Succinylation modification:a potential therapeutic target in stroke

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.

Protein succinylation,hepatic metabolism,and liver diseases

Succinylation is a highly conserved post-translational modication that is processed via enzymatic and non-enzymatic mechanisms.Succinylation exhibits strong effects on protein stability,enzyme activity,and transcriptional regulation.Protein succinylation is extensively present in the liver,and increasing evidence has demonstrated that succinylation is closely related to hepatic metabolism.For instance,histone acetyltransferase 1 promotes liver glycolysis,and the sirtuin 5-induced desuccinylation is involved in the regulation of the hepatic urea cycle and lipid metabolism.Therefore,the effects of succinylation on hepatic glucose,amino acid,and lipid metabolism under the action of various enzymes will be discussed in this work.In addition,how succinylases regulate the progression of different liver diseases will be reviewed,including the desuccinylation activity of sirtuin 7,which is closely associated with fatty liver disease and hepatitis,and the actions of lysine acetyltransferase 2A and histone acetyltransferase 1 that act as succinyltransferases to regulate the succinylation of target genes that influence the development of hepatocellular carcinoma.In view of the diversity and significance of protein succinylation,targeting the succinylation pathway may serve as an attractive direction for the treatment of liver diseases.

Ischemic accumulation of succinate induces Cdc42 succinylation and inhibits neural stem cell proliferation after cerebral ischemia/reperfusion

Ischemic accumulation of succinate causes cerebral damage by excess production of reactive oxygen species. However, it is unknown whether ischemic accumulation of succinate affects neural stem cell proliferation. In this study, we established a rat model of cerebral ischemia/reperfusion injury by occlusion of the middle cerebral artery. We found that succinate levels increased in serum and brain tissue(cortex and hippocampus) after ischemia/reperfusion injury. Oxygen-glucose deprivation and reoxygenation stimulated primary neural stem cells to produce abundant succinate. Succinate can be converted into diethyl succinate in cells. Exogenous diethyl succinate inhibited the proliferation of mouse-derived C17.2 neural stem cells and increased the infarct volume in the rat model of cerebral ischemia/reperfusion injury. Exogenous diethyl succinate also increased the succinylation of the Rho family GTPase Cdc42 but repressed Cdc42 GTPase activity in C17.2 cells. Increasing Cdc42 succinylation by knockdown of the desuccinylase Sirt5 also inhibited Cdc42 GTPase activity in C17.2 cells. Our findings suggest that ischemic accumulation of succinate decreases Cdc42 GTPase activity by induction of Cdc42 succinylation, which inhibits the proliferation of neural stem cells and aggravates cerebral ischemia/reperfusion injury.

Adsorption characteristics of Cd(Ⅱ) and Ni(Ⅱ) from aqueous solution using succinylated hay

An environmentally friendly organic biosorbent was fabricated using hay by succinylation. Metallic cation adsorption tests were performed using synthetic nickel(Ⅱ) and cadmium(Ⅱ) solutions to simulate heavy-metal recovery from aqueous solution. The adsorption efficiency was greater than 98% for both cadmium and nickel ions when the biosorbent concentration was 5.0 g/L and the initial metal concentrations were 50 mg/L. The surface of the biosorbent was characterized using Fourier transform infrared spectroscopy to investigate the changes in the surface functional groups. The functional groups changed according to the surface treatment, resulting in an effective biosorbent. The kinetics of the metals adsorption revealed that the reactions are pseudo-second order, and the adsorption isotherm well followed the Langmuir model. The maximum adsorption capacities predicted by the Langmuir model were 75.19 mg/g and 57.77 mg/g for cadmium and nickel, respectively. The fabricated biosorbent was regenerated using Na Cl multiple times, with 2.1% for Cd and 4.0% for Ni in adsorption capacity after three regeneration cycles. The proposed biosorbent can be a good alternative to resin or other chemical adsorbents for heavy-metal recovery in metallurgical processing or municipal water treatment.

