ArticleLiterature Review

Lysine Acetylation Goes Global: From Epigenetics to Metabolism and Therapeutics

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Abstract

Post-translational acetylation of lysine residues has emerged as a key regulatory mechanism in all eukaryotic organisms. Originally discovered in 1963 as a unique modification of histones, acetylation marks are now found on thousands of nonhistone proteins located in virtually every cellular compartment. Here we summarize key findings in the field of protein acetylation over the past 20 years with a focus on recent discoveries in nuclear, cytoplasmic, and mitochondrial compartments. Collectively, these findings have elevated protein acetylation as a major post-translational modification, underscoring its physiological relevance in gene regulation, cell signaling, metabolism, and disease.

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... The affected residues were either in the histone tail domain (K12 in H1F0; K16 and K17 in HIST2H2BB; K12, K13, K16, and K17 in HIST3H2BA; and K6 in HIST1H4A) or in the histone globular domain (K121 in HIST2BC4 and K55, K60, and K168 in HIST3C14) (Fig. 6). Most of these acetylated sites were reported previously (69,70 (transformation/transcription domain-associated protein) (Fig. 6); the latter two being the components of the NuA4 histone acetyltransferase complex (71). Thus, EFV treatment globally increased protein acetylation in the brain, including histone acetylation, an important secondary CYP46A1 activity effect. ...
... However, when multiple proteins in a pathway have altered acetylation, and the acetylation of multiple Lys residues within the same protein is affected, the net effect of acetylation on protein function and pathway is usually difficult to predict (87). An exception is histone acetylation, which weakens the interaction between DNA and histones and generally increases gene transcription (69,70). Histone acetylation is a part of such important processes in the brain as memory formation, consolidation, and synaptic plasticity; yet it is decreased in both aging and AD (98,99). ...
... Of the nonhistones, tubulin α-4A chain was the first acetylated protein identified (69). The effect of its increased acetylation at K40 (2.6-fold, Fig. 2B) is known, increased protein stability, which improves axonal transport, usually impaired in neurodegenerative disorders (100). ...
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Cytochrome P450 46A1 (CYP46A1) is the CNS-specific cholesterol 24-hydroxylase that controls cholesterol elimination and turnover in the brain. In mouse models, pharmacologic CYP46A1 activation with low-dose efavirenz or by gene therapy mitigates the manifestations of various brain disorders, neurologic, and nonneurologic, by affecting numerous, apparently unlinked biological processes. Accordingly, CYP46A1 is emerging as a promising therapeutic target; however, the mechanisms underlying the multiplicity of the brain CYP46A1 activity effects are currently not understood. We proposed the chain reaction hypothesis, according to which CYP46A1 is important for the three primary (unifying) processes in the brain (sterol flux through the plasma membranes, acetyl-CoA, and isoprenoid production), which in turn affect a variety of secondary processes. We already identified several processes secondary to changes in sterol flux and herein undertook a multiomics approach to compare the brain proteome, acetylproteome, and metabolome of 5XFAD mice (an Alzheimer’s disease model), control and treated with low-dose efavirenz. We found that the latter had increased production of phospholipids from the corresponding lysophospholipids and a globally increased protein acetylation (including histone acetylation). Apparently, these effects were secondary to increased acetyl-CoA production. Signaling of small GTPases due to their altered abundance or abundance of their regulators could be affected as well, potentially via isoprenoid biosynthesis. In addition, the omics data related differentially abundant molecules to other biological processes either reported previously or new. Thus, we obtained unbiased mechanistic insights and identified potential players mediating the multiplicity of the CYP46A1 brain effects and further detailed our chain reaction hypothesis.
... Protein acetylation at ε-NH 2 of lysine residues is a common post-translational modification following phosphorylation [8]. Although acetylation was first discovered in histones [9] to regulates gene expression [10], studies in the past decade by using high-throughput technologies and antiacetyllysine antibody in combination with the mass spectrometry based proteomics have revealed that nonhistone proteins are also commonly acetylated in nature. Currently, more than 1500 proteins with thousands of lysine acetylation sites have been identified from bacteria to plants, animals, and humans [10]. ...
... Although acetylation was first discovered in histones [9] to regulates gene expression [10], studies in the past decade by using high-throughput technologies and antiacetyllysine antibody in combination with the mass spectrometry based proteomics have revealed that nonhistone proteins are also commonly acetylated in nature. Currently, more than 1500 proteins with thousands of lysine acetylation sites have been identified from bacteria to plants, animals, and humans [10]. The reversible protein acetylation regulates enzyme activity, protein stability, intracellular compartmentation, cell signaling, inter-molecule interactions and diseases [10][11][12]. ...
... Currently, more than 1500 proteins with thousands of lysine acetylation sites have been identified from bacteria to plants, animals, and humans [10]. The reversible protein acetylation regulates enzyme activity, protein stability, intracellular compartmentation, cell signaling, inter-molecule interactions and diseases [10][11][12]. All glycolytic enzymes except hexokinase are acetylated [13][14][15]. ...
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It is well known that preslaughter (antemortem) stress such as rough handling, transportation, a negative environment, physical discomfort, lack of consistent routine, and bad feed quality has a big impact on meat quality. The antemortem-induced poor meat quality is characterized by low pH, a pale and exudative appearance, and a soft texture. Previous studies indicate that antemortem stress plays a key role in regulating protein acetylation and glycolysis in postmortem (PM) muscle. However, the underlying molecular and biochemical mechanism is not clearly understood yet. In this study, we investigated the relationship between antemortem and protein acetylation and glycolysis using murine longissimus dorsi muscle isolated from ICR mice and murine muscle cell line C2C12 treated with epinephrine hydrochloride. Because adrenaline secretion increases in stressed animals, epinephrine hydrochloride was intraperitoneally injected epinephrine into mice to simulate pre-slaughter stress in this study to facilitate experimental operations and save experimental costs. Our findings demonstrated that protein acetylation in pyruvate kinase M1 (PKM1) form is significantly reduced by antemortem, and the reduced acetylation subsequently leads to an increase in PKM1 enzymatic activity which causes increased glycolysis in PM muscle. By using molecular approaches, we identified lysine 141 in PKM1 as a critical residue for acetylation. Our results in this study provide useful insight for controlling or improving meat quality in the future.
... [6] Since its discovery, many researchers have focused on investigating this PTM and its specific role in health and disease. [7][8][9] This extensive investigation led, after approximately 50 years, to the development of several FDA approved drugs against Kac, such as Vorinostat (2006) and Belinostat (2014) to treat cutaneous T cell lymphoma, and Selisistat (2009) for the treatment of multiple myeloma. [7] High-resolution liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is the gold standard for the investigation and the identification of protein modifications; [10] significant recent advancements in the proteomic and metabolomic fields allowed for the discovery of novel PTMs. ...
... [7][8][9] This extensive investigation led, after approximately 50 years, to the development of several FDA approved drugs against Kac, such as Vorinostat (2006) and Belinostat (2014) to treat cutaneous T cell lymphoma, and Selisistat (2009) for the treatment of multiple myeloma. [7] High-resolution liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is the gold standard for the investigation and the identification of protein modifications; [10] significant recent advancements in the proteomic and metabolomic fields allowed for the discovery of novel PTMs. Particularly, short-chain (SC) lysine acylations are exponentially attracting the scientists' attention (Figures 1 [6,[11][12][13][14][15][16][17][18][19][20][21][22] ). ...
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Lysine crotonylation (Kcr) is a recently discovered post‐translational modification (PTM). Both histone and non‐histone Kcr‐proteins have been associated with numerous diseases including cancer, acute kidney injury, HIV latency, and cardiovascular disease. Histone Kcr enhances gene expression to a larger extend than the extensively studied lysine acetylation (Kac), suggesting Kcr as a novel potential therapeutic target. Although numerous scientific reports on crotonylation were published in the last years, relevant knowledge gaps concerning this PTM and its regulation still remain. To date, only few selective Kcr‐interacting proteins have been identified and selective methods for the enrichment of Kcr‐proteins in chemical proteomics analysis are still lacking. The development of new techniques to study this underexplored PTM could then clarify its function in health and disease and hopefully accelerate the development of new therapeutics for Kcr‐related disease. Herein we briefly review what is known about the regulation mechanisms of Kcr and the current methods used to identify Kcr‐proteins and their interacting partners. This report aims to highlight the significant potential of Kcr as a therapeutic target and to identify the existing scientific gaps that new research must address.
... To address this, we generated a first-of-its-kind high-resolution temporal overview of the abundance of proteins and various PTMs in CBOs spanning 28 time points from day 17 to day 200. We focused on PTMs involved in intracellular signaling and neuronal development (including selected active amino acids), such as phosphorylation, various forms of cysteines, lysine acetylation, and sialylated N-glycosylation [22][23][24][25][26][27][28][29][30][31]. Using quantitative tandem mass tag (TMT)-based proteomics, PTMomics, and metabolomics, we identified and quantified over 9,300 proteins and various PTM-containing peptides, covering more than 60% of the human proteome. ...
