46 results on '"Paul A. Grimsrud"'
Search Results
2. HGFAC is a ChREBP-regulated hepatokine that enhances glucose and lipid homeostasis
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Ashot Sargsyan, Ludivine Doridot, Sarah A. Hannou, Wenxin Tong, Harini Srinivasan, Rachael Ivison, Ruby Monn, Henry H. Kou, Jonathan M. Haldeman, Michelle Arlotto, Phillip J. White, Paul A. Grimsrud, Inna Astapova, Linus T. Tsai, and Mark A. Herman
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Metabolism ,Medicine - Abstract
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.
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- 2023
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3. Proteomics and phosphoproteomics datasets of a muscle-specific STIM1 loss-of-function mouse model
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Scott P. Lyons, Rebecca J. Wilson, Deborah M. Muoio, and Paul A. Grimsrud
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Mass spectrometry ,Protein phosphorylation ,Protein abundance ,Isobaric tags ,R script ,Calcium homeostasis ,Computer applications to medicine. Medical informatics ,R858-859.7 ,Science (General) ,Q1-390 - Abstract
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].
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- 2022
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4. Disruption of STIM1-mediated Ca2+ sensing and energy metabolism in adult skeletal muscle compromises exercise tolerance, proteostasis, and lean mass
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Rebecca J. Wilson, Scott P. Lyons, Timothy R. Koves, Victoria G. Bryson, Hengtao Zhang, TianYu Li, Scott B. Crown, Jin-Dong Ding, Paul A. Grimsrud, Paul B. Rosenberg, and Deborah M. Muoio
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Skeletal muscle ,STIM1 ,Proteostasis ,Energy metabolism ,Exercise tolerance ,Mitochondria ,Internal medicine ,RC31-1245 - Abstract
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.
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- 2022
- Full Text
- View/download PDF
5. Nicotinamide riboside supplementation confers marginal metabolic benefits in obese mice without remodeling the muscle acetyl-proteome
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Ashley S. Williams, Timothy R. Koves, Yasminye D. Pettway, James A. Draper, Dorothy H. Slentz, Paul A. Grimsrud, Olga R. Ilkayeva, and Deborah M. Muoio
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Physiology ,Proteomics ,Nutrition ,Science - Abstract
Summary: 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.
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- 2022
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6. Lipids Reprogram Metabolism to Become a Major Carbon Source for Histone Acetylation
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Eoin McDonnell, Scott B. Crown, Douglas B. Fox, Betül Kitir, Olga R. Ilkayeva, Christian A. Olsen, Paul A. Grimsrud, and Matthew D. Hirschey
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histone ,acetylation ,epigenetics ,metabolism ,fatty acid ,lipids ,gene expression ,proteomics ,metabolomics ,Biology (General) ,QH301-705.5 - Abstract
Cells integrate nutrient sensing and metabolism to coordinate proper cellular responses to a particular nutrient source. For example, glucose drives a gene expression program characterized by activating genes involved in its metabolism, in part by increasing glucose-derived histone acetylation. Here, we find that lipid-derived acetyl-CoA is a major source of carbon for histone acetylation. Using 13C-carbon tracing combined with acetyl-proteomics, we show that up to 90% of acetylation on certain histone lysines can be derived from fatty acid carbon, even in the presence of excess glucose. By repressing both glucose and glutamine metabolism, fatty acid oxidation reprograms cellular metabolism, leading to increased lipid-derived acetyl-CoA. Gene expression profiling of octanoate-treated hepatocytes shows a pattern of upregulated lipid metabolic genes, demonstrating a specific transcriptional response to lipid. These studies expand the landscape of nutrient sensing and uncover how lipids and metabolism are integrated by epigenetic events that control gene expression.
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- 2016
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7. Respiratory Phenomics across Multiple Models of Protein Hyperacylation in Cardiac Mitochondria Reveals a Marginal Impact on Bioenergetics
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Kelsey H. Fisher-Wellman, James A. Draper, Michael T. Davidson, Ashley S. Williams, Tara M. Narowski, Dorothy H. Slentz, Olga R. Ilkayeva, Robert D. Stevens, Gregory R. Wagner, Rami Najjar, Mathew D. Hirschey, J. Will Thompson, David P. Olson, Daniel P. Kelly, Timothy R. Koves, Paul A. Grimsrud, and Deborah M. Muoio
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Biology (General) ,QH301-705.5 - Abstract
Summary: 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. : Fisher-Wellman et al. use a recently developed mitochondrial diagnostics platform for deep phenotyping of heart mitochondria derived from three disparate genetic models of protein hyperacylation. Their findings oppose the notion that hyperacylation of the mitochondrial proteome leads to broad-ranging vulnerabilities in respiratory function and bioenergetics. Keywords: mitochondrial diagnostics, lysine acylation, malonylation, ATP synthase
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- 2019
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8. The Acetyl Group Buffering Action of Carnitine Acetyltransferase Offsets Macronutrient-Induced Lysine Acetylation of Mitochondrial Proteins
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Michael N. Davies, Lilja Kjalarsdottir, J. Will Thompson, Laura G. Dubois, Robert D. Stevens, Olga R. Ilkayeva, M. Julia Brosnan, Timothy P. Rolph, Paul A. Grimsrud, and Deborah M. Muoio
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Biology (General) ,QH301-705.5 - Abstract
Lysine acetylation (AcK), a posttranslational modification wherein a two-carbon acetyl group binds covalently to a lysine residue, occurs prominently on mitochondrial proteins and has been linked to metabolic dysfunction. An emergent theory suggests mitochondrial AcK occurs via mass action rather than targeted catalysis. To test this hypothesis, we performed mass spectrometry-based acetylproteomic analyses of quadriceps muscles from mice with skeletal muscle-specific deficiency of carnitine acetyltransferase (CrAT), an enzyme that buffers the mitochondrial acetyl-CoA pool by converting short-chain acyl-CoAs to their membrane permeant acylcarnitine counterparts. CrAT deficiency increased tissue acetyl-CoA levels and susceptibility to diet-induced AcK of broad-ranging mitochondrial proteins, coincident with diminished whole body glucose control. Sub-compartment acetylproteome analyses of muscles from obese mice and humans showed remarkable overrepresentation of mitochondrial matrix proteins. These findings reveal roles for CrAT and L-carnitine in modulating the muscle acetylproteome and provide strong experimental evidence favoring the nonenzymatic carbon pressure model of mitochondrial AcK.
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- 2016
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9. Remodeling of the Acetylproteome by SIRT3 Manipulation Fails to Affect Insulin Secretion or β Cell Metabolism in the Absence of Overnutrition
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Brett S. Peterson, Jonathan E. Campbell, Olga Ilkayeva, Paul A. Grimsrud, Matthew D. Hirschey, and Christopher B. Newgard
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Biology (General) ,QH301-705.5 - Abstract
Summary: SIRT3 is a nicotinamide adenine dinucleotide (NAD+)-dependent mitochondrial protein deacetylase purported to influence metabolism through post-translational modification of metabolic enzymes. Fuel-stimulated insulin secretion, which involves mitochondrial metabolism, could be susceptible to SIRT3-mediated effects. We used CRISPR/Cas9 technology to manipulate SIRT3 expression in β cells, resulting in widespread SIRT3-dependent changes in acetylation of key metabolic enzymes but no appreciable changes in glucose- or pyruvate-stimulated insulin secretion or metabolomic profile during glucose stimulation. Moreover, these broad changes in the SIRT3-targeted acetylproteome did not affect responses to nutritional or ER stress. We also studied mice with global SIRT3 knockout fed either standard chow (STD) or high-fat and high-sucrose (HFHS) diets. Only when chronically fed HFHS diet do SIRT3 KO animals exhibit a modest reduction in insulin secretion. We conclude that broad changes in mitochondrial protein acetylation in response to manipulation of SIRT3 are not sufficient to cause changes in islet function or metabolism. : Peterson et al. report that ablation of SIRT3 in 832/13 β cells dramatically alters the mitochondrial acetylproteome but does not affect insulin secretion, metabolomic profile, or β cell survival. Moreover, SIRT3 knockout causes a modest reduction in insulin secretion in mice fed a high-fat and high-sucrose but not a standard chow diet. Keywords: acetylation, metabolism, insulin secretion, pancreatic islets, mitochondria, post-translational modifications, diabetes
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- 2018
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10. Medicago PhosphoProtein Database: a repository for Medicago truncatula phosphoprotein data
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Christopher M. Rose, Muthusubramanian eVenkateshwaran, Paul A. Grimsrud, Michael S. Westphall, Michael R. Sussman, Joshua J. Coon, and Jean-Michel eAné
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Medicago truncatula ,Proteomics ,CAD ,phosphoproteome ,ETD ,Plant culture ,SB1-1110 - Abstract
The ability of legume crops to fix atmospheric nitrogen via a symbiotic association with soil rhizobia makes them an essential component of many agricultural systems. Initiation of this symbiosis requires protein phosphorylation-mediated signaling in response to rhizobial signals named Nod factors. Medicago truncatula (Medicago) is the model system for studying legume biology, making the study of its phosphoproteome essential. Here, we describe the Medicago Phosphoprotein Database (http://phospho.medicago.wisc.edu), a repository built to house phosphoprotein, phosphopeptide, and phosphosite data specific to Medicago. Currently, the Medicago Phosphoprotein Database holds 3,457 unique phosphopeptides that contain 3,404 non-redundant sites of phosphorylation on 829 proteins. Through the web-based interface, users are allowed to browse identified proteins or search for proteins of interest. Furthermore, we allow users to conduct BLAST searches of the database using both peptide sequences and phosphorylation motifs as queries. The data contained within the database are available for download to be investigated at the user’s discretion. The Medicago Phosphoprotein Database will be updated continually with novel phosphoprotein and phosphopeptide identifications, with the intent of constructing an unparalleled compendium of large-scale Medicago phosphorylation data.
