Protein Lysine Research Articles

A cytoplasmic form of EHMT1N methylates viral proteins to enable inclusion body maturation and efficient viral replication.

Protein lysine methyltransferases (PKMTs) methylate histone and non-histone proteins to regulate biological outcomes such as development and disease including viral infection. While PKMTs have been extensively studied for modulating the antiviral responses via host gene regulation, their role in methylation of proteins encoded by viruses and its impact on host-pathogen interactions remain poorly understood. In this study, we discovered distinct nucleo-cytoplasmic form of euchromatic histone methyltransferase 1 (EHMT1N/C), a PKMT, that phase separates into viral inclusion bodies (IBs) upon cytoplasmic RNA-virus infection (Sendai Virus). EHMT1N/C interacts with cytoplasmic EHMT2 and methylates SeV-Nucleoprotein upon infection. Elevated nucleoprotein methylation during infection correlated with coalescence of small IBs into large mature platforms for efficient replication. Inhibition of EHMT activity by pharmacological inhibitors or genetic depletion of EHMT1N/C reduced the size of IBs with a concomitant reduction in replication. Additionally, we also found that EHMT1 condensation is not restricted to SeV alone but was also seen upon pathogenic RNA viral infections caused by Chandipura and Dengue virus. Collectively, our work elucidates a new mechanism by which cytoplasmic EHMT1 acts as proviral host factor to regulate host-pathogen interaction.

Crotonylation modification and its role in diseases

Protein lysine crotonylation is a novel acylation modification discovered in 2011, which plays a key role in the regulation of various biological processes. Thousands of crotonylation sites have been identified in histone and non-histone proteins over the past decades. Crotonylation is conserved and is regulated by a series of enzymes including “writer”, “eraser”, and “reader”. In recent years, crotonylation has received extensive attention due to its breakthrough progress in reproduction, development and pathogenesis of diseases. Here we brief the crotonylation-related enzyme systems, biological functions, and diseases caused by abnormal crotonylation, which provide new ideas for developing disease intervention and treatment regimens.

Blue-Purple evaluation: Chromoproteins facilitate the identification of BioBrick compatibility.

Synthetic BioBricks introduce novel capabilities to manipulate genetic information, direct transcription-translation processes, and program cellular behaviors in living organisms. To maintain the stability and functionality of synthetic BioBricks, assembled DNA fragments should be mutually compatible without inducing negative effects such as metabolic burden or cellular toxicity in host cells. However, a simple, rapid, and reliable method to evaluate BioBrick compatibility remains to be developed. In this study, we report BP (Blue/Purple, Ban/Pick) evaluation, a method utilizing chromoproteins to facilitate the identification of BioBrick compatibility in one-pot reactions. By visualizing and quantifying the ratio of blue to purple Escherichia coli (E. coli) colonies on LB-agar plates, we can easily validate the compatibility of desired BioBrick constructions. To demonstrate our design, we characterized BioBrick assemblies with antitoxin-toxin pair ccdA-ccdB, lysis protein E, or heterologous protein sfGFP. Among these, we successfully identified several compatible assemblies. We anticipate that BP evaluation will enhance biotechnological assessments of BioBrick compatibility in vivo and expand the application of chromoproteins in synthetic biology.

Inhibition of Sirtuin Deacylase Activity by Peroxynitrite.