... In addition, we quantified 29,679 phosphorylated peptides ( Figure 1E), which is one of the most common PTMs that regulate several intracellular signaling pathways [25][26][27]. We identified and quantified 17,012 peptides with a free cysteine residue ( Figure 1F), in which the thiol group directly or indirectly can regulate protein activity [28], and 1,154 peptides containing lysine acetylation ( Figure 1G), which is mostly known for its roles in gene regulation and cellular metabolism [29][30][31]. We furthermore identified and quantified 396 metabolites in the organoid samples ( Figure 1H). ...
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Cerebral organoids (CBOs) are generated from pluripotent stem cells that undergo neuroectoderm specification and neuronal differentiation in three dimensions. The developing neurons in CBOs migrate and self-organize into cerebral cortex-like layers, mimicking human brain development. CBOs develop according to intrinsic signaling mechanisms and offer unique insights into mechanisms of early human brain development. This process requires coordinated spatiotemporal regulation of protein expression and function, where the latter can be achieved by post-translational modifications (PTMs) on proteins. Despite the importance of proteins in brain development and function, profiling of protein abundance and the involvement of PTMs in CBO development remain underexplored. To gain insight into protein and PTM abundance in CBOs, we performed a high-resolution temporal analysis of CBOs up to day 200 using proteomics, PTMomics and metabolomics. We quantified more than 9,300 proteins and various neurodevelopmentally relevant PTMs (including phosphorylation, lysine acetylation, sialylated N-glycosylation, and cysteine modifications). We demonstrate that protein abundance and dynamic PTMs show significant temporal changes during CBO development related to neuronal differentiation and energy metabolism, whereas calcium signaling is mainly regulated by dynamic PTMs. We further show that synaptic protein content correlated with neurotransmitter levels, and we detected astroglia beyond day 100. Lastly, comparative analysis showed proteomic similarities between CBOs and human fetal brain tissue, supporting the physiological relevance of CBOs. Overall, our study presents a temporal atlas of protein and PTM abundance in CBOs and provides a valuable resource for studying neurodevelopment in neural organoids.
... Acetylation involves the transfer of acetyl groups to lysine residues or the N-terminus of proteins catalyzed by acetyltransferases [153]. Lysine acetylation is facilitated by lysine acetyltransferases (KATs), which transfer acetyl groups from acetyl coenzyme A to the ε-amino group of lysine side chains [154]. Acetylation plays a critical role in regulating protein function, chromatin structure, and gene expression [155,156]. ...
... Acetylation plays a critical role in regulating protein function, chromatin structure, and gene expression [155,156]. Histone acetylation facilitates the dissociation of DNA from the histone octamer and relaxation of nucleosome structure, enabling various transcription factors and co-transcription factors to bind specifically to DNA binding sites and activate gene transcription [154,157]. Moreover, the effects of acetylation on cellular chromatin organization and the activation of nuclear transcriptional factors can have implications for tumor development [158]. ...
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While strides in cancer treatment continue to advance, the enduring challenges posed by cancer metastasis and recurrence persist as formidable contributors to the elevated mortality rates observed in cancer patients. Among the multifaceted factors implicated in tumor recurrence and metastasis, cancer stem cells (CSCs) emerge as noteworthy entities due to their inherent resistance to conventional therapies and heightened invasive capacities. Characterized by their notable abilities for self-renewal, differentiation, and initiation of tumorigenesis, the eradication of CSCs emerges as a paramount objective. Recent investigations increasingly emphasize the pivotal role of post-translational protein modifications (PTMs) in governing the self-renewal and replication capabilities of CSCs. This review accentuates the critical significance of several prevalent PTMs and the intricate interplay of PTM crosstalk in regulating CSC behavior. Furthermore, it posits that the manipulation of PTMs may offer a novel avenue for targeting and eliminating CSC populations, presenting a compelling perspective on cancer therapeutics with substantial potential for future applications.
... Histone acetyltransferases (HATs) refer to a group of enzymes that catalyze the transfer of an acetyl group from acetyl CoA to form ε-N-acetyl lysine in histone proteins; this process is well known to play important roles in chromatin remodeling and gene expression [16,17]. While aberrant expression and activity of HATs have been observed in various human cancers [16,18], the potential role of HATs in CCA is largely unknown. ...
... Histone acetyltransferases (HATs) refer to a group of enzymes that catalyze the transfer of an acetyl group from acetyl CoA to form ε-N-acetyl lysine in histone proteins; this process is well known to play important roles in chromatin remodeling and gene expression [16,17]. While aberrant expression and activity of HATs have been observed in various human cancers [16,18], the potential role of HATs in CCA is largely unknown. ...
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Background: Cholangiocarcinoma (CCA) is a highly malignant cancer of the biliary tract with poor prognosis. Further mechanistic insights into the molecular mechanisms of CCA are needed to develop more effective target therapy. Methods: The expression of the histone lysine acetyltransferase KAT2B in human CCA was analyzed in human CCA tissues. CCA xenograft was developed by inoculation of human CCA cells with or without KAT2B overexpression into SCID mice. Western blotting, ChIP-qPCR, qRT-PCR, protein immunoprecipitation, GST pull-down and RNA-seq were performed to delineate KAT2B mechanisms of action in CCA. Results: We identified KAT2B as a frequently downregulated histone acetyltransferase in human CCA. Downregulation of KAT2B was significantly associated with CCA disease progression and poor prognosis of CCA patients. The reduction of KAT2B expression in human CCA was attributed to gene copy number loss. In experimental systems, we demonstrated that overexpression of KAT2B suppressed CCA cell proliferation and colony formation in vitro and inhibits CCA growth in mice. Mechanistically, forced overexpression of KAT2B enhanced the expression of the tumor suppressor gene NF2, which is independent of its histone acetyltransferase activity. We showed that KAT2B was recruited to the promoter region of the NF2 gene via interaction with the transcription factor SP1, which led to enhanced transcription of the NF2 gene. KAT2B-induced NF2 resulted in subsequent inhibition of YAP activity, as reflected by reduced nuclear accumulation of oncogenic YAP and inhibition of YAP downstream genes. Depletion of NF2 was able to reverse KAT2B-induced reduction of nuclear YAP and subvert KAT2B-induced inhibition of CCA cell growth. Conclusions: This study provides the first evidence for an important tumor inhibitory effect of KAT2B in CCA through regulation of NF2-YAP signaling and suggests that this signaling cascade may be therapeutically targeted for CCA treatment.
... In eukaryotes it is known since the 1960s that acetylation of lysine side chains in the unstructured, flexible N-terminal histone tails affects RNA synthesis 39 . Today it is established that histone acetylation is a major regulator of gene expression 39,40 . Regulation of transcription factors by lysine acetylation might exert analogously important functions in bacteria, i.e. to translate the metabolic state via lysine acetylation to alterations of gene expression patterns by modulating transcription factor activity 41 . ...
... Regulation of transcription factors by lysine acetylation might exert analogously important functions in bacteria, i.e. to translate the metabolic state via lysine acetylation to alterations of gene expression patterns by modulating transcription factor activity 41 . Similar mechanisms were shown for the transcriptional regulators such as CRP, RcsB, and others 40,[42][43][44][45][46][47][48][49][50][51][52][53][54] . Lysine acetylation is a post-translational modification that is tightly connected to the cellular metabolic state 43 . ...
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The Escherichia coli TetR-related transcriptional regulator RutR is involved in the coordination of pyrimidine and purine metabolism. Here we report that lysine acetylation modulates RutR function. Applying the genetic code expansion concept, we produced site-specifically lysine-acetylated RutR proteins. The crystal structure of lysine-acetylated RutR reveals how acetylation switches off RutR-DNA-binding. We apply the genetic code expansion concept in E. coli in vivo revealing the consequences of RutR acetylation on the transcriptional level. We propose a model in which RutR acetylation follows different kinetic profiles either reacting non-enzymatically with acetyl-phosphate or enzymatically catalysed by the lysine acetyltransferases PatZ/YfiQ and YiaC. The NAD⁺-dependent sirtuin deacetylase CobB reverses enzymatic and non-enzymatic acetylation of RutR playing a dual regulatory and detoxifying role. By detecting cellular acetyl-CoA, NAD⁺ and acetyl-phosphate, bacteria apply lysine acetylation of transcriptional regulators to sense the cellular metabolic state directly adjusting gene expression to changing environmental conditions.
... KDACs are separated into four classes: class I (KDACs 1, 2, 3, 8), class II (KACs 4-7, 9, 10), class III (Sirtuins 1-7) and class IV (KDAC11). The enzymatic activity of classes I, II, and IV are Zn2+dependent whereas the class III Sirtuins are nicotinamide adenine dinucleotide (NAD+) -dependent (Ali et al., 2018). Lysine acetylation plays a role in modulating protein function through a variety of ways. ...
