Deglutarylation of glutaryl-CoA dehydrogenase by deacylating enzyme SIRT5 promotes lysine oxidation in mice.

A wide range of protein acyl modifications has been identified on enzymes across various metabolic processes; however, the impact of these modifications remains poorly understood. Protein glutarylation is a recently identified modification that can be nonenzymatically driven by glutaryl-CoA. In mammalian systems, this unique metabolite is only produced in the lysine and tryptophan oxidative pathways. To better understand the biology of protein glutarylation, we studied the relationship between enzymes within the lysine/tryptophan catabolic pathways, protein glutarylation, and regulation by the deglutarylating enzyme sirtuin 5 (SIRT5). Here, we identify glutarylation on the lysine oxidation pathway enzyme glutaryl-CoA dehydrogenase (GCDH) and show increased GCDH glutarylation when glutaryl-CoA production is stimulated by lysine catabolism. Our data reveal that glutarylation of GCDH impacts its function, ultimately decreasing lysine oxidation. We also demonstrate the ability of SIRT5 to deglutarylate GCDH, restoring its enzymatic activity. Finally, metabolomic and bioinformatic analyses indicate an expanded role for SIRT5 in regulating amino acid metabolism. Together, these data support a feedback loop model within the lysine/tryptophan oxidation pathway in which glutaryl-CoA is produced, in turn inhibiting GCDH function via glutaryl modification of GCDH lysine residues and can be relieved by SIRT5 deacylation activity.

Extreme Acetylation of the Cardiac Mitochondrial Proteome Does Not Promote Heart Failure.

RATIONALE: Circumstantial evidence links the development of heart failure to posttranslational modifications of mitochondrial proteins, including lysine acetylation (Kac). Nonetheless, direct evidence that Kac compromises mitochondrial performance remains sparse. OBJECTIVE: This study sought to explore the premise that mitochondrial Kac contributes to heart failure by disrupting oxidative metabolism. METHODS AND RESULTS: A DKO (dual knockout) mouse line with deficiencies in CrAT (carnitine acetyltransferase) and Sirt3 (sirtuin 3)-enzymes that oppose Kac by buffering the acetyl group pool and catalyzing lysine deacetylation, respectively-was developed to model extreme mitochondrial Kac in cardiac muscle, as confirmed by quantitative acetyl-proteomics. The resulting impact on mitochondrial bioenergetics was evaluated using a respiratory diagnostics platform that permits comprehensive assessment of mitochondrial function and energy transduction. Susceptibility of DKO mice to heart failure was investigated using transaortic constriction as a model of cardiac pressure overload. The mitochondrial acetyl-lysine landscape of DKO hearts was elevated well beyond that observed in response to pressure overload or Sirt3 deficiency alone. Relative changes in the abundance of specific acetylated lysine peptides measured in DKO versus Sirt3 KO hearts were strongly correlated. A proteomics comparison across multiple settings of hyperacetylation revealed ≈86% overlap between the populations of Kac peptides affected by the DKO manipulation as compared with experimental heart failure. Despite the severity of cardiac Kac in DKO mice relative to other conditions, deep phenotyping of mitochondrial function revealed a surprisingly normal bioenergetics profile. Thus, of the >120 mitochondrial energy fluxes evaluated, including substrate-specific dehydrogenase activities, respiratory responses, redox charge, mitochondrial membrane potential, and electron leak, we found minimal evidence of oxidative insufficiencies. Similarly, DKO hearts were not more vulnerable to dysfunction caused by transaortic constriction-induced pressure overload. CONCLUSIONS: The findings challenge the premise that hyperacetylation per se threatens metabolic resilience in the myocardium by causing broad-ranging disruption to mitochondrial oxidative machinery.

Ablation of Sirtuin5 in the postnatal mouse heart results in protein succinylation and normal survival in response to chronic pressure overload.

