An acetylome peptide microarray reveals specificities and deacetylation substrates for all human sirtuin isoforms

Rauh, David; Fischer, F.; Gertz, M.; Lakshminarasimhan, Mahadevan; Bergbrede, Tim; Aladini, Firouzeh; Kambach, Christian; Becker, Christian F. W.; Zerweck, Johannes; Schutkowski, Mike; Steegborn, Clemens

doi:10.1038/ncomms3327

Cited by 183 publications

(176 citation statements)

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“…This failure might reflect that structural features could be important for substrate recognition by sirtuins. Moreover, we did observe that the five Ran acetylation sites give rise to considerably different signal intensities when detected with a pan-anti-acetyl-lysine (AcK) antibody, which is in line with observations by Rauh et al and other groups (47,48). This primary sequence dependence appears to be a common feature of pan-anti-AcK antibodies and thus has to be taken into account for the evaluation of proteomic screens with affinity-enriched material and quantitative immunoblots of complex protein samples.…”

Section: Discussionsupporting

confidence: 78%

“…Ran AcK71 is specifically deacetylated by Sirt2, whereas Ran AcK37 is a substrate for Sirt1, -2, and -3. Interestingly, in a recent peptide microarray assay, screening sirtuins 1-7 for activity toward all acetylated peptides derived from the Choudhary screen, Rauh et al (47) did not detect any of the described activities. This failure might reflect that structural features could be important for substrate recognition by sirtuins.…”

Section: Discussionmentioning

confidence: 99%

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Small GTP-binding protein Ran is regulated by posttranslational lysine acetylation

Boor

Knyphausen

Kuhlmann

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Ran is a small GTP-binding protein of the Ras superfamily regulating fundamental cellular processes: nucleo-cytoplasmic transport, nuclear envelope formation and mitotic spindle assembly. An intracellular Ran•GTP/Ran•GDP gradient created by the distinct subcellular localization of its regulators RCC1 and RanGAP mediates many of its cellular effects. Recent proteomic screens identified five Ran lysine acetylation sites in human and eleven sites in mouse/rat tissues. Some of these sites are located in functionally highly important regions such as switch I and switch II. Here, we show that lysine acetylation interferes with essential aspects of Ran function: nucleotide exchange and hydrolysis, subcellular Ran localization, GTP hydrolysis, and the interaction with import and export receptors. Deacetylation activity of certain sirtuins was detected for two Ran acetylation sites in vitro. Moreover, Ran was acetylated by CBP/p300 and Tip60 in vitro and on transferase overexpression in vivo. Overall, this study addresses many important challenges of the acetylome field, which will be discussed.Ran | lysine acetylation | genetic code expansion concept | nucleus | nuclear cytosolic transport T he small GTP-binding protein Ran (Ras-related nuclear) is involved in nucleo-cytoplasmic transport processes, nuclear envelope formation, and the formation of the mitotic spindle (1). Ran, furthermore, has a variety of cytosolic functions and is involved in the cross-talk with the actin cytoskeleton. As a member of the Ras superfamily, Ran is structurally composed of a fold known as the G-domain (GTP-binding domain), a central sixstranded β-sheet that is surrounded by α-helices. Ras-family members bind to GTP and GDP nucleotides with high picomolar affinity. However, only in the GTP-bound form and the switch Iand switch II-loops adopt a stable conformation. Ran has been structurally characterized in great detail, including different nucleotide states and various protein complexes (2-4).In interphase cells, about 90% of cellular Ran is nuclear, and only a minor proportion is cytosolic (5). The localization of the guanine-nucleotide exchange factor (GEF) RCC1 (Regulator of chromosome condensation 1) at the nuclear chromatin and the RanGAP (RanGTPase-activating protein) at the cytosolic site of the nuclear pore creates a gradient of Ran•GTP in the nucleus and Ran•GDP in the cytosol (6-8).In the nucleus, Ran•GTP binds to exportins such as CRM1 (Chromosome region maintenance 1) to transport cargo proteins containing a nuclear export signal (NES) into the cytosol (3, 9, 10). Ran•GTP, furthermore, binds to Importin-β•cargo complexes to release the cargo in the nucleus (11-15). In the cytosol, the Importin•Ran•GTP complexes, as well as the ternary exportin•Ran•GTP-cargo complexes, dissociate on binding of RanBP1 and subsequent GTP hydrolysis catalyzed by RanGAP (16,17). The Ran transport cycle closes by translocation of Ran•GDP to the nucleus by the nuclear transport factor 2 (NTF2) (4,(17)(18)(19)(20). Many of these Ran interactions also ...