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The vitamin D endocrine system is responsible for the regulation of many biological processes including bone metabolism, calcium homeostasis, cell proliferation and cell differentiation. Alterations to the vitamin D signaling pathway are associated with several diseases including bone diseases, diabetes, cardiovascular diseases, autoimmune diseases, and cancer. Vitamin D precursors are obtained through diet or synthesized in the skin and must be further chemically modified to become the biologically active hormone, calcitriol. Calcitriol binds to the vitamin D receptor (VDR), a member of the nuclear hormone receptor (NHR) superfamily. VDR forms a heterodimer with retinoid X receptor (RXR) and together they bind to promoters containing vitamin D response elements (VDREs) to activate transcription of target genes. Other NHRs have been shown to accept post-translational modifications that can either increase or decrease their transcriptional output through alterations in protein-protein or protein-DNA interactions. We have generated evidence that two lysines on VDR may be targets of post-translational modifications, and alterations to lysine deacetylase activity will impact VDR transcriptional output through changes in co-activator and co-repressor binding. Together, these data suggest a novel way for the cell to modulate the response of VDR to available vitamin D.
... N-terminal protein acetylation and methionine oxidation were selected as variable modifications, while carbamidomethylation of cysteine was used as a fixed modification. Peptide identifications and sequence coverage were further enhanced by selecting N-terminal acetylation, one of the most prevalent modifications in both prokaryotic and eukaryotic proteins [24,25]. Peptide FDR was set at 1.0%, and proteins with a −10log 10 (P) ≥ 15.0 were considered significant. ...
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Protein identification in complex biological samples using the shotgun mode of LC-MS/MS is typically enhanced by employing longer LC columns and extended gradient times. However, improved identification rates can also be achieved by optimizing MS acquisition frequencies and employing advanced software, without increasing analysis time, thus maintaining the throughput of the method. To date, we found only one study in the literature examining the influence of MS acquisition frequency on protein identification, specifically using two ion trap mass spectrometer models. This study aims to address the gap by analyzing the impact of MS acquisition tuning of the QTOF instrument on the analysis of complex samples. Our findings indicate that increasing acquisition frequency generally improves protein identification, although the extent of improvement depends on the sample type. For CHO cell lysates, protein identifications increased by over 10%, while E. coli and albumin-depleted plasma samples demonstrated gains of 3.6% and 2.6%, respectively. Higher contributions to protein identification were also achieved with extended LC gradients, resulting in improvements of 21.6% for CHO, 18.2% for E. coli, and 10.3% for plasma. Moreover, enabling PEAKS’ deep learning feature significantly boosted identifications, with increases of 22.9% for CHO, 23.2% for E. coli, and 9.2% for plasma.
... The transcriptional activity and stability of ΔNp63α are regulated by post-translational modifications (PTMs) [19,20]. Lysine acetylation, mediated by lysine acetyltransferases (HATs), has been demonstrated to modulate the expression and activity of ΔNp63α [21,22]. ...
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Non-melanoma skin cancer, including basal and squamous cell carcinoma, is the most common form of cancer worldwide, with approximately 5.4 million new cases diagnosed each year in the United States. While the chemotherapeutic drug cisplatin is often used to treat squamous cell carcinoma (SCC) patients, low response rates and disease recurrence are common. In this study, we show that TIP60 and ΔNp63α levels correlate with cisplatin resistance in SCC cell lines, suggesting that TIP60 contributes to the failure of platinum-based drugs in SCC by regulating the stability and transcriptional activity of ΔNp63α. Depletion of endogenous TIP60 or pharmacological inhibition of TIP60 led to a decrease in ΔNp63α protein and acetylation levels in multiple SCC cell lines. We showed that TIP60 upregulates ΔNp63α protein levels in cisplatin-resistant SCC cell lines by protecting it from cisplatin-mediated degradation and increasing its protein stability. Stable expression of TIP60 or ΔNp63α individually promoted resistance to cisplatin and reduced cell death, while loss of either TIP60 or ΔNp63α induced G2/M arrest, increased cell death, and sensitized cells to cisplatin. Moreover, pharmacological inhibition of TIP60 reduced acetylation of ΔNp63α and sensitized resistant cells to cisplatin. Taken together, our study indicates that TIP60-mediated stabilization of ΔNp63α increases cisplatin resistance and provides critical insights into the mechanisms by which ΔNp63α confers cisplatin resistance by promoting cell proliferation and inhibiting apoptosis. Furthermore, our data suggests that inhibition of TIP60 may be therapeutically advantageous in overcoming cisplatin resistance in SCC and other epithelial cancers.
... Notably, targeting acetylation has emerged as a promising therapeutic strategy for treating metabolic diseases [164]. HDAC inhibitors and small-molecule inhibitors targeting histone acetyltransferases (HATs) are two wellestablished strategies that focus on the acetylation pathway in cancer therapy [165]. ...
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Lipid metabolism reprogramming has emerged as a hallmark of malignant tumors. Lipids represent a complex group of biomolecules that not only compose the essential components of biological membranes and act as an energy source, but also function as messengers to integrate various signaling pathways. In tumor cells, de novo lipogenesis plays a crucial role in acquiring lipids to meet the demands of rapid growth. Increasing evidence has suggested that dysregulated lipid metabolism serves as a driver of cancer progression. Posttranslational modifications (PTMs), which occurs in most eukaryotic proteins throughout their lifetimes, affect the activity, abundance, function, localization, and interactions of target proteins. PTMs of crucial molecules are potential intervention sites and are emerging as promising strategies for the cancer treatment. However, there is limited information available regarding the PTMs that occur in cancer lipid metabolism and the potential treatment strategies associated with these PTMs. Herein, we summarize current knowledge of the roles and regulatory mechanisms of PTMs in lipid metabolism. Understanding the roles of PTMs in lipid metabolism in cancer could provide valuable insights into tumorigenesis and progression. Moreover, targeting PTMs in cancer lipid metabolism might represent a promising novel therapeutic strategy.
... Lysine acetylation is a reversible posttranslational modification (PTM) observed in various proteins in all cellular compartments. It is involved in multiple processes such as energy metabolism, protein degradation, protein localization, and cell cycle regulation, among others (Ali et al., 2018;Shvedunova and Akhtar, 2022). The dynamics of acetylation as a regulator of protein function depend on the combined actions of writer (acetylase), eraser (deacetylase), and reader (bromodomain and YEATS [Yaf9,ENL,AF9,Taf14,Sas5] domain) proteins of this PTM. ...
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Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, affects millions globally, with increasing urban cases outside of Latin America. Treatment is based on two compounds, namely, benznidazole (BZ) and nifurtimox, but chronic cases pose several challenges. Targeting lysine acetylation, particularly bromodomain-containing proteins, shows promise as a novel antiparasitic target. Our research focuses on TcBDF3, a cytoplasmic protein, which is crucial for parasite differentiation that recognizes acetylated alpha-tubulin. In our previous study, A1B4 was identified as a high-affinity binder of TcBDF3, showing significant trypanocidal activity with low host toxicity in vitro. In this report, the binding of TcBDF3 to A1B4 was validated using differential scanning fluorescence, fluorescence polarization, and molecular modeling, confirming its specific interaction. Additionally, two new 1,3,4-oxadiazoles derived from A1B4 were identified, which exhibited improved trypanocide activity and cytotoxicity profiles. Furthermore, TcBDF3 was classified for the first time as an atypical divergent member of the bromodomain extraterminal family found in protists and plants. These results make TcBDF3 a unique target due to its localization and known functions not shared with higher eukaryotes, which holds promise for Chagas disease treatment.
... Protein LysAc involves the transfer of an acetyl group from acetyl-CoA to the ε-amino position of a lysine residue within a protein. This process neutralizes the positive charge of the lysine side chain (Ali et al. 2018). LysAc is catalyzed by lysine Communicated by Gerhard Leubner. ...
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Main conclusion This review focuses on HATs and HDACs that modify non-histone proteins, summarizes functional mechanisms of non-histone acetylation as well as the roles of HATs and HDACs in rice and Arabidopsis. Abstract The growth and development of plants, as well as their responses to biotic and abiotic stresses, are governed by intricate gene and protein regulatory networks, in which epigenetic modifying enzymes play a crucial role. Histone lysine acetylation levels, modulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs), are well-studied in the realm of transcriptional regulation. However, the advent of advanced proteomics has unveiled that non-histone proteins also undergo acetylation, with its underlying mechanisms now being clarified. Indeed, non-histone acetylation influences protein functionality through diverse pathways, such as modulating protein stability, adjusting enzymatic activity, steering subcellular localization, influencing interactions with other post-translational modifications, and managing protein–protein and protein–DNA interactions. This review delves into the recent insights into the functional mechanisms of non-histone acetylation in plants. We also provide a summary of the roles of HATs and HDACs in rice and Arabidopsis, and explore their potential involvement in the regulation of non-histone proteins.
