Protein Lysine Acetylation Research Articles

Protein acetylation protects sperm from spontaneous acrosome reaction

In order to penetrate the egg, spermatozoa must undergo the acrosome reaction in close proximity to the egg. This process can take place only after a series of biochemical changes in the sperm, collectively termed capacitation, occur in the female reproductive tract. Sperm cells can undergo spontaneous-acrosome reaction(sAR) before reaching the vicinity of the egg, preventing successful fertilization. Several mechanisms were shown to protect sperm from undergoing sAR, and all of them are involved in proper capacitation. Here, we describe the involvement of protein acetylation in the mechanism that protects bovine spermatozoa from sAR. Incubation of bovine sperm under non-capacitation conditions revealed a strong increase in sAR that was significantly reduced in the presence of deacetylase inhibitors. Protein kinase A (PKA) is an essential key enzyme in sperm capacitation, and its inhibition results in high sAR. The reduction in sAR by hyperacetylation was independent of PKA activity. We previously demonstrated that calmodulin-kinase II (CaMKII) activity protects sperm from sAR, and here we show that its activity is essential for reduction in sAR by hyperacetylation. We further show that the ‘exchange protein directly activated by Camp’ (EPAC) mediates both protein lysine acetylation and the reduced rate of sAR caused by hyperacetylation. In conclusion, these results suggest a PKA-independent and EPAC-CaMKII dependent hyperacetylation mechanism that protects sperm from sAR.

Quantitative Acetylomics Reveals Dynamics of Protein Lysine Acetylation in Mouse Livers During Aging and Upon the Treatment of Nicotinamide Mononucleotide

Lysine acetylation is a reversible and dynamic post-translational modification that plays vital roles in regulating multiple cellular processes including aging. However, acetylome-wide analysis in the aging process remains poorly studied in mammalian tissues. Nicotinamide adenine dinucleotide (NAD+), a hub metabolite, benefits health span at least in part due to the activation of Sirtuins, a family of NAD+-consuming deacetylases, indicating changes in acetylome. Here, we combine two antibodies for the enrichment of acetylated peptides and perform label-free quantitative acetylomic analysis of mouse livers during natural aging and upon the treatment of beta-nicotinamide mononucleotide (NMN), a NAD+ booster. Our study describes previously unknown acetylation sites and reveals the acetylome-wide dynamics with age as well as upon the treatment of NMN. We discover protein acetylation events as potential aging biomarkers. We demonstrate that the life-beneficial effect of NMN could be partially reflected by the changes in age-related protein acetylation. Our quantitative assessment indicates that NMN has mild effects on acetylation sites previously reported as substrates of Sirtuins. Collectively, our data analyze protein acetylation with age, laying critical foundation for the functional study of protein post-translational modification essential for healthy aging and perhaps disease conditions.

GCN5L1 impairs diastolic function in mice exposed to a high fat diet by restricting cardiac pyruvate oxidation.

Left ventricular diastolic dysfunction is a structural and functional condition that precedes the development of heart failure with preserved ejection fraction (HFpEF). The etiology of diastolic dysfunction includes alterations in fuel substrate metabolism that negatively impact cardiac bioenergetics, and may precipitate the eventual transition to heart failure. To date, the molecular mechanisms that regulate early changes in fuel metabolism leading to diastolic dysfunction remain unclear. In this report, we use a diet‐induced obesity model in aged mice to show that inhibitory lysine acetylation of the pyruvate dehydrogenase (PDH) complex promotes energetic deficits that may contribute to the development of diastolic dysfunction in mouse hearts. Cardiomyocyte‐specific deletion of the mitochondrial lysine acetylation regulatory protein GCN5L1 prevented hyperacetylation of the PDH complex subunit PDHA1, allowing aged obese mice to continue using pyruvate as a bioenergetic substrate in the heart. Our findings suggest that changes in mitochondrial protein lysine acetylation represent a key metabolic component of diastolic dysfunction that precedes the development of heart failure.

