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.

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.

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.

SuccSite:Incorporating Amino Acid Composition and Informative k-spaced Amino Acid Pairs to Identify Protein Succinylation Sites

Protein succinylation is a biochemical reaction in which a succinyl group(-CO-CH2-CH2-CO-)is attached to the lysine residue of a protein molecule.Lysine succinylation plays important regulatory roles in living cells.However,studies in this field are limited by the difficulty in experimentally identifying the substrate site specificity of lysine succinylation.To facilitate this process,several tools have been proposed for the computational identification of succinylated lysine sites.In this study,we developed an approach to investigate the substrate specificity of lysine succinylated sites based on amino acid composition.Using experimentally verified lysine succinylated sites collected from public resources,the significant differences in position-specific amino acid composition between succinylated and non-succinylated sites were represented using the Two Sample Logo program.These findings enabled the adoption of an effective machine learning method,support vector machine,to train a predictive model with not only the amino acid composition,but also the composition of k-spaced amino acid pairs.After the selection of the best model using a ten-fold crossvalidation approach,the selected model significantly outperformed existing tools based on an independent dataset manually extracted from published research articles.Finally,the selected model was used to develop a web-based tool,SuccSite,to aid the study of protein succinylation.Two proteins were used as case studies on the website to demonstrate the effective prediction of succinylation sites.We will regularly update SuccSite by integrating more experimental datasets.SuccSite is freely accessible at http://csb.cse.yzu.edu.tw/SuccSite/.

Identifying Lysine Succinylation Sites in Proteins by Broad Learning System and Optimizing Imbalanced Training Dataset via Randomly Labeling Samples

As one important type of post-translational modifications(PTMs),protein lysine succinylation regulates many important biological processes.It is also closely involved with some major diseases in the aspects of Cardiometabolic,liver metabolic,nervous system and so on.Therefore,it is imperative to predict the succinylation sites in proteins for both basic research and drug development.In this paper,a novel predictor called i Succ Lys-BLS was proposed by not only introducing a new machine learning algorithm—Broad Learning System,but also optimizing the imbalanced data by randomly labeling samples.Rigorous cross-validation and independent test indicate that the success rate of i Succ Lys-BLS for positive samples is overwhelmingly higher than its counterparts.

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.

HybridSucc:A Hybrid-learning Architecture for General and Species-specific Succinylation Site Prediction

As an important protein acylation modification,lysine succinylation(Ksucc)is involved in diverse biological processes,and participates in human tumorigenesis.Here,we collected 26,243 non-redundant known Ksucc sites from 13 species as the benchmark data set,combined 10 types of informative features,and implemented a hybrid-learning architecture by integrating deep-learning and conventional machine-learning algorithms into a single framework.We constructed a new tool named HybridSucc,which achieved area under curve(AUC)values of 0.885 and 0.952 for general and human-specific prediction of Ksucc sites,respectively.In comparison,the accuracy of HybridSucc was 17.84%-50.62%better than that of other existing tools.Using HybridSucc,we conducted a proteome-wide prediction and prioritized 370 cancer mutations that change Ksucc states of 218 important proteins,including PKM2,SHMT2,and IDH2.We not only developed a high-profile tool for predicting Ksucc sites,but also generated useful candidates for further experimental consideration.The online service of HybridSucc can be freely accessed for academic research at http://hybridsucc.biocuckoo.org/.

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.

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.

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.

The impact of N-terminal modification of PAA with different chain lengths on the structure,thermal stability and pH sensitivity of succinylated collagen

The limitations of native collagen,such as thermal stability and solubility in physiological environments,can be improved by applying bioconjugation and synthetic chemistry techniques.However,the exquisite control of the modification site of collagen remains a challenge.In this work,pH-responsive poly(acrylic acid)(PAA)with dif-ferent chain lengths was attached to the N-terminal α-amino groups of succinylated collagen using a site-specific modification strategy.Additionally,the structure,thermal stability,and pH sensitivity of succinylated collagen were explored.The modification rate of amino groups in the succinylated collagen-PAA bioconjugate(SPSC-PAA)was eval-uated by the 2,4,6-trinitrobenzene sulfonic acid assay.The impact of N-terminal modification of PAA and its chain length on the thermal stability of collagen was explored by CD and DSC.These techniques revealed that the ther-mal stability of SPSC-Col is pH-responsive and closely related to the chain length of grafted PAA.The pH sensitivity of SPSC-PAA was further explored by rheology and turbidity.Subsquently,the critical pH and isoelectric point of SPSC-PAAs were also examined by turbidity and isoelectric point titration,respectively.This work provides a new insight into the N-terminal modification of collagen on its properties.

