Nε-Lysine Acetylation of the Histone-Like Protein HBsu Regulates the Process of Sporulation and Affects the Resistance Properties of Bacillus subtilis Spores

Bacillus subtilis produces dormant, highly resistant endospores in response to extreme environmental stresses or starvation. These spores are capable of persisting in harsh environments for many years, even decades, without essential nutrients. Part of the reason that these spores can survive such extreme conditions is because their chromosomal DNA is well protected from environmental insults. The α/β-type small acid-soluble proteins (SASPs) coat the spore chromosome, which leads to condensation and protection from such insults. The histone-like protein HBsu has been implicated in the packaging of the spore chromosome and is believed to be important in modulating SASP-mediated alterations to the DNA, including supercoiling and stiffness. Previously, we demonstrated that HBsu is acetylated at seven lysine residues, and one physiological function of acetylation is to regulate chromosomal compaction. Here, we investigate if the process of sporulation or the resistance properties of mature spores are influenced by the acetylation state of HBsu. Using our collection of point mutations that mimic the acetylated and unacetylated forms of HBsu, we first determined if acetylation affects the process of sporulation, by determining the overall sporulation frequencies. We found that specific mutations led to decreases in sporulation frequency, suggesting that acetylation of HBsu at some sites, but not all, is required to regulate the process of sporulation. Next, we determined if the spores produced from the mutant strains were more susceptible to heat, ultraviolet (UV) radiation and formaldehyde exposure. We again found that altering acetylation at specific sites led to less resistance to these stresses, suggesting that proper HBsu acetylation is important for chromosomal packaging and protection in the mature spore. Interestingly, the specific acetylation patterns were different for the sporulation process and resistance properties of spores, which is consistent with the notion that a histone-like code exists in bacteria. We propose that specific acetylation patterns of HBsu are required to ensure proper chromosomal arrangement, packaging, and protection during the process of sporulation.

A novel orally active HDAC6 inhibitor T-518 shows a therapeutic potential for Alzheimer’s disease and tauopathy in mice

Accumulation of tau protein is a key pathology of age-related neurodegenerative diseases such as Alzheimer's disease and progressive supranuclear palsy. Those diseases are collectively termed tauopathies. Tau pathology is associated with axonal degeneration because tau binds to microtubules (MTs), a component of axon and regulates their stability. The acetylation state of MTs contributes to stability and histone deacetylase 6 (HDAC6) is a major regulator of MT acetylation status, suggesting that pharmacological HDAC6 inhibition could improve axonal function and may slow the progression of tauopathy. Here we characterize N-[(1R,2R)-2-{3-[5-(difluoromethyl)-1,3,4-oxadiazol-2-yl]-5-oxo-5H,6H,7H-pyrrolo[3,4-b]pyridin-6-yl}cyclohexyl]-2,2,3,3,3-pentafluoropropanamide (T-518), a novel, potent, highly selective HDAC6 inhibitor with clinically favorable pharmacodynamics. T-518 shows potent inhibitory activity against HDAC6 and superior selectivity over other HDACs compared with the known HDAC6 inhibitors in the enzyme and cellular assays. T-518 showed brain penetration in an oral dose and blocked HDAC6-dependent tubulin deacetylation at Lys40 in mouse hippocampus. A 2-week treatment restored impaired axonal transport and novel object recognition in the P301S tau Tg mouse, tauopathy model, while a 3-month treatment also decreased RIPA-insoluble tau accumulation. Pharmaceutical inhibition of HDAC6 is a potential therapeutic strategy for tauopathy, and T-518 is a particularly promising drug candidate.

IGF2BP2 Promotes Cancer Progression by Degrading the RNA Transcript Encoding a v-ATPase Subunit

Insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) binds to various RNA transcripts and promotes cancer progression, although little is known regarding its regulation. Here we show IGF2BP2 is a substrate of the deacetylase and tumor suppressor sirtuin 1 (SIRT1) and regulates the expression of the vacuolar ATPase subunit ATP6V1A. SIRT1 down-regulation in aggressive cancers leads to increased acetylation of IGF2BP2 which recruits the XRN2 nuclease to degrade the ATP6V1A transcript, decreasing its expression. This impairs lysosomal function and results in the production of a secretome that enhances cancer cell proliferation and metastasis. These findings describe a previously unrecognized role for IGF2BP2 in the degradation of an mRNA transcript essential for lysosomal function and highlight how its sirtuin-regulated acetylation state can have significant biological and disease consequences.

