Acetylation Status Research Articles

Tubulin acetyltransferases access and modify the microtubule luminal K40 residue through anchors in taxane-binding pockets.

Acetylation at α-tubulin K40 is the sole post-translational modification preferred to occur inside the lumen of hollow cylindrical microtubules. However, how tubulin acetyltransferases access the luminal K40 in micrometer-long microtubules remains unknown. Here, we use cryo-electron microscopy and single-molecule reconstitution assays to reveal the enzymatic mechanism for tubulin acetyltransferases to modify K40 in the lumen. One tubulin acetyltransferase spans across the luminal lattice, with the catalytic core docking onto two α-tubulins and the enzyme's C-terminal domain occupying the taxane-binding pockets of two β-tubulins. The luminal accessibility and enzyme processivity of tubulin acetyltransferases are inhibited by paclitaxel, a microtubule-stabilizing chemotherapeutic agent. Characterizations using recombinant tubulins mimicking preacetylated and postacetylated K40 show the crosstalk between microtubule acetylation states and the cofactor acetyl-CoA in enzyme turnover. Our findings provide crucial insights into the conserved multivalent interactions involving α- and β-tubulins to acetylate the confined microtubule lumen.

Restoration of autophagy reduces diabetes-induced endothelial dysfunction

Abstract Introduction Diabetes leads to endothelial dysfunction, a main determinant of several cardiovascular diseases. The molecular mechanisms underlying the effects of hyperglycemia in endothelial cells are not fully characterized. Autophagy, the intracellular process by which the cell digests and recycles senescent or damaged cytoplasmic elements, exerts cardiovascular protective effects, limiting cardiac and vascular injury in the presence of stress. However, it is unclear how hyperglycemia modulates autophagy in endothelial cells. Purpose In this study, we elucidated the role of autophagy in vascular damage induced by diabetes. We also tested whether the restoration autophagy attenuates diabetes-induced endothelial damage. Methods We evaluated the effects of high-glucose (HG, 30 mM for 6 and 24 hours) on markers of endothelial function, autophagy, autophagic flux, and mitophagy in human umbilical endothelial cells (HUVEC) in vitro and in mesenteric arteries from wild-type mice (WT) ex vivo. We tested the effects of autophagy reactivation using the natural polyamine spermidine or ATG7 overexpression (ATG7ov) on apoptosis and angiogenesis in HUVEC treated with HG. Vascular reactivity experiments in the presence of autophagy activation were performed in HG-treated mouse mesenteric arteries and human saphenous veins from patients with peripheral artery disease. Finally, we evaluated the level of autophagy in the mammary arteries of newly discovered diabetic patients undergoing coronary artery bypass graft surgery. Results We found that HG reduces autophagy (0.6 fold p<0.05) and mitophagy (1.5 fold p<0.001) in HUVECs. We also demonstrated that endothelial cells undergoing hyperglycemia fail to activate autophagy under hypoxic conditions (0.6 fold p<0.05). Reactivation of autophagy by ATG7ov rescues apoptosis (0.52 fold p<0.05) and angiogenesis (2.3 fold p<0.05) in HUVEC treated with HG. We further observed that HG exacerbates endothelial dysfunction in a mouse model of genetic inhibition (Beclin 1 +/- mice), compared to WT mice (-19.16 % +-4.97 SEM vs -48.39 % +-2.18 SEM p<0.001). Mechanistically, HG increases cellular acetylation status (0.7 fold p<0.05) and activates p300 (1 fold p<0.05), an autophagy inhibitor. ATG7ov or spermidine, a p300 inhibitor, rescue endothelial-dependent relaxation in mesenteric arteries of WT mice treated with HG (ATG7ov -62.48 % +-5.16 SEM; SP -63.01 +-3.22 SEM; HG -40.67 % +-3.82 p<0.001). Finally, we observed reduced levels of autophagy in mammary arteries from diabetic patients and demonstrated that SP improves vascular function in human saphenous veins (-37.6 % +-12.76 SEM vs -12.52 % +-5.6 SEM p<0.001). Conclusion Our results suggest that the impairment of autophagy is a strong contributor of diabetes induced-endothelial dysfunction. Targeting autophagy may represent a valid therapeutic strategy to counteract the cardiovascular consequences of metabolic stress.