Sirtuins are a class of enzymes that deacylate protein lysine residues using NAD+ as a cosubstrate. Sirtuin deacylase activity has been historically regarded as protective; loss of sirtuin deacylase activity potentially increases susceptibility to aging-related disease development. However, which factors may inhibit sirtuins during aging or disease is largely unknown. Increased oxidant and inflammatory byproduct production damages cellular proteins. Previously, we and others found that sirtuin deacylase activity is inhibited by the nitric oxide (NO)-derived cysteine post-translational modification S-nitrosation. However, the comparative ability of the NO-derived oxidant peroxynitrite (ONOO-) to affect human sirtuin activity had not yet been assessed under uniform conditions. Here, we compare the ability of ONOO- (donated from SIN-1) to post-translationally modify and inhibit SIRT1, SIRT2, SIRT3, SIRT5, and SIRT6 deacylase activity. In response to SIN-1 treatment, inhibition of SIRT1, SIRT2, SIRT3, SIRT5, and SIRT6 deacylase activity correlated with increased tyrosine nitration. Mass spectrometry identified multiple novel tyrosine nitration sites in SIRT1, SIRT3, SIRT5, and SIRT6. As each sirtuin isoform has at least one tyrosine nitration site within the catalytic core, nitration may result in sirtuin inhibition. ONOO- can also react with cysteine residues, resulting in sulfenylation; however, only SIRT1 showed detectable peroxynitrite-mediated cysteine sulfenylation. While SIRT2, SIRT3, SIRT5, and SIRT6 showed no detectable sulfenylation, SIRT6 likely undergoes transient sulfenylation, quickly resolving into an intermolecular disulfide bond. These results suggest that the aging-related oxidant peroxynitrite can post-translationally modify and inhibit sirtuins, contributing to susceptibility to aging-related disease.

Research advances in protein lysine 2-hydroxyisobutyrylation: From mechanistic regulation to disease relevance.

Histone lysine 2-hydroxyisobutyrylation (Khib) was identified as a novel posttranslational modification in 2014. Significant progress has been made in understanding its roles in reproduction, development, and disease. Although 2-hydroxyisobutyrylation shares some overlapping modification sites and regulatory factors with other lysine residue modifications, its unique structure suggests distinct functions. This review summarizes the latest advancements in Khib, including its regulatory mechanisms, roles in mammalian physiological processes, and its relationship with diseases. This provides direction for further research on Khib and offers new perspectives for developing treatment strategies for related diseases.

Symmetrical 2,7-disubstituted 9H-fluoren-9-one as a novel and promising scaffold for selective targeting of SIRT2.

Sirtuin 2 (SIRT2) belongs to the family of silent information regulators (sirtuins), which comprises nicotinamide adenine dinucleotide (NAD+)-dependent protein lysine deacetylases. With a distribution across numerous tissues and organs of the human body, SIRT2 is involved in a wide range of physiological and pathological processes, such as regulating the cell cycle, energy metabolism, DNA repair, and tumorigenesis. Aberrant expression of SIRT2 has been closely associated with particular etiologies of human diseases, positioning SIRT2 as a promising therapeutic target. Herein, we detail the design overview and findings of novel symmetrical 2,7-disubstituted 9H-fluoren-9-one derivatives targeting SIRT2. SG3 displayed the most potent SIRT2-selective inhibitory profile, with an IC50 value of 1.95 , and reduced the cell viability of human breast cancer MCF-7 cells accompanied by hyperacetylation of α-tubulin. Finally, molecular docking, molecular dynamics simulations, and binding free energy calculations using molecular mechanics/generalized born surface area method were performed to verify the binding ability of SG3 to SIRT2. Taken together, these results could enhance our understanding of the structural elements necessary for inhibiting SIRT2 and shed light on the mechanism of inhibition.

Current computational tools for protein lysine acylation site prediction.

As a main subtype of post-translational modification (PTM), protein lysine acylations (PLAs) play crucial roles in regulating diverse functions of proteins. With recent advancements in proteomics technology, the identification of PTM is becoming a data-rich field. A large amount of experimentally verified data is urgently required to be translated into valuable biological insights. With computational approaches, PLA can be accurately detected across the whole proteome, even for organisms with small-scale datasets. Herein, a comprehensive summary of 166 in silico PLA prediction methods is presented, including a single type of PLA site and multiple types of PLA sites. This recapitulation covers important aspects that are critical for the development of a robust predictor, including data collection and preparation, sample selection, feature representation, classification algorithm design, model evaluation, and method availability. Notably, we discuss the application of protein language models and transfer learning to solve the small-sample learning issue. We also highlight the prediction methods developed for functionally relevant PLA sites and species/substrate/cell-type-specific PLA sites. In conclusion, this systematic review could potentially facilitate the development of novel PLA predictors and offer useful insights to researchers from various disciplines.