ROS-stimulated protein lysine acetylation is required for crown root development in rice

IntroductionAs signal molecules in aerobic organisms, locally accumulated ROS have been reported to balance cell division and differentiation in the root meristem. Protein posttranslational modifications such as lysine acetylation play critical roles in controlling a variety of cellular processes. However, the mechanism by which ROS regulate root development is unknown. In addition, how protein lysine acetylation is regulated and whether cellular ROS levels affect protein lysine acetylation remain unclear. ObjectivesWe aimed to elucidate the relationship between ROS and protein acetylation by exploring a rice mutant plant that displays a decreased level of ROS in postembryonic crown root (CR) cells and severe defects in CR development. MethodsFirst, proteomic analysis was used to find candidate proteins responsible for the decrease of ROS detected in the wox11 mutant. Then, biochemical, molecular, and genetic analyses were used to study WOX11-regulated genes involved in ROS homeostasis. Finally, acetylproteomic analysis of wild type and wox11 roots treated with or without potassium iodide (KI) and hydrogen peroxide (H2O2) was used to study the effects of ROS on protein acetylation in rice CR cells. ResultsWe demonstrated that WOX11 was required to maintain ROS homeostasis by upregulating peroxidase genes in the crown root meristem. Acetylproteomic analysis revealed that WOX11-dependent hydrogen peroxide (H2O2) levels in CR cells promoted lysine acetylation of many non-histone proteins enriched for nitrogen metabolism and peptide/protein synthesis pathways. Further analysis revealed that the redox state affected histone deacetylases (HDACs) activity, which was likely related to the high levels of protein lysine acetylation in CR cells. ConclusionWOX11-controlled ROS level in CR meristem cells is required for protein lysine acetylation which represents a mechanism of ROS-promoted CR development in rice.

The sirtuin 1 activator YK 3-237 stimulates capacitation-related events in human spermatozoa

Research questionDoes sirtuin-1 (SIRT1) have a role in the human spermatozoa capacitation process? DesignHuman spermatozoa were incubated for 6 h in a capacitating medium in presence or absence of the specific SIRT1 activator, YK 3-237. Several sperm parameters were determined by flow cytometry: viability, acrosome reaction and mitochondria membrane status. Sperm motility was determined objectively by computer-assisted semen analysis. Sperm capacitation status was evaluated by the extent of protein tyrosine phosphorylation and by the percentage of spermatozoa with the acrosome reacted by a calcium ionophore challenge. ResultsSIRT1 was detected in the connecting piece of human spermatozoa where a lysine acetylation pattern was mainly found along the sperm tail. SIRT1 activation accelerates the occurrence of a phenotype associated with human sperm capacitation, with no differences seen in the lysine acetylation pattern. After 1 h of co-incubation of YK 3-237 with human spermatozoa, tyrosine phosphorylation levels were comparable to control levels after 6 h of incubation in capacitating conditions. In addition, the activator improved sperm responsiveness to a Ca2+ ionophore (A23187) challenge determined by an increase in acrosome-reacted spermatozoa (P = 0.025). Importantly, sperm viability and mitochondrial activity-related parameters assessed by flow cytometry were not affected by YK 3-237. ConclusionYK 3-237 induces capacitation-related events in human spermatozoa such an increase of tyrosine phosphorylation levels and acrosome-reacted spermatozoa after the ionophore challenge. Together, these results show that YK 3-237 affects human spermatozoa capacitation-related events by a mechanism independent of protein lysine acetylation but dependent on bicarbonate and calcium.

