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Δ1‐pyrroline‐5‐carboxylate reductase isoform 1 (PYCR1) is the last enzyme of proline biosynthesis and catalyzes the NAD(P)H‐dependent reduction of Δ1‐pyrroline‐5‐carboxylate to L‐proline. High PYCR1 gene expression is observed in many cancers and linked to poor patient outcomes and tumor aggressiveness. The knockdown of the PYCR1 gene or the inhibition of PYCR1 enzyme has been shown to inhibit tumorigenesis in cancer cells and animal models of cancer, motivating inhibitor discovery. We screened a library of 71 low molecular weight compounds (average MW of 131 Da) against PYCR1 using an enzyme activity assay. Hit compounds were validated with X‐ray crystallography and kinetic assays to determine affinity parameters. The library was counter‐screened against human Δ1‐pyrroline‐5‐carboxylate reductase isoform 3 and proline dehydrogenase (PRODH) to assess specificity/promiscuity. Twelve PYCR1 and one PRODH inhibitor crystal structures were determined. Three compounds inhibit PYCR1 with competitive inhibition parameter of 100 μM or lower. Among these, (S)‐tetrahydro‐2H‐pyran‐2‐carboxylic acid (70 μM) has higher affinity than the current best tool compound N‐formyl‐l‐proline, is 30 times more specific for PYCR1 over human Δ1‐pyrroline‐5‐carboxylate reductase isoform 3, and negligibly inhibits PRODH. Structure‐affinity relationships suggest that hydrogen bonding of the heteroatom of this compound is important for binding to PYCR1. The structures of PYCR1 and PRODH complexed with 1‐hydroxyethane‐1‐sulfonate demonstrate that the sulfonate group is a suitable replacement for the carboxylate anchor. This result suggests that the exploration of carboxylic acid isosteres may be a promising strategy for discovering new classes of PYCR1 and PRODH inhibitors. The structure of PYCR1 complexed with l‐pipecolate and NADH supports the hypothesis that PYCR1 has an alternative function in lysine metabolism. [ABSTRACT FROM AUTHOR]

Simple Summary: The recently identified lysine methyltransferase (KMT) 9 regulates the growth of different types of cancer. While KMT9 was shown to be overexpressed in muscle-invasive bladder cancer (MIBC) tissue samples of patients, a potential functional role of the enzyme in MIBC remains to be clarified. In this study, we show that KMT9 regulates the proliferation, migration, and invasion of various MIBC cell lines as well as the growth of BC tumor organoids and xenografts in mice. Our data provide evidence that tumor cell growth relies on the enzymatic function of KMT9 and that a small-molecule inhibitor of KMT9 impairs BC cell proliferation. These results suggest that KMT9 is a potential novel therapeutic target for MIBC treatment. Novel treatment modalities are imperative for the challenging management of muscle-invasive and metastatic BC to improve patient survival rates. The recently identified KMT9, an obligate heterodimer composed of KMT9α and KMT9β, regulates the growth of various types of tumors such as prostate, lung, and colon cancer. While the overexpression of KMT9α was previously observed to be associated with aggressive basal-like MIBC in an analysis of patients' tissue samples, a potential functional role of KMT9 in this type of cancer has not been investigated to date. In this study, we show that KMT9 regulates proliferation, migration, and invasion of various MIBC cell lines with different genetic mutations. KMT9α depletion results in the differential expression of genes regulating the cell cycle, cell adhesion, and migration. Differentially expressed genes include oncogenes such as EGFR and AKT1 as well as mediators of cell adhesion or migration such as DAG1 and ITGA6. Reduced cell proliferation upon KMT9α depletion is also observed in Pten/Trp53 knockout bladder tumor organoids, which cannot be rescued with an enzymatically inactive KMT9α mutant. In accordance with the idea that the catalytic activity of KMT9 is required for the control of cellular processes in MIBC, a recently developed small-molecule inhibitor of KMT9 (KMI169) also impairs cancer cell proliferation. Since KMT9α depletion also restricts the growth of xenografts in mice, our data suggest that KMT9 is an actionable novel therapeutic target for the treatment of MIBC. [ABSTRACT FROM AUTHOR]

Objective: Preoperative chronic stress (CS) is associated with postoperative brain injury in patients undergoing open heart cardiac surgery. This research is to explore the potential molecular biological mechanisms of brain damage following cardiac surgery in preoperative CS rats by the analyses combining proteomics and metabolomics. Methods: We constructed the chronic unpredictable stress (CUS) and cardiac surgery models in adult rats. We proved the brain injury in CUS cardiac surgery rats by Hematoxylin–Eosin (H&E) staining, followed by separating the hippocampal tissue and investigating the potential mechanisms of brain injury by the methods of data-independent acquisition proteomics and untargeted metabolomics. Results: The signaling pathways of glycoproteins and metabolism of amino acids were the main possible mechanisms of brain injury in CUS rats following cardiac surgery according to the proteomics and metabolomics. In addition, the pathways of animo acids metabolism such as the pathways of lysine degradation and β-alanine metabolism may be the main mechanism of cardiac surgery related brain injury in preoperative CUS rats. Conclusions: The pathways of animo acids metabolism such as lysine degradation and β-alanine metabolism may be the potential mechanisms of brain injury in CUS rats following cardiac surgery. We should focus on the varieties of bioproteins and metabolites in these pathways, and related changes in other signaling pathways induced by the two pathways. [ABSTRACT FROM AUTHOR]

Lysine and its pathway metabolites have been identified as novel biomarkers for metabolic and vascular diseases. The role of them in the identification of moyamoya disease (MMD) has not been elucidated. This study aimed to determine the association between lysine pathway metabolites and the presence of MMD. We prospectively enrolled 360 MMD patients and 89 healthy controls from September 2020 to December 2021 in Beijing Tiantan Hospital. Serum levels of lysine, pipecolic acid and 2-aminoadipic acid were measured by liquid chromatography-mass spectrometry. We employed logistic regression and restricted cubic spline to explore the association between these metabolites and the presence of MMD. Stratified analyses were also conducted to test the robustness of results. We observed that lysine levels in MMD patients were significantly higher and pipecolic acid levels were significantly lower compared to HCs (both p < 0.001), while no difference was found in the level of 2-AAA between both groups. When comparing metabolites by quartiles, elevated lysine levels were linked to increased odds for MMD (the fourth quartile [Q4] vs the first quartile [Q1]: odds ratio, 3.48, 95%CI [1.39–8.75]), while reduced pipecolic acid levels correlated with higher odds (Q4 vs Q1: odds ratio, 0.08; 95 % CI [0.03–0.20]). The restricted cubic spline found a L-shaped relationship between pipecolic acid level and the presence of MMD, with a cutoff point at 2.52 μmol/L. Robust results were also observed across subgroups. Elevated lysine levels were correlated with increased odds of MMD presence, while lower pipecolic acid levels were associated with higher odds of the condition. These results suggest potential new biomarkers for the identification of MMD. URL: https://www.chictr.org.cn/. Unique identifier: ChiCTR2200061889. [ABSTRACT FROM AUTHOR]

Abstract Background Bladder cancer (BLCA) research in Koreans is still lacking, especially in focusing on the prediction of BLCA. The current study aimed to discover metabolic signatures related to BLCA onset and confirm its potential as a biomarker. Methods We designed two nested case-control studies using Korean Cancer Prevention Study (KCPS)-II. Only males aged 35–69 were randomly selected and divided into two sets by recruitment organizations [set 1, BLCA (n = 35) vs. control (n = 35); set 2, BLCA (n = 31) vs. control (n = 31)]. Baseline serum samples were analyzed by non-targeted metabolomics profiling, and OPLS-DA and network analysis were performed. Calculated genetic risk score (GRS) for BLCA from all KCPS participants was utilized for interpreting metabolomics data. Results Critical metabolic signatures shown in the BLCA group were dysregulation of lysine metabolism and tryptophan-indole metabolism. Furthermore, the prediction model consisting of metabolites (lysine, tryptophan, indole, indoleacrylic acid, and indoleacetaldehyde) reflecting these metabolic signatures showed mighty BLCA predictive power (AUC: 0.959 [0.929–0.989]). The results of metabolic differences between GRS-high and GRS-low groups in BLCA indicated that the pathogenesis of BLCA is associated with a genetic predisposition. Besides, the predictive ability for BLCA on the model using GRS and five significant metabolites was powerful (AUC: 0.990 [0.980–1.000]). Conclusion Metabolic signatures shown in the present research may be closely associated with BLCA pathogenesis. Metabolites involved in these could be predictive biomarkers for BLCA. It could be utilized for early diagnosis, prognostic diagnosis, and therapeutic targets for BLCA.