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- 2012
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11. Leveraging proteomics to understand plant-microbe interactions
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Dhileepkumar eJayaraman, Kari L. Forshey, Paul A. Grimsrud, and Jean-Michel eAné
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Plants ,Proteomics ,Symbiosis ,signaling ,Defense ,Plant culture ,SB1-1110 - Abstract
Understanding the interactions of plants with beneficial and pathogenic microbes is a promising avenue to improve crop productivity and agriculture sustainability. Proteomic techniques provide a unique angle to describe these intricate interactions and test hypotheses. The various approaches for proteome analysis generally include protein/peptide separation and identification, but can also provide quantification and the characterization of post-translational modifications. In this review, we discuss how these techniques have been applied to the study to plant-microbe interactions. We also present some areas where this field of study would benefit from the utilization of newly developed methods that overcome previous limitations. Finally, we reinforce the need for expanding, integrating, and curating protein databases, as well as the benefits of combining protein-level datasets with genetics and other high-throughput large-scale approaches for a systems-level understanding of plant-microbe interactions.
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- 2012
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12. Metformin Impairs Intestinal Fructose Metabolism
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Wenxin Tong, Sarah A. Hannou, Ashot Sargsyan, Guo-Fang Zhang, Paul A. Grimsrud, Inna Astapova, and Mark A. Herman
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Article - Abstract
ObjectiveTo investigate the effects of metformin on intestinal carbohydrate metabolismin 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.ResultsMetformin 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.ConclusionMetformin 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.HighlightsMetformin decreases intestinal fructose absorption, metabolism, and fructose delivery to the liver.Metformin reduces intestinal glucose production from fructose-derived metabolites.Metformin reduces protein levels of multiple metabolic enzymes involved in fructose and glucose metabolism in intestinal tissue.
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- 2023
13. Pyruvate-supported flux through medium-chain ketothiolase promotes mitochondrial lipid tolerance in cardiac and skeletal muscles
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Timothy R. Koves, Guo-Fang Zhang, Michael T. Davidson, Alec B. Chaves, Scott B. Crown, Jordan M. Johnson, Dorothy H. Slentz, Paul A. Grimsrud, and Deborah M. Muoio
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Physiology ,Cell Biology ,Molecular Biology - Published
- 2023
14. Extreme Acetylation of the Cardiac Mitochondrial Proteome Does Not Promote Heart Failure
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Deborah M. Muoio, Timothy R. Koves, James A. Draper, Tara M. Narowski, Kelsey H. Fisher-Wellman, Ling Lai, Michael T. Davidson, Dennis M. Abraham, Daniel P. Kelly, and Paul A. Grimsrud
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Male ,Proteomics ,Proteome ,Physiology ,Lysine ,Biology ,Mitochondrion ,Mitochondria, Heart ,Article ,Mitochondrial Proteins ,Sirtuin 3 ,medicine ,Animals ,Myocytes, Cardiac ,Carnitine ,Heart Failure ,Mice, Knockout ,Carnitine O-Acetyltransferase ,Acetylation ,medicine.disease ,Mitochondrial proteome ,Cell biology ,Mice, Inbred C57BL ,Disease Models, Animal ,Oxidative Stress ,Heart failure ,Sirtuin ,biology.protein ,Energy Metabolism ,Cardiology and Cardiovascular Medicine ,Protein Processing, Post-Translational ,medicine.drug - Abstract
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.
- Published
- 2020
15. Nicotinamide riboside supplementation confers marginal metabolic benefits in obese mice without remodeling the muscle acetyl-proteome
- Author
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Ashley S. Williams, Timothy R. Koves, Yasminye D. Pettway, James A. Draper, Dorothy H. Slentz, Paul A. Grimsrud, Olga R. Ilkayeva, and Deborah M. Muoio
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Proteomics ,Multidisciplinary ,Physiology ,Science ,Article ,Nutrition - Abstract
Summary 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., Graphical abstract, Highlights • Dietary NR supplementation (NRS) raises plasma and muscle NAM levels in obese mice • NRS given post obesity slightly improved glucose control and mitochondrial function • NRS did not oppose obesity-induced remodeling of the muscle acetyl-proteome • NRS during weight gain did not protect glucose control and mitochondrial function, Physiology; Proteomics; Nutrition
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- 2021
16. Disruption of STIM1-mediated Ca
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Rebecca J, Wilson, Scott P, Lyons, Timothy R, Koves, Victoria G, Bryson, Hengtao, Zhang, TianYu, Li, Scott B, Crown, Jin-Dong, Ding, Paul A, Grimsrud, Paul B, Rosenberg, and Deborah M, Muoio
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Mice ,Exercise Tolerance ,Proteostasis ,Animals ,Calcium ,Stromal Interaction Molecule 1 ,Energy Metabolism ,Muscle, Skeletal - Abstract
Stromal interaction molecule 1 (STIM1) is a single-pass transmembrane endoplasmic/sarcoplasmic reticulum (E/SR) protein recognized for its role in a store operated CaConditional 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.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.These results show that STIM1 regulates cellular and mitochondrial Ca
- Published
- 2021
17. HGFAC is a ChREBP Regulated Hepatokine that Enhances Glucose and Lipid Homeostasis
- Author
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Michelle Arlotto, Henry H. Kou, Linus Tsai, Harini Srinivasan, Paul A. Grimsrud, Wenxin Tong, Phillip J. White, Ludivine Doridot, Ashot Sargsyan, Rachael Ivison, Jonathan M. Haldeman, Sarah A. Hannou, Ruby Monn, Inna Astapova, and Mark A. Herman
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medicine ,Glucose homeostasis ,Hepatocyte growth factor ,Hepatocyte Growth Factor Activator ,Biology ,Carbohydrate-responsive element-binding protein ,Transcription factor ,Phenotype ,Homeostasis ,Cell biology ,Hormone ,medicine.drug - Abstract
Carbohydrate Responsive 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 (HGF). We demonstrate that HGFAC KO mice have 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 enhances lipid and glucose homeostasis, in part, through actions to activate hepatic PPARγ activity. Together, our studies show that ChREBP mediates an adaptive response to overnutrition via activation of an HGFAC-HGF-PPARγ signaling axis in the liver to preserve glucose and lipid homeostasis.
- Published
- 2021
18. Statin therapy inhibits fatty acid synthase via dynamic protein modifications
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Matthew D. Hirschey, Thompson Jw, Scott B. Crown, Paul A. Grimsrud, Robert Stevens, Gregory R. Wagner, Trub A, Donald S. Backos, Olga Ilkayeva, and Zhang G
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Fatty acid synthase ,Text mining ,biology ,business.industry ,Chemistry ,biology.protein ,lipids (amino acids, peptides, and proteins) ,Statin therapy ,Pharmacology ,business - Abstract
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 reactive HMG-CoA into mevalonate. Recent discoveries revealed HMG-CoA is a highly reactive metabolite that can non-enzymatically modify proteins and impact their activity. Therefore, we predicted that inhibition of HMGCR by statins would increase HMG-CoA levels and subsequent protein modifications. We found a strong increase in HMG-CoA levels, but only a single protein being modified. Fatty acid synthase (FAS) was modified on active site residues and, importantly, the modification is located on non-lysine residues. The dynamic modifications occur only on a subpool 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.
