Succinylation modification: a potential therapeutic target in stroke

Jie Lian,Wenwu Liu,Qin Hu,Xiaohua Zhang

Abstract Stroke is a leading cause of mortality and disability worldwide.Ischemic cell death triggered by the compromised supply of blood oxygen and glucose is one of the major pathophysiology of strokeinduced brain injury.Impaired mitochondrial energy metabolism is observed minutes after stroke and is closely associated with the progression of neuropathology.Recently,a new type of posttranslational modification,known as lysine succinylation,has been recognized to play a significant role in mitochondrial energy metabolism after ischemia.However,the role of succinylation modification in cell metabolism after stroke and its regulation are not well understood.We aimed to review the effects of succinylation on energy metabolism,reactive oxygen species generation,and neuroinflammation,as well as Sirtuin 5 mediated desuccinylation after stroke.We also highlight the potential of targeting succinylation/desuccinylation as a promising strategy for the treatment of stroke.The succinylation level is dynamically regulated by the nonenzymatic or enzymatic transfer of a succinyl group to a protein on lysine residues and the removal of succinyl catalyzed by desuccinylases.Mounting evidence has suggested that succinylation can regulate the metabolic pathway through modulating the activity or stability of metabolic enzymes.Sirtuins,especially Sirtuin 5,are characterized for their desuccinylation activity and have been recognized as a critical regulator of metabolism through desuccinylating numerous metabolic enzymes.Imbalance between succinylation and desuccinylation has been implicated in the pathophysiology of stroke.Pharmacological agents that enhance the activity of Sirtuin 5 have been employed to promote desuccinylation and improve mitochondrial metabolism,and neuroprotective effects of these agents have been observed in experimental stroke studies.However,their therapeutic efficacy in stroke patients should be validated.

Key Words: mitochondria metabolism;neuroprotection;sirtuin 5;stroke;succinylation modification

Introduction

Stroke,caused by the rupture or blockage of a blood vessel to the brain,is a major cause of global disability and mortality (Hollist et al.,2021).Neuronal cell death caused by mitochondrial energy failure,oxidative stress,excitotoxicity,and inflammatory responses contribute considerably to strokeinduced brain damage.However,the pathophysiology of stroke has not been fully elucidated.

Recently,advances in proteomics have uncovered and identified numerous novel post-translational modified molecules that are involved in stroke pathology,and have confirmed that lysine succinylation is a pathological hallmark of metabolic crisis after stroke (Xiao et al.,2021;Xia et al.,2022).The level of succinylation is upregulated in ischemic organs,and is tightly associated with mitochondrial dysfunction and energy depletion (Fukushima et al.,2016;Ali et al.,2020;Chang et al.,2021).Additionally,succinylation might be a promising therapeutical target for the treatment of stroke.Further investigation into the mechanism of succinylation will improve the understanding of neuronal energy metabolism after stroke and provide novel therapeutic targets to protect brain against stroke.

We aimed to review the role of stroke-induced succinylation in energy metabolism,and address the regulation of succinylation by the desuccinylase,Sirtuin 5 (SIRT5).We also present an update on preclinical studies regarding succinylation and discuss the neuroprotective role of SIRT5.

Retrieval Strategy

This review is based on studies retrieved from the PubMed database,covering relevant literature published between January 1993 and December 2022.Our search included a combination of MeSH terms such as ‘succinylation,’ ‘desuccinylation,’ ‘Sirtuin 5/SIRT5,’ ‘ischemia,’ ‘stroke,’ and ‘subarachnoid hemorrhage.’ The results were further screened based on the title and abstract,and the studies that investigated the association between succinylation,SIRT5,and stroke were included.

Discovery of Succinylation and Its Characteristics

Succinylation refers to the modification of a protein with a succinyl group,which most frequently happens to lysine residues.Lysine succinylation is evolutionarily conserved in both bacteria and mammalian cells,and occurs in most compartments of cells (Weinert et al.,2013).In 2011,lysine succinylation was reported as a widespread post-translational modification (PTM) inEscherichia coli,and succinyl-coenzyme A (succinyl-CoA) was identified as a cofactor for lysine succinylation (Zhang et al.,2011).The introduction of succinyl groups can neutralize the inherent positive charge of lysine,resulting in a marked negative charge (-1) due to the presence of a carboxylate group,which is more prominent than that in lysine acetylation (Hirschey and Zhao,2015).This change results in a less compact chromatin structure,allowing for the recruitment of activators or inhibitors of gene transcription (He et al.,2018).Moreover,compared to other well-studied PTMs (such as acetylation and phosphorylation),the size of the succinate group is significantly larger (approximately 100 Da) (Zhang et al.,2011),while the acetyl and phosphoryl groups have smaller masses (42 and 80 Da,respectively) (Parker et al.,2010).Thus,succinylation may alter the structure of the protein and change the function of the protein dramatically.