... Conversely, thioreductases remove the oxygen, sulfide, or nitrate groups. Other PTMs can occur via both enzymatic and spontaneous reactions, e.g., lysine acetylation, where acetyltransferases add acyl groups and deacetylases remove them, while acetyl-CoA or acetyl-phosphate can directly acetylate lysines (9). ...
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The microbiome plays a vital role in human health, with changes in its composition impacting various aspects of the body. Posttranslational modification (PTM) regulates protein activity by attaching chemical groups to amino acids in an enzymatic or non-enzymatic manner. PTMs offer fast and dynamic regulation of protein expression and can be influenced by specific dietary components that induce PTM events in gut microbiomes and their hosts. PTMs on microbiome proteins have been found to contribute to host-microbe interactions. For example, in Escherichia coli, S-sulfhydration of tryptophanase regulates uremic toxin production and chronic kidney disease in mice. On a broader microbial scale, the microbiomes of patients with inflammatory bowel disease exhibit distinct PTM patterns in their metaproteomes. Moreover, pathogens and commensals can alter host PTM profiles through protein secretion and diet-regulated metabolic shifts. The emerging field of metaPTMomics focuses on understanding PTM profiles in the microbiota, their association with lifestyle factors like diet, and their functional effects on host-microbe interactions.
... Acetyltransferase catalyzes the transfer of acetyl group from Ac-CoA to lysine residues on substrate proteins, leading to enzymatic acetylation (59). The in vitro acetylation analysis provided evidence that ActA can acetylate PykF, thereby influencing its enzymatic activity. ...
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Dental caries is associated with microbial dysbiosis caused by the excessive proliferation of Streptococcus mutans in dental biofilms, where oxidative stress serves as the major stressor to microbial communities. The adaptability of S. mutans to oxidative stress is a prerequisite for its proliferation and even for exerting its virulence. Protein acetylation is a reversible and conserved regulatory mechanism enabling bacteria to rapidly respond to external environmental stressors. However, the functions of protein acetylation in regulating oxidative stress adaptability of S. mutans are still unknown. Here, we unveil the impact of acetyltransferase ActA-mediated acetylation on regulating the oxidative stress response of S. mutans. actA overexpression increased the sensitivity of S. mutans to hydrogen peroxide and diminished its competitive ability against Streptococcus sanguinis. In contrast, actA deletion enhanced oxidative stress tolerance and competitiveness of S. mutans. The mass spectrometric analysis identified pyruvate kinase (PykF) as a substrate of ActA, with its acetylation impairing its enzymatic activity and reducing pyruvate production. Supplementation with exogenous pyruvate mitigated oxidative stress sensitivity and restored competitiveness in multi-species biofilms. In vitro acetylation analysis further confirmed that ActA directly acetylates PykF, negatively affecting its enzymatic activity. Moreover, 18 potential lysine-acetylated sites on PykF were identified in vitro, which account for 75% of lysine-acetylated sites detected in vivo. Taken together, our study elucidates a novel regulatory mechanism of ActA-mediated acetylation of PykF in modulating oxidative stress adaptability of S. mutans by influencing pyruvate production, providing insights into the importance of protein acetylation in microbial environmental adaptability and interspecies interactions within dental biofilms. IMPORTANCE Dental caries poses a significant challenge to global oral health, driven by microbial dysbiosis within dental biofilms. The pathogenicity of Streptococcus mutans, a major cariogenic bacterium, is closely linked to its ability to adapt to changing environments and cellular stresses. Our investigation into the protein acetylation mechanisms, particularly through the acetyltransferase ActA, reveals a critical pathway by which S. mutans modulates its adaptability to oxidative stress, the dominant stressor within dental biofilms. By elucidating how ActA affects the oxidative stress adaptability and competitiveness of S. mutans through the regulatory axis of ActA-PykF-pyruvate, our findings provide insights into the dynamic interplay between cariogenic and commensal bacteria within dental biofilms. This work emphasizes the significance of protein acetylation in bacterial stress response and competitiveness, opening avenues for the development of novel strategies to maintain oral microbial balance within dental biofilms.
... Histone acetyltransferases (HATs), also known as bromodomains, play a role in the acetylation of lysine residues in histone tails [116]. Two conserved amino acid residues in the binding pocket of the bromodomain, Asn and Tyr, interact to introduce lysine acetate [117]. ...
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Obesity has become a global issue that affects the emergence of various chronic diseases such as diabetes mellitus, dysplasia, heart disorders, and cancer. In this study, an integration method was developed between the metabolite profile of the active compound of Murraya paniculata and the exploration of the targeting mechanism of adipose tissue using network pharmacology, molecular docking, molecular dynamics simulation, and in vitro tests. Network pharmacology results obtained with the skyline query technique using a block-nested loop (BNL) showed that histone acetyltransferase p300 (EP300), peroxisome proliferator-activated receptor gamma (PPARG), and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PPARGC1A) are potential targets for treating obesity. Enrichment analysis of these three proteins revealed their association with obesity, thermogenesis, energy metabolism, adipocytokines, fat cell differentiation, and glucose homeostasis. Metabolite profiling of M. paniculata leaves revealed sixteen active compounds, ten of which were selected for molecular docking based on drug-likeness and ADME results. Molecular docking results between PPARG and EP300 with the ten active compounds showed a binding affinity value of ≤ -5.0 kcal/mol in all dockings, indicating strong binding. The stability of the protein-ligand complex resulting from docking was examined using molecular dynamics simulations, and we observed the best average root mean square deviation (RMSD) of 0.99 Å for PPARG with trans-3-indoleacrylic acid, which was lower than with the native ligand BRL (2.02 Å). Furthermore, the RMSD was 2.70 Å for EP300 and the native ligand 99E, and the lowest RMSD with the ligand (1R,9S)-5-[(E)-2-(4-Chlorophenyl)vinyl]-11-(5-pyrimidinylcarbonyl)-7,11-diazatricyclo[7.3.1.02,7]trideca-2,4-dien-6-one was 3.33 Å. The in vitro tests to validate the potential of M. paniculata in treating obesity showed that there was a significant decrease in PPARG and EP300 gene expressions in 3T3-L1 mature adipocytes treated with M. paniculata ethanolic extract starting at concentrations 62.5 μg/ml and 15.625 μg/ml, respectively. These results indicate that M. paniculata can potentially treat obesity by disrupting adipocyte maturation and influencing intracellular lipid metabolism.
... Acetylation of the ɛ-amino group of lysine (hereafter acetylation) is a major posttranslational modification (PTM) that was first discovered in histones 1 . Proteome-wide analyses reveal that acetylation is a frequently occurring PTM that targets thousands of sites in mammalian cells [2][3][4][5] . Protein acetylation is implicated in the regulation of diverse cellular processes [6][7][8][9] , but the function of acetylation is most extensively studied in the context of histones and gene regulation. ...
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In all eukaryotes, acetylation of histone lysine residues correlates with transcription activation. Whether histone acetylation is a cause or consequence of transcription is debated. One model suggests that transcription promotes the recruitment and/or activation of acetyltransferases, and histone acetylation occurs as a consequence of ongoing transcription. However, the extent to which transcription shapes the global protein acetylation landscapes is not known. Here, we show that global protein acetylation remains virtually unaltered after acute transcription inhibition. Transcription inhibition ablates the co-transcriptionally occurring ubiquitylation of H2BK120 but does not reduce histone acetylation. The combined inhibition of transcription and CBP/p300 further demonstrates that acetyltransferases remain active and continue to acetylate histones independently of transcription. Together, these results show that histone acetylation is not a mere consequence of transcription; acetyltransferase recruitment and activation are uncoupled from the act of transcription, and histone and non-histone protein acetylation are sustained in the absence of ongoing transcription.
... IV enzymes require zinc for catalysis, while sirtuin proteins (Class III) use nicotinamide adenine dinucleotide (NAD + ) as a cofactor (Ali et al. 2018). Class I HDACs are expressed ubiquitously, they are mainly localized into the nucleus and often found in multi-molecular protein complexes -e.g. ...
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Histone deacetylases (HDACs) are enzymes pivotal for histone modification (i.e. acetylation marks removal), chromatin accessibility and gene expression regulation. Class I HDACs (including HDAC1, 2, 3, 8) are ubiquitously expressed and they often participate in multi-molecular protein complexes. To date, three neurodevelopmental disorders caused by mutations in genes encoding for HDACs (HDAC4, HDAC6 and HDAC8) and thus belonging to the group of chromatinopathies, have been described. We performed whole exome sequencing (WES) for a patient (#249) clinically diagnosed with the chromatinopathy Rubinstein-Taybi syndrome (RSTS) but negative for mutations in RSTS genes, identifying a de novo frameshift variant in HDAC2 gene. We then investigated its molecular effects in lymphoblastoid cell lines (LCLs) derived from the patient compared to LCLs from healthy donors (HD). As the variant was predicted to be likely pathogenetic and to affect the sequence of nuclear localization signal, we performed immunocytochemistry and lysates fractionation, observing a nuclear mis-localization of HDAC2 compared to HD LCLs. In addition, HDAC2 total protein abundance resulted altered in patient, and we found that newly identified variant in HDAC2 affects also acetylation levels, with significant difference in acetylation pattern among patient #249, HD and RSTS cells and in expression of a known molecular target. Remarkably, RNA-seq performed on #249, HD and RSTS cells shows differentially expressed genes (DEGs) common to #249 and RSTS. Interestingly, our reported patient was clinically diagnosed with RSTS, a chromatinopathy which known causative genes encode for enzymes antagonizing HDACs. These results support the role of HDAC2 as causative gene for chromatinopathies, strengthening the genotype-phenotype correlations in this relevant group of disorders.
