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1 Series:[128_TD$IF] Immunometabolism

2 Review Lysine Deacetylases and Regulated 3 in 4

1 1,2 3 1 5Q1 Melanie[TD$FIRSNAME] R.[TD$FIRSNAME.] Shakespear[TD$SURNAME] ,[TD$SURNAME.] Abishek[TD$FIRSNAME] [TD$FIRSNAME.] Iyer[TD$SURNAME] ,[TD$SURNAME.] Catherine[TD$FIRSNAME] Youting[TD$FIRSNAME.] Cheng[TD$SURNAME] ,[TD$SURNAME.] Kausta[TD$FIRSNAME] [TD$FIRSNAME.] Das[TD$SURNAME] Gupta,[TD$SURNAME.] Amit[TD$FIRSNAME] [TD$FIRSNAME.] Singhal[TD$SURNAME] [TD$SURNAME. ,E] 3,4,5 David[TD$FIRSNAME] P.[TD$FIRSNAME.] Fairlie[TD$SURNAME] ,[TD$SURNAME.] 1,2 and Matthew[TD$FIRSNAME] J.[TD$FIRSNAME.] Sweet[TD$SURNAME] [TD$SURNAME.]1,*

Regulated cellular has emerged as a fundamental process con- Highlights trolling functions, but there is still much to uncover about the Metabolic and immune pathways are precise signaling[105_TD$IF] mechanisms involved. Lysine acetylation in[14_TD$IF] macrophages intimately linked in that key metabolic and energy metabolites have regulates the activity, stability,[142_TD$IF] and/or localization of metabolic enzymes, as a direct influence on pro- and anti- well as inflammatory responses.[143_TD$IF] Two protein families, the classical zinc-depen- inflammatory responses of dent deacetylases (HDACs) and the NAD-dependent[14_TD$IF] HDACs (sirtuins, macrophages. SIRTs),[145_TD$IF] mediate lysine deacetylation. We[146_TD$IF] describe here mechanisms by which In response to danger signals, acti- classical HDACs and SIRTs directly regulate specific glycolytic enzymes, as vated macrophages reprogram nutri- [147_TD$IF] ent metabolic pathways, for example well as evidence that links these protein deacetylases to the regulation of enhancing aerobic glycolysis, to meet glycolysis-related . In these contexts, we discuss HDACs and SIRTs heightened energy requirements and as key control points for regulating immunometabolism and inflammatory out- generate sufficient biomolecules to mount an effective innate immune puts from macrophages. response.

Lysine deacetylases are dual regula- 6 Regulated Glycolysis[149_TD$IF] in Myeloid Cell-Mediated Inflammation and Disease tors of both metabolic pathways and 7Q3 Organismal[150_TD$IF]2 survival requires the capacity to combat infection, metabolize nutrients, and store inflammatory responses of 8 energy for times of need. It is therefore not surprising that metabolism and inflammation, two macrophages. 9 fundamental biological processes that are crucial for sustaining life, are intimately linked. The acetylation status of lysine resi- 10 Macrophages are key sentinel cells charged with the task of detecting and responding to dues on some[148_TD$IF] key metabolic enzymes 11 homeostatic alterations such as pathogen invasion, metabolic stress, and/or tissue damage. can control their enzymatic activity, 12 Flexibility in metabolic pathways allows these cells to rapidly respond to changes in their protein stability,[142_TD$IF] and/or subcellular 13 environment. ‘Naïve’ or resting macrophages utilize glycolysis (Box 1) to generate pyruvate, localization. 14 which is oxidized[153_TD$IF] into acetyl-CoA for use by the tricarboxylic[154_TD$IF] acid (TCA) cycle, feeding electrons Understanding the precise molecular 15 in the form of NADH and FADH2 into mitochondrial oxidative (OXPHOS) to basis of metabolic control of macro- 16 generate ATP. However, in response to proin[123_TD$IF] flammatory stimuli, such as those that activate phage inflammatory processes could lead to novel approaches to target immunometabolism and inflammation. Box 1. The Glycolysis[81_TD$IF] Pathway Q12 Glycolysis[82_TD$IF]46 converts one molecule of into two molecules of pyruvate, two molecules of ATP, and two molecules of NADH via a series of intermediate metabolites. Flux through this pathway is regulated in multiple ways: availability of substrate, amount of rate-limiting present, by metabolites, and PTMs of the enzymes involved. Glucose uptake is mediated by cell-surface glucose transporters, and key control points in this pathway are 1Institute for Molecular Bioscience Q2 the irreversible reactions catalyzed by , phosphofructokinase, and . Hexokinase catalyzes (IMB), IMB Centre for Inflammation the first step of glucose metabolism, the rate-limiting ATP-dependent phosphorylation of glucose into glucose-6- and Disease Research, and Australian phosphate [33]. This phosphorylation event also effectively prevents glucose from exiting the cell and helps commit Infectious Diseases Research Centre, glucose-6-phosphate towards energy metabolism pathways such as glycolysis and the pentose phosphate pathway University of Queensland, Brisbane, [33]. Phosphofructokinase catalyzes[89_TD$IF] the third glycolytic reaction:[90_TD$IF] transfer of a phosphoryl group from ATP to fructose-6- QLD 4072, Australia 2 phosphate, yielding fructose-1, 6-bisphosphate[91_TD$IF] (F-1, 6-BP). These two enzymatic reactions consume ATP;[92_TD$IF] however, Australian[135_TD$IF] Research Council (ARC)[136_TD$IF] the second half of glycolysis generates four[93_TD$IF] molecules of ATP, resulting in the net production of two[94_TD$IF] ATP molecules Centre of Excellence in Advanced through this metabolic pathway. Pyruvate kinase, which catalyzes[89_TD$IF] the final step of glycolysis, converting phosphoe- Molecular Imaging, Institute for nolpyruvate into pyruvate, is activated by the upstream metabolite F-1, 6-BP.[95_TD$IF] In mammalian cells, pyruvate then has two Molecular Bioscience, University of Queensland, Brisbane, QLD[137_TD$IF] 4072, fates:[96_TD$IF] it is either reduced by to lactate or is[97_TD$IF] converted by pyruvate dehydrogenase to acetyl-CoA Australia for use in the TCA cycle. 3Singapore Immunology Network