Succinylation occurs primarily in the mitochondrion.The first study that identified natural lysine succinylation confirmed the presence of three succinated proteins,all engaged in metabolism in the mitochondria (Zhang et al.,2011).Succinylation affects metabolic enzyme activity and regulates metabolic pathways according to environmental nutrition availability (Park et al.,2013;Weinert et al.,2013;Chen et al.,2017).In addition,succinylation occurs in the core histones,and lysine succinylation in the histone core is important for gene expression and DNA repair (Smestad et al.,2018).

Regulation of Succinylation

The succinylation reaction can be divided into a nonenzymatic and enzymatic process.Whether succinyl transferases that catalyze succinylation dominate this process or that it is due to a passive reaction between succinyl-CoA with lysine residues on the protein surface remains a point of contention (Yang and Gibson,2019;Sreedhar et al.,2020).By comprehending the process of succinylation and desuccinylation,we can gain insight into the regulatory mechanisms of diverse cellular metabolic pathways.

Nonenzymatic Regulation of Succinylation

Nonenzymatic regulation of succinylation is a passive and spontaneous process,determined by the abundance of succinyl-CoA (Figure 1).Succinyl-CoA is mainly produced in the mitochondria by α-ketoglutarate dehydrogenase complex (KGDHC) or through a nicotinamide adenine dinucleotide (NAD)+dependent reaction of oxoglutarate dehydrogenase complex.Other routes include the catabolism of odd-number chain fatty acids and some amino acids,including valine,isoleucine,methionine,and thymine (Chinopoulos,2021).Succinyl-CoA can be degraded into succinate,forming high-energy phosphates,and undergoes conversion to fumarate via succinate dehydrogenase (SDH) (Chinopoulos,2019).Tissues with elevated succinyl-CoA levels also have a corresponding increase in lysine succinylation (Weinert et al.,2013).The supplementation of α-ketoglutarate or the loss of the key enzyme SDH will cause the mitochondrial accumulation of succinyl-CoA,spontaneously leading to global lysine hypersuccinylation in the mitochondria (Smestad et al.,2018;Zhang et al.,2018).In the nucleus,succinyl-CoA generation is mediated by succinyl-CoA synthetase ADPforming subunit β (SUCLA2),the depletion of which may also cause total cellular protein succinylation (Zhang et al.,2018).However,the enzyme involved in the catalysis of succinyl-CoA has not been found in the cytosolic compartment (Trefely et al.,2020).A possible producer of succinyl-CoA in the cytoplasm is peroxisome.Succinyl-CoA can be produced in the peroxisomes from 3-oxoadipate,long fatty acids,and adipic acid (Chinopoulos,2021) and is transported out of the peroxisomes in the form of succinyl-carnitine (Chinopoulos,2021).These findings provide the evidence that succinyl-CoA which originated from the mitochondria or cytoplasm might serve as the driving force of succinylation.Temperature is another factor that affects succinylation.Succinyl-CoA has a limited half-life (1-2 hours) at room temperature but becomes reactive at physiological temperature (Wagner et al.,2017).Thus,it can be easily transformed into a high-energy anhydride intermediate,which facilitates lysine succinylationin vivo.Unlike protein acetylation by acetyl-CoA,which is highly pH-dependent,succinylation is less dependent on pH.Efficient succinylation is detectable in a pH range of 5-8,and even remains active at a pH range of 4-5 (Wagner and Payne,2013).Taken together,these findings indicate that succinylation can be regulated by succinyl-CoA availability,transportation,and the local microenvironment.

Figure 1｜Regulation of succinylation.

