Understanding the Function of Mammalian Sirtuins and Protein Lysine Acylation.

Protein lysine acetylation is an important posttranslational modification that regulates numerous biological processes. Targeting lysine acetylation regulatory factors, such as acetyltransferases, deacetylases, and acetyl-lysine recognition domains, has been shown to have potential for treating human diseases, including cancer and neurological diseases. Over the past decade, many other acyl-lysine modifications, such as succinylation, crotonylation, and long-chain fatty acylation, have also been investigated and shown to have interesting biological functions. Here, we provide an overview of the functions of different acyl-lysine modifications in mammals. We focus on lysine acetylation as it is well characterized, and principles learned from acetylation are useful for understanding the functions of other lysine acylations. We pay special attention to the sirtuins, given that the study of sirtuins has provided a great deal of information about the functions of lysine acylation. We emphasize the regulation of sirtuins to illustrate that their regulation enables cells to respond to various signals and stresses.

Nucleolin malonylation as a nuclear-cytosol signal exchange mechanism to drive cell proliferation in Hepatocarcinoma by enhancing AKT translation.

Cancer cells undergo metabolic reprogramming that is intricately linked to malignancy. Protein acylations are especially responsive to metabolic changes, influencing signal transduction pathways and fostering cell proliferation. However, as a novel type of acylations, the involvement of malonylation in cancer remains poorly understood. In this study, we observed a significant reduction in malonyl-CoA levels in hepatocellular carcinoma (HCC), which correlated with a global decrease in malonylation. Subsequent nuclear malonylome analysis unveiled nucleolin (NCL) malonylation, which was notably enhanced in HCC biopsies. we demonstrated that NCL undergoes malonylation at lysine residues 124 and 398. This modification triggers the translocation of NCL from the nucleolus to nucleoplasm and cytoplasm, binding to AKT mRNA, and promoting AKT translation in HCC. Silencing AKT expression markedly attenuated HCC cell proliferation driven by NCL malonylation. These findings collectively highlight nuclear signaling in modulating AKT expression, suggesting NCL malonylation as a novel mechanism through which cancer cells drive cell proliferation.

Mycoplasma genitalium membrane lipoprotein induces GAPDH malonylation in urethral epithelial cells to regulate cytokine response.

Membrane lipoproteins serve as primary pro-inflammatory virulence factors in Mycoplasma genitalium. Membrane lipoproteins primarily induce inflammatory responses by activating Toll-like Receptor 2 (TLR2); however, the role of the metabolic status of urethral epithelial cells in inflammatory response remains unclear. In this study, we found that treatment of uroepithelial cell lines with M. genitalium membrane lipoprotein induced metabolic reprogramming, characterized by increased aerobic glycolysis, decreased oxidative phosphorylation, and increased production of the metabolic intermediates acetyl-CoA and malonyl-CoA. The metabolic shift induced by membrane lipoproteins is reversible upon blocking MyD88 and TRAM. Malonyl-CoA induces malonylation of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and malonylated GAPDH could dissociate from the 3' untranslated region of TNF-α and IFN-γ mRNA. This dissociation greatly reduces the inhibitory effect on the translation of TNF-α and IFN-γ mRNA, thus achieving fine-tuning control over cytokine secretion. These findings suggest that GAPDH malonylation following M. genitalium infection is an important inflammatory signal that plays a crucial role in urogenital inflammatory diseases.

Posttranslational Acylations of the Rat Brain Transketolase Discriminate the Enzyme Responses to Inhibitors of ThDP-Dependent Enzymes or Thiamine Transport.