Testing AI predictions that hyperglycemia-induced non-enzymatic glycation of immune system proteins impairs diabetic immunity

People with type 1, type 2 and gestational diabetes, and newborns of diabetic mothers, have increased risks of contracting microbial infections such as SARS-CoV-2, amplifying comorbidity and mortality risks. Hyperglycemia and its associated hyperinflammation mediate these risks but by unknown mechanisms. We hypothesize that hyperglycemia results in glycation (non-enzymatic addition of sugars to free amino groups such as lysines) of antimicrobial and anti-inflammatory proteins and peptides that mediate innate and adaptive immunity. While glycation of hemoglobin A1c is used as a benchmark for glycemic control, studies of the effects of the glycation of immune system molecules are almost non-existent. Three AI protein-glycation prediction models demonstrate that immune system proteins and peptides such as serotransferrin, lactotransferrin, lysozyme, complement proteins, immunoglobulins, Toll-like receptors, etc., probably glycate at multiple sites affecting function. However, the AI-predicted glycation sites differed significantly among the three models employed. Therefore, three tests of these predictions were carried out. Predicted glycation sites were first evaluated in terms of clinically or experimentally identified glycation sites of homologous proteins and known effects of lysine substitutions at predicted glycation sites. Second, mass spectrometry was used to identify glycation sites on lactoferrin and human lysozyme. Third, the effects on the antimicrobial activity of lactotransferrin and lysozyme glycation were tested using a Candida albicans assay. Results of tests demonstrate that protein glycation occurs and interferes with immune function but that AI glycation prediction models are very inaccurate. Suggestions for improving AI accuracy are provided.

Lactylation: An Innovative Approach to Disease Control.

Assessment of tissue microenvironment lactate levels has emerged as a crucial indicator of microcirculation and early organ dysfunction. Lactylation modification, closely associated with lactate concentration, represents a novel post-translational alteration targeting protein lysine residues. Post-translational modifications are chemical changes capable of modulating protein activity and functionality, serving as a rapid mechanism for enhancing proteomic diversity and influencing various life processes. While previous research primarily focused on histone lactylation, recent studies have revealed the occurrence of lactylation on non-histone proteins, exerting significant effects on gene expression and intercellular communication. Lactylation has been implicated in diverse diseases spanning embryonic development, neuronal excitability, inflammatory responses, cardiovascular conditions, tumor progression, invasion, and aging. Hence, lactylation emerges as a pivotal regulator in numerous pathological conditions. This review delves into the mechanisms underlying lactylation and disease pathogenesis, elucidates the multifaceted roles of lactylation in disease progression, and identifies novel therapeutic targets related to lactylation, offering potential avenues for future clinical interventions.

Discovery of a Highly Potent and Selective Inhibitor Targeting Protein Lysine Methyltransferase NSD2.

The histone lysine methyltransferase NSD2 has been recognized as an attractive target for cancer treatment, due to the functional implication of its dysregulation in the initiation and progression of many cancers. Although considerable efforts have been made to develop NSD2 small-molecule inhibitors, highly potent and selective ones are still rarely available till now. Here, we report the discovery of a series of novel NSD2 inhibitors via an extensive SAR exploration of the privileged quinazoline scaffold within compound 8. The most promising compound 42 showed excellent NSD2 enzymatic inhibitory activity and good antiproliferative activity in cells. In addition, it demonstrated favorable pharmacokinetic properties and significantly inhibited the tumor growth in a RS411 tumor xenograft model with good safety. Taken together, compound 42 could be a promising NSD2 inhibitor and deserves further investigation.

Activation and inhibition of sirtuins: From bench to bedside.