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(SIgN), Agency for Science, 17 Toll-like receptors (TLRs), rapid energy[15_TD$IF] and biochemical requirements are met by metabolic [138_TD$IF] [156_TD$IF] Technology, and Research (A*STAR), 18 reprogramming that favors aerobic glycolysis over OXPHOS. This phenomenon, known as the Singapore 19 Warburg effect [1], has been well studied in cancer cells and, more recently, in macrophages 4Lee Kong Chian School of Medicine, Nanyang Technological University, 20 and other immune cells [2,3]. Singapore 308232, Singapore 5Vaccine and Infectious Disease 21 Specific alterations in levels of metabolites, as well as in[157_TD$IF] the functions of metabolic enzymes, are Research Centre (VIDRC), Translational Health Science and 22 associated with metabolic reprogramming in activated macrophages [4]. For example, TLR [140_TD$IF] [105_TD$IF] [158_TD$IF] Technology Institute (THSTI), National 23 signaling instructs the cell to increase glucose uptake and convert it to lactate, whereas OXPHOS Capital Region (NCR) Biotech Science 24 is suppressed by[159_TD$IF] disruptions in the electron transport chain [5]. Carbon-tracing[160_TD$IF] experiments [6] Cluster, Faridabad 121001, Haryana, India 25 demonstrate fragmentation at specific points in the TCA cycle, resulting in a build-up of key 26 metabolites such as citrate that can be used for fatty acid synthesis and/or converted to itaconate, [16_TD$IF] fl 27 which has both antimicrobial [7] and anti-in ammatory [8] properties. Succinate accumulates in *Correspondence: 28 activated macrophages, also as a consequence of TCA cycle disruption, and acts as a molecular [email protected] (M.J. Sweet). 29 switchforthe[162_TD$IF] initiationofinflammatoryresponses; thismetabolitestabilizesthe[163_TD$IF] transcriptionfactor 30 HIF-1a, thus enabling inducible expression of proin[123_TD$IF] flammatory and glycolytic genes [9]. Studies in 31 proliferating cancer cells have been highly informative for understanding how metabolic enzymes 32 control immunometabolism in immune cells. For example, in most cells, pyruvate kinase M (PKM) 33 catalyzes[89_TD$IF] the final step of glycolysis –[164_TD$IF] the conversion of phosphoenolpyruvate to pyruvate. 34 However, in rapidly dividing cells and activated immune cells, an alternatively spliced[165_TD$IF] isoform 35 of PKM, PKM2, acts in an alternative[16_TD$IF] role as a transcriptional coactivator[167_TD$IF] to drive expression of 36 glycolyticand inflammatorygenes.Inthis way,PKM2plays acentralroleindrivingtheshifttowards 37 aerobicglycolysis.Froma therapeuticstandpoint,interferingwiththesemetabolicadaptations,for 38 example by inhibiting glycolytic flux using 2-deoxy-D-glucose [9,10] or by interfering with the 39 transcriptional functions of PKM2 [11,12], reduces macrophage inflammatory outputs and 40 inflammation-related pathology. Nonetheless, our understanding of the precise molecular mech- 41 anisms of metabolic control of innate immune processes is still rudimentary.[168_TD$IF] Deciphering such 42 mechanisms, together with identifying the key signaling[105_TD$IF] metabolites and regulatory enzymes that 43 control this metabolic switch, may unlock new opportunities to target immunometabolic circuits in 44 immune cells to dampen inflammation-associated pathology. Lysine deacetylases are key sus- 45 pects given their well-established roles as cancer- and inflammation-associated regulatory 46 enzymes that can also be modulated pharmacologically. Intriguingly, the capacity of metabolic 47 enzymes to be regulated by lysine acetylation seems to be a common mechanism of control 48 (Figure 1, Key Figure), positing this post-translational modification as a possible molecular switch, 49 and an important pharmacological target, for regulating cellular metabolism and inflammation.

50 Roles for Lysine[169_TD$IF] Deacetylases in Immunometabolism in Macrophages 51Q4 Reversible[170_TD$IF] lysine acetylation is a common post-translational modification (PTM) that is now 52 implicated in both immunological and metabolic pathways. This PTM is controlled by histone 53 deacetylases (HDACs, that remove acetyl group from lysine residues) and histone acetyltrans- 54 ferases (HATs, that add acetyl groups to lysine residues). The HDAC family includes the + 55 classical zinc-dependent HDACs (classes I, II and IV) and the NAD -dependent[17_TD$IF]30 class III HDACs, 56 also known as silent mating-type information regulation 2 (sirtuins or SIRTs). Emerging evi- 57 dence suggests that the classical HDACs and SIRTs modify the activity, stability,[142_TD$IF] and localiza- 58 tion of some metabolic enzymes to regulate immunometabolism. Furthermore, in carrying out 59 their biochemical functions, HDACs also control the availability of certain intermediary metab- 60 olites. These can play important secondary signaling[105_TD$IF] roles, directing the fate and function of a 61 cell in response to external stressors and nutritional cues.

62 Like itaconate and succinate, acetyl-CoA is a metabolite used by cells to link nutrient availability 63 and metabolic status with cellular signaling,[105_TD$IF] chromatin structure,[172_TD$IF] and expression.

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Key Figure Examples of Metabolic Pathways Regulated by Deacetylation

Glucose

K

HK K403 K76 K294

G6PD 6PGD K217 K254 NADPH R5P GAPDH PPP

K117 K227 K251 Glycolysis K305 A cycle TC K321 PKM2 PDH K Pyruvate Acetyl-CoA SDH

K433

PKM2

K Deacetylaon acvates

K Deacetylaon inhibits

K Deacetylaon alters localizaon/structure

Figure 1. Examples are provided of how lysine deacetylation regulates metabolic enzymes that play key gatekeeping roles in metabolic pathways. Abbreviations: GAPDH,[3_TD$IF] glyceraldehyde-3-phosphate dehydrogenase; G6PD,[4_TD$IF] glucose- 6-phosphate[5_TD$IF] dehydrogenase; HK, hexokinase; PDH, pyruvate dehydrogenase; 6PGD, 6-phosphogluconate dehydro- genase; PKM2,[6_TD$IF] pyruvate kinase M2; SDH,[7_TD$IF] succinate dehydrogenase;[8_TD$IF] TCA cycle, tricarboxylic acid cycle.

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64 Numerous metabolic pathways, including glycolysis-derived pyruvate oxidation and b-oxida- 65 tion of fatty acids, generate a mitochondrial pool of acetyl-CoA, which is metabolized[173_TD$IF] via the 66 TCA cycle to yield NADH. Distinct cytosolic and nuclear pools of acetyl-CoA can also be 67 created from mitochondrial export or by other enzymatic reactions [13]. Acetyl-CoA in each 68 compartment is used as the donor for HAT-mediated addition of acetyl groups to lysine 69 residues of target proteins. Classical HDACs, which carry out the reverse reaction of lysine 70 deacetylation, generate acetate that can then be converted by acetyl-CoA synthetases to 71 acetyl-CoA [14,15], thus providing an additional means of generating this metabolite. As 72 discussed in this review, deacetylation of lysine residues on rate-limiting metabolic enzymes 73 could also either inhibit or increase enzyme activity to support nutrient flux through a particular 74 pathway. Thus, by regulating both the cellular acetyl-CoA pool and the acetylation status of 75 metabolic enzymes, HATs and HDACs can control cellular metabolism. In keeping with this 76 concept, small-molecule[174_TD$IF] inhibitors of classical HDACs alter cellular metabolism [16,17]. For 77 example, HDAC inhibition in myeloma cells resulted in decreased glucose uptake over a 48 h[175_TD$IF] 78 period as a result of repression of glucose transporter expression and inhibition of the glycolytic 79 enzyme hexokinase [16]. There is also some evidence to suggest that there is interplay between 80 the metabolic shift and the proin[123_TD$IF] flammatory functions of specific HDACs in activated macro- 81 phages [18].

