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The cell surface of Toxoplasma gondii is rich in glycoconjugates which hold diverse and vital functions in the lytic cycle of this obligate intracellular parasite. Additionally, the cyst wall of bradyzoites, that shields the persistent form responsible for chronic infection from the immune system, is heavily glycosylated. Formation of glycoconjugates relies on activated sugar nucleotides, such as uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). The glucosamine-phosphate-N-acetyltransferase (GNA1) generates N-acetylglucosamine-6-phosphate critical to produce UDP-GlcNAc. Here, we demonstrate that downregulation of T. gondii GNA1 results in a severe reduction of UDP-GlcNAc and a concomitant drop in glycosylphosphatidylinositols (GPIs), leading to impairment of the parasite's ability to invade and replicate in the host cell. Surprisingly, attempts to rescue this defect through exogenous GlcNAc supplementation fail to completely restore these vital functions. In depth metabolomic analyses elucidate diverse causes underlying the failed rescue: utilization of GlcNAc is inefficient under glucose-replete conditions and fails to restore UDP-GlcNAc levels in GNA1-depleted parasites. In contrast, GlcNAc-supplementation under glucose-deplete conditions fully restores UDP-GlcNAc levels but fails to rescue the defects associated with GNA1 depletion. Our results underscore the importance of glucosamine-6-phosphate acetylation in governing T. gondii replication and invasion and highlight the potential of the evolutionary divergent GNA1 in Apicomplexa as a target for the development of much-needed new therapeutic strategies. Author summary: Toxoplasma gondii, Plasmodium, and Cryptosporidium spp., pose serious threats to human health. T. gondii, an intracellular and opportunistic pathogen, effectively avoids the host immune defences by forming long-lasting tissue cysts. Finding potent drugs to eliminate these persisting parasites remains a challenge. The glucosamine-phosphate-N-acetyltransferase (GNA1) catalyses a critical key step in the production of activated sugar nucleotides to build glycoconjugates essential for various functions in the cell. In P. falciparum, this enzyme has been identified as a potential target for antimalarial drugs. In this study, we explored the importance of this pathway in T. gondii and discovered that these sugar-containing compounds play a vital role in the parasite's ability to invade and replicate in host cells–crucial processes for its survival and ability to cause disease. Intriguingly, unlike some organisms that can bypass the pathway, T. gondii relies critically on glucosamine-6-phosphate acetylation. This reliance sheds light on the parasite's distinct metabolic properties and highlights the pathway's potential as a target for new therapeutic strategies. [ABSTRACT FROM AUTHOR]

Post-transcriptional regulation by small RNAs and post-translational modifications (PTM) such as lysine acetylation play fundamental roles in physiological circuits, offering rapid responses to environmental signals with low energy consumption. Yet, the interplay between these regulatory systems remains underexplored. Here, we unveil the cross-talk between sRNAs and lysine acetylation in Streptococcus mutans, a primary cariogenic pathogen known for its potent acidogenic virulence. Through systematic overexpression of sRNAs in S. mutans, we identified sRNA SmsR1 as a critical player in modulating acidogenicity, a key cariogenic virulence feature in S. mutans. Furthermore, combined with the analysis of predicted target mRNA and transcriptome results, potential target genes were identified and experimentally verified. A direct interaction between SmsR1 and 5'-UTR region of pdhC gene was determined by in vitro binding assays. Importantly, we found that overexpression of SmsR1 reduced the expression of pdhC mRNA and increased the intracellular concentration of acetyl-CoA, resulting in global changes in protein acetylation levels. This was verified by acetyl-proteomics in S. mutans, along with an increase in acetylation level and decreased activity of LDH. Our study unravels a novel regulatory paradigm where sRNA bridges post-transcriptional regulation with post-translational modification, underscoring bacterial adeptness in fine-tuning responses to environmental stress. Author summary: Post-transcriptional regulation and post-translational modification are widely present in physiological circuits due to their low energy consumption and rapid response to the environment. sRNA is an important post-transcriptional regulatory factor in bacterial response to environmental stress, and bacteria also use sRNA as a key signal to control multicellular behaviors such as biofilm formation and quorum sensing to regulate virulence. S. mutans is an intractable cariogenic pathogen, but the role of sRNA in S. mutans is poorly understood. Here, we elucidated the virulence regulation and mechanism of sRNA SmsR1 in S. mutans by means of transcriptomics, acetyl-proteomics and animal models, and found its relationship with protein acetylation (post-translational modification), and a new role of sRNA physiological regulatory network was shown. This study presents an innovative perspective that a sRNA in S. mutans is involved in the correlation between post-transcriptional regulation and post-translational modification. [ABSTRACT FROM AUTHOR]

Ferroptosis, a defensive strategy commonly employed by the host cells to restrict pathogenic infections, has been implicated in the development and therapeutic responses of various types of cancer. However, the role of ferroptosis in oncogenic Kaposi's sarcoma-associated herpesvirus (KSHV)-induced cancers remains elusive. While a growing number of non-histone proteins have been identified as acetylation targets, the functions of these modifications have yet to be revealed. Here, we show KSHV reprogramming of host acetylation proteomics following cellular transformation of rat primary mesenchymal precursor. Among them, SERPINE1 mRNA binding protein 1 (SERBP1) deacetylation is increased and required for KSHV-induced cellular transformation. Mechanistically, KSHV-encoded viral interleukin-6 (vIL-6) promotes SIRT3 deacetylation of SERBP1, preventing its binding to and protection of lipoyltransferase 2 (Lipt2) mRNA from mRNA degradation resulting in ferroptosis. Consequently, a SIRT3-specific inhibitor, 3-TYP, suppresses KSHV-induced cellular transformation by inducing ferroptosis. Our findings unveil novel roles of vIL-6 and SERBP1 deacetylation in regulating ferroptosis and KSHV-induced cellular transformation, and establish the vIL-6-SIRT3-SERBP1-ferroptosis pathways as a potential new therapeutic target for KSHV-associated cancers. Author summary: Ferroptosis has been implicated in viral infections and the development of cancers, however, its role in oncogenic Kaposi's sarcoma-associated herpesvirus (KSHV)-induced cancers is still unknown. Although abnormal protein lysine acetylation modification has been strongly implicated in tumorigenesis, the exact role of this modification in KSHV-induced cell transformation remains elusive. Our study uncovers the reduction of acetylation of SERPINE1 mRNA binding protein 1 (SERBP1), which is required for KSHV-induced cellular transformation. KSHV-encoded viral interleukin-6 (vIL-6) promotes SIRT3 deacetylation of SERBP1, preventing its binding to and protection of lipoyltransferase 2 (Lipt2) mRNA from mRNA degradation resulting in ferroptosis. A SIRT3-specific inhibitor, 3-TYP, induces ferroptosis and inhibits survival of KSHV-transformed cells. These findings define a novel mechanism contributing to KSHV-induced cellular transformation and provide a rationale for therapeutically targeting lysine acetylation and the associated enzyme for infectious diseases. [ABSTRACT FROM AUTHOR]

Acetylation of lysine residues is an important and common post-translational regulatory mechanism occurring on thousands of non-histone proteins. Lysine deacetylases (KDACs or HDACs) are a family of enzymes responsible for removing acetylation. To identify the biological mechanisms regulated by individual KDACs, we created HT1080 cell lines containing chromosomal point mutations, which endogenously express either KDAC6 or KDAC8 having single inactivated catalytic domain. Engineered HT1080 cells expressing inactive KDA6 or KDAC8 domains remained viable and exhibited enhanced acetylation on known substrate proteins. RNA-seq analysis revealed that many changes in gene expression were observed when KDACs were inactivated, and that these gene sets differed significantly from knockdown and knockout cell lines. Using GO ontology, we identified several critical biological processes associated specifically with catalytic activity and others attributable to non-catalytic interactions. Treatment of wild-type cells with KDAC-specific inhibitors Tubastatin A and PCI-34051 resulted in gene expression changes distinct from those of the engineered cell lines, validating this approach as a tool for evaluating in-cell inhibitor specificity and identifying off-target effects of KDAC inhibitors. Probing the functions of specific KDAC domains using these cell lines is not equivalent to doing so using previously existing methods and provides novel insight into the catalytic functions of individual KDACs by investigating the molecular and cellular changes upon genetic inactivation. [ABSTRACT FROM AUTHOR]

Arylamine N-acetyltransferase 2 has been related to drug side effects and cancer susceptibility; its protein structure and acetylation capacity results from the polymorphism's arrays on the NAT2 gene. Absorption, distribution, metabolism, and excretion, cornerstones of the pharmacological effects, have shown diversity patterns across populations, ethnic groups, and even interethnic variation. Although the 1000 Genomes Project database has portrayed the global diversity of the NAT2 polymorphisms, several populations and ethnicities remain underrepresented, limiting the comprehensive picture of its variation. The NAT2 clinical entails require a detailed landscape of its striking diversity. This systematic review spans the genetic and acetylation patterns from 164 articles from October 1992 to October 2020. Descriptive studies and controls from observational studies expanded the NAT2 diversity landscape. Our study included 243 different populations and 101 ethnic minorities, and, for the first time, we presented the global patterns in the Middle Eastern populations. Europeans, including its derived populations, and East Asians have been the most studied genetic backgrounds. Contrary to the popular perception, Africans, Latinos and Native Americans have been significantly represented in recent years. NAT2*4, *5B, and *6A were the most frequent haplotypes globally. Nonetheless, the distribution of *5B and *7B were less and more frequent in Asians, respectively. Regarding the acetylator status, East Asians and Native Americans harboured the highest frequencies of the fast phenotype, followed by South Europeans. Central Asia, the Middle East, and West European populations were the major carriers of the slow acetylator status. The detailed panorama presented herein, expands the knowledge about the diversity patterns to genetic and acetylation levels. These data could help clarify the controversial findings between acetylator states and the susceptibility to diseases and reinforce the utility of NAT2 in precision medicine. [ABSTRACT FROM AUTHOR]