The sirtuin family comprises seven NAD+-dependent enzymes which catalyze protein lysine deacylation and mono ADP-ribosylation. Sirtuins act as central regulators of genomic stability and gene expression and control key processes, including energetic metabolism, cell cycle, differentiation, apoptosis, and aging. As a result, all sirtuins play critical roles in cellular homeostasis and organism wellness, and their dysregulation has been linked to metabolic, cardiovascular, and neurological diseases. Furthermore, sirtuins have shown dichotomous roles in cancer, acting as context-dependent tumor suppressors or promoters. Given their central role in different cellular processes, sirtuins have attracted increasing research interest aimed at developing both activators and inhibitors. Indeed, sirtuin modulation may have therapeutic effects in many age-related diseases, including diabetes, cardiovascular and neurodegenerative disorders, and cancer. Moreover, isoform selective modulators may increase our knowledge of sirtuin biology and aid to develop better therapies. Through this review, we provide critical insights into sirtuin pharmacology and illustrate their enzymatic activities and biological functions. Furthermore, we outline the most relevant sirtuin modulators in terms of their modes of action, structure-activity relationships, pharmacological effects, and clinical applications.

Investigation of Protein Lysine Acetylation in Antiviral Innate Immunity.

Protein lysine acetylation involved in the antiviral innate immunity contributes to the regulation of antiviral inflammation responses, including type 1 interferon production and interferon-stimulated gene expression. Thus, investigation of acetylated antiviral proteins is vital for the complete understanding of inflammatory responses to viral infections. Immunoprecipitation (IP) assay with anti-targeted-protein antibody or with acetyl-lysine affinity beads followed by immunoblot provides a classical way to determine the potential modified protein in the antiviral innate pathways, whereas mass spectrometry can be utilized to identify the accurate acetylation lysine residues or explore the acetyl-proteomics. We demonstrate here comprehensive methods of protein lysine acetylation determination in virus-infected macrophages and embryonic fibroblast cells or proteins-overexpressed HEK 293T cells in the context of antiviral innate immunity.

Analysis of genetic variability for nutritional quality traits among maize inbreds adapted to Punjab: A North‐Western province of India

AbstractBackground and ObjectivesThe deficiency of essential amino acids such as methionine, lysine, and tryptophan in maize protein presents a significant challenge in meeting nutritional demand. Comprehensive evaluation of maize germplasm for variability of essential amino acids, protein content, starch content, and β‐carotene contents in grains, along with their relationships with yield and its associated traits, is a prerequisite for strengthening genetic enhancement for nutritional traits. Thus, the current study presents the profiling of 60 maize inbred lines across 11 nutritional quality and yield, aiming to identify variability in these traits and potential donor candidates for commercial breeding.FindingsThe test inbreds exhibited significant variations in the content of grain methionine (0.04%–0.28%), tryptophan (0.019%–0.069%), lysine (0.063%–0.318%), protein (9.31%–11.80%), starch (58.32%–70.88%), and β‐carotene (2.38 ppm–6.79 ppm). Additionally, variations were noted in grain yield (0.87–2.52 t ha−1). Inter‐relationship analyses suggested the feasibility of simultaneous selection of essential amino acids (methionine, tryptophan, lysine) and β‐carotene. The germplasm stock was clustered, and potential diverse donors identified were recommended for utilization in breeding programs. Furthermore, the use of Attenuated Total Reflectance‐Fourier Transform Infrared (ATR‐FTIR) spectroscopy for screening high methionine and high lysine lines in large germplasm based on differences in peak intensity at 2922 cm−1 and 1110 cm−1 was reported, respectively.ConclusionsThe multilocation experiment revealed genetic diversity in both nutritional quality and yield across the 60 inbreds. The absence of a negative correlation between the amino acids and β‐carotene indicates the possibility of the development of hybrids with elevated essential amino‐acid levels combined with high β‐carotene content.Significance and NoveltyThis is the first holistic report on the variability of nutritional quality traits coupled with yield in maize germplasm. Identified nutrient‐rich inbreds will serve as valuable germplasm in maize biofortification programs. AT‐FTIR is proposed as an easy screening technique and the first report, which can be employed for the initial screening of a large set of germplasm for methionine and lysine content.

