z′-factor of 0.77 and a coefficient of variation of 6%, indicating it is a robust assay for HTS. A pilot screen of 1280 pharmacologically active compounds and subsequent validation identified two hits, confirming the application of this fluorescence assay in HTS.

Highlights

N-terminal acetylation has been detected on 80–90% of human cytosolic and transmembrane proteins [1,2], which impacts diverse biological processes, such as regulating protein degradation, protein translocation, and protein–protein interactions [3]
Since the Km values of substrates of N-terminal acetyltransferase D (NatD) are below 10 μM, we report the development of a continuous fluorescence-based assay to dithiobis-(2-nitro- benzoic acid) (DTNB) assay is not suitable for the detection of coenzyme A sodium salt hydrate (CoA) formation under NatD catalyzed quantify CoA production by forming a fluorescent adduct with the thiol fluorescent probe
Recombinant was expressed in E. coli overnight and purified by

Summary

Introduction

N-terminal acetylation has been detected on 80–90% of human cytosolic and transmembrane proteins [1,2], which impacts diverse biological processes, such as regulating protein degradation, protein translocation, and protein–protein interactions [3]. This modification is catalyzed by N-terminal acetyltransferase (NATs), which transfer the acetyl group from acetyl-coenzyme A (AcCoA) to the alpha-N-terminal amino group of the protein substrates [4]. The function of N-terminal acetylation on H4 and H2A remains obscure, NatD has been implicated in cancer cell growth, migration, and invasion in colorectal and lung cancers [10,11,12].

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