Role of Dot1L and H3K79 methylation in regulating somatic hypermutation of immunoglobulin genes.

Somatic hypermutation (SHM) and class-switch recombination (CSR) of the immunoglobulin (Ig) genes allow B cells to make antibodies that protect us against a wide variety of pathogens. SHM is mediated by activation-induced deaminase (AID), occurs at a million times higher frequency than other mutations in the mammalian genome, and is largely restricted to the variable (V) and switch (S) regions of Ig genes. Using the Ramos human Burkitt's lymphoma cell line, we find that H3K79me2/3 and its methyltransferase Dot1L are more abundant on the V region than on the constant (C) region, which does not undergo mutation. In primary naïve mouse B cells examined ex vivo, the H3K79me2/3 modification appears constitutively in the donor Sµ and is inducible in the recipient Sγ1 upon CSR stimulation. Knockout and inhibition of Dot1L in Ramos cells significantly reduces V region mutation and the abundance of H3K79me2/3 on the V region and is associated with a decrease of polymerase II (Pol II) and its S2 phosphorylated form at the IgH locus. Knockout of Dot1L also decreases the abundance of BRD4 and CDK9 (a subunit of the P-TEFb complex) on the V region, and this is accompanied by decreased nascent transcripts throughout the IgH gene. Treatment with JQ1 (inhibitor of BRD4) or DRB (inhibitor of CDK9) decreases SHM and the abundance of Pol II S2P at the IgH locus. Since all these factors play a role in transcription elongation, our studies reinforce the idea that the chromatin context and dynamics of transcription are critical for SHM.

Zebrafish otud6b Negatively Regulates Antiviral Responses by Suppressing K63-Linked Ubiquitination of irf3 and irf7.

Ovarian tumor domain-containing 6B (OTUD6B) belongs to the OTU deubiquitylating enzyme family. In this study, we report that zebrafish otud6b is induced upon viral infection, and overexpression of otud6b suppresses cellular antiviral response. Disruption of otud6b in zebrafish increases the survival rate upon spring viremia of carp virus and grass carp reovirus exposure. Further assays indicate that otud6b interacts with irf3 and irf7 and diminishes traf6-mediated K63-linked polyubiquitination of irf3 and irf7. In addition, the OTU domain is required for otud6b to repress IFN-1 activation and K63-linked polyubiquitination of irf3 and irf7. Moreover, otud6b also attenuates tbk1 to bind to irf3 and irf7, resulting in the impairment of irf3 and irf7 phosphorylation. This study provides, to our knowledge, novel insights into otud6b function and sheds new lights on the regulation of irf3 and irf7 by deubiquitination in IFN-1 signaling.

Structural basis for antigen recognition by methylated lysine-specific antibodies.

Proteins are modulated by a variety of posttranslational modifications including methylation. Despite its importance, the majority of protein methylation modifications discovered by mass spectrometric analyses are functionally uncharacterized, partly owing to the difficulty in obtaining reliable methylsite-specific antibodies. To elucidate how functional methylsite-specific antibodies recognize the antigens and lead to the development of a novel method to create such antibodies, we use an immunized library paired with phage display to create rabbit monoclonal antibodies recognizing trimethylated Lys260 of MAP3K2 as a representative substrate. We isolated several methylsite-specific antibodies that contained unique complementarity determining region sequence. We characterized the mode of antigen recognition by each of these antibodies using structural and biophysical analyses, revealing the molecular details, such as binding affinity toward methylated/nonmethylated antigens and structural motif that is responsible for recognition of the methylated lysine residue, by which each antibody recognized the target antigen. In addition, the comparison with the results of Western blotting analysis suggests a critical antigen recognition mode to generate cross-reactivity to protein and peptide antigen of the antibodies. Computational simulations effectively recapitulated our biophysical data, capturing the antibodies of differing affinity and specificity. Our exhaustive characterization provides molecular architectures of functional methylsite-specific antibodies and thus should contribute to the development of a general method to generate functional methylsite-specific antibodies by de novo design.

Antibodies Isolated from Rheumatoid Arthritis Patients against Lysine-Containing Proteus mirabilis O3 (S1959) Lipopolysaccharide May React with Collagen Type I.

