PI3K drives the de novo synthesis of coenzyme A from vitamin B5.

In response to hormones and growth factors, the class I phosphoinositide-3-kinase (PI3K) signalling network functions as a major regulator of metabolism and growth, governing cellular nutrient uptake, energy generation, reducing cofactor production and macromolecule biosynthesis1. Many of the driver mutations in cancer with the highest recurrence, including in receptor tyrosine kinases, Ras, PTEN and PI3K, pathologically activate PI3K signalling2,3. However, our understanding of the core metabolic program controlled by PI3K is almost certainly incomplete. Here, using mass-spectrometry-based metabolomics and isotope tracing, we show that PI3K signalling stimulates the de novo synthesis of one of the most pivotal metabolic cofactors: coenzyme A (CoA). CoA is the major carrier of activated acyl groups in cells4,5 and is synthesized from cysteine, ATP and the essential nutrient vitamin B5 (also known as pantothenate)6,7. We identify pantothenate kinase 2 (PANK2) and PANK4 as substrates of the PI3K effector kinase AKT8. Although PANK2 is known to catalyse the rate-determining first step of CoA synthesis, we find that the minimally characterized but highly conserved PANK49 is a rate-limiting suppressor of CoA synthesis through its metabolite phosphatase activity. Phosphorylation of PANK4 by AKT relieves this suppression. Ultimately, the PI3K-PANK4 axis regulates the abundance of acetyl-CoA and other acyl-CoAs, CoA-dependent processes such as lipid metabolism and proliferation. We propose that these regulatory mechanisms coordinate cellular CoA supplies with the demands of hormone/growth-factor-driven or oncogene-driven metabolism and growth.

Synthesis and Evaluation of a Stable Isostere of Malonyllysine.

Lysine malonylation is a recently characterized post-translational modification involved in the regulation of energy metabolism and gene expression. One unique feature of this post-translational modification is its potential susceptibility to decarboxylation, which poses possible challenges to its study. As a step towards addressing these challenges, we report the synthesis and evaluation of a stable isostere of malonyllysine. First, we find that synthetic substitution of the malonyl group with a tetrazole isostere results in amino acid's resistant to thermal decarboxylation. Next, we demonstrate that protected variants of this amino acid are readily incorporated into peptides. Finally, we show that tetrazole isosteres of malonyllysine can be recognized by anti-malonyllysine antibodies and histone deacylases, validating their ability to mimic features of the endogenous lysine modification. Overall, this study establishes a new chemical strategy for stably mimicking a metabolite-derived post-translational modification, providing a foothold for tool development and functional analyses.

Acetylation of Cytidine in mRNA Promotes Translation Efficiency.

Generation of the "epitranscriptome" through post-transcriptional ribonucleoside modification embeds a layer of regulatory complexity into RNA structure and function. Here, we describe N4-acetylcytidine (ac4C) as an mRNA modification that is catalyzed by the acetyltransferase NAT10. Transcriptome-wide mapping of ac4C revealed discretely acetylated regions that were enriched within coding sequences. Ablation of NAT10 reduced ac4C detection at the mapped mRNA sites and was globally associated with target mRNA downregulation. Analysis of mRNA half-lives revealed a NAT10-dependent increase in stability in the cohort of acetylated mRNAs. mRNA acetylation was further demonstrated to enhance substrate translation in vitro and in vivo. Codon content analysis within ac4C peaks uncovered a biased representation of cytidine within wobble sites that was empirically determined to influence mRNA decoding efficiency. These findings expand the repertoire of mRNA modifications to include an acetylated residue and establish a role for ac4C in the regulation of mRNA translation.

Bioorthogonal pro-metabolites for profiling short chain fatty acylation.

Short chain fatty acids (SCFAs) play a central role in health and disease. One function of these signaling molecules is to serve as precursors for short chain fatty acylation, a class of metabolically-derived posttranslational modifications (PTMs) that are established by lysine acetyltransferases (KATs) and lysine deacetylases (KDACs). Via this mechanism, short chain fatty acylation serves as an integrated reporter of metabolism as well as KAT and KDAC activity, and has the potential to illuminate the role of these processes in disease. However, few methods to study short chain fatty acylation exist. Here we report a bioorthogonal pro-metabolite strategy for profiling short chain fatty acylation in living cells. Inspired by the dietary component tributyrin, we synthesized a panel of ester-caged bioorthogonal short chain fatty acids. Cellular evaluation of these agents led to the discovery of an azido-ester that is metabolized to its cognate acyl-coenzyme A (CoA) and affords robust protein labeling profiles. We comprehensively characterize the metabolic dependence, toxicity, and histone deacetylase (HDAC) inhibitor sensitivity of these bioorthogonal pro-metabolites, and apply an optimized probe to identify novel candidate protein targets of short chain fatty acids in cells. Our studies showcase the utility of bioorthogonal pro-metabolites for unbiased profiling of cellular protein acylation, and suggest new approaches for studying the signaling functions of SCFAs in differentiation and disease.

