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-catalysed 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 analysed samples revealed the presence of lactoyl-CoA in diverse cell and tissue contexts. In addition, we describe a biosynthetic route to generate 13 C 3 15 N 1 -isotopically labelled lactoyl-CoA, providing a co-eluting internal standard for analysis of this metabolite. We estimate lactoyl-CoA concentrations of 1.14 × 10 −8 pmol per cell in cell culture and 0.0172 pmol mg −1 tissue wet weight in mouse heart. These levels are similar to crotonyl-CoA, but between 20 and 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 in metazoans, and provide a methodological foundation for the interrogation of this novel metabolite in biology and disease.

Edited by Jeffrey E. Pessin Acyl-CoA thioesterases (Acots) hydrolyze fatty acyl-CoA esters. Acots in the mitochondrial matrix are poised to mitigate ␤-oxidation overload and maintain CoA availability. Several Acots associate with mitochondria, but whether they all localize to the matrix, are redundant, or have different roles is unresolved. Here, we compared the suborganellar localization, activity, expression, and regulation among mitochondrial Acots (Acot2,-7,-9, and-13) in mitochondria from multiple mouse tissues and from a model of Acot2 depletion. Acot7,-9, and-13 localized to the matrix, joining Acot2 that was previously shown to localize there. Mitochondria from heart, skeletal muscle, brown adipose tissue, and kidney robustly expressed Acot2,-9, and-13; Acot9 levels were substantially higher in brown adipose tissue and kidney mitochondria, as was activity for C4:0-CoA, a unique Acot9 substrate. In all tissues, Acot2 accounted for about half of the thioesterase activity for C14:0-CoA and C16:0-CoA. In contrast, liver mitochondria from fed and fasted mice expressed little Acot activity, which was confined to long-chain CoAs and due mainly to Acot7 and Acot13 activities. Matrix Acots occupied different functional niches, based on substrate specificity (Acot9 versus Acot2 and-13) and strong CoA inhibition (Acot7,-9, and-13, but not Acot2). Interpreted in the context of ␤-oxidation, CoA inhibition would prevent Acotmediated suppression of ␤-oxidation, while providing a release valve when CoA is limiting. In contrast, CoA-insensitive Acot2 could provide a constitutive siphon for long-chain fatty acyl-CoAs. These results reveal how the family of matrix Acots can mitigate ␤-oxidation overload and prevent CoA limitation. Acyl-CoA thioesterases (Acots) 2 hydrolyze acyl-CoA into CoA and an acyl chain and are classified into two families based on functional domain. Type I Acots are members of the superfamily of ␣/␤-hydrolases, are found only in mammals, and have a high degree of similarity (1). Humans and rodents possess Type I Acots residing within the cytoplasm (Acot1), mitochondria (Acot2), and peroxisomes (Acot3-6 in rodents, Acot3-4 in humans) (1). In contrast, Type II Acots (Acot7-15) share little similarity beyond a hotdog fold domain (2). Some possess StAR-related lipid transfer domains (2) or can interact with a Start domain protein (3) and are found in the cytoplasm (Acot7-14), mitochondria (Acot7-13 and Acot15), and peroxisomes (Acot8) (2). Dual localization is also possible (Acot7,-11, and-13). Type I and II Acots have a signature substrate specificity that includes saturated and unsaturated fatty acyl-CoAs of different chain lengths and, in fewer cases, other CoA esters (3-5). Acots are predicted to have high biological relevance because their substrates, acyl-CoA esters, are also substrates for other enzymes that serve major metabolic pathways, such as mitochondrial ␤-oxidation, and can serve as allosteric or covalent regulators. In fact, genetic manipulation in mice of Type I or II Acots is associated ...

We previously identified the aur1 gene cluster which produces the angucycline antibiotic auricin. Preliminary characterisation of auricin revealed that it is modified by a single aminodeoxysugar, D-forosamine. Here we characterise the D-forosamine-specific genes. The four close tandem genes, aur1TQSV, encoding enzymes involved in the initial steps of the deoxysugar biosynthesis, were located on a large operon with other core auricin biosynthetic genes. Deleting these genes resulted in the absence of auricin and the production of deglycosylated auricin intermediates. The two final D-forosamine biosynthetic genes, sa59, an NDP-hexose aminotransferase, and sa52, an NDP-aminohexose N-dimethyltransferase, are located in a region rather distant from the core auricin genes. A deletion analysis of these genes confirmed their role in D-forosamine biosynthesis. The Δsa59 mutant had a phenotype similar to that of the cluster deletion mutant, while the Δsa52 mutant produced an auricin with a demethylated D-forosamine. Although auricin contains a single deoxyhexose, two glycosyltransferase genes were found to participate in the attachment of D-forosamine to the auricin aglycon. An analysis of the expression of the D-forosamine biosynthesis genes revealed that the initial D-forosamine biosynthetic genes aur1TQSV are regulated together with the other auricin core genes by the aur1Ap promoter under the control of the auricin-specific activator Aur1P. The expression of the other D-forosamine genes, however, is governed by promoters differentially dependent upon the two SARP family auricin-specific activators Aur1PR3 and Aur1PR4. These promoters contain direct repeats similar to the SARP consensus sequence and are involved in the interaction with both regulators.

Streptomyces lavendulae subsp. lavendulae CCM 3239 produces the angucycline antibiotic auricin and was thought to be the type strain of Streptomyces aureofaciens. We report the complete genome sequence of this strain, which consists of a linear chromosome and the linear plasmid pSA3239, and demonstrate it to be S. lavendulae subsp. lavendulae.

