Biology subjects

Trefely, S.

Publications and source records attributed to Trefely, S..

9 recordsLinked to original sources

bioRxiv2022

The carnitine shuttle links mitochondrial metabolism to histone acetylation and lipogenesis

Acetyl-CoA is a central metabolite used for lipid synthesis in the cytosol and histone acetylation in the nucleus, among other pathways. The two major precursors to acetyl-CoA in the nuclear-cytoplasmic compartment are citrate and acetate, which are processed to acetyl-CoA by ATP-citrate lyase (ACLY) and acyl-CoA synthetase short-chain 2 (ACSS2), respectively. While some evidence has suggested the existence of additional routes to nuclear-cytosolic acetyl-CoA, such pathways remain poorly defined. To investigate this, we generated cancer cell lines lacking both ACLY and ACSS2. Unexpectedly, and in contrast to observations in fibroblasts, ACLY and ACSS2 double knockout (DKO) cancer cells remain viable and proliferate, maintain pools of cytosolic acetyl-CoA, and are competent to acetylate proteins in both cytosolic and nuclear compartments. Using stable isotope tracing, we show that both glucose and fatty acids feed acetyl-CoA pools and histone acetylation in DKO cells. Moreover, we provide evidence for the carnitine shuttle and carnitine acetyltransferase (CrAT) as a substantial pathway to transfer two-carbon units from mitochondria to cytosol independent of ACLY. Indeed, in the absence of ACLY, glucose can feed fatty acid synthesis in a carnitine responsive and CrAT-dependent manner. This work defines a carnitine-facilitated route to produce nuclear-cytosolic acetyl-CoA, shedding light on the intricate regulation and compartmentalization of acetyl-CoA metabolism

cell biology↗

bioRxiv2021

O-GlcNAc transferase regulates glioblastoma acetate metabolism via regulation of CDK5-dependent ACSS2 phosphorylation

Glioblastomas (GBMs) preferentially generate acetyl-CoA from acetate as a fuel source to promote tumor growth. O-GlcNAcylation has been shown to be elevated by increasing O-GlcNAc transferase (OGT) in many cancers and reduced O-GlcNAcylation can block cancer growth. Here, we identify a novel mechanism whereby OGT regulates acetate-dependent acetyl-CoA production by regulating phosphorylation of acetyl-CoA synthetase 2 (ACSS2) by cyclin-dependent kinase 5 (CDK5). OGT is required and sufficient for GBM cell growth and regulates acetate conversion to acetyl-CoA. Elevating O-GlcNAcylation in GBM cells increases phosphorylation of ACSS2 on Ser-267 in a CDK5-dependent manner. Importantly, we show that ACSS2 Ser-267 phosphorylation regulates its stability by reducing polyubiquitination and degradation. ACSS2 Ser-267 is critical for OGT-mediated GBM growth as overexpression of ACSS2 Ser-267 phospho-mimetic rescues growth in vitro and in vivo. Importantly, we show that pharmacologically targeting OGT and CDK5 reduces GBM growth ex vivo. Thus, the OGT/CDK5/ACSS2 pathway may be a way to target altered metabolic dependencies in brain tumors.

cancer biology↗

bioRxiv2020

Glutamine deprivation triggers NAGK-dependent hexosamine salvage

Tumors frequently exhibit aberrant glycosylation, which can impact cancer progression and therapeutic responses. The hexosamine biosynthesis pathway (HBP) produces uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), a major substrate for glycosylation in the cell. Prior studies have identified the HBP as a promising therapeutic target in pancreatic ductal adenocarcinoma (PDA). The HBP requires both glucose and glutamine for its initiation. The PDA tumor microenvironment is nutrient poor, however, prompting us to investigate how nutrient limitation impacts hexosamine synthesis. Here, we identify that glutamine limitation in PDA cells suppresses de novo hexosamine synthesis but results in increased free GlcNAc abundance. GlcNAc salvage via N-acetylglucosamine kinase (NAGK) is engaged to feed UDP-GlcNAc pools. NAGK expression is elevated in human PDA, and NAGK deletion from PDA cells impairs tumor growth in mice. Together, these data identify an important role for NAGK-dependent hexosamine salvage in supporting PDA tumor growth.