Enzymatic Catalysis of Succinylation

At present,there is little evidence of an enzyme responsible for global lysine succinylation,but studies have found clues for the existence of succinyltransferase that directly catalyzes this reaction (Figure 1).Through a cellcultured based proteomics approach,Kurmi et al.(2018) reported that the carnitine palmitoyltransferase 1A (CPT1A) has lysine succinyltransferase activityin vivoandin vitro.Li et al.(2020) further revealed that CPT1A could bind to lactate dehydrogenase A (LDHA) and mediate the succinylation at lysine 222,which may decrease lysosomal degradation.CPT1A also mediates succinylation of S100A10 at lysine residue 47 (Wang et al.,2019).In the nucleus,succinylation is catalyzed by lysine acetyltransferase 2A (KAT2A).Succinyl-CoA can be generated from α-ketoglutarate and binds to KAT2A where it executes histone succinylation (Wang et al.,2017b).KAT2A mediated H3K79 succinylation is significantly enriched at gene promoter regions,and can regulate the succinylation of H3K79 in the promoter region of YWHAZ (also named 14-3-3ζ),which facilitates YWHAZ expression (Tong et al.,2020).In a recent study,Yang et al.(2021) reported another form of enzymecatalyzed lysine succinylation,which involves histone acetyltransferase 1 (HAT1);HAT1 was found to regulate lysine succinylation in a range of proteins,including histones and non-histones,especially those targeting glycolysis.HAT1 is,therefore,the enzyme responsible for non-histone succinylation in the mitochondria.However,whether HAT1 acts alone inside mitochondria,where most succinylated targets are identified,remains largely unknown.A hypothesis is that the reaction of lysine succinylation is mostly nonenzymatic,while enzymatic succinylation is only present in specific compartments.Therefore,the specific mechanism involved in the regulation of succinylation requires further research.

Targets of Succinylation to Control Cell Metabolism

The level of mitochondrial succinylation changes dynamically in response to metabolic perturbation (Chen et al.,2017).Succinylation levels may decrease when the tricarboxylic acid (TCA) cycle,glycolysis,and electron transport chain are impaired.Conversely,succinylation may increase when oxygen concentration reduces and high-glucose condition is present (Chen et al.,2017;Yang and Gibson,2019).Growing evidence suggests that lysine succinylation represses enzymes activities that are involved in energy metabolism,including glycolysis,the TCA cycle,electron transport chain,fat acid metabolism,ketone bodies,and ammonia production (Figure 2).

Figure 2｜Succinylation affects cell metabolism after stroke,modulates the function of metabolic-related enzymes,and contributes to energy failure and ROS production,resulting in neuronal death and inflammation.

Glycolysis

The activity of the glycolytic enzyme pyruvate kinase M2 (PKM2) is suppressed by succinylation at lysine 311 (Xiangyun et al.,2017).PKM2 is responsible for the catalysis of the final step of glycolysis,which converts phosphoenolpyruvate to pyruvate.In lipopolysaccharide (LPS)-activated macrophages,upregulating the succinylation of PKM2 by inhibiting desuccinylation enzyme SIRT5 redirects cellular metabolism toward glycolysis (Palmieri,2004).Another enzyme regulated by succinylation is the pyruvate dehydrogenase complex (PDC),which is involved in the oxidation of pyruvate.Succinylation has been shown to upregulate PDC activity,resulting in an increased input in the TCA cycle (Park et al.,2013).

TCA cycle and the electron transport chain

Succinylation can pose opposite effects on different enzymes in the TCA cycle: repressing the activity of isocitrate dehydrogenase 2 (IDH2),which catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate (Zhou et al.,2016);and activating SDH or Complex II,another TCA cycle enzyme complex.The activation of SDH and the consumption of succinate enhances the TCA cycle,thus promoting mitochondrial respiration (Park et al.,2013).The succinylation of mitochondrial membrane proteins also impairs ATP synthase activity.Zhang et al.(2017) reported that the succinylation of the proteinlipid interface of Complex II subunit SDH-B affected ATP synthase activity while desuccinylation of inner mitochondrial membrane proteins enhanced mitochondrial respiration.

Fat acid metabolism and ketone body production

Fatty acid β-oxidation is a metabolic process that involves the dehydrogenation of fatty acyl-CoAs of varying lengths,resulting in the production of nicotinamide adenine dinucleotide (NADH),acetyl-CoA,and flavin adenine dinucleotide (FADH2).In SIRT5 knockout liver tissues,these enzymes are all hypersuccinylated at multiple sites,leading to moderate reduction of β-oxidation and the build-up of medium-and long-chain acylcarnitines (Goetzman et al.,2020).SIRT5,along with SIRT3,also promotes fatty acid β-oxidation by converging upon very long-chain acyl-CoA dehydrogenase and promoting its activity and membrane localization through desuccinylation,which is regulated by the binding of cardiolipin under reversible lysine acylation (Zhang et al.,2015).Succinylation also regulates ketone body formation by modulating the activity of 3-hydroxy-3-methylglutaryl CoA synthase 2,an enzyme that plays a crucial role in the conversion of acetyl-CoA into ketone bodies.Succinylation of 3-hydroxy-3-methylglutaryl CoA synthase 2 impairs its activity and thus affects energy supply in important organs (including the brain and heart) (Rardin et al.,2013).