- Published
- 2021
19. Mitochondrial lysine acylation and cardiometabolic stress: truth or consequence?
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Deborah M Muoio, Ashley S Williams, and Paul A Grimsrud
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Physiology ,Physiology (medical) - Published
- 2022
20. Sirtuin 5 Is Regulated by the SCFCyclin F Ubiquitin Ligase and Is Involved in Cell Cycle Control
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Christine A. Mills, Daniel J. Burke, Xianxi Wang, Dhaval P. Bhatt, Jeanette Gowen Cook, Matthew D. Hirschey, Debojyoti Lahiri, Jacob Peter Matson, Michael J. Emanuele, and Paul A. Grimsrud
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0303 health sciences ,biology ,Cell Biology ,Cell cycle ,Ubiquitin ligase ,Cell biology ,03 medical and health sciences ,0302 clinical medicine ,SCF complex ,Cyclin-dependent kinase ,Skp1 ,biology.protein ,CUL1 ,Cell division control protein 4 ,Molecular Biology ,030217 neurology & neurosurgery ,030304 developmental biology ,Cyclin - Abstract
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.
- Published
- 2021
21. Deglutarylation of glutaryl-CoA dehydrogenase by deacylating enzyme SIRT5 promotes lysine oxidation in mice
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Dhaval P. Bhatt, C. Allie Mills, Kristin A. Anderson, Bárbara J. Henriques, Tânia G. Lucas, Sara Francisco, Juan Liu, Olga R. Ilkayeva, Alexander E. Adams, Shreyas R. Kulkarni, Donald S. Backos, Michael B. Major, Paul A. Grimsrud, Cláudio M. Gomes, and Matthew D. Hirschey
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Mice ,Glutaryl-CoA Dehydrogenase ,Lysine ,Tryptophan ,Animals ,Sirtuins ,Cell Biology ,Oxidation-Reduction ,Protein Processing, Post-Translational ,Molecular Biology ,Biochemistry - Abstract
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.
- Published
- 2022
22. Deglutarylation of GCDH by SIRT5 controls lysine metabolism in mice
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Sara Martins Francisco, Paul A. Grimsrud, Tânia G. Lucas, Dhaval P. Bhatt, Matthew D. Hirschey, Olga Ilkayeva, Cláudio M. Gomes, Alexander E. Adams, C. Allie Mills, Kristin A. Anderson, Shreyas R. Kulkarni, Donald S. Backos, Juan Liu, and Bárbara J. Henriques
- Subjects
chemistry.chemical_classification ,SIRT5 ,biology ,Catabolism ,Metabolite ,Lysine ,Tryptophan ,Oxidative phosphorylation ,complex mixtures ,chemistry.chemical_compound ,Enzyme ,chemistry ,Biochemistry ,Sirtuin ,biology.protein - Abstract
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 non-enzymatically 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). We show increased GCDH glutarylation when glutaryl-CoA production is stimulated by lysine catabolism. Our data reveal glutarylation of GCDH impacts its function, ultimately decreasing lysine oxidation. We then 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 model whereby a feedback loop exists 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.
- Published
- 2020
23. Sirtuin 5 Is Regulated by the SCF
- Author
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Christine A, Mills, Xianxi, Wang, Dhaval P, Bhatt, Paul A, Grimsrud, Jacob Peter, Matson, Debojyoti, Lahiri, Daniel J, Burke, Jeanette Gowen, Cook, Matthew D, Hirschey, and Michael J, Emanuele
- Subjects
SKP Cullin F-Box Protein Ligases ,Saccharomyces cerevisiae Proteins ,F-Box Proteins ,Gene Expression Profiling ,Ubiquitin-Protein Ligases ,Cell Cycle ,Ubiquitination ,Cell Cycle Proteins ,Saccharomyces cerevisiae ,HEK293 Cells ,Gene Expression Regulation, Fungal ,Mutation ,Humans ,Sirtuins ,Genes, Lethal ,Protein Processing, Post-Translational ,HeLa Cells ,Signal Transduction ,Research Article - Abstract
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 (SCF(Cyclin 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 G(1) population and a subsequent increase in both S and G(2)/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.
- Published
- 2020
24. Ablation of Sirtuin5 in the postnatal mouse heart results in protein succinylation and normal survival in response to chronic pressure overload
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Juan Liu, Jason W. Locasale, Kathleen A. Hershberger, Paul A. Grimsrud, Dennis M. Abraham, and Matthew D. Hirschey
- Subjects
0301 basic medicine ,Pressure overload ,Cardiac function curve ,medicine.medical_specialty ,SIRT5 ,biology ,business.industry ,Cell Biology ,Cardiac Ablation ,Biochemistry ,03 medical and health sciences ,Protein succinylation ,Succinylation ,Protein acylation ,030104 developmental biology ,Endocrinology ,Internal medicine ,Sirtuin ,biology.protein ,Medicine ,business ,Molecular Biology - Abstract
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.
- Published
- 2018
25. Lipids Reprogram Metabolism to Become a Major Carbon Source for Histone Acetylation
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Douglas B. Fox, Matthew D. Hirschey, Paul A. Grimsrud, Olga Ilkayeva, Scott B. Crown, Christian A. Olsen, Eoin McDonnell, and Betül Kitir
- Subjects
0301 basic medicine ,Nutrient sensing ,histone ,General Biochemistry, Genetics and Molecular Biology ,Article ,Histones ,lipids ,03 medical and health sciences ,Mice ,proteomics ,Acetyl Coenzyme A ,Cell Line, Tumor ,Animals ,Humans ,Epigenetics ,Amino Acid Sequence ,Beta oxidation ,lcsh:QH301-705.5 ,acetylation ,Regulation of gene expression ,biology ,epigenetics ,Gene Expression Profiling ,Lipid metabolism ,Lipid Metabolism ,metabolomics ,Carbon ,Histone Deacetylase Inhibitors ,030104 developmental biology ,Histone ,Biochemistry ,Gene Expression Regulation ,lcsh:Biology (General) ,Acetylation ,Histone methyltransferase ,biology.protein ,gene expression ,lipids (amino acids, peptides, and proteins) ,fatty acid ,Caprylates ,Oxidation-Reduction ,metabolism - Abstract
SummaryCells integrate nutrient sensing and metabolism to coordinate proper cellular responses to a particular nutrient source. For example, glucose drives a gene expression program characterized by activating genes involved in its metabolism, in part by increasing glucose-derived histone acetylation. Here, we find that lipid-derived acetyl-CoA is a major source of carbon for histone acetylation. Using 13C-carbon tracing combined with acetyl-proteomics, we show that up to 90% of acetylation on certain histone lysines can be derived from fatty acid carbon, even in the presence of excess glucose. By repressing both glucose and glutamine metabolism, fatty acid oxidation reprograms cellular metabolism, leading to increased lipid-derived acetyl-CoA. Gene expression profiling of octanoate-treated hepatocytes shows a pattern of upregulated lipid metabolic genes, demonstrating a specific transcriptional response to lipid. These studies expand the landscape of nutrient sensing and uncover how lipids and metabolism are integrated by epigenetic events that control gene expression.
- Published
- 2016
26. Disruption of Acetyl-Lysine Turnover in Muscle Mitochondria Promotes Insulin Resistance and Redox Stress without Overt Respiratory Dysfunction
- Author
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Deborah M. Muoio, Scott B. Crown, Louise Lantier, Michael T. Davidson, Paul A. Grimsrud, James A. Draper, Tara M. Narowski, Ashley S. Williams, Dorothy H. Slentz, Kelsey H. Fisher-Wellman, Timothy R. Koves, Maria J. Torres, and David H. Wasserman
- Subjects
0301 basic medicine ,Male ,Proteome ,Physiology ,Lysine ,Context (language use) ,Mitochondrion ,Diet, High-Fat ,Article ,Mitochondrial Proteins ,03 medical and health sciences ,Mice ,0302 clinical medicine ,Insulin resistance ,Acetyl Coenzyme A ,Sirtuin 3 ,medicine ,Animals ,Homeostasis ,Insulin ,Respiratory function ,Molecular Biology ,Beta oxidation ,Creatine Kinase ,Membrane Potential, Mitochondrial ,Mice, Knockout ,Carnitine O-Acetyltransferase ,biology ,Chemistry ,Acetylation ,Cell Biology ,Hydrogen Peroxide ,medicine.disease ,Cell biology ,Mitochondria, Muscle ,Oxidative Stress ,030104 developmental biology ,Sirtuin ,biology.protein ,Thermodynamics ,Insulin Resistance ,Energy Metabolism ,Oxidation-Reduction ,030217 neurology & neurosurgery - Abstract
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.