Acetylation-Dependent Compaction of the Histone H4 Tail Ensemble.

Acetylation of the histone H4 tail (H4Kac) has been established as a significant regulator of chromatin architecture and accessibility; however, the molecular mechanisms that underlie these observations remain elusive. Here, we characterize the ensemble features of the histone H4 tail and determine how they change following acetylation on specific sets of lysine residues. Our comprehensive account is enabled by a robust combination of experimental and computational biophysical methods that converge on molecular details including conformer size, intramolecular contacts, and secondary structure propensity. We find that acetylation significantly alters the chemical environment of basic patch residues (16-20) and leads to tail compaction that is partially mediated by transient intramolecular contacts established between the basic patch and N-terminal amino acids. Beyond acetylation, we identify that the protonation state of H18, which is affected by the acetylation state, is a critical regulator of ensemble characteristics, highlighting the potential for interplay between the sequence context and post-translational modifications to define the ensemble features of intrinsically disordered regions. This study elucidates molecular details that could link H4Kac with the regulation of chromatin architecture, illuminating a small piece of the complex network of molecular mechanisms underlying the histone code hypothesis.

Temporal regulation of acetylation status determines PARP1 role in DNA damage response and metabolic homeostasis.

Poly(ADP-ribose) polymerase 1 (PARP1) is an abundant nuclear protein involved in DNA repair, chromatin structure, and transcription. However, the regulation of its different functions remains poorly understood. Here, we report the role of PARP1 acetylation status in modulating its DNA repair and transactivation functions. We demonstrate that histone deacetylase 5 (HDAC5) determines PARP1 acetylation at Lys498 and Lys521 sites. HDAC5-mediated deacetylation at Lys498 site regulates PARP1 DNA damage response and facilitates efficient recruitment of DNA repair factors at damaged sites, thereby promoting cell survival. Additionally, HDAC5-mediated deacetylation at Lys521 site promotes PARP1 coactivator function, resulting in induction of proliferative and metabolic genes in an activating transcription factor 4-dependent manner. Thus, PARP1 induces metabolic adaptation to spur malignant phenotype. Our studies in mouse tumor models suggest that pharmacological inhibition of PARP1 enzymatic activity does not block tumor progression robustly as transactivation function remains unperturbed. These findings provide key mechanistic insights into PARP1 regulation and expand its role in tumor development.

Proteomic analysis of global protein acetylation in response to WSSV infection in a crustacean Cherax quadricarinatus

Protein acetylation, a crucial post-translational modification, plays a significant role in antiviral immunity of host and viral replication processes after infection. Our previous research showed that an acetyltransferase KAT2A significantly enhanced white spot syndrome virus (WSSV) replication in hematopoietic tissue (Hpt) cells from Cherax quadricarinatus, suggesting that alterations in intracellular acetylation states affect viral infection. Nevertheless, the mechanisms by which WSSV invasion regulates intracellular protein acetylation remain unexplored. To explore the connection between protein acetylation and WSSV infection, differentially acetylated proteins (DAcPs) were identified in Hpt cells during WSSV infection by proteomic analysis. The results revealed that 25 DAcPs were identified with primarily involving in transporter activity and proton ATPase activity complex during the early stage of WSSV infection, i.e., the intracellular transport of the virions. Furthermore, that 36 DAcPs associated with chromatin assembly and acetyltransferase complex were identified in the replication stage of WSSV infection, i.e., the process of extensive viral replication. Additionally, acetylation at the K27 and K36 sites of histone H3 was strongly activated during WSSV replication; similarly, the inhibition of histone acetylation by acetyltransferase inhibiter C646 significantly suppressed WSSV replication, implying that WSSV replication requires high levels of histone acetylation, while the acetylation at the K27 and K36 sites of histone H3 probably serves as critical viral regulatory targets. This study deepens the understanding of WSSV-host interaction, and lays the groundwork for future research into the relationship between protein acetylation and viral infection in crustaceans.