DeepDA-Ace: A Novel Domain Adaptation Method for Species-Specific Acetylation Site Prediction

Protein lysine acetylation is an important type of post-translational modification (PTM), and it plays a crucial role in various cellular processes. Recently, although many researchers have focused on developing tools for acetylation site prediction based on computational methods, most of these tools are based on traditional machine learning algorithms for acetylation site prediction without species specificity, still maintained as a single prediction model. Recent studies have shown that the acetylation sites of distinct species have evident location-specific differences; however, there is currently no integrated prediction model that can effectively predict acetylation sites cross all species. Therefore, to enhance the scope of species-specific level, it is necessary to establish a framework for species-specific acetylation site prediction. In this work, we propose a domain adaptation framework DeepDA-Ace for species-specific acetylation site prediction, including Rattus norvegicus, Schistosoma japonicum, Arabidopsis thaliana, and other types of species. In DeepDA-Ace, an attention based densely connected convolutional neural network is designed to capture sequence features, and the semantic adversarial learning strategy is proposed to align features of different species so as to achieve knowledge transfer. The DeepDA-Ace outperformed both the general prediction model and fine-tuning based species-specific model across most types of species. The experiment results have demonstrated that DeepDA-Ace is superior to the general and fine-tuning methods, and its precision exceeds 0.75 on most species. In addition, our method achieves at least 5% improvement over the existing acetylation prediction tools.

The Lysine Acetylation Modification in the Porin Aha1 of Aeromonas hydrophila Regulates the Uptake of Multidrug Antibiotics

Protein lysine acetylation (Kac) modification plays important roles in diverse physiological functions. However, there is little evidence on the role of Kac modification in bacterial antibiotic resistance. Here, we compared the differential expressions of whole-cell proteins and Kac peptides in oxytetracycline sensitive and oxytetracycline resistance (OXYR) strains of Aeromonas hydrophila using quantitative proteomics technologies. We observed a porin family protein Aha1 downregulated in the OXYR strain, which may have an important role in the OXY resistance. Interestingly, seven of eight Kac peptides of Aha1 decreased abundance in OXYR as well. Microbiologic assays showed that the K57R, K187R, and K197R Aha1 mutants significantly increased antibiotic resistance to OXY and reduced the intracellular OXY accumulation in OXY stress. Moreover, these Aha1 mutants displayed multidrug resistance features to tetracyclines and β-lactam antibiotics. The 3D model prediction showed that the Kac states of K57, K187, and K197 sites located at the extracellular pore vestibule of Aha1 may be involved in the uptake of specific types of antibiotics. Overall, our results indicate a novel antibiotic resistance mechanism mediated by Kac modification, which may provide a clue for the development of antibiotic therapy strategies.

Chiral Posttranslational Modification to Lysine ε-Amino Groups.

The sophistication of proteomic analysis has revealed that protein lysine residues are posttranslationally modified by a variety of acyl groups. Protein lysine acetylation regulates metabolism, gene expression, and microtubule formation and has been extensively studied; however, the understanding of the biological significance of other acyl posttranslational modifications (PTMs) is still in its infancy. The acylation of lysine residues is mediated either by acyltransferase "writer" enzymes or by nonenzymatic mechanisms and hydrolase enzymes, termed "erasers", that cleave various acyl PTMs to reverse the modified state. We have studied the human lysine deacylase enzymes, comprising the 11 Zn2+-dependent histone deacetylases (HDACs) and the 7 NAD+-consuming sirtuins (SIRTs), over the past decade. We have thus developed selective inhibitors and molecular probes and have studied the acyl substrate scope of each enzyme using chemically synthesized peptide substrates and photo-cross-linking probes. Recently, we have turned our attention to PTMs containing a stereogenic center, such as ε-N-β-hydroxybutyryllysine (Kbhb) and ε-N-lactyllysine (Kla), that each comprise a pair of mirror image stereoisomers as modifications. Both modifications are found on histones, where they affect gene transcription in response to specific metabolic states, and they are found on cytosolic and mitochondrial enzymes involved in fatty acid oxidation (Kbhb) and glycolysis (Kla), respectively. Thus, chiral modifications to lysine side chains give rise to two distinct diastereomeric products, with separate metabolic origins and potentially different activities exhibited by writer and eraser enzymes. Lysine l-lactylation originates from l-lactate, a major energy carrier produced from pyruvate after glycolysis, and it is highly induced by metabolic states such as the Warburg effect. l-Lactate can possibly be activated by acyl-coenzyme A (CoA) synthetases and transferred to lysine residues by histone acetyltransferases such as p300. d-Lactylation, on the other hand, arises primarily from a nonenzymatic reaction with d-lactylglutathione, an intermediate in the glyoxalase pathway. In addition to their distinct origin, we found that both K(l-la) and K(d-la) modifications are erased by HDACs with different catalytic efficiencies. Also, K(l-bhb) and K(d-bhb) arise from different metabolites but depend on interconnected metabolic pathways, and the two stereoisomers of ε-N-3-hydroxy-3-methylglutaryllysine (Khmg) originate from a single precursor that may then be regulated differently by eraser enzymes. Distinguishing between the individual stereoisomers of PTMs is therefore of crucial importance. In the present Account, we will (1) revisit the long-standing evidence for the distinct production and dynamics of enantiomeric forms of chiral metabolites that serve as ε-N-acyllysine PTMs and (2) highlight the outstanding questions that arise from the recent literature on chiral lysine PTMs resulting from these metabolites.