Posttranslational modification of tubulin increases the dynamic complexity and functional diversity of microtubules. Acetylation of α-tubulin at Lys-40 is a highly conserved posttranslational modification that has been shown to improve the flexibility and resilience of microtubules. Here we studied the in vivo functions of α-tubulin acetylation by knocking-out Atat, the Drosophila α-tubulin acetyltransferase, and by mutating Lys-40 to Arg in α1-tubulin. We found a reduction in the dendritic arborization of larval class I dendritic arborization (da) neurons in both mutants. The dendritic developmental defects in atat mutants could be reversed by enhancing the stability of microtubules either through knocking down the microtubule severing protein Katanin 60 or through overexpressing tubulin-specific chaperone E, suggesting that α-tubulin deacetylation impairsed dendritic morphology by decreasing the stability of microtubules. Using time-lapse recordings, we found that atat and α1-tubulinK40R mutations dramatically increased the number of dendritic protrusions that were likely to be immature dendritic precursors. Finally, we showed that both Atat and α-tubulin acetylation were required in class I da neurons to control larval locomotion. These findings add novel insight into the current knowledge of the role of α-tubulin acetylation in regulating neuronal development and functions. [ABSTRACT FROM AUTHOR]

Increasing evidence highlights the role of bacteria in promoting tumorigenesis. The underlying mechanisms may be diverse and remain poorly understood. Here, we report that Salmonella infection leads to extensive de/acetylation changes in host cell proteins. The acetylation of mammalian cell division cycle 42 (CDC42), a member of the Rho family of GTPases involved in many crucial signaling pathways in cancer cells, is drastically reduced after bacterial infection. CDC42 is deacetylated by SIRT2 and acetylated by p300/CBP. Non-acetylated CDC42 at lysine 153 shows an impaired binding of its downstream effector PAK4 and an attenuated phosphorylation of p38 and JNK, consequently reduces cell apoptosis. The reduction in K153 acetylation also enhances the migration and invasion ability of colon cancer cells. The low level of K153 acetylation in patients with colorectal cancer (CRC) predicts a poor prognosis. Taken together, our findings suggest a new mechanism of bacterial infection-induced promotion of colorectal tumorigenesis by modulation of the CDC42-PAK axis through manipulation of CDC42 acetylation. Author summary: Protein acetylation plays an important role in regulating various aspects of cell life. In this study, we show that Salmonella infection disturbs acetylation of more than 90 proteins, including Rho GTPase cell division cycle 42 (CDC42). CDC42 is involved in many crucial signaling pathways in cancer cells. We find that CDC42 K153 can be deacetylated by the NAD+-dependent deacetylase SIRT2 after Salmonella infection, which causes an impaired binding of its downstream effector PAK4. Low acetylation level of CDC42 K153 is crucial for tumor cell migration, invasion and apoptosis. Furthermore, lower K153 acetylation in tumor tissues compared to adjacent normal tissues of colorectal cancer (CRC) patients is correlated to the poor prognosis of CRC. Collectively, the information derived from this study suggests that bacterial infection could promote CRC tumorigenesis by modulating CDC42 acetylation. [ABSTRACT FROM AUTHOR]

Microtubule acetylation is found in populations of stable, long-lived microtubules, occurring on the conserved lysine 40 (K40) residue of α-tubulin by α-tubulin acetyltransferases (αTATs). α-tubulin K40 acetylation has been shown to stabilize microtubules via enhancing microtubule resilience against mechanical stress. Here we show that a previously uncharacterized αTAT, Drosophila CG17003/leaky (lky), is required for α-tubulin K40 acetylation in early germ cells in Drosophila ovary. We found that loss of lky resulted in a progressive egg chamber fusion phenotype accompanied with mislocalization of germline-specific Vasa protein in somatic follicle cells. The same phenotype was observed upon replacement of endogenous α-tubulin84B with non-acetylatable α-tubulin84BK40A, suggesting α-tubulin K40 acetylation is responsible for the phenotype. Chemical disturbance of microtubules by Colcemid treatment resulted in a mislocalization of Vasa in follicle cells within a short period of time (~30 min), suggesting that the observed mislocalization is likely caused by direct leakage of cellular contents between germline and follicle cells. Taken together, this study provides a new function of α-tubulin acetylation in maintaining the cellular identity possibly by preventing the leakage of tissue-specific gene products between juxtaposing distinct cell types. [ABSTRACT FROM AUTHOR]

The dynamics of neuronal microtubules are essential for brain plasticity. Vesicular transport and synaptic transmission, additionally, requires acetylation of α-tubulin, and aberrant tubulin acetylation and neurobiological deficits are associated. Prolonged exposure to a stressor or consumption of drugs of abuse, like marihuana, lead to neurological changes and psychotic disorders. Here, we studied the effect of psychosocial stress and the administration of cannabinoid receptor type 1 drugs on α-tubulin acetylation in different brain regions of mice. We found significantly decreased tubulin acetylation in the prefrontal cortex in stressed mice. The impact of cannabinoid drugs on stress-induced microtubule disturbance was investigated by administration of the cannabinoid receptor agonist WIN55,212–2 and/or antagonist rimonabant. In both, control and stressed mice, the administration of WIN55,212–2 slightly increased the tubulin acetylation in the prefrontal cortex whereas administration of rimonabant acted antagonistically indicating a cannabinoid receptor type 1 mediated effect. The analysis of gene expression in the prefrontal cortex showed a consistent expression of ApoE attributable to either psychosocial stress or administration of the cannabinoid agonist. Additionally, ApoE expression inversely correlated with acetylated tubulin levels when comparing controls and stressed mice treated with WIN55,212–2 whereas rimonabant treatment showed the opposite. [ABSTRACT FROM AUTHOR]

We previously found that, among human parainfluenza virus type 3 (HPIV3) proteins, the interaction of nucleoprotein (N) and phosphoprotein (P) provides the minimal requirement for the formation of cytoplasmic inclusion bodies (IBs), which are sites of RNA synthesis, and that acetylated α-tubulin enhances IB fusion and viral replication. In this study, using immunoprecipitation and mass spectrometry assays, we determined that vimentin (VIM) specifically interacted with the N–P complex of HPIV3, and that the head domain of VIM was responsible for this interaction, contributing to the inhibition of IB fusion and viral replication. Furthermore, we found that VIM promoted the degradation of α-tubulin acetyltransferase 1 (α-TAT1), through its head region, thereby inhibiting the acetylation of α-tubulin, IB fusion, and viral replication. In addition, we identified a 20-amino-acid peptide derived from the head region of VIM that participated in the interaction with the N–P complex and inhibited viral replication. Our findings suggest that VIM inhibits the formation of HPIV3 IBs by downregulating α-tubulin acetylation via enhancing the degradation of α-TAT1. Our work sheds light on a new mechanism by which VIM suppresses HPIV3 replication. Author summary: Human parainfluenza virus type 3 (HPIV3) is one of the most common pathogenic respiratory RNA viruses, and its replication sites, known as inclusion bodies (IBs), which are formed by nucleoprotein (N) and phosphoprotein (P), are critical in viral RNA synthesis. Exploration of the mechanisms by which viral inclusion bodies are formed in cells may help to identify new antiviral approaches. Our aim was to identify cellular proteins with regulatory effects on the formation of viral IBs. The formation of HPIV3 IBs relies on acetylated α-tubulin, and we found that VIM negatively regulated α-tubulin acetylation by enhancing the degradation of α-TAT1, thereby suppressing the fusion and maturation of viral IBs. Based on the interaction between IBs and VIM, the peptide derived from amino-acid residues 61 to 80 in the VIM head region exerted antiviral effects, providing a new direction for our antiviral study. [ABSTRACT FROM AUTHOR]

Stu2 in S. cerevisiae is a member of the XMAP215/Dis1/CKAP5/ch-TOG family of MAPs and has multiple functions in controlling microtubules, including microtubule polymerization, microtubule depolymerization, linking chromosomes to the kinetochore, and assembly of γ-TuSCs at the SPB. Whereas phosphorylation has been shown to be critical for Stu2 localization at the kinetochore, other regulatory mechanisms that control Stu2 function are still poorly understood. Here, we show that a novel form of Stu2 regulation occurs through the acetylation of three lysine residues at K252, K469, and K870, which are located in three distinct domains of Stu2. Alteration of acetylation through acetyl-mimetic and acetyl-blocking mutations did not impact the essential function of Stu2. Instead, these mutations lead to a decrease in chromosome stability, as well as changes in resistance to the microtubule depolymerization drug, benomyl. In agreement with our in silico modeling, several acetylation-mimetic mutants displayed increased interactions with γ-tubulin. Taken together, these data suggest that Stu2 acetylation can govern multiple Stu2 functions, including chromosome stability and interactions at the SPB. Author summary: Microtubules are proteinaceus polymers that play several important roles in cell division and segregation of the genetic material to each daughter cell. The functions of microtubules are critically dependent upon their dynamic properties in which tubulin subunits are added or removed from the microtubule end, allowing microtubules to grow or shorten in length. These dynamic properties are controlled by several types of microtubule associated proteins. In this study using bakers yeast, we describe our discovery of a previously unappreciated way to regulate the microtubule associated protein Stu2 by a modification called acetylation. When we created mutations in the Stu2 protein that can't be properly acetylated, the cell lost some of its chromosomes. Some of these mutations actually caused the microtubules to be resistant to drugs that normally disassemble the microtubule polymer. As similar versions of the Stu2 protein are found in diverse organisms that range from yeast and fungus, to plants, insects, mammals and humans, our work could provide unique insights into how microtubules malfunction in some human diseases. With further studies, this may provide a new understanding of chromosome loss in birth defects and/or cancer. [ABSTRACT FROM AUTHOR]

