Lysine acetylation is a crucial type of protein post-translational modification, which is involved in many important cellular processes and serious diseases. However, identification of protein acetylated sites through traditional experiment methods is time-consuming and laborious. Those methods are not suitable to identify a large number of acetylated sites quickly. Therefore, computational methods are still very valuable to accelerate lysine acetylated site finding.

Glycoprotein acetylation (GlycA), an emerging inflammatory biomarker, has been used as an indicator of cardiovascular disease. Our research aimed to evaluate the correlation between GlycA and coronary artery disease (CAD) using coronary computed tomography angiography (CCTA).

The SAGA (Spt-Ada-Gcn5 acetyltransferase) complex is an important chromatin modifying complex that can both acetylate and deubiquitinate histones. Sgf29 is a novel component of the SAGA complex. Here, we report the crystal structures of the tandem Tudor domains of Saccharomyces cerevisiae and human Sgf29 and their complexes with H3K4me2 and H3K4me3 peptides, respectively, and show that Sgf29 selectively binds H3K4me2/3 marks. Our crystal structures reveal that Sgf29 harbours unique tandem Tudor domains in its C-terminus. The tandem Tudor domains in Sgf29 tightly pack against each other face-to-face with each Tudor domain harbouring a negatively charged pocket accommodating the first residue alanine and methylated K4 residue of histone H3, respectively. The H3A1 and K4me3 binding pockets and the limited binding cleft length between these two binding pockets are the structural determinants in conferring the ability of Sgf29 to selectively recognize H3K4me2/3. Our in vitro and in vivo functional assays show that Sgf29 recognizes methylated H3K4 to recruit the SAGA complex to its targets sites and mediates histone H3 acetylation, underscoring the importance of Sgf29 in gene regulation.

Partially acetylated chito-oligosaccharides (paCOSs) are bioactive compounds with potential medical applications. Their biological activities are largely dependent on their structural properties, in particular their degree of polymerization (DP) and the position of the acetyl groups along the glycan chain. The production of structurally defined paCOSs in a purified form is highly desirable to better understand the structure/bioactivity relationship of these oligosaccharides. Here, we describe a newly discovered chitinase from Paenibacillus pabuli (PpChi) and demonstrate by mass spectrometry that it essentially produces paCOSs with a DP of three and four that carry a single N-acetylation at their reducing end. We propose that this specific composition of glucosamine (GlcN) and N-acetylglucosamine (GlcNAc) residues, as in GlcN(n)GlcNAc1, is due to a subsite specificity toward GlcN residues at the -2, -3, and -4 positions of the partially acetylated chitosan substrates. In addition, the enzyme is stable, as evidenced by its long shelf life, and active over a large temperature range, which is of high interest for potential use in industrial processes. It exhibits a kcat of 67.2 s-1 on partially acetylated chitosan substrates. When PpChi was used in combination with a recently discovered fungal auxilary activity (AA11) oxidase, a sixfold increase in the release of oligosaccharides from the lobster shell was measured. PpChi represents an attractive biocatalyst for the green production of highly valuable paCOSs with a well-defined structure and the expansion of the relatively small library of chito-oligosaccharides currently available.

Suberoylanilide hydroxamic acid (SAHA) is a well-known histone deacetylase (HDAC) inhibitor and has been used as practical therapy for breast cancer and non-small cell lung cancer (NSCLC). It is previously demonstrated that SAHA treatment could extensively change the profile of acetylome and proteome in cancer cells. However, little is known about the impact of SAHA on other protein modifications and the crosstalks among different modifications and proteome, hindering the deep understanding of SAHA-mediated cancer therapy. In this work, by using SILAC technique, antibody-based affinity enrichment and high-resolution LC-MS/MS analysis, we investigated quantitative proteome, acetylome and ubiquitylome as well as crosstalks among the three datasets in A549 cells toward SAHA treatment. In total, 2968 proteins, 1099 acetylation sites and 1012 ubiquitination sites were quantified in response to SAHA treatment, respectively. With the aid of intensive bioinformatics, we revealed that the proteome and ubiquitylome were negatively related upon SAHA treatment. Moreover, the impact of SAHA on acetylome resulted in 258 up-regulated and 99 down-regulated acetylation sites at the threshold of 1.5 folds. Finally, we identified 55 common sites with both acetylation and ubiquitination, among which ubiquitination level in 43 sites (78.2%) was positive related to acetylation level.

