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Objectives: To investigate a comprehensive proteomic profile of the tear fluid in patients with diabetic retinopathy (DR) and further define non-invasive biomarkers., Methods: A cross-sectional, multicentre study that includes 46 patients with DR, 28 patients with diabetes mellitus (DM), and 30 healthy controls (HC). Tear samples were collected with Schirmer strips. As for the discovery set, data-independent acquisition mass spectrometry was used to characterize the tear proteomic profile. Differentially expressed proteins between groups were identified, with gene ontology enrichment analysis and Kyoto Encyclopedia of Genes and Genomes enrichment analysis further developed. Classifying performance of biomarkers for distinguishing DR from DM was compared by the combination of three machine-learning algorithms. The selected biomarker panel was tested in the validation cohort using parallel reaction monitoring mass spectrometry., Results: Among 3364 proteins quantified, 235 and 88 differentially expressed proteins were identified for DR when compared to HC and DM, respectively, which were fundamentally related to retina homeostasis, inflammation and immunity, oxidative stress, angiogenesis and coagulation, metabolism, and cellular adhesion processes. The biomarker panel consisting of NAD-dependent protein deacetylase sirtuin-2 (SIR2), amine oxidase [flavin-containing] B (AOFB), and U8 snoRNA-decapping enzyme (NUD16) exhibited the best diagnostic performance in discriminating DR from DM, with AUCs of 0.933 and 0.881 in the discovery and validation set, respectively., Conclusions: Tear protein dysregulation is comprehensively revealed to be associated with DR onset. The combination of tear SIR2, AOFB, and NUD16 can be a novel potential approach for non-invasive detection or pre-screening of DR., Clinical Trial Registration: Chinese Clinical Trial Registry Identifier: ChiCTR2100054263. https://www.chictr.org.cn/showproj.html?proj=143177 . Date of registration: 2021/12/12., (© 2024. The Author(s), under exclusive licence to The Royal College of Ophthalmologists.)

Interleukin (IL)-1β is a culprit of adipose tissue inflammation, which in turn causes systematic inflammation and insulin resistance in obese individuals. IL-1β is mainly produced in monocytes and macrophages and marginally in adipocytes, through cleavage of the inactive pro-IL-1β precursor by caspase-1, which is activated via the NLRP3 inflammasome complex. The nuclear factor-κB (NF-κB) transcription factor is the master regulator of inflammatory responses. Brindle berry (Garcinia cambogia) has been widely used as health products for treating obesity and related metabolic disorders, but its active principles remain unclear. We previously found a series of polyisoprenylated benzophenones from brindle berry with anti-inflammatory activities. In this study we investigated whether 14-deoxygarcinol (DOG), a major polyisoprenylated benzophenone from brindle berry, alleviated adipose tissue inflammation and insulin sensitivity in high-fat diet fed mice. The mice were administered DOG (2.5, 5 mg · kg -1  · d -1 , i.p.) for 4 weeks. We showed that DOG injection dose-dependently improved insulin resistance and hyperlipidemia, but not adiposity in high-fat diet-fed mice. We found that DOG injection significantly alleviated adipose tissue inflammation via preventing macrophage infiltration and pro-inflammatory polarization of macrophages, and adipose tissue fibrosis via reducing the abnormal deposition of extracellular matrix. In LPS plus nigericin-stimulated THP-1 macrophages, DOG (1.25, 2.5, 5 μM) dose-dependently suppressed the activation of NLRP3 inflammasome and NF-κB signaling pathway. We demonstrated that DOG bound to and activated the deacetylase Sirtuin 2, which in turn deacetylated and inactivated NLRP3 inflammasome to reduce IL-1β secretion. Moreover, DOG (1.25, 2.5, 5 μM) dose-dependently mitigated inflammatory responses in macrophage conditioned media-treated adipocytes and suppressed macrophage migration toward adipocytes. Taken together, DOG might be a drug candidate to treat metabolic disorders through modulation of adipose tissue remodeling., (© 2022. The Author(s), under exclusive licence to Shanghai Institute of Materia Medica, Chinese Academy of Sciences and Chinese Pharmacological Society.)