Background: Bladder cancer (BLCA) research in Koreans is still lacking, especially in focusing on the prediction of BLCA. The current study aimed to discover metabolic signatures related to BLCA onset and confirm its potential as a biomarker. Methods: We designed two nested case-control studies using Korean Cancer Prevention Study (KCPS)-II. Only males aged 35–69 were randomly selected and divided into two sets by recruitment organizations [set 1, BLCA (n = 35) vs. control (n = 35); set 2, BLCA (n = 31) vs. control (n = 31)]. Baseline serum samples were analyzed by non-targeted metabolomics profiling, and OPLS-DA and network analysis were performed. Calculated genetic risk score (GRS) for BLCA from all KCPS participants was utilized for interpreting metabolomics data. Results: Critical metabolic signatures shown in the BLCA group were dysregulation of lysine metabolism and tryptophan-indole metabolism. Furthermore, the prediction model consisting of metabolites (lysine, tryptophan, indole, indoleacrylic acid, and indoleacetaldehyde) reflecting these metabolic signatures showed mighty BLCA predictive power (AUC: 0.959 [0.929–0.989]). The results of metabolic differences between GRS-high and GRS-low groups in BLCA indicated that the pathogenesis of BLCA is associated with a genetic predisposition. Besides, the predictive ability for BLCA on the model using GRS and five significant metabolites was powerful (AUC: 0.990 [0.980–1.000]). Conclusion: Metabolic signatures shown in the present research may be closely associated with BLCA pathogenesis. Metabolites involved in these could be predictive biomarkers for BLCA. It could be utilized for early diagnosis, prognostic diagnosis, and therapeutic targets for BLCA. [ABSTRACT FROM AUTHOR]

Objective: Many patients with acromegaly, a hormonal disorder with excessive growth hormone (GH) production, report pain in joints. We undertook this study to characterize the joint pathology of mice with overexpression of bovine GH (bGH) or a GH receptor antagonist (GHa) and to investigate the effect of GH on regulation of chondrocyte cellular metabolism. Methods: Knee joints from mice overexpressing bGH or GHa and wild‐type (WT) control mice were examined using histology and micro–computed tomography for osteoarthritic (OA) pathologies. Additionally, cartilage from bGH mice was used for metabolomics analysis. Mouse primary chondrocytes from bGH and WT mice, with or without pegvisomant treatment, were used for quantitative polymerase chain reaction and Seahorse respirometry analyses. Results: Both male and female bGH mice at ~13 months of age had increased knee joint degeneration, which was characterized by loss of cartilage structure, expansion of hypertrophic chondrocytes, synovitis, and subchondral plate thinning. The joint pathologies were also demonstrated by significantly higher Osteoarthritis Research Society International and Mankin scores in bGH mice compared to WT control mice. Metabolomics analysis revealed changes in a wide range of metabolic pathways in bGH mice, including beta‐alanine metabolism, tryptophan metabolism, lysine degradation, and ascorbate and aldarate metabolism. Also, bGH chondrocytes up‐regulated fatty acid oxidation and increased expression of Col10a. Joints of GHa mice were remarkably protected from developing age‐associated joint degeneration, with smooth articular joint surface. Conclusion: This study showed that an excessive amount of GH promotes joint degeneration in mice, which was associated with chondrocyte metabolic dysfunction and hypertrophic changes, whereas antagonizing GH action through a GHa protects mice from OA development. [ABSTRACT FROM AUTHOR]

Background: DNA damage repair is a crucial mechanism highly related to therapy resistance for various therapeutic strategies. Our previous results have shown that the degree of drug resistance in small‐cell lung cancer (SCLC) cell lines was proportional to both the transcription and expression levels of Wee1, indicating that Wee1, an evolutionarily highly conserved kinase, plays a vital role in the therapeutic resistance of SCLC. In the present study, we aim to determine the nonclassical mechanism of Wee1 on DNA repair regulation. Methods: Western blot was conducted to determine the mono‐ubiquitination level of H2Bub. Comet assay was used to evaluate the degree of DNA damage. Immunofluorescence was conducted to determine the DNA repair markers. Co‐immunoprecipitation was utilized to assess the potential interactions with H2BY37ph. MTT assays were used to evaluate the survival rates of SCLC cells. Results: Overexpression of Wee1 increases the level of H2BK120ub and alleviates ionizing radiation (IR)‐induced DNA damage in SCLC cells. Moreover, H2BK120ub is a crucial molecule in Wee1‐mediated double‐strain break (DSB) repair in SCLC cells. Mechanisms study indicated that H2BY37ph is involved in Wee1‐mediated H2BK120ub through interaction with the E3 ubiquitin ligase RNF20–RNF40 complex and upregulates its phosphorylation, mutation of H2BY37 phosphorylation sites attenuated DSB repair and enhanced the sensitivity of IR‐induced SCLC cell death. Conclusion: H2BY37ph produces crosstalk with H2BK120ub in an E3 ubiquitin ligase‐dependent manner, promoting Wee1‐mediated DSB repair in SCLC cells. This study clarifies the nonclassical mechanism of Wee1 regulation of DSB repair, which provides a theoretical basis for the clinical understanding of the regulatory network of Wee1 and its use as a target for overcoming multiple types of therapeutic resistance. [ABSTRACT FROM AUTHOR]

Post-translational modifications on histones are well known to regulate chromatin structure and function, but much less information is available on modifications of the centromeric histone H3 variant and their effect at the kinetochore. Here, we report two modifications on the centromeric histone H3 variant CENP-A/Cse4 in the yeast Saccharomyces cerevisiae, methylation at arginine 143 (R143me) and lysine 131 (K131me), that affect centromere stability and kinetochore function. Both R143me and K131me lie in the core region of the centromeric nucleosome, near the entry/exit sites of the DNA from the nucleosome. Unexpectedly, mutation of Cse4-R143 (cse4-R143A) exacerbated the kinetochore defect of mutations in components of the NDC80 complex of the outer kinetochore (spc25-1) and the MIND complex (dsn1-7). The analysis of suppressor mutations of the spc25-1 cse4-R143A growth defect highlighted residues in Spc24, Ndc80, and Spc25 that localize to the tetramerization domain of the NDC80 complex and the Spc24-Spc25 stalk, suggesting that the mutations enhance interactions among NDC80 complex components and thus stabilize the complex. Furthermore, the Set2 histone methyltransferase inhibited kinetochore function in spc25-1 cse4-R143A cells, possibly by methylating Cse4-K131. Taken together, our data suggest that Cse4-R143 methylation and Cse4-K131 methylation affect the stability of the centromeric nucleosome, which is detrimental in the context of defective NDC80 tetramerization and can be compensated for by strengthening interactions among NDC80 complex components. [ABSTRACT FROM AUTHOR]

In higher fungi, lysine is biosynthesized via the α-aminoadipate (AAA) pathway, which differs from plants, bacteria, and lower fungi. The differences offer a unique opportunity to develop a molecular regulatory strategy for the biological control of plant parasitic nematodes, based on nematode-trapping fungi. In this study, in the nematode-trapping fungus model Arthrobotrys oligospora, we characterized the core gene in the AAA pathway, encoding α-aminoadipate reductase (Aoaar), via sequence analyses and through comparing the growth, and biochemical and global metabolic profiles of the wild-type and Aoaar knockout strains. Aoaar not only has α-aminoadipic acid reductase activity, which serves fungal L-lysine biosynthesis, but it also is a core gene of the non-ribosomal peptides biosynthetic gene cluster. Compared with WT, the growth rate, conidial production, number of predation rings formed, and nematode feeding rate of the ΔAoaar strain were decreased by 40–60%, 36%, 32%, and 52%, respectively. Amino acid metabolism, the biosynthesis of peptides and analogues, phenylpropanoid and polyketide biosynthesis, and lipid metabolism and carbon metabolism were metabolically reprogrammed in the ΔAoaar strains. The disruption of Aoaar perturbed the biosynthesis of intermediates in the lysine metabolism pathway, then reprogrammed amino acid and amino acid-related secondary metabolism, and finally, it impeded the growth and nematocidal ability of A. oligospora. This study provides an important reference for uncovering the role of amino acid-related primary and secondary metabolism in nematode capture by nematode-trapping fungi, and confirms the feasibility of Aoarr as a molecular target to regulate nematode-trapping fungi to biocontrol nematodes. [ABSTRACT FROM AUTHOR]

Background: Diabetic peripheral neuropathy (DPN) is a common neurological complication of diabetes mellitus without efficient interventions. Both lysine demethylase 5B (KDM5B) and sirtuin‐3 (SIRT3) have been found to regulate islet function and glucose homeostasis. KDM5B was predicted to bind to the SIRT3 promoter by bioinformatics. Here, we investigated whether KDM5B affected DPN development via modulating SIRT3. Methods: The db/db mice and high glucose‐stimulated Schwann cells (RSC96) were used as in vivo and in vitro models of DPN, respectively. Glucose level, glucose and insulin tolerance of mice were measured. Neurological function was evaluated by motor nerve conduction velocity (MNCV), tactile allodynia assay and thermal sensitivity assay. Adenosine triphosphate level, oxygen consumption rate, extracellular acidification rate, β‐oxidation rate, acetyl‐CoA level, acetylation levels and activities of long‐chain acyl CoA dehydrogenase (LCAD) and pyruvate dehydrogenase (PDH) were detected. Methyl thiazolyl tetrazolium assay was adopted to determine cell viability. Reactive oxygen species (ROS) production was detected by MitoSox staining. Western blotting for measuring target protein levels. Molecular mechanisms were investigated by co‐immunoprecipitine (Co‐IP), chromatin immunoprecipitation (ChIP) and luciferase reporter assay. Results: KDM5B was up‐regulated, while SIRT3 was down‐regulated in DPN models. SIRT3 overexpression or AMPK activation ameliorated mitochondrial metabolism dysfunction and ROS overproduction during DPN. KDM5B overexpression triggered mitochondrial metabolism disorder and oxidative stress via directly transcriptional inhibiting SIRT3 expression by demethylating H3K4me3 or indirectly repressing AMPK pathway‐regulated SIRT3 expression. Conclusion: KDM5B contributes to DPN via regulating SIRT3‐mediated mitochondrial glucose and lipid metabolism. KDM5B inhibition may be an effective intervention for DPN. [ABSTRACT FROM AUTHOR]