Klebsiella pneumoniae, an emerging multidrug-resistant pathogen, exhibits hypermucoviscosity (HMV) as a critical virulence trait mediated by its capsular polysaccharide (CPS). Recent discoveries have determined acetylation as a significant modification for CPS, although its impact on HMV and virulence was previously unknown. This study elucidates the roles of two enzymes: Klebsiella pneumoniae Acetylated CPS Esterase (KpACE), an esterase that removes acetyl groups from CPS, and WcsU, an acetyltransferase that adds acetyl groups to CPS. KpACE is highly upregulated in an ompR-deficient mutant lacking HMV, and its overexpression consistently reduces HMV and diminishes virulence in a mouse model of pneumonia. The esterase domain-containing KpACE effectively deacetylates model sugar substrates and CPS-K2. Site-directed mutagenesis of the conserved catalytic histidine residue at position 370 significantly reduces its enzymatic activity. This reduction correlates with decreased HMV, affecting key virulence traits including biofilm formation and serum resistance. Similarly, a deficiency in the wcsU gene abolishes CPS acetylation, and reduces HMV and virulence. These results highlight the importance of the delicate balance between CPS acetylation by WcsU and deacetylation by KpACE in regulating the pathogenicity of K. pneumoniae. Understanding this balance provides new insights into the modulation of virulence traits and potential therapeutic targets for combating K. pneumoniae infections., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Wang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Heavy water, containing the heavy hydrogen isotope, is toxic to cells, although the underlying mechanism remains incompletely understood. In addition, certain enzymatic proton transfer reactions exhibit kinetic isotope effects attributed to hydrogen isotopes and their temperature dependencies, indicative of quantum tunneling phenomena. However, the correlation between the biological effects of heavy water and the kinetic isotope effects mediated by hydrogen isotopes remains elusive. In this study, we elucidated the kinetic isotope effects arising from hydrogen isotopes of water and their temperature dependencies in vitro, focusing on deacetylation, DNA cleavage, and protein cleavage, which are crucial enzymatic reactions mediated by hydrolysis. Intriguingly, the intracellular isotope effects of heavy water, related to the in vitro kinetic isotope effects, significantly impeded multiple DNA double-strand break repair mechanisms crucial for cell survival. Additionally, heavy water exposure enhanced histone acetylation and associated transcriptional activation in cells, consistent with the in vitro kinetic isotope effects observed in histone deacetylation reactions. Moreover, as observed for the in vitro kinetic isotope effects, the cytotoxic effect on cell proliferation induced by heavy water exhibited temperature-dependency. These findings reveal the substantial impact of heavy water-induced isotope effects on cellular functions governed by hydrolytic enzymatic reactions, potentially mediated by quantum-level mechanisms underlying kinetic isotope effects., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Yasuda et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The DNA damage response (DDR) constitutes a vital cellular process that safeguards genome integrity. This biological process involves substantial alterations in chromatin structure, commonly orchestrated by epigenetic enzymes. Here, we show that the epigenetic modifier N-terminal acetyltransferase 4 (Nat4), known to acetylate the alpha-amino group of serine 1 on histones H4 and H2A, is implicated in the response to DNA damage in S. cerevisiae. Initially, we demonstrate that yeast cells lacking Nat4 have an increased sensitivity to DNA damage and accumulate more DNA breaks than wild-type cells. Accordingly, upon DNA damage, NAT4 gene expression is elevated, and the enzyme is specifically recruited at double-strand breaks. Delving deeper into its effects on the DNA damage signaling cascade, nat4-deleted cells exhibit lower levels of the damage-induced modification H2AS129ph (γH2A), accompanied by diminished binding of the checkpoint control protein Rad9 surrounding the double-strand break. Consistently, Mec1 kinase recruitment at double-strand breaks, critical for H2AS129ph deposition and Rad9 retention, is significantly impaired in nat4Δ cells. Consequently, Mec1-dependent phosphorylation of downstream effector kinase Rad53, indicative of DNA damage checkpoint activation, is reduced. Importantly, we found that the effects of Nat4 in regulating the checkpoint signaling cascade are mediated by its N-terminal acetyltransferase activity targeted specifically towards histone H4. Overall, this study points towards a novel functional link between histone N-terminal acetyltransferase Nat4 and the DDR, associating a new histone-modifying activity in the maintenance of genome integrity., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Constantinou et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The class I histone deacetylases are essential regulators of cell fate decisions in health and disease. While pan- and class-specific HDAC inhibitors are available, these drugs do not allow a comprehensive understanding of individual HDAC function, or the therapeutic potential of isoform-specific targeting. To systematically compare the impact of individual catalytic functions of HDAC1, HDAC2 and HDAC3, we generated human HAP1 cell lines expressing catalytically inactive HDAC enzymes. Using this genetic toolbox we compare the effect of individual HDAC inhibition with the effects of class I specific inhibitors on cell viability, protein acetylation and gene expression. Individual inactivation of HDAC1 or HDAC2 has only mild effects on cell viability, while HDAC3 inactivation or loss results in DNA damage and apoptosis. Inactivation of HDAC1/HDAC2 led to increased acetylation of components of the COREST co-repressor complex, reduced deacetylase activity associated with this complex and derepression of neuronal genes. HDAC3 controls the acetylation of nuclear hormone receptor associated proteins and the expression of nuclear hormone receptor regulated genes. Acetylation of specific histone acetyltransferases and HDACs is sensitive to inactivation of HDAC1/HDAC2. Over a wide range of assays, we determined that in particular HDAC1 or HDAC2 catalytic inactivation mimicks class I specific HDAC inhibitors. Importantly, we further demonstrate that catalytic inactivation of HDAC1 or HDAC2 sensitizes cells to specific cancer drugs. In summary, our systematic study revealed isoform-specific roles of HDAC1/2/3 catalytic functions. We suggest that targeted genetic inactivation of particular isoforms effectively mimics pharmacological HDAC inhibition allowing the identification of relevant HDACs as targets for therapeutic intervention. Author summary: Histone deacetylases (HDACs) remove acetyl groups from histones and non-histone proteins. Dysregulated acetylation has been attributed to various disease states, including cancer, immunological and neurological diseases. Due to the potential reversibility of epigenetic or post-translational modifications, the pharmacological targeting of HDACs has high potential. However, most HDAC inhibitors used for clinical trials lack specificity which might contribute to side effects. Despite many years of intense research, surprisingly little is known about the substrate specificity of individual HDAC enzymes. To elucidate the consequences of more specific targeting of individual HDAC isoforms, detailed analysis of the catalytic function of each enzyme is required. Albeit HDACs being well studied in general, cross-comparison between the studies is difficult due to the use of different model systems and the frequent use of inhibitors, which lack specificity. Also, commonly used knockout or knockdown models abolish the structural function of the enzymes, potentially not accurately reflecting the situation of inhibitor treatment which only causes enzymatic inactivation. We suggest that the use of catalytic inactive mutants instead of knockouts might be another step towards mimicking isoform specific enzyme inhibition and provide a detailed comparative, side-by-side analysis of class I HDACs. [ABSTRACT FROM AUTHOR]

Retinoblastoma like protein-2 (Rbl2) is functionally regulated by phosphorylation and acetylation. Previously, we demonstrated that lysine K1083 (K1079 in human Rbl2) is a potential target for acetylation but its functional role remains elusive. We investigated alterations in human Rbl2 gene specifically targeting exons 19–22 harbouring acetylatable residues i.e. K1072, K1083 and K1115 through single stranded conformation polymorphism (SSCP) in breast cancer patients. The K1083 was found altered into arginine (R) in 51% of the cases but K1072 and K1115 remained conserved. The 'K1083R' mutation impairs the acetylation potential of this motif that may result in functional inactivation of Rbl2. These patients also showed poor survival outcome that highlights prognostic relevance of this residue. NIH3T3 cells expressing glutamine (K1083Q) mutated Rbl2 could not be arrested in G1 by serum starvation, whereas cells expressing Rbl2 with K1083R showed prolonged G1 arrest in fluorescence activated cell sorting (FACS) analysis. This suggests that K1083 acetylation is important for G1/S transition. Further, we performed molecular dynamic simulations (MDS) to analyse kinetics of residue K1083 with Cyc-D1/CDK4. Mutations at K1083 impaired this binding exposing neighbouring residues S1080, P1081, S1082 and R1084, hence enhancing the possibility of accelerated phosphorylation. S1080 has previously been reported as a promising candidate of cell cycle dependent phosphorylation in Rbl2. This highlights significance of mutations in the pocket domain of Rbl2 gene in breast cancer, and also strengthen the supposition that K1083 acetylation is pre-requisite for its phosphorylation. [ABSTRACT FROM AUTHOR]

Histone acetylation is involved in the regulation of seed germination. The transcription factor ABI5 plays an essential role in ABA- inhibited seed germination. However, the molecular mechanism of how ABI5 and histone acetylation coordinate to regulate gene expression during seed germination is still ambiguous. Here, we show that ENAP1 interacts with ABI5 and they co-bind to ABA responsive genes including ABI5 itself. The hypersensitivity to ABA of ENAP1ox seeds germination is recovered by the abi5 null mutation. ABA enhances H3K9Ac enrichment in the promoter regions as well as the transcription of target genes co-bound by ENAP1 and ABI5, which requires both ENAP1 and ABI5. ABI5 gene is directly regulated by ENAP1 and ABI5. In the enap1 deficient mutant, H3K9Ac enrichment and the binding activity of ABI5 in its own promoter region, along with ABI5 transcription and protein levels are all reduced; while in the abi5-1 mutant, the H3K9Ac enrichment and ENAP1 binding activity in ABI5 promoter are decreased, suggesting that ENAP1 and ABI5 function together to regulate ABI5- mediated positive feedback regulation. Overall, our research reveals a new molecular mechanism by which ENAP1 regulates H3K9 acetylation and mediates the positive feedback regulation of ABI5 to inhibit seed germination. Summary: To optimize the fitness in natural environment, flowering plants initiate seed germination in the favorable environment and maintain seed dormancy under stressful conditions. Precise mechanisms have been evolved to regulate germination timing to ensure plant adaptation to unfavorable environment. ABA, a major stress hormone in plants, induces seed dormancy and represses seed germination. Epigenetic regulation has been known involved in ABA signaling in which the transcription factor ABI5 acts as a regulatory hub. However, the epigenetic regulation such as histone acetylation on ABI5 transcription remains elusive. In this study, we revealed a new molecular mechanism by which histone binding protein ENAP1 regulates H3K9 acetylation, which mediates the positive feedback regulation of ABI5 in an ABI5 dependent manner to inhibit seed germination. [ABSTRACT FROM AUTHOR]

