Regulation of UCP1 and Mitochondrial Metabolism in Brown Adipose Tissue by Reversible Succinylation

Article

Regulation of UCP1 and Mitochondrial Metabolism in Brown Adipose Tissue by Reversible Succinylation Graphical Abstract

Authors GuoXiao Wang, Jesse G. Meyer, Weikang Cai, ..., Christopher Newgard, Birgit Schilling, C. Ronald Kahn

Correspondence [email protected]

In Brief Wang et al. performed succinylproteomics in brown fat (BAT) of normal and Sirt5 KO mice and identified UCP1 as a new target of Sirt5 desuccinylation. UCP1 with succinyl-mimetic mutations displayed reduced activity and stability. Elevated succinylation of mitochondrial protein in Sirt5 KO BAT resulted in altered metabolic flexibility and mitophagy.

Highlights d

Sirt5 regulates mitochondrial protein succinylation and malonylation in brown fat

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Increased succinylation of UCP1 reduces its stability and function

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Sirt5KO in BAT leads to metabolic inflexibility and impairs mitochondrial homeostasis

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These processes are altered by cold exposure and diet

Wang et al., 2019, Molecular Cell 74, 1–14 May 16, 2019 ª 2019 Elsevier Inc. https://doi.org/10.1016/j.molcel.2019.03.021

Please cite this article in press as: Wang et al., Regulation of UCP1 and Mitochondrial Metabolism in Brown Adipose Tissue by Reversible Succinylation, Molecular Cell (2019), https://doi.org/10.1016/j.molcel.2019.03.021

Molecular Cell

Article Regulation of UCP1 and Mitochondrial Metabolism in Brown Adipose Tissue by Reversible Succinylation GuoXiao Wang,1 Jesse G. Meyer,2 Weikang Cai,1 Samir Softic,1 Mengyao Ella Li,1 Eric Verdin,2 Christopher Newgard,3 Birgit Schilling,2 and C. Ronald Kahn1,4,* 1Section

on Integrative Physiology and Metabolism, Joslin Diabetes Center, Harvard Medical School, Boston, MA 02215, USA Institute for Research on Aging, Novato, CA 94945, USA 3Sarah W. Stedman Nutrition and Metabolism Center and Duke Molecular Physiology Institute, Departments of Pharmacology and Cancer Biology and Medicine, Duke University Medical Center, Durham, NC 27708, USA 4Lead Contact *Correspondence: [email protected] https://doi.org/10.1016/j.molcel.2019.03.021 2Buck

SUMMARY

Brown adipose tissue (BAT) is rich in mitochondria and plays important roles in energy expenditure, thermogenesis, and glucose homeostasis. We find that levels of mitochondrial protein succinylation and malonylation are high in BAT and subject to physiological and genetic regulation. BAT-specific deletion of Sirt5, a mitochondrial desuccinylase and demalonylase, results in dramatic increases in global protein succinylation and malonylation. Mass spectrometry-based quantification of succinylation reveals that Sirt5 regulates the key thermogenic protein in BAT, UCP1. Mutation of the two succinylated lysines in UCP1 to acyl-mimetic glutamine and glutamic acid significantly decreases its stability and activity. The reduced function of UCP1 and other proteins in Sirt5KO BAT results in impaired mitochondria respiration, defective mitophagy, and metabolic inflexibility. Thus, succinylation of UCP1 and other mitochondrial proteins plays an important role in BAT and in regulation of energy homeostasis.

INTRODUCTION Brown adipose tissue (BAT) plays a central role in energy balances through its high density of mitochondria and uncoupled respiration. BAT has been shown to be present in all mammals, including humans, and can be activated to increase glucose uptake and energy expenditure (Cypess et al., 2009), making it an attractive therapeutic target for treating obesity and metabolic disease. A critical component of the regulation of BAT is through uncoupling protein 1 (UCP1) and b3-adrenergic receptor stimulation (Cypess et al., 2015), which induces transcription of UCP1 and genes involved in mitochondria biogenesis (Villarroya et al., 2017). UCP1 can also be regulated by interaction with fatty acids (Cannon and Nedergaard, 2004; Fedorenko et al., 2012), as well as by sulfenylation (Chouchani et al., 2016).

Mitochondria dysfunction is an important component in the pathophysiology of a variety of metabolic and cardiovascular diseases (Wallace, 2005). Among all tissues, brown fat relies most heavily on mitochondrial respiration to maintain its normal physiological function of thermogenesis (Cannon and Nedergaard, 2004). In addition to burning its own fat, the sympathetic nervous system drives lipolysis from white fat creating another supply of free fatty acids for BAT heat generation (Schreiber et al., 2017; Shin et al., 2017). The end product of oxidation of these endogenous and exogenous fatty acids in BAT is acetylCoA, which is further oxidized through the tricarboxylic acid cycle and the electron transport chain, and all of these reactions require normal mitochondrial function. Mitochondrial protein functions are subject to multiple reversible post-translational modifications (Stram and Payne, 2016). Examples of such modifications include acetylation, succinylation, malonylation, and glutarylation (Carrico et al., 2018). These reactions are controlled by the level of acyl-donors in the cell and by the action of the mitochondrial sirtuins, Sirt3 and Sirt5, which remove acetyl, and succinyl, malonyl, glutaryl groups, respectively (Giralt and Villarroya, 2012; Hirschey and Zhao, 2015; Park et al., 2013; Tan et al., 2014). Sirtuins are NAD+-dependent protein deacylases homologous to yeast silent information regulator 2 (Sir2). Surtuins require NAD+ as a cofactor, making them sensors of cellular energy status. In addition, sirtuins have been shown to play important roles in controlling metabolism in a variety of organisms (Schwer and Verdin, 2008). Sirt3 and Sirt5 are enriched in and act primarily in mitochondria (Michishita et al., 2005). Knockout of Sirt3 in liver results in increased acetylation of long-chain acyl CoA dehydrogenase (LCAD) and a decrease in its enzymatic activity (Hirschey et al., 2010). Likewise, the majority of Sirt5 substrates for desuccinylation in liver are metabolic enzymes involved in fatty acid beta-oxidation, branched chain amino acid metabolism, tricarboxylic acid (TCA) cycle, and ketone body synthesis (Rardin et al., 2013). In the present study, we demonstrate that there is widespread mitochondrial protein succinylation and malonylation in BAT, and these post-translational modifications are upregulated by knockout of Sirt5. Our results demonstrate hyper-succinylation regulates three important proteins: glutamate dehydrogenase (GDH/GLUD1), succinate dehydrogenase (SDH), and most Molecular Cell 74, 1–14, May 16, 2019 ª 2019 Elsevier Inc. 1

Please cite this article in press as: Wang et al., Regulation of UCP1 and Mitochondrial Metabolism in Brown Adipose Tissue by Reversible Succinylation, Molecular Cell (2019), https://doi.org/10.1016/j.molcel.2019.03.021

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importantly, the unique BAT mitochondrial uncoupling protein UCP1. Acyl-mimetic mutation of the two major succinylation sites in UCP1 significantly decreases its stability and activity. Likewise, hypersuccinylation of GLUD1 and SDH in BAT-specific Sirt5 knockout mice decreases their activities. The cumulative consequence of increased succinylation of these proteins in Sirt5 knockout (KO) BAT is altered mitochondrial homeostasis and metabolic inflexibility. Thus, succinylation of UCP1 and other Sirt5 substrates provide a new and important mechanism regulating mitochondrial function in BAT. RESULTS Protein Acylation Level Is High in BAT and Subject to Physiological and Genetic Regulation To explore the role of protein acylation in BAT, we performed western blot analysis of subcutaneous white adipose tissue (sWAT), visceral or epididymal white adipose tissue (eWAT), BAT, and liver under random fed condition. Levels of protein succinylation and malonylation were highest in BAT, followed by liver, and much lower in sWAT and eWAT (Figure 1A). Both succinylation and malonylation in BAT were regulated by obesity, be-

2 Molecular Cell 74, 1–14, May 16, 2019

Figure 1. Protein Acylation Level Is High in BAT and Subject to Physiological and Genetic Regulation Protein acylation and Sirt5 expression were assessed by western blotting. (A) Total tissue lysates of BAT, sWAT, eWAT, and liver from random-fed, 9-week-old wild type C57/ B6J mice. (B) BAT from 13- to 14-week-old db/+ versus db/ db mice. (C) BAT from wild type mice fed high fat diet (60% fat) for 12 weeks and aged matched chow-fed mice. (D) BAT from wild type mice housed at 22
C, or acclimated to 5
C or 30
C for 10 days, and from wild type mice acutely exposed to 6
C after acclimation at 30
C for 10 days. (E) Total cell lysates and mitochondrial fractions of BAT from mice housed at 22
C or acclimated to 5
C for 10 days. n = 4 to 6 per group. White dashed lines were used to separate groups in a single gel.

ing decreased in genetically obese db/db mice (Figure 1B) and increased in obesity due to high fat diet (HFD) feeding (Figure 1C). This correlated with UCP1 expression (Figures 1B and 1C) and BAT activity, which is reduced in the former and increased in the latter (Alcala´ et al., 2017; Goodbody and Trayhurn, 1981). More importantly, when mice were housed at 5
C–6
C either acutely or chronically, which increases BAT activity, levels of succinylated and malonylated proteins as detected by western blotting increased dramatically (Figure 1D). Conversely, when mice were housed at 30
C, i.e., thermoneutral condition, where BAT activity is low, levels of succinylated and malonylated proteins were markedly reduced (Figure 1D). Interestingly, Sirt5 protein levels positively correlated with the succinylation and/or malonylation levels in BAT under most of these conditions (Figures 1A–1D and S1), indicating that the changes in acylation were not likely due to changes in desuccinylation or demalonylation, but rather reflected changes in rates of formation of these adducts. Indeed, with cold exposure, acetyl, succinyl, and malonyl CoA donors increased 30%–100% (Figure S1E), reflecting a state of carbon stress induced by increased mitochondrial activity (Wagner and Hirschey, 2014) where Sirt5 was induced to alleviate such stress. As expected, both Sirt5 and the acylated proteins it regulates were enriched in the mitochondrial fraction of BAT, and when normalized to mitochondrial mass, levels of succinylation, but not malonylation, were still elevated by cold acclimation (Figure 1E). BAT-Specific Sirt5 Knockout Elevates Protein Succinylation and Malonylation Levels To further assess the regulation of BAT by mitochondrial protein acylation, we generated BAT-specific Sirt5 KO mice (5-BKO) by

Please cite this article in press as: Wang et al., Regulation of UCP1 and Mitochondrial Metabolism in Brown Adipose Tissue by Reversible Succinylation, Molecular Cell (2019), https://doi.org/10.1016/j.molcel.2019.03.021

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(A) H&E staining of BAT from 11-week-old random-fed floxed and 5-BKO mice, scale bar, 100 mm. (B) Ratio of adipose tissue weight versus body weight in 11-week-old chow fed mice, n = 5 versus 6. Data are represented as mean ± SEM. (C) qPCR of BAT from 11-week-old random-fed floxed and 5-BKO mice, n = 6 per group. Data are represented as mean ± SEM, *p < 0.05, Student’s t test. (D) Western blot of BAT from 2-month-old female floxed and 5-BKO mice housed at 22
C or acclimated to 5
C for 10 days. (E) Western blot of BAT cytoplasmic (cyto) and mitochondrial fraction (mito) from 8-week-old, 24 h fasted floxed and 5-BKO mice.