- Published
- 2019
27. Chronic caloric restriction maintains a youthful phosphoproteome in aged skeletal muscle
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Paul A. Grimsrud, James A. Draper, Lauren H. Katz, James P. White, David E Lee, and Akshay Bareja
- Subjects
Proteomics ,0301 basic medicine ,Aging ,medicine.medical_specialty ,Biology ,Article ,Time ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Internal medicine ,medicine ,Animals ,Regeneration ,Rejuvenation ,Muscle, Skeletal ,Protein kinase A ,Caloric Restriction ,Principal Component Analysis ,Caloric theory ,Skeletal muscle ,Phosphoproteins ,Cyclic AMP-Dependent Protein Kinases ,Mice, Inbred C57BL ,030104 developmental biology ,Endocrinology ,medicine.anatomical_structure ,030217 neurology & neurosurgery ,Function (biology) ,Signal Transduction ,Developmental Biology - Abstract
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.
- Published
- 2021
28. Respiratory Phenomics across Multiple Models of Protein Hyperacylation in Cardiac Mitochondria Reveals a Marginal Impact on Bioenergetics
- Author
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Daniel P. Kelly, Michael T. Davidson, Gregory R. Wagner, Paul A. Grimsrud, Dorothy H. Slentz, David P. Olson, J. Will Thompson, Mathew D. Hirschey, Olga Ilkayeva, Deborah M. Muoio, James A. Draper, Tara M. Narowski, Rami Najjar, Timothy R. Koves, Kelsey H. Fisher-Wellman, Robert Stevens, and Ashley S. Williams
- Subjects
0301 basic medicine ,Male ,SIRT5 ,SIRT3 ,Bioenergetics ,Carboxy-Lyases ,Cell Respiration ,General Biochemistry, Genetics and Molecular Biology ,Mitochondria, Heart ,Article ,03 medical and health sciences ,Acyl-CoA ,chemistry.chemical_compound ,Mice ,0302 clinical medicine ,Sirtuin 3 ,Animals ,Sirtuins ,lcsh:QH301-705.5 ,ATP synthase ,biology ,Catabolism ,Acetylation ,Malonyl-CoA decarboxylase ,Mice, Inbred C57BL ,030104 developmental biology ,lcsh:Biology (General) ,chemistry ,Biochemistry ,Proteome ,biology.protein ,Energy Metabolism ,Protein Processing, Post-Translational ,030217 neurology & neurosurgery - Abstract
SUMMARY 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., Graphical Abstract, In Brief Fisher-Wellman et al. use a recently developed mitochondrial diagnostics platform for deep phenotyping of heart mitochondria derived from three disparate genetic models of protein hyperacylation. Their findings oppose the notion that hyperacylation of the mitochondrial proteome leads to broad-ranging vulnerabilities in respiratory function and bioenergetics.
- Published
- 2018
29. Integration of BCAA and Lipid Metabolism by the BCKDH Kinase and Phosphatase
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Guo-Fang Zhang, Christopher B. Newgard, Paul A. Grimsrud, David T. Chuang, Michelle Arlotto, Olga Ilkayeva, Richard M. Wynn, Phillip J. White, Jonathan M. Haldeman, Robert W. McGarrah, and Wen-Hsuan Yang
- Subjects
chemistry.chemical_classification ,medicine.medical_specialty ,Chemistry ,Endocrinology, Diabetes and Metabolism ,Phosphatase ,Lipid metabolism ,Lyase ,law.invention ,Amino acid ,Serine ,Endocrinology ,law ,Internal medicine ,Lipogenesis ,Internal Medicine ,medicine ,Recombinant DNA ,Phosphorylation - Abstract
Strong associations exist between branched chain amino acids (BCAA) and dysregulated glucose and lipid metabolism, but underlying mechanisms are not well understood. One factor contributing to elevated BCAA in obesity is inhibitory phosphorylation of the branched chain keto-acid dehydrogenase complex (BCKDH) in liver. We studied the impact of modulating the activity of the BCKDH kinase (BDK) and phosphatase (PPM1K) on amino acid, glucose and lipid metabolism in Zucker fatty rats (ZFR). Daily administration of a selective inhibitor of BDK, BT2 (20mg.kg-1, IP), for one week or expression of a recombinant adenovirus overexpressing PPM1K significantly lowered BCAA and branched chain keto acid (BCKA) levels in ZFR. Remarkably, these effects were accompanied by strong lowering of liver triacylglycerides and improved glucose tolerance in the absence of weight loss. Proteomics analysis of liver samples from these studies revealed that both inhibition of BDK with BT2 and adenoviral mediated overexpression of PPM1K results in reduced phosphorylation of the lipogenic enzyme ATP-citrate lyase (ACL) on its regulatory serine 454. Phosphorylation of ACL on this site is activating. Sequence analysis revealed the presence of a canonical BDK phosphorylation motif surrounding ser454 in ACL and incubation of ACL with purified BDK resulted in ACL phosphorylation in vitro. Furthermore, adenovirus-mediated overexpression of BDK increased ACL phosphorylation and activated de novo lipogenesis in liver of lean Wistar rats. Together these studies reveal a novel regulatory function for BDK and PPM1K in integration of BCAA and lipid metabolism. Moreover, we show that modulation of this node provides broad protection against metabolic abnormalities associated with obesity. Disclosure P.J. White: None. R.W. McGarrah: None. P.A. Grimsrud: None. W. Yang: None. G. Zhang: None. J.M. Haldeman: None. M. Arlotto: None. O. Ilkayeva: None. R.M. Wynn: None. D.T. Chuang: None. C.B. Newgard: Advisory Panel; Self; Eli Lilly and Company. Research Support; Self; Eli Lilly and Company.
- Published
- 2018
30. Ablation of
- Author
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Kathleen A, Hershberger, Dennis M, Abraham, Juan, Liu, Jason W, Locasale, Paul A, Grimsrud, and Matthew D, Hirschey
- Subjects
Male ,Mice, Knockout ,Proteomics ,Succinic Acid ,Cardiomegaly ,Heart ,Survival Analysis ,Mice, Inbred C57BL ,Mice ,Metabolism ,Gene Expression Regulation ,Pressure ,Animals ,Sirtuins ,Female ,Protein Processing, Post-Translational ,Metabolic Networks and Pathways - Abstract
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.
- Published
- 2018
31. Nicotinamide mononucleotide requires SIRT3 to improve cardiac function and bioenergetics in a Friedreich’s ataxia cardiomyopathy model
- Author
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Kathleen A. Hershberger, Lan Mao, R. Mark Payne, Paul A. Grimsrud, Angelical Martin, Xiaojing Liu, Huaxia Cui, Dhaval P. Bhatt, Jason W. Locasale, Juan Liu, Matthew D. Hirschey, Michael J. Muehlbauer, and Dennis M. Abraham
- Subjects
0301 basic medicine ,Cardiac function curve ,Ataxia ,SIRT3 ,biology ,Chemistry ,Cardiomyopathy ,General Medicine ,Pharmacology ,medicine.disease ,03 medical and health sciences ,030104 developmental biology ,Biochemistry ,Sirtuin ,biology.protein ,medicine ,Protein deacetylase ,NAD+ kinase ,medicine.symptom ,Nicotinamide mononucleotide ,Research Article - Abstract
Increasing NAD+ levels by supplementing with the precursor nicotinamide mononucleotide (NMN) improves cardiac function in multiple mouse models of disease. While NMN influences several aspects of mitochondrial metabolism, the molecular mechanisms by which increased NAD+ enhances cardiac function are poorly understood. A putative mechanism of NAD+ therapeutic action exists via activation of the mitochondrial NAD+-dependent protein deacetylase sirtuin 3 (SIRT3). We assessed the therapeutic efficacy of NMN and the role of SIRT3 in the Friedreich's ataxia cardiomyopathy mouse model (FXN-KO). At baseline, the FXN-KO heart has mitochondrial protein hyperacetylation, reduced Sirt3 mRNA expression, and evidence of increased NAD+ salvage. Remarkably, NMN administered to FXN-KO mice restores cardiac function to near-normal levels. To determine whether SIRT3 is required for NMN therapeutic efficacy, we generated SIRT3-KO and SIRT3-KO/FXN-KO (double KO [dKO]) models. The improvement in cardiac function upon NMN treatment in the FXN-KO is lost in the dKO model, demonstrating that the effects of NMN are dependent upon cardiac SIRT3. Coupled with cardio-protection, SIRT3 mediates NMN-induced improvements in both cardiac and extracardiac metabolic function and energy metabolism. Taken together, these results serve as important preclinical data for NMN supplementation or SIRT3 activator therapy in Friedreich's ataxia patients.