The γ-butyrolactone (GBL) autoregulator-receptor systems play a role in controlling secondary metabolism and/or morphological differentiation in many Streptomyces species. We previously identified the aur1 gene cluster, located on the Streptomyces aureofaciens CCM 3239 large linear plasmid pSA3239, which is responsible for the production of the angucycline antibiotic auricin. Here, we describe the characterisation of two genes, sagA and sagR, encoding GBL autoregulatory signalling homologues, which lie in the upstream part of the aur1 cluster. SagA was similar to GBL synthases and SagR to GBL receptors. The expression of each gene is directed by its own promoter, sagAp for sagA and sagRp for sagR. Both genes were active mainly during the exponential phase, and their transcription was interdependent. The disruption of sagA abolished auricin production, while the disruption of sagR resulted in precocious but dramatically reduced auricin production. Transcription from the aur1Pp and aur1Rp promoters, which direct the expression of auricin-specific cluster-situated regulators (CSRs), was also precocious and increased in the sagR mutant strain. In addition, SagR was also shown to specifically bind both promoters in vitro. These results indicated that the SagA-SagR GBL system regulates auricin production. Unlike many other GBL receptors, SagR does not bind its own promoter, but Aur1R, an auricin-specific repressor from the family of pseudo GBL receptors, does bind both sagAp and sagRp promoters. Moreover, the expression of both promoters was deregulated in an aur1R mutant, indicating that the SagA-SagR GBL system is regulated by a feedback mechanism involving the auricin-specific CSR Aur1R, which regulates downstream.

Friedreich's ataxia is an inherited disorder caused by depletion of frataxin (Fxn), a mitochondrial protein involved in iron-sulfur cluster biogenesis. Cardiac dysfunction is the main cause of death; pathogenesis remains poorly understood but is expected to be linked to an energy deficit. In mice with adult-onset Fxn loss, bioenergetics analysis of heart mitochondria revealed a time-and substrate-dependent decrease in oxidative phosphorylation (oxphos). Oxphos was lower with substrates that depend on Complex I and II, but preserved for lipid substrates, especially through electron entry into Complex III via the electron transfer flavoprotein dehydrogenase. This differential substrate vulnerability is consistent with the half-lives for mitochondrial proteins.Cardiac contractility was preserved, likely due to sustained β-oxidation. Yet, a stress response was stimulated, characterized by activated mTORC1 and the p-eIF2α/ATF4 axis. This study exposes an unrecognized mechanism that maintains oxphos in the Fxn-depleted heart. The stress response that nonetheless occurs suggests energy deficit-independent pathogenesis. KEYWORDSFrataxin; bioenergetics; β-oxidation; cardiac metabolism; mitochondrial disease; integrated stress response cardiac hypertrophy (Huang et al., 2013, Seznec, Simon et al., 2004, Stram, Wagner et al., 2017, Wagner, Pride et al., 2012, which is not easily explained by elevated peIF2α and an ensuing decrease in global translation. Taken together, the mitochondrial disease literature suggests that multiple signaling pathways can be altered to potentially drive phenotypes. Thus, in any given model, it would be useful to broadly understand disrupted signaling. A broader understanding could expand the possible therapeutic targets and also reveal if disease heterogeneity needs to be considered in the context of FRDA treatments. The CKM mouse model of Fxn loss has been useful because it exhibits severe cardiac dysfunction (Huang et al., 2013, Martin, Abraham et al., 2017, Seznec et al., 2004); indeed it has been used to demonstrate the potential of gene replacement therapy in the heart (Belbellaa, Reutenauer et al., 2019, Perdomini, Belbellaa et al., 2014). Yet, this model features complete depletion of Fxn from birth and thus might reflect a developmental response. Moreover, the model has a rapid time course, making it more challenging to disentangle causes from consequences of severe pathology. A recently developed model of adult-onset Fxn depletion (Chandran, Gao et al., 2017) has several advantages for the study of the pathogenesis of cardiomyopathy in FRDA: the model avoids a developmental context, and has a wider window of time without overt major cardiac pathology. We have used this model to investigate the progression of cardiac mitochondrial metabolism and nutrient and stress signaling changes with the goal of obtaining insight into the pathogenesis of Fxn loss in the heart, specifically with regard to the impact on energy metabolism and how changes in energy metabolism might drive pathology. RESUL...

Friedreich’s ataxia (FRDA) is an inherited disorder caused by depletion of frataxin (FXN), a mitochondrial protein required for iron–sulfur cluster (ISC) biogenesis. Cardiac dysfunction is the main cause of death. Yet pathogenesis, and, more generally, how the heart adapts to FXN loss, remain poorly understood, though are expected to be linked to an energy deficit. We modified a transgenic (TG) mouse model of inducible FXN depletion that permits phenotypic evaluation of the heart at different FXN levels, and focused on substrate-specific bioenergetics and stress signaling. When FXN protein in the TG heart was 17% of normal, bioenergetics and signaling were not different from control. When, 8 weeks later, FXN was ~ 97% depleted in the heart, TG heart mass and cardiomyocyte cross-sectional area were less, without evidence of fibrosis or apoptosis. mTORC1 signaling was activated, as was the integrated stress response, evidenced by greater phosphorylation of eIF2α relative to total eIF2α, and decreased protein translation. We interpret these results to suggest that, in TG hearts, an anabolic stimulus was constrained by eIF2α phosphorylation. Cardiac contractility was maintained in the 97%-FXN-depleted hearts, possibly contributed by an unexpected preservation of β-oxidation, though pyruvate oxidation was lower. Bioenergetics alterations were matched by changes in the mitochondrial proteome, including a non-uniform decrease in abundance of ISC-containing proteins. Altogether, these findings suggest that the FXN depleted heart can suppress a major ATP demanding process such as protein translation, which, together with some preservation of β-oxidation, could be adaptive, at least in the short term.

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