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Figure 2. BAT-Specific Sirt5 Deficiency Elevates Protein Succinylation and Malonylation Levels

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breeding Sirt5 floxed mice (Yu et al., 2013) with UCP1-Cre mice (Kong et al., 2014), the latter have been well characterized to be BAT-specific. No Sirt5 deletion was detected in visceral or subcutaneous WAT in 5-BKO mice (Figure S2A). Under standard housing condition (22
C, normal chow), 5-BKO mice had similar body weight and food intake as their littermate controls (Figures S2B and S2C). Likewise, there was no difference in weight or morphology of BAT (Figures 2A and 2B). As expected, there was a 90% decrease in Sirt5 mRNA in BAT from 5-BKO mice, but no differences in the expression of genes involved in browning, fatty acid synthesis or transport, lipolysis, mitochondrial oxidative phosphorylation, or glucose transport (Figure 2C). However, consistent with Sirt5 being a strong desuccinylase or demalonylase, western blotting revealed 2- to 4-fold increases in protein succinylation and malonylation in Sirt5-deficient BAT compared to control, while protein acetylation levels were not

affected (Figure 2D). As in Figure 1D, cold acclimation to 5
C for 10 days significantly increased levels of succinylated and malonylated proteins in BAT of control mice, and Sirt5 deficiency increased these levels even further. The amount of acetylated proteins was also increased by cold acclimation but were not further elevated by Sirt5 deficiency (Figure 2D). As expected, succinylated and malonylated proteins were enriched in mitochondria and were further increased with Sirt5 KO (Figure 2E). Sirt5 Deficiency Leads to Metabolic Inflexibility in BAT An important function of BAT is to defend against cold stress. When floxed and 5-BKO mice were subjected to acute cold exposure under ad libitum fed condition, 5-BKO mice showed similar ability to defend against cold as controls, with both groups losing 1.5
C of body temperature over 5 h (Figure 3A). Interestingly, after an overnight fast, which inactivates BAT but activates Sirt5 (Jokinen et al., 2017; Rothwell et al., 1984), the control mice could still maintain body temperature to acute cold exposure, whereas 5-BKO mice became much more sensitive to cold, losing almost 5
C over 5 h (Figure 3B). This occurred despite the fact that there were no differences in the expression of genes involved in browning, respiration, or lipid and glucose utilization (Figure S2D). Lipid mobilization does not appear to be impaired in Sirt5-BKO mice, as HSL phosphorylation was similar, if not higher in Sirt5 KO BAT compared to control (Figure S2E). Although Sirt5-BKO mice had no problem acclimating to 5
C on HFD, core body temperature was consistently 0.7
C to 1.5
C lower in Sirt5BKO mice than in controls during the feeding period, indicating a defect in BAT thermogenesis through fatty acid oxidation (Figure 3C). Metabolic profiling of 5-BKO and control mice using the

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Figure 3. Sirt5 Deficiency Leads to Metabolic Inflexibility in BAT

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(A) Rectal temperature of 2-month-old floxed and 5-BKO mice during acute cold (from 22
C to 7
C) exposure. Mice were allowed free access to food and water. n = 5 versus 7. Data are represented as mean ± SEM. (B) Rectal temperature of 2.5-month-old floxed and 5-BKO mice during acute cold (from 22
C to 7
C) exposure. Mice were fasted for 18 h. n = 7 versus 8. Data are represented as mean ± SEM. (C) Intra-abdominal temperature of HFD fed floxed and Sirt5-BKO mice after 3 days’ acclimation to 5
C, recorded at 15-min intervals. Dark phase are shaded, measurements occurred between 10:00 p.m. and 2:00 a.m. were boxed. n = 4 versus 6. Data are represented as mean ± SEM. *p < 0.05. (D) Respiration exchange ratio (RER) of 5-monthold floxed and 5-BKO mice on chow diet. n = 6. Data are represented as mean ± SEM. (E) Glucose tolerance test of floxed and 5-BKO mice fed with high fat (60% by calories) diet for 11 weeks. n = 5 versus 8. Data are represented as mean ± SEM. (F) Insulin tolerance tests of floxed and 5-BKO mice fed with high fat diet for 12 weeks. n = 6 versus 8. Data are represented as mean ± SEM. (G) Fasting plasma insulin levels of floxed and 5-BKO mice fed with high fat diet for 13 weeks. n = 6 versus 8. Data are represented as mean ± SEM. *p < 0.05, Student’s t test.

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Comprehensive Lab Animal Monitoring System (CLAMS) revealed that 5-BKO mice had similar O2 consumption and CO2 production as controls (Figures S3A and S3B), but their respiratory exchange ratio (RER) was significantly lower, especially in the early part of the dark period when mice are feeding (Figure 3D). This indicates that 5-BKO mice prefer to use fatty acids as fuel or are incapable of switching from FFA usage to carbohydrates. Consistent with metabolic inflexibility, 5-BKO mice showed normal glucose tolerance on a standard chow diet (Figure S3C) but developed glucose intolerance when challenged with high fat diet (Figure 3E). This occurred without a change in insulin sensitivity as measured by insulin tolerance test (Figure 3F) or any differences in fasting plasma insulin levels between genotypes (Figure 3G). The differences in glucose tolerance were greatest at later time points after glucose injection (Figure 3E) suggesting the defect in 5-BKO mice was due

4 Molecular Cell 74, 1–14, May 16, 2019

to decreased glucose uptake in BAT, as BAT is known to be a major site of glucose utilization in the mouse (Bartelt et al., 2011; Nedergaard et al., 2011). Thus, mice lacking Sirt5 in BAT are refractory in switching fuels from FFA to glucose after overnight fasting. BAT Sirt5 Desuccinylates Proteins in Major Metabolic Pathways To determine cold- and Sirt5-regulated succinylation sites that may contribute to the metabolic regulation of BAT, we assessed protein succinylation in whole BAT from floxed (control) and 5-BKO mice housed at room temperature and mice cold acclimated to 5
C for 10 days using succinyl-lysine affinity enrichment followed by quantitative mass spectrometry analysis (Gillet et al., 2012; Meyer et al., 2017). We identified and quantified a total of 2,404 succinylation sites on 444 proteins (Table S1). For each succinylation site we report here, the unmodified protein was also quantified in a parallel analysis in order to normalize changes in succinylation level to individual protein amount. Of these, 1,387 succinylation sites from 348 proteins were regulated at least 2-fold with a false discovery rate (FDR) <0.01 in at least one condition when normalized to unmodified protein level (Figure S4A). Among the 829 lysine sites whose relative succinylation levels were increased in Sirt5 KO BAT, only 57 were also elevated by cold acclimation (CA) (Figure 4A),

Please cite this article in press as: Wang et al., Regulation of UCP1 and Mitochondrial Metabolism in Brown Adipose Tissue by Reversible Succinylation, Molecular Cell (2019), https://doi.org/10.1016/j.molcel.2019.03.021

CA/RT

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(A) Venn diagram showing lysine sites whose succinylation levels were increased (red) or decreased (blue) by at least 2-fold with FDR <0.01 comparing between Sirt5 KO versus Floxed and cold acclimation (CA) versus room temperature (RT) housed mice. (B) Reactome term enrichment analysis from the list of proteins containing succinylation sites that were identified by mass spectrometry and increased due to Sirt5 KO. (C–F) Succinylation fold changes (F.C.) on individual lysine sites of SDHA (C), SDHB (D), GLUD1 (E), and UCP1 (F). (G) Western blot of BAT from floxed and 5-BKO mice before and after anti-succinyl-K immunoprecipitation.