... The reversible protein acetylation may indirectly affect other PTMs through mechanisms like crosstalk between PTMs, proteinprotein interactions, recruitment of modifying enzymes, etc. In short, zinc-dependent HDACs are evolutionally conserved enzymes that epigenetically regulate gene expression in numerous cellular pathways (reviewed in [81,103]). Reversible protein acetylation and other PTMs, like phosphorylation, methylation, ubiquitination, etc., are the core mechanisms regulating protein function [104]. A large body of evidence indicates that the vast majority of signaling cascades involved in cell cycle progression, proliferation, apoptosis, survival, differentiation, development, angiogenesis, and inflammatory actions are regulated by the zinc-dependent HDACs [82,105,106]. ...
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A monolayer of endothelial cells (ECs) lines the lumen of blood vessels and, as such, provides a semi-selective barrier between the blood and the interstitial space. Compromise of the lung EC barrier due to inflammatory or toxic events may result in pulmonary edema, which is a cardinal feature of acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome (ARDS). The EC functions are controlled, at least in part, via epigenetic mechanisms mediated by histone deacetylases (HDACs). Zinc-dependent HDACs represent the largest group of HDACs and are activated by Zn2+. Members of this HDAC group are involved in epigenetic regulation primarily by modifying the structure of chromatin upon removal of acetyl groups from histones. In addition, they can deacetylate many non-histone histone proteins, including those located in extranuclear compartments. Recently, the therapeutic potential of inhibiting zinc-dependent HDACs for EC barrier preservation has gained momentum. However, the role of specific HDAC subtypes in EC barrier regulation remains largely unknown. This review aims to provide an update on the role of zinc-dependent HDACs in endothelial dysfunction and its related diseases. We will broadly focus on biological contributions, signaling pathways and transcriptional roles of HDACs in endothelial pathobiology associated mainly with lung diseases, and we will discuss the potential of their inhibitors for lung injury prevention.
... For instance, lysine acetyltransferases (KATs) are known for adding an acetyl moiety to the epsilon-amino group of lysine residues in histones and other proteins 3,4 . Within KATs families, GCN5related N-acetyltransferases (GNAT), MYST and p300/CBP families are extensively studied in mammals 5,6 . GNAT catalyze the transfer of an acetyl group from acetyl-coenzyme A (Ac-CoA) to various primary amine substrates, including histones and these are the first identified KATs 7 . ...
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Histone acetylation, a crucial epigenetic modification, is governed by histone acetyltransferases (HATs), that regulate many biological processes. Functions of HATs in insects are not well understood. We identified 27 HATs and determined their functions using RNA interference (RNAi) in the model insect, Tribolium castaneum. Among HATs studied, N-alpha-acetyltransferase 40 (NAA40) knockdown caused a severe phenotype of arrested larval development. The steroid hormone, ecdysone induced NAA40 expression through its receptor, EcR (ecdysone receptor). Interestingly, ecdysone-induced NAA40 regulates EcR expression. NAA40 acetylates histone H4 protein, associated with the promoters of ecdysone response genes: EcR, E74, E75, and HR3, and causes an increase in their expression. In the absence of ecdysone and NAA40, histone H4 methylation by arginine methyltransferase 1 (ART1) suppressed the above genes. However, elevated ecdysone levels at the end of the larval period induced NAA40, promoting histone H4 acetylation and increasing the expression of ecdysone response genes. NAA40 is also required for EcR, and steroid-receptor co-activator (SRC) mediated induction of E74, E75, and HR3. These findings highlight the key role of ecdysone-induced NAA40-mediated histone acetylation in the regulation of metamorphosis.
... Nucleophilic reactions of amines to give various amine-modified products are fundamental and important chemical transformations in fields such as synthetic chemistry and naturally occurring metabolic reactions (1,2). Although amine modifications in organic solvents are classical transformations, reactions in aqueous buffers are not facile. ...
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Amine modification through nucleophilic attack of the amine functionality is a very common chemical transformation. Under biorelevant conditions using acidic-to-neutral pH buffer, however, the nucleophilic reaction of alkyl amines (pKa ≈ 10) is not facile due to the generation of ammonium ions lacking nucleophilicity. Here, we disclose a unique molecular transformation system, c atalysis driven by a myloid– s ubstrate comp l ex (CASL), that promotes amine modifications in acidic buffer. Ammonium ions attached to molecules with amyloid-binding capability were activated through deprotonation due to the close proximity to the amyloid catalyst formed by Ac-Asn-Phe-Gly-Ala-Ile-Leu-NH 2 ( NL6 ), derived from islet amyloid polypeptide (IAPP). Under the CASL conditions, alkyl amines underwent various modifications, i.e., acylation, arylation, cyclization, and alkylation, in acidic buffer. Crystallographic analysis and chemical modification studies of the amyloid catalysts suggested that the carbonyl oxygen of the Phe–Gly amide bond of NL6 plays a key role in activating the substrate amine by forming a hydrogen bond. Using CASL, selective conversion of substrates possessing equivalently reactive amine functionalities was achieved in catalytic reactions using amyloids. CASL provides a unique method for applying nucleophilic conversion reactions of amines in diverse fields of chemistry and biology.
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Lysine acetylation plays a crucial role in cellular processes and is found across various evolutionary organisms. Recent advancements in proteomic techniques revealed the presence of acetylation in thousands of non-histone proteins. Here, we conducted extensive meta-analysis of 48 acetylomes spanning diverse organisms, including archaea, bacteria, fungi, protozoa, worms, plants, insects, crustacea, fish, and mammals. Our analyzes revealed a predominance of a single acetylation site in a protein detected in all studied organisms, and proteins heavily acetylated, with >5-10 acetylated-sites, were represented by Hsp70, histone or transcription GTP-biding domain. Moreover, using gene enrichment approaches we found that ATP metabolic processes, glycolysis, aminoacyl-tRNA synthetase pathways and oxidative stress response are among the most acetylated cellular processes. Finally, to better explore the regulatory function of acetylation in glycolysis and oxidative stress we used aldolase and superoxide dismutase A (SODA) enzymes as model. For aldolase, we found that K147 acetylation, responsible to regulate human enzyme, conserved in all phylogenic clade, suggesting that this acetylation might play the same role in other species; while for SODA, we identified many lysine residues in different species present in the tunnel region, which was demonstrated for human and Trypanosoma cruzi, as negative regulator, also suggesting a conserved regulatory mechanism. In conclusion, this study provides insights into the conservation and functional significance of lysine acetylation in different organisms emphasizing its roles in cellular processes, metabolic pathways, and molecular regulation, shedding light in the extensive function of non-histone lysine acetylation.
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We describe new synthetic routes developed toward a range of substituted analogues of bromo and extra-terminal (BET) bromodomain inhibitors I-BET762/JQ1 based on the triazolo-benzodiazepine scaffold. These new routes allow for the derivatization of the methoxyphenyl and chlorophenyl rings, in addition to the diazepine ternary center and the side chain methylene moiety. Substitution at the level of the side chain methylene afforded compounds targeting specifically and potently engineered BET bromodomains designed as part of a bump and hole approach. We further demonstrate that marked selectivity for the second over the first bromodomain can be achieved with an indole derivative that exploits differential interaction with an aspartate/histidine conservative substitution on the BC loop of BET bromodomains.
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BRD4, the most extensively studied member of BET family, is an epigenetic regulator that localizes to DNA via binding acetylated histones and controls the expression of therapeutically important gene regulatory networks through recruiting transcription factors to form mediator complexes, phosphorylating RNA polymerase II and by its intrinsic histone acetyltransferase activity. Disrupting the protein-protein interactions between BRD4 and acetyl-lysine has been shown to effectively block cell proliferation in cancer, cytokine production in acute inflammation, etc. To date, significant efforts have been devoted to the development of BRD4 inhibitors, and consequently, a dozen have progressed into human clinical trials. Herein, we summarize the advances in drug discovery and development of BRD4 inhibitors by focusing on their chemotypes, in vitro and in vivo activity, selectivity, relevant mechanisms of action and therapeutic potential. Opportunities and challenges to achieve selective and efficacious BRD4 inhibitors as a viable therapeutic strategy for human diseases are also highlighted.
