p300 and GCN5 are two representative lysine acetyltransferases (KATs) in mammalian cells. It was recently reported that they possess multiple acyltransferase activities including acetylation, propionylation, and butyrylation of the ε-amino group of lysine residues of histones and non-histone protein substrates. Although thousands of acetylated substrates and acetylation sites have been identified by mass spectrometry-based proteomic screening, our knowledge about the causative connections between individual KAT members and their corresponding sub-acylomes remain very limited. Herein, we applied 3-azidopropionyl CoA (3AZ-CoA) as a bioorthogonal surrogate of acetyl-, propionyl- and butyryl-CoA for KAT substrate identification. We successfully attached the azide as a chemical warhead to cellular substrates of wild-type p300 and engineered GCN5. The substrates were subsequently labeled with biotin tag through the copper-catalyzed azide-alkyne cycloaddition (CuAAC). Following protein enrichment on streptavidin-coated resin, we conducted LC-MS/MS studies from which more than four hundred proteins were identified as GCN5 or p300 substrate candidates. These proteins are either p300- or GCN5-unique or shared by the two KATs and are extensively involved in various biological events including gene expression, cell cycle, and cellular metabolism. We also experimentally validated two novel substrates of GCN5, that is, IQGAP1 and SMC1. These results demonstrate extensive engagement of GCN5 and p300 in cellular pathways and provide new insights into understanding their functions in specific biological processes.

Exogenous fatty acids provide substrates for energy production and biogenesis of the cytoplasmic membrane, but they also enhance cellular signaling during cancer cell proliferation. However, it remains controversial whether dietary fatty acids are correlated with tumor progression. In this study, we demonstrate that increased Src kinase activity is associated with high-fat diet-accelerated progression of prostate tumors and that Src kinases mediate this pathological process. Moreover, in the prostate regeneration assay, host SCID mice carrying Src(Y529F)-transduced regeneration tissues were fed a low-fat diet or a high-fat diet and treated with vehicle or dasatinib. The high-fat diet not only accelerated Src-induced prostate tumorigenesis in mice but also compromised the inhibitory effect of the anticancer drug dasatinib on Src kinase oncogenic potential We further show that myristoylation of Src kinase is essential to facilitate Src-induced and high-fat diet-accelerated tumor progression. Mechanistically, metabolism of exogenous myristic acid increased the biosynthesis of myristoyl CoA and myristoylated Src and promoted Src kinase-mediated oncogenic signaling in human cells. Of the fatty acids tested, only exogenous myristic acid contributed to increased intracellular myristoyl CoA levels. Our results suggest that targeting Src kinase myristoylation, which is required for Src kinase association at the cellular membrane, blocks dietary fat-accelerated tumorigenesis Our findings uncover the molecular basis of how the metabolism of myristic acid stimulates high-fat diet-mediated prostate tumor progression.

Short-chain acylation of lysine residues recently emerges as a group of reversible posttranslational modifications in mammalian cells. The diversity of acylation further broadens the landscape and complexity of the proteome. Identification of regulatory enzymes and effector proteins for lysine acylation is critical to understand functions of these novel modifications at the molecular level. Here, we report that the MYST family of lysine acetyltransferases (KATs) possesses strong propionyltransferase activity both in vitro and in cellulo. Particularly, the propionyltransferase activity of MOF, MOZ, and HBO1 is as strong as their acetyltransferase activity. Overexpression of MOF in human embryonic kidney 293T cells induced significantly increased propionylation in multiple histone and non-histone proteins, which manifests that the function of MOF goes far beyond its canonical histone H4 lysine-16 acetylation. We also resolved the X-ray co-crystal structure of MOF bound with propionyl coenzyme A, which provides a direct structural basis for the propionyltransferase activity of the MYST KATs. Our data together defines a novel function for the MYST KATs as lysine propionyltransferases and suggests much broader physiological impacts for this family of enzymes.

Short-chain acylations of lysine residues in eukaryotic proteins are recognized as essential posttranslational chemical modifications (PTMs) that regulate cellular processes from transcription, cell cycle, metabolism, to signal transduction. Lysine butyrylation was initially discovered as a normal straight chain butyrylation (Knbu). Here we report its structural isomer, branched chain butyrylation, i.e. lysine isobutyrylation (Kibu), existing as a new PTM on nuclear histones. Uniquely, isobutyryl-CoA is derived from valine catabolism and branched chain fatty acid oxidation which is distinct from the metabolism of n-butyryl-CoA. Several histone acetyltransferases were found to possess lysine isobutyryltransferase activity in vitro, especially p300 and HAT1. Transfection and western blot experiments showed that p300 regulated histone isobutyrylation levels in the cell. We resolved the X-ray crystal structures of HAT1 in complex with isobutyryl-CoA that gleaned an atomic level insight into HAT-catalyzed isobutyrylation. RNA-Seq profiling revealed that isobutyrate greatly affected the expression of genes associated with many pivotal biological pathways. Together, our findings identify Kibu as a novel chemical modification mark in histones and suggest its extensive role in regulating epigenetics and cellular physiology.

To establish a gene delivery system for brain targeting, a low molecular weight polyethylenimine (PEI(10 K)) was modified with myristic acid (MC), and complexed with DNA, yielding MC-PEI(10 K)/DNA nanoparticles successfully. The nanoparticles were observed to be successfully taken up by the brains of mice. The transfection efficiency of the nanoparticles was then investigated, and both the in vitro and in vivo gene expression of MC-PEI(10 K)/DNA nanoparticles is significantly higher than that of unmodified PEI(10 K)/DNA nanoparticles. The anti-glioblastoma effect of MC-PEI(10 K)/pORF-hTRAIL was demonstrated by the survival time of intracranial U87 glioblastoma-bearing mice. The median survival time of the MC-PEI(10 K)/pORF-hTRAIL group (28 days) was significantly longer than that of the PEI(10 K)/pORF-hTRAIL group (24 days), the MC-PEI(10 K)/pGL(3) group (21 days) and the saline group (22 days). Therefore, our results suggested that MC-PEI(10 K) could be potentially used for brain-targeted gene delivery and in the treatment of glioblastoma.