A novel proteomics approach to epigenetic profiling of circulating nucleosomes

Alteration of epigenetic modifications plays an important role in human cancer. Notably, the dysregulation of histone post-translational modifications (PTMs) has been associated with several cancers including colorectal cancer (CRC). However, the signature of histone PTMs on circulating nucleosomes is still not well described. We have developed a fast and robust enrichment method to isolate circulating nucleosomes from plasma for further downstream proteomic analysis. This method enabled us to quantify the global alterations of histone PTMs from 9 CRC patients and 9 healthy donors. Among 54 histone proteoforms identified and quantified in plasma samples, 13 histone PTMs were distinctive in CRC. Notably, methylation of histone H3K9 and H3K27, acetylation of histone H3 and citrullination of histone H2A1R3 were upregulated in plasma of CRC patients. A comparative analysis of paired samples identified 3 common histone PTMs in plasma and tumor tissue including the methylation and acetylation state of lysine 27 of histone H3. Moreover, we highlight for the first time that histone H2A1R3 citrulline is a modification upregulated in CRC patients. This new method presented herein allows the detection and quantification of histone variants and histone PTMs from circulating nucleosomes in plasma samples and could be used for biomarker discovery of cancer.

Differential profiles of HDAC1 substrates and associated proteins in breast cancer cells revealed by trapping

Histone deacetylase (HDAC) proteins, which regulate the acetylation state of proteins, are the targets of multiple clinical drugs for cancer treatment. Due to the heterogeneity of tumors, HDAC proteins play...

Mitochondria-Targeted Inhibitors of the Human SIRT3 Lysine Deacetylase

<p>Sirtuin 3 (SIRT3) is the major protein lysine deacetylase in the mitochondria. This hydrolase regulates a wide range of metabolically involved enzymes and has been considered as a potential drug target in certain cancers. Investigation of pharmacological intervention has been challenging due to a lack of potent and selective inhibitors of SIRT3. Here, we developed a strategy for selective inhibition of SIRT3 in cells, over its structurally similar isozymes that localize primarily to nucleus (SIRT1) and cytoplasm (SIRT2). This was achieved by directing the inhibitors straight to the mitochondria through incorporation of sequences inspired by previously described mitochondria-targeting peptides. Our inhibitors exhibited excellent mitochondrial localization in HeLa cells as indicated by fluorophore-conjugated versions and target engagement was demonstrated by a thermal shift assay of SIRT3 using western blotting. The acetylation state of documented SIRT3 target MnSOD was shown to be perturbed in cells with little effect on known targets of SIRT1 and SIRT2, showing that our lead compound exhibits selectivity for SIRT3 in cells. We expect that the developed inhibitor will now enable a more detailed investigation of SIRT3 as a potential drug target and help shed further light on the diverse biology regulated by this enzyme. </p>

Mitochondria-Targeted Inhibitors of the Human SIRT3 Lysine Deacetylase

<p>Sirtuin 3 (SIRT3) is the major protein lysine deacetylase in the mitochondria. This hydrolase regulates a wide range of metabolically involved enzymes and has been considered as a potential drug target in certain cancers. Investigation of pharmacological intervention has been challenging due to a lack of potent and selective inhibitors of SIRT3. Here, we developed a strategy for selective inhibition of SIRT3 in cells, over its structurally similar isozymes that localize primarily to nucleus (SIRT1) and cytoplasm (SIRT2). This was achieved by directing the inhibitors straight to the mitochondria through incorporation of sequences inspired by previously described mitochondria-targeting peptides. Our inhibitors exhibited excellent mitochondrial localization in HeLa cells as indicated by fluorophore-conjugated versions and target engagement was demonstrated by a thermal shift assay of SIRT3 using western blotting. The acetylation state of documented SIRT3 target MnSOD was shown to be perturbed in cells with little effect on known targets of SIRT1 and SIRT2, showing that our lead compound exhibits selectivity for SIRT3 in cells. We expect that the developed inhibitor will now enable a more detailed investigation of SIRT3 as a potential drug target and help shed further light on the diverse biology regulated by this enzyme. </p>

