18F-FDG PET/CT of Intracholecystic Papillary Neoplasm in Cystic Neck and Duct.

ABSTRACT: We report 18F-FDG PET/CT appearances of intracholecystic papillary neoplasm (ICPN) in the gallbladder neck and duct of a 74-year-old woman with a history of hepatitis B cirrhosis. The lesion presented with a large and sessile soft mass in the neck and duct of gallbladder with obvious glucose metabolism on PET/CT images, which was confirmed pathologically as ICPN (gastric foveolar type) with high-grade intraepithelial neoplasia. ICPN localized in the gallbladder neck and duct is extremely rare, and is easily misdiagnosed as gallbladder carcinoma. Our report aids in the application of PET/CT in the differential diagnosis of ICPN and guiding early surgery.

Substrate and functional characterization of the lysine acetyltransferase MsKat and deacetylase MsCobB in Mycobacterium smegmatis.

Tuberculosis (TB) is a serious cause of infectious death worldwide. Recent studies have reported that about 30% of the Mtb proteome was modified post-translationally, indicating that their functions are essential for drug resistance, mycobacterial survival, and pathogenicity. Among them, lysine acetylation, reversibly regulated by acetyltransferase and deacetylase, has important roles involved in energy metabolism, cellular adaptation, and protein interactions. However, the substrate and biological functions of these two important regulatory enzymes remain unclear. Herein, we utilized the non-pathogenic M. smegmatis strain as a model and systematically investigated the dynamic proteome changes in response to the overexpressing of MsKat/MsCobB in mycobacteria. A total of 4179 proteins and 1236 acetylated sites were identified in our data. Further analysis of the dynamic changes involved in proteome and acetylome showed that MsKat/MsCobB played a regulatory role in various metabolic pathways and nucleic acid processes. After that, the quantitative mass spectrometric method was utilized and proved that the AMP-dependent synthetase, Citrate synthase, ATP-dependent specificity component of the Clp protease, and ATP-dependent DNA/RNA helicases were identified to be the substrates of MsKat. Overall, our study provided an important resource underlying the substrates and functions of the acetylation regulatory enzymes in mycobacteria. SIGNIFICANCE: In this study, we systematically analyzed the dynamic molecular changes in response to the MsKat/MsCobB overexpression in mycobacteria at proteome and lysine acetylation level by using a TMT-based quantitative proteomic approach. Pathways related with glycolysis, degradation of branched chain amino acids, phosphotransferase system were affected after disturbance of the two regulates enzymes involved in lysine acetylation. We also proved that AMP-dependent synthetase Clp protease, ATP-dependent DNA/RNA helicases and citrate synthase was the substrate of MsKat according to our proteomic data and biological validation. Together, our study underlined the substrates and functions of the acetylation regulatory enzymes in mycobacteria.

Characterization of diverse lysine acylations in Bacillus thuringiensis: Substrate profiling and functional exploration.

Lysine acylation has been extensively investigated due to its regulatory role in a diverse range of biological functions across prokaryotic and eukaryotic species. In-depth acylomic profiles have the potential to enhance comprehension of the biological implications of organisms. However, the extent of research on global acylation profiles in microorganisms is limited. Here, four lysine acylomes were conducted in Bacillus thuringiensis by using the LC-MS/MS based proteomics combined with antibody-enrichment strategies, and a total of 3438 acetylated sites, 5797 propionylated sites, 1705 succinylated sites, and 925 malonylated sites were identified. The motif analysis of these modified proteins revealed a high conservation of glutamate in acetylation and propionylation, whereas such conservation was not observed in succinylation and malonylation modifications. Besides, conservation analysis showed that homologous acylated proteins in Bacillus subtilis and Escherichia coli were connected with ribosome and aminoacyl-tRNA biosynthesis. Further biological experiments showed that lysine acylation lowered the RNA binding ability of CodY and impaired the in vivo protein activity of MetK. In conclusion, our study expanded the current understanding of the global acylation in Bacillus, and the comparative analysis demonstrated that shared acylation proteins could play important roles in regulating both metabolism and RNA transcription progression.