RPS6KA1 is a histone acetylation-related oncoprotein in acute myeloid leukemia which is targeted by afzelin

BackgroundHistone acetylation plays a critical role in the progression of acute myeloid leukemia (AML). This study aimed to explore the prognostic significance and biological implications of histone acetylation-related genes in AML and to identify potential oncoproteins and therapeutic compounds.MethodsGenes associated with AML and histone acetylation were identified using the TCGA-LAML and IMEx Interactome databases. A histone acetylation-related risk model was developed using the least absolute shrinkage and selection operator method. The prognostic value of the model was evaluated through Kaplan-Meier survival analysis, time-dependent receiver operating characteristic curve, univariate and multivariate Cox regression, and nomogram calibration. Key genes were identified using random forest, support vector machine, and multivariate Cox analysis. Molecular docking was employed to assess the binding affinity between ribosomal protein S6 kinase A1 (RPS6KA1) and potential compounds. Furthermore, the effects of RPS6KA1 and afzelin on the malignant behaviors and downstream pathways of AML cells were validated through in vitro experiments.ResultsA risk model composed of 6 genes, including HDAC6, CREB3, KLF13, GOLGA2, RPS6KA1 and ZMIZ2, was established, demonstrating strong prognostic predictive capability. Among these, RPS6KA1 emerged as a key risk factor linked to histone acetylation status in AML. Elevated RPS6KA1 expression was observed in AML samples and was associated with poor prognosis. RPS6KA1 knockdown suppressed AML cell proliferation, migration, and invasion, induced G0/G1 phase arrest, and promoted apoptosis. Additionally, RPS6KA1 was identified as a potential target for afzelin, which exhibited anti-AML activity by inactivating RPS6KA1.ConclusionHistone acetylation status is closely associated with AML patient prognosis. RPS6KA1 acts as an oncoprotein in AML, facilitating disease progression. Afzelin may represent a novel therapeutic agent for AML by targeting RPS6KA1, which requires validation by clinical trials.

Palmitic acid causes hepatocyte inflammation by suppressing the BMAL1-NAD+-SIRT2 axis.

Palmitic acid is the most abundant saturated fatty acid in circulation and causes hepatocyte toxicity and inflammation. As saturated fatty acid can also disrupt the circadian rhythm, the present work evaluated the connection between clock genes and NAD+ dependent Sirtuins in protecting hepatocytes from lipid-induced damage. Hepatocytes (immortal cells PH5CH8, hepatoma cells HepG2) treated with higher doses of palmitic acid (400-600μM) showed typical features of steatosis accompanied with growth inhibition and increased level of inflammatory markers (IL-6 IL-8, IL-1α and IL-1β) together with decline in NAD+ levels. Palmitic acid treated hepatocytes showed significant decline in not only the protein levels of SIRT2 but also its activity as revealed by the acetylation status of its downstream targets (Tubulin and NF-ƙB). Additionally, the circadian expression of both SIRT2 and BMAL1 was inhibited in presence of palmitic acid in only the non-cancerous hepatocytes, PH5CH8 cells. Clinical specimens obtained from subjects with NASH-associated fibrosis, ranging from absent (F0) to cirrhosis (F4), showed a significant decline in levels of SIRT2 and BMAL1, especially in the cirrhotic liver. Ectopic expression of BMAL1 or activating SIRT2 by supplementation with nicotinamide riboside (precursor of NAD+) dampened the palmitic acid induced lipoinflammation and lipotoxicity more effectively in PH5CH8 cells as compared to HepG2 cells. Mechanistically, palmitic acid caused transcriptional suppression of SIRT2 by disrupting the chromatin occupancy of BMAL1 at its promoter site. Overall, the work suggested that SIRT2 is a clock-controlled gene that is transcriptionally regulated by BMAL1. In conclusion the activation of the BMAL1-NAD+-SIRT2 axis shows hepatoprotective effects by preventing lipotoxicity and dampening inflammation.