Transketolase (TKT) is an essential thiamine diphosphate (ThDP)-dependent enzyme of the non-oxidative branch of the pentose phosphate pathway, with the glucose-6P flux through the pathway regulated in various medically important conditions. Here, we characterize the brain TKT regulation by acylation in rats with perturbed thiamine-dependent metabolism, known to occur in neurodegenerative diseases. The perturbations are modeled by the administration of oxythiamine inhibiting ThDP-dependent enzymes in vivo or by reduced thiamine availability in the presence of metformin and amprolium, inhibiting intracellular thiamine transporters. Compared to control rats, chronic administration of oxythiamine does not significantly change the modification level of the two detected TKT acetylation sites (K6 and K102) but doubles malonylation of TKT K499, concomitantly decreasing 1.7-fold the level of demalonylase sirtuin 5. The inhibitors of thiamine transporters do not change average levels of TKT acylation or sirtuin 5. TKT structures indicate that the acylated residues are distant from the active sites. The acylations-perturbed electrostatic interactions may be involved in conformational shifts and/or the formation of TKT complexes with other proteins or nucleic acids. Acetylation of K102 may affect the active site entrance/exit and subunit interactions. Correlation analysis reveals that the action of oxythiamine is characterized by significant negative correlations of K499 malonylation or K6 acetylation with TKT activity, not observed upon the action of the inhibitors of thiamine transport. However, the transport inhibitors induce significant negative correlations between the TKT activity and K102 acetylation or TKT expression, absent in the oxythiamine group. Thus, perturbations in the ThDP-dependent catalysis or thiamine transport manifest in the insult-specific patterns of the brain TKT malonylation and acetylations.

Integrated proteome and malonylome analyses reveal the potential meaning of TLN1 and ACTB in end-stage renal disease.

BACKGROUND: End-stage renal disease (ESRD) is a condition that is characterized by the loss of kidney function. ESRD patients suffer from various endothelial dysfunctions, inflammation, and immune system defects. Lysine malonylation (Kmal) is a recently discovered post-translational modification (PTM). Although Kmal has the ability to regulate a wide range of biological processes in various organisms, its specific role in ESRD is limited. METHODS: In this study, the affinity enrichment and liquid chromatography-tandem mass spectrometry (LC-MS/MS) techniques have been used to create the first global proteome and malonyl proteome (malonylome) profiles of peripheral blood mononuclear cells (PBMCs) from twenty patients with ESRD and eighty-one controls. RESULTS: On analysis, 793 differentially expressed proteins (DEPs) and 12 differentially malonylated proteins (DMPs) with 16 Kmal sites were identified. The Rap1 signaling pathway and platelet activation pathway were found to be important in the development of chronic kidney disease (CKD), as were DMPs TLN1 and ACTB, as well as one malonylated site. One conserved Kmal motif was also discovered. CONCLUSIONS: These findings provided the first report on the Kmal profile in ESRD, which could be useful in understanding the potential role of lysine malonylation modification in the development of ESRD.

The Enzyme Lysine Malonylation of Calvin Cycle and Gluconeogenesis Regulated Glycometabolism in Nostoc flagelliforme to Adapt to Drought Stress.

Lysine malonylation (Kmal) is an evolutionarily conserved post-translational modification (PTM) that has been demonstrated to be involved in cellular and organismal metabolism. However, the role that Kmal plays in response to drought stress of the terrestrial cyanobacteria N. flagelliforme is still unknown. In this study, we performed the first proteomic analysis of Kmal in N. flagelliforme under different drought stresses using LC-MS/MS. In total, 421 malonylated lysine residues were found in 236 different proteins. GO and KEGG enrichment analysis indicated that these malonylated proteins were highly enriched in several metabolic pathways, including carbon metabolism and photosynthesis. Decreased malonylation levels were found to hinder the reception and transmission of light energy and CO2 fixation, which led to a decrease in photosynthetic activity. Kmal was also shown to inhibit the flux of the TCA cycle and activate the gluconeogenesis pathway in response to drought stress. Furthermore, malonylated antioxidant enzymes and antioxidants were synergistically involved in reactive oxygen species (ROS) scavenging. Malonylation was involved in lipid degradation and amino acid biosynthesis as part of drought stress adaptation. This work represents the first comprehensive investigation of the role of malonylation in dehydrated N. flagelliforme, providing an important resource for understanding the drought tolerance mechanism of this organism.