Mitochondrial Sirtuin 5 (SIRT5) is an NAD+-dependent demalonylase, desuccinylase, and deglutarylase that controls several metabolic pathways. A number of recent studies point to SIRT5 desuccinylase activity being important in maintaining cardiac function and metabolism under stress. Previously, we described a phenotype of increased mortality in whole-body SIRT5KO mice exposed to chronic pressure overload compared with their littermate WT controls. To determine whether the survival phenotype we reported was due to a cardiac-intrinsic or cardiac-extrinsic effect of SIRT5, we developed a tamoxifen-inducible, heart-specific SIRT5 knockout (SIRT5KO) mouse model. Using our new animal model, we discovered that postnatal cardiac ablation of Sirt5 resulted in persistent accumulation of protein succinylation up to 30 weeks after SIRT5 depletion. Succinyl proteomics revealed that succinylation increased on proteins of oxidative metabolism between 15 and 31 weeks after ablation. Heart-specific SIRT5KO mice were exposed to chronic pressure overload to induce cardiac hypertrophy. We found that, in contrast to whole-body SIRT5KO mice, there was no difference in survival between heart-specific SIRT5KO mice and their littermate controls. Overall, the data presented here suggest that survival of SIRT5KO mice may be dictated by a multitissue or prenatal effect of SIRT5.

Quantification of mitochondrial acetylation dynamics highlights prominent sites of metabolic regulation.

Lysine acetylation is rapidly becoming established as a key post-translational modification for regulating mitochondrial metabolism. Nonetheless, distinguishing regulatory sites from among the thousands identified by mass spectrometry and elucidating how these modifications alter enzyme function remain primary challenges. Here, we performed multiplexed quantitative mass spectrometry to measure changes in the mouse liver mitochondrial acetylproteome in response to acute and chronic alterations in nutritional status, and integrated these data sets with our compendium of predicted Sirt3 targets. These analyses highlight a subset of mitochondrial proteins with dynamic acetylation sites, including acetyl-CoA acetyltransferase 1 (Acat1), an enzyme central to multiple metabolic pathways. We performed in vitro biochemistry and molecular modeling to demonstrate that acetylation of Acat1 decreases its activity by disrupting the binding of coenzyme A. Collectively, our data reveal an important new target of regulatory acetylation and provide a foundation for investigating the role of select mitochondrial protein acetylation sites in mediating acute and chronic metabolic transitions.

Rapid phosphoproteomic and transcriptomic changes in the rhizobia-legume symbiosis.

Symbiotic associations between legumes and rhizobia usually commence with the perception of bacterial lipochitooligosaccharides, known as Nod factors (NF), which triggers rapid cellular and molecular responses in host plants. We report here deep untargeted tandem mass spectrometry-based measurements of rapid NF-induced changes in the phosphorylation status of 13,506 phosphosites in 7739 proteins from the model legume Medicago truncatula. To place these phosphorylation changes within a biological context, quantitative phosphoproteomic and RNA measurements in wild-type plants were compared with those observed in mutants, one defective in NF perception (nfp) and one defective in downstream signal transduction events (dmi3). Our study quantified the early phosphorylation and transcription dynamics that are specifically associated with NF-signaling, confirmed a dmi3-mediated feedback loop in the pathway, and suggested "cryptic" NF-signaling pathways, some of them being also involved in the response to symbiotic arbuscular mycorrhizal fungi.

A proteogenomic survey of the Medicago truncatula genome.

Peptide sequencing by computational assignment of tandem mass spectra to a database of putative protein sequences provides an independent approach to confirming or refuting protein predictions based on large-scale DNA and RNA sequencing efforts. This use of mass spectrometrically-derived sequence data for testing and refining predicted gene models has been termed proteogenomics. We report herein the application of proteogenomic methodology to a database of 10.9 million tandem mass spectra collected over a period of two years from proteolytically generated peptides isolated from the model legume Medicago truncatula. These spectra were searched against a database of predicted M. truncatula protein sequences generated from public databases, in silico gene model predictions, and a whole-genome six-frame translation. This search identified 78,647 distinct peptide sequences, and a comparison with the publicly available proteome from the recently published M. truncatula genome supported translation of 9,843 existing gene models and identified 1,568 novel peptides suggesting corrections or additions to the current annotations. Each supporting and novel peptide was independently validated using mRNA-derived deep sequencing coverage and an overall correlation of 93% between the two data types was observed. We have additionally highlighted examples of several aspects of structural annotation for which tandem MS provides unique evidence not easily obtainable through typical DNA or RNA sequencing. Proteogenomic analysis is a valuable and unique source of information for the structural annotation of genomes and should be included in such efforts to ensure that the genome models used by biologists mirror as accurately as possible what is present in the cell.