SDHA Ksuc 20

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Figure 4. BAT Sirt5 Desuccinylates Proteins in Major Metabolic Pathways

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functional term enrichment analysis of the 249 proteins whose succinylation sites 5 increased due to Sirt5 KO revealed 0 enrichment of several mitochondrial pathways, including complex I biogenesis, thermogenesis by uncoupling proteins, electron transport chain (ETC), UCP1 Ksuc Glud1 Ksuc SDHB Ksuc E D F mitochondrial fatty acid beta oxidation, 22.6 4.7 25 20 17.2 16.3 5 and TCA cycle (Figures 4B, S5A, and Floxed 20 4 15 15 S5B). This was observed in both room 5-BKO 15 3 2.1 2 10 temperature (RT) and cold acclimated 10 2 4.7 4.7 3.6 (CA) mice (Figures S4A, S5A, and S5B). 5 2.4 5 1 Among the most highly succinylated 0 0 0 proteins in Sirt5 KO BAT were the mitochondrial enzymes succinate dehydrogenase A and B (SDHA and SDHB), glutamate dehydrogenase (GLUD1), and Floxed 5-BKO the mitochondrial uncoupling protein G Floxed 5-BKO Input IgG succinyl-K UCP1. Follow-up experiments to the IP IgG succinyl-K mass spectrometry results suggested GLUD1/2 WB GLUD1/2 SDHA that these proteins had relatively high SDHA SDHB stoichiometries of succinylation. Thus, SDHB UQCRC2 immunoprecipitation with anti-succinylUQCRC2 NDFUB8 NDUFB8 lysine antibody in Sirt5 KO BAT lysates UCP1 UCP1 resulted in depletion of 9% of SDHA, Sirt5 17% of SDHB, 24% of UCP1, and 38% of GLUD1 in the lysate (Figure S4B). In indicating a fine level of tuning of succinylation by CA which is not addition, mass spectrometry revealed five lysine sites on likely simply due to changes in Sirt5 activity. Likewise, when SDHA whose relative succinylation levels increased over 4-fold normalized to individual protein amount, only 102 lysine sites in response to Sirt5 loss in mice housed at room temperature had increased relative succinylation in response to CA, indi- (Figure 4C), and for two of these (K179 and K485), the increase cating the global increase of relative succinylation induced by was more than 10-fold. Likewise, SDHB had three lysine sites CA observed by western blotting (Figures 1D and 1E) was due whose relative succinylation levels were increased by more to a combination of significant increases in the level of the mito- than 2-fold in Sirt5 KO BAT compared to control, one of which, chondrial protein and increases in the level of succinylation, and K169, had a close to 5-fold increase (Figure 4D). GLUD1 was that the effects of cold acclimation were selective. Similarly, CA also a very confident target of Sirt5 in BAT; four of its lysines significantly decreased relative succinylation levels in 657 lysine showed more than 2-fold increase in relative succinylation in sites, while only 60 sites, with 17 of them overlapping with CA, response to Sirt5 deficiency, and for two (K90, K480), the inwere similarly affected by Sirt5 deficiency (Figure 4A). Reactome crease was more than 15-fold (Figure 4E). Mass spectrometry 10

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and immunoaffinity enrichment by anti-succinyl-lysine antibody also identified UQCRC2 of mitochondria ETC complex III as a target of Sirt5 (Figure 4G; Table S1). In addition, NDUFB8 of complex I showed increased pull-down by anti-succinyl-K antibody, although NDUFB8 itself was not identified as a Sirt5 target by mass spectrometry, potentially because NDUFB8 is in strong association with other subunits in complex I, which are succinylated targets of Sirt5. Perhaps the most interesting of the succinylated proteins observed in BAT was UCP1. UCP1 is unique to brown fat and critical for its thermogenic function (Cannon and Nedergaard, 2004). UCP1 has not been previously identified as being succinylated or a Sirt5 target, however, in 5-BKO mice housed at room temperature, mass spectrometry revealed 4- and 15-fold increases in UCP1 relative succinylation levels at K56 and K151 as compared to control (Figure 4F). Similar fold increases in relative succinylation on these two sites also occurred when 5-BKO mice were housed under cold acclimated condition (Figure 4F). These changes in the succinylation of UCP1, as well as SDHA/ SDHB and GLUD1, were confirmed by western blotting with protein-specific antibodies after immunoprecipitation with anti-succinyl-K antibody (Figure 4G). Hypersuccinylation Due to Sirt5 Deficiency Impairs Mitochondria Respiration and Enzymatic Activity To determine if the increased succinylation and/or malonylation of mitochondrial OxPhos proteins altered their functions, we performed Seahorse analysis on mitochondria isolated from BAT of 5-BKO and floxed control mice. When compared to control, mitochondria from 5-BKO mice had a 30% reduction in oxygen consumption rate (OCR) when pyruvate and malate were used as substrates for complex I (Figure 5A). With addition of rotenone to block complex I and succinate as substrate for complex II, OCR was borderline significantly lower in Sirt5 deficient BAT mitochondria compared to control, indicating somewhat impaired complex II activity (Figure 5B). However, in the presence of antimycin to block complex III and N, N, N0 , N0 -tetramethyl-p-phenylenediamine (TMPD) as an electron donor for cytochrome c in complex IV, OCR only trended to be lower in BAT mitochondria lacking in Sirt5 without reaching significance (Figure 5C), consistent with the fact that complex I had the most Sirt5 targets for de-

succinylation while complex IV had much fewer (Figures 4B and S5A; Table S1). SDH, as part of complex II, not only relays electrons in respiration chain, but also participates in the TCA cycle, converting succinate to fumarate. Metabolomic analysis of BAT extracts revealed that Sirt5 deficiency led to a significant 27% decrease in fumarate levels, with no significant change in succinate concentration (Figure 5D, E), consistent with a decrease in SDH activity. Indeed, direct assessment of SDH activity in isolated mitochondria from Sirt5 KO BAT showed a 20% reduction in SDH activity compared to control (Figure 5F). Sirt5 Deficiency Impairs Brown Adipocyte FAO, GDH Activity, and Metabolic Flexibility To determine the cell autonomous effects of Sirt5 deficiency on BAT function, we immortalized brown preadipocytes from neonatal Sirt5-floxed mice with SV40 large-T antigen (Fasshauer et al., 2000) and transduced them with GFP or GFP-Cre adenovirus to induce Sirt5 KO in vitro and allow isolation of the transduced cells by fluorescence-activated cell sorting (FACS). Sirt5 deficiency did not affect brown fat differentiation, as shown by normal oil red O staining (Figure 5G) and high levels of PPARg protein expression following differentiation (Figure 5H). Given that many enzymes in the fatty acid oxidation pathway were substrates of Sirt5 for desuccinylation (Figures 4B and S5B), we compared the ability of Sirt5 KO and floxed brown adipocytes to oxidize 14C-palmitate. This revealed an 30% reduction in 14 CO2 release in Sirt5 KO cells versus controls (Figure 5I). Furthermore, this was incomplete oxidation, as indicated by a lower ratio of radiolabeled CO2 to radiolabeled acid soluble metabolites (ASM) in the Sirt5 KO cells (Figure 5I). A similar fatty acid oxidation defect was also observed in the isolated mitochondria from Sirt5 KO BAT (Figure 5J). Likewise, Sirt5 KO cells showed impaired glutamate oxidation under basal conditions (Kreb-Ringer HEPES buffer with 100 mM glutamate). Thus, Sirt5 KO brown adipocytes had a nearly 40% reduction in 14CO2 release derived from 14 C-glutamic acid (Figure 5K). Interestingly, addition of leucine, an allosteric activator of GLUD1 (Tomita et al., 2011), resulted in 90% rescue of 14CO2 release from Sirt5 KO brown adipocytes compared to control, whereas glutamate oxidation in the floxed cells was not affected by addition of leucine

Figure 5. Hypersuccinylation Due to Sirt5 Deficiency Impairs Mitochondria Respiration and Enzymatic Activity (A–C) Oxygen consumption rate (OCR) in Seahorse flux assay using isolated mitochondria of BAT from overnight fasted floxed and 5-BKO mice using pyruvate (A), succinate (B), or TMPD (C) as substrate. n = 10 wells from 5 mice per genotype. Data are represented as mean ± SEM. (D and E) TMPD: N, N, N0 , N0 -tetramethyl-p-phenylenediamine (D) Succinate and (E) fumarate concentrations in 50% aqueous acetonitrile homogenates of BAT from random fed floxed and 5-BKO mice. n = 5 versus 6. Data are represented as mean ± SEM. (F) Succinate dehydrogenase (SDH) activity in BAT mitochondria isolated from floxed and 5-BKO mice. n = 7 versus 5. Data are represented as mean ± SEM. (G) Oil Red O staining of day 6 mature brown adipocytes. (H) Western blot of day 6 mature brown adipocytes. (I) Fatty acid oxidation assay using C14-palmitic acid in day 7 mature floxed and Sirt5 KO brown adipocytes. n = 6. Data are represented as mean ± SD. ASM, acid soluble metabolite. (J) Oxygen consumption rate (OCR) in Seahorse flux assay using isolated mitochondria of BAT from overnight fasted female floxed and 5-BKO mice. n = 10 wells from 5 pools (2 mice per pool) per genotype. Data are represented as mean ± SEM. (K and L) Glutamate oxidation assay (K) and glutamate uptake assay (L) in the presence or absence of leucine in day 7 mature floxed and Sirt5 KO brown adipocytes. n = 6. Data are represented as mean ± SD. *p < 0.05, Student’s t test with Bonferroni correction. (M and N) OCR (M) and ECAR (N) of Seahorse flux assay in day 5 floxed and Sirt5 KO brown adipocytes. n = 10 wells per group, normalized to protein amount. Data are represented as mean ± SEM. Glc, glucose; 2-DG, 2-deoxy-glucose; Eto, etomoxir; R/A, rotenone+antimycin. *p < 0.05, Student’s t test.