Profiling and Quantification of Anthocyanins in Purple-Pericarp Sweetcorn and Purple-Pericarp Maize.

Purple-pericarp sweetcorn accessions, derived from crossing purple-pericarp maize with white shrunken2 sweetcorn, were assessed for differences in anthocyanin profile at both sweetcorn eating stage and at full kernel maturity. The 'Tim1' sweetcorn line developed a similar total anthocyanin concentration to its 'Costa Rica' parent when assessed at sweetcorn-eating stage. At full maturity it surpassed the purple maize parent, but this was mainly due to the presence of starch diluting the anthocyanin concentration of the latter. The anthocyanin/colour relationship was affected by both total anthocyanin concentration and the ratio of cyanidin- to pelargonidin-based anthocyanins. Malonylation of anthocyanins was also found to vary and did not appear to be linked with either cyanidin:pelargonidin ratio or total anthocyanin concentration. In addition, anthocyanin synthesis was affected by kernel maturity at harvest, with colour development increasing in conjunction with a progression of anthocyanin development across the kernel surface. Pigmentation was present in the aleurone, pericarp and vitreous endosperm of kernels of the purple-pericarp maize parent and purple-pericarp sweetcorn accessions when fully mature, but pigmentation was only apparent in the pericarp at sweetcorn-eating stage. Importantly for consumers, anthocyanin pigmentation covered almost the entire kernel surface at sweetcorn-eating stage.

The acyltransferase PMAT1 malonylates brassinolide glucoside.

Brassinosteroids (BRs) are steroid hormones of plants that coordinate fundamental growth and development processes. Their homeostasis is controlled by diverse means, including glucosylation of the bioactive BR brassinolide (BL), which is catalyzed by the UDP-glycosyltransferases (UGTs) UGT73C5 and UGT73C6 and occurs mainly at the C-23 position. Additional evidence had suggested that the resultant BL-23-O-glucoside (BL-23-O-Glc) can be malonylated, but the physiological significance of and enzyme required for this reaction had remained unknown. Here, we show that in Arabidopsis thaliana malonylation of BL-23-O-Glc is catalyzed by the acyltransferase phenolic glucoside malonyl-transferase 1 (PMAT1), which is also known to malonylate phenolic glucosides and lipid amides. Loss of PMAT1 abolished BL-23-O-malonylglucoside formation and enriched BL-23-O-Glc, showing that the enzyme acts on the glucoside. An overexpression of PMAT1 in plants where UGT73C6 was also overexpressed, and thus, BL-23-O-Glc formation was promoted, enhanced the symptoms of BR-deficiency of UGT73C6oe plants, providing evidence that PMAT1 contributes to BL inactivation. Based on these results, a model is proposed in which PMAT1 acts in the conversion of both endogenous and xenobiotic glucosides to adjust metabolic homeostasis in spatial and temporal modes.

Synthesis and Evaluation of a Stable Isostere of Malonyllysine.

Lysine malonylation is a recently characterized post-translational modification involved in the regulation of energy metabolism and gene expression. One unique feature of this post-translational modification is its potential susceptibility to decarboxylation, which poses possible challenges to its study. As a step towards addressing these challenges, we report the synthesis and evaluation of a stable isostere of malonyllysine. First, we find that synthetic substitution of the malonyl group with a tetrazole isostere results in amino acid's resistant to thermal decarboxylation. Next, we demonstrate that protected variants of this amino acid are readily incorporated into peptides. Finally, we show that tetrazole isosteres of malonyllysine can be recognized by anti-malonyllysine antibodies and histone deacylases, validating their ability to mimic features of the endogenous lysine modification. Overall, this study establishes a new chemical strategy for stably mimicking a metabolite-derived post-translational modification, providing a foothold for tool development and functional analyses.

Systematic analysis of the lysine malonylome in Sanghuangporus sanghuang.