cancer biology↗

bioRxiv2020

The deacylase SIRT5 supports melanoma viability by regulating chromatin dynamics

Cutaneous melanoma remains the most lethal skin cancer, and ranks third among all malignancies in terms of years of life lost. Despite the advent of immune checkpoint and targeted therapies, only roughly half of patients with advanced melanoma achieves a durable remission. SIRT5 is a member of the sirtuin family of protein deacylases that regulate metabolism and other biological processes. Germline Sirt5 deficiency is associated with mild phenotypes in mice. Here we show that SIRT5 is required for proliferation and survival across all cutaneous melanoma genotypes tested, as well as uveal melanoma, a genetically distinct melanoma subtype that arises in the eye and is incurable once metastatic. Likewise, SIRT5 is required for efficient tumor formation by melanoma xenografts and in an autochthonous mouse Braf;Pten-driven melanoma model. Via metabolite and transcriptomic analyses, we find that SIRT5 is required to maintain histone acetylation and methylation levels in melanoma cells, thereby promoting proper gene expression. SIRT5-dependent genes notably include MITF, a key lineage-specific survival oncogene in melanoma, and the c-MYC proto-oncogene. SIRT5 may represent a novel, druggable genotype-independent addiction in melanoma.

cancer biology↗

bioRxiv2020

Quantitative sub-cellular acyl-CoA analysis reveals distinct nuclear regulation

Quantitative sub-cellular metabolomic measurements can yield crucial insights into the roles of metabolites in cellular processes, but are subject to multiple confounding factors. We developed Stable Isotope Labeling of Essential nutrients in cell Culture - Sub-cellular Fractionation (SILEC-SF), which uses isotope labeled internal standard controls that are present throughout fractionation and processing to quantify acyl-Coenzyme A thioesters in sub-cellular compartments by liquid chromatography-mass spectrometry. We tested SILEC-SF in a range of sample types and examined the compartmentalized responses to oxygen tension, cellular differentiation, and nutrient availability. Application of SILEC-SF to the challenging analysis of the nuclear compartment revealed a nuclear acyl-CoA profile distinct from that of the cytosol, with notable nuclear enrichment of propionyl-CoA. Using isotope tracing we identified the branched chain amino acid (BCAA) isoleucine as a major metabolic source of nuclear propionyl-CoA and histone propionylation, thus revealing a new mechanism of crosstalk between metabolism and the epigenome.

biochemistry↗

bioRxiv2020

Quantitative prospective and retrospective mass spectrometry of lactoyl-CoA in mammalian cells and tissues

Lysine lactoylation is a recently described protein post-translational modification (PTM). However, the biochemical pathways responsible for this acylation remain unclear. Two metabolite-dependent mechanisms have been proposed: enzymatic histone lysine lactoylation derived from lactoyl-coenzyme A (lactoyl-CoA, also termed lactyl-CoA), and non-enzymatic lysine lactoylation resulting from acyl-transfer via lactoyl-glutathione. While the former has precedent in the form of enzyme-catalyzed lysine acylation, the lactoyl-CoA metabolite has not been previously quantified in mammalian systems. Here we use liquid chromatography-high resolution mass spectrometry (LC-HRMS) together with a synthetic standard to detect and validate the presence of lactoyl-CoA in cell and tissue samples. Conducting a retrospective analysis of data from previously analyzed samples revealed the presence of lactoyl-CoA in diverse cell and tissue contexts. In addition, we describe a biosynthetic route to generate 13C3 15N1 -isotopically-labeled lactoyl-CoA, providing a co-eluting internal standard for analysis of this metabolite. We estimate lactoyl-CoA concentrations of 1.14x10-8 pmol/cell in cell culture and 0.0172 pmol/mg tissue wet weight in mouse heart. These levels are similar to crotonyl-CoA, but between 20-350 times lower than predominant acyl-CoAs such as acetyl-, propionyl-, and succinyl-CoA. Overall our studies provide the first quantitative measurements of lactoyl-CoA and provide a methodological foundation for the interrogation of this novel metabolite in biology and disease. Highlights- Detection of lactoyl-CoA at picomole concentrations across tissues and cells - Lactoyl-CoA was detected at concentrations similar to crontonyl-CoA within HepG2 cells - Isotopically labeled 13C315N1-lactoyl-CoA can be prepared by SILEC

biochemistry↗

bioRxiv2019

Direct anabolic three carbon metabolism to a six carbon metabolite occurs in human heart and in vivo across mouse tissues