Cells undergoing developmental processes are characterized by persistent non-genetic alterations in chromatin, termed epigenetic changes, represented by distinct patterns of DNA methylation and histone post-translational modifications. Sirtuins, a group of conserved NAD(+)-dependent deacetylases or ADP-ribosyltransferases, promote longevity in diverse organisms; however, their molecular mechanisms in ageing regulation remain poorly understood. Yeast Sir2, the first member of the family to be found, establishes and maintains chromatin silencing by removing histone H4 lysine 16 acetylation and bringing in other silencing proteins. Here we report an age-associated decrease in Sir2 protein abundance accompanied by an increase in H4 lysine 16 acetylation and loss of histones at specific subtelomeric regions in replicatively old yeast cells, which results in compromised transcriptional silencing at these loci. Antagonizing activities of Sir2 and Sas2, a histone acetyltransferase, regulate the replicative lifespan through histone H4 lysine 16 at subtelomeric regions. This pathway, distinct from existing ageing models for yeast, may represent an evolutionarily conserved function of sirtuins in regulation of replicative ageing by maintenance of intact telomeric chromatin.

Acetylation within the globular core domain of histone H3 on lysine 56 (H3K56) has recently been shown to have a critical role in packaging DNA into chromatin following DNA replication and repair in budding yeast. However, the function or occurrence of this specific histone mark has not been studied in multicellular eukaryotes, mainly because the Rtt109 enzyme that is known to mediate acetylation of H3K56 (H3K56ac) is fungal-specific. Here we demonstrate that the histone acetyl transferase CBP (also known as Nejire) in flies and CBP and p300 (Ep300) in humans acetylate H3K56, whereas Drosophila Sir2 and human SIRT1 and SIRT2 deacetylate H3K56ac. The histone chaperones ASF1A in humans and Asf1 in Drosophila are required for acetylation of H3K56 in vivo, whereas the histone chaperone CAF-1 (chromatin assembly factor 1) in humans and Caf1 in Drosophila are required for the incorporation of histones bearing this mark into chromatin. We show that, in response to DNA damage, histones bearing acetylated K56 are assembled into chromatin in Drosophila and human cells, forming foci that colocalize with sites of DNA repair. Furthermore, acetylation of H3K56 is increased in multiple types of cancer, correlating with increased levels of ASF1A in these tumours. Our identification of multiple proteins regulating the levels of H3K56 acetylation in metazoans will allow future studies of this critical and unique histone modification that couples chromatin assembly to DNA synthesis, cell proliferation and cancer.

Sirtuin 1 (Sirt1) and Sirtuin 2 (Sirt2) belong to the family of NAD+ (nicotinamide adenine dinucleotide-positive)-dependent class III histone deacetylases and are involved in regulating lifespan. As cancer is a disease of ageing, targeting Sirtuins is emerging as a promising antitumour strategy. Here we present Salermide (N-{3-[(2-hydroxy-naphthalen-1-ylmethylene)-amino]-phenyl}-2-phenyl-propionamide), a reverse amide with a strong in vitro inhibitory effect on Sirt1 and Sirt2. Salermide was well tolerated by mice at concentrations up to 100 muM and prompted tumour-specific cell death in a wide range of human cancer cell lines. The antitumour activity of Salermide was primarily because of a massive induction of apoptosis. This was independent of global tubulin and K16H4 acetylation, which ruled out a putative Sirt2-mediated apoptotic pathway and suggested an in vivo mechanism of action through Sirt1. Consistently with this, RNA interference-mediated knockdown of Sirt1, but not Sirt2, induced apoptosis in cancer cells. Although p53 has been reported to be a target of Sirt1, genetic p53 knockdowns showed that the Sirt1-dependent proapoptotic effect of Salermide is p53-independent. We were finally able to ascribe the apoptotic effect of Salermide to the reactivation of proapoptotic genes epigenetically repressed exclusively in cancer cells by Sirt1. Taken together, our results underline Salermide's promise as an anticancer drug and provide evidence for the molecular mechanism through which Sirt1 is involved in human tumorigenesis.