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(Figure 5K). Because glutamate uptake was similar between Sirt5 KO and control brown adipocytes (Figure 5L), the decreased glutamate oxidation in Sirt5 KO brown adipocytes is most likely due to impaired GLUD1 activity, as its allosteric activator rescued the defects. The metabolic inflexibility and impaired mitochondria respiration of Sirt5 KO BAT was also recapitulated in vitro. Thus, in glucose- and serum-free DMEM, mimicking the fasting condition, Sirt5 KO brown adipocytes exhibited significantly lower basal OCR than control cells (Figure 5M). After addition of 10 mM glucose, OCR in control cells dropped significantly, while OCR in KO cells remained stable. This may be due to the control cells being more glycolytic under basal conditions, as reflected by higher extracellular acidification rate (ECAR) (Figure 5N), indicating that they could quickly switch to glycolysis to generate energy. In contrast, Sirt5 KO brown adipocytes failed to quickly adapt to this change (Figure 5M), despite a complete recovery of ECAR after addition of glucose (Figure 5L). Inhibiting glycolysis by 2-deoxyglucose had modest effect on OCR in both Sirt5 KO and control brown adipocytes. Finally, when etomoxir, a CPT1a inhibitor that blocks lipid utilization, was added, there was a major increase of OCR in floxed cells, but again OCR in the KO cells remained stable or decreased slightly. With both glucose and fatty acid utilization blocked, brown adipocytes rely on amino acids, such as glutamate (Yelamanchi et al., 2016). The fact that Sirt5 KO brown adipocytes had significantly lower OCR compared to control when glutamate was the major fuel further suggested that hypersuccinylation of GLUD1 due to Sirt5 deficiency impaired its activity. Taken together, in contrast to robust changes in OCR in control cells in response to different energy sources, the OCR curve in Sirt5 KO brown adipocytes remained relatively flat despite changes in substrate usage, demonstrating metabolic inflexibility of these cells. Overacylation Due to Sirt5 Deficiency Impairs UCP1 Activity and Stability UCP1 plays a critical role in BAT thermogenesis by allowing mitochondria to dissipate the mitochondrial proton gradient, thereby producing heat instead of ATP. As noted above, in Sirt5-KO BAT there is hypersuccinylation of two lysine residues in UCP1, K56, and K151 (Figure 4F). These two lysines in UCP1 are conserved among most placental mammals (Figure S6) and localize to unstructured loops of the molecule in the mitochondria matrix (Figure 6A). To determine whether acylation at those two sites affects UCP1 activity, acyl-mimetic K to Q mutations were made at both lysines and the wild type (WT) or 2KQ mutant forms of UCP1 were stably expressed in 3T3-L1 cells by retroviral transduction. The expression of WT and 2KQ UCP1 were similar at mRNA levels (Figure 6B). Western blotting for protein was complicated by the fact that some antibodies to UCP1 have been raised to peptides in the region of potential acylation. Thus, the relative abundance of 2KQ-UCP1 protein appeared less than WT protein when AB10983 was used for immunoblotting, which recognizes a domain encompassing the K151 site (Figure 6C), whereas mutant and WT UCP1 protein levels appeared comparable using an antibody to other regions of UCP1 such as sc-6528 (Figure 6C), consistent with the mRNA

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data. Seahorse analysis of 3T3-L1 cells expressing either empty vector, WT, or the 2KQ mutant form of UCP1 revealed no effect of UCP1 overexpression on basal oxygen consumption rate in the presence of 25 mM glucose (Figures 6D and 6E). As expected, oligomycin treatment decreased OCR in all three cell types, but the absolute OCR value remained significantly higher in cells expressing WT UCP1 compared to the other two (Figures 6D and 6E), indicating increased basal proton leak. Addition of 50 mM free fatty acids (FFA, oleate:palmitate = 2:1), which is known to activate UCP1 (Li et al., 2014; Nicholls and Rial, 1999), had modest effect on OCR in cells transduced with empty vector, but was able to by-pass the inhibition of oligomycin and robustly activated uncoupled respiration in cells expressing WT UCP1. Cells expressing the 2KQ mutant also responded, but the OCR increase in response to FFA was only 40% of that in cells expressing WT-UCP1. Addition of FCCP to all these cell lines further increased OCR to similar levels of maximal respiration capacity, and all cell lines showed similar non-mitochondrial respiration after rotenone/antimycin treatment (Figures 6D and 6E). Similarly, reduced UCP1 activity was observed when K56 and K151 were both mutated to the succinyl-mimetic glutamic acid (E) (Figures 6H and 6I). However, despite similar mRNA expression (Figure 6F), the protein level of 2KE-UCP1 seemed to be significantly less than WT when either anti-UCP1 antibody (AB10983 or sc-6528) was used (Figure 6G). This could indicate that either the glutamic acid mutation is antigenically more different from lysine than glutamine, or that mutation of these lysines to glutamic acid decreased stability of the UCP1 protein. To test the latter possibility, 3T3-L1 preadipocytes expressing WT or the mutant forms of UCP1 were treated with cycloheximide, a protein synthase inhibitor, and the UCP1 turnover rate assessed by western blotting (Figure 6J). Indeed, both the 2KQ and 2KE mutated forms of UCP1 showed significantly reduced half-lives decreasing from around 24 h in the WT to around 3.5 h with the mutant protein (Figure 6J). Despite significantly shortened half-life, the steady-state protein amount of 2KQ-UCP1 looked similar to WT indicating that the decreased activity in the 2KQ mutant is largely attributable to decreased function of the protein. Taken together, these results demonstrate that acylation of K56 and K151 constitutes an important layer of regulation for UCP1 affecting both protein turnover and function. Overacylation Due to Sirt5 Deficiency Leads to Autophagy and/or Mitophagy Defect To determine if Sirt5 deficiency affects mitochondria homeostasis in times of changing energy availability, 5-BKO and floxed littermates were subjected to a 24-h fast. While all animals lost similar amounts of weight when fasted and had similar changes in blood glucose and plasma insulin (Figures S7A–S7C), after fasting BAT weight (as a percentage of body weight) was significantly lower in 5-BKO mice versus controls (Figure S7D). Western blot analysis showed that protein succinylation was significantly decreased by fasting in control BAT, but not in 5-BKO BAT (Figure S7E). Similarly, Mito OxPhos protein levels were decreased in the control, but not in 5-BKO, BAT by fasting (Figures 7A and 7B). This retention of Mito OxPhos proteins in Sirt5 KO BAT during fasting was post-transcriptional, as mRNA

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(A) Mouse UCP1 structure model showing location of K56 and K151. (B) UCP1 mRNA expression in confluent 3T3-L1 preadipocytes stably infected with retrovirus expressing MSCV-puro vector or wild type or 2KQ mutant of UCP1. n = 4 in each group. Data are represented as mean ± SD. (C) UCP1 protein expression in confluent 3T3-L1 pre-adipocytes as in (B). (D) Seahorse Flux assay using confluent 3T3-L1 preadipocytes transduced as in (B). Oligo, oligomycin; R/A, rotenone+antimycin. (E) Quantitation of OCR for Seahorse in (D). n = 6 for vector, n = 7 for WT and 2KQ UCP1. Data are represented as mean ± SD. *p < 0.05, Student’s t test with Bonferroni correction. (F) UCP1 mRNA expression in confluent 3T3-L1 preadipocytes stably infected with retrovirus expressing MSCV-puro vector, or wild-type or 2KE mutant of UCP1. n = 4 in each group. Data are represented as mean ± SD. (G) UCP1 protein expression in confluent 3T3-L1 pre-adipocytes as in (F). (H) Seahorse Flux assay using confluent 3T3-L1 preadipocytes transduced as in (F). (I) Quantitation of OCR for Seahorse in (H). n = 6 for vector, n = 7 for WT and 2KE UCP1. Data are represented as mean ± SD. *p < 0.05, Student’s t test with Bonferroni correction. (J) Western blot of 3T3-L1 preadipocytes transduced with WT-UCP1 or the 2KQ/2KE mutant and treated with 20 mg/mL cycloheximide (CHX) for 0, 3.5, and 7 h. Global ubiquitination were used as positive control for CHX effect.

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expression of these proteins was not different between floxed and KO BAT (Figure 7C). qPCR analysis of genes involved in mitophagy and autophagy revealed that autophagy markers, such as Bnip3 and LC3, were significantly induced by fasting in both genotypes, but the induction was much greater in Sirt5 KO BAT (Figure 7D). Expression of mitochondrial fission 1 (Fis1) was also significantly higher in Sirt5 KO BAT compared to control after fasting, indicating more mitochondrial fission. Consistent with this, electron microscopy of BAT from fasted mice revealed that Sirt5 deficiency shifted BAT mitochondrial size distribution to smaller mitochondria (Figure 7E), without affecting mitochondrial structure (Figure 7F). LC3 is important in both autophagy and mitophagy (Tanida et al., 2008). It is synthesized as pro-LC3, cleaved into LC3I, which after conjugation with phosphatidylethanolamine becomes LC3II that inserts into the membrane of the autophago-

7

some. When autophagosomes fuse with lysosomes, LC3II is degraded together with the inner membrane. Western blotting Ubiquitin showed that LC3II protein levels were UCP1 (sc-6528) significantly higher in Sirt5 KO BAT Tubulin compared to the controls, especially under fasting condition (Figures 7A and 7B). The fact that Mito OxPhos proteins accumulated more in Sirt5 KO BAT under fasting condition suggests the accumulation of LC3II was due to defects in autophagosome degradation. Consistent with this, when 5-BKO and floxed mice were fasted for 24 h, more LC3II accumulated in Sirt5-KO BAT than in controls (Figures 7G and 7H). If, however, the lysosome inhibitor leupeptin (Juha´sz, 2012) was given 3 h before sacrifice, LC3II accumulated to the same level in both control and Sirt5-KO BAT (Figures 7G and 7H), indicating that the increase in LC3II in Sirt5 KO BAT was due to a defect in autophagosome degradation. Interestingly, leupeptin treatment significantly decreased global succinylation in BAT from floxed mice but not from Sirt5 KO BAT (Figure S7F), indicating either that blockade of autophagy created a cellular signal mimicking starvation activating Sirt5 and decreasing levels of succinylation, or that autophagy blockade reduced abundance of succinyl-CoA that is required to non-enzymatically succinylate proteins. hrs of CHX (20ug/mL)