Lysine acetylation is a frequently occurring post-translational modification (PTM), emerging as an important metabolic regulatory mechanism in prokaryotes. This process is achieved enzymatically by the protein acetyltransferase (KAT) to specifically transfer the acetyl group, or non-enzymatically by direct intermediates (acetyl phosphate or acetyl-CoA). Although lysine acetylation modification of glucosyltransferases (Gtfs), the important virulence factor in Streptococcus mutans, was reported in our previous study, the KAT has not been identified. Here, we believe that the KAT ActG can acetylate Gtfs in the enzymatic mechanism. By overexpressing 15 KATs in S. mutans, the synthesized water-insoluble extracellular polysaccharides (EPS) and biofilm biomass were measured, and KAT (actG) was identified. The in-frame deletion mutant of actG was constructed to validate the function of actG. The results showed that actG could negatively regulate the water-insoluble EPS synthesis and biofilm formation. We used mass spectrometry (MS) to identify GtfB and GtfC as the possible substrates of ActG. This was also demonstrated by in vitro acetylation assays, indicating that ActG could increase the acetylation levels of GtfB and GtfC enzymatically and decrease their activities. We further found that the expression level of actG in part explained the virulence differences in clinically isolated strains. Moreover, overexpression of actG in S. mutans attenuated its cariogenicity in the rat caries model. Taken together, our study demonstrated that the KAT ActG could induce the acetylation of GtfB and GtfC enzymatically in S. mutans, providing insights into the function of lysine acetylation in bacterial virulence and pathogenicity. Author summary: Lysine acetylation is a regulatory post-translational modification (PTM) important in physiological processes across all domains of life. Although it has been well studied and characterized in eukaryotes, new insights into the lysine acetylation in bacteria have gained momentum in recent years, and hundreds to thousands of protein acetylation processes have been identified in various bacteria with novel enrichment strategies. However, the specific mechanisms of regulating lysine acetylation and function are still poorly understood. Therefore, we screened for the KAT mediating Gtfs acetylation by constructing 15 strains of S. mutans that overexpressed the GCN5-related N-acetyltransferases (GNAT) family members. Eventually, we identified and characterized ActG, a GNAT family member, that could catalyze the acetylation of GtfB and GtfC in S. mutans by the enzymatic mechanism, inversely related to their enzymatic activities, subsequently affecting the water-insoluble EPS synthesis and biofilm formation. In addition, ActG impaired the cariogenicity of S. mutans in a rat caries model. Thus, this study provides significant insights into the effect of lysine acetylation on S. mutans virulence and pathogenicity by regulating target protein functions and relative physiological processes. [ABSTRACT FROM AUTHOR]

The ultraviolet (UV) radiation triggers a pigmentation response in human skin, wherein, melanocytes rapidly activate divergent maturation and proliferation programs. Using single-cell sequencing, we demonstrate that these 2 programs are segregated in distinct subpopulations in melanocytes of human and zebrafish skin. The coexistence of these 2 cell states in cultured melanocytes suggests possible cell autonomy. Luria-Delbrück fluctuation test reveals that the initial establishment of these states is stochastic. Tracking of pigmenting cells ascertains that the stochastically acquired state is faithfully propagated in the progeny. A systemic approach combining single-cell multi-omics (RNA+ATAC) coupled to enhancer mapping with H3K27 acetylation successfully identified state-specific transcriptional networks. This comprehensive analysis led to the construction of a gene regulatory network (GRN) that under the influence of noise, establishes a bistable system of pigmentation and proliferation at the population level. This GRN recapitulates melanocyte behaviour in response to external cues that reinforce either of the states. Our work highlights that inherent stochasticity within melanocytes establishes dedicated states, and the mature state is sustained by selective enhancers mark through histone acetylation. While the initial cue triggers a proliferation response, the continued signal activates and maintains the pigmenting subpopulation via epigenetic imprinting. Thereby our study provides the basis of coexistence of distinct populations which ensures effective pigmentation response while preserving the self-renewal capacity., Competing Interests: RSG is the co-founder of Vyome Biosciences Pvt Ltd., a biopharmaceutical company working in the area of dermatology. Other authors declare no competing interest., (Copyright: © 2024 Aggarwal et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Expression of the androgen receptor is key to the response of cells and tissues to androgenic steroids, such as testosterone or dihydrotestosterone, as well as impacting the benefit of hormone-dependent therapies for endocrine diseases and hormone-dependent cancers. However, the mechanisms controlling androgen receptor expression are not fully understood, limiting our ability to effectively promote or inhibit androgenic signalling therapeutically. An autoregulatory loop has been described in which androgen receptor may repress its own expression in the presence of hormone, although the molecular mechanisms are not fully understood. In this work, we elucidate the mechanisms of autoregulation and demonstrate, for the first time, that a similar repression of the AR gene is facilitated by the progesterone receptor. We show that the progesterone receptor, like the androgen receptor binds to response elements within the AR gene to effect transcriptional repression in response to hormone treatment. Mechanistically, this repression involves hormone-dependent histone deacetylation within the AR 5'UTR region and looping between sequences in intron 2 and the transcription start site (TSS). This novel pathway controlling AR expression in response to hormone stimulation may have important implications for understanding cell or tissue selective receptor signalling., Competing Interests: The authors have no conflicts of interest to declare., (Copyright: © 2024 Gillen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Rheumatoid arthritis (RA) is a systemic immune-mediated disease characterized by joint inflammation and destruction. The disease typically affects small joints in the hands and feet, later progressing to involve larger joints such as the knees, shoulders, and hips. While the reasons for these joint-specific differences are unclear, distinct epigenetic patterns associated with joint location have been reported. In this study, we evaluated the unique epigenetic landscapes of fibroblast-like synoviocytes (FLS) from hip and knee synovium in RA patients, focusing on the expression and regulation of Homeobox (HOX) transcription factors. These highly conserved genes play a critical role in embryonic development and are known to maintain distinct expression patterns in various adult tissues. We found that several HOX genes, especially HOXD10, were differentially expressed in knee FLS compared with hip FLS. Epigenetic differences in chromatin accessibility and histone marks were observed in HOXD10 promoter between knee and hip FLS. Histone modification, particularly histone acetylation, was identified as an important regulator of HOXD10 expression. To understand the mechanism of differential HOXD10 expression, we inhibited histone deacetylases (HDACs) with small molecules and siRNA. We found that HDAC1 blockade or deficiency normalized the joint-specific HOXD10 expression patterns. These observations suggest that epigenetic differences, specifically histone acetylation related to increased HDAC1 expression, play a crucial role in joint-specific HOXD10 expression. Understanding these mechanisms could provide insights into the regional aspects of RA and potentially lead to therapeutic strategies targeting specific patterns of joint involvement during the course of disease., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Lee et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The plant pathogen Pseudomonas syringae secretes multiple effectors that modulate plant defenses. Some effectors trigger defenses due to specific recognition by plant immune complexes, whereas others can suppress the resulting immune responses. The HopZ3 effector of P. syringae pv. syringae B728a (PsyB728a) is an acetyltransferase that modifies not only components of plant immune complexes, but also the Psy effectors that activate these complexes. In Arabidopsis, HopZ3 acetylates the host RPM1 complex and the Psy effectors AvrRpm1 and AvrB3. This study focuses on the role of HopZ3 during tomato infection. In Psy-resistant tomato, the main immune complex includes PRF and PTO, a RIPK-family kinase that recognizes the AvrPto effector. HopZ3 acts as a virulence factor on tomato by suppressing AvrPto1Psy-triggered immunity. HopZ3 acetylates AvrPto1Psy and the host proteins PTO, SlRIPK and SlRIN4s. Biochemical reconstruction and site-directed mutagenesis experiments suggest that acetylation acts in multiple ways to suppress immune signaling in tomato. First, acetylation disrupts the critical AvrPto1Psy-PTO interaction needed to initiate the immune response. Unmodified residues at the binding interface of both proteins and at other residues needed for binding are acetylated. Second, acetylation occurs at residues important for AvrPto1Psy function but not for binding to PTO. Finally, acetylation reduces specific phosphorylations needed for promoting the immune-inducing activity of HopZ3's targets such as AvrPto1Psy and PTO. In some cases, acetylation competes with phosphorylation. HopZ3-mediated acetylation suppresses the kinase activity of SlRIPK and the phosphorylation of its SlRIN4 substrate previously implicated in PTO-signaling. Thus, HopZ3 disrupts the functions of multiple immune components and the effectors that trigger them, leading to increased susceptibility to infection. Finally, mass spectrometry used to map specific acetylated residues confirmed HopZ3's unusual capacity to modify histidine in addition to serine, threonine and lysine residues. Author summary: By secreting virulence proteins (effectors) into their hosts, pathogenic bacteria hijack host cellular processes to promote bacterial colonization and disease development. For the plant pathogen Pseudomonas syringae, the coordinated action of effectors often mediates modifications of host defense proteins to inhibit their function. However, plants have evolved the ability to induce innate immunity upon recognition of effector-induced modifications of host proteins. How do pathogens circumvent the immune-inducing activity of certain effectors? They deploy more effectors to suppress these defenses. HopZ3, an acetyltransferase from P. syringae, is unique among plant pathogen effectors characterized so far in its ability to modify not only multiple components of the effector-triggered immune pathway, but also the triggering effector itself. Through the direct acetylation of residues involved in the interaction and activation of the bacterial effector AvrPto1Psy and tomato kinase PTO, HopZ3 modifications disrupt their binding and block phosphorylations necessary for immune induction. Additionally, HopZ3 acetylates other possible components in the PTO signaling pathway, including activation sites in SlRIPK kinase, leading to suppression of its activity and reduced phosphorylation of SlRIN4s. Our study emphasizes the importance of HopZ3-dependent acetylation of immune complexes and bacterial effectors across plant species in the suppression of effector-induced immunity. [ABSTRACT FROM AUTHOR]

The cAMP-CRP regulon coordinates transcription regulation of several energy-related genes, the lac operon among them. Lactose, or IPTG, induces the lac operon expression by binding to the LacI repressor, and releasing it from the promoter sequence. At the same time, the expression of the lac operon requires the presence of the CRP-cAMP complex, which promotes the binding of the RNA polymerase to the promoter region. The modified nucleotide cAMP accumulates in the absence of glucose and binds to the CRP protein, but its ability to bind to DNA can be impaired by lysine-acetylation of CRP. Here we add another layer of control, as acetylation of CRP seems to be modified by ppGpp. In cells grown in glycerol minimal media, ppGpp seems to repress the expression of lacZ, where ΔrelA mutants show higher expression of lacZ than in WT. These differences between the WT and ΔrelA strains seem to depend on the levels of acetylated CRP. During the growth in minimal media supplemented with glycerol, ppGpp promotes the acetylation of CRP by the Nε-lysine acetyltransferases YfiQ. Moreover, the expression of the different genes involved in the production and degradation of Acetyl-phosphate (ackA-pta) and the enzymatic acetylation of proteins (yfiQ) are stimulated by the presence of ppGpp, depending on the growth conditions. [ABSTRACT FROM AUTHOR]