Proteomics analysis of histone deacetylase inhibitor-resistant solid tumors reveals resistant signatures and potential drug combinations.

Histone deacetylase inhibitors (HDACis) are important drugs for cancer therapy, but the indistinct resistant mechanisms of solid tumor therapy greatly limit their clinical application. In this study we conducted HDACi-perturbated proteomics and phosphoproteomics analyses in HDACi-sensitive and -resistant cell lines using a tandem mass tag (TMT)-based quantitative proteomic strategy. We found that the ribosome biogenesis proteins MRTO4, PES1, WDR74 and NOP16 vital to tumorigenesis might regulate the tumor sensitivity to HDACi. By integrating HDACi-perturbated protein signature with previously reported proteomics and drug sensitivity data, we predicted and validated a series of drug combination pairs potentially to enhance the sensitivity of HDACi in diverse solid tumor. Functional phosphoproteomic analysis further identified the kinase PDK1 and ROCK as potential HDACi-resistant signatures. Overall, this study reveals the potential HDACi-resistant signatures and may provide promising drug combination strategies to attenuate the resistance of solid tumor to HDACi.

A proteomic landscape of pharmacologic perturbations for functional relevance.

Pharmacological perturbation studies based on protein-level signatures are fundamental for drug discovery. In the present study, we used a mass spectrometry (MS)-based proteomic platform to profile the whole proteome of the breast cancer MCF7 cell line under stress induced by 78 bioactive compounds. The integrated analysis of perturbed signal abundance revealed the connectivity between phenotypic behaviors and molecular features in cancer cells. Our data showed functional relevance in exploring the novel pharmacological activity of phenolic xanthohumol, as well as the noncanonical targets of clinically approved tamoxifen, lovastatin, and their derivatives. Furthermore, the rational design of synergistic inhibition using a combination of histone methyltransferase and topoisomerase was identified based on their complementary drug fingerprints. This study provides rich resources for the proteomic landscape of drug responses for precision therapeutic medicine.

Prognostic biomarker discovery based on proteome landscape of Chinese lung adenocarcinoma.

Despite recent innovations in imaging and genomic screening promotes advance in diagnosis and treatment of lung adenocarcinoma (LUAD), there remains high mortality of LUAD and insufficient understanding of LUAD biology. Our previous study performed an integrative multi-omic analysis of LUAD, filling the gap between genomic alterations and their biological proteome effects. However, more detailed molecular characterization and biomarker resources at proteome level still need to be uncovered. In this study, a quantitative proteomic experiment of patient-derived benign lung disease samples was carried out. After that, we integrated the proteomic data with previous dataset of 103 paired LUAD samples. We depicted the proteomic differences between non-cancerous and tumor samples and among diverse pathological subtypes. We also found that up-regulated mitophagy was a significant characteristic of early-stage LUAD. Additionally, our integrative analysis filtered out 75 potential prognostic biomarkers and validated two of them in an independent LUAD serum cohort. This study provided insights for improved understanding proteome abnormalities of LUAD and the novel prognostic biomarker discovery offered an opportunity for LUAD precise management.

Microorganism-regulated autophagy in gastrointestinal cancer.

Gastrointestinal cancer has always been one of the most urgent problems to be solved, and it has become a major global health issue. Microorganisms in the gastrointestinal tract regulate normal physiological and pathological processes. Accumulating evidence reveals the role of the imbalance in the microbial community during tumorigenesis. Autophagy is an important intracellular homeostatic process, where defective proteins and organelles are degraded and recycled under stress. Autophagy plays a dual role in tumors as both tumor suppressor and tumor promoter. Many studies have shown that autophagy plays an important role in response to microbial infection. Here, we provide an overview on the regulation of the autophagy signaling pathway by microorganisms in gastrointestinal cancer.