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A

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DISCUSSION Protein acylation is an important post-translational modification that can regulate protein function (Stram and Payne, 2016). This is especially true in mitochondria, where acetylation, malonylation, succinylation, and glutarylation can be regulated by the mitochondrial sirtuins, Sirt3 and Sirt5 (Hirschey et al., 2010; Nishida et al., 2015; Park et al., 2013; Rardin et al., 2013; Tan et al., 2014). BAT is highly enriched in mitochondria, and mitochondria in this tissue play an especially important role in regulating energy balance. Much of the regulation of BAT occurs through the activation of UCP1, which dissipates the mitochondrial proton gradient to generate heat (Cannon and Nedergaard, 2004; Matthias et al., 2000; Nedergaard et al., 2001). In BAT, both the number of mitochondria and the level of UCP1 expression are highly regulated by both the state of differentiation of the cell and the level of b-adrenergic stimulation (Cannon and Nedergaard, 2004; Villarroya et al., 2017). UCP1 has also been shown to be regulated allosterically by association with free fatty acids (Divakaruni et al., 2012) and by sulfenylation of Cys253 in response to the ROS generated when UCP1 is activated (Chouchani et al., 2016). In this study, we show that UCP1 can also undergo succinylation, which results in a decrease in UCP1 stability, as both acyl-mimetic 2KQ and succinyl-mimetic 2KE mutant of UCP1 have markedly decreased half-lives compared to their WT counterparts. Succinylation at K56 and K151 is also important in regulating UCP1 function, as the acyl-mimetic 2KQ mutant of UCP1, which were expressed at similar levels compared to WT UCP1, has a 60% decrease in free fatty acid induced UCP1 activation. Although UCP1 succinylation is not increased by cold acclimation when normalized to unmodified UCP1 protein amount, unpublished data from BioRxiv (http://biorxiv.org/lookup/doi/ 10.1101/445718) showed that UCP1 acetylation at K56, K73, K151, and K67 were significantly increased by severe cold, and the acyl-mimetic 4KQ mutation significantly decreased its stability. These results suggest that decreased stability and function of acylated UCP1 may serve as a break to dampen the effect of cold-induced BAT activation so that once the mice were transferred back to warm temperature from cold, the extra UCP1 can be quickly degraded to prevent overheating of the mice. In line with this, cold exposure has been shown to induce a major increase in succinate flux, which activates BAT (Mills et al., 2018). The increased succinyl-CoA that succinylates the newly synthesized mitochondria proteins may also negatively affect their stability and function, serving as a negative

feedback in the system. When Sirt5 is knocked out, the negative feedback gets amplified, which leads to impaired BAT thermogenic function and results in lower core body temperature in Sirt5 KO mice compared to control when acclimated to 5
C. Succinylation of UCP1 is part of a broader program of succinylation-mediated regulation of other mitochondrial enzymes in BAT. Indeed, using mass spectrometry, we have identified a total of 2,404 succinylation sites on 444 proteins in BAT, of which over one-third were upregulated by at least 2-fold with Sirt5 deficiency. These proteins include a broad range of proteins involved in fatty acid oxidation, electron transport chain, and amino acid metabolism. In addition to UCP1, two enzymes identified as important functional targets of succinylation in mitochondrial of BAT are GLUD1 and SDH. Glutamate dehydrogenase 1 (GLUD1) is heavily succinylated in Sirt5 KO BAT, as it is in the liver (Rardin et al., 2013), and this leads to a 45% decrease in its activity, as measured by 14 CO2 release from 14C-glutamic acid. Interestingly, the impaired 14 CO2 release in Sirt5 KO brown adipocytes is rescued by addition of leucine, a known allosteric activator of GLUD1 (Tomita et al., 2011), indicating that leucine binding to GLUD1 outweighs the inhibitory effect of increased succinylation. We also find significant succinylation of two subunits of succinate dehydrogenase in BAT of Sirt5 KO mice. This is associated with a 20% decrease in SDH activity in isolated mitochondria. In vivo, metabolic profiling reveals a decrease in SDH activity with reduced levels of fumarate in Sirt5 KO BAT, while succinate levels remain similar. Moreover, complex II respiration is also impaired in Sirt5 KO BAT mitochondria from overnight fasted mice compared to control. The consistent effect of Sirt5 deficiency on SDH activity in this study, in contrast to the variable effects reported in the liver (Park et al., 2013; Zhang et al., 2017), may reflect the generally higher level of stoichiometry of protein succinylation observed in BAT compared to other tissues. Consistent with other studies on acylation, the effect of elevated succinylation in response to Sirt5 deficiency in BAT is primarily inhibitory. Indeed, functions of enzyme clusters such as complexes I and II of the respiration chain and fatty acid oxidation are also impaired in Sirt5 KO BAT and brown adipocytes, and this correlates with increased succinylation on multiple lysine sites of these proteins. The cumulative effect of impaired functions of these highly acylated mitochondria proteins and enzymes is metabolic inflexibility in 5-BKO mice. In vivo, metabolic inflexibility is manifested as acute cold intolerance after overnight fast, decreased RER at the onset of dark phase, and glucose intolerance in diet-induced obesity.

Figure 7. Overacylation Due to Sirt5 Deficiency Leads to Autophagy and/or Mitophagy Defect (A) Western blot of selected mitochondrial and autophagy and/or mitophagy markers in BAT extracts from 2.5-month-old random fed and 24 h fasted floxed and 5-BKO mice. n = 4 per group. (B) Quantitation of the 24 h fasted group in (A). n = 4. Data are represented as mean ± SEM. *p < 0.05, Student’s t test. (C) qPCR analysis of Mito Oxphos genes in BAT from 2.5-month-old random fed (same mice as in Figure 2C) and 24 h fasted floxed and 5-BKO mice. n = 5 to 6 per group. Data are represented as mean ± SEM. *p < 0.05, Student’s t test. (D) qPCR analysis of autophagy and mitophagy genes in BAT as in (C). (E) Histogram of BAT mitochondria size quantification from 24 h fasted chow fed 2-month-old floxed and 5-BKO mice. Electron microscopic (EM) images of 600 mitochondria from 4 mice per genotype were quantified using ImageJ. Data are represented as mean ± SEM. *p < 0.05, Student’s t test. (F) Representative EM pictures showing mitochondria size and morphology from each genotype, scale bar, 500 nm. (G) Western blot of BAT from 2-month-old floxed and 5-BKO mice 3 h after saline or leupeptin (40 mg/kg) injection following 21 h fast. n = 3. (H) Quantification of LC3II protein amount in (G), normalized to vinculin. Data are represented as mean ± SEM. *p < 0.05, Student’s t test.

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In vitro, metabolic inflexibility is displayed as blunted response to different energy stimuli. Metabolic inflexibility is a common feature for animals with mitochondrial sirtuin deficiency, as Sirt3 KO mice are also less metabolically flexible than their WT littermates and exhibited multiple features of the metabolic syndrome as they age (Hirschey et al., 2010; Jing et al., 2011, 2013). One interesting observation is how Sirt5 deficiency in one tissue may partially compensate for defects in other tissues. For example, while there is clear glucose intolerance in Sirt5-BKO mice when challenged with high fat diet, Sirt5 whole body KO mice tend to be more glucose tolerant than controls (Yu et al., 2013). This is likely due to decreased hepatic gluconeogenesis in Sirt5 whole body KO mice, as gluconeogenesis pathway is an important target of Sirt5 for demalonylation in liver (Nishida et al., 2015), and this may counter the effect of decreased glucose usage in Sirt5 KO BAT. Although Sirt5 whole body KO mice display relatively mild phenotypes under normal physiological conditions (Yu et al., 2013), a number of studies have shown that Sirt5-mediated desuccinylation, as well as demalonylation and deglutarylation, can lead to increased enzymatic activity of the acylated proteins (Kumar and Lombard, 2018) and alter phenotypes of mice under stress conditions. Thus, Sirt5 demalonylates glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and other glycolytic enzymes to promote glycolytic flux in liver (Nishida et al., 2015). Sirt5 also desuccinylates and activates isocitrate dehydrogenase 2 (IDH2) (Zhou et al., 2016), and the rate-limiting ketogenic enzyme 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2) (Rardin et al., 2013), and deglutarylates carbamoyl-phosphate synthase 1 (CPS1) to enhance its activity (Tan et al., 2014). In vivo, Sirt5 KO mice display increased mortality in response to kainite-induced seizures (Li and Liu, 2016), are more susceptible to chemically induced nigrostriatal dopaminergic degeneration (Liu et al., 2015), and display more severe cortical degeneration following ischemic injury (Morris-Blanco et al., 2016). Likewise, Sirt5 knockout in heart results in hypertrophic cardiomyopathy, with increased long-chain acyl-CoAs and decreased ATP levels in heart under fasting condition (Sadhukhan et al., 2016). Similar to heart and neuron, phenotypes of brown fat-specific Sirt5 KO mice are also observed under levels of physiological stress. These poor adaptations to extreme conditions in the absence of Sirt5 are all manifestations of metabolic inflexibility. Both fatty acid oxidation and branched chain amino acid metabolism are important pathways targeted by Sirt5 for desuccinylation, their impaired function likely made the Sirt5 KO BAT more starved during fasting, and thus led to further induction of autophagy genes such as Bnip3 and LC3. Higher Fis1 expression and smaller mitochondria are also observed in Sirt5 KO BAT compared to control BAT after fasting, and smaller mitochondria are known to be more depolarized and more susceptible to mitophagy (Gomes et al., 2011; Twig and Shirihai, 2011). Similar increases in mitochondrial fission have been shown in Sirt5 KO mouse embryonic fibroblasts (MEFs) compared to control MEFs after nutrient starvation (Guedouari et al., 2017). Despite higher induction of autophagy genes and smaller mitochondria, fasting induced autophagy or mitophagy is defective in Sirt5 KO BAT, represented by accumulation of Mito OxPhos proteins and LC3II. Although none of the mitophagy/

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autophagy mediators were identified as Sirt5 targets for desuccinylation in our mass spectrometry analysis, we cannot rule out the possibility that they are Sirt5 targets for demalonylation and/ or deglutarylation. Another potential explanation for the mitophagy defect is that oversuccinylation and/or malonylation in Sirt5 KO BAT makes the mitochondria less recognizable by the autophagy machinery. In conclusion, mitochondrial protein succinylation and Sirt5 play important roles in regulating BAT mitochondria homeostasis. Increased mitochondrial protein succinylation in Sirt5 deficient BAT results in impaired mitochondrial enzyme activity and respiration, defects in mitophagy during nutrient deprivation and metabolic inflexibility. Reversible succinylation also serves as an important regulator of UCP1 function and stability. Malonylation and glutarylation, although not studied in this manuscript, also likely contribute to impaired mitochondrial protein function in Sirt5 KO BAT. STAR+METHODS Detailed methods are provided in the online version of this paper and include the following: d d d

d

d d

KEY RESOURCES TABLE CONTACT FOR REAGENT AND RESOURCE SHARING EXPERIMENTAL MODEL AND SUBJECT DETAILS B MICE B CELL LINE METHOD DETAILS B Acute cold exposure B Core body temperature measurement with implanted sensor B Glucose and insulin tolerance test B Mitochondria isolation B Mitochondria functional assays B Brown adipocyte differentiation and functional assay B Fatty acid oxidation B Glutamate oxidation and uptake B UCP1 KE and KQ mutation B Electron Microscopy B RNA extraction and qPCR analysis B Protein extraction, immunoblot, and immunoprecipitation B Metabolomics analyses QUANTIFICATION AND STATISTICAL ANALYSIS FOR NON-MASS SPEC EXPERIMENTS DATA AND SOFTWARE AVAILABILITY B MASS SPECTROMETRY