SIRT6 loss causes intervertebral disc degeneration in mice by promoting senescence and SASP status.

Intervertebral disc degeneration is a major risk factor contributing to chronic low back and neck pain. While the etiological factors for disc degeneration vary, age is still one of the most important risk factors. Recent studies have shown the promising role of SIRT6 in mammalian aging and skeletal tissue health, however its role in the intervertebral disc health remains unexplored. We investigated the contribution of SIRT6 to disc health by studying the age-dependent spinal phenotype of mice with conditional deletion of Sirt6 in the disc (Acan CreERT2 ; Sirt6 fl/fl ). Histological studies showed a degenerative phenotype in knockout mice compared to Sirt6 fl/fl control mice at 12 months which became pronounced at 24 months. RNA-Seq analysis of NP and AF tissues, quantitative histone analysis, and in vitro multiomics employing RNA-seq with ATAC-seq revealed that SIRT6-loss resulted in changes in acetylation and methylation status of specific Histone 3 lysine residues, thereby affecting DNA accessibility and transcriptomic landscape. A decrease in autophagy and an increase in DNA damage were also noted in Sirt6-deficient cells. Further mechanistic insights revealed that loss of SIRT6 increased senescence and SASP burden in the disc characterized by increased p21, γH2AX, IL-6, and TGF-β abundance. Taken together our study highlights the contribution of SIRT6 in modulating DNA damage, autophagy and cell senescence, and its importance in maintaining disc health during aging thereby underscoring it as a potential therapeutic target to treat intervertebral disc degeneration.

Fluoride Alters Gene Expression via Histone H3K27 Acetylation in Ameloblast-like LS8 Cells.

Excessive fluoride ingestion during tooth development can cause dental fluorosis. Previously, we reported that fluoride activates histone acetyltransferase (HAT) to acetylate p53, promoting fluoride toxicity in mouse ameloblast-like LS8 cells. However, the roles of HAT and histone acetylation status in fluoride-mediated gene expression remain unidentified. Here, we demonstrate that fluoride-mediated histone modification causes gene expression alterations in LS8 cells. LS8 cells were treated with or without fluoride followed by ChIP-Seq analysis of H3K27ac. Genes were identified by differential H3K27ac peaks within ±1 kb from transcription start sites. The levels of mRNA of identified genes were assessed using rea-time PCR (qPCR). Fluoride increased H3K27ac peaks associated with Bax, p21, and Mdm2 genes and upregulated their mRNA levels. Fluoride decreased H3K27ac peaks and p53, Bad, and Bcl2 had suppressed transcription. HAT inhibitors (Anacardic acid or MG149) suppressed fluoride-induced mRNA of p21 and Mdm2, while fluoride and the histone deacetylase (HDAC) inhibitor sodium butyrate increased Bad and Bcl2 expression above that of fluoride treatment alone. To our knowledge, this is the first study that demonstrates epigenetic regulation via fluoride treatment via H3 acetylation. Further investigation is required to elucidate epigenetic mechanisms of fluoride toxicity in enamel development.

Loss of NAMPT and SIRT2 but not SIRT1 attenuate GLO1 expression and activity in human skeletal muscle