BACKGROUND: Sanghuangporus sanghuang is a well-known traditional medicinal mushroom associated with mulberry. Despite the properties of this mushroom being known for many years, the regulatory mechanisms of bioactive compound biosynthesis in this medicinal mushroom are still unclear. Lysine malonylation is a posttranslational modification that has many critical functions in various aspects of cell metabolism. However, at present we do not know its role in S. sanghuang. In this study, a global investigation of the lysine malonylome in S. sanghuang was therefore carried out. RESULTS: In total, 714 malonyl modification sites were matched to 255 different proteins. The analysis indicated that malonyl modifications were involved in a wide range of cellular functions and displayed a distinct subcellular localization. Bioinformatics analysis indicated that malonylated proteins were engaged in different metabolic pathways, including glyoxylate and dicarboxylate metabolism, glycolysis/gluconeogenesis, and the tricarboxylic acid (TCA) cycle. Notably, a total of 26 enzymes related to triterpene and polysaccharide biosynthesis were found to be malonylated, indicating an indispensable role of lysine malonylation in bioactive compound biosynthesis in S. sanghuang. CONCLUSIONS: These findings suggest that malonylation is associated with many metabolic pathways, particularly the metabolism of the bioactive compounds triterpene and polysaccharide. This paper represents the first comprehensive survey of malonylation in S. sanghuang and provides important data for further study on the physiological function of lysine malonylation in S. sanghuang and other medicinal mushrooms.

Computational analysis and prediction of lysine malonylation sites by exploiting informative features in an integrative machine-learning framework.

As a newly discovered post-translational modification (PTM), lysine malonylation (Kmal) regulates a myriad of cellular processes from prokaryotes to eukaryotes and has important implications in human diseases. Despite its functional significance, computational methods to accurately identify malonylation sites are still lacking and urgently needed. In particular, there is currently no comprehensive analysis and assessment of different features and machine learning (ML) methods that are required for constructing the necessary prediction models. Here, we review, analyze and compare 11 different feature encoding methods, with the goal of extracting key patterns and characteristics from residue sequences of Kmal sites. We identify optimized feature sets, with which four commonly used ML methods (random forest, support vector machines, K-nearest neighbor and logistic regression) and one recently proposed [Light Gradient Boosting Machine (LightGBM)] are trained on data from three species, namely, Escherichia coli, Mus musculus and Homo sapiens, and compared using randomized 10-fold cross-validation tests. We show that integration of the single method-based models through ensemble learning further improves the prediction performance and model robustness on the independent test. When compared to the existing state-of-the-art predictor, MaloPred, the optimal ensemble models were more accurate for all three species (AUC: 0.930, 0.923 and 0.944 for E. coli, M. musculus and H. sapiens, respectively). Using the ensemble models, we developed an accessible online predictor, kmal-sp, available at http://kmalsp.erc.monash.edu/. We hope that this comprehensive survey and the proposed strategy for building more accurate models can serve as a useful guide for inspiring future developments of computational methods for PTM site prediction, expedite the discovery of new malonylation and other PTM types and facilitate hypothesis-driven experimental validation of novel malonylated substrates and malonylation sites.

Cellular carbon stress is a mediator of obesity-associated osteoarthritis development.

OBJECTIVE: 'Carbon stress' is a newly found mechanism that links obesity and dysregulated metabolism. It is defined as the cellular accumulation of metabolites during obesity post-translationally modifying metabolic proteins and decreasing their enzymatic activity. The objective of this study was to investigate if 'carbon stress' also occurs in cartilage and contributes to obesity associated OA development. METHODS: We histologically evaluated for OA pathology in wild-type (WT) and hyperphagic mice (Pomc-neuron specific enhancer one deficient, PomcΔ1) that were subjected to standard chow (Chow, n = 6 for both genotypes) or high-fat feeding (HFD, n = 7 for both genotypes). Joints were stained and quantified for 'carbon stress' markers, including succinyl-lysine (SCK), malonyl-lysine (MAK), and acetyl-lysine (ACK). Lastly, we used a mouse model with deletion of Sirt5 (n = 7), which is an enzyme that removes SCK and MAK, to test if changing the abundance of 'carbon stress' would affect OA pathogenesis. RESULTS: Both HFD and Pomc deficiency associated obesity induced cartilage degeneration as well as greater abundance of SCK and MAK in the cartilage. PomcΔ1-HFD mice did not have exacerbated OA pathology as compared to PomcΔ1-Chow mice. ACK was mildly increased in the obese groups comparing to WT-Chow. Sirt5-/- mice developed early-OA like phenotype at 40 weeks of age as characterized by cartilage fibrillation and more hypertrophic chondrocytes. Cartilage from Sirt5-/- mice also had increased SCK and MAK, while ACK remained unchanged comparing to WT mice. CONCLUSION: Our data suggests that carbon stress also occurs in cartilage tissue during obesity and can potentially contribute to obesity-associated OA.