Cysteine S-acetylation is a post-translational modification involved in metabolic regulation.

Cysteine is a reactive amino acid central to the catalytic activities of many enzymes. It is also a common target of post-translational modifications (PTMs), such as palmitoylation. This longchain acyl PTM can modify cysteine residues and induce changes in protein subcellular localization. We hypothesized that cysteine could also be modified by short-chain acyl groups, such as cysteine S-acetylation. To test this, we developed sample preparation and non-targeted mass spectrometry protocols to analyze the mouse liver proteome for cysteine acetylation. Our findings revealed hundreds of sites of cysteine acetylation across multiple tissue types, revealing a previously uncharacterized cysteine acetylome. Cysteine acetylation shows a marked cytoplasmic subcellular localization signature, with tissue-specific acetylome patterns and specific changes upon metabolic stress. This study uncovers a novel aspect of cysteine biochemistry, highlighting short-chain modifications alongside known long-chain acyl PTMs. These findings enrich our understanding of the landscape of acyl modifications and suggest new research directions in enzyme activity regulation and cellular signaling in metabolism.

Ketone flux through BDH1 supports metabolic remodeling of skeletal and cardiac muscles in response to intermittent time-restricted feeding.

Time-restricted feeding (TRF) has gained attention as a dietary regimen that promotes metabolic health. This study questioned if the health benefits of an intermittent TRF (iTRF) schedule require ketone flux specifically in skeletal and cardiac muscles. Notably, we found that the ketolytic enzyme beta-hydroxybutyrate dehydrogenase 1 (BDH1) is uniquely enriched in isolated mitochondria derived from heart and red/oxidative skeletal muscles, which also have high capacity for fatty acid oxidation (FAO). Using mice with BDH1 deficiency in striated muscles, we discover that this enzyme optimizes FAO efficiency and exercise tolerance during acute fasting. Additionally, iTRF leads to robust molecular remodeling of muscle tissues, and muscle BDH1 flux does indeed play an essential role in conferring the full adaptive benefits of this regimen, including increased lean mass, mitochondrial hormesis, and metabolic rerouting of pyruvate. In sum, ketone flux enhances mitochondrial bioenergetics and supports iTRF-induced remodeling of skeletal muscle and heart.

"Metformin Impairs Intestinal Fructose Metabolism".

Objective: To investigate the effects of metformin on intestinal carbohydrate metabolism in vivo. Method: Male mice preconditioned with a high-fat, high-sucrose diet were treated orally with metformin or a control solution for two weeks. Fructose metabolism, glucose production from fructose, and production of other fructose-derived metabolites were assessed using stably labeled fructose as a tracer. Results: Metformin treatment decreased intestinal glucose levels and reduced incorporation of fructose-derived metabolites into glucose. This was associated with decreased intestinal fructose metabolism as indicated by decreased enterocyte F1P levels and diminished labeling of fructose-derived metabolites. Metformin also reduced fructose delivery to the liver. Proteomic analysis revealed that metformin coordinately down-regulated proteins involved carbohydrate metabolism including those involved in fructolysis and glucose production within intestinal tissue. Conclusion: Metformin reduces intestinal fructose metabolism, and this is associated with broad-based changes in intestinal enzyme and protein levels involved in sugar metabolism indicating that metformin's effects on sugar metabolism are pleiotropic.

HGFAC is a ChREBP-regulated hepatokine that enhances glucose and lipid homeostasis.

Carbohydrate response element-binding protein (ChREBP) is a carbohydrate-sensing transcription factor that regulates both adaptive and maladaptive genomic responses in coordination of systemic fuel homeostasis. Genetic variants in the ChREBP locus associate with diverse metabolic traits in humans, including circulating lipids. To identify novel ChREBP-regulated hepatokines that contribute to its systemic metabolic effects, we integrated ChREBP ChIP-Seq analysis in mouse liver with human genetic and genomic data for lipid traits and identified hepatocyte growth factor activator (HGFAC) as a promising ChREBP-regulated candidate in mice and humans. HGFAC is a protease that activates the pleiotropic hormone hepatocyte growth factor. We demonstrate that HGFAC-KO mice had phenotypes concordant with putative loss-of-function variants in human HGFAC. Moreover, in gain- and loss-of-function genetic mouse models, we demonstrate that HGFAC enhanced lipid and glucose homeostasis, which may be mediated in part through actions to activate hepatic PPARγ activity. Together, our studies show that ChREBP mediated an adaptive response to overnutrition via activation of HGFAC in the liver to preserve glucose and lipid homeostasis.