A Comprehensive Analysis of the Lysine Acetylome in the Aquatic Animals Pathogenic Bacterium Vibrio mimicus

Protein lysine acetylation is an evolutionarily conserved post-translational modification (PTM), which is dynamic and reversible, playing a crucial regulatory role in almost every aspect of metabolism, of both eukaryotes and prokaryotes. Several global lysine acetylome studies have been carried out in various bacteria, but thus far, there have been no reports of lysine acetylation for the commercially important aquatic animal pathogen Vibrio mimicus. In the present study, we used anti-Ac-K antibody beads to highly sensitive immune-affinity purification and combined high-resolution LC-MS/MS to perform the first global lysine acetylome analysis in V. mimicus, leading to the identification of 1,097 lysine-acetylated sites on 582 proteins, and more than half (58.4%) of the acetylated proteins had only one site. The analysis of acetylated modified peptide motifs revealed six significantly enriched motifs, namely, KacL, KacR, L(-2) KacL, LKacK, L(-7) EKac, and IEKac. In addition, bioinformatic assessments state clearly that acetylated proteins have a hand in many important biological processes in V. mimicus, such as purine metabolism, ribosome, pyruvate metabolism, glycolysis/gluconeogenesis, the TCA cycle, and so on. Moreover, 13 acetylated proteins were related to the virulence of V. mimicus. To sum up, this is a comprehensive analysis whole situation protein lysine acetylome in V. mimicus and provides an important foundation for in-depth study of the biological function of lysine acetylation in V. mimicus.

Presence of acetylated α-tubulin in human sperm nuclei: A contributor to sperm heterogeneity

The global rise in male infertility necessitates a constant search for underlying causes, diagnostics and corrective treatments. Sperm cells are irreplaceable cells for the process of reproduction. Intrauterine insemination (IUI) is a simple and non-invasive technique, most commonly used to overcome both unexplained and mild male-factor infertility. Even though spermatozoa may be classified as “normal” in terms of morphology, motility and concentration, they could be defective at the subcellular level. Protein lysine acetylation within human sperm, a major post-translational modification of tubulin, is suspected to be a cause of sperm abnormality but is not fully understood. An examination of α-tubulin and acetylated α-tubulin immunopresence, spatial distribution and co-localisation in normozoospermic and asthenozoospermic semen samples was conducted to determine if acetylated α-tubulin could be a biomarker of sperm heterogeneity to better predict sperm fertility potential for IUI outcome. Acetylated α-tubulin was present in both sperm subcompartments, tail and nucleus of normozoospermic samples. We found more immunopositivity for acetylated α-tubulin in the sperm tail and less in nucleus of asthenozoospermic compared to normozoospermic samples. Hence, the acetylated α-tubulin in sperm may contribute to sperm nuclei heterogeneity and serve as a novel molecular biomarker.

GCN5 maintains muscle integrity by acetylating YY1 to promote dystrophin expression.