Malonyl-proteome profiles of Staphylococcus aureus reveal lysine malonylation modification in enzymes involved in energy metabolism.

BACKGROUND: Protein lysine malonylation, a novel post-translational modification (PTM), has been recently linked with energy metabolism in bacteria. Staphylococcus aureus is the third most important foodborne pathogen worldwide. Nonetheless, substrates and biological roles of malonylation are still poorly understood in this pathogen. RESULTS: Using anti-malonyl-lysine antibody enrichment and high-resolution LC-MS/MS analysis, 440 lysine-malonylated sites were identified in 281 proteins of S. aureus strain. The frequency of valine in position - 1 and alanine at + 2 and + 4 positions was high. KEGG pathway analysis showed that six categories were highly enriched, including ribosome, glycolysis/gluconeogenesis, pentose phosphate pathway (PPP), tricarboxylic acid cycle (TCA), valine, leucine, isoleucine degradation, and aminoacyl-tRNA biosynthesis. In total, 31 malonylated sites in S. aureus shared homology with lysine-malonylated sites previously identified in E. coli, indicating malonylated proteins are highly conserved among bacteria. Key rate-limiting enzymes in central carbon metabolic pathways were also found to be malonylated in S. aureus, namely pyruvate kinase (PYK), 6-phosphofructokinase, phosphoglycerate kinase, dihydrolipoyl dehydrogenase, and F1F0-ATP synthase. Notably, malonylation sites were found at or near protein active sites, including KH domain protein, thioredoxin, alanine dehydrogenase (ALD), dihydrolipoyl dehydrogenase (LpdA), pyruvate oxidase CidC, and catabolite control protein A (CcpA), thus suggesting that lysine malonylation may affect the activity of such enzymes. CONCLUSIONS: Data presented herein expand the current knowledge on lysine malonylation in prokaryotes and indicate the potential roles of protein malonylation in bacterial physiology and metabolism.

Functions of the sirtuin deacylase SIRT5 in normal physiology and pathobiology.

Sirtuins are NAD+-dependent protein deacylases/ADP-ribosyltransferases that have emerged as candidate targets for new therapeutics to treat metabolic disorders and other diseases, including cancer. The sirtuin SIRT5 resides primarily in the mitochondrial matrix and catalyzes the removal of negatively charged lysine acyl modifications; succinyl, malonyl, and glutaryl groups. Evidence has now accumulated to document the roles of SIRT5 as a significant regulator of cellular homeostasis, in a context- and cell-type specific manner, as has been observed previously for other sirtuin family members. SIRT5 regulates protein substrates involved in glycolysis, the TCA cycle, fatty acid oxidation, electron transport chain, ketone body formation, nitrogenous waste management, and ROS detoxification, among other processes. SIRT5 plays pivotal roles in cardiac physiology and stress responses and is involved in the regulation of numerous aspects of myocardial energy metabolism. SIRT5 is implicated in neoplasia, as both a tumor promoter and suppressor in a context-specific manner, and may serve a protective function in the setting of neurodegenerative disorders. Here, we review the current understanding of functional impacts of SIRT5 on its metabolic targets, and its molecular functions in both normal and pathological conditions. Finally, we will discuss the potential utility of SIRT5 as a drug target and also summarize the current status, progress, and challenges in developing small molecule compounds to modulate SIRT5 activity with high potency and specificity.