Mitochondria-Targeted Inhibitors of the Human SIRT3 Lysine Deacetylase

<p>Sirtuin 3 (SIRT3) is the major protein lysine deacetylase in the mitochondria. This hydrolase regulates a wide range of metabolically involved enzymes and has been considered as a potential drug target in certain cancers. Investigation of pharmacological intervention has been challenging due to a lack of potent and selective inhibitors of SIRT3. Here, we developed a strategy for selective inhibition of SIRT3 in cells, over its structurally similar isozymes that localize primarily to nucleus (SIRT1) and cytoplasm (SIRT2). This was achieved by directing the inhibitors straight to the mitochondria through incorporation of sequences inspired by previously described mitochondria-targeting peptides. Our inhibitors exhibited excellent mitochondrial localization in HeLa cells as indicated by fluorophore-conjugated versions and target engagement was demonstrated by a thermal shift assay of SIRT3 using western blotting. The acetylation state of documented SIRT3 target MnSOD was shown to be perturbed in cells with little effect on known targets of SIRT1 and SIRT2, showing that our lead compound exhibits selectivity for SIRT3 in cells. We expect that the developed inhibitor will now enable a more detailed investigation of SIRT3 as a potential drug target and help shed further light on the diverse biology regulated by this enzyme. </p>

The Mitochondrial Protein VDAC1 at the Crossroads of Cancer Cell Metabolism: The Epigenetic Link

Carcinogenesis is a complicated process that involves the deregulation of epigenetics, resulting in cellular transformational events, such as proliferation, differentiation, and metastasis. Most chromatin-modifying enzymes utilize metabolites as co-factors or substrates and thus are directly dependent on such metabolites as acetyl-coenzyme A, S-adenosylmethionine, and NAD+. Here, we show that using specific siRNA to deplete a tumor of VDAC1 not only led to reprograming of the cancer cell metabolism but also altered several epigenetic-related enzymes and factors. VDAC1, in the outer mitochondrial membrane, controls metabolic cross-talk between the mitochondria and the rest of the cell, thus regulating the metabolic and energetic functions of mitochondria, and has been implicated in apoptotic-relevant events. We previously demonstrated that silencing VDAC1 expression in glioblastoma (GBM) U-87MG cell-derived tumors, resulted in reprogramed metabolism leading to inhibited tumor growth, angiogenesis, epithelial–mesenchymal transition and invasiveness, and elimination of cancer stem cells, while promoting the differentiation of residual tumor cells into neuronal-like cells. These VDAC1 depletion-mediated effects involved alterations in transcription factors regulating signaling pathways associated with cancer hallmarks. As the epigenome is sensitive to cellular metabolism, this study was designed to assess whether depleting VDAC1 affects the metabolism–epigenetics axis. Using DNA microarrays, q-PCR, and specific antibodies, we analyzed the effects of si-VDAC1 treatment of U-87MG-derived tumors on histone modifications and epigenetic-related enzyme expression levels, as well as the methylation and acetylation state, to uncover any alterations in epigenetic properties. Our results demonstrate that metabolic rewiring of GBM via VDAC1 depletion affects epigenetic modifications, and strongly support the presence of an interplay between metabolism and epigenetics.

Acetylation of Phenylalanine Hydroxylase and Tryptophan 2,3-Dioxygenase Alters Hepatic Aromatic Amino Acid Metabolism in Weaned Piglets

Weaning significantly alters hepatic aromatic amino acid (AAA) metabolism and physiological functions. However, less is known about the regulating mechanism of hepatic AAA metabolism after weaning. A total of 200 21-day-old piglets (Duroc × Landrace) were assigned randomly to the control group and the weaning group. In this study, weaning significantly decreased the concentration of phenylalanine, tryptophan, and tyrosine in piglet livers (p < 0.05). Additionally, through the detection of liver AAA metabolites and metabolic enzyme activity, it was observed that hepatic tryptophan catabolism was enhanced, while that of phenylalanine was weakened (p < 0.05). Intriguingly, acetyl-proteome profiling of liver from weaned piglets showed that weaning exacerbated the acetylation of phenylalanine hydroxylase (PAH) and the deacetylation of tryptophan 2,3-dioxygenase (TDO). Analysis of PAH and TDO acetylation in Chang liver cells showed that acetylation decreased the PAH activity, while deacetylation increased the TDO activity (p < 0.05). Furthermore, metabolites of AAAs and the acetylation statuses of PAH and TDO in primary hepatocytes from weaned piglets were consistent with the results in vivo. These findings indicated that weaning altered the PAH and TDO activity by affecting the acetylation state of the enzyme in piglets’’ livers. Lysine acetylation may be a potential regulatory mechanism for AAA metabolism in response to weaning.