- Published
- 2017
32. A Class of Reactive Acyl-CoA Species Reveals the Non-Enzymatic Origins of Protein Acylation
- Author
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Gregory R. Wagner, Donald S. Backos, Robert Stevens, Hao Yang, Dhaval P. Bhatt, Matthew D. Hirschey, Grant A. Mitchell, Paul A. Grimsrud, J. Will Thompson, Thomas M. O’Connell, Olga Ilkayeva, and Laura G. Dubois
- Subjects
0301 basic medicine ,Physiology ,Acylation ,Lysine ,Quantitative proteomics ,Reactive intermediate ,Chemical biology ,Cell Biology ,Biology ,Proteomics ,Malate dehydrogenase ,Article ,Citric acid cycle ,03 medical and health sciences ,Protein acylation ,030104 developmental biology ,0302 clinical medicine ,Biochemistry ,lipids (amino acids, peptides, and proteins) ,Molecular Biology ,Protein Processing, Post-Translational ,030217 neurology & neurosurgery - Abstract
The mechanisms underlying the formation of acyl protein modifications remain poorly understood. By investigating the reactivity of endogenous acyl-CoA metabolites, we found a class of acyl-CoAs that undergoes intramolecular catalysis to form reactive intermediates which non-enzymatically modify proteins. Based on this mechanism, we predicted, validated, and characterized a protein modification: 3-hydroxy-3-methylglutaryl(HMG)-lysine. In a model of altered HMG-CoA metabolism, we found evidence of two additional protein modifications: 3-methylglutaconyl(MGc)-lysine and 3-methylglutaryl(MG)-lysine. Using quantitative proteomics, we compared the ‘acylomes’ of two reactive acyl-CoA species, namely HMG-CoA and glutaryl-CoA, which are generated in different pathways. We found proteins that are uniquely modified by each reactive metabolite, as well as common proteins and pathways. We identified the tricarboxylic acid cycle as a pathway commonly regulated by acylation, and validated malate dehydrogenase as a key target. These data uncover a fundamental relationship between reactive acyl-CoA species and proteins, and define a new regulatory paradigm in metabolism.
- Published
- 2017
33. A Quantitative Map of the Liver Mitochondrial Phosphoproteome Reveals Posttranslational Control of Ketogenesis
- Author
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Michael S. Westphall, Derek J. Bailey, Donald S. Stapleton, Brian S. Yandell, Natalie M. Niemi, Alan D. Attie, Shane L. Hubler, Paul A. Grimsrud, Joshua J. Coon, Adam Jochem, David J. Pagliarini, Joshua J. Carson, Alexander S. Hebert, and Mark P. Keller
- Subjects
Hydroxymethylglutaryl-CoA Synthase ,Phosphopeptides ,Proteomics ,Databases, Factual ,Proteome ,Physiology ,Mice, Obese ,Mitochondria, Liver ,Ketone Bodies ,Mitochondrion ,Biology ,Article ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Tandem Mass Spectrometry ,Organelle ,Ketogenesis ,Animals ,Humans ,Phosphorylation ,Molecular Biology ,030304 developmental biology ,0303 health sciences ,Mechanism (biology) ,HEK 293 cells ,Cell Biology ,HEK293 Cells ,Biochemistry ,030217 neurology & neurosurgery - Abstract
SummaryMitochondria are dynamic organelles that play a central role in a diverse array of metabolic processes. Elucidating mitochondrial adaptations to changing metabolic demands and the pathogenic alterations that underlie metabolic disorders represent principal challenges in cell biology. Here, we performed multiplexed quantitative mass spectrometry-based proteomics to chart the remodeling of the mouse liver mitochondrial proteome and phosphoproteome during both acute and chronic physiological transformations in more than 50 mice. Our analyses reveal that reversible phosphorylation is widespread in mitochondria, and is a key mechanism for regulating ketogenesis during the onset of obesity and type 2 diabetes. Specifically, we have demonstrated that phosphorylation of a conserved serine on Hmgcs2 (S456) significantly enhances its catalytic activity in response to increased ketogenic demand. Collectively, our work describes the plasticity of this organelle at high resolution and provides a framework for investigating the roles of proteome restructuring and reversible phosphorylation in mitochondrial adaptation.
- Published
- 2012
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34. SIRT4 Is a Lysine Deacylase that Controls Leucine Metabolism and Insulin Secretion
- Author
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Jonathan E. Campbell, Michelle F. Green, Brett S. Peterson, Christian A. Olsen, Jonathan D. Douros, Donald S. Backos, John A. Capra, Matthew D. Hirschey, Andreas Stahl Madsen, Gregory R. Wagner, R. Michael Sivley, Kelsey H. Fisher-Wellman, Frank K. Huynh, Olga Ilkayeva, Robert Stevens, Kristin A. Anderson, Paul A. Grimsrud, J. Will Thompson, J. Darren Stuart, and Deborah M. Muoio
- Subjects
0301 basic medicine ,Models, Molecular ,Physiology ,medicine.medical_treatment ,Biology ,Article ,Amidohydrolases ,Mitochondrial Proteins ,03 medical and health sciences ,Insulin resistance ,Leucine ,Insulin Secretion ,medicine ,Glucose homeostasis ,Animals ,Homeostasis ,Humans ,Insulin ,Sirtuins ,Amino Acid Sequence ,Molecular Biology ,Phylogeny ,Mice, Knockout ,Lysine ,Cell Biology ,Metabolism ,medicine.disease ,Metabolic Flux Analysis ,Mice, Inbred C57BL ,030104 developmental biology ,Glucose ,HEK293 Cells ,Biochemistry ,Carbon-Carbon Ligases ,Sirtuin ,biology.protein ,NAD+ kinase ,Insulin Resistance ,Flux (metabolism) - Abstract
Sirtuins are NAD+-dependent protein deacylases that regulate several aspects of metabolism and aging. In contrast to the other mammalian sirtuins, the primary enzymatic activity of mitochondrial sirtuin 4 (SIRT4) and its overall role in metabolic control have remained enigmatic. Using a combination of phylogenetics, structural biology, and enzymology, we show that SIRT4 removes three acyl moieties from lysine residues: methylglutaryl (MG)-, hydroxymethylglutaryl (HMG)-, and 3-methylglutaconyl (MGc)-lysine. The metabolites leading to these post-translational modifications are intermediates in leucine oxidation, and we show a primary role for SIRT4 in controlling this pathway in mice. Furthermore, we find that dysregulated leucine metabolism in SIRT4KO mice leads to elevated basal and stimulated insulin secretion, which progressively develops into glucose intolerance and insulin resistance. These findings identify a robust enzymatic activity for SIRT4, uncover a mechanism controlling branched-chain amino acid flux, and position SIRT4 as a crucial player maintaining insulin secretion and glucose homeostasis during aging.