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Lysine acetylation has emerged as a dominant post-translational modification (PTM) regulating tau proteins in Alzheimer’s disease (AD) and related tauopathies. Mass spectrometry studies indicate that tau acetylation sites cluster within the microtubule-binding region (MTBR), a region that is highly conserved among tau, MAP2, and MAP4 family members, implying that acetylation could represent a conserved regulatory mechanism for MAPs beyond tau. Here, we combined mass spectrometry, biochemical assays, and cell-based approaches to demonstrate that the tau family members MAP2 and MAP4 are also subject to reversible acetylation. We identify a cluster of lysines in the MAP2 and MAP4 MTBR that undergo CBP-catalyzed acetylation, many of which are conserved in tau. Similar to tau, MAP2 acetylation can occur in a cysteine-dependent auto-regulatory manner in the presence of acetyl-CoA. Furthermore, tubulin reduced MAP2 acetylation, suggesting tubulin binding dictates MAP acetylation status. Taken together, these results uncover a striking conservation of MAP2/Tau family post-translational modifications that could expand our understanding of the dynamic mechanisms regulating microtubules.
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Bromodomains (BRDs) have emerged as compelling targets for cancer therapy. The development of selective and potent BET (bromo and extra-terminal) inhibitors and their significant activity in diverse tumor models have rapidly translated into clinical studies and have motivated drug development efforts targeting non-BET BRDs. However, the complex multidomain/subunit architecture of BRD protein complexes complicates predictions of the consequences of their pharmacological targeting. To address this issue, we developed a promiscuous BRD inhibitor [bromosporine (BSP)] that broadly targets BRDs (including BETs) with nanomolar affinity, creating a tool for the identification of cellular processes and diseases where BRDs have a regulatory function. As a proof of principle, we studied the effects of BSP on leukemic cell lines known to be sensitive to BET inhibition and found, as expected, strong antiproliferative activity. Comparison of the modulation of transcriptional profiles by BSP after a short exposure to the inhibitor resulted in a BET inhibitor signature but no significant additional changes in transcription that could account for inhibition of other BRDs. Thus, nonselective targeting of BRDs identified BETs, but not other BRDs, as master regulators of context-dependent primary transcription response.
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Recognition of histone covalent modifications by 'reader' modules constitutes a major mechanism for epigenetic regulation. A recent upsurge of newly discovered histone lysine acylations, such as crotonylation (Kcr), butyrylation (Kbu), and propionylation (Kpr), greatly expands the coding potential of histone lysine modifications. Here we demonstrate that the histone acetylation-binding double PHD finger (DPF) domains of human MOZ (also known as KAT6A) and DPF2 (also known as BAF45d) accommodate a wide range of histone lysine acylations with the strongest preference for Kcr. Crystal structures of the DPF domain of MOZ in complex with H3K14cr, H3K14bu, and H3K14pr peptides reveal that these non-acetyl acylations are anchored in a hydrophobic 'dead-end' pocket with selectivity for crotonylation arising from intimate encapsulation and an amide-sensing hydrogen bonding network. Immunofluorescence and chromatin immunoprecipitation (ChIP)-quantitative PCR (qPCR) showed that MOZ and H3K14cr colocalize in a DPF-dependent manner. Our studies call attention to a new regulatory mechanism centered on histone crotonylation readout by DPF family members.
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Bromodomains (BRDs) have emerged as compelling targets for cancer therapy. The development of selective and potent BET (bromo and extra-terminal) inhibitors and their significant activity in diverse tumor models have rapidly translated into clinical studies and have motivated drug development efforts targeting non-BET BRDs. However, the complex multidomain/subunit architecture of BRD protein complexes complicates predictions of the consequences of their pharmacological targeting. To address this issue, we developed a promiscuous BRD inhibitor [bromosporine (BSP)] that broadly targets BRDs (including BETs) with nanomolar affinity, creating a tool for the identification of cellular processes and diseases where BRDs have a regulatory function. As a proof of principle, we studied the effects of BSP on leukemic cell lines known to be sensitive to BET inhibition and found, as expected, strong antiproliferative activity. Comparison of the modulation of transcriptional profiles by BSP after a short exposure to the inhibitor resulted in a BET inhibitor signature but no significant additional changes in transcription that could account for inhibition of other BRDs. Thus, nonselective targeting of BRDs identified BETs, but not other BRDs, as master regulators of context-dependent primary transcription response.
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Pygopus 2 (Pygo2) is a chromatin effector that plays an essential role in canonical Wnt signaling associated with development and stem cell growth. Its function is to facilitate histone acetylation by recruitment of histone acetyltransferases (HATs) at active sites of ?-catenin-mediated transcription. In this study, we report that Pygo2 itself, is transiently acetylated when bound to the activated TCF/?-catenin transcription complex, which correlated with ?-catenin binding and Axin2 gene activation. The HATs CBP/p300, but not GCN5/PCAF targeted specific Lysine residues of the N-terminal homology domain of Pygo2 for acetylation. Functional analyses revealed that the presence of CBP and p300 increased association of Pygo2 with GCN5, independent of Pygo2 acetylation status. Finally, while acetylation of Pygo2 had little effect on active ?-catenin complex formation, p300 mediated Pygo2 acetylation resulted in the displacement of Pygo2 from the nucleus to the cytoplasm by targeting specific Lysine residues in the Pygo2 nuclear localization sequence. Together, these findings are consistent with a model in which acetylation of Pygo2 by CBP/p300 family members in the active TCF/?-catenin complex occurs coincident with histone acetylation and may be required for the recycling of Pygo2 complexes away from the complex subsequent to target gene activation.
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Restoration of anti-tumor immunity by blocking PD-L1 signaling through the use of antibodies has proven to be beneficial in cancer therapy. Here, we show that BET bromodomain inhibition suppresses PD-L1 expression and limits tumor progression in ovarian cancer. CD274 (encoding PD-L1) is a direct target of BRD4-mediated gene transcription. In mouse models, treatment with the BET inhibitor JQ1 significantly reduced PD-L1 expression on tumor cells and tumor-associated dendritic cells and macrophages, which correlated with an increase in the activity of anti-tumor cytotoxic T cells. The BET inhibitor limited tumor progression in a cytotoxic T-cell-dependent manner. Together, these data demonstrate a small-molecule approach to block PD-L1 signaling. Given the fact that BET inhibitors have been proven to be safe with manageable reversible toxicity in clinical trials, our findings indicate that pharmacological BET inhibitors represent a treatment strategy for targeting PD-L1 expression.
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Lysine acetylation is an important post-translational modification in cell signaling. In acetylome studies, a high-quality pan-acetyl-lysine antibody is key to successful enrichment of acetylated peptides for subsequent mass spectrometry analysis. Here we show an alternative method to generate polyclonal pan-acetyl-lysine antibodies using a synthesized random library of acetylated peptides as the antigen. Our antibodies are tested to be specific for acetyl-lysine peptides/proteins via ELISA and dot blot. When pooled, five of our antibodies show broad reactivity to acetyl-lysine peptides, complementing a commercial antibody in terms of peptide coverage. The consensus sequence of peptides bound by our antibody cocktail differs slightly from that of the commercial antibody. Lastly, our antibodies are tested in a proof-of-concept to analyze the acetylome of HEK293 cells. In total we identified 1557 acetylated peptides from 416 proteins. We thus demonstrated that our antibodies are well-qualified for acetylome studies and can complement existing commercial antibodies.
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The sixth author’s name is missing a character. The complete, correct name is: David A Sinclair. The correct citation is: Riepsamen A, Wu L, Lau L, Listijono D, Ledger W, Sinclair DA, et al. (2015) Nicotinamide Impairs Entry into and Exit from Meiosis I in Mouse Oocytes. PLoS ONE 10(5): e0126194. doi:10.1371/journal.pone.0126194
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Acetylation of proteins as a post-translational modification is gaining rapid acceptance as a cellular control mechanism on par with other protein modification mechanisms such as phosphorylation and ubiquitination. Through genetic manipulations and evolving proteomic technologies, identification and consequences of transcription factor acetylation is beginning to emerge. In this review, we summarize the field and discuss newly unfolding mechanisms and consequences of transcription factor acetylation in normal and stressed hearts. This article is part of a Special Issue entitled: The role of post-translational protein modifications on heart and vascular metabolism edited by Jason R.B. Dyck & Jan F.C. Glatz.
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N-terminal (Nt) acetylation is known to be a highly abundant co-translational protein modification, but the recent discovery of Golgi- and chloroplast-resident N-terminal acetyltransferases (NATs) revealed that it can also be added post-translationally. Nt-acetylation may act as a degradation signal in a novel branch of the N-end rule pathway, whose functions include the regulation of human blood pressure. Nt-acetylation also modulates protein interactions, targeting, and folding. In plants, Nt-acetylation plays a role in the control of resistance to drought and in regulation of immune responses. Mutations of specific human NATs that decrease their activity can cause either the lethal Ogden syndrome or severe intellectual disability and cardiovascular defects. In sum, recent advances highlight Nt-acetylation as a key factor in many biological pathways.