Profiling Cytidine Acetylation with Specific Affinity and Reactivity.

The human acetyltransferase NAT10 has recently been shown to catalyze formation of N4-acetylcytidine (ac4C), a minor nucleobase known to alter RNA structure and function. In order to better understand the role of RNA acetyltransferases in biology and disease, here we report the development and application of chemical methods to study ac4C. First, we demonstrate that ac4C can be conjugated to carrier proteins using optimized protocols. Next, we describe methods to access ac4C-containing RNAs, enabling the screening of anti-ac4C antibodies. Finally, we validate the specificity of an optimized ac4C affinity reagent in the context of cellular RNA by demonstrating its ability to accurately report on chemical deacetylation of ac4C. Overall, these studies provide a powerful new tool for studying ac4C in biological contexts, as well as new insights into the stability and half-life of this highly conserved RNA modification. More broadly, they demonstrate how chemical reactivity may be exploited to aid the development and validation of nucleobase-targeting affinity reagents designed to target the emerging epitranscriptome.

Discovering Targets of Non-enzymatic Acylation by Thioester Reactivity Profiling.

Non-enzymatic protein modification driven by thioester reactivity is thought to play a major role in the establishment of cellular lysine acylation. However, the specific protein targets of this process are largely unknown. Here we report an experimental strategy to investigate non-enzymatic acylation in cells. Specifically, we develop a chemoproteomic method that separates thioester reactivity from enzymatic utilization, allowing selective enrichment of non-enzymatic acylation targets. Applying this method to cancer cell lines identifies numerous candidate targets of non-enzymatic acylation, including several enzymes in lower glycolysis. Functional studies highlight malonyl-CoA as a reactive thioester metabolite that can modify and inhibit glycolytic enzyme activity. Finally, we show that synthetic thioesters can be used as novel reagents to probe non-enzymatic acylation in living cells. Our studies provide new insights into the targets and drivers of non-enzymatic acylation, and demonstrate the utility of reactivity-based methods to experimentally investigate this phenomenon in biology and disease.

Co-opting a Bioorthogonal Reaction for Oncometabolite Detection.

Dysregulated metabolism is a hallmark of many diseases, including cancer. Methods to fluorescently detect metabolites have the potential to enable new approaches to cancer detection and imaging. However, fluorescent sensing methods for naturally occurring cellular metabolites are relatively unexplored. Here we report the development of a chemical approach to detect the oncometabolite fumarate. Our strategy exploits a known bioorthogonal reaction, the 1,3-dipolar cycloaddition of nitrileimines and electron-poor olefins, to detect fumarate via fluorescent pyrazoline cycloadduct formation. We demonstrate hydrazonyl chlorides serve as readily accessible nitrileimine precursors, whose reactivity and spectral properties can be tuned to enable detection of fumarate and other dipolarophile metabolites. Finally, we show this reaction can be used to detect enzyme activity changes caused by mutations in fumarate hydratase, which underlie the familial cancer predisposition syndrome hereditary leiomyomatosis and renal cell cancer. Our studies define a novel intersection of bioorthogonal chemistry and metabolite reactivity that may be harnessed to enable biological profiling, imaging, and diagnostic applications.

Modular synthesis of cell-permeating 2-ketoglutarate esters.

Cell-permeating esters of 2-ketoglutarate (2-KG) have been synthesized through a convergent sequence from two modules in two and three steps, respectively. This route provides access to a full series of mono- and disubstituted 2-KG esters, enabling us to define the effect of regioisomeric masking on metabolite release and antihypoxic activity in cell-based assays. In addition to providing insight into the biological activity of cell permeable 2-KG esters, the straightforward and modular nature of this synthetic route may prove useful for the development of next-generation 2-KG analogues for diagnostic and therapeutic applications.