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.

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.

Facile semisynthesis of histone H3 enables nucleosome probes for investigation of histone H_(3)K79 modifications

Histone H3K79 modifications are essential to regulate chromatin structure and gene transcription,but understanding of the molecular mechanisms is limited.Because H3K79 is at globular domain,short histone peptide cannot mimic H3K79 in chromatin.Instead,reconstituted nucleosome-based chemical tools are ideally used to investigate H3K79 modifications.In consequence,H3K79-modified histone H3 with additional chemical handles are required,but such synthesis is challenging and laborious.Here we report a facile semisynthesis method that enables multifunctional histone H3 readily available.H3K79-containing fragment is short for straight peptide synthesis that was later ligated to recombinant expressed H3 fragments for full-length product in large scale.As a result,nucleosomes with H3K79 modifications as well as photo-reactive group and affinity tag were obtained to investigate potential binding proteins.We believe this method that enhances synthetic accessibility of nucleosome probes will accelerate understanding of the underexplored H3K79 modifications.

Accessory gland protein regulates pairing process and oviposition in the subterranean termite Reticulitermes chinensis after swarming

Swarming and pairing behaviors are significant to population dispersal of termites.Tandem running is a key process in pairing behavior of dealates to find a mate.Succinylation can lead to significant changes in protein structure and function,which is widely involved in metabolism and behavior regulation in many organisms.However,whether succinylation modification regulates termites’tandem running is currently unknown.In this research,we performed quantitative modified proteomics of the subterranean termite Reticulitermes chinensis Snyder before and after alate swarming.The succinylation levels of accessory gland protein(ACP)were significantly altered after alate swarming.We found that ACP is enriched in male accessory gland and female oocytes of termites.The acetylation and succinylation sites of ACP affected tandem running of dealates.The transcriptome and metabolome analyses of alates injected with ACP and its mutant proteins showed thatβ-alanine metabolism pathway was the major downstream pathway of ACP.Silencing the significantly differentially expressed genes in theβ-alanine metabolic pathway(acyl-CoA dehydrogenase,enoyl-CoA hydratase,3-hydroxyisobutyrate dehydrogenase,methylmalonate-semialdehyde dehydrogenase)suppressed tandem running and altered oviposition of paired dealates.These findings demonstrate that protein translation modification is an important regulator of tandem running behavior of termites,which implies that the succinylation and acetylation modification sites of ACP could be potential targets for insecticide action.Our research offers a potential approach for developing novel dispersal inhibitors against social insect pests.

NF-κB/HDAC1/SREBP1c pathway mediates the inflammation signal in progression of hepatic steatosis

The transcription factor nuclear factor kappa B(NF-κB)is activated in hepatoctes in the pathogenesis of hepatic steatosis.However,the action mechanism of NF-κB remains to be established in the hepatic steatosis.In this study,the P50 subunit of NF-κB was found to promote the hepatic steatosis through regulation of histone deacetylase 1(HDAC1)in hepatocytes.The activity was supported by the phenotypes of P50 knockout(P50-KO)mice and P65 knockout(P65-KO)mice.Hepatic steatosis was reduced in the P50-KO mice,but not in the P65-KO mice.The reduction was a result of inhibition of HDAC1 activity in the P50-KO cells.Knockdown of Hdac1 gene led to suppression of hepatocyte steatosis in HepG2 cells.A decrease in sterol-regulatory element binding protein lc(SREBP1 c)protein was observed in the liver of P50-KO mice and in cell with Hdac1 knockdown.The decrease was associated with an increase in succinylation of SREBP1 c protein.The study suggests that P50 stabilizes HDAC1 to support the SREBP1 c activity in hepatic steatosis in the pathophysiological condition.Interruption of this novel pathway in the P50-KO,but not the P65-KO mice,may account for the difference in hepatic phenotypes in the two lines of transgenic mice.