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Background Neurons are highly polarized cells in which asymmetric axonal-dendritic distribution of proteins is crucial for neuronal function. Loss of polarized distribution of the axonal protein tau is an early sign of Alzheimer’s disease (AD) and other neurodegenerative disorders. The cytoskeletal network in the axon initial segment (AIS) forms a barrier between the axon and the somatodentritic compartment, contributing to axonal retention of tau. Although perturbation of the AIS cytoskeleton has been implicated in neurological disorders, the molecular triggers and functional consequence of AIS perturbation are incompletely understood. Results Here we report that tau acetylation and consequent destabilization of the AIS cytoskeleton promote the somatodendritic mislocalization of tau. AIS cytoskeletal proteins, including ankyrin G and βIV-spectrin, were downregulated in AD brains and negatively correlated with an increase in tau acetylated at K274 and K281. AIS proteins were also diminished in transgenic mice expressing tauK274/281Q, a tau mutant that mimics K274 and K281 acetylation. In primary neuronal cultures, the tauK274/281Q mutant caused hyperdynamic microtubules (MTs) in the AIS, shown by live-imaging of MT mobility and fluorescence recovery after photobleaching. Using photoconvertible tau constructs, we found that axonal tauK274/281Q was missorted into the somatodendritic compartment. Stabilizing MTs with epothilone D to restore the cytoskeletal barrier in the AIS prevented tau mislocalization in primary neuronal cultures. Conclusions Together, these findings demonstrate that tau acetylation contributes to the pathogenesis of neurodegenerative disease by compromising the cytoskeletal sorting machinery in the AIS. Electronic supplementary material The online version of this article (doi:10.1186/s13024-016-0109-0) contains supplementary material, which is available to authorized users.
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Significance Inhibitors of bromodomain and extraterminal domain family proteins (BETi) have generated considerable excitement and are in clinical trials for treatment of several cancers. Cancers treated with targeted therapies eventually become resistant, yet molecular mechanisms underlying resistance to BETi are poorly understood. To discover novel molecular mechanisms mediating resistance to BETi, we performed a shRNA-based genetic screen. We found that loss of tripartite motif-containing protein 33 (TRIM33), a chromatin-associated E3 ubiquitin ligase, confers resistance to BETi. TRIM33 loss diminished BETi-mediated reduction in MYC expression and enhanced TGF-β signaling. Notably, inhibition of TGF-β signaling increased sensitivity of cells to the antiproliferative effects of BETi. In particular, a TGF-β receptor inhibitor potentiated growth suppression by BETi, suggesting a clinically viable strategy for combination therapy.
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Tau proteins are abnormally aggregated in a range of neurodegenerative tauopathies including Alzheimer's disease (AD). Recently, tau has emerged as an extensively post-translationally modified protein, among which lysine acetylation is critical for normal tau function and its pathological aggregation. Here, we demonstrate that tau isoforms have different propensities to undergo lysine acetylation, with auto-acetylation occurring more prominently within the lysine-rich microtubule-binding repeats. Unexpectedly, we identified a unique intrinsic property of tau in which auto-acetylation induces proteolytic tau cleavage, thereby generating distinct N- and C-terminal tau fragments. Supporting a catalytic reaction-based mechanism, mapping and mutagenesis studies showed that tau cysteines, which are required for acetyl group transfer, are also essential for auto-proteolytic tau processing. Further mass spectrometry analysis identified the C-terminal 2nd and 4th microtubule binding repeats as potential sites of auto-cleavage. The identification of acetylation-mediated auto-proteolysis provides a new biochemical mechanism for tau self-regulation and warrants further investigation into whether auto-catalytic functions of tau are implicated in AD and other tauopathies.
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Many histone acetyltransferases undergo autoacetylation, either through chemical or enzymatic means, to potentiate enzymatic cognate substrate lysine acetylation, although the mode and molecular role of such autoacetylation is poorly understood. The MYST family of histone acetyltransferases is autoacetylated at an active site lysine residue to facilitate cognate substrate lysine binding and acetylation. Here, we report on a detailed molecular investigation of K274 autoacetylation of the human MYST protein Males Absent on the First (hMOF). A mutational scan of hMOF K274 reveals that all amino acid substitutions of this residue are able to bind cofactor but are significantly destabilized, both in vitro and in cells, and are catalytically inactive for cognate histone H4 peptide lysine acetylation. The X-ray crystal structure of a hMOF K274P mutant suggests that the reduced stability and catalytic activity stems from a disordering of the residue 274-harboring α2-β7 loop. We also provide structural evidence that a C316S/E350Q mutant, which is defective for cognate substrate lysine acetylation; and biochemical evidence that a K268M mutant, that is defective for K274 chemical acetylation in the context of a K274-peptide, can still undergo quantitative K274 autoacetylation. Together, these studies point to the critical and specific role of hMOF K274 autoacetylation in hMOF stability and cognate substrate acetylation and argues that binding of Ac-CoA to hMOF likely drives K274 autoacetylation for subsequent cognate substrate acetylation.
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Gcn5 is a conserved acetyltransferase that regulates transcription by acetylating the N-terminal tails of histones. Motivated by recent studies identifying a chemically diverse array of lysine acyl modifications in vivo, the acyl-chain specificity of the acetyltransferase human Gcn5 (Gcn5L2) was examined. Whereas Gcn5L2 robustly catalyzes lysine acetylation, the acyltransferase activity of Gcn5L2 becomes progressively weaker with increasing acyl-chain length. To understand how Gcn5 discriminates between different acyl-CoA molecules, structures of the catalytic domain of human Gcn5L2 bound to propionyl-CoA and butyryl-CoA were determined. Although the active site of Gcn5L2 can accommodate propionyl-CoA and butyryl-CoA without major structural rearrangements, butyryl-CoA adopts a conformation incompatible with catalysis that obstructs the path of the incoming lysine residue and acts as a competitive inhibitor of Gcn5L2 versus acetyl-CoA. These structures demonstrate how Gcn5L2 discriminates between acyl-chain donors and explain why Gcn5L2 has weak activity for acyl moieties that are larger than an acetyl group.
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672 Long-term disease-free survival is achieved in over 80% of children with B-cell acute lymphoblastic leukemia (B-ALL) but only 40% of adults. Particular genomic alterations in B-ALL, including rearrangements of MLL and the cytokine receptor subunit CRLF2, confer a poor prognosis in both children and adults. In addition, current therapies for B-ALL are associated with significant short- and long-term toxicities, highlighting the critical need for new therapeutics. The novel compound JQ1 inhibits the BET class of human bromodomain proteins from mediating the assembly of macromolecular protein complexes that are required for transcriptional activation and polymerase elongation. In hematologic and epithelial tumors, JQ1 can downregulate the expression of c-MYC and thereby suppress malignant growth and survival. We investigated the therapeutic potential of JQ1 across multiple genetically-defined subsets of B-ALL. JQ1 potently induced apoptotic cell death (IC50∼30–300 nM) in all B-ALL cell lines (697, CEMO-1 NALM-6, MHH-CALL4, MUTZ-5, Reh, RS4;11, SEMK2) studied. Among the most sensitive lines (IC50<50 nM) were MUTZ-5 and MHH-CALL4, which both harbor IGH@-CRLF2 translocations as well as activating mutations in JAnus Kinase 2 (JAK2). CRLF2 heterodimerizes with the IL7 receptor (IL7R) subunit in response to thymic stromal lymphopoietin, which induces JAK/STAT, MAP kinase and AKT signaling. To identify mechanisms through which JQ1 induces cell death in MUTZ-5 and MHH-CALL4 cells, we quantified transcript and protein levels for relevant targets in the presence of JQ1 500 nM or vehicle (DMSO). Chromatin immunoprecipitation was also performed with antibodies against BRD4 followed by PCR to determine the effects of JQ1 on BRD4 binding at relevant promoters. As previously observed, JQ1 induced the downregulation of MYC mRNA, loss of BRD4 at the MYC promoter, and reduced the expression of c-Myc target genes. Immunoblotting with phospho-specific antibodies demonstrated almost complete loss of JAK2 and STAT5 phosphorylation in cells treated with JQ1, suggesting that JQ1 also blocks signaling downstream of CRLF2/IL7R. While the levels of CRLF2 mRNA were unaffected by JQ1 in MUTZ-5 and MHH-CALL4 cells, JQ1 markedly downregulated IL7R mRNA and depleted BRD4 from the IL7R promoter in both lines. The reduction in IL7R mRNA levels led to dramatic decreases in IL7R surface expression. Genome-wide expression profiling demonstrated a highly restricted effect of JQ1, with IL7R and MYC being the 7th and 23rd most downregulated genes, respectively. In fact, IL7R was the only cytokine receptor in both CRLF2-rearranged B-ALL lines that was significantly downregulated by JQ1 treatment. In addition, JQ1 potently reduced IL7R mRNA across other B-ALL cell lines with diverse cytogenetics. To determine whether JQ1 could suppress the growth of human B-ALL in vivo, we xenografted a human CRLF2-rearranged B-ALL primary sample into Nod.SCID.IL2RG−/− mice. Upon the development of >30% bone marrow involvement by human CD45+/CRLF2+ B-ALL cells, mice were randomized to receive JQ1 (50mg/kg intraperitoneally daily) or vehicle (DMSO). After 5 days of treatment, sentinel mice were sacrificed for pharmacodynamic endpoints. Spleens from mice treated with JQ1 had markedly reduced c-Myc expression and STAT5 phosphorylation compared with spleens from vehicle-treated mice. In survival cohorts (n=9 per arm), treatment with JQ1 significantly prolonged overall survival (p=0.0002) compared with vehicle. These results demonstrate that BET bromodomain inhibition is a promising therapeutic strategy for patients with B-ALL, including subsets with high-risk cytogenetics. Moreover, the surprising finding that JQ1 also targets IL7R expression suggests that bromodomain inhibitors may be especially useful in malignant and nonmalignant disorders dependent on IL7R. Disclosures Bradner: Tensha Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Acetylon Pharmaceuticals: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; SHAPE Pharmaceuticals: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Weinstock:Novartis: Consultancy, Research Funding.