Post-translational modification (PTM) is an important biochemical process. which includes six most well-studied types: phosphorylation, acetylation, methylation, sumoylation, ubiquitylation and glycosylation. PTM is involved in various cell signaling pathways and biological processes. Abnormal PTM status is closely associated with severe diseases (such as cancer and neurologic diseases) by regulating protein functions, such as protein-protein interactions (PPIs). A set of databases was constructed separately for PTM sites and PPI; however, the resource of regulation for PTM on PPI is still unsolved.

Cancer-associated fibroblasts (CAFs) play critical roles in cancer progression by regulating tumor cell proliferation, angiogenesis, and metastasis. Recent studies demonstrated that CAFs induce inhibitory immune cell infiltration and chemotherapy resistance in gastric cancer by activating the NF-κB signaling pathway to secrete IL6, IL8, and other inflammatory factors. Inhibition of the NF-κB signaling pathway in CAFs might be a potential therapeutic strategy in gastric cancer. However, how the NF-κB pathway is activated in CAFs remains unclear. We showed that mesenchymal stem cells (MSCs) differentiated into CAFs, induced by the exosomes derived from gastric cancer cells. During the process of differentiation from MSCs into CAFs, we showed that nuclear PKM2 expression was continuously upregulated and associated with NF-κB P65 acetylation, contributing to P65 nuclear retention in CAFs and constant transcription of IL-6, IL-8, and other inflammatory factors, thus promoting gastric cancer cell proliferation. We showed that NF-κB P65 acetylation was induced by P300. We showed that nuclear PKM2 was derived from exosomes of gastric cancer cell lines and the positive feedback loop induced by PKM2-P65 combination. It is also proved that P300 inhibitors can inhibit tumor proliferation in an AGS subcutaneous xenograft tumor model. Our study showed that gastric cancer cells influence the continuous activation of the NF-κB signaling pathway in CAFs by secreting gastric cancer exosomes containing PKM2, thus inducing abnormal metabolism and inflammation activation. This study provides a new therapeutic target for CAF normalization or deactivation strategies.

Reverting activated hepatic stellate cells (HSCs) to less activation or quiescent status is a promising strategy for liver fibrosis. Histone deacetylase inhibitor (HDACI) could suppress HSCs activation. Our previous study demonstrated a critical role of miRNAs in HSCs activation. Here, we explored the involvement of miRNAs in HDACI induced HSCs deactivation. Human cell line LX2 that resembled activated HSCs was treated with an HDACI - valproic acid (VPA). The effects of VPA on the protein and miRNA profile of LX2 were comprehensively analyzed by iTraq quantitative proteomics and miRNA microarray. The interaction between miRNA and proteins was investigated systematically. The biofunctions of differentially expressed proteins and miRNA targeted proteins were annotated. VPA treatment attenuated the activation phenotype of LX2. In VPA treated LX2, among 1548 quantified proteins, only 86 proteins were differentially expressed (VPA-proteins). While among 282 high-abundance miRNAs, 123 were differentially expressed (VPA-miRNAs), with 104 down-regulated and 19 up-regulated. The top biofunctions of VPA-proteins were closely related to HSCs activation, including cell death and survival, cell movement, cellular growth and proliferation. Furthermore, 22 out of the 36 VPA-proteins involved in cell death and survival, and 19 out of the 30 VPA-proteins involved in cellular movement were predicted targets of VPA-miRNAs. A direct regulatory effect of histone acetylation on miRNA expression was also established. In conclusion, our data provided the molecular mechanisms for VPA induced HSCs deactivation at the protein level and suggested crosstalk between histone acetylation and miRNAs in the inhibitory effects of HDACI on HSCs activation.