Mitochondrial trifunctional protein (TFP) is a membrane-associated heterotetramer that catalyzes three of the four reactions needed to chain-shorten long-chain fatty acids inside the mitochondria. TFP is known to be heavily modified by acetyllysine and succinyllysine post-translational modifications (PTMs), many of which are targeted for reversal by the mitochondrial sirtuin deacylases SIRT3 and SIRT5. However, the functional significance of these PTMs is not clear, with some reports showing TFP gain-of-function and some showing loss-of-function upon increased acylation. Here, we mapped the known SIRT3/SIRT5-targeted lysine residues onto the recently solved TFP crystal structure which revealed that many of the target sites are involved in substrate channeling within the TFPα subunit. To test the effects of acylation on substate channeling through TFPα, we enzymatically synthesized the physiological long-chain substrate (2E)-hexadecenoyl-CoA. Assaying TFP in SIRT3 and SIRT5 knockout mouse liver and heart mitochondria with (2E)-hexadecenoyl-CoA revealed no change in enzyme activity. Finally, we investigated the effects of lysine acylation on TFP membrane binding in vitro. Acylation did not alter recombinant TFP binding to cardiolipin-containing liposomes. However, the presence of liposomes strongly abrogated the acylation reaction between succinyl-CoA and TFP lysine residues. Thus, TFP in the membrane-bound state may be protected against lysine acylation. [ABSTRACT FROM AUTHOR]

The final stage of Ebola virus (EBOV) replication is budding from host cells, where the matrix protein VP40 is essential for driving this process. Many post-translational modifications such as ubiquitination are involved in VP40 egress, but acetylation has not been studied yet. Here, we characterize NEDD4 is acetylated at a conserved Lys667 mediated by the acetyltransferase P300 which drives VP40 egress process. Importantly, P300-mediated NEDD4 acetylation promotes NEDD4-VP40 interaction which enhances NEDD4 E3 ligase activity and is essential for the activation of VP40 ubiquitination and subsequent egress. Finally, we find that Zaire ebolavirus production is dramatically reduced in P300 knockout cell lines, suggesting that P300-mediated NEDD4 acetylation may have a physiological effect on Ebola virus life cycle. Thus, our study identifies an acetylation-dependent regulatory mechanism that governs VP40 ubiquitination and provides insights into how acetylation controls EBOV VP40 egress. Author summary: Ebola virus (EBOV) is one of the deadliest pathogens, causing fatal hemorrhagic fever diseases in humans and primates. In this study, we find that P300-mediated NEDD4 acetylation facilitates EBOV egress. Acetylation promotes NEDD4-VP40 interactions which enhances NEDD4 E3 ligase activity and is essential for the activation of VP40 ubiquitination and subsequent egress. This study implies that inhibitory effect of acetylation can be regarded as an attractive candidate of drug target for the treatment of Ebola virus disease. [ABSTRACT FROM AUTHOR]

The acetylation of H4 lysine 16 (H4 K16Ac) in Saccharomyces cerevisiae counteracts the binding of the heterochromatin complex SIR to chromatin and inhibits gene silencing. Contrary to other histone acetylation marks, the H4 K16Ac level is high on genes with low transcription, whereas highly transcribed genes show low H4 K16Ac. Approximately 60% of cellular H4 K16Ac in S. cerevisiae is provided by the SAS-I complex, which consists of the MYST-family acetyltransferase Sas2, Sas4 and Sas5. The absence of SAS-I causes inappropriate spreading of the SIR complex and gene silencing in subtelomeric regions. Here, we investigated the genome-wide dynamics of SAS-I dependent H4 K16Ac during DNA replication. Replication is highly disruptive to chromatin and histone marks, since histones are removed to allow progression of the replication fork, and chromatin is reformed with old and new histones after fork passage. We found that H4 K16Ac appears in chromatin immediately upon replication. Importantly, this increase depends on the presence of functional SAS-I complex. Moreover, the appearance of H4 K16Ac is delayed in genes that are strongly transcribed. This indicates that transcription counteracts SAS-I-mediated H4 K16 acetylation, thus "sculpting" histone modification marks at the time of replication. We furthermore investigated which acetyltransferase acts redundantly with SAS-I to acetylate H4 K16Ac. esa1Δ sds3Δ cells, which were also sas2Δ sir3Δ in order to maintain viability, contained no detectable H4 K16Ac, showing that Esa1 and Sas2 are redundant for cellular H4 K16 acetylation. Furthermore, esa1Δ sds3Δ sas2Δ sir3Δ showed a more pronounced growth defect compared to the already defective esa1Δ sds3Δ sir3Δ. This indicates that SAS-I has cellular functions beyond preventing the spreading of heterochromatin. [ABSTRACT FROM AUTHOR]

TRIM32 is an E3 ligase implicated in diverse biological pathways and pathologies such as muscular dystrophy and cancer. TRIM32 are expressed both as full-length proteins, and as a truncated protein. The mechanisms for regulating these isoforms are poorly understood. Here we identify a PEST sequence in TRIM32 located in the unstructured region between the RING-BBox-CoiledCoil domains and the NHL repeats. The PEST sequence directs cleavage of TRIM32, generating a truncated protein similarly to the short isoform. We map three lysine residues that regulate PEST mediated cleavage and auto-ubiquitylation activity of TRIM32. Mimicking acetylation of lysine K247 completely inhibits TRIM32 cleavage, while the lysines K50 and K401 are implicated in auto-ubiquitylation activity. We show that the short isoform of TRIM32 is catalytic inactive, suggesting a dominant negative role. These findings uncover that TRIM32 is regulated by post-translational modifications of three lysine residues, and a conserved PEST sequence. [ABSTRACT FROM AUTHOR]

The Aggregated mHTT panel for R6/2 hippocampus in Fig 5A and the Soluble mHTT panel for WT hippocampus in Fig 5B are duplicate images. The Aggregated mHTT panels for cortex in Fig 5A and 5B, and the Soluble mHTT panel for WT cortex in Fig 5B are incorrectly taken from a blot of hippocampus samples. [Extracted from the article]

Rocky desertification is a bottleneck that reduces ecological and environmental security in karst areas. Paper mulberry, a unique deciduous tree, shows good performance in rocky desertification areas. Its resistance mechanisms are therefore of high interest. In this study, a lysine acetylation proteomics analysis of paper mulberry seedling leaves was conducted in combination with the purification of acetylated protein by high-precision nano LC-MS/MS. We identified a total of 7130 acetylation sites in 3179 proteins. Analysis of the modified sites showed a predominance of nine motifs. Six positively charged residues: lysine (K), arginine (R), and histidine (H), serine (S), threonine (T), and tyrosine (Y) occurred most frequently at the +1 position, phenylalanine (F) was both detected both upstream and downstream of the acetylated lysines; and the sequence logos showed a strong preference for lysine and arginine around acetylated lysines. Functional annotation revealed that the identified enzymes were mainly involved in translation, transcription, ribosomal structure and biological processes, showing that lysine acetylation can regulate various aspects of primary carbon and nitrogen metabolism and secondary metabolism. Acetylated proteins were enriched in the chloroplast, cytoplasm, and nucleus, and many stress response-related proteins were also discovered to be acetylated, including PAL, HSP70, and ERF. HSP70, an important protein involved in plant abiotic and disease stress responses, was identified in paper mulberry, although it is rarely found in woody plants. This may be further examined in research in other plants and could explain the good adaptation of paper mulberry to the karst environment. However, these hypotheses require further verification. Our data can provide a new starting point for the further analysis of the acetylation function in paper mulberry and other plants. [ABSTRACT FROM AUTHOR]

As a versatile genome editing tool, the CRISPR-Cas9 system induces DNA double-strand breaks at targeted sites to activate mainly two DNA repair pathways: HDR which allows precise editing via recombination with a homologous template DNA, and NHEJ which connects two ends of the broken DNA, which is often accompanied by random insertions and deletions. Therefore, how to enhance HDR while suppressing NHEJ is a key to successful applications that require precise genome editing. Histones are small proteins with a lot of basic amino acids that generate electrostatic affinity to DNA. Since H2A.X is involved in DNA repair processes, we fused H2A.X to Cas9 and found that this fusion protein could improve the HDR/NHEJ ratio by suppressing NHEJ. As various post-translational modifications of H2A.X play roles in the regulation of DNA repair, we also fused H2A.X mimicry variants to replicate these post-translational modifications including phosphorylation, methylation, and acetylation. However, none of them were effective to improve the HDR/NHEJ ratio. We further fused other histone variants to Cas9 and found that H2A.1 suppressed NHEJ better than H2A.X. Thus, the fusion of histone variants to Cas9 is a promising option to enhance precise genome editing., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Kato-Inui et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Recombinant Francisella tularensis universal stress protein with a C-terminal histidine-tag (rUsp/His6) was expressed in Escherichia coli. Endogenous F. tularensis Usp has a predicted molecular mass of 30 kDa, but rUsp/His6 had an apparent molecular weight of 33 kDa based on Western blot analyses. To determine the source of the higher molecular weight for rUsp/His6, post translational modifications were examined. Tryptic peptides of purified rUsp/His6 were subjected to liquid chromatography tandem mass spectrometry (LC-MS/MS) and fragmentation spectra were searched for acetylated lysines and polyaminated glutamines. Of the 24 lysines in rUsp/His6, 10 were acetylated (K63, K68, K72, K129, K175, K201, K208, K212, K233, and K238) and three of the four glutamines had putrescine, spermidine and spermine adducts (Q55, Q60 and Q267). The level of post-translational modification was substoichiometric, eliminating the possibility that these modifications were the sole contributor to the 3 kDa extra mass of rUsp/His6. LC-MS/MS revealed that stop codon readthrough had occurred resulting in the unexpected addition of 20 extra amino acids at the C-terminus of rUsp/His6, after the histidine tag. Further, the finding of polyaminated glutamines in rUsp/His6 indicated that E. coli is capable of transglutaminase activity., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Girardo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Ageing and chronic diseases lead to muscle loss and impair the regeneration of skeletal muscle. Thus, it's crucial to seek for effective intervention to improve the muscle regeneration. Tid1, a mitochondrial co-chaperone, is important to maintain mitochondrial membrane potential and ATP synthesis. Previously, we demonstrated that mice with skeletal muscular specific Tid1 deficiency displayed muscular dystrophy and postnatal lethality. Tid1 can interact with STAT3 protein, which also plays an important role during myogenesis. In this study, we used GMI, immunomodulatory protein of Ganoderma microsporum, as an inducer in C2C12 myoblast differentiation. We observed that GMI pretreatment promoted the myogenic differentiation of C2C12 myoblasts. We also showed that the upregulation of mitochondria protein Tid1 with the GMI pre-treatment promoted myogenic differentiation ability of C2C12 cells. Strikingly, we observed the concomitant elevation of STAT3 acetylation (Ac-STAT3) during C2C12 myogenesis. Our study suggests that GMI promotes the myogenic differentiation through the activation of Tid1 and Ac-STAT3. [ABSTRACT FROM AUTHOR]