Global landscape of lysine acylomes in Bacillus subtilis.

Lysine acetylation is a common posttranslational modification that regulates numerous biochemical functions in both eukaryotic and prokaryotic species. In addition, several new non-acetyl acylations are structurally different from lysine acetylation and participate in diverse physiological functions. Here, a comprehensive analysis of several lysine acylomes was performed by combining the high-affinity antibody enrichment with high-resolution LC-MS/MS. In total, we identified 2536 lysine acetylated sites, 4723 propionylated sites, 2150 succinylated sites and 3001 malonylated sites in Bacillus subtilis, respectively. These acylated proteins account for 35.8% of total protein in this bacterium. The four lysine acylomes showed a motif preference for glutamate surrounding the modified lysine residues, and a functional preference for several metabolic pathways, such as carbon metabolism, fatty acid metabolism, and ribosome. In addition, more protein-protein interaction clusters were identified in the propionylated substrates than other three lysine acylomes. In summary, our study presents a global landscape of acylation in the Gram-positive model organism Bacillus and their potential functions in metabolism and physiology.

An Integrative Proteome-Based Pharmacologic Characterization and Therapeutic Strategy Exploration of SAHA in Solid Malignancies.

Targeting histone epigenetic modification is an important strategy for anticancer therapy. Histone deacetylase inhibitors (HDACis) have been clinically approved in the treatment of diverse hematological cancers, but mechanisms of drug resistance and poor therapeutic efficacy in solid malignancies remain largely unknown. In this study, we applied a mass spectrometry-based quantitative proteomic strategy to investigate the molecular differences in HDACi vorinostat (SAHA) sensitive and resistant cell lines. The proteomic results revealed that the glycolysis pathway was highly enriched after vorinostat treatment in the resistant cell line, leading to the prediction of a new drug combination, SAHA and hexokinase inhibitor (2-deoxyglucose). The efficacy of this combination was further verified in several solid tumor cell lines. Quantitative proteomics revealed that alterations in the transcription process and protein homeostasis could play roles in the synergetic utilization of these two compounds. Our study showed the application of proteomics in elucidating the drug mechanism and predicting drug combination and the potential of expanding the utilization of HDACi.

Aberrant ROS Mediate Cell Cycle and Motility in Colorectal Cancer Cells Through an Oncogenic CXCL14 Signaling Pathway.

Background: Reactive oxygen species (ROS) act as signal mediators to induce tumorigenesis. Objective: This study aims to explore whether chemokine CXCL14 is involved in the proliferation and migration of ROS-induced colorectal cancer (CRC) cells. Methods: The proliferative and migratory capacities of CRC cells treated with or without H2O2 were measured by various methods, including the CKK-8 assay, colony formation assay, flow cytometry, wounding healing assay, and migration assay. Results: The results revealed that H2O2 promoted the proliferation and migration of CRC cells by regulating the cell cycle progression and the epithelial to mesenchymal transition (EMT) process. Furthermore, we noted that the expression level of CXCL14 was elevated in both HCT116 cells and SW620 cells treated with H2O2. An antioxidant N-Acetyl-l-cysteine (NAC) pretreatment could partially suppress the CXCL14 expression in CRC cells treated with H2O2. Next, we constructed CRC cell lines stably expressing CXCL14 (HCT116/CXCL14 and SW620/CXCL14) and CRC cell lines with empty plasmid vectors (HCT116/Control and SW620/Control) separately. We noted that both H2O2 treatment and CXCL14 over-expression could up-regulate the expression levels of cell cycle-related and EMT-related proteins. Moreover, the level of phosphorylated ERK (p-ERK) was markedly higher in HCT116/CXCL14 cells when compared with that in HCT116/Control cells. CXCL14-deficiency significantly inhibited the phosphorylation of ERK compared with control (i.e., scrambled shNCs). H2O2 treatment could partially restore the expression levels of CXCL14 and p-ERK in HCT116/shCXCL14 cells. Conclusion: Our studies thus suggest that aberrant ROS may promote colorectal cancer cell proliferation and migration through an oncogenic CXCL14 signaling pathway.