Ammonia production

In the mitochondria,glutaminase catalyzes the synthesis of glutamine from glutamate and ammonia.The accumulation of glutamate exacerbates brain damage following hemorrhage and is associated with long-term neurological deficits due to glutamate-related excitotoxicity (Sun et al.,2021b).SIRT5 and glutaminase coimmunoprecipitate,and the increased succinylation of glutaminase in case of SIRT5 knockout,reverses the reaction and increases ammonia production,thereby augmenting mitophagy and autophagy in the SIRT5 silenced cells (Polletta et al.,2015).

Modulation of desuccinylation

Sirtuins and lysine desuccinylation

The first prokaryotic desuccinylation enzyme,CobB,was identified inEscherichia coli,and is a Sir2-like bacterial lysine deacetylase with lysine desuccinylation and deacetylation activities (Colak et al.,2013).In mammals,this desuccinylation process is primarily mediated by sirtuins,which are categorized as class III histone deacetylases.Sirtuin mediated catalysis requires NAD+as a co-substrate and may generate two byproducts: nicotinamide and 2’-O-acetyl-ADP-ribose (Sauve et al.,2006).The sirtuin family has seven isoforms,from SIRT1 to SIRT7,all of which possess a conserved catalytic domain of 275 amino acids (Haigis and Sinclair,2010).Among these isoforms,SIRT5 and SIRT7 have shown powerful abilities of desuccinylation (Li et al.,2016).SIRT5 is present in all cell compartments,especially the mitochondria matrix,where the TCA cycle and other energy-generating metabolic actions are preformed (Nakagawa et al.,2009;Anderson et al.,2014).In addition,a significant fraction of endogenous SIRT5 is present cytosolically,while a very small amount of SIRT5 is also detectable in the nucleus (Park et al.,2013).In contrast,SIRT7 is only present in the nucleus,and has an affinity to nuclear histones (Li et al.,2016).SIRT7-catalyzed desuccinylation is predominant in the nucleus and is critical for the response to DNA damage and cell survival (Li et al.,2016).SIRT5 is the main enzyme for desuccinylation in mammalian cells and SIRT7 plays a supplementary role in desuccinylation in the nucleus.

SIRT5-mediated lysine desuccinylation

SIRT5 can catalyze the cleavage of an acyl group from a lysine residue and is particularly responsible for protein desuccinylation due to its unique affinity to negatively charged acyl lysine modifications.These characteristics also make SIRT5 effective for demalonylation,deglutarylation,and other lysine modifications (Du et al.,2011).In murine models with SIRT5 knockout,lysine succinylation increases dramatically,particularly in the mitochondria,the primary compartment where SIRT5 functions,indicating the significant role of this enzyme in modulating the overall succinylation level (Du et al.,2011).Histone succinylation also increases in SIRT5 knockout animals,especially in the proteins related to DNA damage and stress (Park et al.,2013;Carrico et al.,2018;Liu et al.,2022).The change of succinylation level corresponds to the finding that SIRT5 overexpression increases oxygen consumption as well as oxidative respiratory function,which implies a close relationship between SIRT5 and mitochondrial function (Buler et al.,2014;Gut et al.,2020).

Regulation of SIRT5

Sirtuin activity is regulated through various mechanisms,including enzymatic substrate availability,transcriptional regulation,and PTM.However,how sirtuins contribute to SIRT5 expression and activity,particularlyin vivo,is still unclear (Figure 3).Small molecules can also modulate sirtuin activity,which provides an opportunity for therapeutic interventions.

Figure 3｜Regulation of SIRT5 expression and activity,mediated by substrate,transcriptional regulators,miRNA,and post-transcriptional modification.

Substrate level:The function of SIRT5 is dependent upon the cofactor NAD+;therefore,the availability of NAD+is crucial for SIRT5 reactivity.AMP-activated protein kinase (AMPK) can increase the NAD+/NADH ratio by inducing mitochondrial β-oxidation,which increases sirtuin activity (Canto et al.,2009).LPS-induced NADH generation also reduces SIRT5 activity,leading to protein succinylation (Tannahill et al.,2013).Other methods,such as promoting biosynthesis or regulating the activity of NAD+-depleting enzymes,are also effective for the modulation of SIRT5 activity (Houtkooper et al.,2010,2012).However,these methods can regulate the activities of all sirtuins,and thus this regulation is not confined to SIRT5.