Protein lysine acetylation is a post-translational modification that regulates protein structure and function. It is targeted to proteins by lysine acetyltransferases (KATs) or removed by lysine deacetylases. This work identifies a role for the KAT enzyme general control of amino acid synthesis protein 5 (GCN5; KAT2A) in regulating muscle integrity by inhibiting DNA binding of the transcription factor/repressor Yin Yang 1 (YY1). Here we report that a muscle-specific mouse knockout of GCN5 (Gcn5skm-/-) reduces the expression of key structural muscle proteins, including dystrophin, resulting in myopathy. GCN5 was found to acetylate YY1 at two residues (K392 and K393), disrupting the interaction between the YY1 zinc finger region and DNA. These findings were supported by human data, including an observed negative correlation between YY1 gene expression and muscle fiber diameter. Collectively, GCN5 positively regulates muscle integrity through maintenance of structural protein expression via acetylation-dependent inhibition of YY1. This work implicates the role of protein acetylation in the regulation of muscle health and for consideration in the design of novel therapeutic strategies to support healthy muscle during myopathy or aging.

Acetylproteomics analyses reveal critical features of lysine-\u03b5-acetylation in Arabidopsis and a role of 14-3-3 protein acetylation in alkaline response

Lysine-ε-acetylation (Kac) is a post-translational modification (PTM) that is critical for metabolic regulation and cell signaling in mammals. However, its prevalence and importance in plants remain to be determined. Employing high-resolution tandem mass spectrometry, we analyzed protein lysine acetylation in five representative Arabidopsis organs with 2 ~ 3 biological replicates per organ. A total of 2887 Kac proteins and 5929 Kac sites were identified. This comprehensive catalog allows us to analyze proteome-wide features of lysine acetylation. We found that Kac proteins tend to be more uniformly expressed in different organs, and the acetylation status exhibits little correlation with the gene expression level, indicating that acetylation is unlikely caused by stochastic processes. Kac preferentially targets evolutionarily conserved proteins and lysine residues, but only a small percentage of Kac proteins are orthologous between rat and Arabidopsis. A large portion of Kac proteins overlap with proteins modified by other PTMs including ubiquitination, SUMOylation and phosphorylation. Although acetylation, ubiquitination and SUMOylation all modify lysine residues, our analyses show that they rarely target the same sites. In addition, we found that “reader” proteins for acetylation and phosphorylation, i.e., bromodomain-containing proteins and GRF (General Regulatory Factor)/14-3-3 proteins, are intensively modified by the two PTMs, suggesting that they are main crosstalk nodes between acetylation and phosphorylation signaling. Analyses of GRF6/14-3-3λ reveal that the Kac level of GRF6 is decreased under alkaline stress, suggesting that acetylation represses plant alkaline response. Indeed, K56ac of GRF6 inhibits its binding to and subsequent activation of the plasma membrane H+-ATPase AHA2, leading to hypersensitivity to alkaline stress. These results provide valuable resources for protein acetylation studies in plants and reveal that protein acetylation suppresses phosphorylation output by acetylating GRF/14-3-3 proteins.

A Proteomic Approach for the Quantification of Posttranslational Protein Lysine Acetylation in Candida albicans.

Candida albicans is a normal component of the human microflora that colonizes mucosal/epithelial surfaces and the gastrointestinal tract as a commensal organism. However, in an immunocompromised host, it can cause life-threatening infections of high mortality and morbidity. Virulence as well as antifungal drug resistance of C. albicans is often regulated by posttranslational modifications (PTM) of proteins via lysine acetylation by lysine acetyltransferases. Here, we report an experimental approach using tandem mass tag (TMT) labeling for the detection and quantification of lysine acetylation of histone and nonhistone proteins in C. albicans.