Protein domains often recognize short linear protein motifs composed of a core conserved consensus sequence surrounded by less critical, modulatory positions. PTEN, a lipid phosphatase involved in phosphatidylinositol 3-kinase (PI3K) pathway, contains such a short motif located at the extreme C-terminus capable to recognize PDZ domains. It has been shown that the acetylation of this motif could modulate the interaction with several PDZ domains. Here we used an accurate experimental approach combining high-throughput holdup chromatographic assay and competitive fluorescence polarization technique to measure quantitative binding affinity profiles of the PDZ domain-binding motif (PBM) of PTEN. We substantially extended the previous knowledge towards the 266 known human PDZ domains, generating the full PDZome-binding profile of the PTEN PBM. We confirmed that inclusion of N-terminal flanking residues, acetylation or mutation of a lysine at a modulatory position significantly altered the PDZome-binding profile. A numerical specificity index is also introduced as an attempt to quantify the specificity of a given PBM over the complete PDZome. Our results highlight the impact of modulatory residues and post-translational modifications on PBM interactomes and their specificity. [ABSTRACT FROM AUTHOR]

Lysine acetylation (Kac) plays a critical role in the regulation of many important cellular processes. However, little is known about Kac in Solenopsis invicta, which is among the 100 most dangerous invasive species in the world. Kac in S. invicta was evaluated for the first time in this study. Altogether, 2387 Kac sites were tested in 992 proteins. The prediction of subcellular localization indicated that most identified proteins were located in the cytoplasm, mitochondria, and nucleus. Venom allergen Sol i 2, Sol i 3, and Sol i 4 were found to be located in the extracellular. The enriched Kac site motifs included Kac H, Kac Y, Kac G, Kac F, Kac T, and Kac W. H, Y, F, and W frequently occurred at the +1 position, whereas G, Y, and T frequently occurred at the –1 position. In the cellular component, acetylated proteins were enriched in the cytoplasmic part, mitochondrial matrix, and cytosolic ribosome. Furthermore, 25 pathways were detected to have significant enrichment. Interestingly, arginine and proline metabolism, as well as phagosome, which are related to immunity, involved several Kac proteins. Sequence alignment analyses demonstrated that V-type proton ATPase subunit G, tubulin alpha chain, and arginine kinase, the acetylated lysine residues, were evolutionarily conserved among different ant species. In the investigation of the interaction network, diverse interactions were adjusted by Kac. The results indicated that Kac may play an important role in the sensitization, cellular energy metabolism, immune response, nerve signal transduction, and response to biotic and abiotic stress of S. invicta. It may be useful to confirm the functions of Kac target proteins for the design of specific and effective drugs to prevent and control this dangerous invasive species. [ABSTRACT FROM AUTHOR]

Histone acetylation, balanced by histone acetyltransferase (HAT) and histone deacetylase (HDAC) complexes, affects dynamic transitions of chromatin structure to regulate transcriptional accessibility. However, little is known about the interplay between HAT and HDAC complexes in Fusarium graminearum, a causal agent of Fusarium Head Blight (FHB) that uniquely contains chromosomal regions enriched for house-keeping or infection-related genes. In this study, we identified the ortholog of the human inhibitor of growth (ING1) gene in F. graminearum (FNG1) and found that it specifically interacts with the FgEsa1 HAT of the NuA4 complex. Deletion of FNG1 led to severe growth defects and blocked conidiation, sexual reproduction, DON production, and plant infection. The fng1 mutant was normal in H3 acetylation but significantly reduced in H4 acetylation. A total of 34 spontaneous suppressors of fng1 with faster growth rate were isolated. Most of them were still defective in sexual reproduction and plant infection. Thirty two of them had mutations in orthologs of yeast RPD3, SIN3, and SDS3, three key components of the yeast Rpd3L HDAC complex. Four mutations in these three genes were verified to suppress the defects of fng1 mutant in growth and H4 acetylation. The rest two suppressor strains had a frameshift or nonsense mutation in a glutamine-rich hypothetical protein that may be a novel component of the FgRpd3 HDAC complex in filamentous fungi. FgRpd3, like Fng1, localized in euchromatin. Deletion of FgRPD3 resulted in severe growth defects and elevated H4 acetylation. In contract, the Fgsds3 deletion mutant had only a minor reduction in growth rate but FgSIN3 appeared to be an essential gene. RNA-seq analysis revealed that 48.1% and 54.2% of the genes with altered expression levels in the fng1 mutant were recovered to normal expression levels in two suppressor strains with mutations in FgRPD3 and FgSDS3, respectively. Taken together, our data showed that Fng1 is important for H4 acetylation as a component of the NuA4 complex and functionally related to the FgRpd3 HDAC complex for transcriptional regulation of genes important for growth, conidiation, sexual reproduction, and plant infection in F. graminearum. Author summary: Fusarium graminearum is the major causal agent of Fusarium Head Blight, a devastating disease of wheat and barley worldwide. Epigenetic regulation related to histone acetylation is involved in fungal development and invasive growth. Here, we functionally characterized the ortholog of the human inhibitor of growth (ING1) gene in F. graminearum (FNG1) and revealed its role in histone acetylation. By interacting with the FgEsa1 HAT of the NuA4 complex, Fng1 mediated H4 acetylation and was important for growth, conidiation, sexual development and pathogenicity. The fng1 mutant was unstable and a total of 34 spontaneous suppressors were isolated. Suppressor mutations were identified in four genes. While three of them, FgRPD3, FgSIN3, and FgSDS3, are key components of the Rpd3 HDAC complex, the other one encodes a glutamine-rich protein appeared to be a novel component of the Rpd3 HDAC complex in filamentous ascomycetes. Nevertheless, none of the mutation occurred in components of other HDAC complexes. Most of spontaneous suppressors were still defective in sexual reproduction and plant infection, indicating a stage-specific relationship between Fng1 and the Rpd3 HDAC complex. FgRpd3 and FgSds3 likely co-localized with Fng1 in euchromatin and played a critical role in vegetative growth. Approximately half of the genes with altered expression levels in the fng1 mutant were recovered to normal expression levels in two suppressor strains with mutations in FgRPD3 and FgSDS3. Most of these genes had no homologs in yeast, suggesting Fng1 and Rpd3 HDAC complex likely regulates genes unique to F. graminearum and filamentous fungi and with high genetic variations. Taken together, our data showed the functional relationship between Fng1 and the Rpd3 HDAC complex in H4 acetylation and hyphal growth, which has not been reported in other fungi. [ABSTRACT FROM AUTHOR]

Hormone-dependent activation of enhancers includes histone hyperacetylation and mediator recruitment. Histone hyperacetylation is mostly explained by a bimodal switch model, where histone deacetylases (HDACs) disassociate from chromatin, and histone acetyl transferases (HATs) are recruited. This model builds on decades of research on steroid receptor regulation of transcription. Yet, the general concept of the bimodal switch model has not been rigorously tested genome wide. We have used a genomics approach to study enhancer hyperacetylation by the thyroid hormone receptor (TR), described to operate as a bimodal switch. H3 acetylation, HAT and HDAC ChIP-seq analyses of livers from hypo- and hyperthyroid wildtype, TR deficient and NCOR1 disrupted mice reveal three types of thyroid hormone (T3)-regulated enhancers. One subset of enhancers is bound by HDAC3-NCOR in the absence of hormone and constitutively occupy TR and HATs irrespective of T3 levels, suggesting a poised enhancer state in absence of hormone. In presence of T3, HDAC3-NCOR1 dissociates from these enhancers leading to histone hyperacetylation, suggesting a histone acetylation rheostat function of HDACs at poised enhancers. Another subset of enhancers, not occupied by HDACs, is hyperacetylated in a T3-dependent manner, where TR is recruited to chromatin together with HATs. Lastly, a subset of enhancers, is not occupied directly by TR yet require TR for histone hyperacetylation. This indirect enhancer activation involves co-association with TR bound enhancers within super-enhancers or topological associated domains. Collectively, this demonstrates various mechanisms controlling hormone-dependent transcription and adds significant details to the otherwise simple bimodal switch model. Author summary: Thyroid hormone (T3) is a central regulator of growth, thermogenesis, heart rate and metabolism. In the liver T3 binds thyroid hormone receptor beta (TRβ) controlling expression of genes involved in processes such as lipid and cholesterol metabolism. The molecular mechanisms controlling TR-dependent gene regulation are centred on a bimodal switch model. In the absence of T3 co-repressors bind TR reducing gene expression. When hormone binds TR, co-repressors dissociate, and co-activators are recruited inducing gene expression. This model predominates the current understanding of T3-regulated gene expression. However, only a few studies have tested this model by genome-wide approaches. We have quantified Histone3 acetylation genome-wide in the liver of hypo- and hyperthyroid mice and identified gene regulatory regions regulated by T3. Probing TR and co-regulators at these regulatory regions, and analysing Histone3 acetylation in mouse models for disrupted co-repressor and TR activity, reveal additional insights to the mechanisms regulating T3-dependent gene expression. We suggest a revision of the prevailing bimodal switch model which helps understanding T3-regulated gene expression in tissues such as liver. We hope that this study, together with future studies, will add new perspectives on nuclear receptor-mediated transcriptional regulation to reveal general principles. [ABSTRACT FROM AUTHOR]