Aberrant expression of long non-coding RNAs (lncRNA) is associated with altered DNA methylation and histone modifications during carcinogenesis. However, identifying epigenetically dysregulated lncRNAs and characterizing their functional mechanisms in different cancer subtypes are still major challenges for cancer studies. In this study, we systematically analyzed the epigenetic alterations of lncRNAs at important regulatory elements in three breast cancer subtypes. We identified 87, 691, and 1,197 epigenetically dysregulated lncRNAs in luminal, basal, and claudin-low subtypes of breast cancer, respectively. The landscape of epigenetically dysregulated lncRNAs at enhancer elements revealed that epigenetic changes of the majority of lncRNAs occurred in a subtype-specific manner and contributed to subtype-specific biological functions. We identified six acetylation of lysine 27 on histone H3 (H3K27ac)-dysregulated lncRNAs and three DNA methylation-dysregulated lncRNAs (CTC-303L1.2, RP11-738B7.1, and SLC26A4-AS1) as prognostic biomarkers of basal subtype. These lncRNAs were involved in immune response-related biological functions. Treatment of the basal breast cancer cell line MDA-MB-468 with CREBBP/EP300 bromodomain inhibitors downregulated H3K27 acetylation levels and caused a decrease in the expression of five H3K27ac-dysregulated lncRNAs (LINC00393, KB-1836B5.1, RP1-140K8.5, AC005162.1, and AC020916.2) and inhibition of the growth of breast cancer cells. One epigenetically dysregulated lncRNA (LINC01983) and four lncRNA regulators (UCA1, RP11-221J22.2, RP11-221J22.1, and RP1-212P9.3) were identified as prognostic biomarkers of the luminal molecular subtype of breast cancer by controlling the tumor necrosis factor (TNF) signaling pathway, T helper (Th)17 cell differentiation, and T cell migration. Finally, our results highlighted a profound role of enhancer-related H3K27ac-dysregulated lncRNAs, DNA methylation-dysregulated lncRNAs, and lncRNA regulators in breast cancer subtype carcinogenesis and their potential prognostic value.

The silk-spinning and Lepidopteran model insect Bombyx mori (Bombycidae) is a mulberry specialist. The BmSuc1 gene is the first β-fructofuranosidase (β-FFase) encoding gene identified in animals, and β-FFase acts as an essential sucrase for glycometabolism modulation in the silkworm larvae, involved in resistance to mulberry alkaloids. Glyphodes pyloalis Walker (Lepidoptera: Pyralidae) is an important mulberry pest leading to heavy economic loss of sericulture. However, no molecular or biochemical information is available about G. pyloalis β-FFase homologs. In this study, five β-FFase homologous genes in G. pyloalis were obtained. The genes GpSuc1a and GpSuc2c were expressed in the midgut; GpSuc2c encodes a truncated polypeptide. The expression and the localization of GpSUC1a in the midgut was characterized. Whereas recombinant GpSUC1a expressed in both Escherichia coli and BmN cells displayed little activity as compared with higher activity of BmSUC1, β-FFase activity in the larval midgut of G. pyloalis and GpSUC1a purified from the midgut were both confirmed. The data suggested that the activation of GpSUC1a is probably controlled by a more complicated post-translational regulation system in G. pyloalis larvae than that of BmSUC1 in B. mori. To study post-translational modifications (PTMs), GpSUC1a and BmSUC1 were purified from larval midguts using immunoprecipitation and subjected to LC-MS to perform PTMs analysis. Some putative N-glycosylated sites were found in GpSUC1a but none in BmSUC1, while there was more methylation in BmSUC1 than in GpSUC1a, indicating that such PTMs were supporting the differential β-FFases activities in these two mulberry feeding caterpillars.