Succinylated whey protein isolate as a sustained-release excipient of puerarin derivative oral tablets:Preparation,optimization and pharmacokinetics

This work was done to investigate succinylated commercial whey protein isolate(S-WPI)as an oral sustained-release delivery carrier for puerarin 5(PR-5). The succinylation condi-tions were established for S-WPIs by optimization of single factor study and Box–Beehnkendesign. The effect of succinylation degree on S-WPIs solubility was evaluated. Physicochem-ical properties of S-WPIs dried by different three methods on their flow ability, particle size,morphology and in vitro release behavior were studied. After preparing PR-5 sustained re-lease protein tablets with S-WPIs as the carrier by direct powder compression method, thedrug release were studied in vitro and the oral pharmacokinetics and bioavailability wasevaluated using in vivo dog model. It was observed that concentration of substrate has asignificant effect on succinylation. Release behavior in vitro showed spry dried S-WPIs with100% succinylation rate and 30% drug loading would be applied to the preparation of PR-5 sustained-release protein tablets based on the swelling mechanism(protein loss). Comparedwith PR-5 conventional tablet with oral administration, T max value of PR-5 sustained-releaseprotein tablets was approximately 1.58 fold greater than those of the conventional tablets asfurther evidenced by the significantly prolonged MRT and T 1/2. The findings demonstratedthat spray-dried S-WPIs has potential as a promising functional excipient for the design of PR-5 oral sustained-release tablets which can fully improve sustained-release effect and oral bioavailability.

Synthesis of N-Succinyl-chitosan(Suc-Chi) and Preparation of Oxymatrine(OM)/N-Succinyl-chitosannanoparticles

Oxymatrine （OM）/N-succinyl-chitosan （Suc-Chi, with a degree of substitution being 0. 32） was synthesized via the ring-opening reaction of succinic anhydride with chitosan in dimethyl sulfoxide. OM-loaded Suc-Chi nanoparticles were prepared by an ionotropic gelation process and OM was quantified via the HPLC method： The influences of the initial OM concentration on the nanoparticle characteristics and OM release behavior were evaluated. The nanoparticles were found to have a mean diameter within a range of 267-392 nm, a positive surface charge, and a zeta potential in the range of 19-27 inV. The formulation with an initial OM concentration of 100μg/mL provided the highest loaded capacity（0. 77% ） and the highest extent of the released OM （68% at 24 h）, suggesting the possibility to achieve a therapeutic dose. According to the data obtained, this Suc-Chi-based nanotechnology will open up new and interesting prospects for the development of new anticancer drugs.

Alteration of mitochondrial protein succinylation against cellular oxidative stress in cancer

Dear Editor,Lysine residue succinylation is a novel post-translational modification that recently attracted extensive attention.Succinylation is achieved by non-enzymatic processes or by a series of enzymes[like p300,lysine acetyltransferase 2A(KAT2A)]that transfer the succinyl groups from succinyl-coenzyme A(CoA)to the specific lysine,modulating protein function in various physiological processes[1].As a high-energy metabolite,succinyl-CoA is mainly produced within the mitochondrial matrix and peroxisomes.Its high-rate generation in the tricarboxylic acid(TCA)cycle and its impermeability across the mitochondrial inner membrane(due to its negative charge property)enhance succinyl-CoA accumulation within mitochondria.

Succinylated soybean protein as novel excipient for matrix sustained release microspheres

Many native proteins possess limited functionality, and modification such as succinylation is often performed to expand the range of functional properties available for pharmaceutical dosage form. Succinylation could be useful for modulating protein-based system swelling and drug delivery properties especially for sustained controlled release dosage form like microsphere. A well designed controlled drug delivery system can overcome the problems of conventional drug therapy and gives better therapeutic efficacy of a drug.

The Efficacy and Safety of Succinylated Atelocollagen and Adenosine for the Treatment of Periorbital Wrinkles

The degradation of structural collagen contributes to the characteristic appearance of wrinkles. The anti-wrinkle effects of a variety of substances have been studied, but the potential anti-wrinkle effects of topical applications of collagen for periorbital wrinkles have not been investigated. To evaluate the effects of topical application of succinylated atelocollagen on periorbital wrinkles and to compare the results of treatment with adenosine, a clinical study on Korean participants was carried out. Each participant’s right or left periorbital area was treated with either a solution containing succinylated atelocollagen and adenosine or a solution containing only succinylated atelocollagen for two months. A placebo solution was applied to the opposite periorbital area of each patient’s face for two months. Based on objective and subjective measurements of clinical improvement, the assessment scores for treated sites were statistically significantly higher than scores for placebo sites after two months of treatment. Analysis of silicone casts of periorbital wrinkles demonstrated partial effects of succinylated atelocollagen on periorbital wrinkles. However, we did not observe any effects of adenosine on periorbital wrinkles. Succinylated atelocollagen may be an effective treatment option for periorbital wrinkles, but further studies including a longer treatment period and larger subject group are needed to verify these results.