- Published
- 2016
35. The BCKDH Kinase and Phosphatase Integrate BCAA and Lipid Metabolism via Regulation of ATP-Citrate Lyase
- Author
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Inna Astapova, Robert W. McGarrah, Lyra B. Olson, Wen-Hsuan Yang, Tabitha George, Phillip J. White, David T. Chuang, Paul A. Grimsrud, Jie An, Olga Ilkayeva, Grenier-Larouche Thomas, R. Max Wynn, Shih Chia Tso, Sarah A. Hannou, Michelle Arlotto, Amanda L. Lapworth, Mark A. Herman, Michelle Lai, Jonathan M. Haldeman, Guo-Fang Zhang, and Christopher B. Newgard
- Subjects
Adult ,Male ,0301 basic medicine ,medicine.medical_specialty ,Adolescent ,ATP citrate lyase ,Physiology ,Phosphatase ,030209 endocrinology & metabolism ,Article ,3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide) ,03 medical and health sciences ,0302 clinical medicine ,Internal medicine ,Genetic model ,medicine ,Animals ,Humans ,Obesity ,Rats, Wistar ,Molecular Biology ,Aged ,Aged, 80 and over ,Basic Helix-Loop-Helix Leucine Zipper Transcription Factors ,Chemistry ,Kinase ,Catabolism ,Lipogenesis ,Lipid metabolism ,Cell Biology ,Middle Aged ,medicine.disease ,Rats ,Rats, Zucker ,Protein Phosphatase 2C ,Disease Models, Animal ,HEK293 Cells ,030104 developmental biology ,Endocrinology ,ATP Citrate (pro-S)-Lyase ,Female ,Steatosis ,Amino Acids, Branched-Chain - Abstract
Summary Branched-chain amino acids (BCAA) are strongly associated with dysregulated glucose and lipid metabolism, but the underlying mechanisms are poorly understood. We report that inhibition of the kinase (BDK) or overexpression of the phosphatase (PPM1K) that regulates branched-chain ketoacid dehydrogenase (BCKDH), the committed step of BCAA catabolism, lowers circulating BCAA, reduces hepatic steatosis, and improves glucose tolerance in the absence of weight loss in Zucker fatty rats. Phosphoproteomics analysis identified ATP-citrate lyase (ACL) as an alternate substrate of BDK and PPM1K. Hepatic overexpression of BDK increased ACL phosphorylation and activated de novo lipogenesis. BDK and PPM1K transcript levels were increased and repressed, respectively, in response to fructose feeding or expression of the ChREBP-β transcription factor. These studies identify BDK and PPM1K as a ChREBP-regulated node that integrates BCAA and lipid metabolism. Moreover, manipulation of the BDK:PPM1K ratio relieves key metabolic disease phenotypes in a genetic model of severe obesity.
- Published
- 2018
36. Downregulation of Adipose Glutathione S-Transferase A4 Leads to Increased Protein Carbonylation, Oxidative Stress, and Mitochondrial Dysfunction
- Author
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Olga Ilkayeva, Xin Xu, Wendy Wright, Deborah E. Muoio, Brian M. Wiczer, Paul A. Grimsrud, David A. Bernlohr, David W. Graham, Edgar A. Arriaga, Katherine Cianflone, Jonathan R. Brestoff, Jessica Curtis, and Rocio Foncea
- Subjects
Mitochondrial ROS ,medicine.medical_specialty ,Endocrinology, Diabetes and Metabolism ,Protein Carbonylation ,Adipose tissue ,Down-Regulation ,Mitochondrion ,Biology ,03 medical and health sciences ,chemistry.chemical_compound ,Mice ,0302 clinical medicine ,Insulin resistance ,Downregulation and upregulation ,Adipocyte ,Internal medicine ,3T3-L1 Cells ,Internal Medicine ,medicine ,Animals ,Humans ,Obesity ,030304 developmental biology ,Glutathione Transferase ,Oligonucleotide Array Sequence Analysis ,chemistry.chemical_classification ,Mice, Knockout ,0303 health sciences ,Reactive oxygen species ,medicine.disease ,Mitochondria ,Mice, Inbred C57BL ,Oxidative Stress ,Endocrinology ,Metabolism ,chemistry ,Original Article ,Insulin Resistance ,030217 neurology & neurosurgery - Abstract
OBJECTIVE Peripheral insulin resistance is linked to an increase in reactive oxygen species (ROS), leading in part to the production of reactive lipid aldehydes that modify the side chains of protein amino acids in a reaction termed protein carbonylation. The primary enzymatic method for lipid aldehyde detoxification is via glutathione S-transferase A4 (GSTA4) dependent glutathionylation. The objective of this study was to evaluate the expression of GSTA4 and the role(s) of protein carbonylation in adipocyte function. RESEARCH DESIGN AND METHODS GSTA4-silenced 3T3-L1 adipocytes and GSTA4-null mice were evaluated for metabolic processes, mitochondrial function, and reactive oxygen species production. GSTA4 expression in human obesity was evaluated using microarray analysis. RESULTS GSTA4 expression is selectively downregulated in adipose tissue of obese insulin-resistant C57BL/6J mice and in human obesity-linked insulin resistance. Tumor necrosis factor-α treatment of 3T3-L1 adipocytes decreased GSTA4 expression, and silencing GSTA4 mRNA in cultured adipocytes resulted in increased protein carbonylation, increased mitochondrial ROS, dysfunctional state 3 respiration, and altered glucose transport and lipolysis. Mitochondrial function in adipocytes of lean or obese GSTA4-null mice was significantly compromised compared with wild-type controls and was accompanied by an increase in superoxide anion. CONCLUSIONS These results indicate that downregulation of GSTA4 in adipose tissue leads to increased protein carbonylation, ROS production, and mitochondrial dysfunction and may contribute to the development of insulin resistance and type 2 diabetes.
- Published
- 2010
37. Carbonylation of Adipose Proteins in Obesity and Insulin Resistance
- Author
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Matthew J. Picklo, Paul A. Grimsrud, David A. Bernlohr, and Timothy J. Griffin
- Subjects
medicine.medical_specialty ,biology ,Chemistry ,Protein Carbonylation ,Adipose tissue ,White adipose tissue ,Biotin hydrazide ,Biochemistry ,Analytical Chemistry ,Insulin receptor ,chemistry.chemical_compound ,Endocrinology ,Lipotoxicity ,Internal medicine ,Adipocyte ,biology.protein ,medicine ,adipocyte protein 2 ,Molecular Biology - Abstract
Obesity is a state of mild inflammation correlated with increased oxidative stress. In general, pro-oxidative conditions lead to production of reactive aldehydes such as trans-4-hydroxy-2-nonenal (4-HNE) and trans-4-oxo-2-nonenal implicated in the development of a variety of metabolic diseases. To investigate protein modification by 4-HNE as a consequence of obesity and its potential relationship to the development of insulin resistance, proteomics technologies were utilized to identify aldehyde-modified proteins in adipose tissue. Adipose proteins from lean insulin-sensitive and obese insulin-resistant C57Bl/6J mice were incubated with biotin hydrazide and detected using horseradish peroxidase-conjugated streptavidin. High carbohydrate, high fat feeding of mice resulted in a approximately 2-3-fold increase in total adipose protein carbonylation. Consistent with an increase in oxidative stress in obesity, the abundance of glutathione S-transferase A4 (GSTA4), a key enzyme responsible for metabolizing 4-HNE, was decreased approximately 3-4-fold in adipose tissue of obese mice. To identify specific carbonylated proteins, biotin hydrazide-modified adipose proteins from obese mice were captured using avidin-Sepharose affinity chromatography, proteolytically digested, and subjected to LC-ESI MS/MS. Interestingly enzymes involved in cellular stress response, lipotoxicity, and insulin signaling such as glutathione S-transferase M1, peroxiredoxin 1, glutathione peroxidase 1, eukaryotic elongation factor 1alpha-1 (eEF1alpha1), and filamin A were identified. The adipocyte fatty acid-binding protein, a protein implicated in the regulation of insulin resistance, was found to be carbonylated in vivo with 4-HNE. In vitro modification of adipocyte fatty acid-binding protein with 4-HNE was mapped to Cys-117, occurred equivalently using either the R or S enantiomer of 4-HNE, and reduced the affinity of the protein for fatty acids approximately 10-fold. These results indicate that obesity is accompanied by an increase in the carbonylation of a number of adipose-regulatory proteins that may serve as a mechanistic link between increased oxidative stress and the development of insulin resistance.