Autism spectrum disorders (ASDs) are highly associated with oxidative stress. We have recently shown that Disconnected-interacting protein homolog 2 A (DIP2A) functions in ASD pathophysiology by regulating cortactin acetylation for spine development and synaptic transmission. However, its role is not fully understood in the context of its abundant expression in mitochondria. In this paper, we found that DIP2A was involved in superoxide dismutase (SOD)-mediated antioxidative reactions. In mice, DIP2A knockout inhibited SOD activity and increased reactive oxygen species (ROS) levels in the cerebral cortex. In vitro gain-of-function experiments further confirmed the positive role of DIP2A in scavenging ROS upon oxidative stress. Moreover, DIP2A knockout caused irregular mitochondrial morphology in the cerebral cortex and impaired mitochondrial metabolism with an over consumption of lipids for energy supply. Taken together, these results revealed unrecognized functions of DIP2A in antioxidative protection, providing another possible explanation for DIP2A-mediated ASD pathophysiology.

Metabolic reprogramming of cancer cells is essential for tumorigenesis in which pyruvate kinase M2 (PKM2), the low activity isoform of pyruvate kinase, plays a critical role. Herein, we describe the identification of a nature-product-derived micheliolide (MCL) that selectively activates PKM2 through the covalent binding at residue cysteine424 (C424), which is not contained in PKM1. This interaction promotes more tetramer formation, inhibits the lysine433 (K433) acetylation, and influences the translocation of PKM2 into the nucleus. In addition, the pro-drug dimethylaminomicheliolide (DMAMCL) with similar properties as MCL significantly suppresses the growth of leukemia cells and tumorigenesis in a zebrafish xenograft model. Cell-based assay with knock down PKM2 expression verifies that the effects of MCL are dependent on PKM2 expression. DMAMCL is currently in clinical trials in Australia. Our discovery may provide a valuable pharmacological mechanism for clinical treatment and benefit the development of new anticancer agents.

EGFR activates phosphatidylinositide 3-kinase (PI3K), but the mechanism underlying this activation is not completely understood. We demonstrated here that EGFR activation resulted in lysine acetyltransferase 5 (KAT5)-mediated K395 acetylation of the platelet isoform of phosphofructokinase 1 (PFKP) and subsequent translocation of PFKP to the plasma membrane, where the PFKP was phosphorylated at Y64 by EGFR. Phosphorylated PFKP binds to the N-terminal SH2 domain of p85α, which is distinct from binding of Gab1 to the C-terminal SH2 domain of p85α, and recruited p85α to the plasma membrane resulting in PI3K activation. PI3K-dependent AKT activation results in enhanced phosphofructokinase 2 (PFK2) phosphorylation and production of fructose-2,6-bisphosphate, which in turn promotes PFK1 activation. PFKP Y64 phosphorylation-enhanced PI3K/AKT-dependent PFK1 activation and GLUT1 expression promoted the Warburg effect, tumor cell proliferation, and brain tumorigenesis. These findings underscore the instrumental role of PFKP in PI3K activation and enhanced glycolysis through PI3K/AKT-dependent positive-feedback regulation.

Numerous pieces of evidence have identified that the NLRP3 inflammasome plays a pivotal role in the development and pathogenesis of colitis. Targeting the NLRP3 inflammasome represents a potential therapeutic treatment. Our previous studies have suggested that acetylation of NLRP3 is indispensable to NLRP3 inflammasome activation, and some acetyltransferase inhibitors could suppress the NLRP3 inflammasome activation. Here, we identified that C646, an inhibitor of histone acetyltransferase p300, exerts anti-inflammatory effects in DSS-induced colitis mice by targeting the NLRP3 inflammasome. Mechanistically, C646 not only inhibits NF-κB activation, leading to the decreased expression of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) and NLRP3, but also suppresses the NLRP3 inflammasome assembly by disrupting the interaction between NLRP3 and ASC. In addition, C646 attenuated the LPS-induced acute systemic inflammation model. Thus, our results demonstrate the ability of C646 to suppress the NLRP3 inflammasome activity and its potential application in the treatment of inflammatory bowel disease.