Microtubules are a major cytoskeletal component of neurites, and the regulation of microtubule stability is essential for neurite morphogenesis. βPix (ARHGEF7) is a guanine nucleotide exchange factor for the small GTPases Rac1 and Cdc42, which modulate the organization of actin filaments and microtubules. βPix is expressed as alternatively spliced variants, including the ubiquitous isoform βPix-a and the neuronal isoforms βPix-b and βPix-d, but the function of the neuronal isoforms remains unclear. Here, we reveal the novel role of βPix neuronal isoforms in regulating tubulin acetylation and neurite outgrowth. At DIV4, hippocampal neurons cultured from βPix neuronal isoform knockout (βPix-NIKO) mice exhibit defects in neurite morphology and tubulin acetylation, a type of tubulin modification which often labels stable microtubules. Treating βPix-NIKO neurons with paclitaxel, which stabilizes the microtubules, or reintroducing either neuronal βPix isoform to the KO neurons overcomes the impairment in neurite morphology and tubulin acetylation, suggesting that neuronal βPix isoforms may promote microtubule stabilization during neurite development. βPix-NIKO neurons also exhibit lower phosphorylation levels for Stathmin1, a microtubule-destabilizing protein, at Ser16. Expressing either βPix neuronal isoform in the βPix-NIKO neurons restores Stathmin1 phosphorylation levels, with βPix-d having a greater effect than βPix-b. Furthermore, we find that the recovery of neurite length and Stathmin1 phosphorylation via βPix-d expression requires PAK kinase activity. Taken together, our study demonstrates that βPix-d regulates the phosphorylation of Stathmin1 in a PAK-dependent manner and that neuronal βPix isoforms promote tubulin acetylation and neurite morphogenesis during neuronal development. [ABSTRACT FROM AUTHOR]

Chromatin structure plays a decisive role in gene regulation through the actions of transcriptional activators, coactivators, and epigenetic machinery. These trans-acting factors contribute to gene expression through their interactions with chromatin structure. In yeast INO1 activation, transcriptional activators and coactivators have been defined through intense study but the mechanistic links within these trans-acting factors and their functional implications are not yet fully understood. In this study, we examined the crosstalk within transcriptional coactivators with regard to the implications of Snf2p acetylation during INO1 activation. Through various biochemical analysis, we demonstrated that both Snf2p and Ino80p chromatin remodelers accumulate at the INO1 promoter in the absence of Snf2p acetylation during induction. Furthermore, nucleosome density and histone acetylation patterns remained unaffected by Snf2p acetylation status. We also showed that cells experience increased sensitivity to copper toxicity when remodelers accumulate at the INO1 promoter due to the decreased CUP1 expression. Therefore, our data provide evidence for crosstalk within transcriptional co-activators during INO1 activation. In light of these findings, we propose a model in which acetylation-driven chromatin remodeler recycling allows for efficient regulation of genes that are dependent upon limited co-activators. [ABSTRACT FROM AUTHOR]

Chitin deacetylases (CDAs) emerge as a valuable tool to produce chitosans with a nonrandom distribution of N-acetylglucosamine (GlcNAc) and glucosamine (GlcN) units. We hypothesized before that CDAs tend to bind certain sequences within the substrate matching their subsite preferences for either GlcNAc or GlcN units. Thus, they deacetylate or N-acetylate their substrates at nonrandom positions. To understand the molecular basis of these preferences, we analyzed the binding site of a CDA from Pestalotiopsis sp. (PesCDA) using a detailed activity screening of a site-saturation mutagenesis library. In addition, molecular dynamics simulations were conducted to get an in-depth view of crucial interactions along the binding site. Besides elucidating the function of several amino acids, we were able to show that only 3 residues are responsible for the highly specific binding of PesCDA to oligomeric substrates. The preference to bind a GlcNAc unit at subsite -2 and -1 can mainly be attributed to N75 and H199, respectively. Whereas an exchange of N75 at subsite -2 eliminates enzyme activity, H199 can be substituted with tyrosine to increase the GlcN acceptance at subsite -1. This change in substrate preference not only increases enzyme activity on certain substrates and changes composition of oligomeric products but also significantly changes the pattern of acetylation (PA) when N-acetylating polyglucosamine. Consequently, we could clearly show how subsite preferences influence the PA of chitosans produced with CDAs., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Bonin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Dendritic spine development is crucial for the establishment of excitatory synaptic connectivity and functional neural circuits. Alterations in spine morphology and density have been associated with multiple neurological disorders. Autism candidate gene disconnected-interacting protein homolog 2 A (DIP2A) is known to be involved in acetylated coenzyme A (Ac-CoA) synthesis and is primarily expressed in the brain regions with abundant pyramidal neurons. However, the role of DIP2A in the brain remains largely unknown. In this study, we found that deletion of Dip2a in mice induced defects in spine morphogenesis along with thin postsynaptic density (PSD), and reduced synaptic transmission of pyramidal neurons. We further identified that DIP2A interacted with cortactin, an activity-dependent spine remodeling protein. The binding activity of DIP2A-PXXP motifs (P, proline; X, any residue) with the cortactin-Src homology 3 (SH3) domain was critical for maintaining the level of acetylated cortactin. Furthermore, Dip2a knockout (KO) mice exhibited autism-like behaviors, including excessive repetitive behaviors and defects in social novelty. Importantly, acetylation mimetic cortactin restored the impaired synaptic transmission and ameliorated repetitive behaviors in these mice. Altogether, our findings establish an initial link between DIP2A gene variations in autism spectrum disorder (ASD) and highlight the contribution of synaptic protein acetylation to synaptic processing. [ABSTRACT FROM AUTHOR]

Mitochondria have been increasingly recognized as a central regulatory nexus for multiple metabolic pathways, in addition to ATP production via oxidative phosphorylation (OXPHOS). Here we show that inducing mitochondrial DNA (mtDNA) stress in Drosophila using a mitochondrially-targeted Type I restriction endonuclease (mtEcoBI) results in unexpected metabolic reprogramming in adult flies, distinct from effects on OXPHOS. Carbohydrate utilization was repressed, with catabolism shifted towards lipid oxidation, accompanied by elevated serine synthesis. Cleavage and translocation, the two modes of mtEcoBI action, repressed carbohydrate rmetabolism via two different mechanisms. DNA cleavage activity induced a type II diabetes-like phenotype involving deactivation of Akt kinase and inhibition of pyruvate dehydrogenase, whilst translocation decreased post-translational protein acetylation by cytonuclear depletion of acetyl-CoA (AcCoA). The associated decease in the concentrations of ketogenic amino acids also produced downstream effects on physiology and behavior, attributable to decreased neurotransmitter levels. We thus provide evidence for novel signaling pathways connecting mtDNA to metabolism, distinct from its role in supporting OXPHOS. [ABSTRACT FROM AUTHOR]

Expression of FosB gene in striatum is essential in addiction establishment. Activated glucocorticoid receptors (GRs) induce FosB gene expression in response to stressor. Therefore, elevation of FosB expression in striatum serves as one mechanism by which stress increases risk for addiction. In this study, adult male Sprague-Dawley rats were used to investigate whether chronic stress result in histone modifications at FosB gene promoter in striatum and how these histone modifications affect FosB expression and the establishment of addiction behavior after administration of drugs of abuse. Animals were randomly assigned to three groups: Electric foot shock (EFS) group received 7-day EFS to induce chronic stress; electric foot shock plus mifepristone (EFS + Mif) group were injected with mifepristone, a nonspecific GRs antagonist, before EFS; control group did not receive any EFS. All groups then received 2-day conditioned place preference (CPP) training with morphine (5 mg/kg body weight) to test vulnerability to drug addiction. Before and after morphine administration, FosB mRNA in striatum was quantified by real-time RT-PCR. Levels of histone H3/H4 acetylation and histone H3K4 dimethylation at FosB promoter in striatum after morphine administration were measured by using chromatin immunoprecipitation (ChIP) plus real-time PCR. EFS group had stronger place preference to morphine and had significantly higher level of FosB mRNA in striatum than the other two groups. H3K4 dimethylation was 2.6-fold higher in EFS group than control group, while no statistical difference in H3/H4 acetylation. Mifepristone administration before EFS decreased histone H3K4 dimethylation and FosB mRNA in striatum, and also diminished morphine-induced conditioned place preference. Altogether, increased level of H3K4 dimethylation at FosB promoter in striatum is partially dependent on the activation of GR and responsible for the elevated level of morphine-induced FosB mRNA in chronic stressed animals. [ABSTRACT FROM AUTHOR]

RAS is the founding member of a superfamily of GTPases and regulates signaling pathways involved in cellular growth control. While recent studies have shown that the activation state of RAS can be controlled by lysine ubiquitylation and acetylation, the existence of lysine methylation of the RAS superfamily GTPases remains unexplored. In contrast to acetylation, methylation does not alter the side chain charge and it has been challenging to deduce its impact on protein structure by conventional amino acid substitutions. Herein, we investigate lysine methylation on RAS and RAS-related GTPases. We developed GoMADScan (Go language-based Modification Associated Database Scanner), a new user-friendly application that scans and extracts posttranslationally modified peptides from databases. The GoMADScan search on PhosphoSitePlus databases identified methylation of conserved lysine residues in the core GTPase domain of RAS superfamily GTPases, including residues corresponding to RAS Lys-5, Lys-16, and Lys-117. To follow up on these observations, we immunoprecipitated endogenous RAS from HEK293T cells, conducted mass spectrometric analysis and found that RAS residues, Lys-5 and Lys-147, undergo dimethylation and monomethylation, respectively. Since mutations of Lys-5 have been found in cancers and RASopathies, we set up molecular dynamics (MD) simulations to assess the putative impact of Lys-5 dimethylation on RAS structure. Results from our MD analyses predict that dimethylation of Lys-5 does not significantly alter RAS conformation, suggesting that Lys-5 methylation may alter existing protein interactions or create a docking site to foster new interactions. Taken together, our findings uncover the existence of lysine methylation as a novel posttranslational modification associated with RAS and the RAS superfamily GTPases, and putative impact of Lys-5 dimethylation on RAS structure. [ABSTRACT FROM AUTHOR]