Most rheumatic diseases, including rheumatoid arthritis (RA), are characterized by immune disorders that affect antibody activity. In the present study, using Dot blot and ELISA assay, we showed that patients with rheumatic disease produced significantly more antibodies against lipopolysaccharide (LPS) P. mirabilis O3 compared to healthy donors (p < 0.05), and affinity purified antibodies against LPS O3 may cross-react with collagen type I. It was demonstrated that purified of antibodies isolated from RA patients sera, reacted stronger with the collagen than healthy donors (p = 0.015), and cross-reaction was correlated with level of anti-citrullinated peptide antibodies (r = 0.7, p = 0.003). Moreover, using six different lipopolysaccharides were demonstrated the significant correlations in sera reactivity among lysine-containing lipopolysaccharides observed in patients' sera (p < 0.05). Using Attenuated Total Reflection Fourier Transform Infrared Spectroscopy (ATR-FTIR) it was shown that unique wavenumbers of sera spectra correlate with reactivity with lipopolysaccharides allowing distinguish patients from healthy blood donors. Antibodies adsorption by synthetic antigens shows that in patients' group anti-LPS O3 antibodies can be adsorbed by both amides of galacturonic acid and lysine or threonine, which suggests less specificity of antibodies binding with non-carbohydrate LPS component. The observed correlations suggest that non-carbohydrate components of LPS may be an important epitope for less specific anti-LPS antibodies, which might lead to cross-reactions and affect disease development.

Natural T Cell Epitope Containing Methyl Lysines on Mycobacterial Heparin-Binding Hemagglutinin.

T cell epitopes are mostly nonmodified peptides, although posttranslationally modified peptide epitopes have been described, but they originated from viral or self-proteins. In this study, we provide evidence of a bacterial methylated T cell peptide epitope. The mycobacterial heparin-binding hemagglutinin (HBHA) is a protein Ag with a complex C-terminal methylation pattern and is recognized by T cells from humans latently infected with Mycobacterium tuberculosis By comparing native HBHA with recombinant HBHA produced in Mycobacterium smegmatis (rHBHA-Ms), we could link antigenic differences to differences in the methylation profile. Peptide scan analyses led to the discovery of a peptide containing methyl lysines recognized by a mAb that binds to native HBHA ∼100-fold better than to rHBHA-Ms This peptide was also recognized by T cells from latently infected humans, as evidenced by IFN-γ release upon peptide stimulation. The nonmethylated peptide did not induce IFN-γ, arguing that the methyl lysines are part of the T cell epitope.

Lysine Bioconjugation on Native Albumin with a Sulfonyl Acrylate Reagent.

This protocol details a novel bioconjugation strategy that uses a methanesulfonyl acrylate reagent that is directed to the most reactive lysine on human serum albumin, which enables the construction of chemically defined and stable bioconjugates. The reaction proceeds rapidly and a regioselective modification is achieved using a single molar equivalent of the reagent under biocompatible conditions (37 °C, pH 8.0). Importantly, the bioconjugate retains both the secondary structural content and function of the unmodified protein. During the reaction of the amino group of lysine and the sulfonyl acrylate reagent, methanesulfinic acid is released after the conjugate addition, which then generates an electrophilic acrylate moiety on the protein. This acrylate can be further used for site-specific protein labeling using a synthetic molecule bearing a reactive amine under biocompatible conditions (21 °C, pH 8.0).

Engineering Dual Variable Domains for the Generation of Site-Specific Antibody-Drug Conjugates.

Site-specific antibody-drug conjugate (ADC) technologies are highly desirable for the production of therapeutics with well-defined biochemical and pharmacological characteristics. We have developed a strategy to produce site-specific ADCs using a highly reactive lysine residue embedded in a dual-variable-domain (DVD) format. Here we provide protocols for the engineering, expression, and purification of the DVDs used for this strategy. We also provide a protocol for DVD-drug conjugation and describe methods for their biochemical characterization, including a catalytic assay to monitor conjugation efficiency.

Engineering Antibodies with C-Terminal Sortase-Mediated Modification for Targeted Nanomedicine.

