Biology subjects

Ho, Y.-H.

Publications and source records attributed to Ho, Y.-H..

3 recordsLinked to original sources

bioRxiv2021

Effects of Oncohistone Mutations and PTM Crosstalk on the N-terminal Acetylation Activities of NatD

Acetylation at the -N-terminus (N) is one of the most abundant modifications detected on histone H4 and H2A, which is catalyzed by N-terminal acetyltransferase D (NatD). NatD substrate recognition motif, N-terminal SGRGK, is enriched with frequent oncohistone mutations and post-translational modifications (PTMs). However, there is no information on how oncohistone mutations and other PTMs affect NatD-catalyzed acetylation. Herein, we determined how changes of local chemical environment on the N-terminal SGRGK sequence regulate NatD-catalyzed N acetylation on histone H4/H2A. Our studies indicate that all oncohistone mutations at SGRG suppressed the catalytic efficiency of NatD. Meanwhile, H4 serine 1 phosphorylation and arginine 3 methylation also negatively affect the NatD activity, but the lysine 5 acetylation has a marginal effect on NatD. This work reveals the impacts of oncohistone mutations on NatD activity and unravels the crosstalk between NatD and PTMs. To our knowledge, this is the first report on the potential regulatory mechanism of NatD, highlighting different revenues to interrogate the NatD-mediated pathway in the future.

biochemistry↗

bioRxiv2021

Novel bisubstrate inhibitors for protein N-terminal acetyltransferase D

Protein N-terminal acetyltransferase D (NatD, NAA40) that specifically acetylates the alpha-N-terminus of histone H4 and H2A has been implicated in various diseases, but no inhibitor has been reported for this important enzyme. Based on the acetyl transfer mechanism of NatD, we designed and prepared a series of highly potent NatD bisubstrate inhibitors by covalently linking coenzyme A to different peptide substrates via an acetyl or propionyl spacer. The most potent bisubstrate inhibitor displayed an apparent Ki value of 1.0 nM. Biochemical studies indicated that bisubstrate inhibitor is competitive to the peptide substrate and noncompetitive to the cofactor, suggesting NatD undergoes an ordered Bi-Bi mechanism. We also demonstrated that these inhibitors are highly specific towards NatD, displaying about 1,000-fold selectivity over other closely related acetyltransferases. High-resolution crystal structures of NatD bound to two of these inhibitors revealed the molecular basis for their selectivity and inhibition mechanism, providing a rational path for future inhibitor development.

biochemistry↗

bioRxiv2020

Development of a continuous fluorescence-based assay for N-terminal acetyltransferase D

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.

biochemistry↗