Integrative Proteomic Characterization of Human Lung Adenocarcinoma.

Genomic studies of lung adenocarcinoma (LUAD) have advanced our understanding of the disease's biology and accelerated targeted therapy. However, the proteomic characteristics of LUAD remain poorly understood. We carried out a comprehensive proteomics analysis of 103 cases of LUAD in Chinese patients. Integrative analysis of proteome, phosphoproteome, transcriptome, and whole-exome sequencing data revealed cancer-associated characteristics, such as tumor-associated protein variants, distinct proteomics features, and clinical outcomes in patients at an early stage or with EGFR and TP53 mutations. Proteome-based stratification of LUAD revealed three subtypes (S-I, S-II, and S-III) related to different clinical and molecular features. Further, we nominated potential drug targets and validated the plasma protein level of HSP 90ß as a potential prognostic biomarker for LUAD in an independent cohort. Our integrative proteomics analysis enables a more comprehensive understanding of the molecular landscape of LUAD and offers an opportunity for more precise diagnosis and treatment.

Dynamic Characterization of Protein and Posttranslational Modification Levels in Mycobacterial Cholesterol Catabolism.

Cholesterol of the host macrophage membrane is vital for mycobacterial infection, replication, and persistence. During chronic infection within host lung tissues, cholesterol facilitates the phagocytosis of mycobacteria into macrophages. Cholesterol degradation leads to increased flux of acetyl-coenzyme A (CoA) and propionyl-CoA, providing energy and building blocks for virulence macromolecules as well as donors for global protein acylation. Potential functions of lysine acylation are gradually revealed in bacterial survival and pathogenesis. However, the mycobacterial proteome and posttranslational modification (PTM) changes involved in the cholesterol catabolism bioprocess remain unclear. Here, we used nonpathogenic Mycobacterium smegmatis as a model and simultaneously monitored mycobacterial proteome and acetylome changes in the presence of glucose and cholesterol. We discovered that cholesterol metabolic enzymes were upregulated with respect to both protein expression levels and lysine acylation levels during the metabolic shift from glucose to cholesterol. After that, adenylating enzymes related to cholesterol metabolism were proven to be precisely regulated at the propionylation level by mycobacterial acyltransferase M. smegmatis Kat (MsKat) in response to cellular propionyl-CoA accumulation. Furthermore, the kinase expression and phosphorylation levels were also changed along with fluctuations in cholesterol levels. Our results expanded current knowledge of acylation regulation in the cholesterol catabolism of mycobacteria and provided references for possible antimycobacterium strategy.IMPORTANCE Cholesterol assimilation is a critical step in mycobacterial chronic infection. However, knowledge from the dynamic characterization of cholesterol metabolism in mycobacteria at the protein expression and PTM levels remains limited. Our study uncovered the landscape of protein expression, lysine acetylation, lysine propionylation, and S/T/Y phosphorylation during the metabolic changes from glucose to cholesterol in mycobacteria. The data showed that cholesterol-induced carbon shift resulted in the elevation of protein expression and lysine acylation in diverse metabolic enzymes involved in cholesterol degradation and that the presence of cholesterol also promoted the perturbations at the phosphorylation level in the kinase system in mycobacteria. This study systematically characterized the regulation of cholesterol catabolism at several different levels, which provided the detailed references in mycobacterial proteome and potential antimycobacterial strategies.

Comparative evaluation of label-free quantification strategies.