Pharmacokinetic Evaluation of Chitosan-Succinyl-Prednisolone Conjugate Microparticles as a Colonic Delivery System: Comparison with Enteric-Coated Conjugate Microparticles

In the previous study, chitosan-succinyl-prednisolone conjugate microparticles (MP) were found to exhibit good efficacy and reduced toxicity nearly as well as their Eudragit L-coated microparticles (MP/EuL). This proposes a question whether enteric-coating of MP is necessary or not. Although MP/EuL were already examined for their pharmacokinetic and gastrointestinal behaviors, MP have not been done yet. Therefore, in this study, MP were evaluated by investigating pharmacokinetic features in detail. MP with the in vitro features equivalent to those of the previous conjugate microparticles could be produced more readily in the modified preparative method. Pharmacokinetic and gastrointestinal behaviors of MP were investigated by intragastric dosing (5 mg PD eq./kg) to rats with 2,4,6-trinitrobenzenesulfonic acid-induced ulcerative colitis. The plasma concentration was suppressed extensively in MP as well as MP/EuL, supporting the reduction of PD systemic toxic side effects. However, the plasma level increased gradually up to 7 h in MP, but not in MP/EuL. At 8 h after dosing, MP were detected in the stomach to a fair extent, and free PD was found there, indicating that MP were subjected to trapping in the stomach probably due to positive charge of chitosan molecules. For MP, such prolonged localization and slow release of PD in the stomach were probably associated with the gradual increase in plasma concentration. Therefore, MP/EuL were evaluated to be superior to MP for effective targeting to ulcerative colitis. It is concluded that enteric-coating is very important for the targeting system using MP.

Preparation, Swelling and Antibacterial Behaviors of N-Succinyl Chitosan-g-Poly(Acrylic Acid-co-Acrylamide) Superabsorbent Hydrogels

Superabsorbent hydrogels were prepared successfully from N-succinyl chitosan grafted poly（acrylic acid-co-acrylamide）. The potassium persulfate（KPS）, N, N＇-methylenebisacrylamide（MBA） were used as the initiator and crosslinker, respectively. Fourier transform infrared spectroscopy（FTIR） and scanning electron microscopy（SEM） were used to confirm the porous network structure of superabsorbent hydrogel. The effects of reaction parameters on the swelling behaviors of the superabsorbent hydrogels were investigated. The results indicated that water absorbency increased first, and then decreased gradually with the increase in the contents of monomer（AA＋AM）, KPS, MBA or acrylamide. The product had excellent water absorbency of 1375 g/g in distilled water and 83 g/g in 0.9wt% NaCl solution. Simultaneously, the superabsorbent hydrogels were p H sensitive. The antibacterial activities of the hydrogels against Escherichia coli（E. coli） were improved effectively because of polyamidoamine（PAMAM） dendrimer absorbed in the hydrogels.

Lysine succinylation,the metabolic bridge between cancer and immunity

Lysine succinylation is a naturally occurring post-translational modification(PTM)that regulates the stability and function of proteins.It can be regulated by enzymes such as SIRT5 and SIRT7.Recently,the effect and significance of lysine succinylation in cancer and its implication in immunity have been extensively explored.Lysine succinylation is involved in the malignant phenotype of cancer cells.Abnormal regulation of lysine succinylation occurs in different cancers,and inhibitors targeting lysine succinylation regulatory enzymes can be used as potential anti-cancer strategies.Therefore,this review focused on the target protein lysine succinylation and its functions in cancer and immunity,in order to provide a reference for finding more potential clinical cancer targets in the future.

LDHA Desuccinylase Sirtuin 5 as A Novel Cancer Metastatic Stimulator in Aggressive Prostate Cancer

Prostate cancer(PCa)is the most commonly diagnosed genital cancer in men worldwide.Around 80%of the patients who developed advanced PCa suffered from bone metastasis,with a sharp drop in the survival rate.Despite great efforts,the detailed mechanisms underlying castration-resistant PCa(CRPC)remain unclear.Sirtuin 5(SIRT5),an NAD+-dependent desuccinylase,is hypothesized to be a key regulator of various cancers.However,compared to other SIRTs,the role of SIRT5 in cancer has not been extensively studied.Here,we revealed significantly decreased SIRT5 levels in aggressive PCa cells relative to the PCa stages.The correlation between the decrease in the SIRT5 level and the patient’s reduced survival rate was also confirmed.Using quantitative global succinylome analysis,we characterized a significant increase in the succinylation at lysine 118(K118su)of lactate dehydrogenase A(LDHA),which plays a role in increasing LDH activity.As a substrate of SIRT5,LDHA-K118su significantly increased the migration and invasion of PCa cells and LDH activity in PCa patients.This study reveals the reduction of SIRT5 protein expression and LDHA-K118su as a novel mechanism involved in PCa progression,which could serve as a new target to prevent CPRC progression for PCa treatment.