The occurence and development of tumors is a complicated process, which not only depends on the mutation or deletion of genes, but also is affected by epigenetic regulation. Accumulating evidences have shown that epigenetic modifications play fundamental roles in transcriptional regulation, heterochromatin formation, X chromosome inactivation, DNA damage response and tumor development. SET domain containing lysine methyltransferase 7 (SETD7) was initially identified as an important lysine methyltransferase, which methylated histone and non-histone proteins. These modifications play fundamental roles. Once this modification disorders, it can directly lead to cell abnormalities and cause many diseases. Studies have shown that SETD7 is related to the occurence and development of various tumors, but the methylation sites of SETD7 and its regulatory mechanism have not been fully elucidated. Tis article summarizes the research progress of the role of SETD7 on histone and non-histone methylation modification in tumors and the molecular mechanism, in order to provide new therapeutic targets for tumor pathogenesis and diagnosis. [ABSTRACT FROM AUTHOR]

The transition from lean to obese states involves systemic metabolic remodeling that impacts insulin sensitivity, lipid partitioning, inflammation, and glycemic control. Here, we have taken a pharmacological approach to test the role of a nutrient-regulated chromatin modifier, lysine-specific demethylase (LSD1), in obesity-associated metabolic reprogramming. We show that systemic administration of an LSD1 inhibitor (GSK-LSD1) reduces food intake and body weight, ameliorates nonalcoholic fatty liver disease (NAFLD), and improves insulin sensitivity and glycemic control in mouse models of obesity. GSK-LSD1 has little effect on systemic metabolism of lean mice, suggesting that LSD1 has a context-dependent role in promoting maladaptive changes in obesity. In analysis of insulin target tissues we identified white adipose tissue as the major site of insulin sensitization by GSK-LSD1, where it reduces adipocyte inflammation and lipolysis. We demonstrate that GSK-LSD1 reverses NAFLD in a non-hepatocyte-autonomous manner, suggesting an indirect mechanism potentially via inhibition of adipocyte lipolysis and subsequent effects on lipid partitioning. Pair-feeding experiments further revealed that effects of GSK-LSD1 on hyperglycemia and NAFLD are not a consequence of reduced food intake and weight loss. These findings suggest that targeting LSD1 could be a strategy for treatment of obesity and its associated complications including type 2 diabetes and NAFLD. [ABSTRACT FROM AUTHOR]

Background: Although eradication therapy for chronic Helicobacter pylori (H. pylori) reduces the risk of gastric cancer (GC), its effectiveness is not complete. Therefore, it is also critically important to identifying those patients who remain at high risk after H. pylori eradication therapy. Accumulation of protein methylation is strongly implicated in cancer, and recent study showed that dimethylation of eEF1A lysine 55 (eEF1AK55me2) promotes carcinogenesis in vivo. We aimed to investigate the relationship between eEF1A dimethylation and H. pylori status, efficacy of eradication therapy, and GC risk in H. pylori-eradicated mucosa, and to reveal the potential downstream molecules of eEF1A dimethylation.Methods: Records of 115 patients (11 H. pylori-negative, 29 H. pylori-positive, 75 post-eradication patients) who underwent upper gastrointestinal endoscopy were retrospectively reviewed. The eEF1A dimethyl level was evaluated in each functional cell type of gastric mucosa by immunofluorescent staining. We also investigated the relationship between eEF1AK55me2 downregulation by CRISPR/Cas9 mediated deletion of Mettl13, which is known as a dimethyltransferase of eEF1AK55me2.Results: The level of eEF1A dimethylation significantly increased in the surface and basal areas of H. pylori-positive mucosa compared with the negative mucosa (surface, p = 0.0031; basal, p = 0.0036, respectively). The eEF1A dimethyl-levels in the surface area were significantly reduced by eradication therapy (p = 0.005), but those in the basal area were maintained even after eradication therapy. Multivariate analysis revealed that high dimethylation of eEF1A in the basal area of the mucosa was the independent factor related to GC incidence (odds ratio = 3.6611, 95% confidence interval = 1.0350-12.949, p = 0.0441). We also showed the relationship between eEF1A dimethylation and expressions of reprogramming factors, Oct4 and Nanog, by immunohistochemistry and in vitro genome editing experiments.Conclusions: The results indicated that H. pylori infection induced eEF1A dimethylation in gastric mucosa. The accumulation of dimethyl-eEF1A in the basal area of the mucosa might contribute to GC risk via regulation of reprograming factors in H. pylori eradicated-gastric mucosa. [ABSTRACT FROM AUTHOR]

Simple Summary: The histone methyltransferase SET domain-containing protein 8 (SETD8) methylates histone H4 lysine 20 and non-histone proteins such as p53. Our aim was to determine the involvement of SETD8 in endometrial cancer and its therapeutic potential and identify the downstream genes regulated by SETD8 via H4K20 methylation and the p53 signaling pathway. We confirmed that SETD8 expression was elevated in endometrial cancer tissues. Our results suggest that the suppression of SETD8 using siRNA or a selective inhibitor attenuated cell proliferation and promoted the apoptosis of endometrial cancer cells. In these cells, SETD8 regulates genes via H4K20 methylation and the p53 signaling pathway. We also identified the prognostically important genes related to apoptosis, such as those encoding KIAA1324 and TP73, in endometrial cancer. SETD8 is an important gene for carcinogenesis and progression of endometrial cancer via H4K20 methylation. The histone methyltransferase SET domain-containing protein 8 (SETD8), which methylates histone H4 lysine 20 (H4K20) and non-histone proteins such as p53, plays key roles in human carcinogenesis. Our aim was to determine the involvement of SETD8 in endometrial cancer and its therapeutic potential and identify the downstream genes regulated by SETD8 via H4K20 methylation and the p53 signaling pathway. We examined the expression profile of SETD8 and evaluated whether SETD8 plays a critical role in the proliferation of endometrial cancer cells using small interfering RNAs (siRNAs). We identified the prognostically important genes regulated by SETD8 via H4K20 methylation and p53 signaling using chromatin immunoprecipitation sequencing, RNA sequencing, and machine learning. We confirmed that SETD8 expression was elevated in endometrial cancer tissues. Our in vitro results suggest that the suppression of SETD8 using siRNA or a selective inhibitor attenuated cell proliferation and promoted the apoptosis of endometrial cancer cells. In these cells, SETD8 regulates genes via H4K20 methylation and the p53 signaling pathway. We also identified the prognostically important genes related to apoptosis, such as those encoding KIAA1324 and TP73, in endometrial cancer. SETD8 is an important gene for carcinogenesis and progression of endometrial cancer via H4K20 methylation. [ABSTRACT FROM AUTHOR]

Simple Summary: Epigenetic events, which comprise post-translational modifications of histone tails or DNA methylation, control gene expression by altering chromatin structure without change in the DNA sequence. Histone tails modifications are driven by specific cellular enzymes such as histone methyltransferases or histone acetylases, which play a key role in regulating diverse biological processes. Their alteration may have consequences on growth and tumorigenesis. Epigenetic regulations, that comprise histone modifications and DNA methylation, are essential to processes as diverse as development and cancer. Among the histone post-translational modifications, lysine methylation represents one of the most important dynamic marks. Here, we focused on methyltransferases of the nuclear binding SET domain 1 (NSD) family, that catalyze the mono- and di-methylation of histone H3 lysine 36. We review the loss of function mutations of NSD1 in humans that are the main cause of SOTOS syndrome, a disease associated with an increased risk of developing cancer. We then report the role of NSD1 in triggering tumor suppressive or promoter functions according to the tissue context and we discuss the role of NSD1 in melanoma. Finally, we examine the ongoing efforts to target NSD1 signaling in cancers. [ABSTRACT FROM AUTHOR]