Transcriptional regulation:SIRT5 expression is influenced by a variety of cellular regulators,among which AMPK and peroxisome proliferatoractivated receptor coactivator-1α (PGC-1α) are important.PGC-1α overexpression increases mRNA and protein expression of SIRT5 in mouse primary hepatocytes,indicating a positive relationship between PGC-1α activation and SIRT5 expression.Conversely,when AMPK is overexpressed or activated by metformin,SIRT5 expression is inhibited,suggesting a negative relationship between AMPK and SIRT5 (Buler et al.,2014).Furthermore,some transcription factors have been identified in the core promoter region of SIRT5,including CCAAT enhancer binding protein beta (CEBPβ),Kruppellike factor 2 (KLF2),E2F transcription factor 4 (E2F4),peroxisome proliferatoractivated receptor alpha (PPARα),myogenic differentiation 1 (MYOD),Kruppel-like factor 6 (KLF6),and nuclear respiratory factor 1 (NRF1) (Hong et al.,2018,2019;Sun et al.,2021a).Among them,CEBPβ and KLF6 are negative factors,while E2F4,PPARα,MYOD,NRF1,and KLF2 are positive.Interestingly,demethylation of the SIRT5 promoter can enhance the activity thereof,potentially leading to an increase in the expression of the SIRT5 gene.Additionally,demethylation may facilitate the binding of transcription factors such as E2F4 and KLF6 to the promoter region,further contributing to the regulation of SIRT5 expression (Hong et al.,2019).

Post-translational modifications:SIRT5 is regulated by ubiquitination,an important PTM responsible for protein quality control.Specific mitochondrial proteins are ubiquitinated and subsequently degraded by the proteasome to counter stress,which maintains mitochondrial homeostasis (Sulkshane et al.,2020).Cyclin F,the first member of the F-box family,serves as a substrate adapter for the SCF E3 ubiquitin ligase as well as an important factor involved in cell cycle transition,which does not bind to or activate a cyclindependent kinase (D’Angiolella et al.,2013).Cyclin F-bound Skp1,CUL1,and F-box protein complexes (SCFCyclin F) promote the ubiquitination of SIRT5 under stress,and are thus destroyed by the proteasome,which is potentially important for cells to maintain a normal cell cycle (Mills et al.,2021).

MicroRNAs (miRNAs):these molecules regulate mRNA levels by either degrading the primary transcript or inhibiting translation.There are studies reporting that miR-212-3p (Shang et al.,2021),miR-145-5p (Chang et al.,2021),miR-656-3p (Tang and Yang,2020),miR-3677-3p (Tang and Yang,2020),microRNA-19b (Sun et al.,2016) and miR-299-3p (Dang et al.,2018) can suppress SIRT5 expression.

Small-molecule activators:The first sirtuin activator is resveratrol-related polyphenol (Howitz et al.,2003).Resveratrol is a plant metabolite,naturally produced in some plants such as grapes,which activates yeast Sir2 and human SIRT1 and promotes longevity (Baur et al.,2006).Resveratrol and its metabolite,piceatannol,significantly affect the activity of SIRT5in vitro.Specifically,these compounds are able to interact with the FdL fluorophore within SIRT5,influencing the binding mode of the substrate peptide (Gertz et al.,2012).This interaction between resveratrol and SIRT5 is of particular interest because it is involved in the regulation of key metabolic processes (such as protein desuccinylation) that are modulated by this enzyme.Furthermore,resveratrol is able to increase the desuccinylation activity of SIRT5,but has no influence on its protein expression,highlighting the potential therapeutic benefits of targeting this enzyme (Xiao et al.,2021).

However,despite the potential benefits as a sirtuin modulator,resveratrol faces challenges that make it unsuitable for clinical application as a selective drug.The metabolic characters of resveratrol contribute to its limited bioavailability,which hampers its efficacy as a therapeutic agent.Additionally,the low potency of resveratrol in affecting SIRT5 (Gertz et al.,2012) and the lack of specificity raise concerns about potential side effects,and further question its suitability for clinical application.Therefore,researchers are exploring alternative compounds and derivatives of resveratrol that can overcome these limitations and provide more targeted and effective treatment options for various diseases.Mitochondria-targeted resveratrol derivative is generated through conjugating polyphenol kernel with a butyl-triphenylphosphonium lipophilic cation,that can pass through biological membranes,while its accumulation in compartments (such as the mitochondrial matrix) produces more negative potentials (Biasutto et al.,2021).These mitochondriotropic analogues exhibit improved solubility and stability compared to resveratrol,and have been found to demonstrate cytotoxic effects that specifically target rapidly dividing cells (Naoi et al.,2019),while ischemic cerebral injury is not the case.Further research is needed to investigate their efficacy and safety for clinical application.Apart from naturally occurring molecules and their derivatives,some synthetic compounds (previously recognized as SIRT1 activators) also exhibit effects on SIRT5 (Mori et al.,2022).For instance,BML-217,a stilbene derivative,is a potent activator of SIRT5 and can increase the activity of SIRT5 13.6-fold.Another compound,dipyridamole,which is used to prevent blood clot formation,has also been identified as an activator of SIRT5.Additionally,ZM336372,an inhibitor of c-Raf,has been identified as an agonist of SIRT5.More recently,a new small-molecule SIRT5 agonist,MC3138,was developed by Hu et al.,and is a selective agonist of SIRT5 over SIRT1/3,with 50% inhibiting concentration (IC50) ranging from 25.4-236.9 mM (Hu et al.,2021).