Pyruvate-supported flux through medium-chain ketothiolase promotes mitochondrial lipid tolerance in cardiac and skeletal muscles.

Even-chain acylcarnitine (AC) metabolites, most of which are generated as byproducts of incomplete fatty acid oxidation (FAO), are viewed as biomarkers of mitochondrial lipid stress attributable to one or more metabolic bottlenecks in the ß-oxidation pathway. The origins and functional implications of FAO bottlenecks remain poorly understood. Here, we combined a sophisticated mitochondrial phenotyping platform with state-of-the-art molecular profiling tools and multiple two-state mouse models of respiratory function to uncover a mechanism that connects AC accumulation to lipid intolerance, metabolic inflexibility, and respiratory inefficiency in skeletal muscle mitochondria. These studies also identified a short-chain carbon circuit at the C4 node of FAO wherein reverse flux of glucose-derived acetyl CoA through medium-chain ketothiolase enhances lipid tolerance and redox stability in heart mitochondria by regenerating free CoA and NAD+. The findings help to explain why diminished FAO capacity, AC accumulation, and metabolic inflexibility are tightly linked to poor health outcomes.

Proteomics and phosphoproteomics datasets of a muscle-specific STIM1 loss-of-function mouse model.

STIM1 is an ER/SR transmembrane protein that interacts with ORAI1 to activate store operated Ca2+ entry (SOCE) upon ER/SR depletion of calcium. Normally highly expressed in skeletal muscle, STIM1 deficiency causes significant changes to mitochondrial ultrastructure that do not occur with loss of ORAI1 or other components of SOCE. The datasets in this article are from large-scale proteomics and phosphoproteomics experiments in an inducible mouse model of skeletal muscle-specific STIM1 knock out (KO). These data reveal statistically significant changes in the relative abundance of specific proteins and sites of protein phosphorylation in STIM1 KO gastrocnemius. Protein samples from five biological replicates of each condition (+/- STIM1) were enzymatically digested, the resulting peptides labeled with tandem mass tag (TMT) reagents, mixed, and fractionated. Phosphopeptides were enriched and a small amount of each input retained for protein abundance analysis. All phosphopeptide and input fractions were analyzed by nano LC-MS/MS on a Q Exactive Plus Orbitrap mass spectrometer, searched with Proteome Discoverer software, and processed with in-house R-scripts for data normalization and statistical analysis. Article published in Molecular Metabolism [1].

Disruption of STIM1-mediated Ca2+ sensing and energy metabolism in adult skeletal muscle compromises exercise tolerance, proteostasis, and lean mass.

OBJECTIVE: Stromal interaction molecule 1 (STIM1) is a single-pass transmembrane endoplasmic/sarcoplasmic reticulum (E/SR) protein recognized for its role in a store operated Ca2+ entry (SOCE), an ancient and ubiquitous signaling pathway. Whereas STIM1 is known to be indispensable during development, its biological and metabolic functions in mature muscles remain unclear. METHODS: Conditional and tamoxifen inducible muscle STIM1 knock-out mouse models were coupled with multi-omics tools and comprehensive physiology to understand the role of STIM1 in regulating SOCE, mitochondrial quality and bioenergetics, and whole-body energy homeostasis. RESULTS: This study shows that STIM1 is abundant in adult skeletal muscle, upregulated by exercise, and is present at SR-mitochondria interfaces. Inducible tissue-specific deletion of STIM1 (iSTIM1 KO) in adult muscle led to diminished lean mass, reduced exercise capacity, and perturbed fuel selection in the settings of energetic stress, without affecting whole-body glucose tolerance. Proteomics and phospho-proteomics analyses of iSTIM1 KO muscles revealed molecular signatures of low-grade E/SR stress and broad activation of processes and signaling networks involved in proteostasis. CONCLUSION: These results show that STIM1 regulates cellular and mitochondrial Ca2+ dynamics, energy metabolism and proteostasis in adult skeletal muscles. Furthermore, these findings provide insight into the pathophysiology of muscle diseases linked to disturbances in STIM1-dependent Ca2+ handling.