The current advances in nanoengineered materials coupled with the precise targeting capability of recombinant antibodies can create nanoscale diagnostics and therapeutics which show enhanced accumulation and extended retention at a target tissue. Smaller antibodies such as single-chain variable fragments (scFv) preserve the selective and strong binding of their parent antibody to their antigen with the benefits of low immunogenicity, more efficient tissue penetration and easy introduction of functional residues suitable for site-specific conjugation. This is of high importance as nonspecific antibody modification often involves attachment to free cysteine or lysine amino acids which may reside in the active site, leading to reduced antigen binding.In this chapter, we outline a facile and versatile chemoenzymatic approach for production of targeted nanocarrier scFv conjugates using the bacterial trans-peptidase Sortase A (Srt A). Srt A efficiently mediates sequence-specific peptide ligation under mild conditions and has few undesirable side reactions. We first describe the production, purification and characterization of Srt A enzyme and a scFv construct which targets activated platelets, called scFvanti-GPIIb/IIIa. Following this, our protocol illustrates the chemoenzymatic modification of the antibody at the C-terminus with an orthogonal click chemistry linker. This avoids any random attachment to the biologically active antigen binding site of the antibody. Finally, we describe the modification of a nanoparticle surface with scFv attachment via two methods: (1) direct Sortase-mediated conjugation; or (2) a two-step system which consists of scFv Sortase-mediated conjugation followed by strain promoted azide-alkyne cycloaddition. Finally, methodology is described to assess the successful assembly of targeted particles.

Iron-dependent histone 3 lysine 9 demethylation controls B cell proliferation and humoral immune responses.

Trace elements play important roles in human health, but little is known about their functions in humoral immunity. Here, we show an important role for iron in inducing cyclin E and B cell proliferation. We find that iron-deficient individuals exhibit a significantly reduced antibody response to the measles vaccine when compared to iron-normal controls. Mice with iron deficiency also exhibit attenuated T-dependent or T-independent antigen-specific antibody responses. We show that iron is essential for B cell proliferation; both iron deficiency and α-ketoglutarate inhibition could suppress cyclin E1 induction and S phase entry of B cells upon activation. Finally, we demonstrate that three demethylases, KDM2B, KDM3B and KDM4C, are responsible for histone 3 lysine 9 (H3K9) demethylation at the cyclin E1 promoter, cyclin E1 induction and B cell proliferation. Thus, our data reveal a crucial role of H3K9 demethylation in B cell proliferation, and the importance of iron in humoral immunity.

Differential Effects of Dry vs. Wet Heating of ß-Lactoglobulin on Formation of sRAGE Binding Ligands and sIgE Epitope Recognition.

The effect of glycation and aggregation of thermally processed ß-lactoglobulin (BLG) on binding to sRAGE and specific immunoglobulin E (sIgE) from cow milk allergic (CMA) patients were investigated. BLG was heated under dry conditions (water activity < 0.7) and wet conditions (in phosphate buffer at pH 7.4) at low temperature (<73 °C) and high temperatures (>90 °C) in the presence or absence of the milk sugar lactose. Nε-(carboxymethyl)-l-lysine (CML) western blot and glycation staining were used to directly identify glycation structures on the protein fractions on SDS-PAGE. Western blot was used to specify sRAGE and sIgE binding fractions. sRAGE binding was highest under wet-heated BLG independent of the presence of the milk sugar lactose. Under wet heating, high-molecular-weight aggregates were most potent and did not require the presence of CML to generate sRAGE binding ligands. In the dry system, sRAGE binding was observed only in the presence of lactose. sIgE binding affinity showed large individual differences and revealed four binding profiles. Dependent on the individual, sIgE binding decreased or increased by wet heating independent of the presence of lactose. Dry heating required the presence of lactose to show increased binding to aggregates in most individuals. This study highlights an important role of heating condition-dependent protein aggregation and glycation in changing the immunogenicity and antigenicity of cow's milk BLG.

Quantitative proteome and lysine succinylome analyses provide insights into metabolic regulation in breast cancer.

BACKGROUND: Breast cancer, the most common invasive cancer and cause of cancer-related death in women worldwide, is a multifactorial, complex disease, and many molecular players and mechanisms underlying the complexity of its clinical behavior remain unknown. METHODS: To explore the molecular features of breast cancer, quantitative proteome and succinylome analyses in breast cancer were extensively studied using quantitative proteomics techniques, anti-succinyl lysine antibody-based affinity enrichment, and high-resolution LC-MS/MS analysis. RESULTS: Our study is the first to detect the regulation of lysine succinylation in breast cancer progression. We identified a novel mechanism by which the pentose phosphate pathway and the endoplasmic reticulum protein processing pathway might be regulated via lysine succinylation in their core enzymes. CONCLUSIONS: These results expand our understanding of tumorigenesis mechanisms and provide a basis for further characterization of the pathophysiological roles in breast cancer progression, laying a foundation for innovative and novel breast cancer drugs and therapies.

Dietary Advanced Glycation Endproducts Induce an Inflammatory Response in Human Macrophages in Vitro.