Glyoxalase I (GLO1) is the primary enzyme for detoxification of the reactive dicarbonyl methylglyoxal (MG). Loss of GLO1 promotes accumulation of MG resulting in a recapitulation of diabetic phenotypes. We previously demonstrated attenuated GLO1 protein in skeletal muscle from individuals with type 2 diabetes (T2D). However, whether GLO1 attenuation occurs prior to T2D and the mechanisms regulating GLO1 abundance in skeletal muscle are unknown. GLO1 expression and activity were determined in skeletal muscle tissue biopsies from 15 lean healthy individuals (LH, BMI: 22.4 ± 0.7) and 5 individuals with obesity (OB, BMI: 32.4 ± 1.3). GLO1 protein was attenuated by 26 ± 0.3 % in OB compared to LH skeletal muscle (p = 0.019). Similar reductions for GLO1 activity were observed (p = 0.102). NRF2 and Keap1 expression were equivocal between groups despite a 2-fold elevation in GLO1 transcripts in OB skeletal muscle (p = 0.008). GLO1 knock-down (KD) in human immortalized myotubes promoted downregulation of muscle contraction and organization proteins indicating the importance of GLO1 expression for skeletal muscle function. SIRT1 KD had no effect on GLO1 protein or activity whereas, SIRT2 KD attenuated GLO1 protein by 28 ± 0.29 % (p < 0.0001) and GLO1 activity by 42 ± 0.12 % (p = 0.0150). KD of NAMPT also resulted in attenuation of GLO1 protein (28 ± 0.069 %, p = 0.003), activity (67 ± 0.09 %, p = 0.011) and transcripts (50 ± 0.13 %, p = 0.049). Neither the provision of the NAD+ precursors NR nor NMN were able to prevent this attenuation in GLO1 protein. However, NR did augment GLO1 specific activity (p = 0.022 vs NAMPT KD). These perturbations did not alter GLO1 acetylation status. SIRT1, SIRT2 and NAMPT protein levels were all equivocal in skeletal muscle tissue biopsies from individuals with obesity and lean individuals. These data implicate NAD+-dependent regulation of GLO1 in skeletal muscle independent of altered GLO1 acetylation and provide rationale for exploring NR supplementation to rescue attenuated GLO1 abundance and activity in conditions such as obesity.

Abstract Tu103: SIRT2 Inhibition Decreases Glycolysis and Attenuates Hypertrophic Response in H9c2 Cardiomyocytes

Introduction: Myocardial glycolysis increases in hypertrophic and failing hearts. Hyperacetylation also occurs in the failing heart, and many glycolytic enzymes are known to be subject to acetylation. However, it is generally considered that acetylation has inhibitory effects on glycolysis. As a result, it is not clear whether acetylation changes directly contribute to glycolysis changes in cardiac hypertrophy. We therefore determined whether changes in the acetylation of glycolytic enzymes and the activity of the cytosolic deacetylase SIRT2 regulate cardiac glycolysis. Methods: Glycolysis rates were directly measured in rat heart-derived H9c2 cardiomyocytes perfused with 5 mM [5- 3 H] glucose, 0.8 mM palmitate, and 4% bovine serum albumin. Before these metabolic measurements, H9c2 cells were treated with either a SIRT2 inhibitor (10 µM AGK2) or a vehicle for 24 hours. In separate experiments, SIRT2 was also knocked down in H9c2 cells using siRNA, followed by glycolysis rate determinations. The impact of SIRT2 inhibition or SIRT2 knockdown on the acetylation status of glycolytic enzyme was also assessed. Furthermore, the effects of SIRT2 inhibition on hypertrophic signalling were assessed by treating H9c2 cells with phenylephrine. Results: SIRT2 inhibition markedly decreased glycolysis rates in H9c2 cells compared to vehicle-treated cells (524±108 vs 2631±372 nmol . g dry wt -1. min -1 , p&lt;0.05). Similarly, SIRT2 knockdown resulted in a significant reduction in glycolysis rates compared to scrambled siRNA-treated H9c2 cells (745±31 vs 1659±168 nmol . g dry wt -1. min -1 , p&lt;0.05). The decrease in SIRT2 was accompanied by an increase in the acetylation status of the glycolytic enzyme glyceraldehyde phosphate dehydrogenase (GPDH). Moreover, a trend towards increased phosphofructokinase (PFK) acetylation was also observed in SIRT2 knockdown H9c2 cells compared to scrambled siRNA-treated cells. Lastly, AGK2 treatment also attenuated phenylephrine-mediated hypertrophic responses in H9c2 cells. Conclusions: Increased acetylation of glycolytic enzymes is associated with a decrease in glycolysis, and SIRT2 inhibition or deletion in H9c2 cells significantly decreases glycolysis rates and attenuates hypertrophy. SIRT2 may therefore contribute to the increased glycolysis seen in hypertrophy and heart failure.