A novel lytic phage infecting MDR Salmonella enterica and its application as effective food biocontrol.

Salmonella enterica is a foodborne pathogen associated with both typhoid and non-typhoid illness in humans and animals. This problem is further exacerbated by the emergence of antibiotic-resistant strains of Salmonella enterica. Therefore, to meet public health and safety, there is a need for an alternative strategy to tackle antibiotic-resistant bacteria. Bacteriophages or (bacterial viruses), due to their specificity, self-dosing, and antibiofilm activity, serve as a better approach to fighting against drug-resistant bacteria. In the current study, a broad-host range lytic phage phiSalP219 was isolated against multidrug-resistant Salmonella enterica serotypes Paratyphi from a pond water sample. Salmonella phage phiSalP219 was able to lyse 28/30 tested strains of Salmonella enterica. Salmonella phage phiSalP219 exhibits activity in acidic environments (pH3) and high temperatures (70°C). Electron microscopy and genome analysis revealed that phage phiSalP219 is a member of class Caudoviricetes. The genome of Salmonella phage phiSalP219 is 146Kb in size with 44.5% GC content. A total of 250 Coding Sequence (CDS) and 25 tRNAs were predicted in its genome. Predicted open reading frames (ORFs) were divided into five groups based on their annotation results: (1) nucleotide metabolism, (2) DNA replication and transcription, (3) structural proteins, (4) lysis protein, and (5) other proteins. The absence of lysogeny-related genes in their genome indicates that Salmonella phage phiSalP219 is lytic in nature. Phage phiSalP219 was also found to be microbiologically safe (due to the absence of toxin or virulence-related genes) in the control of Salmonella enterica serovar Typhimurium infections in the ready-to-eat meat and also able to eradicate biofilm formed by the same bacterium on the borosilicate glass surface.

Posttranslationally modified self-peptides promote hypertension in mouse models.

Posttranslational modifications can enhance immunogenicity of self-proteins. In several conditions, including hypertension, systemic lupus erythematosus, and heart failure, isolevuglandins (IsoLGs) are formed by lipid peroxidation and covalently bond with protein lysine residues. Here, we show that the murine class I major histocompatibility complex (MHC-I) variant H-2Db uniquely presents isoLG-modified peptides and developed a computational pipeline that identifies structural features for MHC-I accommodation of such peptides. We identified isoLG-adducted peptides from renal proteins, including sodium glucose transporter 2, cadherin 16, Kelch domain-containing protein 7A, and solute carrier family 23, that are recognized by CD8+ T cells in tissues of hypertensive mice, induce T cell proliferation in vitro, and prime hypertension after adoptive transfer. Finally, we find patterns of isoLG-adducted antigen restriction in class I human leukocyte antigens that are similar to those in murine analogs. Thus, we have used a combined computational and experimental approach to define likely antigenic peptides in hypertension.

Sirt5 regulates chondrocyte metabolism and osteoarthritis development through protein lysine malonylation.

