ε-Poly-l-lysine: A Naturally Occurring Biodegradable Polypeptide for Selective Detection of 5-Nitroimidazole Antibiotics in Animal Products and Living Cells via Fluorescence.

The 5-nitroimidazole (5-NI) class of antibiotics, such as metronidazole, ornidazole, secnidazole, and tinidazole, are widely used to prevent bacterial infection in humans and livestock industries. However, their overuse contaminates the farmed animal products and water bodies. Hence, a selective, sensitive, and cost-effective method to detect 5-NI antibiotics is the need of the hour. Herein, we report a rapid, inexpensive, and efficient sensing system to detect 5-NI drugs using an as-prepared solution of ε-poly-l-lysine (ε-PL), a naturally occurring and biodegradable homopolypeptide that has an intrinsic fluorescence via clustering-triggered emission. The low nanomolar detection limit (3.25-3.97 nM) for the aforementioned representative 5-NI drugs highlights the sensitivity of the system, outperforming most of the reported sensors alike. The resulting fluorescence quenching was found to be static in nature. Importantly, excellent recovery (100.26-104.41%) was obtained for all real samples and animal products tested. Visual detection was demonstrated by using paper strips and silica gel for practical applications. Furthermore, ε-PL could detect 5-NI antibiotics in living 3T3-L1 mouse fibroblast cells via cellular imaging. Taken together, the present work demonstrates the detection of 5-NI antibiotics using a biocompatible natural polypeptide, ε-PL, and represents a simple and inexpensive analytical tool for practical application.

Identification of selective plant-derived natural carotenoid and flavonoids as the potential inhibitors of DHHC-mediated protein S-palmitoylation: an in silico study.

Protein S-palmitoylation mediated by DHHCs is recognized as a distinct and reversible form of lipid modification connected with several health perturbations, including neurodegenerative disorders, cancer, and autoimmune conditions. However, the pharmacological characteristics of current pan-DHHC inhibitors, particularly their toxicity and off-target effects, have hindered their in-depth cellular investigations. The therapeutic properties of the natural compounds, with minimal side effects, allowed us to evaluate them as DHHC-targeting inhibitors. Here, we performed an insilico screening of 115 phytochemicals to assess their interactions with the DHHC20 binding site. Among these compounds, lutein, 5-hydroxyflavone, and 6-hydroxyflavone exhibited higher binding energy (-9.2, -8.5, and -8.5 kcal/mol) in the DHHC20 groove compared to pan-DHHC inhibitor 2-BP (-7.0 kcal/mol). Furthermore, we conducted a 100 ns MD simulation to evaluate the stability of these complexes under physiological conditions. The MDsimulation results indicated that DHHC20 formed a more stable conformation with lutein compared to 5-hydroxyflavone and 6-hyroxyflavone via hydrophobic and H-bond interactions. Conclusively, these results could serve as a promising starting point for exploring the use of these natural molecules as DHHC20 inhibitors.Communicated by Ramaswamy H. Sarma.

Novel molecular hybrids of EGCG and quinoxaline: Potent multi-targeting antidiabetic agents that inhibit α-glucosidase, α-amylase, and oxidative stress.

Diabetes mellitus is a multifactorial disease and its effective therapy often demands several drugs with different modes of action. Herein, we report a rational design and synthesis of multi-targeting novel molecular hybrids comprised of EGCG and quinoxaline derivatives that can effectively inhibit α-glucosidase, α-amylase as well as control oxidative stress by scavenging ROS. The hybrids showed superior inhibition of α-glucosidase along with similar α-amylase inhibition as compared to standard drug, acarbose. Most potent compound, 15c showed an IC50 of 0.50 µM (IC50 of acarbose 190 µM) against α-glucosidase. Kinetics studies with 15c revealed a competitive inhibition against α-glucosidase. Binding affinity of 15c (-9.5 kcal/mol) towards α-glucosidase was significantly higher than acarbose (-7.7 kcal/mol). 15c exhibited remarkably high antioxidant activity (IC50 = 18.84 µM), much better than vitamin C (IC50 = 33.04 µM). Of note, acarbose shows no antioxidant activity. Furthermore, α-amylase activity was effectively inhibited by 15c with an IC50 value of 16.35 µM. No cytotoxicity was observed for 15c (up to 40 µM) in MCF-7 cells. Taken together, we report a series of multi-targeting molecular hybrids capable of inhibiting carbohydrate hydrolysing enzymes as well as reducing oxidative stress, thus representing an advancement towards effective and novel therapeutic approaches for diabetes.