Histone acetylation is one of the most important posttranslational modifications that contribute to transcriptional initiation and chromatin remodeling. In the present study, we aimed to investigate the effect of sodium butyrate (NaBu), a natural histone deacetylase inhibitor (HDACi), on the maturation of oocytes, preimplantation embryonic development, and expression of important developmental genes. The results indicated that NaBu decreased the rates of GVBD and the first polar body extrusion (PBE) in vitro in a dose-dependent manner. Meanwhile, NaBu treatment led to an abnormality in the spindle apparatus in oocytes in MI. However, the ratio of phosphor-extracellular signal-regulated kinases (p-ERK)/ERK significantly decreased in oocytes treated with 2.0 mM NaBu for 8 h. Furthermore, NaBu treatment at 2.0 mM improved the quality of embryos and the mRNA expression levels of important developmental genes such as HDAC1, Sox2, and Pou5f1. These data suggest that although a high concentration NaBu will impede the meiosis of oocytes, 2.0 mM NaBu will promote the development of embryos in vitro. Further investigation is needed to clarify the direct/indirect effects of NaBu on the regulation of important developmental genes and their subsequent impacts on full-term development in mammals. [ABSTRACT FROM AUTHOR]

The Pseudomonas syringae acetyltransferase HopZ1a is delivered into host cells by the type III secretion system to promote bacterial growth. However, in the model plant host Arabidopsis thaliana, HopZ1a activity results in an effector-triggered immune response (ETI) that limits bacterial proliferation. HopZ1a-triggered immunity requires the nucleotide-binding, leucine-rich repeat domain (NLR) protein, ZAR1, and the pseudokinase, ZED1. Here we demonstrate that HopZ1a can acetylate members of a family of ‘receptor-like cytoplasmic kinases’ (RLCK family VII; also known as PBS1-like kinases, or PBLs) and promote their interaction with ZED1 and ZAR1 to form a ZAR1-ZED1-PBL ternary complex. Interactions between ZED1 and PBL kinases are determined by the pseudokinase features of ZED1, and mutants designed to restore ZED1 kinase motifs can (1) bind to PBLs, (2) recruit ZAR1, and (3) trigger ZAR1-dependent immunity in planta, all independently of HopZ1a. A ZED1 mutant that mimics acetylation by HopZ1a also triggers immunity in planta, providing evidence that effector-induced perturbations of ZED1 also activate ZAR1. Overall, our results suggest that interactions between these two RLCK families are promoted by perturbations of structural features that distinguish active from inactive kinase domain conformations. We propose that effector-induced interactions between ZED1/ZRK pseudokinases (RLCK family XII) and PBL kinases (RLCK family VII) provide a sensitive mechanism for detecting perturbations of either kinase family to activate ZAR1-mediated ETI. [ABSTRACT FROM AUTHOR]

Canine urothelial carcinoma (cUC) is the most common tumor of the lower urinary tract in dogs. Although chemotherapy and radical surgery have improved the overall survival, most dogs with cUC succumb to metastasis or recurrence. Therefore, the development of an effective systematic therapy is warranted. In this study, a comprehensive drug screening test using a cUC cell line was performed and the anti-tumor effect of a histone deacetylase (HDAC) inhibitor was evaluated. Comprehensive drug screening was performed on cUC cells. Based on this screening, the anti-proliferation effect of vorinostat, an HDAC inhibitor clinically applied in humans, was evaluated using several cUC cell lines in sulforhodamine B and flow cytometry assays. Western blot analysis was also performed to evaluate the degree of acetylation of histone H3 as well as the expression and phosphorylation of cell cycle-related molecules. The anti-tumor effect of vorinostat in vivo was evaluated using a xenograft model. Finally, immunohistochemistry was performed on acetyl-histone H3 in cUC and the relationship between the degree of acetylation and prognosis was examined using Kaplan–Meier survival analysis. Drug screening revealed that HDAC inhibitors consistently inhibited the growth of cUC cells. Vorinostat inhibited the growth of 6 cUC cell lines in a dose-dependent manner and induced G0/G1 cell cycle arrest. Western blot analysis showed that vorinostat mediated the acetylation of histone H3, the dephosphorylation of p-Rb, and the upregulation of p21 upon exposure to vorinostat. Furthermore, inhibition of tumor growth was observed in the xenograft model. In clinical cUC cases, neoplastic urothelium showed significant deacetylation of histones compared to the normal control, where lower histone acetylation levels were associated with a poor prognosis. In conclusion, the therapeutic potential of vorinostat was demonstrated in cUC. Histone deacetylation may be related to cUC tumor progression. [ABSTRACT FROM AUTHOR]

Gram-negative pathogens resistant to amikacin and other aminoglycosides of clinical relevance usually harbor the 6’-N-acetyltransferase type Ib [AAC(6')-Ib], an enzyme that catalyzes inactivation of the antibiotic by acetylation using acetyl-CoA as donor substrate. Inhibition of the acetylating reaction could be a way to induce phenotypic conversion to susceptibility in these bacteria. We have previously observed that Zn2+ acts as an inhibitor of the enzymatic acetylation of aminoglycosides by AAC(6')-Ib, and in complex with ionophores it effectively reduced the levels of resistance in cellulo. We compared the activity of 8-hydroxyquinoline, three halogenated derivatives, and 5-[N-Methyl-N-Propargylaminomethyl]-8-Hydroxyquinoline in complex with Zn2+ to inhibit growth of amikacin-resistant Acinetobacter baumannii in the presence of the antibiotic. Two of the compounds, clioquinol (5-chloro-7-iodo-8-hydroxyquinoline) and 5,7-diiodo-8-hydroxyquinoline, showed robust inhibition of growth of the two A. baumannii clinical isolates that produce AAC(6')-Ib. However, none of the combinations had any activity on another amikacin-resistant A. baumannii strain that possesses a different, still unknown mechanism of resistance. Time-kill assays showed that the combination of clioquinol or 5,7-diiodo-8-hydroxyquinoline with Zn2+ and amikacin was bactericidal. Addition of 8-hydroxyquinoline, clioquinol, or 5,7-diiodo-8-hydroxyquinoline, alone or in combination with Zn2+, and amikacin to HEK293 cells did not result in significant toxicity. These results indicate that ionophores in complex with Zn2+ could be developed into potent adjuvants to be used in combination with aminoglycosides to treat Gram-negative pathogens in which resistance is mediated by AAC(6')-Ib and most probably other related aminoglycoside modifying enzymes. [ABSTRACT FROM AUTHOR]

Early exposure to some mild stresses can slow down the aging process and extend lifespan, raising the question of how early life stress might impact the somatic health of aged animals. Here, we reveal that early life heat experience triggers the establishment of epigenetic memory in soma, which promotes long-lasting stress responses and longevity in C. elegans. Unlike lethal heat shock, mild heat activates a unique transcriptional program mimicking pathogen defense responses, characterized by the enhanced expression of innate immune and detoxification genes. Surprisingly, the expression of defense response genes persists long after heat exposure, conferring enhanced stress resistance even in aged animals. Further studies identify the histone acetyltransferase CBP-1 and the chromatin remodeling SWI/SNF complex as epigenetic modulators of the long-lasting defense responses. Histone acetylation is elevated by heat stress and maintained into agedness thereafter. Accordingly, histone acetylation levels were increased on the promoters of defense genes. Moreover, disruption of epigenetic memory abrogates the longevity response to early hormetic heat stress, indicating that long-lasting defense responses are crucial for the survival of aged animals. Together, our findings provide mechanistic insights into how temperature stress experienced in early life provides animals with lifetime health benefits. [ABSTRACT FROM AUTHOR]

The inherent capacity of somatic cells to switch their phenotypic status in response to damage stimuli in vivo might have a pivotal role in ageing and cancer. However, how the entry-exit mechanisms of phenotype reprogramming are established remains poorly understood. In an attempt to elucidate such mechanisms, we herein introduce a stochastic model of combined epigenetic regulation (ER)-gene regulatory network (GRN) to study the plastic phenotypic behaviours driven by ER heterogeneity. To deal with such complex system, we additionally formulate a multiscale asymptotic method for stochastic model reduction, from which we derive an efficient hybrid simulation scheme. Our analysis of the coupled system reveals a regime of tristability in which pluripotent stem-like and differentiated steady-states coexist with a third indecisive state, with ER driving transitions between these states. Crucially, ER heterogeneity of differentiation genes is for the most part responsible for conferring abnormal robustness to pluripotent stem-like states. We formulate epigenetic heterogeneity-based strategies capable of unlocking and facilitating the transit from differentiation-refractory (stem-like) to differentiation-primed epistates. The application of the hybrid numerical method validates the likelihood of such switching involving solely kinetic changes in epigenetic factors. Our results suggest that epigenetic heterogeneity regulates the mechanisms and kinetics of phenotypic robustness of cell fate reprogramming. The occurrence of tunable switches capable of modifying the nature of cell fate reprogramming might pave the way for new therapeutic strategies to regulate reparative reprogramming in ageing and cancer. [ABSTRACT FROM AUTHOR]

Sec61p is the channel-forming subunit of the heterotrimeric Sec61 complex that mediates co-translational protein import into the endoplasmic reticulum (ER). In yeast, proteins can also be post-translationally translocated by the hetero-heptameric Sec complex, composed of the Sec61 and the Sec63 complexes. The Sec61 channel is also a candidate for the dislocation channel for misfolded proteins from the ER to the cytosol during ER-associated degradation (ERAD). The structure of the Sec61 complex is highly conserved, but the roles of its N-terminal acetylation and its amphipathic N-terminal helix are unknown so far. To gain insight into the function of the Sec61p N-terminus, we mutated its N-acetylation site, deleted its amphipathic helix, or both the helix and the N-acetylation site. Mutation of the N-acetylation site on its own had no effect on protein import into the ER in intact cells, but resulted in an ERAD defect. Yeast expressing sec61 without the N-terminal amphipathic helix displayed severe growth defects and had profound defects in post-translational protein import into the ER. Nevertheless the formation of the hetero-heptameric Sec complex was not affected. Instead, the lack of the N-terminal amphipathic helix compromised the integrity of the heterotrimeric Sec61 complex. We conclude that the N-terminal helix of Sec61p is required for post-translational protein import into the ER and Sec61 complex stability, whereas N-terminal acetylation of Sec61p plays a role in ERAD. [ABSTRACT FROM AUTHOR]