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Transcription activation involves RNA polymerase II (Pol II) recruitment and release from the promoter into productive elongation, but how specific chromatin regulators control these steps is unclear. Here, we identify a novel activity of the histone acetyltransferase p300/CREB-binding protein (CBP) in regulating promoter-proximal paused Pol II. We find that Drosophila CBP inhibition results in "dribbling" of Pol II from the pause site to positions further downstream but impedes transcription through the +1 nucleosome genome-wide. Promoters strongly occupied by CBP and GAGA factor have high levels of paused Pol II, a unique chromatin signature, and are highly expressed regardless of cell type. Interestingly, CBP activity is rate limiting for Pol II recruitment to these highly paused promoters through an interaction with TFIIB but for transit into elongation by histone acetylation at other genes. Thus, CBP directly stimulates both Pol II recruitment and the ability to traverse the first nucleosome, thereby promoting transcription of most genes.
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Discovered in the beginning of the 20(th) century, nicotinamide adenine dinucleotide (NAD(+)) has evolved from a simple oxidoreductase cofactor to being an essential cosubstrate for a wide range of regulatory proteins that include the sirtuin family of NAD(+)-dependent protein deacylases, widely recognized regulators of metabolic function and longevity. Altered NAD(+) metabolism is associated with aging and many pathological conditions, such as metabolic diseases and disorders of the muscular and neuronal systems. Conversely, increased NAD(+) levels have shown to be beneficial in a broad spectrum of diseases. Here, we review the fundamental aspects of NAD(+) biochemistry and metabolism and discuss how boosting NAD(+) content can help ameliorate mitochondrial homeostasis and as such improve healthspan and lifespan.
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The transcription factor signal transducer and activator of transcription (STAT) 3 is activated downstream of cytokines, growth factors and oncogenes to mediate their functions under both physiological and pathological conditions. In particular, aberrant/unrestrained STAT3 activity is detected in a wide variety of tumors, driving multiple pro-oncogenic functions. For that, STAT3 is widely considered as an oncogene and is the object of intense translational studies. One of the distinctive features of this factor is however, its ability to elicit different and sometimes contrasting effects under different conditions. In particular, STAT3 activities have been shown to be either pro-oncogenic or tumor-suppressive according to the tumor aetiology/mutational landscape, suggesting that the molecular bases underlining its functions are still incompletely understood. Here we discuss some of the properties that may provide the bases to explain STAT3 heterogeneous functions, and in particular how post-translational modifications contribute shaping its sub-cellular localization and activities, the cross talk between these activities and cell metabolic conditions, and finally how its functions can control the behaviour of both tumor and tumor microenvironment cell populations.
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Cancer cells are characterized by aberrant epigenetic landscapes and often exploit chromatin machinery to activate oncogenic gene expression programs. Recognition of modified histones by 'reader' proteins constitutes a key mechanism underlying these processes; therefore, targeting such pathways holds clinical promise, as exemplified by the development of bromodomain and extra-terminal (BET) inhibitors. We recently identified the YEATS domain as an acetyl-lysine-binding module, but its functional importance in human cancer remains unknown. Here we show that the YEATS domain-containing protein ENL, but not its paralogue AF9, is required for disease maintenance in acute myeloid leukaemia. CRISPR-Cas9-mediated depletion of ENL led to anti-leukaemic effects, including increased terminal myeloid differentiation and suppression of leukaemia growth in vitro and in vivo. Biochemical and crystal structural studies and chromatin-immunoprecipitation followed by sequencing analyses revealed that ENL binds to acetylated histone H3, and co-localizes with H3K27ac and H3K9ac on the promoters of actively transcribed genes that are essential for leukaemia. Disrupting the interaction between the YEATS domain and histone acetylation via structure-based mutagenesis reduced the recruitment of RNA polymerase II to ENL-target genes, leading to the suppression of oncogenic gene expression programs. Notably, disrupting the functionality of ENL further sensitized leukaemia cells to BET inhibitors. Together, our data identify ENL as a histone acetylation reader that regulates oncogenic transcriptional programs in acute myeloid leukaemia, and suggest that displacement of ENL from chromatin may be a promising epigenetic therapy, alone or in combination with BET inhibitors, for aggressive leukaemia.
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Growing evidence suggests that changes in histone acetylation in specific sites of the chromatin modulate neuronal plasticity and contribute to antidepressant-like action. Sirtuin 2 (SIRT2) is a class III NAD⁺-dependent histone deacetylase involved in transcriptional repression of genes regulating synaptic plasticity. Importantly, a key role for the glutamate system in prefrontal cortex (PFC) synaptic plasticity changes induced by antidepressants has been suggested. Here, we asked whether SIRT2 could be a pharmacological target for depression therapy. The compound 2-{3-(3-fluorophenethyloxy)phenylamino}benzamide (33i), a selective SIRT2 inhibitor in vitro, was studied in mice (C57Bl6). Firstly, the inhibitory effect of subchronic 33i (5–15 mg/kg, 10 days) on SIRT2 activity in the PFC was evaluated. Moreover, the effect of SIRT2 inhibition on the expression of synaptic plasticity markers linked to glutamate neurotransmission (VGLUT1, synaptophysin, mGluR4, GluA1, GluN2B, GluN2A) and on serotonin levels was studied. Further, neurochemical and behavioral effects of chronic (5 weeks) 33i (15 mg/kg) on the chronic mild stress (CMS) model were analyzed. Subchronic 33i inhibited SIRT2, increased GluN2A, GluN2B and serotonin levels in the PFC. Moreover, chronic 33i reverted CMS-induced anhedonia and social avoidance. Moreover, 33i upregulated postsynaptic GluN2B and phosphorylated form of GluA1 (p-GluA1), suggesting that SIRT2 inhibition enhance synaptic strength. Yet, CMS also increased both GluN2A and GluN2B in the postsynaptic fraction. These results suggest that Sirt2 inhibition induce antidepressant-like action and this effect could be mediated by modulation of glutamate and serotonin system in the PFC. Moreover, it highlights the therapeutic potential of SIRT2 inhibitors as new antidepressant agents.
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Resveratrol (Resv), a natural polyphenol, is suggested to have various health benefits including improved insulin sensitivity. Resv activates Sirtuin (Sirt1) in several species and tissues. Sirt1 is a protein deacetylase with an important role in ageing, metabolism and β-cell function. In insulinoma β-cells (INS-1E), Resv is previously shown to improve glucose-stimulated insulin secretion in a Sirt1-dependent mechanism and to protect against β-cell dedifferentiation in non-human primates, while inducing hypertrophy in myoblasts. Mammalian (mechanistic) Target of Rapamycin (mTOR), is a key regulator of cellular metabolism and regulates the cell size, β-cell survival and proliferation.
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Pathogenesis in tauopathies involves the accumulation of tau in the brain and progressive synapse loss accompanied by cognitive decline. Pathological tau is found at synapses, and it promotes synaptic dysfunction and memory deficits. The specific role of toxic tau in disrupting the molecular networks that regulate synaptic strength has been elusive. A novel mechanistic link between tau toxicity and synaptic plasticity involves the acetylation of two lysines on tau, K274, and K281, which are associated with dementia in Alzheimer's disease (AD). We propose that an increase in tau acetylated on these lysines blocks the expression of long-term potentiation at hippocampal synapses leading to impaired memory in AD. Acetylated tau could inhibit the activity-dependent recruitment of postsynaptic AMPA-type glutamate receptors required for plasticity by interfering with the postsynaptic localization of KIBRA, a memory-associated protein. Strategies that reduce the acetylation of tau may lead to effective treatments for cognitive decline in AD.