Recently immunoglobulins (Igs) have been found to be expressed by cells other than B lymphocytes, including various human carcinoma cells. Sarcomas are derived from mesenchyme, and the knowledge about the occurrence of Ig production in sarcoma cells is very limited. Here we investigated the phenomenon of immunoglobulin G (IgG) expression and its molecular basis in 3 sarcoma cell lines. The mRNA transcripts of IgG heavy chain and kappa light chain were detected by RT-PCR. In addition, the expression of IgG proteins was confirmed by Western blot and immunofluorescence. Immuno-electron microscopy localized IgG to the cell membrane and rough endoplasmic reticulum. The essential enzymes required for gene rearrangement and class switch recombination, and IgG germ-line transcripts were also identified in these sarcoma cells. Chromatin immunoprecipitation results demonstrated histone H3 acetylation of both the recombination activating gene and Ig heavy chain regulatory elements. Collectively, these results confirmed IgG expression in sarcoma cells, the mechanism of which is very similar to that regulating IgG expression in B lymphocytes.

Neointimal hyperplasia/proliferation (IH) is the primary etiology of vascular stenosis. Epigenomic studies concerning IH have been largely confined to in vitro models, and IH-underlying epigenetic mechanisms remain poorly understood. This study integrates information from in vivo epigenomic mapping, conditional knockout, gene transfer and pharmacology in rodent models of IH. The data from injured (IH-prone) rat arteries revealed a surge of genome-wide occupancy by histone-3 lysine-27 trimethylation (H3K27me3), a gene-repression mark. This was unexpected in the traditional view of prevailing post-injury gene activation rather than repression. Further analysis illustrated a shift of H3K27me3 enrichment to anti-proliferative genes, from pro-proliferative genes where gene-activation mark H3K27ac(acetylation) accumulated instead. H3K27ac and its reader BRD4 (bromodomain protein) co-enriched at Ezh2; conditional BRD4 knockout in injured mouse arteries reduced H3K27me3 and its writer EZH2, which positively regulated another pro-IH chromatin modulator UHRF1. Thus, results uncover injury-induced loci-specific H3K27me3 redistribution in the epigenomic landscape entailing BRD4→EZH2→UHRF1 hierarchical regulations. Given that these players are pharmaceutical targets, further research may help improve treatments of IH.

Histone deacetylase 6 (HDAC6) is the specific subtype of HDACs which preferentially located in the cytoplasm, and is crucial in insulin signalling. However, the role of HDAC6 in type 2 diabetic nephropathy (DN) remains undefined. In current study, we observed that HDAC6 was markedly activated in the kidneys of type 2 diabetic patients and db/db mice with albuminuria, along with the advanced glycation end products (AGE)-treated podocytes. Selective inhibition of HDAC6 activity protected kidneys from hyperglycaemia in db/db mice. Notably, overexpressing HDAC6 inhibited autophagy and promoted motility aside from the apoptosis of podocytes exposed to AGE. We further determined that HDAC6 regulated the autophagy partially by decreasing the acetylation of α-tubulin at the residue of lysine 40. In contrast, we confirmed that there was no interaction of HDAC6 with α-tubulin at the sites of lysine 112 and lysine 352. Consistently, inhibiting HDAC6 by siRNA or the selective inhibitor, tubacin, restored the autophagy level and motility of podocytes and rescued podocytes from AGE stimulation. We provide strong evidence of an unexpected role of HDAC6 in the cascade that modulates podocytes autophagy and motility, enlightening that HDAC6 may be a promising therapeutic target for DN treatment.