Pentosidine (PEN), an advanced glycation end product (AGE), is associated with various age-related diseases and schizophrenia. This study aimed to identify the natural compounds that inhibit PEN synthesis from glucuronic acid using an in vitro system. A screening of 93 natural compounds revealed 47 that reduced PEN synthesis by > 50 %, with eight inhibiting it by > 80 %. The top five inhibitors were anthocyanins, with petunidin chloride showing the strongest effect, inhibiting PEN synthesis by approximately 90 %. These compounds directly inhibited PEN synthesis without degrading or capturing the synthesized PEN. Petunidin chloride had an IC 50 value approximately 85 times lower than that of pyridoxamine, an AGE inhibitor. A correlation between the antioxidant capacity of the compounds and their PEN-inhibitory effects was observed, suggesting that antioxidant properties may contribute to the inhibition mechanism. This study provides potential new therapeutic strategies for diseases associated with PEN accumulation, including schizophrenia, and highlights the potential of anthocyanins in the development of safer preventive interventions., Competing Interests: Declaration of competing interest We have no conflicts of interest to disclose., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Cationic polymers that bind with the plasmids to form polyplexes protect the DNA from enzymatic degradation and improve cellular and tissue uptake. Complete or near complete gel retardation of the polyplex is an important assay to determine the optimal polymer: plasmid ratio for in vitro and in vivo studies. Nevertheless, despite minimal to moderate gel retardation of histidine-lysine (HK) polyplexes formed with low peptide: plasmid DNA ratios (1:2 and 1:4; w:w), the polyplexes effectively targeted the tumor in vivo. To understand the lack of predictability of the initial gel mobility shift assays, we revisited the retardation and stability of polyplexes with these electrophoresis assays. Because the histidine component with a pKa of about 6.0 will have a greater positive charge and may bind plasmids with a higher affinity at lower pHs, we compared the retardation of the two HK polyplexes when the pH of the running buffer of the gel mobility shift assay was altered. Both HK polyplexes were retarded significantly more when the running buffer had a pH of 7.3 instead of the standard pH of 8.3. Indeed, the HK polyplexes formed at the 1:2 ratio showed complete retardation at pH 7.3. Consequently, while both HK polyplexes formed at these low ratios targeted the tumor, the polyplex formed with the 1:2 ratio had reduced tumor gene expression variability and lower lung and liver values. Thus, the selection of the optimal ratios for the linear HK and plasmid for transfection studies in vivo was improved with a running buffer pH of 7.3., Competing Interests: Declaration of competing interest The authors of this manuscript declare that there are no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase (cGAS)-dependent pathway is a key DNA-sensing pathway that recognizes cytosolic DNA and plays a crucial role in initiating innate immune responses against pathogenic microbes and cancer. Various molecules have been identified as regulators of the cGAS-dependent pathway that controls innate immune responses. However, despite the important roles of Stimulator-of-interferon genes (STING) in the cGAS-dependent pathway, the regulation of its activation has not been elucidated. Here, we show that the E3 ubiquitin ligase, RING finger protein 39 (RNF39), interacts with STING in macrophages and HERK293T cells. Moreover, RNF39 accelerates DNA-sensing pathways by promoting lysine (K)63-linked ubiquitination of STING, and then facilitating the formation of STING-TBK1 complex. Concordantly, Rnf39 deficiency inhibits innate immune responses triggered by DNA viral infection and accelerates viral replication. Furthermore, herpes simplex virus-1 (HSV-1) infection induces RNF39 expression in an IFN-I-dependent manner. Thus, we outline a novel mechanism for controlling STING activation and a feedback mechanism for controlling antiviral immune responses. RNF39 could be a priming intervention target for the prevention and treatment of viral diseases, especially DNA viral infections., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Microalgae are emerging as a novel single-cell protein source that can substitute traditional plant protein feeds. In this investigation, lysine and protein accumulation in Chlorella pyrenoidosa were significantly enhanced under red light-emitting diode light, addressing challenge of limiting amino acid in plant proteins. The study employed targeted metabolomics, HPLC, and qRT-PCR to validate the light-induced pathway triggering lysine biosynthesis. Specifically, the pathway involves Ca 2+ -CaM as an intermediary in signal transduction, which directly inhibits PEPC activity. This inhibition directs a significant carbon flux towards central carbon metabolism, resulting in increased pyruvate levels-a critical precursor for lysine biosynthesis via the diaminopimelate pathway. Ultimately, the content of protein and lysine under red light increased by 36.02 % and 99.56 %, respectively, compared to those under white light. These findings provide a novel orientation for the precise regulation of lysine accumulation in microalgae, and moreover lay a solid theoretical foundation for producing microalgal proteins., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Microbial biofilms provide advantages in fermentation processes. However, Corynebacterium glutamicum (C. glutamicum) usually exhibits relatively poor biofilm formation compared to other industrial strains. To develop a biofilm-enhanced fermentation process for C. glutamicum, seven genes potentially related to biofilm formation in C. glutamicum were systematically investigated, which include ppk2B, glgC, virB11, cslA, NCgl2909, NCgl0350 and exeR. Deletion of the NCgl0350, NCgl2909 genes and heterologous expression of the cslA gene were found to increase biofilm amounts by 16.9%, 21.2% and 135%, respectively, compared to the wild-type strain. Meanwhile, the production of L-lysine by engineered strains was assessed in biofilm-based continuous fermentation. The most notable result was observed for the cslA-expressing strain, which produced an average of 26.1% higher L-lysine compared with that of wild-type strain in 6-L bioreactors. In conclusion, this study offers valuable insights into the biofilm formation in C. glutamicum and its application in continuous fermentation processes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The human lysine acetyltransferases KAT3A ( CREBBP ) and KAT3B ( EP300 ) are essential enzymes in gene regulation in the nucleus. Their ubiquitous expression in metazoan cell types controls cell proliferation and differentiation during development. This comprehensive review delves into the biological roles of KAT3A and KAT3B in neurodevelopment, shedding light on how alterations in their regulation or activity can potentially contribute to a spectrum of neurodegenerative diseases (e.g., Huntington's and Alzheimer's). We explore the pathophysiological implications of KAT3 function loss in these disorders, considering their conserved protein domains and biochemical functions in chromatin regulation. The discussion also underscores the crucial role of KAT3 proteins and their substrates in supporting the integration of key cell signaling pathways. Furthermore, the narrative highlights the interdependence of KAT3-mediated lysine acetylation with lysine methylation and arginine methylation. From a cellular perspective, KAT3-dependent signal integration at subnuclear domains is mediated by liquid-liquid phase separation in response to KAT3-mediated lysine acetylation. The disruption of these finely tuned regulatory processes underscores their pathological roles in neurodegeneration. This review also points to the exciting potential for future research in this field, inspiring further investigation and discovery in the area of neurodevelopment and neurodegenerative diseases., Competing Interests: The authors declare that they have no conflicts of interest.

Post-translational modifications (PTMs), as epigenetic modifications, are significant in the interaction between virus and its host. However, it is unclear whether rotavirus (RV) causes changes in both the host cell epigenetic protein modification and the regulatory mechanism of viral replication. Here, we analyzed the proteome of Caco-2 cells to determine if acetylation modification occurred within the cells after RV infection. We found that glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a protein involved in glycolysis, was deacetylated at lysine 219 via histone deacetylase 9 (HDAC9) in 50 h after the RV infection. Remarkably, the deacetylation of GAPDH promoted RV replication. Finally, we found that glycolysis was alterable in Caco-2 cells by RV or the deacetylation of GAPDH lysine 219, using the Seahorse XF Glycolysis Stress Test. In conclusion, our results demonstrate for the first time that RV infection promoted deacetylation of GAPDH at lysine 219 in order to increase its own viral replication in Caco-2 cells., Competing Interests: Declarations Conflict of interest No conflict of interest exits in the submission of this manuscript, and manuscript is approved by all authors for publication. I would like to declare on behalf of my co-authors that the work described was original research that has not been published previously, and not under consideration for publication elsewhere, in whole or in part., (© 2024. The Author(s).)