+ 82 NAD[176_TD$IF] is another metabolite used as a coenzyme in metabolic reactions, as well as a co- + 83 substrate for the SIRTs. During glycolysis, NAD [17_TD$IF] is reduced to NADH, and then oxidized[178_TD$IF] back to + 84 NAD during reduction of pyruvate to lactate or during mitochondrial OXPHOS. Cells therefore + 85 use the NAD /NADH[179_TD$IF] ratio to monitor metabolic activity and state. SIRT-mediated[180_TD$IF] + 86 deacetylation of target proteins is coupled to the cleavage of NAD [18_TD$IF] to nicotinamide (NAM),[145_TD$IF] + 87 and the O-acetyl-ADP-ribose moiety of NAD [182_TD$IF] acts as the acceptor of the acetyl group. Dynamic + 88 changes occur in the NAD /NADH[179_TD$IF] ratio during TLR activation of macrophages, and these are 89 sensed by SIRTs to instruct the metabolic shift that supports early-[183_TD$IF] and late-phase responses 90 [19]. NAM acts as an endogenous feedback inhibitor of SIRT activity and has been shown in + 91 multiple systems to inhibit inflammatory pathways. For example, adequate NAD [184_TD$IF] was required 92 for optimal lipopolysaccharide[185_TD$IF] (LPS)-induced tumor necrosis factor (TNF) production and[186_TD$IF] 93 NAM-inhibited SIRT activity to reduce, via translational control, the synthesis of this important 94 inflammatory cytokine [20]. Thus, the[187_TD$IF] acetylation status of metabolic enzymes, as well as levels 95 of metabolic intermediates that are generated by the protein deacetylase activity of classical 96 HDACs (acetyl-CoA) and SIRTs (NAM), control metabolic responses and inflammatory outputs 97 from macrophages.

98 The Lysine[18_TD$IF] Deacetylase Family 99 Classical HDACs 100 Members[189_TD$IF] of the classical HDAC family (classes[190_TD$IF] I, II and IV), encompassing HDAC1[9_TD$IF] –11, utilize 101 zinc as a cofactor in the catalytic site and are subdivided[192_TD$IF] based on the homology of their 102 deacetylase domains [21]. These enzymes developed before[193_TD$IF] the evolution of ,[194_TD$IF] and it 103 is now known that lysine deacetylation is a PTM that regulates many non-histone proteins. 104 Class I HDACs (1, 2, 3,[195_TD$IF] and 8) are mostly restricted to the nuclear compartment and are 105 responsible for histone deacetylation and epigenetic control of . Class IIa 106 HDACs (4, 5, 7,[196_TD$IF] and 9) share high homology in their C-terminal deacetylase domains,[197_TD$IF] and also 107 contain an N-terminal protein interaction region and nuclear export and import sequences. 108 These proteins shuttle between the nucleus and in response to a variety of cell 109 signals such as growth factors. Some reports have also demonstrated that members of this 110 subfamily can localize to other cellular compartments such as the mitochondria [22]. These 111 properties suggest that class IIa HDACs likely exert at least some effects by acting on non-

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112 histone substrates. HDAC6 and HDAC10, which comprise the class IIb subfamily, are 113 cytoplasmic enzymes that are distinguished by their duplicated deacetylase domains. 114 HDAC6 is known to deacetylate multiple cytosolic proteins such as tubulin, HSP90,[198_TD$IF] and 115 peroxiredoxins. The sole class IV member, HDAC11,[19_TD$IF] shares sequence similarity with[20_TD$IF] both 116 class I and II HDACs. Although it has not been as extensively studied as the other classical 117 HDACs, recent reports have demonstrated important functions for this enzyme in immune 118 cells, for example in[201_TD$IF] acting to constrain effector T cell function [23], to repress IL-10 in antigen 119 presenting cells [24], and to regulate proin[123_TD$IF] flammatory cytokines in [25]. Small-[20_TD$IF] 120 molecule inhibitors of classical HDACs are approved clinically for oncological applications and 121 have shown therapeutic promise in experimental models of inflammatory diseases [21].The 122 most widely studied of these are broad-spectrum inhibitors that target all or many of the 123 classical HDACs by binding to zinc within the catalytic site. More recently, class or isoform- 124 selective HDAC inhibitors have been developed by modification of the moiety that interacts 125 with the rim region of the active site [26] or by taking advantage of residue differences in the 126 active sites[203_TD$IF] of class I and IIb versus IIa HDAC enzymes [27]. An in-depth understanding of how 127 HDACs regulate metabolic pathways in immune cells may provide insights into the molecular 128 basis for anti-inflammatory effects of broad-spectrum HDAC inhibitors, and should also help 129 to[204_TD$IF] guide the development of more specific inhibitors targeting individual HDACs for appli- 130 cations in inflammation-related diseases.

131 Sirtuins (SIRTs) 132 The sirtuins,[205_TD$IF] or class III HDACs, are highly conserved from bacteria to humans [28].They + 133 are NAD -dependent[17_TD$IF] protein deacetylases and/or mono-ADP-ribosyltransferases. Mam- 134 mals contain seven sirtuins (SIRT1[206_TD$IF] –7) that have high across their + 135 catalytic and NAD -binding[207_TD$IF] domains [29].However,eachmemberdiffersinsubcellular 136 localization and catalytic activity (Table 1). SIRT1 and SIRT2 shuttle between the nucleus 137 and cytoplasm in a cell cycle- and cell-type dependent manner. SIRT3[208_TD$IF] –5aremitochon- 138 drial sirtuins, while SIRT6 is found exclusively in the nucleus. SIRT7 is found in both the 139 nucleus and cytoplasm. Substrates of nuclear sirtuins include histones and non-histone 140 proteins such as nuclear transcription factors and cofactors, while cytoplasmic and 141 mitochondrial sirtuins play important roles in deacetylating key enzymes involved in 142 glycolysis, fatty acid oxidation (FAO), the TCA cycle,andotheroxidativeandmetabolic[209_TD$IF] 143 pathways [29]. Because[210_TD$IF] sirtuins are expressed throughout the body, they function as 144 cellular energy sensors and modulate a wide range of physiological and metabolic 145 processes. Dysregulation of their activity has been associated with various metabolic, 146 infectious,andneurologicaldiseases,whiletheiractivationreportedlyhasbene[21_TD$IF] ficial 147 effects in some preclinical rodent models [30],makingthempromisingtargetsforphar- 148 macological intervention in human diseases.

149 HDACs as Signaling[21_TD$IF] Hubs in Regulating Immunometabolism 150 Given that the functions of some key metabolic enzymes are regulated by lysine acetylation 151 (below), and that metabolites generated through the actions of lysine deacetylases also control 152 immune processes, these enzymes are likely to be important in controlling cellular metabolism 153 and inflammatory responses. Numerous studies have linked specific classical HDACs [21] and 154 SIRTs [31] to inflammatory responses, but whether these effects relate to effects on metabolic 155 pathways has not yet been widely investigated. Below we describe studies across different 156 biological systems that implicate regulated lysine deacetylation in the control of aerobic 157 glycolysis and the pentose phosphate pathway, and describe how some of these modifications 158 may be involved in regulating inflammatory outputs from innate immune cells such as 159 macrophages.