The sigma-2 receptor (S2R) has long been pharmacologically targeted for antipsychotic treatment and tumor imaging. Only recently was it known for its coding gene and for its role implicated in cholesterol homeostasis. Here, we have investigated the transcriptional control of S2R by the Bromo/ExtraTerminal epigenetic reader family (BETs, including BRD2, 3, and 4) upon cholesterol perturbation. Cholesterol deprivation was induced in ARPE19 cells using a blocker of lysosomal cholesterol export. This condition up-regulated S2R mRNA and protein, and also SREBP2 but not SREBP1, both transcription factors key to cholesterol/fatty acid metabolism. Silencing BRD2 but not BRD3 or BRD4 (though widely deemed a master regulator) averted S2R up-regulation that was induced by cholesterol deprivation. Silencing SREBP2 but not SREBP1 diminished S2R expression. Furthermore, endogenous BRD2 co-immunoprecipitated with the transcription-active N-terminal half of SREBP2, and chromatin immunoprecipitation-qPCR signified co-occupancy of BRD2, H3K27ac (histone acetylation), and SREBP2Nterm at the S2R gene promoter. In summary, this study reveals a previously unrecognized BRD2/SREBP2 cooperative regulation of S2R transcription, thus shedding new light on signaling in response to cholesterol deprivation.

Epigenetically switched, proliferative vascular smooth muscle cells (SMCs) form neointima, engendering stenotic diseases. Histone-3 lysine-27 trimethylation (H3K27me3) and acetylation (H3K27ac) marks are associated with gene repression and activation, respectively. The polycomb protein embryonic ectoderm development (EED) reads H3K27me3 and also enhances its deposition, hence is a canonical gene repressor. However, herein we found an unexpected role for EED in activating the bona fide pro-proliferative gene Ccnd1 (cyclinD1). EED overexpression in SMCs increased Ccnd1 mRNA, seemingly contradicting its gene-repressing function. However, consistently, EED co-immunoprecipitated with gene-activating H3K27ac reader BRD4, and they co-occupied at both mitogen-activated Ccnd1 and mitogen-repressed P57 (bona fide anti-proliferative gene), as indicated by chromatin immunoprecipitation qPCR. These results were abolished by an inhibitor of either the EED/H3K27me3 or BRD4/H3K27ac reader function. In accordance, elevating BRD4 increased H3K27me3. In vivo, while EED was upregulated in rat and human neointimal lesions, selective EED inhibition abated angioplasty-induced neointima and reduced cyclinD1 in rat carotid arteries. Thus, results uncover a previously unknown role for EED in Ccnd1 activation, likely via its cooperativity with BRD4 that enhances each other's reader function; i.e., activating pro-proliferative Ccnd1 while repressing anti-proliferative P57. As such, this study confers mechanistic implications for the epigenetic intervention of neointimal pathology.

It has long been accepted that immunoglobulins (Igs) were produced by B lymphoid cells only. Recently Igs have been found to be expressed in various human cancer cells and promote tumor growth. Recombination activating gene 1 (RAG1) and RAG2, which are essential enzymes for initiating variable-diversity-joining segment recombination, have also been found to be expressed in cancer cells. However, the mechanism of RAG activation in these cancer cells has not been elucidated. Here, we investigated the regulatory mechanism of RAG expression in four human cancer cell lines by analyzing transcription factors that induce RAG activation in B cells. By RT-PCR, Western blot and immunofluorescence, we found that transcription factors E2A, FOXO1 and FOXP1 were expressed and localized to the nuclei of these cancer cells. Over-expression of E2A, FOXO1 or Foxp1 increased RAG expression, while RNA interference of E2A, FOXO1 or FOXP1 decreased RAG expression in the cancer cells. Chromatin immunoprecipitation experiments showed acetylation of RAG enhancer (Erag) and E2A, FOXO1 or FOXP1 were bound to Erag in vivo. These results indicate that in these cancer cells the transcription factors E2A, FOXO1 and FOXP1 regulate RAG expression, which initiates Ig gene rearrangement much in the way similar to B lymphocytes.