Starch and proteins are main storage product to determine the appearance, cooking, texture, and nutritional quality of rice (Oryza sativa L.). OsNAC20 and OsNAC26, as pivotal transcription factors, redundantly regulate the expression of genes responsible for starch and protein synthesis in the rice endosperm. Any knockout of OsNAC20 or OsNAC26 did not result in visible endosperm defects. In this study, we had isolated and characterized a mutant named as floury endosperm25 (flo25). The caryopsis of the flo25 mutant exhibits a floury endosperm, accompanied by reductions in both the 1000-grain weight and grain length, as well as diminished levels of total starch and protein. Through map-based cloning, it was determined that FLO25 encodes a NAM, ATAF, and CUC (NAC) transcription factors, namely OsNAC26, with a lysine to asparagine substitution at position 98 in the flo25 mutant. Remarkably, lysine 98 is conserved across plants species, and this mutation does not alter the subcellular localization of OsNAC26 but significantly attenuates its transcriptional activity and its ability to activate downstream target genes. Furthermore, the mutant protein encoded by OsNAC26 -flo25 could interact with OsNAC20, disrupting the native interaction between OsNAC20 proteins. Additionally, when lysine 98 is substituted with asparagine in OsNAC20, the resulting mutant protein, OsNAC20(K98N), similarly disrupts the interaction between OsNAC26 proteins. Collectively, these findings underscore the pivotal role of Lysine 98 (K) in modulating the transcriptional activity of NAC20/NAC26 within the rice endosperm., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Evidence suggests that fish are more tolerant than mammals to imbalanced dietary amino acid profiles. However, the behavioral and physiological responses of fish to individual deficiencies in dietary indispensable amino acids (IDAA) remain unclear. This study examined how stomachless fish respond to diets deficient in limiting IDAA (lysine, methionine, and threonine), using Zebrafish (Danio rerio) as a model. The response to deficient diets was assessed based on; 1) growth performance and feeding efficiency; 2) feed intake; 3) expression of appetite-regulating hormones and nutrient-sensing receptors; and 4) muscle postprandial free amino acid (FAA) levels. There were 6 treatments, each with 3 replicate tanks. A semi-purified diet was formulated for each group. The CG diet was based on casein and gelatin, while the FAA50 diet had 50 % of dietary protein supplied with crystalline amino acids. Both were formulated to contain matching, balanced amino acid profiles. The remaining diets were formulated the same as the FAA50 diet, with minor adjustments to create deficiencies in selected IDAA. The (-) Lys, (-) Met, and (-) Thr diets had lysine, methionine, and threonine withheld from the FAA mix, respectively, and the Def diet was deficient in all three. The juvenile Zebrafish were fed to satiation 3 times daily from 21 to 50 days-post-hatch. Results showed that 50 % replacement of dietary protein with crystalline amino acids significantly reduced growth of juvenile Zebrafish. There were no significant differences in growth between the FAA50 group and groups that received deficient diets. The deficiency of singular IDAA did not induce significant changes in feed intake; however, the combined deficiency in the Def diet caused a significant increase in feed intake. This increased feed intake led to decreased feeding efficiency. A significant decrease in feeding efficiency was also observed in the (-) Lys group. There was an observed upregulation of neuropeptide Y (NPY), an orexigenic hormone, in the Def group. Overall, results from this study suggest stomachless fish increase feed intake when challenged with IDAA-deficient diets, and the regulation of NPY might play a role in this response., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Targeting the lysine residue of protein kinases to develop covalent inhibitors is an emerging hotspot. Herein, we have reported an approach to develop lysine-targeted covalent inhibitors of PI3Kδ by in situ interaction upgradation of the H-bonding to covalent bonding. Several warhead groups were introduced and screened in situ, leading to lysine-targeted covalent inhibitors bearing aromatic esters with high bioactivity and PI3Kδ selectivity. Compound A11 bearing phenolic ester was finally optimized to show a long duration of action in SU-DHL-6 cells by multiple assays. Docking simulation and further protein mass spectrometry confirmed that A11 bound to PI3Kδ by covalent-bonding interactions with Lys779. Furthermore, A11 exhibited potently antitumor efficacy without obvious toxicity in the SU-DHL-6 and Pfeiffer xenograft mouse models. This study identified A11 to be a much more effective antitumor agent in vitro and in vivo as a lysine-targeted covalent inhibitor, and it also provided a practical approach for the development of lysine-targeted covalent inhibitors.

Tight control over cell identity gene expression is necessary for proper adult form and function. The opposing activities of Polycomb and trithorax complexes determine the on/off state of cell identity genes such as the Hox factors. Polycomb group complexes repress target genes, whereas trithorax group complexes are required for their expression. Although trithorax and its orthologs function as methyltransferases specific to histone H3 lysine 4 (H3K4), there is no direct evidence that H3K4 regulates Polycomb group target genes in vivo. Using histone gene replacement in Drosophila , we provide evidence of two key roles for replication-dependent histone H3.2K4 in Polycomb target gene control. First, we found that H3.2K4 mutants mimic H3.2K4me3 in antagonizing methyltransferase activity of the PRC2 Polycomb group complex. Second, we found that H3.2K4 is also required for proper activation of Polycomb targets. We conclude that H3.2K4 directly regulates Polycomb target gene expression., (© 2024 Anyetei-Anum et al.; Published by Cold Spring Harbor Laboratory Press.)

Protein post-translational modifications (PTMs) regulate protein functions but remain poorly characterized in Nannochloropsis . This study examined three PTMs: lysine acetylation (Kac), lysine β-hydroxybutyrylation (Kbhb), and phosphorylation. Using LC-MS/MS, we identified 4571 Kac sites, 7812 Kbhb sites, and 6237 phosphorylation sites across 2455, 3109, and 2786 proteins, respectively. Subcellular localization analysis revealed significant overlaps between Kac and Kbhb proteins, primarily in the chloroplast, cytosol, and nucleus, while phosphorylated proteins were predominantly located in the nucleus and chloroplast. Motif analysis highlighted specific amino acid enrichments around modification sites, with several motifs conserved. Additionally, 529 proteins harbored all three PTMs, underscoring the potential regulatory interplay. Kac, Kbhb, and phosphorylated proteins were particularly abundant in glycolysis, the TCA cycle, carbon fixation, and lipid metabolism pathways, influencing energy production and lipid accumulation. Based on previous transcriptome data under nutrient-limited conditions, these frequently modified key enzymes appear to be vital components in the response to abiotic stress. The presence of histone modifications related to Kac and Kbhb might also point to the epigenetic regulation in gene expression and stress adaptation. This comprehensive PTM landscape in N. oceanica provides a foundation of valuable insights into future metabolic engineering and biotechnological applications.

Cellular prion protein (PrP C ) is a glycosylphosphatidylinositol (GPI)-anchored cell-surface protein. The mature cell-surface PrP C is internalized and subsequently degraded by lysosomes. Although, proteasomes are proposed to be involved, the precise mechanism of PrP C degradation remains uncertain. Given that proteins are ubiquitinated primarily on lysine residues, we sought to determine whether lysine residues within PrP C are involved in the ubiquitination and subsequent degradation of PrP C . We generated a plasmid vector expressing a mutant PrP C (called lysine-null PrP C ) in which all lysine residues were replaced with arginine residues. Subsequently, we established stably transformed cell lines (designated HpL2-1 PrP-WT and HpL2-1 PrP-K/R, respectively) using the mouse PrP C -deficient neuronal cell line (HpL2-1) and plasmids expressing wild-type (WT) or lysine-null PrP C (PrP-K/R). We found that HpL2-1 PrP-WT and HpL2-1 PrP-K/R cells correctly expressed their respective PrP C which translocated efficiently to the plasma membrane. Subsequently, using immunoblotting and confocal microscopy, we found that treatment with cycloheximide (CHX; a protein synthesis inhibitor) significantly reduced PrP C expression in both these transformed cell lines, indicating that WT and lysine-null PrP C are degraded similarly. Taken together, these results indicate that the lysine residues of PrP C do not regulate its degradation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Lysine acylation can direct protein function, localization, and interactions. Sirtuins deacylate lysine toward maintaining cellular homeostasis, and their aberrant expression contributes to the pathogenesis of multiple conditions, including cancer. Measuring sirtuins' activity is essential to exploring their potential as therapeutic targets, but accurate quantification is challenging. We developed "SIRTify", a high-sensitivity assay for measuring sirtuin activity in vitro and in vivo. SIRTify is based on a split-version of the NanoLuc luciferase consisting of a truncated, catalytically inactive N-terminal moiety (LgBiT) that complements with a high-affinity C-terminal peptide (p86) to form active luciferase. Acylation of two lysines within p86 disrupts binding to LgBiT and abates luminescence. Deacylation by sirtuins reestablishes p86 and restores binding, generating a luminescence signal proportional to sirtuin activity. Measurements accurately reflect reported sirtuin specificity for lysine-acylations and confirm the effects of sirtuin modulators. SIRTify quantifies lysine deacylation dynamics and may be adaptable to monitoring additional post-translational modifications., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Metabolic changes during tumour development lead to acidification of the extracellular environment and a smaller increase of intracellular pH. Searches for somatic missense mutations that could reveal adaptation to altered pH have focussed on arginine to histidine changes, part of a general arginine depletion that originates from DNA mutational mechanisms. Analysis of mutations to histidine, potentially a simple route to the introduction of pH-sensing, shows no clear biophysical separation overall of subsets that are more and less frequently mutated in cancer genomes. Within the more frequently mutated subset, individual sites predicted to mediate pH-dependence upon mutation include NDST1 (a Golgi-resident heparan sulphate modifying enzyme), the HLA-C chain of MHCI complex, and the water channel AQP-7. Arginine depletion is a general feature that persists in the more frequently mutated subset, and is complemented by over-representation of mutations to lysine. Arginine to lysine balance is a known factor in determining protein solubility, with higher lysine content being more favourable. Proteins with greater change in arginine to lysine balance are enriched for cell periphery location, where proteostasis is likely to be challenged in tumour cells. Somatic missense mutations in a cancer genome number only in the 10s typically, although can be much higher. Whether the altered arginine to lysine balance is of sufficient scale to play a role in tumour development is unknown., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Sallah, Warwicker. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Alzheimer's disease (AD), an age-related neurodegenerative disorder, is characterized by irreversible brain tissue degeneration. The amyloid-β (Aβ) cascade hypothesis stands as the predominant paradigm explaining AD pathogenesis. This study aimed to elucidate the mechanisms underlying Aβ-induced pyroptosis in AD. AD models were established using amyloid precursor protein/presenilin 1 (APP/PS1) transgenic mice and Aβ-treated BV-2 cells (5 µM, 24 h). NEK7 expression was evaluated in vitro and in vivo. Cell pyroptosis was assessed before and after NEK7 expression was inhibited in BV-2 cells. Adeno-associated virus (AAV) vectors carrying short hairpin RNA (shRNA) against NEK7 (AAV-sh-NEK7) were administered to mice to knockdown NEK7 in vivo. Spatial learning and memory abilities were evaluated using the Morris water maze test. The interaction between NEK7 and histone H4 lysine 12 lactylation (H4K12la) were then investigated. The results suggested that NEK7 expression was markedly elevated in both in vitro and in vivo AD models. Treatment with Aβ significantly reduced cell viability and enhanced pyroptosis in BV-2 cells; these effects were reversed by inhibiting NEK7. Furthermore, AD mice with NEK7 knockdown exhibited shorter escape latencies and increased time spent in the target quadrant, suggesting that NEK7 inhibition improved cognitive function and memory retention. Mechanistically, Aβ treatment induced histone lactylation in BV-2 cells, and suppression of lactylation attenuated NEK7 transcriptional activity and mRNA levels. In summary, elevated NEK7 expression promoted histone lactylation in BV-2 cells, thereby facilitating pyroptosis. Inhibition of NEK7 conferred protection against Aβ-induced cellular damage and enhanced cognitive performance and memory retention in AD model mice. Collectively, targeting NEK7 represents a potential therapeutic strategy for alleviating AD symptoms., Competing Interests: Declarations Ethical approval The study was approved by the Ethics Committee of Tongren Hospital, Shanghai Jiao Tong University School of Medicine. All experiments were performed in accordance with the ARRIVE guidelines. Consent for publication All authors approved the final manuscript and the submission to this journal. Competing interests The authors confirm that no conflicts of interest exist in this work., (© 2024. The Author(s).)