The selection of a data processing method for use in mass spectrometry-based label-free proteome quantification contributes significantly to its accuracy and precision. In this study, we comprehensively evaluated 7 commonly-used label-free quantification methods (MaxQuant-Spectrum count, MaxQuant-iBAQ, MaxQuant-LFQ, MaxQuant-LFAQ, Proteome Discoverer, MetaMorpheus, TPP-StPeter) with a focus on missing values, precision, accuracy, selectivity, and reproducibility of low abundance protein quantification in both single shot and fractionation. Our results showed that among the tested strategies, MaxQuant in MaxLFQ mode outperformed other strategies in terms of accuracy and precision in both whole proteome and low abundance proteome quantification, whereas the Proteome Discoverer (PD) strategy using SEQUEST as a search engine performed better in terms of quantifiable low abundance proteome coverage. We subsequently applied the PD and MaxLFQ strategies in a blood proteomic dataset and found that many FDA-approved tumor prognostic biomarkers could be identified as well as quantified using the PD strategy, indicating the potential advantage of PD in label-free quantification studies. These results provide a reference for method choice in label-free quantification data analysis. SIGNIFICANCE: Mass spectrometry-based label-free quantification methods play an important role in label-free proteome data analysis. In this study, we evaluated 7 commonly-used label-free quantification methods with respect to the following aspects: missing values, precision, accuracy, selectivity, and reproducibility for low abundance protein quantification. The results showed that, among the strategies evaluated, the PD strategy with SEQUEST as a search engine performed better in terms of low abundance protein coverage. This study provides a reference for method choice in label-free quantification data analysis.

Global characterization of proteome and lysine methylome features in EZH2 wild-type and mutant lymphoma cell lines.

Lysine methylation is a widespread protein post-translational modification showing essentialities in versatile cellular process. EZH2, a methyltransferase specifically trimethylates the lysine 27 of histone H3 and its aberrance in several cancers promotes the development of its inhibitors against hematological tumors. In this study, we presented a deep exploration of lysine mono-, di- and trimethylomes in EZH2 wild-type and Y641 mutant lymphoma cell lines. Our results showed that several substrates were modified in different methylation levels. Moreover, these methylated lysine residues could also undergo other types of PTMs. Combined with the differences proved in protein expression, lysine acetylation, lysine ubiquitylation and protein N-termianl acetylation level, our study underlined the substrate specificity of lysine methylation and its crosstalk with other types of PTMs. Totally, our study raised new insights into the global cellular methylation features in hematological cell lines, which provided further inspects into the distribution and function of lysine methylation. SIGNIFICANCE: Our study showed the global landscape of mono-, di- and trimethylomes in the EZH2-aberrant DLBCL cell lines, revealing the molecular characteristics of lysine methylation. Combined with the protein abundance and potential crosstalk among different types of PTMs, our study raised new insights into the global cellular methylation features in hematological tumors and provided further inspects into the distribution and function of lysine methylation.

LysargiNase and Chemical Derivatization Based Strategy for Facilitating In-Depth Profiling of C-Terminome.

Global identification of protein C-termini is highly challenging due to their low abundance in conventional shotgun proteomics. Several enrichment strategies have been developed to facilitate the detection of C-terminal peptides. One major issue of previous approaches is the limited C-terminome coverage. Herein, we integrated LysargiNase digestion, chemical acetylation on neo-N-terminus, and a-ion-aided peptide matching into poly(allylamine)-based C-terminomics (termed as LAACTer). In this strategy, we leveraged LysargiNase, a protease with cleavage specificity N-terminal to Lys and Arg residues, to cover previously unidentifiable C-terminome and employed chemical acetylation and a-ion-aided peptide matching to efficiently boost peptide identifications. Triplicates of LAACTer identified a total of 834 C-termini from proteome of 293T cell, which expanded the coverage by 164% (643 more unique C-termini) compared with the parallel experiments using the original workflow. Compared with the largest human C-terminome data sets (containing 800-900 C-termini), LAACTer not only achieved comparable profiling depth but also yielded 465 previously unidentified C-termini. In a SILAC (stable isotope labeling with amino acids in cell culture)-based quantitative study for identification of GluC-cleaved products, LAACTer quantified 300% more C-terminal peptides than the original workflow. Using LAACTer and the original workflow, we performed global analysis for the C-terminal sequences of 293T cell. The original and processed C-termini displayed distinct sequence patterns, implying the "C-end rules" that regulates protein stability could be more complex than just amino acid motifs. In conclusion, we reason LAACTer could be a powerful proteomic tool for in-depth C-terminomics and would benefit better functional understanding of protein C-termini.