SUPPLEMENTAL INFORMATION Supplemental Information can be found online at https://doi.org/10.1016/j. molcel.2019.03.021. ACKNOWLEDGMENTS We thank Joslin flow cytometry core for sorting Sirt5 floxed and KO brown preadipocytes, the Harvard Medical School Electron Microscopy Core for processing BAT for EM analysis, Histology Core for H&E staining of BAT, and all

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the Kahn lab members for discussion. This project was funded by R24 (5R24DK085610-09), an ADA-Pfizer postdoc fellowship (9-17-CMF-016), an S10 instrument grant (1S10 OD016281), and two NIH T32 grants (5T32DK007260-42 and 4T32AG000266-19). AUTHOR CONTRIBUTIONS G.W. and C.R.K. conceived the project and designed the research. G.W. performed metabolic and molecular studies. W.C. cloned the UCP1 2KQ mutant. J.G.M. performed mass spectrometry data collection and analysis and helped in writing, reviewing, and editing the manuscript. B.S. supervised and provided instrumentation and reagents for mass spectrometry. E.V. provided the Sirt5 floxed mice. C.N. performed metabolomics analysis. G.W. and C.R.K. wrote the manuscript. All authors helped edit the manuscript. DECLARATION OF INTERESTS The authors declare no competing interests. Received: July 24, 2018 Revised: February 6, 2019 Accepted: March 20, 2019 Published: April 15, 2019 REFERENCES Akie, T.E., and Cooper, M.P. (2015). Determination of Fatty Acid Oxidation and Lipogenesis in Mouse Primary Hepatocytes. J. Vis. Exp. (102), e52982.

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14 Molecular Cell 74, 1–14, May 16, 2019

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STAR+METHODS KEY RESOURCES TABLE

REAGENT or RESOURCE

SOURCE

IDENTIFIER

Pan anti-succinyllysine antibody

PTM Biolabs

PTM-401, RRID: AB_2687628

acetylated-K

Cell Signaling Technology

9441, RRID: AB_331805

malonyl-K

PTM Biolabs

PTM-901, RRID: AB_2687947

Sirt5

Cell Signaling Technology

8782, RRID: AB_2716763

UCP1

Santa Cruz and Abcam

sc-6528, RRID: AB_2304265; ab10983, RRID: AB_2241462

Mito Oxphos

Abcam

MS604, RRID: AB_2629281

Glud1

Santa Cruz

sc-515542

SDHA

Abcam

ab14715, RRID: AB_301433

Vinculin

Millipore

MAB3574, RRID: AB_2304338

Tubulin

Cell Signaling Technology

2146, RRID: AB_2210545

LC3A/B

Cell Signaling Technology

12741, RRID: AB_2617131

HSL

Cell Signaling Technology

4107, RRID: AB_2296900

Antibodies

phospho-HSL (Ser563

Cell Signaling Technology

4139, RRID: AB_2135495

A/G Plus-Agarose

Santa Cruz

sc-2003, RRID: AB_10201400

PTMScan Succinyl-Lysine Motif [Succ-K] Kit

Cell Signaling Technology

13764

PTMScan Acety-Lysine Motif [Ac-K] Kit

Cell Signaling Technology

13416

Chemicals, Peptides, and Recombinant Proteins Oligomycin

Calbiochem

Cat#: 495455

FCCP

Sigma

C2920-10MG

TMPD

Sigma

87890-5G

Rotenone

Sigma

R8875-1g

Antimycin A

Sigma

A8674-25MG

L-Glutamic acid

Sigma

G1501-100G

sodium succinate

Sigma

S2378-100G

L-Carnitine

Sigma

C0158-5G

ATP

Thermo Fisher

R0441

Coenzyme A

Sigma

C3144-25MG

Rosiglitazone

Sigma

R2408-50MG

2-Deoxy-D-glucose

Sigma

D8375-5G

L-Leucine

Sigma

L8000-25G

Guanosine 50 -diphosphate sodium salt

Sigma

G7127-100MG

Oleic acid

Sigma

O1008-5G

(+)-Etomoxir sodium salt hydrate

Sigma

E1905-5MG

IBMX

Sigma

I5879-100MG

Dexamethasone

Sigma

D4902-25MG

Leupeptin

Cayman Chemical

103476-89-7

3,30 ,5-Triiodo-L-thyronine sodium salt

Sigma

T6397-100MG

L-Ascorbic acid

Sigma

A0278-25G

DL-Malic acid

Sigma

M0875-100G

Indomethacin

Fisher Scientific

ICN19021705

PALMITIC ACID, [1-14C]

PerkinElmer

NEC075H250UC

L-[14C(U)]-Glutamic Acid

PerkinElmer

NEC290E050UC (Continued on next page)

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Continued REAGENT or RESOURCE

SOURCE

IDENTIFIER

10x RIPA Lysis buffer

Millipore

20-188

Protease Inhibitor Cocktail

Biotool

B14003

Phosphatase Inhibitor Cocktail

Biotool

BG1428

Tris (hydroxymethyl) aminomethane

Sigma

252859-500G

Sequanal grade Urea

Thermo Scientific

29700

Halt protease inhibitor

Thermo Fisher

78430

5 mM Trichostatin A

Sigma

T1952

Nicotinamide

Sigma

N0636-100G

Dithiothreitol

Sigma

D9779

Iodoacetamide

Sigma

I1149

Trypsin (sequencing grade)

Promega

V5113

LC-MS grade Formic Acid

Sigma

94318

Oasis HLB 1cc vac cartridges 30mg

Waters

186003908

LC-MS grade TFA

VWR

85183

Empore sorbent disks

3M

98060402173

2.0 mL microcentrifuge tubes

Thermo Fisher

21-402-905

0.65 mL microcentrifuge tubes

VWR International

53550-970

Low-retention pipette tips

VWR International

53503-290

Critical Commercial Assays Mouse insulin ELISA kit

Crystal Chem

90080

SDH activity assay kit

Sigma

MAK197

Pierce BCA Protein Assay Kit

Thermo Fisher Scientific

Cat# 23225

High Capacity cDNA Reverse Transcription Kit

Applied Biosystems

Cat#: 4368813

Succinylated-K sites and fold change, see Table S1

This paper

N/A

Raw western image

https://data.mendeley.com/ datasets/djwj4vff8y/1

N/A

Raw mass spectrometric data

https://massive.ucsd.edu/ProteoSAFe/ static/massive.jsp; ftp://massive.ucsd.edu/ MSV000082491

ID: MSV000082491

Raw mass spectrometric data

http://proteomecentral.proteomexchange.org/ cgi/GetDataset?ID=PXD010205

ID PXD010205

Peptide spectral library and raw quantitative data

http://panoramaweb.org/project/ Panorama%20Public/2018/Schilling%20-% 20BrownAdiposeTissue/begin.view?

N/A

3T3-L1 cells

ATCC

CL-173

Brown preadipocytes

Isolated in the lab

N/A

Mouse: Sirt5 fl/fl

Eric Verdin (PMID:24076663)

N/A

Mouse: UCP1-Cre

The Jackson Laboratory

Stock No:024670

This paper

N/A

MSCV-puro vector

Clontech

PT3303-5

MSCV-puro mUCP1 (WT)

This paper

N/A

Deposited Data

Experimental Models: Cell Lines

Experimental Models: Organisms/Strains

Oligonucleotides Q-PCR primer sequences, see Table S2 Recombinant DNA

(Continued on next page)

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Continued REAGENT or RESOURCE

SOURCE

IDENTIFIER

MSCV-puro mUCP1 (2KE)

This paper

N/A

MSCV-puro mUCP1(2KQ)

This paper

N/A

ImageJ

https://imagej.nih.gov/ij/

N/A

SWATHTuner

PMID: 26302369 https://sourceforge.net/p/ swathtuner/wiki/Home/

N/A

MaxQuant

https://maxquant.org/

N/A

Spectronaut

https://www.biognosys.com/shop/ spectronaut-x

N/A

Skyline

https://skyline.ms/

N/A

PIQED

https://github.com/jgmeyerucsd/PIQEDia

N/A

R

https://github.com/jgmeyerucsd/pRoteomics

N/A

Chow diet

PharmaServ

Mouse Diet 9F

High-fat diet

Research diets

D12492

Software and Algorithms

Other

CONTACT FOR REAGENT AND RESOURCE SHARING Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, C.R. Kahn ([email protected]) EXPERIMENTAL MODEL AND SUBJECT DETAILS MICE Sirt5fl/fl and Sirt5fl/fl; UCP1-Cre mice were maintained on C57BL/6J background. All mice were group housed at 22
C on a 12-hour light/12-hour dark cycle with ad libitum access to food and water. In vivo metabolic parameters were measured utilizing Comprehensive Lab Animal Monitoring System (CLAMS) performed by Joslin Diabetes Center animal physiology core. All animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) and were in accordance with NIH guidelines. CELL LINE 3T3-L1 preadipocytes were purchased from ATCC. Brown preadipocyte were isolated in the lab as previously described (Klein et al., 2002). Briefly, SV40 T-large antigen immortalized brown preadipocytes from Sirt5 floxed neonatal pups were infected with adenovirus expressing GFP or GFP-Cre. Two days after infection, cells were sorted for GFP signal by Joslin Flow Cytometry Core. METHOD DETAILS Acute cold exposure For acute cold exposure, 2 to 2.5 months old male mice on chow diet were transferred from 22
C to 7
C cold room, either with free access to food and water, or pre-fasted overnight and with free access to water only. Body temperature were measured every 1hr using a rectal probe (TH-5 thermalert monitoring thermometer). Core body temperature measurement with implanted sensor Subcue mini Dataloggers (http://www.subcue.com/mini.htm) were programed (delay 16 days before measurement starts and measure every 15 minutes for up to 21 days) and sealed with glass sealer two days before experiments. On the day of surgery, HFD fed Sirt5-BKO and floxed mice were given I.P. injection of 100uL/10 g BW of ketamine/xylazine mix (100 mg/kg Ketamine, 16 mg/kg Xylazine). Furs next to the lower left midline of the belly were shaved and the bare skin cleaned with 70% ethanol soaked cotton ball. The skin and muscle layer were sequentially cut to create a 1cm long opening with a scalper and fine scissor, followed by insertion of the sensor into the abdominal cavity. Muscle layers were closed by suture (ethicon 639. size: 6-0) and skin layers were closed up by skin clips (Clay Adams brand MikRon Autoclip 9mm. Cat No: 427631). Mice were allowed two weeks of recovery before temperature measurement starts and the cold acclimation starts 16 days after surgery. Mice were sacrificed 10 days after being housed at 5
C.