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Section: Discussionsupporting

confidence: 78%

Section: Discussionmentioning

confidence: 99%

Small GTP-binding protein Ran is regulated by posttranslational lysine acetylation

Boor

Knyphausen

Kuhlmann

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…Sirtuins are protein deacetylases involved in cell cycle regulation, genomic stability and energy metabolism (Choudhary et al, 2009(Choudhary et al, , 2014. In contrast to other sirtuins, Sirt2 localizes predominantly in the cytosol (North et al, 2003;Rauh et al, 2013). This suggests that Sirt2 might link AMPK phosphorylation to Glut1 activation.…”

Section: Discussionmentioning

confidence: 99%

Acute stimulation of glucose influx by mitoenergetic dysfunction requires LKB1, AMPK, Sirt2 and mTOR/RAPTOR

Liemburg-Apers

Wagenaars

Smeitink

et al. 2016

Journal of Cell Science

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Mitochondria play a central role in cellular energy production, and their dysfunction can trigger a compensatory increase in glycolytic flux to sustain cellular ATP levels. Here, we studied the mechanism of this homeostatic phenomenon in C2C12 myoblasts. Acute (30 min) mitoenergetic dysfunction induced by the mitochondrial inhibitors piericidin A and antimycin A stimulated Glut1-mediated glucose uptake without altering Glut1 (also known as SLC2A1) mRNA or plasma membrane levels. The serine/threonine liver kinase B1 (LKB1; also known as STK11) and AMP-activated protein kinase (AMPK) played a central role in this stimulation. In contrast, ataxiatelangiectasia mutated (ATM; a potential AMPK kinase) and hydroethidium (HEt)-oxidizing reactive oxygen species (ROS; increased in piericidin-A-and antimycin-A-treated cells) appeared not to be involved in the stimulation of glucose uptake. Treatment with mitochondrial inhibitors increased NAD + and NADH levels (associated with a lower NAD + :NADH ratio) but did not affect the level of Glut1 acetylation. Stimulation of glucose uptake was greatly reduced by chemical inhibition of Sirt2 or mTOR-RAPTOR. We propose that mitochondrial dysfunction triggers LKB1-mediated AMPK activation, which stimulates Sirt2 phosphorylation, leading to activation of mTOR-RAPTOR and Glut1-mediated glucose uptake.

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“…Among the seven mammalian sirtuins (SIRT1-7), SIRT3-5 are located in mitochondria (12,13). Unlike SIRT3, both SIRT4 and SIRT5 have very weak deacetylase activities (14). SIRT5 possesses unique enzymatic activity on hydrolyzing negatively charged lysine modifications such as lysine succinylation, malonylation, and glutarylation (1,4,8).…”

mentioning

confidence: 99%

Metabolomics-assisted proteomics identifies succinylation and SIRT5 as important regulators of cardiac function

Sadhukhan

Liu

Ryu

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

263

244

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Cellular metabolites, such as acyl-CoA, can modify proteins, leading to protein posttranslational modifications (PTMs). One such PTM is lysine succinylation, which is regulated by sirtuin 5 (SIRT5). Although numerous proteins are modified by lysine succinylation, the physiological significance of lysine succinylation and SIRT5 remains elusive. Here, by profiling acyl-CoA molecules in various mouse tissues, we have discovered that different tissues have different acyl-CoA profiles and that succinyl-CoA is the most abundant acyl-CoA molecule in the heart. This interesting observation has prompted us to examine protein lysine succinylation in different mouse tissues in the presence and absence of SIRT5. Protein lysine succinylation predominantly accumulates in the heart when Sirt5 is deleted. Using proteomic studies, we have identified many cardiac proteins regulated by SIRT5. Our data suggest that ECHA, a protein involved in fatty acid oxidation, is a major enzyme that is regulated by SIRT5 and affects heart function. Sirt5 knockout (KO) mice have lower ECHA activity, increased longchain acyl-CoAs, and decreased ATP in the heart under fasting conditions. Sirt5 KO mice develop hypertrophic cardiomyopathy, as evident from the increased heart weight relative to body weight, as well as reduced shortening and ejection fractions. These findings establish that regulating heart metabolism and function is a major physiological function of lysine succinylation and SIRT5.sirtuin | lysine succinylation | fatty acid metabolism | desuccinylation | hypertrophic cardiomyopathy P rotein posttranslational modifications (PTMs) contribute toward the functional diversity of proteomes through regulating their activity, stability, and cellular localization. Many novel PTMs have been identified recently that result from enzymatic or nonenzymatic reactions with metabolites (1-5). Lysine, being the most frequently posttranslationally modified amino acid, has become the target of various PTMs such as acetylation, methylation, propionylation, butyrylation, crotonylation, succinylation, malonylation, glutarylation, long-chain fatty acylation, ubiquitination, and 2-hydroxyisobutyrylation (1, 3-9). Unlike lysine acetylation, lysine succinylation is a relatively new PTM and the succinyl donor is presumably succinyl-CoA. Acetylation on lysine neutralizes the positive charge of lysine side chain and is known to affect the structure and function of chromatin (10) as well as cellular metabolism (11). However, succinylation on lysine undergoes a complete charge reversal by changing a positively charged side chain to a negatively charged one. Regarding the change in charge, lysine succinylation is similar to phosphorylation, producing a two-unit charge shift in the modified residues. So, it can be anticipated that lysine succinylation would have a significant role in metabolic pathways, as was previously found for acetylation or phosphorylation.Sirtuins are an evolutionarily conserved family of NAD-dependent lysine deacylases. Among the seven mammalia...

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An acetylome peptide microarray reveals specificities and deacetylation substrates for all human sirtuin isoforms

Cited by 183 publications

References 57 publications

Small GTP-binding protein Ran is regulated by posttranslational lysine acetylation

Small GTP-binding protein Ran is regulated by posttranslational lysine acetylation

Acute stimulation of glucose influx by mitoenergetic dysfunction requires LKB1, AMPK, Sirt2 and mTOR/RAPTOR

Metabolomics-assisted proteomics identifies succinylation and SIRT5 as important regulators of cardiac function

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