Protein Acylation is a General Regulatory Mechanism in Biosynthetic Pathway of Acyl-CoA-Derived Natural Products.

Coenzyme A (CoA) esters of short fatty acids (acyl-CoAs) function as key precursors for the biosynthesis of various natural products and the dominant donors for lysine acylation. Herein, we investigated the functional interplay between beneficial and adverse effects of acyl-CoA supplements on the production of acyl-CoA-derived natural products in microorganisms by using erythromycin-biosynthesized Saccharopolyspora erythraea as a model: accumulation of propionyl-CoA benefited erythromycin biosynthesis, but lysine propionylation inhibited the activities of important enzymes involved in biosynthetic pathways of erythromycin. The results showed that the overexpression of NAD+-dependent deacylase could circumvent the inhibitory effects of high acyl-CoA concentrations. In addition, we demonstrated the similar lysine acylation mechanism in other acyl-CoA-derived natural product biosynthesis, such as malonyl-CoA-derived alkaloid and butyryl-CoA-derived bioalcohol. These observations systematically uncovered the important role of protein acylation on interaction between the accumulation of high concentrations of acyl-CoAs and the efficiency of their use in metabolic pathways.

Characterization of the Lysine Acylomes and the Substrates Regulated by Protein Acyltransferase in Mycobacterium smegmatis.

Protein acylation plays important roles in bacterial pathogenesis through regulation of enzymatic activity, protein stability, nucleic acid binding ability, and protein-protein interactions. Mycobacteria, a genus including invasive pathogens known to cause serious diseases, shapes its pathogenicity through adaptation of its energy metabolism to microenvironments encountered within mammalian hosts. In this process, acetyl-CoA and propionyl-CoA function as important intermediates. However, the function of acetyl-CoA/propionyl-CoA driven protein acylation remains to be elucidated. Herein, we systematically investigated protein acetylome/propionylome in the nonpathogenic Mycobacterium smegmatis through antibody-enrichment-based proteomic analysis in which 146 acetylated sites on 121 proteins and 26 propionylated sites on 25 proteins were identified. After that, characteristic differences of the two acylomes were elucidated through such bioinformatic methods as motif analysis, protein-protein analysis, Gene Ontology analysis, and KEGG analysis. In addition, quantitative mass spectrometric method was used to evaluate the site-specific and motif-biased catalytic mechanism mediated by the cAMP-dependent acetyltransferase MsKat in M. smegmatis. Furthermore, we raised the possibility that both O-serine and Nε-lysine acetylation might coregulate the propionyl-CoA synthetase. This study described the landscape of acetylome and propionylome in the M. smegmatis, showing an unexpected role of protein acylation regulation in mycobacteria.

Protein Acylation Affects the Artificial Biosynthetic Pathway for Pinosylvin Production in Engineered E. coli.

The effect of regulatory system on the engineered biosynthetic pathway in chassis cells remains incompletely understood in microorganisms. Acyl-CoAs function as key precursors for the biosynthesis of various natural products and the dominant donors for protein acylation. The polyphenol pinosylvin, with high antimicrobial and antifungal activities, is biosynthesized with malonyl-CoA as its direct precursors. But correlation between lysine malonylation and pinosylvin biosynthesis remains unknown. Herein, we found that the malonyl-CoA-driven lysine malonylation plays an important role in interaction between the engineered pathway of pinosylvin synthesis and E. coli chassis cell. Oversupply of malonyl-CoA leads to an increase in malonylation level of global proteome as well as the enzymes in the artificial pathway, thereby decreasing yield of pinosylvin. The results revealed that the intricate balance of cellular acyl-CoA concentrations is critical for the yields of acyl-CoA-derived natural products. We next modified the enzymes in the biosynthetic pathway to adjust their acylation level and successfully improved the yield of pinosylvin. Our study uncovers the effect of protein acylation on the biosynthetic pathway, helps optimization of synthetic constructs, and provides new strategies in metabolic engineering and synthetic biology at the protein post-translational level.