Protein acetylation-mediated cross regulation of acetic acid and ethanol synthesis in the gas-fermenting Clostridium ljungdahlii

The autotrophic acetogen Clostridium ljungdahlii has emerged as a major candidate in the biological conversion of one-carbon gases (CO2/CO) to bulk chemicals and fuels. Nevertheless, the regulatory pathways and downstream metabolic changes responsible for product formation and distribution in this bacterium remain minimally explored. Protein lysine acetylation (PLA), a prevalent posttranslational modification, controls numerous crucial cellular functions. Herein, we revealed a novel cross-regulatory mechanism that uses both the PLA system and transcription factors to regulate the carbon flow distribution for product formation in C. ljungdahlii. The dominant acetylation/deacetylation system (At2/Dat1) in C. ljungdahlii was found to regulate the ratio of two major products, acetic acid and ethanol. Subsequent genetic and biochemical analyses revealed that the activities of Pta and AdhE1, two crucial enzymes responsible for acetic acid and ethanol synthesis, respectively, were greatly affected by their levels of PLA. We found that the acetylation statuses of Pta and AdhE1 underwent significant dynamic changes during the fermentation process, leading to differential synthesis of acetic acid and ethanol. Furthermore, the crucial redox-sensing protein Rex was shown to be regulated by PLA, which subsequently altered its transcriptional regulation on genes responsible for acetic acid and ethanol formation and distribution. Based on our understanding of this cross-regulatory module, we optimized the ethanol synthetic pathway by modifying the acetylation status (deacetylation-mimicked mutations of crucial lysine residues) of the related key enzyme, achieving significantly increased titer and yield of ethanol, an important chemical and fuel, by C. ljungdahlii in gas fermentation.

Lysine acetylation regulates the interaction between proteins and membranes

Lysine acetylation regulates the function of soluble proteins in vivo, yet it remains largely unexplored whether lysine acetylation regulates membrane protein function. Here, we use bioinformatics, biophysical analysis of recombinant proteins, live-cell fluorescent imaging and genetic manipulation of Drosophila to explore lysine acetylation in peripheral membrane proteins. Analysis of 50 peripheral membrane proteins harboring BAR, PX, C2, or EHD membrane-binding domains reveals that lysine acetylation predominates in membrane-interaction regions. Acetylation and acetylation-mimicking mutations in three test proteins, amphiphysin, EHD2, and synaptotagmin1, strongly reduce membrane binding affinity, attenuate membrane remodeling in vitro and alter subcellular localization. This effect is likely due to the loss of positive charge, which weakens interactions with negatively charged membranes. In Drosophila, acetylation-mimicking mutations of amphiphysin cause severe disruption of T-tubule organization and yield a flightless phenotype. Our data provide mechanistic insights into how lysine acetylation regulates membrane protein function, potentially impacting a plethora of membrane-related processes.

Assays to Study Enzymatic and Non‐Enzymatic Protein Lysine Acetylation In Vitro

Proteins can be lysine-acetylated both enzymatically, by lysine acetyltransferases (KATs), and non-enzymatically, by acetyl-CoA and/or acetyl-phosphate. Such modification can be reversed by lysine deacetylases classified as NAD+ -dependent sirtuins or by classical Zn2+ -dependent deacetylases (KDACs). The regulation of protein lysine acetylation events by KATs and sirtuins/KDACs, or by non-enzymatic processes, is often assessed only indirectly by mass spectrometry or by mutational studies in cells. Mutational approaches to study lysine acetylation are limited, as these often poorly mimic lysine acetylation. Here, we describe protocols to assess the direct regulation of protein lysine acetylation by both sirtuins/KDACs and KATs, as well as non-enzymatically. We first describe a protocol for the production of site-specific lysine-acetylated proteins using a synthetic biological approach, the genetic code expansion concept (GCEC). These natively folded, lysine-acetylated proteins can then be used as direct substrates for sirtuins and KDACs. This approach addresses various limitations encountered with other methods. First, results from sirtuin/KDAC-catalyzed deacetylation assays obtained using acetylated peptides as substrates can vary considerably compared to experiments using natively folded substrate proteins. In addition, producing lysine-acetylated proteins for deacetylation assays by using recombinantly expressed KATs is difficult, as these often do not yield proteins that are homogeneously and quantitatively lysine acetylated. Moreover, KATs are often huge multi-domain proteins, which are difficult to recombinantly express and purify in soluble form. We also describe protocols to study the direct regulation of protein lysine acetylation, both enzymatically, by sirtuins/KDACs and KATs, and non-enzymatically, by acetyl-CoA and/or acetyl-phosphate. The latter protocol also includes a section that explains how specific lysine acetylation sites can be detected by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The protocols described here can be useful for providing a more detailed understanding of the enzymatic and non-enzymatic regulation of lysine acetylation sites, an important aspect to judge their physiological significance. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of N-(ε)-lysine-acetylated proteins using the genetic code expansion concept (GCEC) Basic Protocol 2: In vitro sirtuin (SIRT)-catalyzed deacetylation of lysine-acetylated proteins prepared by the GCEC Basic Protocol 3: In vitro KDAC/HDAC-catalyzed deacetylation of lysine-acetylated proteins Basic Protocol 4: In vitro lysine acetylation of recombinantly expressed proteins by lysine acetyltransferases (KATs) Basic Protocol 5: In vitro non-enzymatic lysine acetylation of proteins by acetyl-CoA and/or acetyl-phosphate.