Cortactin binds F-actin and promotes cell migration. We showed earlier that cortactin is acetylated. Here, we identify SIRT1 (a class III histone deacetylase) as a cortactin deacetylase and p300 as a cortactin acetylase. We show that SIRT1 deacetylates cortactin in vivo and in vitro and that the SIRT1 inhibitor EX-527 increases amounts of acetylated cortactin in ovarian cancer cells. We also show that p300 acetylates cortactin in vivo and that cells lacking or depleted of p300 express less-acetylated cortactin than do control cells. Deletion analysis mapped the SIRT1-binding domain of cortactin to its repeat region, which also binds F-actin. Mouse embryo fibroblasts (MEFs) lacking sir2alpha (the mouse homolog of SIRT1) migrated more slowly than did wild-type cells. The expression of SIRT1 in sir2alpha-null cells restored migratory capacity, as did expression of a deacetylation-mimetic mutant of cortactin. SIRT1 and cortactin were more abundant in breast tumor tissue than in their normal counterparts, whereas SIRT1 expression inversely correlates with the ratio of acetylation cortactin versus total cortactin. These data suggest that deacetylation of cortactin is associated with high levels of SIRT1 and tumorigenesis. Finally, breast and ovarian cancer cell lines expressing an acetylation mimetic mutant of cortactin are less motile than that of control cells, whereas cells expressing the deacetylation mimetic mutant of cortactin migrate faster than that of control cells in Transwell migration assays. In summary, our results suggest that cortactin is a novel substrate for SIRT1 and p300 and, for the first time, a possible role for SIRT1 in cell motility through deacetylation of cortactin.

Modifications on histones control important biological processes through their effects on chromatin structure. Methylation at lysine 4 on histone H3 (H3K4) is found at the 5' end of active genes and contributes to transcriptional activation by recruiting chromatin-remodelling enzymes. An adjacent arginine residue (H3R2) is also known to be asymmetrically dimethylated (H3R2me2a) in mammalian cells, but its location within genes and its function in transcription are unknown. Here we show that H3R2 is also methylated in budding yeast (Saccharomyces cerevisiae), and by using an antibody specific for H3R2me2a in a chromatin immunoprecipitation-on-chip analysis we determine the distribution of this modification on the entire yeast genome. We find that H3R2me2a is enriched throughout all heterochromatic loci and inactive euchromatic genes and is present at the 3' end of moderately transcribed genes. In all cases the pattern of H3R2 methylation is mutually exclusive with the trimethyl form of H3K4 (H3K4me3). We show that methylation at H3R2 abrogates the trimethylation of H3K4 by the Set1 methyltransferase. The specific effect on H3K4me3 results from the occlusion of Spp1, a Set1 methyltransferase subunit necessary for trimethylation. Thus, the inability of Spp1 to recognize H3 methylated at R2 prevents Set1 from trimethylating H3K4. These results provide the first mechanistic insight into the function of arginine methylation on chromatin.

We previously identified SIRT2, an nicotinamide adenine dinucleotide (NAD)-dependent tubulin deacetylase, as a protein downregulated in gliomas and glioma cell lines, which are characterized by aneuploidy. Other studies reported SIRT2 to be involved in mitotic progression in the normal cell cycle. We herein investigated whether SIRT2 functions in the mitotic checkpoint in response to mitotic stress caused by microtubule poisons. By monitoring chromosome condensation, the exogenously expressed SIRT2 was found to block the entry to chromosome condensation and subsequent hyperploid cell formation in glioma cell lines with a persistence of the cyclin B/cdc2 activity in response to mitotic stress. SIRT2 is thus a novel mitotic checkpoint protein that functions in the early metaphase to prevent chromosomal instability (CIN), characteristics previously reported for the CHFR protein. We further found that histone deacetylation, but not the aberrant DNA methylation of SIRT2 5'untranslated region is involved in the downregulation of SIRT2. Although SIRT2 is normally exclusively located in the cytoplasm, the rapid accumulation of SIRT2 in the nucleus was observed after treatment with a nuclear export inhibitor, leptomycin B and ionizing radiation in normal human fibroblasts, suggesting that nucleo-cytoplasmic shuttling regulates the SIRT2 function. Collectively, our results suggest that the further study of SIRT2 may thus provide new insights into the relationships among CIN, epigenetic regulation and tumorigenesis.

