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AP-MS analysis of sirtuin-4 interactions in human fibroblasts

ABSTRACT: Sirtuins (SIRTs) form a critical family of nicotinamide adenine dinucleotide (NAD)-dependent enzymes that govern genome regulation, metabolism, and aging. Despite conserved deacetylase domains, SIRTs4-7 have little to no deacetylase activity, and a robust catalytic activity for mitochondrial SIRT4 has remained elusive. Moreover, in vitro characterization of SIRT4 has been hampered by difficulty in maintaining soluble and active recombinant protein. Therefore, to investigate potential cellular substrates of SIRT4, we used proteomics to define its mitochondrial protein interactions in human fibroblasts.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture, Fibroblast

SUBMITTER: Todd Greco

LAB HEAD: Ileana Cristea

PROVIDER: PXD001447 | Pride | 2015-01-05

REPOSITORIES: Pride

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			Action	DRS
	GFP_MITO_MOCK1_01.raw	Raw
	GFP_MITO_MOCK1_02.raw	Raw
	GFP_MITO_MOCK1_03.raw	Raw
	GFP_MITO_MOCK1_04.raw	Raw
	GFP_MITO_MOCK1_05.raw	Raw

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Publications

Sirtuin 4 is a lipoamidase regulating pyruvate dehydrogenase complex activity.

Mathias Rommel A RA Greco Todd M TM Oberstein Adam A Budayeva Hanna G HG Chakrabarti Rumela R Rowland Elizabeth A EA Kang Yibin Y Shenk Thomas T Cristea Ileana M IM

Cell 20141201 7

Sirtuins (SIRTs) are critical enzymes that govern genome regulation, metabolism, and aging. Despite conserved deacetylase domains, mitochondrial SIRT4 and SIRT5 have little to no deacetylase activity, and a robust catalytic activity for SIRT4 has been elusive. Here, we establish SIRT4 as a cellular lipoamidase that regulates the pyruvate dehydrogenase complex (PDH). Importantly, SIRT4 catalytic efficiency for lipoyl- and biotinyl-lysine modifications is superior to its deacetylation activity. PD ...[more]

PMID: 25525879

Similar Datasets

AP-MS analysis of sirtuin-4 interactions in human fibroblasts

Project description:Sirtuins (SIRTs) form a critical family of nicotinamide adenine dinucleotide (NAD)-dependent enzymes that govern genome regulation, metabolism, and aging. Despite conserved deacetylase domains, SIRTs4-7 have little to no deacetylase activity, and a robust catalytic activity for mitochondrial SIRT4 has remained elusive. Moreover, in vitro characterization of SIRT4 has been hampered by difficulty in maintaining soluble and active recombinant protein. Therefore, to investigate potential cellular substrates of SIRT4, we used proteomics to define its mitochondrial protein interactions in human fibroblasts.

2016-03-04 | MSV000079557 | MassIVE

The Role of NAD+-dependent lysine deacetylases (Sirtuin) and their Inhibitors in Aspergillus fumigatus acetylome and virulence

Project description:Protein acetylation is a posttranslational modification that orchestrates gene regulation and various biological processes, such as fungal pathogenesis. One promising strategy for treating fungal infections is targeting the enzymes that regulate protein acetylation. Sirtuins, an NAD+-dependent lysine deacetylase, have been described as histone deacetylase and as regulators of secondary metabolism in various Aspergillus spp. However, the roles of sirtuin remain unclear. We employed a comprehensive set of experimental techniques, including gene deletion, phenotyping, in vivo virulence assays, metabolome analysis, transcriptome analysis, and acetylome analysis. Our findings reveal that sirtuins in A. fumigatus are intricately involved in crucial cellular processes such as cell wall integrity, secondary metabolite (SM) production, protease secretion, thermotolerance, and virulence. Notably, among the six sirtuins studied, AFSirE exerts a major influence on the phenotype of A. fumigatus. Furthermore, through acetylome analysis, we identified 42 and 260 proteins exhibiting differential acetylation in the AfSirE and SIRTko strains, respectively. Transcriptome data obtained from RNA sequencing (RNA-seq) demonstrated that sirtuins play a regulatory role in the expression of genes associated with SM production, cell wall component biosynthesis, and a variety of virulence factors.

2024-06-20 | PXD044964 | Pride

Genome-wide Analysis of Functional Sirtuin Chromatin Targets in Yeast

Project description:Sir2 and the homologous proteins, Hst1, Hst2, Hst3, and Hst4 from Saccharomyces cerevisiae are NAD+-dependent histone deacetylases of the sirtuin protein family. Sir2 functions in transcriptional silencing at the silent mating-type loci, telomeres, and rDNA locus, but also promotes replicative lifespan. To gain a better understanding of the chromatin-regulatory roles carried out by Sir2 and the Hst proteins, we performed ChIP-sequencing analysis on all five sirtuins and Sum1, the DNA binding partner for Hst1. Sir2, Hst1, and Sum1 were abundantly, and functionally co-enriched at several major targets, including the telomeric repeats, where they were required for maintaining proper telomere repeat length. At tRNA target genes they were required for efficient cohesin and condensin deposition. Across the open reading frames of glycolytic and ribosomal protein genes, Sir2 and Hst1 functioned in NAD+-dependent transcriptional repression at the diauxic shift, directly linking Sir2 to glucose metabolism, which could have implications for longevity. Six factors and Input ChIP-seq samples were analyzed in Saccharomyces eerevisiae.