Anabolic metabolism of carbon in mammals is mediated via the one and two carbon carriers S-adenosyl methionine and acetyl-coenzyme A (acetyl-CoA). In contrast, anabolic metabolism using three carbon units via propionate is not thought to occur. Mammals are primarily thought to oxidize the 3-carbon short chain fatty acid propionate by shunting propionyl-CoA to succinyl-CoA for entry into the TCA cycle. We found that this may not be absolute and that in mammals one non-oxidative fate of two units of propionyl-CoA is to condense to a six-carbon trans-2-methyl-2-pentenoyl-CoA (2M2PE-CoA). We confirmed this pathway using purified protein extracts provided limited substrates and confirmed the product with a synthetic standard. In whole-body in vivo stable isotope tracing with infusion of 13C-labeled valine achieving steady state, 2M2PE-CoA formed via propionyl-CoA in multiple murine tissues including heart, kidney, and to a lesser degree in brown adipose tissue, liver, and tibialis anterior muscle. Using ex vivo isotope tracing, we found that 2M2PE-CoA formed in human myocardial tissue incubated with propionate to a limited extent. While the complete enzymology of this pathway remains to be elucidated, these results confirm the in vivo existence of at least one anabolic three to six carbon reaction conserved in humans and mice that utilizes three carbons via propionate. Highlights- Synthesis and confirmation of structure 2-methyl-2-pentenoyl-CoA - In vivo fate of valine across organs includes formation of a 6-carbon metabolite from propionyl-CoA - Ex vivo metabolism of propionate in the human heart includes direct anabolism to a 6-carbon product - In both cases, this reaction occurred at physiologically relevant concentrations of propionate and valine - In vitro this pathway requires propionyl-CoA and NADH/NADPH as substrates

biochemistry↗

bioRxiv2019

Subcellular metabolic pathway kinetics are revealed by correcting for artifactual post harvest metabolism

OBJECTIVEThe dynamic regulation of metabolic pathways can be monitored by stable isotope tracing. Yet, many metabolites are part of distinct processes within different subcellular compartments. Standard isotope tracing experiments relying on analyses in whole cells may not accurately reflect compartmentalized metabolic processes. Analysis of compartmentalized metabolism and the dynamic interplay between compartments can potentially be achieved by stable isotope tracing followed by subcellular fractionation. Although it is recognized that metabolism can take place during biochemical fractionation of cells, a clear understanding of how such post-harvest metabolism impacts the interpretation of subcellular isotope tracing data and methods to correct for this are lacking. We set out to directly assess artifactual metabolism, enabling us to develop and test strategies to correct for it. We apply these techniques to examine the compartment-specific metabolic kinetics of 13C-labeled substrates targeting central metabolic pathways.\n\nMETHODSWe designed a stable isotope tracing strategy to interrogate post-harvest metabolic activity during subcellular fractionation using liquid chromatography-mass spectrometry (LC-MS).\n\nRESULTSWe show that post-harvest metabolic activity occurs rapidly (within seconds) upon cell harvest. With further characterization we reveal that this post-harvest metabolism is enzymatic, and reflects the metabolic capacity of the sub-cellular compartment analyzed; but is limited in the extent of its propagation into downstream metabolites in metabolic pathways. We also propose and test a post-labeling strategy to assess the amount of post-harvest metabolism occurring in an experiment and then to adjust data to account for this. We validate this approach for both mitochondrial and cytosolic metabolic analyses.\n\nCONCLUSIONSOur data indicate that isotope tracing coupled with sub-cellular fractionation can reveal distinct and dynamic metabolic features of cellular compartments, and that confidence in such data can be improved by applying a post-labeling correction strategy. We examine compartmentalized metabolism of acetate and glutamine and show that acetyl-CoA is turned over rapidly in the cytosol and acts as a pacemaker of anabolic metabolism in this compartment.

biochemistry↗

bioRxiv2018

Artefactual formation of pyruvate from in-source conversion of lactate

RationaleLactate and pyruvate are high abundance products of glucose metabolism. Analysis of both molecules as part metabolomics studies in cellular metabolism and physiology have been aided by advances in liquid chromatography-mass spectrometry (LC-MS).\n\nMethodsWe used ion pairing-chromatography and negative ion mode ESI on an QExactive HF to perform stable isotope assisted metabolomics profiling of lactate and pyruvate metabolism.\n\nResultsUsing an LC-MS method for polar metabolite analysis we discovered an artefactual formation of pyruvate from in-source fragmentation of lactate. Surprisingly, this in-source fragmentation has not been previously described, thus we report this identification to warn other investigators. This artefact was detected by baseline chromatographic resolution of lactate and pyruvate by LC with confirmation of this artefact by stable isotope labeling of lactate and pyruvate.\n\nConclusionsThese findings have immediate implications for metabolomics studies by LC-MS and direct infusion MS, especially in negative ion mode, whereby users should resolve lactate from pyruvate or robustly quantify the potential formation of pyruvate from higher abundance lactate in their assays.

biochemistry↗