Protein post-translational modification by lysine succinylation:Biochemistry,biological implications,and therapeutic opportunities

Lysine succinylation(Ksuc)is a novel protein post-translational modification(PTM)wherein a succinyl group modifies a lysine residue.Ksuc leads to significant chemical and struc-tural changes to the modified protein.Recent studies have shown that Ksuc might play an important role in organism physiology and some pathophysiological processes,such as tumor-igenesis and metabolic diseases.To provide an understanding of the molecular mechanism and functions of Ksuc in different organisms,we reviewed the current literature about Ksuc,mainly summarizing the research advances in eukaryotes and prokaryotes based on both traditional study methods and site prediction tools.We also discussed inhibitors or activators associated with Ksuc that may contribute to proteomic studies and could be useful in future clinical prac-tice.A deeper understanding of Ksuc may shed new light on life science at the protein level and could lead to novel therapeutic strategies for various diseases.

Sodium butyrate activates HMGCS2 to promote ketone body production through SIRT5-mediated desuccinylation

Ketone bodies have beneficial metabolic activities,and the induction of plasma ketone bodies is a health promotion strategy.Dietary supplementation of sodium butyrate(SB)is an effective approach in the induction of plasma ketone bodies.However,the cellular and molecular mechanisms are unknown.In this study,SB was found to enhance the catalytic activity of 3-hydroxy-3-methylglutaryl-CoA synthase 2(HMGCS2),a rate-limiting enzyme in ketogenesis,to promote ketone body production in hepatocytes.SB administrated by gavage or intraperitoneal injection significantly induced bloodβ-hydroxybutyrate(BHB)in mice.BHB production was induced in the primary hepatocytes by SB.Protein succinylation was altered by SB in the liver tissues with down-regulation in 58 proteins and up-regulation in 26 proteins in the proteomics analysis.However,the alteration was mostly observed in mitochondrial proteins with 41%down-and 65%up-regulation,respectively.Succinylation status of HMGCS2 protein was altered by a reduction at two sites(K221 and K358)without a change in the protein level.The SB effect was significantly reduced by a SIRT5 inhibitor and in Sirt5-KO mice.The data suggests that SB activated HMGCS2 through SIRT5-mediated desuccinylation for ketone body production by the liver.The effect was not associated with an elevation in NAD+/NADH ratio according to our metabolomics analysis.The data provide a novel molecular mechanism for SB activity in the induction of ketone body production.

Comprehensive analysis of the gut microbiome and posttranslational modifications elucidates the route involved in microbiota-host interactions

The gut microbiome interacts with the host to maintain body homeostasis,with gut microbial dysbiosis implicated in many diseases.However,the underlying mechanisms of gut microbe regulation of host behavior and brain functions remain unclear.This study aimed to elucidate the influence of gut microbiota on brain functions via post-translational modification mechanisms in the presence or absence of bacteria without any stimulation.We conducted succinylome analysis of hippocampal proteins in germ-free(GF)and specific pathogen-free(SPF)mice and metagenomic analysis of feces from SPF mice.These results were integrated with previously reported hippocampal acetylome and phosphorylome data from the same batch of mice.Subsequent bioinformatics analyses revealed 584 succinylation sites on 455 proteins,including 54 up-regulated succinylation sites on 91 proteins and 99 down-regulated sites on 51 proteins in the GF mice compared to the SPF mice.We constructed a panoramic map of gut microbiota-regulated succinylation,acetylation,and phosphorylation,and identified cross-talk and relative independence between the different types of post-translational modifications in modulating complicated intracellular pathways.Pearson correlation analysis indicated that 13 taxa,predominantly belonging to the Bacteroidetes phylum,were correlated with the biological functions of post-translational modifications.Positive correlations between these taxa and succinylation and negative correlations between these taxa and acetylation were identified in the modulation of intracellular pathways.This study highlights the hippocampal physiological changes induced by the absence of gut microbiota,and proteomic quantification of succinylation,phosphorylation,and acetylation,contributing to our understanding of the role of the gut microbiome in brain function and behavioral phenotypes.