Assessing N-terminal acetylation status of cellular proteins via an antibody specific for acetylated methionine

N-terminal acetylation is being recognized as a factor affecting protein lifetime and proteostasis. It is a modification where an acetyl group is added to the N-terminus of proteins, and this occurs in 80 % of the human proteome. N-terminal acetylation is catalyzed by enzymes called N-terminal acetyltransferases (NATs). The various NATs acetylate different N-terminal amino acids, and methionine is a known target for some of the NATs. Currently, the acetylation status of most proteins can only be assessed with a limited number of methods, including mass spectrometry, which although powerful and robust, remains laborious and can only survey a fraction of the proteome. We here present testing of an antibody that was developed to specifically recognize Nt-acetylated methionine-starting proteins. We have used dot blots with synthetic acetylated and non-acetylated peptides in addition to protein analysis of lysates from NAT KO cell lines to assess the specificity and application of this anti-Nt-acetylated methionine antibody (anti-NtAc-Met). Our results demonstrate that this antibody is indeed NtAc-specific and further show that it has selectivity for some subtypes of methionine-starting N-termini, specifically potential substrates of the NatC, NatE and NatF enzymes. We propose that this antibody may be a powerful tool to identify NAT substrates or to analyse changes in N-terminal acetylation for specific cellular proteins of interest.

Editing of endogenous tubulins reveals varying effects of tubulin posttranslational modifications on axonal growth and regeneration

Tubulin posttranslational modifications (PTMs) modulate the dynamic properties of microtubules and their interactions with other proteins. However, the effects of tubulin PTMs were often revealed indirectly through the deletion of modifying enzymes or the overexpression of tubulin mutants. In this study, we directly edited the endogenous tubulin loci to install PTM-mimicking or -disabling mutations and studied their effects on microtubule stability, neurite outgrowth, axonal regeneration, cargo transport, and sensory functions in the touch receptor neurons of Caenorhabditis elegans. We found that the status of β-tubulin S172 phosphorylation and K252 acetylation strongly affected microtubule dynamics, neurite growth, and regeneration, whereas α-tubulin K40 acetylation had little influence. Polyglutamylation and detyrosination in the tubulin C-terminal tail had more subtle effects on microtubule stability likely by modulating the interaction with kinesin-13. Overall, our study systematically assessed and compared several tubulin PTMs for their impacts on neuronal differentiation and regeneration and established an in vivo platform to test the function of tubulin PTMs in neurons.

Editing of endogenous tubulins reveals varying effects of tubulin posttranslational modifications on axonal growth and regeneration.

Tubulin posttranslational modifications (PTMs) modulate the dynamic properties of microtubules and their interactions with other proteins. However, the effects of tubulin PTMs were often revealed indirectly through the deletion of modifying enzymes or the overexpression of tubulin mutants. In this study, we directly edited the endogenous tubulin loci to install PTM-mimicking or -disabling mutations and studied their effects on microtubule stability, neurite outgrowth, axonal regeneration, cargo transport, and sensory functions in the touch receptor neurons of Caenorhabditis elegans. We found that the status of β-tubulin S172 phosphorylation and K252 acetylation strongly affected microtubule dynamics, neurite growth, and regeneration, whereas α-tubulin K40 acetylation had little influence. Polyglutamylation and detyrosination in the tubulin C-terminal tail had more subtle effects on microtubule stability likely by modulating the interaction with kinesin-13. Overall, our study systematically assessed and compared several tubulin PTMs for their impacts on neuronal differentiation and regeneration and established an in vivo platform to test the function of tubulin PTMs in neurons.

Histone H3K18 & H3K23 acetylation directs establishment of MLL-mediated H3K4 methylation