Advanced glycation endproducts (AGEs) can be found in protein- and sugar-rich food products processed at high temperatures, which make up a vast amount of the Western diet. The effect of AGE-rich food products on human health is not yet clear and controversy still exists due to possible contamination of samples with endotoxin and the use of endogenous formed AGEs. AGEs occur in food products, both as protein-bound and individual molecules. Which form exactly induces a pro-inflammatory effect is also unknown. In this study, we exposed human macrophage-like cells to dietary AGEs, both in a protein matrix and individual AGEs. It was ensured that all samples did not contain endotoxin concentrations > 0.06 EU/mL. The dietary AGEs induced TNF-alpha secretion of human macrophage-like cells. This effect was decreased by the addition of N(ε)-carboxymethyllysine (CML)-antibodies or a receptor for advanced glycation endproducts (RAGE) antagonist. None of the individual AGEs induce any TNF-alpha, indicating that AGEs should be bound to proteins to exert an inflammatory reaction. These findings show that dietary AGEs directly stimulate the inflammatory response of human innate immune cells and help us define the risk of regular consumption of AGE-rich food products on human health.

A novel monoclonal antibody targeting carboxymethyllysine, an advanced glycation end product in atherosclerosis and pancreatic cancer.

Advanced glycation end products are formed by non-enzymatic reactions between proteins and carbohydrates, causing irreversible lysine and arginine alterations that severely affect protein structure and function. The resulting modifications induce inflammation by binding to scavenger receptors. An increase in advanced glycation end products is observed in a number of diseases e.g. atherosclerosis and cancer. Since advanced glycation end products also are present in healthy individuals, their detection and quantification are of great importance for usage as potential biomarkers. Current methods for advanced glycation end product detection are though limited and solely measure total glycation. This study describes a new epitope-mapped single chain variable fragment, D1-B2, against carboxymethyllysine, produced from a phage library that was constructed from mouse immunizations. The phage library was selected against advanced glycation end product targets using a phage display platform. Characterization of its binding pattern was performed using large synthetic glycated peptide and protein libraries displayed on microarray slides. D1-B2 showed a preference for an aspartic acid, three positions N-terminally from a carboxymethyllysine residue and also bound to a broad collection of glycated proteins. Positive immunohistochemical staining of mouse atherosclerotic plaques and of a tissue microarray of human pancreatic tumors confirmed the usability of the new scFv for advanced glycation end product detection in tissues. This study demonstrates a promising methodology for high-throughput generation of epitope-mapped monoclonal antibodies against AGE.

Effects of dietary lysine restriction on inflammatory responses in piglets.

The aim of this study was to investigate the effects of lysine restriction on inflammatory responses in piglets. 38 male piglets with similar body weight of 9.62 kg were randomly divided into control group (basal diet) and lysine-restricted group (diet containing 70% lysine of the control diet). The results showed that lysine restriction increased the serum concentration of IgG an IgM. Piglets fed the lysine-restricted diet exhibited overexpression of interleukin-8 (IL-8) in the kidney (P < 0.05) and IL-6 and IL-4 in the spleen (P < 0.05). The mRNA abundances of IL-4 in the kidney (P < 0.05) and IL-10 in the liver (P < 0.05) were significantly lower in the lysine-restricted group compared with the control group. Meanwhile, lysine restriction increased the mRNA level of Tlr8 in the kidney (P < 0.05) but decreased the mRNA level of Tlr8 in the liver (P < 0.05). Finally, lysine restriction markedly enhanced extracellular signal regulated kinases 1/2 (ERK1/2) phosphorylation in the kidney and liver and nuclear transcription factor kappa B (NF-κB) was activated in the liver and spleen in response to dietary lysine restriction. In conclusion, lysine restriction affected inflammatory responses in the kidney, liver, and spleen via mediating serum antibody volume, inflammatory cytokines, Tlrs system, and ERK1/2 and NF-κB signals in piglets.

The lysine deacetylase Sirtuin 1 modulates the localization and function of the Notch1 receptor in regulatory T cells.