Sirtuin 3 deficiency exacerbates age-related periodontal disease.

BackgroundSirtuin 3 (SIRT3), a mitochondrial NAD+‐dependent deacetylase, has received much attention for its effect on metabolism and aging. However, the role of SIRT3 in periodontal disease remains unknown.ObjectiveThis study aimed to investigate the functional role of SIRT3 in age‐related periodontal disease and underlying mechanisms.MethodsSixteen mice were randomly assigned into four groups: the young wild type (WT), the aged WT, the young SIRT3‐knockout (KO), and the aged SIRT3‐KO. SIRT3 and cyclophilin D (CypD) expression and protein lysine acetylation levels in alveolar bones were detected by western blot. The bone architecture and the distance of CEJ‐ABC were assessed using micro‐CT and HE staining. The osteoclast number was observed through tartrate‐resistant acid phosphatase (TRAP) staining. Mitochondrial morphology in SIRT3 knockdown MC3T3‐E1 osteoblastic cells was analyzed by Immunofluorescence staining. In gingival tissues, the NAD+/NADH ratio was measured, and oxidative stress was detected by MitoSOX staining, HO‐1 staining, and MnSOD expression. Mitochondrial biogenesis was measured by PGC‐1α expression and oxygen consumption rate (OCR).ResultsIn parallel with the imbalanced NAD+/NADH ratio, the SIRT3 expression was significantly decreased in the alveolar bones of the aged mice, accompanied by a global elevation of protein acetylation levels. The aged SIRT3‐KO group showed the highest rate of bone resorption and the largest number of TRAP‐positive osteoclasts among the four groups. Moreover, the reactive oxygen species level was up‐regulated in the young and the aged SIRT3‐KO groups. SIRT3 deficiency promoted mitochondrial fission and increased the CypD expression. Furthermore, the lack of SIRT3 reduced the PGC‐1α expression in gingival tissues and exhibited a significant reduction in maximal OCR.ConclusionReduced SIRT3 abundance contributes to aged‐related periodontal disease via the exacerbation of oxidative stress and mitochondrial dysfunction.

STALLION: a stacking-based ensemble learning framework for prokaryotic lysine acetylation site prediction.

Protein post-translational modification (PTM) is an important regulatory mechanism that plays a key role in both normal and disease states. Acetylation on lysine residues is one of the most potent PTMs owing to its critical role in cellular metabolism and regulatory processes. Identifying protein lysine acetylation (Kace) sites is a challenging task in bioinformatics. To date, several machine learning-based methods for the in silico identification of Kace sites have been developed. Of those, a few are prokaryotic species-specific. Despite their attractive advantages and performances, these methods have certain limitations. Therefore, this study proposes a novel predictor STALLION (STacking-based Predictor for ProkAryotic Lysine AcetyLatION), containing six prokaryotic species-specific models to identify Kace sites accurately. To extract crucial patterns around Kace sites, we employed 11 different encodings representing three different characteristics. Subsequently, a systematic and rigorous feature selection approach was employed to identify the optimal feature set independently for five tree-based ensemble algorithms and built their respective baseline model for each species. Finally, the predicted values from baseline models were utilized and trained with an appropriate classifier using the stacking strategy to develop STALLION. Comparative benchmarking experiments showed that STALLION significantly outperformed existing predictor on independent tests. To expedite direct accessibility to the STALLION models, a user-friendly online predictor was implemented, which is available at: http://thegleelab.org/STALLION.