- Published
- 2007
38. Further Insights into Quinone Cofactor Biogenesis: Probing the Role of mauG in Methylamine Dehydrogenase Tryptophan Tryptophylquinone Formation
- Author
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Arwen R. Pearson, Paul A. Grimsrud, Sudha Marimanikkupam, M. Elizabeth Graichen, Sean A. Agger, Carrie M. Wilmot, Limei Hsu Jones, Yongting Wang, Teresa De la Mora-Rey, and Victor L. Davidson
- Subjects
Spectrometry, Mass, Electrospray Ionization ,Molecular Sequence Data ,Coenzymes ,Biochemistry ,Cofactor ,chemistry.chemical_compound ,Tryptophan tryptophylquinone ,Chymotrypsin ,Histidine ,Trypsin ,Methylamine dehydrogenase ,Amino Acid Sequence ,Indolequinones ,Heme ,Paracoccus denitrificans ,Indole test ,Oxidoreductases Acting on CH-NH Group Donors ,biology ,Hydrolysis ,Tryptophan ,Valine ,Cytochrome-c Peroxidase ,biology.organism_classification ,Heterotetramer ,Kinetics ,Protein Subunits ,chemistry ,Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ,Mutagenesis, Site-Directed ,biology.protein ,Electrophoresis, Polyacrylamide Gel ,Protein Processing, Post-Translational - Abstract
Paracoccus denitrificans methylamine dehydrogenase (MADH) is an enzyme containing a quinone cofactor tryptophan tryptophylquinone (TTQ) derived from two tryptophan residues (betaTrp(57) and betaTrp(108)) within the polypeptide chain. During cofactor formation, the two tryptophan residues become covalently linked, and two carbonyl oxygens are added to the indole ring of betaTrp(57). Expression of active MADH from P. denitrificans requires four other genes in addition to those that encode the polypeptides of the MADH alpha(2)beta(2) heterotetramer. One of these, mauG, has been shown to be involved in TTQ biogenesis. It contains two covalently attached c-type hemes but exhibits unusual properties compared to c-type cytochromes and diheme cytochrome c peroxidases, to which it has some sequence similarity. To test the role that MauG may play in TTQ maturation, the predicted proximal histidine to each heme (His(35) and His(205)) has each been mutated to valine, and wild-type MADH was expressed in the background of these two mauG mutants. The resultant MADH has been characterized by mass spectrometry and electrophoretic and kinetic analyses. The majority species is a TTQ biogenesis intermediate containing a monohydroxylated betaTrp(57), suggesting that this is the natural substrate for MauG. Previous work has shown that MADH mutated at the betaTrp(108) position (the non-oxygenated TTQ partner) is predominantly also this intermediate, and work on these mutants is extended and compared to the MADH expressed in the background of the histidine to valine mauG mutations. In this study, it is unequivocally demonstrated that MauG is required to initiate the formation of the TTQ cross-link, the conversion of a single hydroxyl located on betaTrp(57) to a carbonyl, and the incorporation of the second oxygen into the TTQ ring to complete TTQ biogenesis. The properties of MauG, which are atypical of c-type cytochromes, are discussed in the context of these final stages of TTQ biogenesis.
- Published
- 2004
39. Mitochondrial DNA variant in COX1 subunit significantly alters energy metabolism of geographically divergent wild isolates in Caenorhabditis elegans
- Author
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David J. Pagliarini, Xiaowu Gai, Alexander S. Hebert, Amy B. Rosenfeld, Joshua J. Coon, Icksoo Lee, Satish Srinivasan, Eiko Nakamaru-Ogiso, Erzsebet Polyak, Marni J. Falk, Maik Hüttemann, Julian Ostrovsky, Theodore G. Schurr, Rui Xiao, Paul A. Grimsrud, Mary A. Selak, Stephen D. Dingley, Mai Tsukikawa, and Young Joon Kwon
- Subjects
Male ,Models, Molecular ,Mitochondrial DNA ,Cell Respiration ,Mitochondrion ,Biology ,Genome ,DNA, Mitochondrial ,Article ,Animals, Genetically Modified ,Electron Transport Complex IV ,Structural Biology ,Genetic variation ,Animals ,Caenorhabditis elegans ,Caenorhabditis elegans Proteins ,Molecular Biology ,mtDNA control region ,Genetics ,Geography ,Genetic Variation ,biology.organism_classification ,Mitochondria ,Amino Acid Substitution ,Mitochondrial matrix ,Adaptation ,Energy Metabolism - Abstract
Mitochondrial DNA (mtDNA) sequence variation can influence the penetrance of complex diseases and climatic adaptation. While studies in geographically defined human populations suggest that mtDNA mutations become fixed when they have conferred metabolic capabilities optimally suited for a specific environment, it has been challenging to definitively assign adaptive functions to specific mtDNA sequence variants in mammals. We investigated whether mtDNA genome variation functionally influences Caenorhabditis elegans wild isolates of distinct mtDNA lineages and geographic origins. We found that, relative to N2 (England) wild-type nematodes, CB4856 wild isolates from a warmer native climate (Hawaii) had a unique p.A12S amino acid substitution in the mtDNA-encoded COX1 core catalytic subunit of mitochondrial complex IV (CIV). Relative to N2, CB4856 worms grown at 20 °C had significantly increased CIV enzyme activity, mitochondrial matrix oxidant burden, and sensitivity to oxidative stress but had significantly reduced lifespan and mitochondrial membrane potential. Interestingly, mitochondrial membrane potential was significantly increased in CB4856 grown at its native temperature of 25 °C. A transmitochondrial cybrid worm strain, chpIR (M, CB4856 > N2), was bred as homoplasmic for the CB4856 mtDNA genome in the N2 nuclear background. The cybrid strain also displayed significantly increased CIV activity, demonstrating that this difference results from the mtDNA-encoded p.A12S variant. However, chpIR (M, CB4856 > N2) worms had significantly reduced median and maximal lifespan relative to CB4856, which may relate to their nuclear– mtDNA genome mismatch. Overall, these data suggest that C. elegans wild isolates of varying geographic origins may adapt to environmental challenges through mtDNA variation to modulate critical aspects of mitochondrial energy metabolism.
- Published
- 2013
40. Quantification of Mitochondrial Acetylation Dynamics Highlights Prominent Sites of Metabolic Regulation*
- Author
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Joshua J. Coon, Alan D. Attie, Brendan J. Floyd, Drew R. Gunderson, John M. Denu, David J. Pagliarini, Kristin E. Dittenhafer-Reed, Alexander S. Hebert, Amelia Still, Brendan K. Dolan, Mark P. Keller, Paul A. Grimsrud, Joshua J. Carson, Michael S. Westphall, Craig A. Bingman, and Donald S. Stapleton
- Subjects
SIRT3 ,Proteome ,Mice, Obese ,Mitochondria, Liver ,Biology ,Proteomics ,Biochemistry ,complex mixtures ,Protein acylation ,Mice ,Acetyl Coenzyme A ,Sirtuin 3 ,Animals ,Acetyl-CoA C-Acetyltransferase ,skin and connective tissue diseases ,Molecular Biology ,ACAT1 ,Acetylation ,Cell Biology ,Metabolic pathway ,Metabolism ,Acetyltransferase ,bacteria ,sense organs - Abstract
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.
- Published
- 2013
41. A Proteogenomic Survey of the Medicago truncatula Genome*
- Author
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Paul A. Grimsrud, Michael R. Sussman, Maegen Howes-Podoll, Derek J. Bailey, Jean-Michel Ané, Joshua J. Coon, Jeremy D. Volkening, Michael S. Westphall, Muthusubramanian Venkateshwaran, and Christopher M. Rose
- Subjects
Proteomics ,Proteome ,In silico ,Molecular Sequence Data ,Computational biology ,Biology ,Biochemistry ,Genome ,Deep sequencing ,Mass Spectrometry ,Analytical Chemistry ,Medicago truncatula ,Amino Acid Sequence ,Databases, Protein ,Molecular Biology ,Gene ,Plant Proteins ,Genetics ,Information Dissemination ,Research ,Sequence Analysis, DNA ,Proteogenomics ,biology.organism_classification ,Peptides ,Algorithms ,Genome, Plant - Abstract
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.
- Published
- 2012
42. Rapid phosphoproteomic and transcriptomic changes in the rhizobia-legume symbiosis
- Author
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Paul A. Grimsrud, Michael R. Sussman, Désirée den Os, Jeremy D. Volkening, Li Huey Yeun, Joshua J. Coon, Derek J. Bailey, Muthusubramanian Venkateshwaran, Christopher M. Rose, Junko Maeda, Michael S. Westphall, Maegen Howes-Podoll, Kwanghyun Park, and Jean-Michel Ané
- Subjects
Lipopolysaccharides ,Biochemistry ,Analytical Chemistry ,Rhizobia ,Transcriptome ,Symbiosis ,Transcription (biology) ,Tandem Mass Spectrometry ,Mycorrhizae ,Botany ,Medicago truncatula ,Phosphorylation ,Molecular Biology ,Plant Proteins ,Sinorhizobium meliloti ,biology ,Research ,fungi ,food and beverages ,biology.organism_classification ,Phosphoproteins ,Cell biology ,Rhizobium ,Signal Transduction - Abstract
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.
