bims-meprid 2022-07-24 papers

Curr Protoc. 2022 Jul;2(7): e497

Identification and Profiling of Histone Acetyltransferase Substrates by Bioorthogonal Labeling.

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.

Keywords: bioorthogonal labeling; chemical proteomics; click chemistry; histone acetyltransferase; lysine acetylation; protein substrates

DOI: https://doi.org/10.1002/cpz1.497

Cell Chem Biol. 2022 Jul 21. pii: S2451-9456(22)00161-1. [Epub ahead of print]29(7): 1232-1244.e5

Native chemical ligation approach to sensitively probe tissue acyl-CoA pools.

Andrew M James, Abigail A I Norman, Jack W Houghton, Hiran A Prag, Angela Logan, Robin Antrobus, Richard C Hartley, Michael P Murphy.

During metabolism, carboxylic acids are often activated by conjugation to the thiol of coenzyme A (CoA). The resulting acyl-CoAs comprise a group of ∼100 thioester-containing metabolites that could modify protein behavior through non-enzymatic N-acylation of lysine residues. However, the importance of many potential acyl modifications remains unclear because antibody-based methods to detect them are unavailable and the in vivo concentrations of their respective acyl-CoAs are poorly characterized. Here, we develop cysteine-triphenylphosphonium (CysTPP), a mass spectrometry probe that uses "native chemical ligation" to sensitively detect the major acyl-CoAs present in vivo through irreversible modification of its amine via a thioester intermediate. Using CysTPP, we show that longer-chain (C13-C22) acyl-CoAs often constitute ∼60% of the acyl-CoA pool in rat tissues. These hydrophobic longer-chain fatty acyl-CoAs have the potential to non-enzymatically modify protein residues.

Keywords: acyl-CoA; acylation; coenzyme A; cysteine; native chemical ligation; thioester; thiol; triphenylphosphonium

DOI: https://doi.org/10.1016/j.chembiol.2022.04.005

Cell Rep. 2022 Jul 19. pii: S2211-1247(22)00926-3. [Epub ahead of print]40(3): 111120

Methionine metabolism regulates pluripotent stem cell pluripotency and differentiation through zinc mobilization.

Erinn Zixuan Sim, Takayuki Enomoto, Nobuaki Shiraki, Nao Furuta, Soshiro Kashio, Taiho Kambe, Tomonori Tsuyama, Akihiro Arakawa, Hiroki Ozawa, Mizuho Yokoyama, Masayuki Miura, Shoen Kume.

Pluripotent stem cells (PSCs) exhibit a unique feature that requires S-adenosylmethionine (SAM) for the maintenance of their pluripotency. Methionine deprivation in the medium causes a reduction in intracellular SAM, thus rendering PSCs in a state potentiated for differentiation. In this study, we find that methionine deprivation triggers a reduction in intracellular protein-bound Zn content and upregulation of Zn exporter SLC30A1 in PSCs. Culturing PSCs in Zn-deprived medium results in decreased intracellular protein-bound Zn content, reduced cell growth, and potentiated differentiation, which partially mimics methionine deprivation. PSCs cultured under Zn deprivation exhibit an altered methionine metabolism-related metabolite profile. We conclude that methionine deprivation potentiates differentiation partly by lowering cellular Zn content. We establish a protocol to generate functional pancreatic β cells by applying methionine and Zn deprivation. Our results reveal a link between Zn signaling and methionine metabolism in the regulation of cell fate in PSCs.

Keywords: CP: Stem cell research; induced pluripotent stem cells; in vitro differentiation; methionine deprivation; pancreatic differentiation; transporter; zinc

DOI: https://doi.org/10.1016/j.celrep.2022.111120

iScience. 2022 Jul 15. 25(7): 104669

Gut microbe-derived metabolite trimethylamine N-oxide activates PERK to drive fibrogenic mesenchymal differentiation.

Seok-Jo Kim, Swarna Bale, Priyanka Verma, Qianqian Wan, Feiyang Ma, Johann E Gudjonsson, Stanley L Hazen, Paul W Harms, Pei-Suen Tsou, Dinesh Khanna, Lam C Tsoi, Nilaksh Gupta, Karen J Ho, John Varga.

Intestinal dysbiosis is prominent in systemic sclerosis (SSc), but it remains unknown how it contributes to microvascular injury and fibrosis that are hallmarks of this disease. Trimethylamine (TMA) is generated by the gut microbiome and in the host converted by flavin-containing monooxygenase (FMO3) into trimethylamine N-oxide (TMAO), which has been implicated in chronic cardiovascular and metabolic diseases. Using cell culture systems and patient biopsies, we now show that TMAO reprograms skin fibroblasts, vascular endothelial cells, and adipocytic progenitor cells into myofibroblasts via the putative TMAO receptor protein R-like endoplasmic reticulum kinase (PERK). Remarkably, FMO3 was detected in skin fibroblasts and its expression stimulated by TGF-β1. Moreover, FMO3 was elevated in SSc skin biopsies and in SSc fibroblasts. A meta-organismal pathway thus might in SSc link gut microbiome to vascular remodeling and fibrosis via stromal cell reprogramming, implicating the FMO3-TMAO-PERK axis in pathogenesis, and as a promising target for therapy.

Keywords: Cell biology; Microbial metabolism; Microbiome

DOI: https://doi.org/10.1016/j.isci.2022.104669