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Q11 Table 1. Human[28_TD$IF] Sirtuins: Localization, Enzymatic Activity, and Roles in Glycolysis Sirtuin Location[29_TD$IF] Main enzymatic activities Substrates involved in glycolysis and functions Refs[30_TD$IF]

SIRT1 Nucleus, cytoplasm[31_TD$IF] Deacetylation, ADP-ribosylation Activates PGC-1[32_TD$IF] a, which attenuates [65,71,94][35_TD$IF] transcription of glycolytic genes. Regulates transcription of the[3_TD$IF] HIF1A gene by deacetylating H3K14 and also deacetylates HIF-[34_TD$IF] 1a directly, leading to decreased glycolysis. Inhibits glycolysis by deacetylating the glycolytic enzyme PGAM-1

SIRT2 Nucleus, cytoplasm[31_TD$IF] Deacetylation Deacetylates[36_TD$IF] PEPCK that is required for [54,66,95] maintaining glucose homeostasis. Deacetylates[37_TD$IF] K305 of PKM2. Deacetylates[38_TD$IF] HIF-1a

SIRT3 Nucleus, mitochondria[39_TD$IF] Deacetylation Represses glycolysis by destabilizing HIF-1[40_TD$IF] a. [62,67,96] Overexpression of SIRT3[41_TD$IF] increases glycolysis and mitochondrial respiration. Deacetylates K321 of PDH, leading to activation[42_TD$IF] of the PDC

SIRT4 Mitochondria Deacetylation, ADP-ribosylation ADP-ribosylates glutamate dehydrogenase [96,97][4_TD$IF] (GDH), downregulating its activity and promoting glutamine/glutamate metabolism and insulin secretion. Overexpression of SIRT4[43_TD$IF] increases glycolysis and mitochondrial respiration

SIRT5 Mitochondria Demalonylation, deacetylation[45_TD$IF] Protein demalonylation by SIRT5 maintains [96,98][4_TD$IF] glycolysis, and malonylation of K184 of GAPDH results in reduced glycolysis. Overexpression of SIRT5[46_TD$IF] increases glycolysis and mitochondrial respiration

SIRT6 Nucleus Deacetylation, ADP-ribosylation Deacetylates H3K9 at the promoter of the HIF1A[47_TD$IF] [52,84][49_TD$IF] gene, thereby repressing HIF1A[48_TD$IF] transcriptional activity leading to diminished glycolysis under homeostatic conditions. Deacetylates K433 of dimeric PKM2

SIRT7 Nucleus, cytoplasm[31_TD$IF] Deacetylation No current evidence for regulation of glycolysis

160 Lysine Deacetylases[213_TD$IF] and the Control of Glycolysis 161 The Glycolysis Pathway 162 Although glycolysis is a relatively inefficient means of generating ATP, heightened flux 163 through this pathway in macrophages is essential for the[214_TD$IF] rapid generation of energy, 164 biosynthetic intermediates,andcofactorsforenzymes,allofwhicharerequiredforinduc-[215_TD$IF] 165 ible inflammatory responses. Lysine acetylation/deacetylation has a well-established role in 166 controlling metabolic enzymes in the TCA cycle, particularly through the actions of sirtuins[216_TD$IF] 167 (Box 2). Although somewhat less studied, this PTM has also been implicated in regulating 168 the stability and/or activity of some glycolytic enzymes (Figure 2). Below we describe known 169 roles for specificglycolyticenzymesinthecontrolofinnateimmunefunctionsandevidence 170 for regulated acetylation of these enzymes (Table[217_TD$IF] 2 and Figure 3), as well as gene regulatory 171 mechanisms by which classical HDACs and SIRTs influence glycolysis. Interestingly, the 172 pentose phosphate pathway is coupled to glycolysis and inducible effector functions of 173 macrophages, and enzymes in this pathway are also regulated by lysine deacetylases (Box 174 3 and Figure 2).

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Box 2. Acetylation, the TCA Cycle,[98_TD$IF] and Immunometabolism In the mitochondria, eight enzymatic reactions in the TCA cycle utilize[9_TD$IF] pyruvate-derived acetyl-CoA to transfer electrons

to NAD or FAD, generating two ATP, six NADH molecules,[10_TD$IF] and two FADH2 per glucose molecule for use by the ETC.[10_TD$IF] Every TCA enzyme can be acetylated [85] and,[102_TD$IF] similarly to glycolytic enzymes, distinct changes in TCA cycle enzyme expression and/or function are crucial for the[103_TD$IF] accumulation of metabolic intermediates such as succinate, itaconate, malate,[104_TD$IF] and fumarate that act as signaling[105_TD$IF] molecules to regulate macrophage immunometabolism. For example, citrate exported from the mitochondria can be converted into cytosolic acetyl-CoA and used for histone acetylation and the[106_TD$IF] expression of glycolytic genes [76]. Citrate can also be converted to itaconate, which inhibits succinate dehydrogenase (SDH) to promote accumulation of succinate [8]. This provides a mechanism to promote proin[107_TD$IF] flammatory (e.g., IL-1b) and reduce anti-inflammatory (e.g.,[108_TD$IF] IL-10) gene expression [9,86], as well as to[109_TD$IF] amplify ROS production [86]. SDH has dual functions:[10_TD$IF] within the TCA cycle it oxidizes[1_TD$IF] succinate to fumarate but also operates as complex[12_TD$IF] II of the ETC. Acetylation of SDH at the active site interferes with substrate binding and decreases enzymatic activity [87], whereas deacetylation by mitochondrial[13_TD$IF] SIRT3 increases SDH enzymatic activity [87,88]. Other mitochondrial enzymes that can be targeted by SIRT3 include acetyl-CoA synthetase 2, isocitrate dehydrogenase,[14_TD$IF] and glutamate dehydrogenase. Thus, regulated acetylation provides a means of controlling the activity of SDH and other mitochondrial enzymes, and,[15_TD$IF] in turn, immunometabolism.

175 (HKs) 176 The four isoforms of hexokinase (HK-I, HK-II, HK-III,[218_TD$IF] and HK-IV, encoded by HK1, HK2, HK3,[17_TD$IF] 177 and GCK, respectively) vary in tissue expression and subcellular location,[219_TD$IF] and also differ in 178 their affinity towards glucose [32]. HK enzyme activity is inhibited by its enzymatic product, 179 glucose-6-phosphate, enabling tight regulation through feedback inhibition [33]. Thus, dys- 180 regulated expression and/or activity of HKs is a major contributor to the[20_TD$IF] increased glucose 181 gorging and elevated glycolysis that facilitate cancer[21_TD$IF] cell proliferation and metastasis [33,34]. 182 HKs also have key functions in innate immunity. HK-I-dependent glycolysis was required for 183 activation of the NLRP3 inflammasome in LPS-primed mouse macrophages [35]. By[2_TD$IF] contrast, 184 HK-II plays a dominant role in TLR-inducible glycolysis in mouse dendritic cells [36], implying 185 that different HKs may control the glycolytic shift in different immune cell populations. This 186 study also showed that localization of HK-II to mitochondria[23_TD$IF] was important for the glycolytic 187 shift in DC. Intriguingly, another study showed that peptidoglycan activated the NLRP3 188 inflammasome through N[24_TD$IF] -acetylglucosamine-mediated inhibition of HK, resulting in its dis- 189 sociation from mitochondrial outer membranes [37]. Thus, it would appear that the subcellular[25_TD$IF] 190 localization of HKs dictate their immunological functions. Whether such functions are con- 191 trolled by lysine acetylation remains[26_TD$IF] be determined. However, a global acetylomics screen 192 identified HK-I and HK-II as acetylated proteins in human cell lines [38]. Furthermore, broad-[27_TD$IF] 193 spectrum classical HDAC inhibition in multiple myeloma cells decreased HK enzymatic 194 activity [16], suggesting that one or more of the classical HDACs may contribute to deace- 195 tylation-mediated activation of this crucial[15_TD$IF] step in the glycolytic pathway. These studies give 196 impetus to future investigations on regulated deacetylation of HK during macrophage 197 activation.