Nicotinamide riboside supplementation confers marginal metabolic benefits in obese mice without remodeling the muscle acetyl-proteome.

Nicotinamide riboside supplements (NRS) have been touted as a nutraceutical that promotes cardiometabolic and musculoskeletal health by enhancing nicotinamide adenine dinucleotide (NAD+) biosynthesis, mitochondrial function, and/or the activities of NAD-dependent sirtuin deacetylase enzymes. This investigation examined the impact of NRS on whole body energy homeostasis, skeletal muscle mitochondrial function, and corresponding shifts in the acetyl-lysine proteome, in the context of diet-induced obesity using C57BL/6NJ mice. The study also included a genetically modified mouse model that imposes greater demand on sirtuin flux and associated NAD+ consumption, specifically within muscle tissues. In general, whole body glucose control was marginally improved by NRS when administered at the midpoint of a chronic high-fat diet, but not when given as a preventative therapy upon initiation of the diet. Contrary to anticipated outcomes, the study produced little evidence that NRS increases tissue NAD+ levels, augments mitochondrial function, and/or mitigates diet-induced hyperacetylation of the skeletal muscle proteome.

Statin therapy inhibits fatty acid synthase via dynamic protein modifications.

Statins are a class of drug widely prescribed for the prevention of cardiovascular disease, with pleiotropic cellular effects. Statins inhibit HMG-CoA reductase (HMGCR), which converts the metabolite HMG-CoA into mevalonate. Recent discoveries have shown HMG-CoA is a reactive metabolite that can non-enzymatically modify proteins and impact their activity. Therefore, we predicted that inhibition of HMGCR by statins might increase HMG-CoA levels and protein modifications. Upon statin treatment, we observe a strong increase in HMG-CoA levels and modification of only a single protein. Mass spectrometry identifies this protein as fatty acid synthase (FAS), which is modified on active site residues and, importantly, on non-lysine side-chains. The dynamic modifications occur only on a sub-pool of FAS that is located near HMGCR and alters cellular signaling around the ER and Golgi. These results uncover communication between cholesterol and lipid biosynthesis by the substrate of one pathway inhibiting another in a rapid and reversible manner.

Large-scale phosphoprotein analysis in Medicago truncatula roots provides insight into in vivo kinase activity in legumes.

Nitrogen fixation in legumes requires the development of root organs called nodules and their infection by symbiotic rhizobia. Over the last decade, Medicago truncatula has emerged as a major model plant for the analysis of plant-microbe symbioses and for addressing questions pertaining to legume biology. While the initiation of symbiosis and the development of nitrogen-fixing root nodules depend on the activation of a protein phosphorylation-mediated signal transduction cascade in response to symbiotic signals produced by the rhizobia, few sites of in vivo phosphorylation have previously been identified in M. truncatula. We have characterized sites of phosphorylation on proteins from M. truncatula roots, from both whole cell lysates and membrane-enriched fractions, using immobilized metal affinity chromatography and tandem mass spectrometry. Here, we report 3,457 unique phosphopeptides spanning 3,404 nonredundant sites of in vivo phosphorylation on 829 proteins in M. truncatula Jemalong A17 roots, identified using the complementary tandem mass spectrometry fragmentation methods electron transfer dissociation and collision-activated dissociation. With this being, to our knowledge, the first large-scale plant phosphoproteomic study to utilize electron transfer dissociation, analysis of the identified phosphorylation sites revealed phosphorylation motifs not previously observed in plants. Furthermore, several of the phosphorylation motifs, including LxKxxs and RxxSxxxs, have yet to be reported as kinase specificities for in vivo substrates in any species, to our knowledge. Multiple sites of phosphorylation were identified on several key proteins involved in initiating rhizobial symbiosis, including SICKLE, NUCLEOPORIN133, and INTERACTING PROTEIN OF DMI3. Finally, we used these data to create an open-access online database for M. truncatula phosphoproteomic data.