In an unmodified state, positively charged histone N-terminal tails engage nucleosomal DNA in a manner which restricts access to not only the underlying DNA, but also key tail residues subject to binding and/or modification. Charge-neutralizing modifications, such as histone acetylation, serve to disrupt this DNA-tail interaction, facilitating access to such residues. We previously showed that a polyacetylation-mediated chromatin “switch” governs the read-write capability of H3K4me3 by the MLL1 methyltransferase complex. Here, we discern the relative contributions of site-specific acetylation states along the H3 tail and extend our interrogation to other chromatin modifiers. We show that the contributions of H3 tail acetylation to H3K4 methylation by MLL1 are highly variable, with H3K18 and H3K23 acetylation exhibiting robust stimulatory effects, and that this extends to the related H3K4 methyltransferase complex, MLL4. We show that H3K4me1 and H3K4me3 are found preferentially co-enriched with H3 N-terminal tail proteoforms bearing dual H3K18 and H3K23 acetylation (H3{K18acK23ac}). We further show that this effect is specific to H3K4 methylation, while methyltransferases targeting other H3 tail residues (H3K9, H3K27, & H3K36), a methyltransferase targeting the nucleosome core (H3K79), and a kinase targeting a residue directly adjacent to H3K4 (H3T3) are insensitive to tail acetylation. Together, these findings indicate a unique and robust stimulation of H3K4 methylation by H3K18 and H3K23 acetylation and provide key insight into why H3K4 methylation is often associated with histone acetylation in the context of active gene expression.

Selective HDAC6 Inhibition Has the Potential for Anti-Cancer Effect in Renal Cell Carcinoma.

Despite significant advancements in systemic therapy for renal cell carcinoma (RCC), the prognosis for patients with metastatic RCC remains poor, as they are often incurable. Consequently, there is an urgent need for innovative therapeutic strategies to further enhance the efficacy of RCC treatment and improve patient outcomes. One such promising avenue lies in targeting histone deacetylase (HDAC) 6, a protein known to regulate numerous crucial biological processes implicated in cancer progression by modulating the acetylation status of various cytoplasmic proteins. To explore the therapeutic potential of HDAC6 inhibition in RCC, our study focused on investigating the effects of HDAC6 inhibitors on cultured RCC cells. Utilizing a panel of 12 small molecule selective HDAC6 inhibitors and employing genetic knockdown techniques, we examined the impact of HDAC6 inhibition on RCC cellular dynamics. Our findings revealed that HDAC6 inhibition exerted a profound effect on RCC cells, resulting in decreased cell viability and DNA replication. Importantly, this effect was attributed to the induction of apoptosis. Our study provides valuable insights into the mechanisms underlying the anticancer effects of selective HDAC6 inhibitors on RCC. A detailed understanding of the molecular mechanisms underlying the anticancer effects of HDAC6 inhibition is important to explore new therapeutic strategies for metastatic RCC.

Sirtuin E deacetylase is required for full virulence of Aspergillus fumigatus

Aspergillus fumigatus represents a public health problem due to the high mortality rate in immunosuppressed patients and the emergence of antifungal-resistant isolates. Protein acetylation is a crucial post-translational modification that controls gene expression and biological processes. The strategic manipulation of enzymes involved in protein acetylation has emerged as a promising therapeutic approach for addressing fungal infections. Sirtuins, NAD+-dependent lysine deacetylases, regulate protein acetylation and gene expression in eukaryotes. However, their role in the human pathogenic fungus A. fumigatus remains unclear. This study constructs six single knockout strains of A. fumigatus and a strain lacking all predicted sirtuins (SIRTKO). The mutant strains are viable under laboratory conditions, indicating that sirtuins are not essential genes. Phenotypic assays suggest sirtuins’ involvement in cell wall integrity, secondary metabolite production, thermotolerance, and virulence. Deletion of sirE attenuates virulence in murine and Galleria mellonella infection models. The absence of SirE alters the acetylation status of proteins, including histones and non-histones, and triggers significant changes in the expression of genes associated with secondary metabolism, cell wall biosynthesis, and virulence factors. These findings encourage testing sirtuin inhibitors as potential therapeutic strategies to combat A. fumigatus infections or in combination therapy with available antifungals.

Modulation of histone acetylation enables fully mechanized hybrid rice breeding.