198 Glyceraldehyde-3-Phosphate[28_TD$IF] Dehydrogenase (GAPDH) 199 GAPDH catalyzes the conversion of glyceraldehyde-3-phosphate (G3P) to 1,3-bisphospho-D- 200 glycerate. This enzyme was originally thought to reside solely in the cytoplasm as a tetramer of 201 four identical 37 kDa subunits that perform its well-characterized glycolytic function [39]. 202 However, based on its oligomerization[29_TD$IF] state, PTM, and subcellular localization,[230_TD$IF] GAPDH can 203 execute a multitude of other important cellular functions. Interestingly, in mammalian cancer 204 cells GAPDH[231_TD$IF] was recently predicted to be a key enzyme in regulating aerobic glycolysis [40]. 205 That study proposed that the regulation of energy metabolism and cellular homeostasis by 206 GAPDH is much more complex than was[23_TD$IF] initially thought. Emerging evidence suggests that 207 PTMs[23_TD$IF] of GAPDH may control its subcellular localization[230_TD$IF] and functions, acting as a molecular 208 switch for both inducible glycolysis and proin[123_TD$IF] flammatory mediator production.

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(A) Enzymes in glycolysis

HK HDACi

HK Glucose G6P

ATP ADP

GAPDH PCAF ac-K217 PCAF ac-K254

ac-K117/227/251 Acvity

K254 PKM2 HDAC5 PCAF SIRT2

ac-K305 PKM2 PKM2 HSC70

PKM2 Degradaon

p300

PKM2 ac-K433 SIRT6 K433

K433 TF

HDAC

(B) Enzymes in the PPP

NADP+ NADPH HDAC4 ac-K403 SIRT2 ac-K76 ac-K294 G6PD 6PGD G6P 6PGL 6PG Ru5P

NADP+ NADPH

Q9 Figure 2. [10_TD$IF]Acetylation Regulates Glycolytic Enzyme Form and Function. (A) Examples of glycolytic enzymes regulated by acetylation, as well as the specific acetyltransferases/deacetylases implicated in these processes. Hex- okinase (HK), a rate-limiting enzyme in the glycolysis pathway, phosphorylates glucose to generate glucose-6-phosphate (G6P). This enzyme is known to be lysine-acetylated [38], and histone[1_TD$IF] deacetylase inhibition (HDACi) reduced the activity of this enzyme [16][12_TD$IF] , implicating HK deacetylation in inducible glycolysis in activated macrophages. Multiple lysine residues of (Figure legend continued on the bottom of the next page.)

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209 In macrophages, binding of GAPDH to the 3[24_TD$IF] 0-untranslated region (UTR) of TNF mRNA couples 210 expression of this key inflammatory cytokine with the cellular metabolic state. When glycolytic 211 rate is low, binding of GAPDH represses TNF expression through post-transcriptional mecha- 212 nisms. Enhanced glycolysis dissociates the complex, presumably by increasing the amount of 213 GAPDH substrate (G3P) to compete at the enzymatic site, simultaneously enabling inducible 214 TNF expression and regulated immunometabolism [41,42]. A similar mechanism occurs in T 215 cells, where GAPDH directly binds to mRNA encoding IFN-[235_TD$IF] g and prevents its translation [43]. 216 When T cells are activated, increased glucose influx promotes the disassociation of GAPDH 217 from IFN-[236_TD$IF] g mRNA, allowing translation and secretion of this effector cytokine, as well as 218 amplified aerobic glycolysis that is required for T cell proliferation [43]. Intriguingly, another study 219 showed that GAPDH was recruited to the plasma membrane in activated macrophages. At[237_TD$IF] the 220 membrane it functioned as an immune receptor for plasminogen, controlling the recruitment of 221 macrophages to sites of inflammation [44]. Collectively, these studies suggest that the cellular 222 metabolic state and subcellular localization of GAPDH dictates its role in regulating 223 immunometabolism.

224 Emerging evidence suggests that regulated lysine acetylation of GAPDH may be an important 225 switch that controls its dual metabolic and immune regulatory functions. For example, in 226 response to systemic bacterial infection, acetylation[238_TD$IF] of GAPDH on lysine (K) 217 in mouse + 227 memory CD8 T cells supported GAPDH enzymatic activity. The enhanced glycolytic rate was 228 required for optimal memory recall responses to Listeria monocytogenes [45]. In cancer cells, 229 high glucose levels promoted the acetylation of GAPDH at K254, enhancing its enzymatic 230 activity to support proliferation [46]. This inducible acetylation was mediated by the acetyl- 231 transferase PCAF, whereas glucose deprivation promoted an interaction with HDAC5, its 232 deacetylation, and a reduction in GAPDH enzymatic activity [46]. Acetylation at other residues 233 (K117, 227,[240_TD$IF] and 251) was also required for GAPDH nuclear translocation under apoptotic 234 stress in NIH3T3 cells [47]. Whether the regulation of GAPDH acetylation by glucose availability 235 in macrophages alters its subcellular localization and/or interaction with cytokine mRNAs is 236 unknown at this stage. Nonetheless, the effects of stimuli that control immunometabolism in 237 macrophages, such as TLR ligands, on GAPDH acetylation/deacetylation warrant further 238 investigation.

239 The Pyruvate[241_TD$IF] Kinase M Isoform PKM2 240 The various non-glycolytic functions of PKM2 in inflammatory cells position it as a hallmark 241 enzyme that bridges metabolism and immunity. In the glycolytic pathway, tetrameric PKM2 242 catalyzes[89_TD$IF] the rate-limiting final step of glycolysis by transferring the phosphate group of 243 phosphoenolpyruvate to ADP, producing pyruvate and ATP. This tetrameric form relies on 244 the availability of the upstream intermediate fructose-1,6-bisphosphate (F-1,6-BP), and

GAPDH can be acetylated (ac).[13_TD$IF] PCAF-mediated acetylation of GAPDH on K117/227/251 was required for nuclear translocation and subsequent moonlighting functions in gene regulation. Acetylation of lysine[14_TD$IF] (K) 217 [45] and K254 [46] enhanced the glycolytic enzymatic activity of GAPDH,[15_TD$IF] whereas deacetylation by HDAC5 reduced it [46]. Acetylation of lysine residues on PKM2 can regulate its functions in multiple ways. PCAF-mediated acetylation of K305 promotes its[16_TD$IF] association with chaperone HSC70, targeting PKM2 for lysosomal degradation [53],[17_TD$IF] and SIRT2 can deacetylate this residue to promote tetramer formation [54]. PCAF-mediated acetylation on K433 of the cytosolic, tetrameric form of PKM2 interferes with FBP binding and promotes PKM2 dimer formation [51]. Nuclear SIRT6 can deacetylate dimeric PKM2, enhancing its nuclear export [52].(B)[18_TD$IF] Key enzymes in the pentose phosphate pathway (PPP) are[19_TD$IF] regulated by acetylation. Acetylation of K403 on G6PD inhibited enzymatic activity, whereas deacetylation by SIRT2 enhanced its activity [91]. 6PGD can be regulated by acetylation at two sites; K76 acetylation promotes NADP+[20_TD$IF] binding and K294 acetylation Q10 promotes active dimer formation. HDAC4 can deacetylate both of these residues [93]. Abbreviations:[21_TD$IF] G6P, glucose-6- phosphate; 6PGL, 6-phosphogluconolactone; 6PG, 6-phosphogluconate; Ru5P, ribulose-5-phosphate.