Chondrocyte metabolic dysfunction plays an important role in osteoarthritis (OA) development during aging and obesity. Protein post-translational modifications (PTMs) have recently emerged as an important regulator of cellular metabolism. We aim to study one type of PTM, lysine malonylation (MaK) and its regulator Sirt5 in OA development. Human and mouse cartilage tissues were used to measure SIRT5 and MaK levels. Both systemic and cartilage-specific conditional knockout mouse models were subject to high-fat diet (HFD) treatment to induce obesity and OA. Proteomics analysis was performed in Sirt5 -/- and WT chondrocytes. SIRT5 mutation was identified in the Utah Population Database (UPDB). We found that SIRT5 decreases while MAK increases in the cartilage during aging. A combination of Sirt5 deficiency and obesity exacerbates joint degeneration in a sex dependent manner in mice. We further delineate the malonylome in chondrocytes, pinpointing MaK's predominant impact on various metabolic pathways such as carbon metabolism and glycolysis. Lastly, we identified a rare coding mutation in SIRT5 that dominantly segregates in a family with OA. The mutation results in substitution of an evolutionally invariant phenylalanine (Phe-F) to leucine (Leu-L) (F101L) in the catalytic domain. The mutant protein results in higher MaK level and decreased expression of cartilage ECM genes and upregulation of inflammation associated genes. We found that Sirt5 mediated MaK is an important regulator of chondrocyte cellular metabolism and dysregulation of Sirt5-MaK could be an important mechanism underlying aging and obesity associated OA development.

Lactylome Analysis Unveils Lactylation-Dependent Mechanisms of Stemness Remodeling in the Liver Cancer Stem Cells.

Lactate plays a critical role as an energy substrate, metabolite, and signaling molecule in hepatocellular carcinoma (HCC). Intracellular lactate-derived protein lysine lactylation (Kla) is identified as a contributor to the progression of HCC. Liver cancer stem cells (LCSCs) are believed to be the root cause of phenotypic and functional heterogeneity in HCC. However, the impact of Kla on the biological processes of LCSCs remains poorly understood. Here enhanced glycolytic metabolism, lactate accumulation, and elevated levels of lactylation are observed in LCSCs compared to HCC cells. H3K56la was found to be closely associated with tumourigenesis and stemness of LCSCs. Notably, a comprehensive examination of the lactylome and proteome of LCSCs and HCC cells identified the ALDOA K230/322 lactylation, which plays a critical role in promoting the stemness of LCSCs. Furthermore, this study demonstrated the tight binding between aldolase A (ALDOA) and dead box deconjugate enzyme 17 (DDX17), which is attenuated by ALDOA lactylation, ultimately enhancing the regulatory function of DDX17 in maintaining the stemness of LCSCs. This investigation highlights the significance of Kla in modulating the stemness of LCSCs and its impact on the progression of HCC. Targeting lactylation in LCSCs may offer a promising therapeutic approach for treating HCC.

Stacking-Kcr: A Stacking Model for Predicting the Crotonylation Sites of Lysine by Fusing Serial and Automatic Encoder

Background: Protein lysine crotonylation (Kcr), a newly discovered important posttranslational modification (PTM), is typically localized at the transcription start site and regulates gene expression, which is associated with a variety of pathological conditions such as developmental defects and malignant transformation. Objective: Identifying Kcr sites is advantageous for the discovery of its biological mechanism and the development of new drugs for related diseases. However, traditional experimental methods for identifying Kcr sites are expensive and inefficient, necessitating the development of new computational techniques. Methods: In this work, to accurately identify Kcr sites, we propose a model for ensemble learning called Stacking-Kcr. Firstly, extract features from sequence information, physicochemical properties, and sequence fragment similarity. Then, the two characteristics of sequence information and physicochemical properties are fused using automatic encoder and serial, respectively. Finally, the fused two features and sequence fragment similarity features are then respectively input into the four base classifiers, a meta classifier is constructed using the first level prediction results, and the final forecasting results are obtained. Results: The five-fold cross-validation of this model has achieved an accuracy of 0.828 and an AUC of 0.910. This shows that the Stacking-Kcr method has obvious advantages over traditional machine learning methods. On independent test sets, Stacking-Kcr achieved an accuracy of 84.89% and an AUC of 92.21%, which was higher than 1.7% and 0.8% of other state-of-the-art tools. Additionally, we trained Stacking-Kcr on the phosphorylation site, and the result is superior to the current model. Conclusion: These outcomes are additional evidence that Stacking-Kcr has strong application potential and generalization performance.

Alcohol- and Low-Iron Induced Changes in Antioxidant and Energy Metabolism Associated with Protein Lys Acetylation.