Lysine residues on protein surfaces are abundant and often found in enzyme active sites, making them critical targets for studying undruggable proteins. However, the varied microenvironment surrounding lysine residues results in a wide range of p K a values, complicating site-specific covalent binding. In this study, we address the challenges posed by the diverse reactivity of amino side chains by modulating the amide reaction activity of heteroaromatic activated esters. By fine-tuning the type, position, and number of heteroatoms, we successfully rationalized the regulation of their amide reaction activity, leading to the design of probes for selective lysine labeling within the proteome for profiling purposes. Systematic optimization of these esters' reactivity and selectivity has yielded a series of effective probes suitable for both in vitro and cellular applications. These findings significantly enhance our understanding of protein functions and mechanisms, facilitated by the precise identification and analysis of protein labeling and profiling.

Similar to ubiquitin, the ubiquitin-like protein NEDD8 is not only conjugated to other proteins but is itself subject to posttranslational modifications including lysine acetylation. Yet, compared to ubiquitin, only little is known about the biochemical and structural consequences of site-specific NEDD8 acetylation. Here, we generated site-specifically mono-acetylated NEDD8 variants for each known acetylation site by genetic code expansion. We show that, in particular, acetylation of K11 has a negative impact on the usage of NEDD8 by the NEDD8-conjugating enzymes UBE2M and UBE2F and that this is likely due to electrostatic and steric effects resulting in conformational changes of NEDD8. Finally, we provide evidence that p300 acts as a position-specific NEDD8 acetyltransferase., (© 2024 The Author(s). ChemBioChem published by Wiley-VCH GmbH.)

Diverse post-translational modifications have been shown to play important roles in regulating protein function in eukaryotes. By contrast, the roles of post-translational modifications in bacteria are not so well understood, particularly as they relate to pathogenesis. Here, we demonstrate post-translational protein modification by covalent addition of lactate to lysine residues (lactylation) in the human pathogen Staphylococcus aureus. Lactylation is dependent on lactate concentration and specifically affects alpha-toxin, in which a single lactylated lysine is required for full activity and virulence in infection models. Given that lactate levels typically increase during infection, our results suggest that the pathogen can use protein lactylation as a mechanism to increase toxin-mediated virulence during infection., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Heterozygous mutations in the histone lysine acetyltransferase gene KAT6B ( MYST4/MORF/QKF ) underlie neurodevelopmental disorders, but the mechanistic roles of KAT6B remain poorly understood. Here, we show that loss of KAT6B in embryonic neural stem and progenitor cells (NSPCs) impaired cell proliferation, neuronal differentiation, and neurite outgrowth. Mechanistically, loss of KAT6B resulted in reduced acetylation at histone H3 lysine 9 and reduced expression of key nervous system development genes in NSPCs and the developing cortex, including the SOX gene family, in particular Sox2 , which is a key driver of neural progenitor proliferation, multipotency and brain development. In the fetal cortex, KAT6B occupied the Sox2 locus. Loss of KAT6B caused a reduction in Sox2 promoter activity in NSPCs. Sox2 overexpression partially rescued the proliferative defect of Kat6b -/- NSPCs. Collectively, these results elucidate molecular requirements for KAT6B in brain development and identify key KAT6B targets in neural precursor cells and the developing brain., (© 2024 Bergamasco et al.)

Single walled carbon nanotubes (SWCNT) have recently been demonstrated as modular, near-infrared (nIR) probes for reporting hydrolase activity; however, these have been limited to naturally amphipathic substrate targets used to noncovalently functionalize the hydrophobic nanoparticles. Many relevant substrate targets are hydrophobic (such as recalcitrant biomass) and pose a challenge for modular functionalization. In this work, a facile mechanochemistry approach was used to couple insoluble substrates, such as lignin, to SWCNT using l-lysine amino acid as a linker and tip sonication as the mechanochemical energy source. The proposed coupling mechanism is ion pairing between the lysine amines and lignin carboxylic acids, as evidenced by FTIR, NMR, SEM, and elemental analyses. The limits of detection for the lignin-lysine-SWCNT (LLS) probe were established using commercial enzymes and found to be 0.25 ppm (volume basis) of the formulated product. Real-world use of the LLS probes was shown by evaluating soil hydrolase activities of soil samples gathered from different corn root proximal locations and soil types. Additionally, the probes were used to determine the effect of storage temperature on the measured enzyme response. The modularity of this mechanochemical functionalization approach is demonstrated with other substrates such as zein and 9-anthracenecarboxylic acid, which further corroborate the mechanochemical mechanism.

Outer mitochondrial membrane fusion, a vital cellular process, is mediated by mitofusins. However, the underlying molecular mechanism remains elusive. We have performed extensive multiscale molecular dynamics simulations to predict a model of the transmembrane (TM) domain of the yeast mitofusin Fzo1. Coarse-grained simulations of the two TM domain helices, TM1 and TM2, reveal a stable interface, which is controlled by the charge status of residue Lys716. Atomistic replica-exchange simulations further tune our model, which is confirmed by a remarkable agreement with an independent AlphaFold2 (AF2) prediction of Fzo1 in complex with its fusion partner Ugo1. Furthermore, the presence of the TM domain destabilizes the membrane, even more if Lys716 is charged, which can be an asset for initiating fusion. The functional role of Lys716 was confirmed with yeast experiments, which show that mutating Lys716 to a hydrophobic residue prevents mitochondrial fusion., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Breast cancer (BC) is the leading cause of death among cancer patients worldwide. In 2020, almost 12% of all cancers were diagnosed with BC. Therefore, it is important to search for new potential markers of cancer progression that could be helpful in cancer diagnostics and successful anti-cancer therapies. In this study, we investigated the potential role of the lysine acetyltransferases KAT6A and KAT6B in the outcome of patients with invasive breast carcinoma. The expression profiles of KAT6A/B in 495 cases of IDC and 38 cases of mastopathy (FBD) were examined by immunohistochemistry. KAT6A/B expression was also determined in the breast cancer cell lines MCF-7, BT-474, SK-BR-3, T47D, MDA-MB-231, and MDA-MB-231/BO2, as well as in the human epithelial mammary gland cell line hTERT-HME1 - ME16C, both at the mRNA and protein level. Statistical analysis of the results showed that the nuclear expression of KAT6A/B correlates with the estrogen receptor status: KAT6A NUC vs. ER r = 0.2373 and KAT6B NUC vs. ER r = 0.1496. Statistical analysis clearly showed that KAT6A cytoplasmic and nuclear expression levels were significantly higher in IDC samples than in FBD samples (IRS 5.297 ± 2.884 vs. 2.004 ± 1.072, p < 0.0001; IRS 5.133 ± 4.221 vs. 0.1665 ± 0.4024, p < 0.0001, respectively). Moreover, we noticed strong correlations between ER and PR status and the nuclear expression of KAT6A and KAT6B (nucKAT6A vs. ER, p = 0.0048; nucKAT6A vs. PR p = 0.0416; nucKAT6B vs. ER p = 0.0306; nucKAT6B vs. PR p = 0.0213). Significantly higher KAT6A and KAT6B expression was found in the ER-positive cell lines T-47D and BT-474, whereas significantly lower expression was observed in the triple-negative cell lines MDA-MB-231 and MDA-MB-231/BO2. The outcomes of small interfering RNA (siRNA)-mediated suppression of KAT6A/B genes revealed that within estrogen receptor (ER) positive and negative cell lines, MCF-7 and MDA-MB-231, attenuation of KAT6A led to concurrent attenuation of KAT6A, whereas suppression of KAT6B resulted in simultaneous attenuation of KAT6A. Furthermore, inhibition of KAT6A/B genes resulted in a reduction in estrogen receptor (ER) mRNA and protein expression levels in MCF-7 and MDA-MMB-231 cell lines. Based on our findings, the lysine acetyltransferases KAT6A and KAT6B may be involved in the progression of invasive ductal breast cancer. Further research on other types of cancer may show that KAT6A and KAT6B could serve as diagnostic and prognostic markers for these types of malignancies., (© 2024. The Author(s).)