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Table[50_TD$IF] 2. Identification of Lysine[51_TD$IF] Residues in Glycolytic Enzymes Regulated by Acetylation Enzyme[52_TD$IF] Acetyl-lysine residue(s) Functional[53_TD$IF] consequence of lysine acetylation/ Refs[54_TD$IF] deacetylation

Hexokinase (HK)

Hexokinase[5_TD$IF] domain-containing 1 K173a, K174a Currently unknown, residues identified by global [85] proteomic screening

HK-I K453a[57_TD$IF]6 Currently unknown, residues identified by global [38][56_TD$IF] proteomic screening HK-II K337a,[58_TD$IF] K346a

Phosphofructokinase-1 (PFK1)

6-Phosphofructokinase type C K797a[57_TD$IF]9 Currently unknown, residues identified by global [85][60_TD$IF] proteomic screening Phosphofructokinase, muscle K113a[61_TD$IF]

Phosphofructokinase, platelet K776a[62_TD$IF]

6-Phosphofructokinase, type K762a[63_TD$IF]

Aldolase K12, K41, K146 Acetylation of K146 is[64_TD$IF] predicted to decrease [99][65_TD$IF] enzyme activity

Triosephosphate isomerase (TPI) K222a[57_TD$IF] Currently unknown, residues identified by global [99][56_TD$IF] proteomic screening

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) K117, K227, K251, K254 Acetylation of K117, K227, and[6_TD$IF] K251 is required [46,47][65_TD$IF] for nuclear translocation of GAPDH. HDAC5- mediated deacetylation of K254 decreases enzyme activity

Phosphoglycerate kinase-1 (PGK1) K388 ARD1-mediated K388 acetylation of PGK1 [100][67_TD$IF] increases kinase activity to phosphorylate Beclin1 S30 and promote autophagy initiation and tumorigenesis

Phosphoglycerate mutase (PGAM)

PGAM1[68_TD$IF] K251, K253, K254 Acetylation[69_TD$IF] enhances enzyme activity. SIRT1- [94][70_TD$IF] mediated deacetylation decreases enzyme activity

PGAM2 K100 Acetylation[71_TD$IF] decreases enzyme activity. SIRT2- [101][70_TD$IF] mediated deacetylation increases enzyme activity

PGAM1/2 K100, K106, K113, K138 SIRT2-mediated[72_TD$IF] deacetylation decreases [102][70_TD$IF] enzyme activity

Enolase K71a[73_TD$IF] Currently unknown, residues identified by global [99][74_TD$IF] proteomic screening

Pyruvate kinase M2 (PKM2) K305, K433 K305[75_TD$IF] acetylation decreases enzyme activity and [51,53][70_TD$IF] increases lysosomal degradation. K433 acetylation prevents activation and promotes nuclear accumulation and kinase activity

Pyruvate dehydrogenase (PDHA1) K321 SIRT3-mediated[72_TD$IF] deacetylation increases PDHA1 [62][76_TD$IF] enzyme activity and attenuates the Warburg effect

Lactate dehydrogenase A (LDHA) K5 Acetylation[7_TD$IF] decreases enzyme activity. SIRT2- [103,104][78_TD$IF] mediated deacetylation increases enzyme activity. LDHA acetylation decreases cell proliferation and migration. SIRT3-mediated deacetylation increases enzyme activity aLysine residues identified using global proteomic screening in the indicated reference;[79_TD$IF] peptide sequences were matched to reference sequences to identify[80_TD$IF] the specific lysine residue(s) indicated here.

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[23_TD$IF]46 HDACs/SIRTs Figure 3. Deacetylases as Potential Links Between Regulated Metabo- Proin lism and Inflammatory Outputs of fl Individual HDACs and An ammat Macrophages. (e.g., cyt fl c enzymes microbial response SIRTs have both pro- and anti-in amma- ory response tory functions in macrophages. In the (e.g., ROS) okines) Glycoly context of the former, these lysine dea- (e.g., HK, PKM2) PPP enzymes cetylases positively regulate the function (e.g., G6PD) or expression of key enzymes in the gly- colysis pathway (e.g., HK, PKM2) and the PPP (e.g., G6PD), as well as specific func- tional responses of macrophages (e.g., proinflammatory cytokine production, antimicrobial responses). Thus, deacety- Inducible glycolysis Inflammaon lases are strong candidates as molecular links between regulated metabolism and and PPP and host defense biological responses of macrophages (teal arrows). In addition, HDACs and SIRTs are also likely to regulate some macrophage functions in host defense and inflammation independently of meta- bolic control (orange arrow). Abbrevia- tions: HDAC, histone deacetylase; PPP, pentose phosphate pathway; ROS, reac- tive oxygen species; SIRT, sirtuin.

245 dissociation of the tetramer to a dimeric form occurs when concentrations of this intermediate 246 are low [48] or when phosphorylation of Y105 disrupts binding of F-1,6-BP [48]. The reduced 247 enzymatic activity of dimeric PKM2 allows build-up of key glycolytic intermediates to be used as 248 precursors for protein, lipid,[24_TD$IF] and synthesis. Extracellular signals such as growth 249 factors and inflammatory stimuli can also induce[243_TD$IF] the formation of dimeric PKM2, which 250 reportedly has functions that are independent of its role as a glycolytic enzyme, including 251 as a nuclear protein kinase and a coactivator[167_TD$IF] of transcription factors [11]. For example, TLR4 252 signaling[105_TD$IF] in macrophages induced association of PKM2 with HIF-1a in the nucleus to drive 253 expression of glycolytic (e.g.,[24_TD$IF] Ldha) and inflammatory genes (e.g.,[245_TD$IF] Il1b) [11]. In addition, in 254 macrophages from patients with coronary artery disease, nuclear translocation of dimeric

Box 3. Control of the Pentose[16_TD$IF] Phosphate Pathway (PPP) by Lysine Acetylation The[17_TD$IF] PPP utilizes glycolysis-derived glucose-6-phosphate (G6P) to generate reducing equivalents in the form of NADPH and ribose-5-phosphate for nucleotide synthesis. In TLR-activated macrophages, flux through the PPP is enhanced to provide NADPH for the oxidative respiratory[18_TD$IF] burst and to generate glutathione as a buffer for ROS. Glucose-6- phosphate dehydrogenase (G6PD), the first and rate-limiting enzyme in the PPP, has been extensively implicated in inflammatory responses and is regulated by acetylation. G6PD catalyzes[89_TD$IF] the oxidation of G6P into 6-phosphoglu- cono-d-lactone, with concomitant reduction of NADP+[19_TD$IF] to NADPH. Active G6PD exists as a dimer of two identical monomers, with each monomer containing a G6P-binding site and a catalytic coenzyme site that[120_TD$IF] binds NADP+. LPS and free fatty acids stimulate G6PD expression in macrophages, and its expression is elevated in adipose tissue macrophages from obese patients. The elevated expression of this enzyme promoted oxidative stress and activated the p38 MAPK and NF-[12_TD$IF] kB signaling pathways to drive expression of proin[123_TD$IF] flammatory cytokines [89]. Conversely, deletion of G6PD attenuated macrophage inflammatory responses, improved insulin sensitivity,[124_TD$IF] and reduced chronic tissue inflammation in a model of diet-induced obesity [90]. Targeting aberrant expression and/or activity of G6PD would thus be predicted to reduce inflammation-associated pathology. Proteomic acetylome profiling has identified seven lysine residues on G6PD that can be acetylated (K89, K171, K386, K403, K432, K497,[125_TD$IF] and K514), and subsequent studies have identified a regulatory role for[126_TD$IF] K403 acetylation. In HEK293 cells, active dimer formation was prevented by K403 acetylation, thereby reducing G6PD enzymatic activity [91]. Conversely,[127_TD$IF] deacetylation of this residue by SIRT2 activated enzyme activity and enhanced NADPH production [91,92]. Given the importance of the respiratory burst for antimicrobial responses, a detailed understanding of how acetylation regulates PPP enzyme activity in macrophages may have implications for host-directed antimicrobial therapies.