Molecular Cell 74, 1–14.e1–e7, May 16, 2019 e3

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Glucose and insulin tolerance test For glucose tolerance test, mice were fasted overnight and intraperitoneally (i.p.) injected with 1-2 g glucose per kg of body mass. Insulin tolerance tests were performed in nonfasted mice by i.p. injection of 1.5 to 2 mU insulin per kg of body mass. Blood glucose levels were measured at 0, 15, 30, 60, and 120 minutes using a glucose meter (Infinity, US Diagnostics). Mitochondria isolation Mitochondria isolation was performed following published protocols (Cannon and Nedergaard, 2008; Frezza et al., 2007; Sun et al., 2014). Briefly, BAT from overnight fasted floxed and 5-BKO mice were dissected and homogenized using a glass-teflon homogenizer in the isolating buffer containing 300 mM sucrose, 1 mM EDTA, 5 mM MOPS, 5 mM KH2PO4, and 0.2% fatty acid free-BSA (pH7.2). The homogenate were filtered using 100 mm filter mesh, spin down at 800xg for 10 min. The supernatant containing mitochondria were transferred to another tube and spun down again at 8500xg for 10 min. The supernatants were then decanted and walls of the tube were wiped with paper to remove adhering fat. The pellet containing mitochondria were resuspended again in isolation buffer and transferred to a 1.5 mL eppendoff tube, spun down at 8500xg again for 10 min. The cleaned mitochondria pellet was expanded in ROS buffer containing 120 mM KCl, 10 mM HEPES, 5 mM MgCl2, and 2 mM K2HPO4 (pH7.2), spun down again at 8500xg for 10 min and resuspended in minimal amount of ROS buffer prior to determination of protein concentrations using a BCA assay (Pierce). All isolation steps were carried out in the cold room. For western blot and Succinate dehydrogenase (SDH) activity, protease inhibitor cocktails were added in isolation buffer, and the mitochondria pellet were resuspended in RIPA buffer or SDH activity assay buffer for activity measurement using a commercial kit (Sigma MAK197). Mitochondria functional assays The oxygen consumption rates (OCRs) were determined using the XF24 Extracellular Flux Analyzer (Seahorse Bioscience, MA, USA) following the manufacturers’ protocols. For the electron-flow (EF) measurements, isolated mitochondria were seeded at 5 mg of protein per well in XF24 V7 cell-culture microplates (Seahorse Bioscience), then pelleted by centrifugation (2,000x g for 20 min at 4
C) in 1X MAS buffer (70 mM sucrose, 220 mM mannitol, 10 mM KH2PO4, 5 mM MgCl2, 2 mM HEPES, and 1 mM EGTA in 0.2% FA-free BSA; pH 7.2) supplemented with 10 mM pyruvate (no pyruvate if it’s for fatty acid oxidation), 5 mM malate, with a final volume of 500 mL per well. The XF24 plate was then transferred to a temperature-controlled (37
C) Seahorse analyzer and subjected to a 12-min equilibration period and 2 assay cycles to measure the basal rate, comprising of a 1 min mix, and a 3 min measure period each; and compounds were added by automatic pneumatic injection followed by a single assay cycle after each. Concentration of drugs used in the assay for Figures 5A–5C: Rotenone: 2 mM, succinate: 5 mM, antimycin: 4 mM, N, N, N0 , N0 -tetramethyl-p-phenylenediamine (TMPD): 100 mM in 10 mM ascorbate. For fatty acid oxidation (Figure 5J), Basal: 5 mM Malate. FFA: 100 mM Oleate:palmitate (2:1), plus 2 mM L-carnitine, plus 100 mM ATP, plus 5 mM CoA. GDP: 0.5 mM. Brown adipocyte differentiation and functional assay Brown preadipocytes were cultured in DMEM with 10% FBS until two days post confluence (denoted as day 0 of differentiation). BAC differentiation was induced by adding a cocktail containing 0.5 mM IBMX, 125 mM indomethacin, 1 mM dexamethasone and 5 mM rosiglitazone to maintenance medium containing 10% FBS, 20 nM insulin and 1 nM T3. Two days after induction, cells were cultured in the maintenance medium plus 5 mM rosiglitazone. Seahorse flux assay were carried out on day 5 to day 6 differentiated brown adipocytes in XF24 V7 cell-culture microplates following manufacture’s protocol. To make the seahorse running buffer, DMEM base (Sigma D5030) were dissolved in 500 mL millipure water, 1.85 g NaCl (Sigma S3014) were separately dissolved in 500 mL water, the two were combined and 15 mg Phenol Red (Sigma P-5530) were added. 10 mL were removed from above media and 10 mL 100X GlutaMax-1 (GIBCO 35050-061) was added. Assay dependent amount of glucose (G8270) were added and the media was warmed to 37
C before pH adjustment to 7.4 with 10 M NaOH from Sigma. The media was then filter sterilized and stored at 4
C. For mature brown adipocytes, basal and after port injection measurements were looped 4 times except for rotenone/antimycin injection, which is looped twice. Each loop comprises a 4 min mix, 2 min delay and 2 min measure time. OCR and ECAR results were normalized to protein content in each well. Concentration of drugs used for Figures 5M and 5N: Basal: glucose free DMEM running buffer. Glc: 10 mM glucose. 2-DG: 22 mM 2-deoxy-glucose. Eto: 40 mM etomoxir. R/A: 0.1 mM rotenone plus 2.5 mM antimycin. Fatty acid oxidation Fatty acid oxidation assay were performed on day 7 differentiated brown adipocytes following a protocol from Journal of Visualized Experiments (Akie and Cooper, 2015). Glutamate oxidation and uptake Glutamate oxidation assay were performed in a similar way as fatty acid oxidation. Briefly, day 7 differentiated floxed and Sirt5 KO brown adipocytes were starved in DMEM with 1 g/L glucose and 0.5% FBS for 3 hr before switching to Kreb-Ringer HEPES buffer (10 mM NaHCO3, 120 mM NaCl, 4 m M KH2PO4, 1 mM MgSO4, 1 mM CaCl2, 30 mM HEPES, pH7.4) containing 100 mM glutamate. For half number of the wells, 1mM leucine was added 10 min before incubation with 14C labeled glutamic acid for 40 min. Media from

e4 Molecular Cell 74, 1–14.e1–e7, May 16, 2019

Please cite this article in press as: Wang et al., Regulation of UCP1 and Mitochondrial Metabolism in Brown Adipose Tissue by Reversible Succinylation, Molecular Cell (2019), https://doi.org/10.1016/j.molcel.2019.03.021