In diverse organisms, calorie restriction slows the pace of ageing and increases maximum lifespan. In the budding yeast Saccharomyces cerevisiae, calorie restriction extends lifespan by increasing the activity of Sir2 (ref. 1), a member of the conserved sirtuin family of NAD(+)-dependent protein deacetylases. Included in this family are SIR-2.1, a Caenorhabditis elegans enzyme that regulates lifespan, and SIRT1, a human deacetylase that promotes cell survival by negatively regulating the p53 tumour suppressor. Here we report the discovery of three classes of small molecules that activate sirtuins. We show that the potent activator resveratrol, a polyphenol found in red wine, lowers the Michaelis constant of SIRT1 for both the acetylated substrate and NAD(+), and increases cell survival by stimulating SIRT1-dependent deacetylation of p53. In yeast, resveratrol mimics calorie restriction by stimulating Sir2, increasing DNA stability and extending lifespan by 70%. We discuss possible evolutionary origins of this phenomenon and suggest new lines of research into the therapeutic use of sirtuin activators.

Calorie restriction extends lifespan in a broad range of organisms, from yeasts to mammals. Numerous hypotheses have been proposed to explain this phenomenon, including decreased oxidative damage and altered energy metabolism. In Saccharomyces cerevisiae, lifespan extension by calorie restriction requires the NAD+-dependent histone deacetylase, Sir2 (ref. 1). We have recently shown that Sir2 and its closest human homologue SIRT1, a p53 deacetylase, are strongly inhibited by the vitamin B3 precursor nicotinamide. Here we show that increased expression of PNC1 (pyrazinamidase/nicotinamidase 1), which encodes an enzyme that deaminates nicotinamide, is both necessary and sufficient for lifespan extension by calorie restriction and low-intensity stress. We also identify PNC1 as a longevity gene that is responsive to all stimuli that extend lifespan. We provide evidence that nicotinamide depletion is sufficient to activate Sir2 and that this is the mechanism by which PNC1 regulates longevity. We conclude that yeast lifespan extension by calorie restriction is the consequence of an active cellular response to a low-intensity stress and speculate that nicotinamide might regulate critical cellular processes in higher organisms.

Calorie restriction (CR) extends lifespan in a wide spectrum of organisms and is the only regimen known to lengthen the lifespan of mammals. We established a model of CR in budding yeast Saccharomyces cerevisiae. In this system, lifespan can be extended by limiting glucose or by reducing the activity of the glucose-sensing cyclic-AMP-dependent kinase (PKA). Lifespan extension in a mutant with reduced PKA activity requires Sir2 and NAD (nicotinamide adenine dinucleotide). In this study we explore how CR activates Sir2 to extend lifespan. Here we show that the shunting of carbon metabolism toward the mitochondrial tricarboxylic acid cycle and the concomitant increase in respiration play a central part in this process. We discuss how this metabolic strategy may apply to CR in animals.

In Caenorhabditis elegans, mutations that reduce the activity of an insulin-like receptor (daf-2) or a phosphatidylinositol-3-OH kinase (age-1) favour entry into the dauer state during larval development and extend lifespan in adults. Downregulation of this pathway activates a forkhead transcription factor (daf-16), which may regulate targets that promote dauer formation in larvae and stress resistance and longevity in adults. In yeast, the SIR2 gene determines the lifespan of mother cells, and adding an extra copy of SIR2 extends lifespan. Sir2 mediates chromatin silencing through a histone deacetylase activity that depends on NAD (nicotinamide adenine dinucleotide) as a cofactor. We have surveyed the lifespan of C. elegans strains containing duplications of chromosomal regions. Here we report that a duplication containing sir-2.1-the C. elegans gene most homologous to yeast SIR2-confers a lifespan that is extended by up to 50%. Genetic analysis indicates that the sir-2.1 transgene functions upstream of daf-16 in the insulin-like signalling pathway. Our findings suggest that Sir2 proteins may couple longevity to nutrient availability in many eukaryotic organisms.