Small-molecule inhibitors:A variety of studies have investigated the inhibitors of SIRT5.Since SIRT5 requires NAD+to react on polypeptides,it is not surprising that most inhibitors target the binding pocket of NAD+(Villalba and Alcain,2012).Suramin has a huge,symmetric diarylurea structure that can block both substrate-binding sites of SIRT5,which have made it a star molecule as a SIRT5 inhibitor (Schuetz et al.,2007).However,Suramin’s poor ability to diffuse across biological membranes limits its application,and it affects too many targets besides sirtuins (Wiedemar et al.,2020).Thus,some new compounds are being developed and examined.Inhibitors targeting the acyl-Lys peptide binding site present a particularly promising approach for the development of drugs that target SIRT5,given its distinct succinyl substrate preference.Notably,thio-succinylated peptides have shown great success in inhibiting SIRT5 with an IC50of 5 µM.Moreover,at a concentration of 100 µM,these peptides have no discernible inhibitory effect on SIRT1-3,highlighting the selectivity of this inhibitor for SIRT5 (He et al.,2012).These findings suggest that thio-succinylated peptides targeting the acyl-Lys peptide binding site may serve as promising candidates for further development of specific SIRT5 inhibitors.

Thiourea compounds are also promising inhibitors of SIRT5.Zang et al.(2015) reported Nε-carboxyethyl-thiocarbamoyl lysine was also a SIRT5 inhibitor.Its inhibitory effect is particularly strong and specific (IC50of 5 mM) and,for SIRT5,was found to be over 20 times stronger than that observed for other sirtuin members such as SIRT1 and SIRT6.Furthermore,the diversity of acyl moieties provides the potential for the advancement of SIRT5-inhibiting peptides,such as 3-methyl-3-phenyl-succinyl-CPS1,which exhibits the highest potency for SIRT5 inhibition and displays notable selectivity for SIRT5 (Roessler et al.,2014).

NAD+is necessary for sirtuins to catalyze acylation by binding the nicotinamide moiety inside the C-pocket,while free nicotinamide can rebind to the C-pocket afterwards and reverse the reaction (Gertz et al.,2013).The productive binding of NAD+is critical for the enzymatic activity of sirtuins and provides a basis for the development of NAD+-mimicking compounds that can modulate sirtuin activity.GW5074,a 4-hydroxybenzylidene indolinone compound which has potentials in the treatment of viral infection and autoimmune diseases (Yang et al.,2019),can inhibit SIRT5-dependent desuccinylation efficiently and exclusively (Suenkel et al.,2013),and thus it may become a promising inhibitor of SIRT5.

Targeting Succinylation Modification in Stroke

Succinylation is a hallmark of impaired cell metabolism in the brain (Park et al.,2013).In a study regarding succinylome of the brain in healthy subjects,succinylated proteins were mainly present in the mitochondria and were related to metabolic processes such as the TCA cycle and glycolysis (Yang et al.,2022).In pathological conditions,the role of succinylation modification has been investigated in tumorigenesis (Kumar and Lombard,2018) and neurodegenerative diseases such as Alzheimer’s disease (Li et al.,2015;Wu et al.,2021;He et al.,2022).Recent studies have revealed the potential relationship between SIRT5-regulated succinylation and cerebrovascular diseases,particularly stroke (Diaz-Canestro et al.,2018;Wang et al.,2020;Deng et al.,2021;Xiao et al.,2021;Xia et al.,2022).

Ischemic stroke

Ischemic stroke is a consequence of reduced regional cerebral blood flow (Ferrari et al.,2022;Noh et al.,2023).The reduction of regional cerebral blood flow causes irreversible injury to the core of the infarct area if it is not corrected within 4-6 hours (Powers et al.,2018).Ischemia in the central nervous system may damage the blood-brain barrier (BBB),leading to local edema or recruitment of inflammatory cells in the subacute phase (Han et al.,2020;Li et al.,2022).In vitroandin vivoexperiments have shown an elevated level of succinylation in the brain cells after ischemia.In neuronal cell lines and primary neurons,hypoxia has been found to induce succinylation (Gibson et al.,2015;Chen et al.,2017).In a rat bilateral common carotid artery occlusion model,Wang et al.(2020) reported increased global succinylation in the hippocampus as compared to the sham rats.However,pathological specimens from stroke patients are still needed for further succinylomic analysis to support these findings under physiological conditions.