each well were transferred to another plate and snap frozen in liquid nitrogen (Original plate with the cells were lysed with RIPA buffer for OD protein concentration). 100 mL of 70% perchloric acid were added to each well of the media plate, and the released hot CO2 were trapped in a filter paper plate pre-wetted with NaOH. Glutamate uptake assay were performed in a separate plate, where the cells were incubated with 14C labeled glutamic acid for only 5 minutes, followed by addition of 50 mL of 200 mM cold glutamate to stop the reaction. Then the cells were washed three times with ice cold PBS, and lysed with 0.4 mL RIPA lysis buffer. 250 mL lysates were added into 4 mL scintillation fluid for counting and 4 mL lysates were used to OD protein concentration. UCP1 KE and KQ mutation Figure 6A of UCP1 structure showing the 2 succinyl-K residues is adapted from https://www.proteinmodelportal.org/? pid=modelDetail&provider=MODBASE&template=1okcA&pmpuid=1001082818542&range_from=1&range_to=307&ref_ac= P12242&mapped_ac=P12242&zid=async). Open reading frame of WT and 2KE/2KQ mutant UCP1 were directly synthesized from Integrated DNA Technologies and ligated into MSCVpuro vector. 3T3-L1 cell lines expressing MSCVpuro vector, WT-UCP1 and 2KE/2KQ-UCP1 were generated through retrovirus infection. UCP1 activities were measured by seahorse flux assay in XF24 V7 cell-culture microplates. For confluent 3T3-L1 preadipocytes, basal and after port injection measurements were looped 3 times except for rotenone/antimycin injection, which was looped twice. Each loop comprises a 3 min mix, 2 min delay and 3 min measure time. The 100 mM FFA (Oleate to palmitate 2:1) stock was made by dissolving 400 mg of albumin (BSA) in 10 mL of H2O, followed by adding 209 mL of Oleic acid and 85.47 mg of Palmitic acid to BSA. The suspension was mixed vigorously and heated up in water bath to 60
C for 30 min. Samples were aliquoted and stored at 20
C. Drug concentrations for Seahorse Flux Assay testing UCP1 activity in Figures 6D and 6H, Basal: 25 mM glucose in DMEM running buffer. Oligomycin: 4 mM, FFA: 50 mM for the 2KQ set and 25 mM for the 2KE set. FCCP: 0.25 mM, R/A: 0.1 mM rotenone plus 2.5 mM antimycin. Electron Microscopy Freshly dissected BAT from 24 hour fasted floxed and 5-BKO mice were fixed in 0.2 M Cacodylate buffer containing 2.5% paraformaldehyde, 5% glutaraldehyde and 0.06% picric acid. The EM processing and imaging were carried out at Harvard EM core. Quantification of mitochondria size was performed using ImageJ (NIH). RNA extraction and qPCR analysis mRNA was extracted by homogenizing brown adipose tissues in TRIzol, treating with chloroform, and precipitating in 70% ethanol. cDNA was made using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, catalog 4368813). qPCR was performed utilizing C1000 Thermal Cycler (BioRad, catalog CFX384).). Primer sequences used are listed in Table S2. Protein extraction, immunoblot, and immunoprecipitation For immunoblot, tissues were homogenized in RIPA buffer with protease and phosphatase inhibitor cocktail. For immunoprecipitation, BAT were homogenized in ICK lysis buffer containing 50 mM Tris (pH 7.5), 150 mM NaCl, 5 mM NaF, 25 mM b-glycerol phosphate, 1 mM sodium orthovanadate, 10% glycerol, 1% Triton X-100, and freshly added protease inhibitors. 2 mg of primary antibody was agitated overnight in a cold room, and protein was pulled with protein A/G Plus-Agarose. Proteins were separated using SDSPAGE and transferred to PVDF membrane (Millipore). Quantification of immunoblots was performed using ImageJ (NIH). Metabolomics analyses Previously frozen BAT tissues were homogenized in 50% aqueous acetonitrile containing 0.3% formic acid (50 mg/mL). BAT amino acids, acyl CoAs, and organic acids were analyzed using stable isotope dilution techniques. Amino acid measurements were made by flow injection MS/MS using sample preparation methods described previously (An et al., 2004). The data were acquired using a Micromass Quattro Micro system equipped with a model 2777 autosampler, a model 1525 HPLC solvent delivery system, and a data system controlled by the MassLynx 4.1 operating system (Waters). Organic acids were quantified using methods described previously with Trace Ultra GC coupled to a Trace DSQ MS operating under Xcalibur 1.4 (Thermo Fisher Scientific) (Jensen et al., 2006). Acyl CoAs were measured by LC-MS/MS (where LC indicates liquid chromatography) as described previously (White et al., 2016). QUANTIFICATION AND STATISTICAL ANALYSIS FOR NON-MASS SPEC EXPERIMENTS Two tailed Student’s t test is used for statistical analysis between Sirt5 KO and floxed groups. If more than two groups are compared together, bonferroni correction is applied. All statistical details including the method, exact value of n, dispersion and precision measures can be found in the figure legend. p < 0.05 is considered significant. DATA AND SOFTWARE AVAILABILITY Raw western images can be found in https://data.mendeley.com/datasets/djwj4vff8y/1

Molecular Cell 74, 1–14.e1–e7, May 16, 2019 e5

Please cite this article in press as: Wang et al., Regulation of UCP1 and Mitochondrial Metabolism in Brown Adipose Tissue by Reversible Succinylation, Molecular Cell (2019), https://doi.org/10.1016/j.molcel.2019.03.021

MASS SPECTROMETRY Sample preparation for mass spectrometry experiments BAT from Sirt5 floxed mice and brown fat-specific Sirt5 KO mice (5-BKO) housed at RT and cold acclimated to 5
C were subjected to succinylation affinity enrichment and subsequent mass spectrometric analysis. For each of the 4 mouse groups, we analyzed BAT from 5 mice. We analyzed the samples using an adapted form of the automated PTM identification and quantification using exclusively DIA (PIQED) workflow as described previously (Meyer et al., 2017). Sample preparation and data collection was done blinded with anonymized labels, and unblinded before final data analysis. BAT tissue samples were homogenized in 50 mM Tris buffer pH 7.6 containing 1x HALT protease inhibitor (Pierce), 150 mM NaCl, 8 M Urea, 1 mM trichostatin A (TSA), and 3 mM Nicotinamide. Protein concentrations were determined using the BCA assay, and aliquots of lysate containing 2 mg of soluble protein from each sample were prepared in parallel. Soluble proteins were reduced with 4.5 mM dithioerytrol (DTT) for 30 minutes at 37
C, cooled to room temperature, alkylated with 10 mM iodoacetamide for 30 minutes in the dark, and then enzymatic protein hydrolysis was initiated by addition of trypsin (1:50, w:w, enzyme:substrate). The following day, enzymatically-catalyzed hydrolysis was quenched by addition of formic acid (FA) to 1% final concentration, and samples were frozen at 20
C overnight. The following day, the peptide mixtures were thawed on ice and desalted using Waters’ Oasis HLB 1cc Vac cartridges (30 mg sorbent), and peptides were eluted using 80% acetonitrile (ACN), 0.2% FA, and 19.8% water. Eluted peptides were dried completely in a speedvac and stored at 80
C until further processing. Extra protein beyond the 2 mg per sample used for quantitative comparisons was pooled into one 2.8 mg aliquot, and one 10 mg aliquot, and those protein aliquots were processed as described above for building a BAT-specific PTM spectral library. One-pot Immunoprecipitation of Peptides containing Acetyl-Lysine and Succinyl-Lysine Dried peptides were resuspended in 1.4 mL IAP buffer (50 mM MOPS–NaOH, pH 7.2, 10 mM Na2HPO4, 50 mM NaCl) and dissolved by pipetting. Samples were then simultaneously enriched for both acetyl-lysine and succinyl-lysine as previously described (Basisty et al., 2018). Briefly, resuspended peptide samples were incubated with 10 mL each of antibody-bead conjugates specific for acetyllysine and succinyl-lysine (Cell Signaling Technologies, PTMScan kits #13416 and #13764, respectively). Immunoprecipitation was allowed to proceed overnight at 4
C with gentle mixing. The following day, beads were washed twice with 1 mL ice-cold IAP buffer, and then thrice with 1 mL ice-cold IAP buffer. Bound peptides were eluted sequentially with 45 mL and then 55 mL of 0.15% TFA for 5 minutes each at RT. Eluted PTM peptides were then directly loaded onto in-house made C18 StageTips, desalted with 0.2% FA in water, and eluted with solution containing 50% ACN, 49.8% water, and 0.2% FA. Eluted peptides were dried completely and stored at 80
C until further analysis. Quantitative and qualitative peptide analysis by mass spectrometry All mass spectrometry data were collected using a SCIEX TripleTOF 5600 system coupled to an Eksigent nanoflow liquid chromatography pump and a cHiPLC chromatography system. All online peptide separations were done using a linear gradient from 5% mobile phase B to 35% mobile phase B over 80 minutes. Mobile phase B was then ramped to 80% over 5 minutes, held at 80% B for 8 minutes before returning to 5% B for 25-minute re-equilibration. Data-dependent acquisition was used to identify peptides. Every cycle consisted of one 250 ms precursor ion scan followed by isolating the top 10 most abundant precursor ions between 400-1,250 m/z with signal over 150 counts per second. Tandem mass spectra were accumulated for up to 100 ms collecting fragment masses between 100-2,000 m/z. Dynamic exclusion was enabled for 20 s. Data-Independent acquisition (DIA) was used to quantify peptides. Every DIA cycle consisted of 250 ms precursor ion scan followed by 64 variable-width isolation windows to produce fragment ion spectra. For protein-level quantification using non-enriched peptides, variable windows were the same as recently reported (Collins et al., 2017). For PTM quantification, variable windows were determined based on the distribution of identified peptides using SWATHTuner (Zhang et al., 2015), which are available as part of the Skyline document on panorama (see location below). To identify peptides and build spectral libraries for subsequent protein quantification, non-enriched peptides from each sample were analyzed using nanoflow liquid chromatography, data-dependent acquisition tandem mass spectrometry (nLCDDA-MS/MS). To build spectral libraries for quantification of protein acetylation and succinylation sites, peptides from separate, pooled one-pot enrichments were analyzed by nLC-DDA-MS/MS. For quantification of proteins and PTMs each non-enriched and one-pot PTM enriched sample was analyzed by nanoflow liquid chromatography, data-independent acquisition tandem mass spectrometry (nLC-DIA-MS/MS) as described previously (Basisty et al., 2018; Meyer et al., 2017). Mass spectrometry data analysis Proteins and PTM-containing peptides were identified by database search against the mouse proteome (downloaded from Uniprot on August 10th, 2015) with MaxQuant. Database searches used the default parameters except for the analysis of PTM enrichments variable acetylation and succinylation were allowed. Protein identifications from MaxQuant were imported into Spectronaut to build a spectral library, which was used to quantify proteins. PTM identifications from MaxQuant were imported into Skyline to build a spectral library, and PTMs were quantified as described previously, including use of the protein quantity correction module of PIQED (1). All identifications were filtered to 1% FDR. Tables of MaxQuant identification detail output are available on massive (see link below). Further downstream analysis was done with custom code written in R available from https://github.com/jgmeyerucsd/pRoteomics.

e6 Molecular Cell 74, 1–14.e1–e7, May 16, 2019

Please cite this article in press as: Wang et al., Regulation of UCP1 and Mitochondrial Metabolism in Brown Adipose Tissue by Reversible Succinylation, Molecular Cell (2019), https://doi.org/10.1016/j.molcel.2019.03.021

Mass spectrometry data availability ‘‘Raw mass spectrometry data, spectral libraries as well as tables of identifications and quantification are available on massive (https://massive.ucsd.edu/ProteoSAFe/static/massive.jsp, ID: MSV000082491, direct link: https://massive.ucsd.edu/ProteoSAFe/ dataset.jsp?task=a40b6049135c4865b9aedba5f24bafe7). The Skyline document containing the spectral library and quantitative data is available from panorama (https://panoramaweb.org/project/Schilling/BrownAdiposeTissue/begin.view? and at http:// panoramaweb.org/project/Panorama%20Public/2018/Schilling%20-%20BrownAdiposeTissue/begin.view?).

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