- Published
- 2012
43. X-ray crystallographic analysis of adipocyte fatty acid binding protein (aP2) modified with 4-hydroxy-2-nonenal
- Author
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Douglas H. Ohlendorf, Andrew C. Kruse, David A. Bernlohr, Leonard J. Banaszak, Paul A. Grimsrud, and Kristina Hellberg
- Subjects
Models, Molecular ,Stereochemistry ,Protein Conformation ,Molecular Sequence Data ,Peptide ,Cysteine Proteinase Inhibitors ,Crystallography, X-Ray ,Fatty Acid-Binding Proteins ,Ligands ,Biochemistry ,Fatty acid-binding protein ,Article ,chemistry.chemical_compound ,Mice ,Side chain ,Adipocytes ,Non-covalent interactions ,Animals ,Carboxylate ,Molecular Biology ,chemistry.chemical_classification ,Aldehydes ,Fatty acid ,Oxidative Stress ,chemistry ,Covalent bond ,lipids (amino acids, peptides, and proteins) ,Target protein ,Dimerization - Abstract
Fatty acid binding proteins (FABP) have been characterized as facilitating the intracellular solubilization and transport of long-chain fatty acyl carboxylates via noncovalent interactions. More recent work has shown that the adipocyte FABP is also covalently modified in vivo on Cys117 with 4-hydroxy-2-nonenal (4-HNE), a bioactive aldehyde linked to oxidative stress and inflammation. To evaluate 4-HNE binding and modification, the crystal structures of adipocyte FABP covalently and noncovalently bound to 4-HNE have been solved to 1.9 A and 2.3 A resolution, respectively. While the 4-HNE in the noncovalently modified protein is coordinated similarly to a carboxylate of a fatty acid, the covalent form show a novel coordination through a water molecule at the polar end of the lipid. Other defining features between the two structures with 4-HNE and previously solved structures of the protein include a peptide flip between residues Ala36 and Lys37 and the rotation of the side chain of Phe57 into its closed conformation. Representing the first structure of an endogenous target protein covalently modified by 4-HNE, these results define a new class of in vivo ligands for FABPs and extend their physiological substrates to include bioactive aldehydes.
- Published
- 2010
44. Oxidative Stress and Covalent Modification of Protein with Bioactive Aldehydes*S⃞
- Author
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Paul A. Grimsrud, Hongwei Xie, David A. Bernlohr, and Timothy J. Griffin
- Subjects
Proteomics ,Antioxidant ,Protein Carbonylation ,medicine.medical_treatment ,Lysine ,medicine.disease_cause ,Biochemistry ,Mass Spectrometry ,chemistry.chemical_compound ,medicine ,Animals ,Humans ,Electrophoresis, Gel, Two-Dimensional ,Molecular Biology ,Histidine ,chemistry.chemical_classification ,Reactive oxygen species ,Aldehydes ,Proteins ,Minireviews ,Cell Biology ,Oxidative Stress ,chemistry ,Hydroxyl radical ,Reactive Oxygen Species ,Oxidative stress ,Cysteine - Abstract
The term “oxidative stress” links the production of reactive oxygen species to a variety of metabolic outcomes, including insulin resistance, immune dysfunction, and inflammation. Antioxidant defense systems down-regulated due to disease and/or aging result in oxidatively modified DNA, carbohydrates, proteins, and lipids. Increased production of hydroxyl radical leads to the formation of lipid hydroperoxides that produce a family of α,β-unsaturated aldehydes. Such reactive aldehydes are subject to Michael addition reactions with the side chains of lysine, histidine, and cysteine residues, referred to as “protein carbonylation.” Although not widely appreciated, reactive lipids can accumulate to high levels in cells, resulting in extensive protein modification leading to either loss or gain of function. The use of mass spectrometric methods to identify the site and extent of protein carbonylation on a proteome-wide scale has expanded our view of how oxidative stress can regulate cellular processes.
- Published
- 2008
45. Carbonylation of adipose proteins in obesity and insulin resistance: identification of adipocyte fatty acid-binding protein as a cellular target of 4-hydroxynonenal
- Author
-
Paul A, Grimsrud, Matthew J, Picklo, Timothy J, Griffin, and David A, Bernlohr
- Subjects
Mice, Inbred C57BL ,Protein Carbonylation ,Proteomics ,Aldehydes ,Mice ,Adipose Tissue ,Tandem Mass Spectrometry ,Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ,Animals ,Obesity ,Insulin Resistance ,Fatty Acid-Binding Proteins ,Glutathione Transferase - Abstract
Obesity is a state of mild inflammation correlated with increased oxidative stress. In general, pro-oxidative conditions lead to production of reactive aldehydes such as trans-4-hydroxy-2-nonenal (4-HNE) and trans-4-oxo-2-nonenal implicated in the development of a variety of metabolic diseases. To investigate protein modification by 4-HNE as a consequence of obesity and its potential relationship to the development of insulin resistance, proteomics technologies were utilized to identify aldehyde-modified proteins in adipose tissue. Adipose proteins from lean insulin-sensitive and obese insulin-resistant C57Bl/6J mice were incubated with biotin hydrazide and detected using horseradish peroxidase-conjugated streptavidin. High carbohydrate, high fat feeding of mice resulted in a approximately 2-3-fold increase in total adipose protein carbonylation. Consistent with an increase in oxidative stress in obesity, the abundance of glutathione S-transferase A4 (GSTA4), a key enzyme responsible for metabolizing 4-HNE, was decreased approximately 3-4-fold in adipose tissue of obese mice. To identify specific carbonylated proteins, biotin hydrazide-modified adipose proteins from obese mice were captured using avidin-Sepharose affinity chromatography, proteolytically digested, and subjected to LC-ESI MS/MS. Interestingly enzymes involved in cellular stress response, lipotoxicity, and insulin signaling such as glutathione S-transferase M1, peroxiredoxin 1, glutathione peroxidase 1, eukaryotic elongation factor 1alpha-1 (eEF1alpha1), and filamin A were identified. The adipocyte fatty acid-binding protein, a protein implicated in the regulation of insulin resistance, was found to be carbonylated in vivo with 4-HNE. In vitro modification of adipocyte fatty acid-binding protein with 4-HNE was mapped to Cys-117, occurred equivalently using either the R or S enantiomer of 4-HNE, and reduced the affinity of the protein for fatty acids approximately 10-fold. These results indicate that obesity is accompanied by an increase in the carbonylation of a number of adipose-regulatory proteins that may serve as a mechanistic link between increased oxidative stress and the development of insulin resistance.
- Published
- 2007
46. The Acetyl Group Buffering Action of Carnitine Acetyltransferase Offsets Macronutrient-Induced Lysine Acetylation of Mitochondrial Proteins
- Author
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Robert Stevens, Paul A. Grimsrud, J. Will Thompson, Lilja Kjalarsdottir, Laura G. Dubois, Olga Ilkayeva, M. Julia Brosnan, Michael N. Davies, Deborah M. Muoio, and Timothy P. Rolph
- Subjects
0301 basic medicine ,endocrine system ,Glucose control ,Lysine ,Biology ,Article ,General Biochemistry, Genetics and Molecular Biology ,Mitochondrial Proteins ,Mice ,03 medical and health sciences ,Carnitine Acetyltransferase ,Animals ,Humans ,Mitochondrial protein ,lcsh:QH301-705.5 ,chemistry.chemical_classification ,Carnitine O-Acetyltransferase ,Acetylation ,Lysine residue ,030104 developmental biology ,Enzyme ,Biochemistry ,chemistry ,lcsh:Biology (General) ,Mitochondrial matrix - Abstract
SummaryLysine acetylation (AcK), a posttranslational modification wherein a two-carbon acetyl group binds covalently to a lysine residue, occurs prominently on mitochondrial proteins and has been linked to metabolic dysfunction. An emergent theory suggests mitochondrial AcK occurs via mass action rather than targeted catalysis. To test this hypothesis, we performed mass spectrometry-based acetylproteomic analyses of quadriceps muscles from mice with skeletal muscle-specific deficiency of carnitine acetyltransferase (CrAT), an enzyme that buffers the mitochondrial acetyl-CoA pool by converting short-chain acyl-CoAs to their membrane permeant acylcarnitine counterparts. CrAT deficiency increased tissue acetyl-CoA levels and susceptibility to diet-induced AcK of broad-ranging mitochondrial proteins, coincident with diminished whole body glucose control. Sub-compartment acetylproteome analyses of muscles from obese mice and humans showed remarkable overrepresentation of mitochondrial matrix proteins. These findings reveal roles for CrAT and L-carnitine in modulating the muscle acetylproteome and provide strong experimental evidence favoring the nonenzymatic carbon pressure model of mitochondrial AcK.
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