Methods for modifying intact peptides are useful but can be unselective with regard to amino acid position and sequence context. In this work, we used in vitro selection to identify DNAzymes that acylate a Lys residue of a short peptide in sequence-dependent fashion. The DNAzymes do not acylate Lys when placed at other residues in the peptide, and the acylation activity depends on the Lys sequence context. A high acylation yield is observed on the preparative nanomole scale. These findings are promising for further development of DNAzymes for broader application to top-down Lys acylation of peptide and protein substrates., (© 2024 The Author(s). ChemBioChem published by Wiley-VCH GmbH.)

Recently discovered epigenetic modification lysine lactylation contributes to tumor development and progression in several types of cancer. In addition to the tumor-intrinsic effects, histone lactylation may mediate tumor microenvironment remodeling and immune evasion. In this study, we observed elevated pan-lysine lactylation and histone H3 lysine 18 lactylation (H3K18la) levels in non-small cell lung cancer (NSCLC) tissues, which was positively correlated with poor patient prognosis. Interruption of glycolysis by 2-deoxy-D-glucose and oxamate treatment and silencing of lactate dehydrogenase A and lactate dehydrogenase B reduced H3K18la levels and circumvented immune evasion of NSCLC cells by enhancing CD8+ T-cell cytotoxicity. Mechanistically, H3K18la directly activated the transcription of pore membrane protein 121 (POM121), which enhanced MYC nuclear transport and direct binding to the CD274 promoter to induce PD-L1 expression. In a mouse NSCLC xenograft model, combination therapy with a glycolysis inhibitor and an anti-PD-1 antibody induced intratumoral CD8+ T-cell function and exhibited strong antitumor efficacy. Overall, this work revealed that H3K18la potentiates the immune escape of NSCLC cells by activating the POM121/MYC/PD-L1 pathway, which offers insights into the role of posttranslational modifications in carcinogenesis and provides a rationale for developing an epigenetic-targeted strategy for treating NSCLC. Significance: H3K18 lactylation supports immunosuppression in non-small cell lung cancer by inducing POM121 to increase MYC activity and PD-L1 expression, which can be reversed by metabolic reprogramming and immunotherapy treatment., (©2024 American Association for Cancer Research.)

Background: The vegetable-based diet alone does not provide the lysine (Lys) needed to maximize poultry productive performance., Objectives: This experiment aimed to evaluate the effects of dietary digestible Lys (dLys) level on productive and reproductive performance, egg quality, blood metabolites and immune responses in breeding Japanese quails (Coturnix japonica)., Methods: The experiment was conducted in a completely randomized design with 6 treatments, 5 replicates and 15 (12 females and 3 meals) 10-week-old breeding Japanese quails each. A basal diet was formulated to meet nutritional requirements of breeding quails except dLys. The basal diet was supplemented with graded (+0.82 g/kg) levels of l-Lys-HCl, corresponding to dietary dLys levels of 0.690%, 0.755%, 0.820%, 0.885%, 0.950% and 1.015%. The experiment lasted for 12 weeks, which was divided into 3-4-week periods., Results: Significant differences were observed for egg production (EP), egg mass (EM) and feed efficiency (FE) in response to increasing dietary dLys concentration with quadratic trends. The highest traits were observed in the birds fed with a diet containing 0.885% dLys. However, feed intake, egg quality, reproductive performance, blood metabolites and immune responses against sheep red blood cell inoculation were not significantly affected by increasing dietary dLys concentrations. The dLys requirements during 11-14, 15-18, 19-22 and 11-22 (overall) weeks of age for optimal EP, EM and FE, based on the quadratic broken-line regression analysis, were estimated 272, 265, 250 and 266; 293, 285, 264 and 279; and 303, 294, 281 and 293 mg/bird/day, respectively., Conclusions: The dLys requirements vary depending on the EP phase and the trait being optimized. The estimated dLys requirement for FE was higher than those for EP and EM. During the peak stage of the first laying cycle, the dietary dLys level of 0.932% and a daily intake of 303 mg dLys/bird are sufficient for optimal performance., (© 2024 The Author(s). Veterinary Medicine and Science published by John Wiley & Sons Ltd.)

Hypoxia that occurs during the luteinization process of granulosa cells (GC) contributes to the formation of lactate in follicles. Lysine lactylation (Kla), a post-translational modification directly regulated by lactate levels, is a metabolic sensor that converts metabolic information into gene expression patterns. In this study, we employed human chorionic gonadotropin (hCG) to induce GCs luteinization and discovered that hypoxia enhances hCG-mediated GCs luteinization by stimulating lactate production/lactylation. The elevated levels of luteinization markers (including progesterone synthesis, expression of CYP11A1 and STAR) were accompanied by increased lactate production as well as enhanced lactylation in mouse ovarian GCs after the injection of hCG in vivo. By treating GCs with hypoxia in vitro, we found that hypoxia accelerated hCG-induced GCs luteinization, which was inhibited after blocking lactate production/lactylation. Further investigations revealed that H3K18la might contribute to hCG-induced luteinization in hypoxic GCs by upregulating CYP11A1 and STAR transcription. Additionally, we identified that CREB K136la is also required for hCG-induced GCs luteinization under hypoxia. Finally, the in vitro findings were verified in vivo, which showed impaired GCs luteinization and corpus luteum formation after blocking the lactate/lactylation by intraperitoneal injection of oxamate/C646 in mice. Taken together, this study uncovered a novel role of protein lactylation in the regulation of GCs luteinization., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024. Published by Elsevier B.V.)

Dietary advanced glycation end-products (dAGEs) accumulate in organs and are thought to initiate chronic low-grade inflammation (CLGI), induce glycoxidative stress, drive immunosenescence, and influence gut microbiota. Part of the toxicological interest in glycation products such as dietary carboxymethyl-lysine (dCML) relies on their interaction with receptor for advanced glycation end-products (RAGE). It remains uncertain whether early or lifelong exposure to dAGEs contributes physiological changes and whether such effects are reversible or permanent. Our objective was to examine the physiological changes in Wild-Type (WT) and RAGE KO mice that were fed either a standard diet (STD - 20.8 ± 5.1 µg dCML/g) or a diet enriched with dCML (255.2 ± 44.5 µg dCML/g) from the perinatal period for up to 70 weeks. Additionally, an early age (6 weeks) diet switch (dCML→STD) was explored to determine whether potential harmful effects of dCML could be reversed. Previous dCML accumulation patterns described by our group were confirmed here, with significant RAGE-independent accumulation of dCML in kidneys, ileum and colon over the 70-week dietary intervention (respectively 3-fold, 17-fold and 20-fold increases compared with controls). Diet switching returned tissue dCML concentrations to their baseline levels. The dCML-enriched diet had no significative effect on endogenous glycation, inflammation, oxidative stress or senescence parameters. The relative expression of TNFα, VCAM1, IL6, and P16 genes were all upregulated (∼2-fold) in an age-dependent manner, most notably in the kidneys of WT animals. RAGE knockout seemed protective in this regard, diminishing age-related renal expression of TNFα. Significant increases in TNFα expression were detectable in the intestinal tract of the Switch group (∼2-fold), suggesting a higher sensitivity to inflammation perhaps related to the timing of the diet change. Minor fluctuations were observed at family level within the caecal microbiota, including Eggerthellaceae, Anaerovoracaceae and Marinifilaceae communities, indicating slight changes in composition. Despite chronic dCML consumption resulting in higher free CML levels in tissues, there were no substantial increases in parameters related to inflammageing. Age was a more important factor in inflammation status, notably in the kidneys, while the early-life dietary switch may have influenced intestinal susceptibility to inflammation. This study affirms the therapeutic potential of RAGE modulation and corroborates evidence for the disruptive effect of dietary changes occurring too early in life. Future research should prioritize the potential influence of dAGEs on disease aetiology and development, notably any exacerbating effects they may have upon existing health conditions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Cyclin-dependent kinase 9 (CDK9), a catalytic subunit of the positive transcription elongation factor b (P-TEFb) complex, is a global transcriptional elongation factor associated with cell proliferation. CDK9 activity is regulated by certain histone acetyltransferases, such as p300, GCN5 and P/CAF. However, the impact of males absent on the first (MOF) (also known as KAT8 or MYST1) on CDK9 activity has not been reported. Therefore, the present study aimed to elucidate the regulatory role of MOF on CDK9. We present evidence from systematic biochemical assays and molecular biology approaches arguing that MOF interacts with and acetylates CDK9 at the lysine 35 (i.e. K35) site, and that this acetyl-group can be removed by histone deacetylase HDAC1. Notably, MOF-mediated acetylation of CDK9 at K35 promotes the formation of the P-TEFb complex through stabilizing CDK9 protein and enhancing its association with cyclin T1, which further increases RNA polymerase II serine 2 residues levels and global transcription. Our study reveals for the first time that MOF promotes global transcription by acetylating CDK9, providing a new strategy for exploring the comprehensive mechanism of the MOF-CDK9 axis in cellular processes., (© 2024 Federation of European Biochemical Societies.)