Mal-Prec: computational prediction of protein Malonylation sites via machine learning based feature integration : Malonylation site prediction.

BACKGROUND: Malonylation is a recently discovered post-translational modification that is associated with a variety of diseases such as Type 2 Diabetes Mellitus and different types of cancers. Compared with experimental identification of malonylation sites, computational method is a time-effective process with comparatively low costs. RESULTS: In this study, we proposed a novel computational model called Mal-Prec (Malonylation Prediction) for malonylation site prediction through the combination of Principal Component Analysis and Support Vector Machine. One-hot encoding, physio-chemical properties, and composition of k-spaced acid pairs were initially performed to extract sequence features. PCA was then applied to select optimal feature subsets while SVM was adopted to predict malonylation sites. Five-fold cross-validation results showed that Mal-Prec can achieve better prediction performance compared with other approaches. AUC (area under the receiver operating characteristic curves) analysis achieved 96.47 and 90.72% on 5-fold cross-validation of independent data sets, respectively. CONCLUSION: Mal-Prec is a computationally reliable method for identifying malonylation sites in protein sequences. It outperforms existing prediction tools and can serve as a useful tool for identifying and discovering novel malonylation sites in human proteins. Mal-Prec is coded in MATLAB and is publicly available at https://github.com/flyinsky6/Mal-Prec , together with the data sets used in this study.

Lysine malonylation and propionylation are prevalent in human lens proteins.

Acylated lysine residues represent major chemical modifications in proteins. We investigated the malonylation and propionylation of lysine residues (MalK, PropK) in the proteins of aging human lenses. Western blot results showed that the two modifications are present in human lens proteins. Liquid chromatography-mass spectrometry (LC-MS/MS) results showed 4-18 and 4-32 pmol/mg protein of MalK and PropK, respectively, in human lens proteins with no apparent changes related to aging. Mass spectrometry results revealed that MalK- and PropK-modified lysine residues are present in all major crystallins, other cytosolic proteins, and membrane and cytoskeletal proteins of the lens. Several mitochondrial and cytosolic proteins in cultured human lens epithelial cells showed MalK and PropK modifications. Sirtuin 3 (SIRT3) and sirtuin 5 (SIRT5) were present in human lens epithelial and fiber cells. Moreover, lens epithelial cell lysate deacylated propionylated and malonylated lysozyme. The absence of SIRT3 and SIRT5 led to higher PropK and MalK levels in mouse lenses. Together, these data suggest that MalK and PropK are widespread modifications in lens and SIRT3 and SIRT5 could regulate their levels in lens epithelial cells.

Allosteric, transcriptional and post-translational control of mitochondrial energy metabolism.

The heart is the organ with highest energy turnover rate (per unit weight) in our body. The heart relies on its flexible and powerful catabolic capacity to continuously generate large amounts of ATP utilizing many energy substrates including fatty acids, carbohydrates (glucose and lactate), ketones and amino acids. The normal health mainly utilizes fatty acids (40-60%) and glucose (20-40%) for ATP production while ketones and amino acids have a minor contribution (10-15% and 1-2%, respectively). Mitochondrial oxidative phosphorylation is the major contributor to cardiac energy production (95%) while cytosolic glycolysis has a marginal contribution (5%). The heart can dramatically and swiftly switch between energy-producing pathways and/or alter the share from each of the energy substrates based on cardiac workload, availability of each energy substrate and neuronal and hormonal activity. The heart is equipped with a highly sophisticated and powerful mitochondrial machinery which synchronizes cardiac energy production from different substrates and orchestrates the rate of ATP production to accommodate its contractility demands. This review discusses mitochondrial cardiac energy metabolism and how it is regulated. This includes a discussion on the allosteric control of cardiac energy metabolism by short-chain coenzyme A esters, including malonyl CoA and its effect on cardiac metabolic preference. We also discuss the transcriptional level of energy regulation and its role in the maturation of cardiac metabolism after birth and cardiac adaptability for different metabolic conditions and energy demands. The role post-translational modifications, namely phosphorylation, acetylation, malonylation, succinylation and glutarylation, play in regulating mitochondrial energy metabolism is also discussed.