Understanding the role of iron in ethanol-derived hepatic stress could help elucidate the efficacy of dietary or clinical interventions designed to minimize liver damage from chronic alcohol consumption. We hypothesized that normal levels of iron are involved in ethanol-derived liver damage and reduced dietary iron intake would lower the damage caused by ethanol. We used a pair-fed mouse model utilizing basal Lieber-DeCarli liquid diets for 22 weeks to test this hypothesis. In our mouse model, chronic ethanol exposure led to mild hepatic stress possibly characteristic of early-stage alcoholic liver disease, seen as increases in liver-to-body weight ratios. Dietary iron restriction caused a slight decrease in non-heme iron and ferritin (FeRL) expression while it increased transferrin receptor 1 (TfR1) expression without changing ferroportin 1 (FPN1) expression. It also elevated protein lysine acetylation to a more significant level than in ethanol-fed mice under normal dietary iron conditions. Interestingly, iron restriction led to an additional reduction in nicotinamide adenine dinucleotide (NAD+) and NADH levels. Consistent with this observation, the major mitochondrial NAD+-dependent deacetylase, NAD-dependent deacetylase sirtuin-3 (SIRT3), expression was significantly reduced causing increased protein lysine acetylation in ethanol-fed mice at normal and low-iron conditions. In addition, the detection of superoxide dismutase 1 and 2 levels (SOD1 and SOD2) and oxidative phosphorylation (OXPHOS) complex activities allowed us to evaluate the changes in antioxidant and energy metabolism regulated by ethanol consumption at normal and low-iron conditions. We observed that the ethanol-fed mice had mild liver damage associated with reduced energy and antioxidant metabolism. On the other hand, iron restriction may exacerbate certain activities of ethanol further, such as increased protein lysine acetylation and reduced antioxidant metabolism. This metabolic change may prove a barrier to the effectiveness of dietary reduction of iron intake as a preventative measure in chronic alcohol consumption.

Global profiling of protein lactylation in microglia in experimental high-altitude cerebral edema

BackgroundHigh-altitude cerebral edema (HACE) is considered an end-stage acute mountain sickness (AMS) that typically occurs in people after rapid ascent to 2500 m or more. While hypoxia is a fundamental feature of the pathophysiological mechanism of HACE, emerging evidence suggests that inflammation serves as a key risk factor in the occurrence and development of this disease. However, little is known about the molecular mechanism underlying their crosstalk.MethodsA mouse HACE model was established by combination treatment with hypobaric hypoxia exposure and lipopolysaccharides (LPS) stimulation. Lactylated-proteomic analysis of microglia was performed to reveal the global profile of protein lactylation. Molecular modeling was applied to evaluate the 3-D modeling structures. A combination of experimental approaches, including western blotting, quantitative real-time reverse transcriptionpolymerase chain reaction (qRT-PCR), and enzyme-linked immunosorbent assay (ELISA), confocal microscopy and RNA interference, were used to explore the underlying molecular mechanisms.ResultsWe found that hypoxia exposure increased the lactate concentration and lactylation in mouse HACE model. Moreover, hypoxia aggravated the microglial neuroinflammatory response in a lactate-dependent manner. Global profiling of protein lactylation has shown that a large quantity of lysine-lactylated proteins are induced by hypoxia and preferentially occur in protein complexes, such as the NuRD complex, ribosome biogenesis complex, spliceosome complex, and DNA replication complex. The molecular modeling data indicated that lactylation could affect the 3-D theoretical structure and increase the solvent accessible surface area of HDAC1, MTA1 and Gatad2b, the core members of the NuRD complex. Further analysis by knockdown or selectively inhibition indicated that the NuRD complex is involved in hypoxia-mediated aggravation of inflammation.ConclusionsThese results revealed a comprehensive profile of protein lactylation in microglia and suggested that protein lysine lactylation plays an important role in the regulation of protein function and subsequently contributes to the neuroinflammatory response under hypoxic conditions.