Since SIRT5 is the main desuccinylation enzyme in human cells and the loss of SIRT5 may cause global succinylation,studies have investigated the roles of SIRT5 and related desuccinylation in ischemia diseases.Recent studies have indicated that SIRT5 may exert a neuroprotective effect on stroke by restoring energy metabolism (Buler et al.,2014;Morris-Blanco et al.,2016;Xiao et al.,2021).Protein kinase C epsilon (PKC-ε) can promote SIRT5-mediated desuccinylation in brain cells by enhancing nicotinamide phosphoribosyl transferase (Morris-Blanco et al.,2016).The rise of NAD+levels may thus improve SIRT5 activity,enhancing desuccinylation in the mitochondrial energy production of PKC-ε-activated cells (Koronowski et al.,2018).Some target proteins concerning glycolysis,TCA cycle,mitochondrial respiration,fatty acid β-oxidation,and ketogenesis are displayed inTable 1.PKC-ε can prevent cortical degeneration in middle cerebral artery occlusion mice only when SIRT5 is activated,implying a neuroprotective effect (Morris-Blanco et al.,2016).

Table 1｜Succinylation effects on mitochondrial metabolism

Reactive oxygen species (ROS) accumulation is an important factor related to neuronal death after ischemia (Forrester et al.,2018;Mahan,2021;Schipke et al.,2022;Wang et al.,2022).SIRT5 targets enzymes that play important roles in ROS detoxification,exerting a protective effect on ischemic injury (Singh et al.,2018).NADPH,as the major reductant in cells,plays an important role in maintaining the amount of glutathione,which can effectively scavenge ROS and protect cells from oxidative damage (Margis et al.,2008).Interestingly,SIRT5 has been shown to desuccinylate two key enzymes,glucose-6-phosphate dehydrogenase (G6PD) and isocitrate dehydrogenase 2 (IDH2),resulting in their activation,which may promote NADPH production,ultimately maintaining cellular redox homeostasis (Zhou et al.,2016).In contrast,SIRT5 depletion causes hypersuccinylation and inhibition of IDH2 and G6PD,which,in turn,reduces NADPH production.This leads to a decline in the ability to scavenge ROS,making cells more vulnerable to oxidative stress (Zhou et al.,2016).Copper-zinc superoxide dismutase (SOD) is another target of SIRT5-mediated desuccinylation in the cytoplasm,and is a crucial antioxidant enzyme in the mitochondria responsible for the conversion of O2-to H2O2.The produced H2O2is then detoxified to H2O by catalases,providing an essential defense mechanism against harmful ROS and oxidative stress (Okado-Matsumoto and Fridovich,2001;Fukai and Ushio-Fukai,2011).Cytosolic Cu/Zn-SOD is succinylated at K123 in SIRT5 knockout mice,and can moderately reduce cellular ROS,whereas SIRT5-mediated desuccinylation can significantly boost the ability of Cu/Zn-SOD to reduce cellular ROS (Lin et al.,2013).Thus,the activation of SIRT5 and its desuccinylation activity protect the TCA cycle from ROS damage due to ischemic-induced mitochondrial dysfunction,which maintains energy generation in ischemic cells.Inflammation is another important contributor to the pathophysiology of stroke.While the inflammatory response is necessary for neurogenesis,it is also a key contributor to secondary brain injury after stroke (Rahman et al.,2021;Xue et al.,2021).In cases of ischemia,microglia/macrophages are activated,while peripheral macrophages and T cells are recruited to the peri-infarct regions 24-72 hours after stroke (Liesz et al.,2011;Gliem et al.,2012;Hu et al.,2012;Beuker et al.,2021).Notably,protein succinylation has a potential regulatory effect on inflammation after stroke (Mills and O’Neill,2014;Xia et al.,2022).Recently,a study revealed that SIRT5 expression in the microglia increased in the early phase of ischemic stroke,which desuccinylated more target proteins in the microglia (Xia et al.,2022).Desuccinylation of ANXA1 blocks membrane recruitment and extracellular secretion,resulting in the hyperactivation of microglia and excessive expression of pro-inflammatory cytokines and chemokines,which may cause neuronal injury by inducing neuroinflammation.Similarly,PKM2 is also a succinylated target which can be regulated by SIRT5 (Park et al.,2013).Succinylation of PKM2 at K311 impairs glycolytic activity and induces nuclear translocation,increasing the production of pro-inflammatory cytokine interleukin-1β in macrophages (Palsson-McDermott et al.,2015;Wang et al.,2017a).Succinylation of PKM2 inhibits pyruvate kinase activity by promoting tetramer-to-dimer transition,which suppresses the hypoxiainducible factor 1α/interleukin-1βmediated pro-inflammatory response in the macrophages (Wang et al.,2017a).Furthermore,SIRT5 expression increases in the peripheral blood macrophages/mononuclear cells of patients 6 hours after symptom onset (Diaz-Canestro et al.,2018).These findings link protein succinylation to neuroinflammation after stroke and further investigations into this aspect are required.