The ability to tune cellular functions in response to nutrient availability has important consequences for immune homeostasis. The activity of the receptor Notch in regulatory T (Treg) cells, which suppress the functions of effector T cells, is indispensable for Treg cell survival under conditions of diminished nutrient supply. Anti-apoptotic signaling induced by the Notch1 intracellular domain (NIC) originates from the cytoplasm and is spatially decoupled from the nuclear, largely transcriptional functions of NIC. We showed that Sirtuin 1 (Sirt1), which is an NAD+ (nicotinamide adenine dinucleotide)-dependent lysine deacetylase that inhibits NIC-dependent gene transcription, stabilized NIC proximal to the plasma membrane to promote the survival and function of activated Treg cells. Sirt1 was required for NIC-dependent protection from apoptosis in cell lines but not for the activity of the anti-apoptotic protein Bcl-xL. In addition, a variant NIC protein in which four lysines were mutated to arginines (NIC4KR) retained anti-apoptotic activity, but was not regulated by Sirt1, and reconstituted the functions of nonnuclear NIC in Notch1-deficient Treg cells. Loss of Sirt1 compromised Treg cell survival, resulting in antigen-induced T cell proliferation and inflammation in two mouse models. Thus, the Sirt1-Notch interaction may constitute an important checkpoint that tunes noncanonical Notch1 signaling.

Polycomb Repressive Complex 2-Mediated Chromatin Repression Guides Effector CD8+ T Cell Terminal Differentiation and Loss of Multipotency.

Understanding immunological memory formation depends on elucidating how multipotent memory precursor (MP) cells maintain developmental plasticity and longevity to provide long-term immunity while other effector cells develop into terminally differentiated effector (TE) cells with limited survival. Profiling active (H3K27ac) and repressed (H3K27me3) chromatin in naive, MP, and TE CD8+ T cells during viral infection revealed increased H3K27me3 deposition at numerous pro-memory and pro-survival genes in TE relative to MP cells, indicative of fate restriction, but permissive chromatin at both pro-memory and pro-effector genes in MP cells, indicative of multipotency. Polycomb repressive complex 2 deficiency impaired clonal expansion and TE cell differentiation, but minimally impacted CD8+ memory T cell maturation. Abundant H3K27me3 deposition at pro-memory genes occurred late during TE cell development, probably from diminished transcription factor FOXO1 expression. These results outline a temporal model for loss of memory cell potential through selective epigenetic silencing of pro-memory genes in effector T cells.

Biochemical Principles and Functional Aspects of Pipecolic Acid Biosynthesis in Plant Immunity.

The nonprotein amino acid pipecolic acid (Pip) regulates plant systemic acquired resistance and basal immunity to bacterial pathogen infection. In Arabidopsis (Arabidopsis thaliana), the lysine (Lys) aminotransferase AGD2-LIKE DEFENSE RESPONSE PROTEIN1 (ALD1) mediates the pathogen-induced accumulation of Pip in inoculated and distal leaf tissue. Here, we show that ALD1 transfers the α-amino group of l-Lys to acceptor oxoacids. Combined mass spectrometric and infrared spectroscopic analyses of in vitro assays and plant extracts indicate that the final product of the ALD1-catalyzed reaction is enaminic 2,3-dehydropipecolic acid (DP), whose formation involves consecutive transamination, cyclization, and isomerization steps. Besides l-Lys, recombinant ALD1 transaminates l-methionine, l-leucine, diaminopimelate, and several other amino acids to generate oxoacids or derived products in vitro. However, detailed in planta analyses suggest that the biosynthesis of 2,3-DP from l-Lys is the major in vivo function of ALD1. Since ald1 mutant plants are able to convert exogenous 2,3-DP into Pip, their Pip deficiency relies on the inability to form the 2,3-DP intermediate. The Arabidopsis reductase ornithine cyclodeaminase/µ-crystallin, alias SYSTEMIC ACQUIRED RESISTANCE-DEFICIENT4 (SARD4), converts ALD1-generated 2,3-DP into Pip in vitro. SARD4 significantly contributes to the production of Pip in pathogen-inoculated leaves but is not the exclusive reducing enzyme involved in Pip biosynthesis. Functional SARD4 is required for proper basal immunity to the bacterial pathogen Pseudomonas syringae Although SARD4 knockout plants show greatly reduced accumulation of Pip in leaves distal to P. syringae inoculation, they display a considerable systemic acquired resistance response. This suggests a triggering function of locally accumulating Pip for systemic resistance induction.

Autoantibodies against mono- and tri-methylated lysine display similar but also distinctive characteristics.