Free radicals produce in DNA a large variety of base and deoxyribose lesions that are corrected by the base excision DNA repair (BER) system. However, the C1'-oxidized abasic residue 2-deoxyribonolactone (dL) traps DNA repair lyases in covalent DNA-protein crosslinks (DPC), including the core BER enzyme DNA polymerase beta (Polβ). Polβ-DPC are rapidly processed in mammalian cells by proteasome-dependent digestion. Blocking the proteasome causes oxidative Polβ-DPC to accumulate in a ubiquitylated form, and this accumulation is toxic to human cells. In the current study, we investigated the mechanism of Polβ-DPC processing in cells exposed to the dL-inducing oxidant 1,10-copper-ortho-phenanthroline. Alanine substitution of either or both of two Polβ C-terminal residues, lysine-206 and lysine-244, enhanced the accumulation of mutant Polβ-DPC relative to the wild-type protein, and removal of the mutant DPC was diminished. Substitution of the N-terminal lysines 41, 61, and 81 did not affect Polβ-DPC processing. For Polβ with the C-terminal lysine substitutions, the amount of ubiquitin in the stabilized DPC was lowered by ∼40 % relative to wild-type Polβ. Suppression of the HECT domain-containing E3 ubiquitin ligase TRIP12 augmented the formation of oxidative Polβ-DPC and prevented Polβ-DPC removal in oxidant-treated cells. Consistent with the toxicity of accumulated oxidative Polβ-DPC, TRIP12 knockdown increased oxidant-mediated cytotoxicity. Thus, ubiquitylation of lysine-206 and lysine-244 by TRIP12 is necessary for digestion of Polβ-DPC by the proteasome as the rapid first steps of DPC repair to prevent their cytotoxic accumulation. Understanding how DPC formed with Polβ or other AP lyases are repaired in vivo is an important step in revealing how cells cope with the toxic potential of such adducts., Competing Interests: Declaration of Competing Interest R.W.S. is co-founder of Canal House Biosciences, LLC, is on the Scientific Advisory Board, and has an equity interest. Canal House Biosciences was not involved in the preparation of this study nor of this manuscript. The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The effect of dietary energy and lysine levels on laying persistency and body composition in brown and white hens was studied. Dietary treatments with 2 Metabolizable Energy levels (ME lay; constant or reduction over time) and 2 apparent fecal digestible Lys levels (AFD Lys; constant or reduction over time), were fed to Lohmann white or brown hens, from 17 to 75 wk of age, in a 2 × 2 × 2 factorial design. Data were subjected to mixed model analyses. The egg production curve was modeled using a non-linear regression function. White hens showed an improved laying persistency and a higher number of total eggs per hen compared to brown hens, indicated by a shorter peak production phase but a significant lower slope of decline after peak (P < 0.05). Similarly, hensfed a reduced instead of a constant ME Lay diet had a better laying persistency, indicated by a shorter peak production phase and a significant smaller slope of decline after peak (P < 0.05). This improved laying persistency was probably related to a higher ADFI and nutrient intake of hens fed the reduced ME Lay diets (P < 0.05). A segmented regression analysis showed reasonable correlation between egg mass production and ME Lay intake, with an R 2 adjusted of 0.78 for the overall model. The egg mass production was not increased for intakes above 330 kcal of ME Lay intake for white hens and 324 kcal for brown hens. Limited effects of dietary treatments on body crude fat and crude protein were found. Both energy and protein requirement seemed to increase over time, indicated by higher voluntary nutrient intake and a reducing body crude fat after wk 43 in all treatments. The egg mass production correlated poorly with the AFD Lys intake, with an R 2 of 0.22. In conclusion, laying persistency was mostly influenced by breed and dietary ME Lay level, but not dietary AFD Lys level., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Interaction of β 2 -glycoprotein I (β 2 GPI) with anionic membranes is crucial in antiphospholipid syndrome (APS), implicating the role of its membrane-binding domain, domain V (DV). The mechanism of DV binding to anionic lipids is not fully understood., Objectives: This study aimed to elucidate the molecular details of β 2 GPI DV binding to anionic membranes., Methods: We utilized molecular dynamics simulations to investigate the structural basis of anionic lipid recognition by DV. To corroborate the membrane-binding mode identified in the highly mobile membrane mimetic simulations, we conducted additional simulations using a full membrane model., Results: The study identified critical regions in DV, namely the lysine-rich loop and the hydrophobic loop, which are essential for membrane association via electrostatic and hydrophobic interactions, respectively. A novel lysine pair contributing to membrane binding was also discovered, providing new insights into β 2 GPI's membrane interaction. Simulations revealed 2 distinct binding modes of DV to the membrane, with mode 1 characterized by the insertion of the hydrophobic loop into the lipid bilayer, suggesting a dominant mechanism for membrane association. This interaction is pivotal for the pathogenesis of APS, as it facilitates the recognition of β 2 GPI by antiphospholipid antibodies., Conclusion: The study advances our understanding of the molecular interactions between β 2 GPI's DV and anionic membranes, which are crucial for APS pathogenesis. It highlights the importance of specific regions in DV for membrane binding and reveals a predominant binding mode. These findings have significant implications for APS diagnostics and therapeutics, offering a deeper insight into the molecular basis of the syndrome., Competing Interests: Declaration of competing interests There are no competing interests to disclose., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Intracellular AA regulate milk protein synthesis within the mammary gland by modifying mammary plasma flow (MPF) and AA transporter activity. Amino acid transporters catalyze translocation using Na + gradient, substrate gradient (uniporters), and exchange mechanisms; further, they exhibit specificity for individual AA or groups of AA with similar side-chain properties within each transport system. Nonessential AA are actively transported through Na + -dependent transporters and, thus, are often used as intracellular currencies for EAA transport through exchange transporters. Therefore, it was hypothesized that individual EAA supplementation would compete with other EAA for shared transporters, and supplementation with Ala, Gln, and Gly would stimulate EAA transport through exchange transporters. Ten primiparous lactating dairy cows were divided into 2 groups based on milk production and were randomly assigned to treatment sequences within 2 balanced 5 × 5 Latin squares by group. Period length was 14 d. Treatments were 9-d jugular infusions of (1) saline; (2) 34.5 g of Val per day; (3) a ratio of 32.7 g of Ala to 40 g of Gln to 26.7 g of Gly per day (AQG); (4) 43 g of Lys per day; or (5) 33.5 g of Ile per day. All cows were fed a common base diet formulated to contain 15.0% CP. Infusions of Ile, Lys, or AQG did not affect milk protein or milk production; however, Val infusion decreased both. The effects of Val infusion on milk protein production appeared to be partially driven by decreased DMI. The decline in milk protein percentage indicated that milk lactose production was also affected. Additionally, Val infusion increased MPF efficiency (MPF/milk; L/L) by approximately 44%. Infusion of Val tended to decrease or decreased mammary net uptakes of Lys, Leu, Met, and total AA. Infusion of Ile tended to increase its mammary net uptakes but did not affect any other AA. Infusions of Lys and AQG did not affect any mammary net uptakes. Infusion of Val tended to decrease Phe and total NEAA mammary clearance rates. Infusion with AQG stimulated Tyr clearance rates and tended to decline system N mammary clearance rates. Ratios of branched-chain AA mammary uptake to milk protein output (U:O) did not differ from 1 for Val-infused cows, which indicated that little intramammary catabolism was occurring. Additionally, the average NEAA U:O in response to all treatments except Val was 0.70, but Val-infused cows had NEAA U:O that averaged 0.09, indicating increased synthesis within the glands. The effects of Val on mammary net clearance rates of multiple EAA support the incorporation of AA limitations in ration optimizers to prevent AA imbalances. It is possible that oversupplementation of EAA other than Val may also decrease DMI and mammary activity. Identifying efficiency apexes for each of the EAA will allow more precise diet formulation and supplementation, leading to improved production efficiency., (The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Purpose: Diabetic kidney disease (DKD) is a serious complication of diabetes mellitus and a leading cause of chronic kidney disease and end-stage renal disease. One potential mechanism underlying cellular dysfunction contributing to kidney disease is aberrant protein post-translational modifications. Lysine acetylation is associated with cellular metabolic flux and is thought to be altered in patients with diabetes and dysfunctional renal metabolism., Experimental Design: A novel extraction and LC-MS/MS approach was adapted to quantify sites of lysine acetylation from formalin-fixed paraffin-embedded (FFPE) kidney tissue and from patients with DKD and non-diabetic donors (n = 5 and n = 7, respectively)., Results: Analysis of FFPE tissues identified 840 total proteins, with 225 of those significantly changing in patients with DKD. Acetylomic analysis quantified 289 acetylated peptides, with 69 of those significantly changing. Pathways impacted in DKD patients revealed numerous metabolic pathways, specifically mitochondrial function, oxidative phosphorylation, and sirtuin signaling. Differential protein acetylation in DKD patients impacted sirtuin signaling, valine, leucine, and isoleucine degradation, lactate metabolism, oxidative phosphorylation, and ketogenesis., Conclusions and Clinical Relevance: A quantitative acetylomics platform was developed for protein biomarker discovery in formalin-fixed and paraffin-embedded biopsies of kidney transplant patients suffering from DKD., (© 2024 Wiley‐VCH GmbH.)