Hemorrhagic stroke

Hemorrhagic stroke includes intracerebral hemorrhage,intraventricular hemorrhage,and subarachnoid hemorrhage (SAH).Although hemorrhagic stroke constitutes only 10% of all strokes,the mortality rate of hemorrhagic stroke patients within 30 days is as high as 50% (Magid-Bernstein et al.,2022).The pathophysiology of hemorrhagic stroke is complex;it is caused by the initial hematoma’s mass effect and hydrocephalus,and the activation of injurious pathways in the subacute phase,concerning inflammation,iron and blood-related toxicity,and oxidative stress (Chen et al.,2022;Magid-Bernstein et al.,2022;Zhou et al.,2023).

The role of lysine succinylation in hemorrhagic stroke is still poorly understood.According to a transcriptome analysis of protein succinyl-lysine modifications in the context of intracerebral hemorrhage,the succinylated proteins are primarily located in mitochondrial and synapse-related subcellular compartments and participate in especially energy metabolism.Notably,the expression of these proteins is at a high level in neurons,endothelial cells,and astrocytes (Deng et al.,2021).A recent study on SAH also highlights the effect of succinylation on metabolism-related enzymes.Decreased SIRT5 expression after SAH causes hypersuccinylation of citrate synthase,which spontaneously represses ATP synthase but increases ROS production,resulting in neuronal cell death and neurological deficits in the acute phase of SAH (Xiao et al.,2021).After administration of resveratrol,SIRT5,as well as desuccinylation of ATP synthase,is activated,which restores mitochondrial metabolism and alleviates early brain injury (Xiao et al.,2021).These findings suggest that SIRT5-mediated desuccinylation is required for neuroprotection against cerebrovascular accident-induced ischemic injury by desuccinylating key enzymes that maintain mitochondrial respiration in the neuronal cells.However,more efforts are required to identify the mechanisms which may guide drug design.

Some targets of SIRT5 may have negative effect after stroke,since SIRT5 also has acetylation,malonylation,and glutarylation functions (Tan et al.,2014).Several studies have shown that SIRT5 may increase BBB permeability (Diaz-Canestro et al.,2018),and blunt the fibrinolytic system (Liberale et al.,2021).This is supported by clinical findings that the expression of SIRT5 increases in acute coronary syndrome patients who are also susceptible to ischemic stroke (Liberale et al.,2021).Thus,the role of SIRT5 in the pathogenesis of stroke remains controversial and more studies are needed to understand the therapeutic potential of SIRT5 mediated desuccinylation.

Limitations

There are several limitations to note in our review.Firstly,investigations in this field are still at an early stage and limited data on targeting succinylation in stroke treatment are available.Secondly,most findings included in this review are from preclinical studies,and more studies are warranted to elucidate whether these results can be translated to a clinical setting.Thirdly,this review focused on the role of succinylation in stroke pathophysiology and other factors that contribute to stroke-induced brain injury were not involved.Finally,only English articles were included in our search,which may exclude relevant studies in other languages.

Conclusion and Perspectives

Succinylation is a PTM occurring on many metabolic enzymes under normal and pathological conditions.The dynamic regulation of succinylation and desuccinylation is important for the homeostasis of cerebral metabolism.Abnormal succinylation has been recognized as an indicator of compromised energy metabolism after stroke.There are also many unknown substrate targets and factors that affect the level of lysine succinylation in subcellular compartments.As an important desuccinylation enzyme,SIRT5 modulates a variety of metabolic pathways and improves cell adaptation to hypoxia/ischemia.It is very possible that targeting SIRT5-mediated desuccinylation would be a promising therapeutic strategy for the treatment of stroke.Pharmacological agents that enhance the activity of SIRT5 have been employed to promote desuccinylation and improve mitochondrial metabolism,and neuroprotective effects of these agents have been observed in experimental stroke studies.However,their therapeutic efficacy in stroke patients should be validated.

Author contributions:JL and WL collected the data and wrote the manuscript.QH and XZ revised the manuscript.All authors read and approved the final manuscript.

Conflicts of interest:None declared.

Data availability statement:Not applicable.

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