2013-05-10 | E-GEOD-41415 | biostudies-arrayexpress

The pan-Sirtuin inhibitor MC2494 regulates mitochondrial function in a leukemia cell line

Project description:The involvement of sirtuins (SIRTs) in modulating metabolic and stress response pathways is attracting growing scientific interest. Some SIRT family members are located in mitochondria, dynamic organelles that perform several crucial functions essential for eukaryotic life. Mitochondrial dysfunction has emerged as having a key role in a number of human diseases, including cancer. Here, we investigated mitochondrial damage resulting from treatment with a recently characterized pan-SIRT inhibitor, MC2494. MC2494 was able to block mitochondrial biogenesis and function in terms of ATP synthesis and energy metabolism, suggesting that it might orchestrate cell response to metabolic stress and thereby interfere with tumorigenesis. Targeting mitochondrial function could thus be considered a potential anticancer strategy for use in clinical therapy.

2021-09-08 | PXD017508 | Pride

AP-MS analysis of sirtuin-2 interactions and whole-cell proteome analysis in human fibroblasts

Project description:Sirtuins are NAD+-dependent deacylases with diverse enzymatic activities and substrates. They were implicated in regulation of cancer progression, neurodegeneration, aging, and viral infection. Sirtuin-2 localizes to the cytoplasm and is known to deacetylate alpha-tubulin. Current knowledge on sirtuin-2 interactions and its effect on gene expression in normal cells is very limited. We used proteomics approach to define its interactions and its effect on global protein levels in human fibroblasts.

2016-08-12 | PXD004212 | Pride

Sustained activation of AMPK enhances differentiation of human iPSC-derived cardiomyocytes via sirtuin activation

Project description:Lee and colleagues demonstrate that sustained activation of AMPK enhances differentiation of iPSC-derived cardiomyocytes. Sustained AMPK activation decreased histone acetylation at known target sites for nuclear-localized sirtuins, suggesting that AMPK activation enhances sirtuin activity. AMPK-induced sirtuin-mediated deacetylation of histone proteins may regulate chromatin accessibility and enhance cardiomyocyte differentiation.

2020-06-12 | GSE152265 | GEO

GNPS_Untarget metabolomic analyses of Aspergillus fumigatus sirtuin knockout strains

Project description:Aspergillus fumigatus sirtuins knockout mutants metabolomics.

2021-05-05 | MSV000087365 | GNPS

A synthetic non-histone substrate to study substrate targeting by the Gcn5 HAT and sirtuin HDACs

Project description:Gcn5 and sirtuins are conserved HAT and HDAC enzymes that were first characterised as regulators of gene expression via histone acetylation. Although histone tails are important substrates of these enzymes, they also target non-histone proteins in diverse processes. Previously, we used SILAC-based mass spectrometry to identify novel non-histone substrates of Gcn5 and sirtuins in yeast. We found a shared target consensus sequence. In our latest work we used a synthetic biology approach to demonstrate that this consensus sequence can direct acetylation and deacetylation targeting by these enzymes in vivo. We used the synthetic substrate as a tool to prioritize SILAC-based acetylome analyses of SAGA mutants. Presented here are analyses of ada3∆ mutants analyzed in this work versus wild-type control. The strains and labels used are described in the BioRxiv report linked in the methods sections below.

2019-02-25 | PXD012608 | Pride

Yeast expression data from conditions that inhibit sirtuins

Project description:Sir2 is an NAD+-dependent histone deacetylase, and is the founding member of a large, phylogentically conserved, family of such deacetylases called the Sirtuins. The budding yeast, Saccharomyces cerevisiae, harbors 4 paralogs of Sir2, known as Hst1, Hst2, Hst3, and Hst4. Reducing the intracellular NAD+ concentration is inhibitory for the Sirtuins, and raising the intracellular nicotinamide (NAM) concentration is inhibitory. Microarray gene expression analysis was used to identify novel classes of yeast genes whose expression is altered when either NAD+ concentration is reduced or NAM is elevated. A subset of genes involved in thiamine biosynthesis was identified as being upregulated when Sir2 or Hst1 was inactivated. Several mutants in the NAD+ biosynthesis and salvage pathways were tested, along with WT and WT grown in the presence of 5 mM nicotinamide, which inhibits the Sirtuins.

2010-05-11 | GSE18488 | GEO

Dysregulated cellular redox status during hyperammonemia causes mitochondrial dysfunction by inhibiting sirtuin mediated deacetylation

Project description:Perturbed metabolism of ammonia, an endogenous cytotoxic molecule, causes mitochondrial dysfunction with decreased nicotinamide adenine dinucleotide (NAD+) in skeletal muscle. Consistent with our previous report of enrichment of NAD metabolism and sirtuin pathways during hyperammonemia, NAD+-dependent Sirtuin3 (Sirt3) expression and deacetylase activity weredecreased with increased muscle protein acetylation in murine and human skeletal muscle/myotubes. Acetylomics and cell fractions showed hyperammonemia-induced hyperacetylation of critical mitochondrial and signaling molecules. Overexpression of mitochondrial targeted Lactobacillus brevis NADH oxidase (MitoLbNOX) reversed ammonia-induced oxidative dysfunction, electron transport chain (ETC) supercomplex disassembly, lower ATP content, NAD+, and redox ratio (NAD+/NADH). Protein hyperacetylation, post-mitotic senescence and lower mitochondrial sirtuin (Sirt3) expressionduring hyperammonemia were also reversed by MitoLbNOX. Sirt3 overexpression alone did not reverse ammonia-induced redox or mitochondrial dysfunction but reversed hyperacetylation and senescence. These data show that targeting redox ratio, rather than Sirt3, more consistently restores mitochondrial homeostasis and protein acetylation during hyperammonemia.

2023-10-24 | PXD033430 | Pride

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