Hybrid rice has achieved high grain yield and greatly contributes to food security, but the manual-labour-intensive hybrid seed production process limits fully mechanized hybrid rice breeding. For next-generation hybrid seed production, the use of small-grain male sterile lines to mechanically separate small hybrid seeds from mixed harvest is promising. However, it is difficult to find ideal grain-size genes for breeding ideal small-grain male sterile lines without penalties in the number of hybrid seeds and hybrid rice yield. Here we report that the use of small-grain alleles of the ideal grain-size gene GSE3 in male sterile lines enables fully mechanized hybrid seed production and dramatically increases hybrid seed number in three-line and two-line hybrid rice systems. The GSE3 gene encodes a histone acetyltransferase that binds histones and influences histone acetylation levels. GSE3 is recruited by the transcription factor GS2 to the promoters of their co-regulated grain-size genes and influences the histone acetylation status of their co-regulated genes. Field trials demonstrate that genome editing of GSE3 can be used to immediately improve current elite male sterile lines of hybrid rice for fully mechanized hybrid rice breeding, providing a new perspective for mechanized hybrid breeding in other crops.

Synergistic cytotoxicity of histone deacetylase and poly-ADP ribose polymerase inhibitors and decitabine in pancreatic cancer cells: Implications for novel therapy.

Histone deacetylase inhibitors (HDACi) can modulate the acetylation status of proteins, influencing the genomic instability exhibited by cancer cells. Poly (ADP ribose) polymerase (PARP) inhibitors (PARPi) have a direct effect on protein poly (ADP-ribosyl)ation, which is important for DNA repair. Decitabine is a nucleoside cytidine analogue, which when phosphorylated gets incorporated into the growing DNA strand, inhibiting methylation and inducing DNA damage by inactivating and trapping DNA methyltransferase on the DNA, thereby activating transcriptionally silenced DNA loci. We explored various combinations of HDACi and PARPi +/- decitabine (hypomethylating agent) in pancreatic cancer cell lines BxPC-3 and PL45 (wild-type BRCA1 and BRCA2) and Capan-1 (mutated BRCA2). The combination of HDACi (panobinostat or vorinostat) with PARPi (talazoparib or olaparib) resulted in synergistic cytotoxicity in all cell lines tested. The addition of decitabine further increased the synergistic cytotoxicity noted with HDACi and PARPi, triggering apoptosis (evidenced by increased cleavage of caspase 3 and PARP1). The 3-drug combination treatments (vorinostat, talazoparib, and decitabine; vorinostat, olaparib, and decitabine; panobinostat, talazoparib, and decitabine; panobinostat, olaparib, and decitabine) induced more DNA damage (increased phosphorylation of histone 2AX) than the individual drugs and impaired the DNA repair pathways (decreased levels of ATM, BRCA1, and ATRX proteins). The 3-drug combinations also altered the epigenetic regulation of gene expression (NuRD complex subunits, reduced levels). This is the first study to demonstrate synergistic interactions between the aforementioned agents in pancreatic cancer cell lines and provides preclinical data to design individualized therapeutic approaches with the potential to improve pancreatic cancer treatment outcomes.

Regulation of TSC2 lysosome translocation and mitochondrial turnover by TSC2 acetylation status

Sirtuin1 (SIRT1) activity decreases the tuberous sclerosis complex 2 (TSC2) lysine acetylation status, inhibiting the mechanistic target of rapamycin complex 1 (mTORC1) signalling and concomitantly, activating autophagy. This study analyzes the role of TSC2 acetylation levels in its translocation to the lysosome and the mitochondrial turnover in both mouse embryonic fibroblast (MEF) and in mouse insulinoma cells (MIN6) as a model of pancreatic β cells. Resveratrol (RESV), an activator of SIRT1 activity, promotes TSC2 deacetylation and its translocation to the lysosome, inhibiting mTORC1 activity. An improvement in mitochondrial turnover was also observed in cells treated with RESV, associated with an increase in the fissioned mitochondria, positive autophagic and mitophagic fluxes and an enhancement of mitochondrial biogenesis. This study proves that TSC2 in its deacetylated form is essential for regulating mTORC1 signalling and the maintenance of the mitochondrial quality control, which is involved in the homeostasis of pancreatic beta cells and prevents from several metabolic disorders such as Type 2 Diabetes Mellitus.