Searches for genes involved in the ageing process have been made in genetically tractable model organisms such as yeast, the nematode Caenorhabditis elegans, Drosophila melanogaster fruitflies and mice. These genetic studies have established that ageing is indeed regulated by specific genes, and have allowed an analysis of the pathways involved, linking physiology, signal transduction and gene regulation. Intriguing similarities in the phenotypes of many of these mutants indicate that the mutations may also perturb regulatory systems that control ageing in higher organisms.

Yeast Sir2 is a heterochromatin component that silences transcription at silent mating loci, telomeres and the ribosomal DNA, and that also suppresses recombination in the rDNA and extends replicative life span. Mutational studies indicate that lysine 16 in the amino-terminal tail of histone H4 and lysines 9, 14 and 18 in H3 are critically important in silencing, whereas lysines 5, 8 and 12 of H4 have more redundant functions. Lysines 9 and 14 of histone H3 and lysines 5, 8 and 16 of H4 are acetylated in active chromatin and hypoacetylated in silenced chromatin, and overexpression of Sir2 promotes global deacetylation of histones, indicating that Sir2 may be a histone deacetylase. Deacetylation of lysine 16 of H4 is necessary for binding the silencing protein, Sir3. Here we show that yeast and mouse Sir2 proteins are nicotinamide adenine dinucleotide (NAD)-dependent histone deacetylases, which deacetylate lysines 9 and 14 of H3 and specifically lysine 16 of H4. Our analysis of two SIR2 mutations supports the idea that this deacetylase activity accounts for silencing, recombination suppression and extension of life span in vivo. These findings provide a molecular framework of NAD-dependent histone deacetylation that connects metabolism, genomic silencing and ageing in yeast and, perhaps, in higher eukaryotes.

Transcriptional silencing in Saccharomyces cerevisiae at the HM mating-type loci and telomeres occurs through the formation of a heterochromatin-like structure. HM silencing is regulated by cis-acting elements, termed silencers, and by trans-acting factors that bind to the silencers. These factors attract the four SIR (silent information regulator) proteins, three of which (SIR2-4) spread from the silencers to alter chromatin, hence silencing nearby genes. We show here that an HMR locus with a defective silencer can be silenced by anchoring the locus to the nuclear periphery. This was accomplished by fusing integral membrane proteins to the GAL4 DNA-binding domain and overproducing the hybrid proteins, causing them to accumulate in the endoplasmic reticulum and the nuclear membrane. We expressed the hybrid proteins in a strain carrying an HMR silencer with GAL4-binding sites (UAS(G)) replacing silencer elements, causing the silencer to become anchored to the nuclear periphery and leading to silencing of a nearby reporter gene. This silencing required the hybrids of the GAL4 DNA-binding domain with membrane proteins, the UAS(G) sites and the SIR proteins. Our results indicate that perinuclear localization helps to establish transcriptionally silent chromatin.

Transcriptional silencing of the HM mating-type loci in the yeast Saccharomyces cerevisiae is caused by the localized formation of an altered chromatin structure, analogous to heterochromatin in higher eukaryotes. Silencing depends on cis-acting sequences, termed silencers, as well as several trans-acting factors, including histones H4 and H3, proteins RAP1 and ABF1, and the four SIR proteins (SIR1-4). Each of the four HM silencers contains an autonomously replicating sequence (ARS) to which the origin replication complex (ORC) binds. This six-protein complex is required for initiation of DNA replication, as well as for silencing. Efficient establishment of the silenced state requires both passage through the S phase of the cell cycle and SIR1 protein. Previous experiments suggested that SIR1 might be localized to the silencers by binding to ORC and/or RAP1. Here we report that SIR1 can bind directly to ORC1, the largest of the ORC subunits, and that targeting of SIR1 to ORC1 at a silencer is sufficient to establish a silenced state.