Phase 1 study of the histone deacetylase inhibitor entinostat plus clofarabine for poor-risk Philadelphia chromosome-negative (newly diagnosed older adults or adults with relapsed refractory disease) acute lymphoblastic leukemia or biphenotypic leukemia

PURPOSEDespite advances in immunotherapies, the prognosis for adults with Philadelphia chromosome-negative, newly diagnosed (ND) or relapsed/refractory (R/R) acute lymphoblastic leukemia/acute biphenotypic leukemia (ALL/ABL) remains poor. The benzamide derivative entinostat inhibits histone deacetylase and induces histone hyperacetylation. The purine nucleoside analogue clofarabine is FDA-approved for R/R ALL in children 1−21 years of age. Low doses of clofarabine have been reported to induce DNA hypomethylation. We conducted a phase 1 study of low dose clofarabine with escalating doses of entinostat in adults with ND or R/R ALL/ABL. EXPERIMENTAL DESIGNAdults ≥60 years with ND ALL/ABL or ≥21 years with R/R ALL/ABL received repeated cycles every 3 weeks of entinostat (4 mg, 6 mg or 8 mg orally days 1 and 8) and clofarabine (10 mg/m2/day IV for 5 days, days 3–7) (Arm A). Adults aged 40–59 years with ND ALL/ABL or age ≥21 years in first relapse received entinostat and clofarabine prior to traditional chemotherapy on day 11 (Arm B). Changes in DNA damage, global protein lysine acetylation, myeloid-derived suppressor cells and monocytes were measured in PBMCs before and during therapy. RESULTSTwenty-eight patients were treated at three entinostat dose levels with the maximum administered dose being entinostat 8 mg. The regimen was well tolerated with infectious and metabolic derangements more common in the older population versus the younger cohort. There was no severe hyperglycemia and no peripheral neuropathy in this small study. There were 2 deaths (1 sepsis, 1 intracranial bleed). Overall response rate was 32 %; it was 50 % for ND ALL/ABL. Entinostat increased global protein acetylation and inhibited immunosuppressive monocyte subpopulations, while clofarabine induced DNA damage in all cell subsets examined. CONCLUSIONEntinostat plus clofarabine appears to be tolerable and active in older adults with ND ALL/ABL, but less active in R/R patients. Further evaluation of this regimen in ND ALL/ABL appears warranted.

Global analysis of lysine acetylation in soybean leaves

Protein lysine acetylation (Kac) is an important post-translational modification in both animal and plant cells. Global Kac identification has been performed at the proteomic level in various species. However, the study of Kac in oil and resource plant species is relatively limited. Soybean is a globally important oil crop and resouce plant. In the present study, lysine acetylome analysis was performed in soybean leaves with proteomics techniques. Various bioinformatics analyses were performed to illustrate the structure and function of these Kac sites and proteins. Totally, 3148 acetylation sites in 1538 proteins were detected. Motif analysis of these Kac modified peptides extracted 17 conserved motifs. These Kac modified protein showed a wide subcellular location and functional distribution. Chloroplast is the primary subcellular location and cellular component where Kac proteins were localized. Function and pathways analyses indicated a plenty of biological processes and metabolism pathways potentially be influenced by Kac modification. Ribosome activity and protein biosynthesis, carbohydrate and energy metabolism, photosynthesis and fatty acid metabolism may be regulated by Kac modification in soybean leaves. Our study suggests Kac plays an important role in soybean physiology and biology, which is an available resource and reference of Kac function and structure characterization in oil crop and resource plant, as well as in plant kingdom.