Chronic caloric restriction maintains a youthful phosphoproteome in aged skeletal muscle.

Caloric restriction (CR) can prolong aged skeletal muscle function, yet the molecular mechanisms are not completely understood. We performed phosphoproteomic analysis on muscle from young and old mice fed an ad libitum diet, and old mice fed a CR diet. CR promoted a youthful phosphoproteomic signature, suppressing several known "pro-aging" pathways including Protein kinase A (PKA). This study validates global signaling changes in skeletal muscle during CR.

Sirtuin 5 Is Regulated by the SCFCyclin F Ubiquitin Ligase and Is Involved in Cell Cycle Control.

The ubiquitin-proteasome system is essential for cell cycle progression. Cyclin F is a cell cycle-regulated substrate adapter F-box protein for the Skp1, CUL1, and F-box protein (SCF) family of E3 ubiquitin ligases. Despite its importance in cell cycle progression, identifying cyclin F-bound SCF complex (SCFCyclin F) substrates has remained challenging. Since cyclin F overexpression rescues a yeast mutant in the cdc4 gene, we considered the possibility that other genes that genetically modify cdc4 mutant lethality could also encode cyclin F substrates. We identified the mitochondrial and cytosolic deacylating enzyme sirtuin 5 (SIRT5) as a novel cyclin F substrate. SIRT5 has been implicated in metabolic processes, but its connection to the cell cycle is not known. We show that cyclin F interacts with and controls the ubiquitination, abundance, and stability of SIRT5. We show SIRT5 knockout results in a diminished G1 population and a subsequent increase in both S and G2/M. Global proteomic analyses reveal cyclin-dependent kinase (CDK) signaling changes congruent with the cell cycle changes in SIRT5 knockout cells. Together, these data demonstrate that SIRT5 is regulated by cyclin F and suggest a connection between SIRT5, cell cycle regulation, and metabolism.

Disruption of Acetyl-Lysine Turnover in Muscle Mitochondria Promotes Insulin Resistance and Redox Stress without Overt Respiratory Dysfunction.

This study sought to examine the functional significance of mitochondrial protein acetylation using a double knockout (DKO) mouse model harboring muscle-specific deficits in acetyl-CoA buffering and lysine deacetylation, due to genetic ablation of carnitine acetyltransferase and Sirtuin 3, respectively. DKO mice are highly susceptible to extreme hyperacetylation of the mitochondrial proteome and develop a more severe form of diet-induced insulin resistance than either single KO mouse line. However, the functional phenotype of hyperacetylated DKO mitochondria is largely normal. Of the >120 measures of respiratory function assayed, the most consistently observed traits of a markedly heightened acetyl-lysine landscape are enhanced oxygen flux in the context of fatty acid fuel and elevated rates of electron leak. In sum, the findings challenge the notion that lysine acetylation causes broad-ranging damage to mitochondrial quality and performance and raise the possibility that acetyl-lysine turnover, rather than acetyl-lysine stoichiometry, modulates redox balance and carbon flux.

Respiratory Phenomics across Multiple Models of Protein Hyperacylation in Cardiac Mitochondria Reveals a Marginal Impact on Bioenergetics.

Acyl CoA metabolites derived from the catabolism of carbon fuels can react with lysine residues of mitochondrial proteins, giving rise to a large family of post-translational modifications (PTMs). Mass spectrometry-based detection of thousands of acyl-PTMs scattered throughout the proteome has established a strong link between mitochondrial hyperacylation and cardiometabolic diseases; however, the functional consequences of these modifications remain uncertain. Here, we use a comprehensive respiratory diagnostics platform to evaluate three disparate models of mitochondrial hyperacylation in the mouse heart caused by genetic deletion of malonyl CoA decarboxylase (MCD), SIRT5 demalonylase and desuccinylase, or SIRT3 deacetylase. In each case, elevated acylation is accompanied by marginal respiratory phenotypes. Of the >60 mitochondrial energy fluxes evaluated, the only outcome consistently observed across models is a ∼15% decrease in ATP synthase activity. In sum, the findings suggest that the vast majority of mitochondrial acyl PTMs occur as stochastic events that minimally affect mitochondrial bioenergetics.