Autoantibodies can be either harmful or beneficial to the body. The beneficial autoantibodies play important roles in immunosurveillance, clearance of body waste and maintenance of immune homeostasis. Despite their importance, however, people's knowledge on the protective autoantibodies is still very limited. In the current study, we examined two autoantibodies that recognized epitopes with only one amino acid. One was against mono-methylated lysine (Kme) and the other was against tri-methylated lysine (Kme3). We found that the antibodies were highly specific and not polyreactive. They did not cross-react each other. Although anti-Kme antibodies were IgM only, a large proportion of the anti-Kme3 antibodies were switched to the IgG isotype. Mass spectrometric analysis showed that both of the antibodies were mainly derived from IGHV 3-7 and/or IGHV3-74 germ line genes with conserved CDR2. De novo sequencing showed that there was a mutation at either of the SS positions on the CDR1 region, which changed one of the serine residues to a basic amino acid, i.e., arginine or lysine. We also found that neither of the antibodies was expressed at birth, and their earliest appearance was approximately 5 months after birth. All healthy human beings expressed the antibodies when they reached age two and maintained the expression thereafter throughout their life. Patients with systemic lupus erythematosus had lower levels of the IgM isotype antibodies. Serum levels of the two IgM antibodies were closely correlated, implying that they were produced by cells from the same B cell subset. We also found that both anti-Kme and anti-Kme3 antibodies could bind and might take part in the clearance of neutrophil extracellular traps released from activated cells. In conclusion, although anti-Kme and anti-Kme3 antibodies share many similarities in their origins, they are different antibodies and have different characteristics.

Antimodified protein antibody response pattern influences the risk for disease relapse in patients with rheumatoid arthritis tapering disease modifying antirheumatic drugs.

OBJECTIVE: To perform a detailed analysis of the autoantibody response against post-translationally modified proteins in patients with rheumatoid arthritis (RA) in sustained remission and to explore whether its composition influences the risk for disease relapse when tapering disease modifying antirheumatic drug (DMARD) therapy. METHODS: Immune responses against 10 citrullinated, homocitrullinated/carbamylated and acetylated peptides, as well as unmodified vimentin (control) and cyclic citrullinated peptide 2 (CCP2) were tested in baseline serum samples from 94 patients of the RETRO study. Patients were classified according to the number of autoantibody reactivities (0-1/10, 2-5/10 and >5/10) or specificity groups (citrullination, carbamylation and acetylation; 0-3) and tested for their risk to develop relapses after DMARD tapering. Demographic and disease-specific parameters were included in multivariate logistic regression analysis for defining the role of autoantibodies in predicting relapse. RESULTS: Patients varied in their antimodified protein antibody response with the extremes from recognition of no (0/10) to all antigens (10/10). Antibodies against citrullinated vimentin (51%), acetylated ornithine (46%) and acetylated lysine (37%) were the most frequently observed subspecificities. Relapse risk significantly (p=0.011) increased from 18% (0-1/10 reactivities) to 34% (2-5/10) and 55% (>5/10). With respect to specificity groups (0-3), relapse risk significantly (p=0.021) increased from 18% (no reactivity) to 28%, 36% and finally to 52% with one, two or three antibody specificity groups, respectively. CONCLUSIONS: The data suggest that the pattern of antimodified protein antibody response determines the risk of disease relapse in patients with RA tapering DMARD therapy. TRIAL REGISTRATION NUMBER: 2009-015740-42; Results.

Site-Specific Acetyl Lysine Antibodies Reveal Differential Regulation of Histone Acetylation upon Kinase Inhibition.

Lysine acetylation regulates diverse biological functions for the modified proteins. Mass spectrometry-based proteomic approaches have identified thousands of lysine acetylation sites in cells and tissues. However, functional studies of these acetylation sites were limited by the lack of antibodies recognizing the specific modification sites. Here, we generated 55 site-specific acetyl lysine antibodies for the detection of this modification in cell lysates and evaluated the quality of these antibodies. Based on the immunoblotting analyses, we found that the nature of amino acid sequences adjacent to the modification sites affected the specificity of the site-specific acetyl lysine antibodies. Amino acids with charged, hydrophilic, small, or flexible side chains adjacent to the modification sites increase the likelihood of obtaining high quality site-specific acetyl lysine antibodies. This result may provide valuable insights in fine-tuning the amino acid sequences of the epitopes for the generation of site-specific acetyl lysine antibodies. Using the site-specific acetyl lysine antibodies, we further discovered that acetylation of histone 3 at four lysine residues was differentially regulated by kinase inhibitors. This result demonstrates the potential application of these antibodies in the study of new signaling pathways that lysine acetylation may participate in.