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255 PKM2 facilitated binding with STAT3 to promote[246_TD$IF] the expression of IL-1b and IL-6 [12]. Despite 256 such evidence (and that described below), the protein kinase activity of PKM2 remains 257 somewhat controversial; for example, Hosios et[247_TD$IF] al. [49] could find no evidence of direct transfer 258 of phosphate from ATP to protein by PKM2. Interestingly, dimeric PKM2 was also detected in 259 the circulation of cancer patients, and was shown to promote [50]. The contri- 260 butions of extracellular PKM2 to inflammation are not well understood at this stage, and indeed 261 there[248_TD$IF] is still much to uncover about the specific molecular mechanisms by which intracellular 262 PKM2 drives macrophage inflammatory responses.

263 Several lysine residues in PKM family members can be regulated by acetylation. K433 is located 264 in PKM2-specific 10 and has been linked to the switch between its cytoplasmic metabolite 265 kinase activity and nuclear protein kinase function. Acetylation of this residue by the HAT p300 266 interfered with the ability of F-1,6-BP to bind to[249_TD$IF] PKM2, driving the switch to a dimer and 267 promoting nuclear accumulation and its protein kinase activity [51]. In cancer cells, this 268 promoted cell proliferation and tumorigenesis, and cotreatment[250_TD$IF] with a pan inhibitor of classical 269 HDACs (trichostatin A) and NAM enhanced this modification, implicating both HDACs and 270 SIRTs in its regulation. Additional studies in cancer cells showed that nucleus-localized[251_TD$IF] SIRT6 271 can deacetylate K433 of dimeric PKM2 to promote nuclear export and loss of its protein kinase 272 activity [52]. Also in cancer cells, PCAF-mediated acetylation of K305 in response to high 273 glucose decreased classical pyruvate kinase activity, enhanced the[103_TD$IF] accumulation of glycolytic 274 intermediates,[25_TD$IF] and promoted autophagy-mediated degradation of PKM2 [53]. SIRT2 deace- 275 tylated this residue to promote tetrameric pyruvate kinase activity [54], and treatment with 276 trichostatin A plus NAM decreased PKM2 protein expression [53], indicating that deacetylation 277 of this residue is able to stabilize[253_TD$IF] the protein. Finally, in breast cancer cells, another pan classical 278 HDAC inhibitor, SAHA, induced acetylation of K305 [55]. No functional outcome of this was 279 described,[254_TD$IF] and a clear delineation of how classical HDACs and SIRTs regulate PKM2 acetyla- 280 tion in specific cell types is still required. Residues of PKM2 regulated by acetylation are yet to 281 be identified in immune cells;[25_TD$IF] however, a recent study in macrophages demonstrated that 282 succinylation of K311 promoted formation of the nuclear dimer, enabling association with HIF- 283 1a and inducible IL-1b expression [56]. SIRT5 was shown to be required for desuccinylation of 284 this residue, inducing the tetrameric form and preventing inflammatory mediator production. In / 285 keeping with this regulatory role, Sirt[256_TD$IF] À À[239_TD$IF] mice were highly susceptible to DSS-induced colitis 286 [56]. Lysine succinylation frequently overlaps with acetylation [57],[17_TD$IF] and this opens up the 287 interesting possibility of crosstalk between different lysine modifications (e.g.,[23_TD$IF] acetylation, 288 succinylation) on K311 or other lysine residues of PKM2 to control its stability and/or function. 289 In this regard, HDACs/SIRTs may act as gatekeepers, deacetylating lysine residues to enable 290 alternative PTMs.

291 Pyruvate Dehydrogenase[257_TD$IF] (PDH) 292 The pyruvate dehydrogenase complex (PDC) is formed by the association of three catalytic 293 enzymes; PDH[258_TD$IF] (E1), dihydrolipoamide transacetylase (E2),[259_TD$IF] and dihydrolipoamide dehydroge- 294 nase (E3), as well as the E3-binding protein that tethers them together [58]. When localized[260_TD$IF] in 295 the mitochondrial membrane, the PDC decarboxylates pyruvate into acetyl-CoA for use by the 296 TCA cycle, linking glycolysis to the TCA cycle and determining whether pyruvate is used for 297 OXPHOS or is reduced to lactate to sustain aerobic glycolysis. Intriguingly, despite its large size, 298 intact and functional PDC can translocate across the mitochondrial membrane and shuttle to 299 the nucleus, where it provides a localized[261_TD$IF] nuclear pool of acetyl-CoA for histone acetylation and 300 the[26_TD$IF] control of gene expression [59]. Because[263_TD$IF] PDH catalyzes the rate-limiting step of pyruvate 301 decarboxylation, the activity of this subunit determines the rate of flux, which in turn replenishes 302 the TCA cycle. Proin[264_TD$IF] flammatory macrophages sustain pyruvate oxidation through PDH and use

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303 it to generate citrate in the mitochondria, providing substrate for the production of itaconate and 304 for lipogenesis [60]. The major inhibitory control point of PDC activity is phosphorylation on 305 three residues of PDH by pyruvate dehydrogenase kinase (PDK) [61]. SIRT3-mediated 306 deacetylation of PDH at K321 increased its enzymatic activity in cancer cells [62],[17_TD$IF] and 307 acetylation at this residue inhibited activity by recruiting PDK [63]. Thus, it would appear that 308 deacetylation of PDH provides a means to[265_TD$IF] activate the PDC. There is other evidence of indirect 309 control of PDH activity via regulated lysine acetylation in cancer cells. Acetylation of K202 of 310 pyruvate dehydrogenase phosphatase 1 (PDP1) inhibits its interaction with PDH, preventing 311 dephosphorylation-mediated activation of PDH [63]. Thus, deacetylation of PDP1 would be 312 predicted to favor[26_TD$IF] PDH activation. ACAT1 and SIRT3 were identified as the responsible lysine 313 acetylase and deacetylase, respectively, in this pathway.