Protein Acetylation and Butyrylation Regulate the Phenotype and Metabolic Shifts of the Endospore-forming Clostridium acetobutylicum.

Clostridium acetobutylicum is a strict anaerobic, endospore-forming bacterium, which is used for the production of the high energy biofuel butanol in metabolic engineering. The life cycle of C. acetobutylicum can be divided into two phases, with acetic and butyric acids being produced in the exponential phase (acidogenesis) and butanol formed in the stationary phase (solventogenesis). During the transitional phase from acidogenesis to solventogenesis and latter stationary phase, concentration peaks of the metabolic intermediates butyryl phosphate and acetyl phosphate are observed. As an acyl group donor, acyl-phosphate chemically acylates protein substrates. However, the regulatory mechanism of lysine acetylation and butyrylation involved in the phenotype and solventogenesis of C. acetobutylicum remains unknown. In our study, we conducted quantitative analysis of protein acetylome and butyrylome to explore the dynamic change of lysine acetylation and butyrylation in the exponential phase, transitional phase, and stationary phase of C. acetobutylicum Total 458 lysine acetylation sites and 1078 lysine butyrylation sites were identified in 254 and 373 substrates, respectively. Bioinformatics analysis uncovered the similarities and differences between the two acylation modifications in C. acetobutylicum Mutation analysis of butyrate kinase and the central transcriptional factor Spo0A was performed to characterize the unique role of lysine butyrylation in the metabolic pathway and sporulation process of C. acetobutylicum Moreover, quantitative proteomic assays were performed to reveal the relationship between protein features (e.g. gene expression level and lysine acylation level) and metabolites in the three growth stages. This study expanded our knowledge of lysine acetylation and butyrylation in Clostridia and constituted a resource for functional studies on lysine acylation in bacteria.

Functional Investigation of a GRIN2A Variant Associated with Rolandic Epilepsy.

N-methyl-D-aspartate receptors (NMDARs), a subtype of glutamate-gated ion channels, play a central role in epileptogenesis. Recent studies have identified an increasing number of GRIN2A (a gene encoding the NMDAR GluN2A subunit) mutations in patients with epilepsy. Phenotypes of GRIN2A mutations include epilepsy-aphasia disorders and other epileptic encephalopathies, which pose challenges in clinical treatment. Here we identified a heterozygous GRIN2A mutation (c.1341T>A, p.N447K) from a boy with Rolandic epilepsy by whole-exome sequencing. The patient became seizure-free with a combination of valproate and lamotrigine. Functional investigation was carried out using recombinant NMDARs containing a GluN2A-N447K mutant that is located in the ligand-binding domain of the GluN2A subunit. Whole-cell current recordings in HEK 293T cells revealed that the N447K mutation increased the NMDAR current density by ~1.2-fold, enhanced the glutamate potency by 2-fold, and reduced the sensitivity to Mg2+ inhibition. These results indicated that N447K is a gain-of-function mutation. Interestingly, alternative substitutions by alanine and glutamic acid at the same residue (N447A and N447E) did not change NMDAR function, suggesting a residual dependence of this mutation in altering NMDAR function. Taken together, this study identified human GluN2A N447K as a novel mutation associated with epilepsy and validated its functional consequences in vitro. Identification of this mutation is also helpful for advancing our understanding of the role of NMDARs in epilepsy and provides new insights for precision therapeutics in epilepsy.