Systematic analysis of the lysine malonylome in common wheat.

BACKGROUND: Protein lysine malonylation, a newly discovered post-translational modification (PTM), plays an important role in diverse metabolic processes in both eukaryotes and prokaryotes. Common wheat is a major global cereal crop. However, the functions of lysine malonylation are relatively unknown in this crop. Here, a global analysis of lysine malonylation was performed in wheat. RESULTS: In total, 342 lysine malonylated sites were identified in 233 proteins. Bioinformatics analysis showed that the frequency of arginine (R) in position + 1 was highest, and a modification motif, KmaR, was identified. The malonylated proteins were located in multiple subcellular compartments, especially in the cytosol (45%) and chloroplast (30%). The identified proteins were found to be involved in diverse pathways, such as carbon metabolism, the Calvin cycle, and the biosynthesis of amino acids, suggesting an important role for lysine malonylation in these processes. Protein interaction network analysis revealed eight highly interconnected clusters of malonylated proteins, and 137 malonylated proteins were mapped to the protein network database. Moreover, five proteins were simultaneously modified by lysine malonylation, acetylation and succinylation, suggesting that these three PTMs may coordinately regulate the function of many proteins in common wheat. CONCLUSIONS: Our results suggest that lysine malonylation is involved in a variety of biological processes, especially carbon fixation in photosynthetic organisms. These data represent the first report of the lysine malonylome in common wheat and provide an important dataset for further exploring the physiological role of lysine malonylation in wheat and likely all plants.

Tissue distribution, subcellular localization, and enzymatic activity analysis of human SIRT5 isoforms.

SIRT5 is one of the seven mammalian sirtuins which are NAD+-dependent deacylases. In human beings, SIRT5 gene encodes for four SIRT5 protein isoforms, namely SIRT5iso1, SIRT5iso2, SIRT5iso3, and SIRT5iso4. Previous studies have focused mostly on SIRT5iso1. Characteristics regarding localization, activity and tissue distribution of the other three SIRT5 isoforms remain unclear. In the present study, we characterized these properties of these SIRT5 isoforms. We found that SIRT5iso1-3 were mitochondria-localized, while SIRT5iso4 localized mainly in cytoplasm. SIRT5iso2-4 had little deacylase activity comparing with SIRT5iso1. Although cDNAs of all SIRT5 isoforms were readily detected in multiply tissues according to EST database, proteins of SIRT5iso2-4 were seldom observed in human cell lines. Altogether, we dissected the four isoforms of human SIRT5 protein.

Predicting lysine-malonylation sites of proteins using sequence and predicted structural features.

Malonylation is a recently discovered post-translational modification (PTM) in which a malonyl group attaches to a lysine (K) amino acid residue of a protein. In this work, a novel machine learning model, SPRINT-Mal, is developed to predict malonylation sites by employing sequence and predicted structural features. Evolutionary information and physicochemical properties are found to be the two most discriminative features whereas a structural feature called half-sphere exposure provides additional improvement to the prediction performance. SPRINT-Mal trained on mouse data yields robust performance for 10-fold cross validation and independent test set with Area Under the Curve (AUC) values of 0.74 and 0.76 and Matthews' Correlation Coefficient (MCC) of 0.213 and 0.20, respectively. Moreover, SPRINT-Mal achieved comparable performance when testing on H. sapiens proteins without species-specific training but not in bacterium S. erythraea. This suggests similar underlying physicochemical mechanisms between mouse and human but not between mouse and bacterium. SPRINT-Mal is freely available as an online server at: http://sparks-lab.org/server/SPRINT-Mal/. © 2018 Wiley Periodicals, Inc.