琥珀酰化修饰对甘油醛-3-磷酸脱氢酶活性的影响

通过定点突变和生物学分析,研究了赖氨酸琥珀酰化对GAPDH活性的影响。结果表明:发菜GAPDH基因全长为1014 bp,由338个氨基酸组成,其中K264位点在藻类中高度保守。将K264位点的氨基酸K(AAA)突变为R(AGA),野生型和突变型GAPDH在大肠杆菌中表达,获得一个36.61 ku的外源蛋白。纯化后蛋白活性测定发现,发生琥珀酰化修饰比未发生琥珀酰化修饰的GAPDH(K264)活性显著降低,表明琥珀酰化修饰参与发菜GAPDH活性的调节。研究结果为深入研究发菜GAPDH的分子信息和生物学功能提供理论参考。

Enzymatic and metabolic regulation of lysine succinylation

Lysine succinylation(Ksucc),defined as a transfer of a succinyl group to a lysine residue of a protein,is a newly identified protein post-translational modification^1-3.This chemical modification is reversible,dynamic,and evolutionarily conserved^4 where it has been comprehensively studied in both bacterial and mammalian cells^5-7.Numerous proteins involved in the regulation of various cellular and biological processes have been shown to be heavily succinylated^5-7.Emerging clinical data provides evidence that dysregulation of Ksucc is correlated with the development of several diseases,including cardiovascular diseases and cancer^7-9.Therefore,an in-depth understanding of Ksucc and its regulation is important not only for understanding its physiological function but also for developing drug therapies and targeted agents for these diseases.In this review,we highlight some of the recent advances in understanding the role of Ksucc and desuccinylation under physiological and pathological conditions.

蛋白质琥珀酰化参与丁酸抑制胃癌细胞增殖及迁移的作用

目的:研究丁酸对胃癌细胞增殖及代谢的影响,并探讨其潜在作用机制。方法:使用5 mmol/L丁酸钠处理胃癌细胞48 h后,通过EdU染色及Transwell法检测细胞增殖、迁移情况;收集丁酸处理后的胃癌细胞,提取代谢物进行代谢组学分析筛选出其中显著富集的代谢通路以及上调的代谢物;采用Western blot以及细胞免疫荧光检测丁酸钠处理后胃癌细胞蛋白质琥珀酰化程度,同时采用RT-qPCR检测SIRT5、SUCLG1、SUCLG2和SUCLA2的mRNA水平。结果:丁酸钠处理48 h后胃癌细胞增殖被显著抑制,细胞中检测到三羧酸循环及氧化磷酸化等代谢通路显著富集,其中三羧酸循环代谢物L-苹果酸、琥珀酰辅酶A及延胡索酸显著上调(均P<0.05);Western blot和免疫荧光显示丁酸处理后胃癌细胞蛋白琥珀酰化水平上调(P<0.05)。同时RT-qPCR证实了丁酸处理上调了琥珀酰辅酶A合成酶编码基因SUCLG1、SUCLG2和SUCLA2的mRNA表达水平(均P<0.05),但并未影响SIRT5表达(P>0.05)。结论:丁酸能够抑制胃癌细胞增殖及迁移,其可能通过促进SUCLs表达以及上调琥珀酰辅酶A水平并进一步促进胃癌细胞蛋白质琥珀酰化实现。

琥珀酰化修饰对马氏珠母贝植核免疫的影响

【目的】研究蛋白质琥珀酰化修饰在马氏珠母贝(Pinctada fucata martensii)植核后免疫应答中的作用,为阐明珍珠贝植核反应的分子调控机制提供理论依据。【方法】通过对马氏珠母贝注射琥珀酸钠模拟提高琥珀酰化修饰水平,检测马氏珠母贝植核后鳃组织总蛋白琥珀酰化程度、免疫相关基因表达、抗氧化相关酶酶活以及珍珠贝的存活率和留核率。【结果与结论】注射琥珀酸钠后,鳃组织琥珀酰化修饰水平在12 h和72 h显著上升;使用实时荧光定量PCR检测胁迫应激相关基因的时序表达,胁迫应激相关因子SOD和Caspase2在6 h表达量显著上调,NF-κB和IRAK1在48 h表达量显著上调,TRAF3和IκK的表达量在96 h显著上调(P <0.05),表明琥珀酰化修饰水平提高后受体贝机体细胞免疫增强;抗氧化相关酶、过氧化物酶和谷胱甘肽活性都在96 h出现显著上调(P <0.05),表明受体贝体液免疫增强。与对照组[磷酸盐缓冲溶液(PBS)+植核]和植核组相比,实验组(琥珀酸钠+植核)中珍珠贝的存活率在7 d和45 d存在显著差异,在60 d留核率显著上升(P <0.05)。结果说明,琥珀酰化修饰参与了马氏珠母贝的植核免疫反应,注射琥珀酸钠可以提高受体贝的免疫活力,并提高其在植核后的存活率和留核率。