314 HDAC/SIRT-Mediated[267_TD$IF] Control of Glycolysis Through Gene Regulation 315 In addition to acetylation/deacetylation-mediated control of the activity of glycolytic enzymes, 316 HDACs and SIRTs can also influence glycolysis by modulating the expression of glycolysis- 317 associated genes, either by deacetylating transcription factors or histones (epigenetic mecha- 318 nisms). For example, HIF-1a, a key at the convergence of inflammatory and 319 metabolic signals, regulates the[106_TD$IF] expression of glycolytic genes such as glucose transporters 320 (Glut1), Hk2, Pkm,[17_TD$IF] and Ldha, thereby enhancing glycolytic flux [64]. HIF-1a is known to be 321 regulated by acetylation; SIRT1 [65] and SIRT2 [66] can each directly bind to, deacetylate,[268_TD$IF] and 322 inactivate HIF-1a, while SIRT3-dependent (ROS) production stabilizes[163_TD$IF] 323 HIF-1a by targeting the prolyl hydroxylases that induce degradation [67]. Class I and II classical 324 HDACs can also regulate HIF-1a localization,[269_TD$IF] stability, and function to control glycolytic and 325 inflammatory gene expression. Furthermore, the[187_TD$IF] enzymatic activity of classical HDACs was 326 required for HIF-1a-dependent transcriptional responses in TLR-activated macrophages [68]. 327 The peroxisome proliferator-activated receptor g[270_TD$IF] coactivator 1a (PGC-1a) and Forkhead box[271_TD$IF] O 328 (FoxO) transcription factors are other deacetylase-modulated targets that control glycolytic 329 gene expression and regulate TLR4 signaling[105_TD$IF] in macrophages [69,70]. In hepatocytes, PGC-1a 330 is deacetylated by SIRT1 to modulate the balance of glycolytic and gluconeogenic genes [71]. 331 Also in the liver, class IIa HDACs are necessary[27_TD$IF] to recruit HDAC3 for deacetylation and the[273_TD$IF] 332 activation of FoxO transcription factors, thus enabling the[274_TD$IF] expression of enzymes involved in 333 [72]. In adipocytes, sirtuins[275_TD$IF] can also directly deacetylate FoxO transcription 334 factors to regulate cell metabolism; SIRT1-mediated deacetylation of FoxO1 resulted in 335 increased adiponectin transcription [73], which in turn modulated glucose metabolism, FAO,[276_TD$IF] 336 and insulin levels. Whether these specific HDACs also deacetylate PGC-1a and/or FoxO to 337 control regulated metabolism in macrophages remains an open question.

338 Numerous studies have demonstrated that metabolic input is integrated into changes at the 339 level of chromatin structure. Lactate, the end-product[27_TD$IF] of aerobic glycolysis, inhibits HDAC 340 activity [74], thus providing a[278_TD$IF] molecular mechanism by which the metabolic state of the cell can 341 exert epigenetic control. In keeping with this, glycolytic metabolism promoted global histone 342 acetylation and an open chromatin configuration in cancer cells [75], likely by providing 343 additional acetyl-CoA for HATs [76,77] and by increased production of HDAC-inhibitory lactate 344 [74]. Furthermore, extracellular signals that influence macrophage activation status can regu- 345 late gene expression through control of metabolites. For example, IL-4 signaling[105_TD$IF] induced a 346 nuclear pool of acetyl-CoA that was necessary for histone acetylation and the[279_TD$IF] expression of 347 genes associated with alternatively activated macrophages [78]. In addition, epigenetic reprog- 348 ramming of monocytes in response to Candida albicans required high glucose consumption + 349 coupled with elevated lactate production and a high NAD /NADH[179_TD$IF] ratio [79], with SIRT1[280_TD$IF] and 6 350 being implicated in this process [19,79]. With respect to specific epigenetic mechanisms,

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351 SIRT1 directly regulates transcription via deacetylation of histones such as H1 [80], H3 [80,81],[17_TD$IF] Outstanding Questions 352 and H4 [80]. By deacetylating H3K14 at[281_TD$IF] the promoter of the HIF1A[28_TD$IF] gene, SIRT1 inhibited HIF- Which HDACs and SIRTs control reg- ulated glycolysis in macrophages, and 353 1a expression [82]. Similarly, in activated stem cells, increased glycolysis was via modifications on which metabolic 354 associated with reduced SIRT1-mediated deacetylation of H4K16 [83]. SIRT6 also regulates enzymes? How does this differ from 355 glucose homeostasis by deacetylation of H3K9 to inhibit the expression of various HIF-1a- cancer cells? 356 dependent glycolytic genes [19,84]. Thus, multiple SIRT family members appear to be particu- 357 larly important for the[283_TD$IF] epigenetic control of glycolysis in[284_TD$IF] several cellular systems. In response to infectious or inflamma- tory stimuli, what are the molecular [285_TD$IF] mechanisms that control lysine deace- 358 Concluding Remarks tylase enzyme activity in 359 There is very strong evidence that[286_TD$IF] specific HDAC/SIRT enzymes deacetylate[287_TD$IF] metabolic macrophages? 360 enzymes and regulate[28_TD$IF] metabolic pathways in cancer cells. While aerobic glycolysis is 361 utilized[289_TD$IF] by both cancer cells and activated macrophages, there are many questions that remain How does lysine acetylation regulate [290_TD$IF] the subcellular[29_TD$IF] localization of meta- 362 about how HDACs and SIRTs control macrophage immunometabolism (Outstanding Ques- [293_TD$IF] [230_TD$IF] bolic enzymes in macrophages and, 363 tions). For example, there are likely to be many differences in the expression and/or localization conversely, do metabolites influence 364 of individual lysine deacetylases between cancer cell lines and primary immune cells. Thus, the[294_TD$IF] subcellular localization of lysine 365 detailed mechanistic studies will[291_TD$IF] now be necessary to better understand the roles of individual deacetylases in macrophages? 366 HDAC/SIRT enzymes in regulating glycolysis in macrophages. Some of these enzymes have Do HDACs, metabolic enzymes,[295_TD$IF] and 367 already been linked to the regulation of macrophage inflammatory responses, for example key transcription factors function fl 368 HDAC7 that promotes macrophage-mediated in ammatory responses [68] and SIRT5 that together in protein complexes to regu- 369 plays a regulatory role by constraining the inflammatory functions of PKM2 [56]. A more late macrophage immunometabolism? [296_TD$IF] [297_TD$IF] 370 comprehensive molecular understanding of how specific lysine deacetylases control specific Do deacetylases function as scaffolds or as enzymes in such complexes? 371 glycolytic enzymes in resting and activated macrophages is likely to deliver new opportunities 372 for manipulating immunometabolism in these cells as an anti-inflammatory strategy for man- Does the ability[298_TD$IF] of lysine deacetylases 373 aging disease. to influence other PTMs[23_TD$IF] such as suc- cinylation, malonylation,[29_TD$IF] and SUMOy- 374 Acknowledgments lation regulate inducible glycolysis in macrophages? 375Q5 This work was supported by an ARC[30_TD$IF] Discovery Project to M.J.S. (DP170102321), and a National Health and Medical 376 Research Council of Australia (NHMRC) project[301_TD$IF] grant to M.J.S. and D.P.F. (ID 1125316). M.J.S. is supported by an Do pharmacological agents that target 377 NHMRC Senior Research Fellowship (ID 1107914), D.P.F.[302_TD$IF] is supported by an NHMRC Senior Principal Research lysine deacetylases exert some of their [30_TD$IF] [304_TD$IF] 378 Fellowship (ID 1117017), M.R.S. is supported by a University of Queensland Postdoctoral Research Fellowship. A.I. anti-inflammatory effects via regulation 379 was supported by a University of Queensland Postdoctoral Research Fellowship,[305_TD$IF] and D.P.F. and A.I. are supported by the of immunometabolism? 380 ARC[306_TD$IF] Centre of Excellence in Advanced Molecular Imaging[307_TD$IF] (CE140100011). C.Y.C. is supported by Biomedical Research 381 Council A*STAR Young Investigator[308_TD$IF] Grant 1518251030, and A.S. is supported by SIgN A*STAR, A*STAR JCO-CDA[309_TD$IF] 382 15302FG151, and Singapore-India joint grant 1518224018.

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