4″-Alkyl EGCG Derivatives Induce Cytoprotective Autophagy Response by Inhibiting EGFR in Glioblastoma Cells.

Epidermal growth factor receptor (EGFR)-targeted therapy has been proven vital in the last two decades for the treatment of multiple cancer types, including nonsmall cell lung cancer, glioblastoma, breast cancer and head and neck squamous cell carcinoma. Unfortunately, the majority of approved EGFR inhibitors fall into the drug resistance category because of continuous mutations and acquired resistance. Recently, autophagy has surfaced as one of the emerging underlying mechanisms behind resistance to EGFR-tyrosine kinase inhibitors (TKIs). Previously, we developed a series of 4″-alkyl EGCG (4″-Cn EGCG, n = 6, 8, 10, 12, 14, 16, and 18) derivatives with enhanced anticancer effects and stability. Therefore, the current study hypothesized that 4″-alkyl EGCG might induce cytoprotective autophagy upon EGFR inhibition, and inhibition of autophagy may lead to improved cytotoxicity. In this study, we have observed growth inhibition and caspase-3-dependent apoptosis in 4″-alkyl EGCG derivative-treated glioblastoma cells (U87-MG). We also confirmed that 4″-alkyl EGCG could inhibit EGFR in the cells, as well as mutant L858R/T790M EGFR, through an in vitro kinase assay. Furthermore, we have found that EGFR inhibition with 4″-alkyl EGCG induces cytoprotective autophagic responses, accompanied by the blockage of the AKT/mTOR signaling pathway. In addition, cytotoxicity caused by 4″-C10 EGCG, 4″-C12 EGCG, and 4″-C14 EGCG was significantly increased after the inhibition of autophagy by the pharmacological inhibitor chloroquine. These findings enhance our understanding of the autophagic response toward EGFR inhibitors in glioblastoma cells and suggest a potent combinatorial strategy to increase the therapeutic effectiveness of EGFR-TKIs.

Protein S-palmitoylation is markedly inhibited by 4″-alkyl ether lipophilic derivatives of EGCG, the major green tea polyphenol: In vitro and in silico studies.

S-palmitoylation is a dynamic lipid-based protein post-translational modification facilitated by a family of protein acyltransferases (PATs) commonly known as DHHC-PATs or DHHCs. It is the only lipid modification that is reversible, and this very fact uniquely qualifies it for therapeutic interventions through the development of DHHC inhibitors. Herein, we report that 4″-alkyl ether lipophilic derivatives of EGCG can effectively inhibit protein S-palmitoylation in vitro. With the help of metabolic labeling followed by copper(I)-catalyzed azide-alkyne cycloaddition Click reaction, we demonstrate that 4″-C14 EGCG and 4″-C16 EGCG markedly inhibited S-palmitoylation in various mammalian cells including HEK 293T, HeLa, and MCF-7 using both in gel fluorescence as well as confocal microscopy. Further, these EGCG derivatives were able to attenuate the S-palmitoylation to the basal level in DHHC3-overexpressed cells, suggesting that they are plausibly targeting DHHCs. Confocal microscopy data qualitatively reflected spatial and temporal distribution of S-palmitoylated proteins in different sub-cellular compartments and the inhibitory effects of 4″-C14 EGCG and 4″-C16 EGCG were clearly observed in the native cellular environment. Our findings were further substantiated by in silico analysis which revealed promising binding affinity and interactions of 4″-C14 EGCG and 4″-C16 EGCG with key amino acid residues present in the hydrophobic cleft of the DHHC20 enzyme. We also demonstrated the successful inhibition of S-palmitoylation of GAPDH by 4″-C16 EGCG. Taken together, our in vitro and in silico data strongly suggest that 4″-C14 EGCG and 4″-C16 EGCG can act as potent inhibitors for S-palmitoylation and can be employed as a complementary tool to investigate S-palmitoylation.

Mitochondria-targeting EGCG derivatives protect H9c2 cardiomyocytes from H2O2-induced apoptosis: design, synthesis and biological evaluation.

Pathologies related to cardiovascular diseases mostly emerge as a result of oxidative stress buildup in cardiomyocytes. The heavy load of mitochondrial oxidative phosphorylation in cardiac tissues corresponds to a surge in oxidative stress leading to mitochondrial dysfunction and cellular apoptosis. Thus, scavenging the reactive oxygen species (ROS) linked to mitochondria can significantly improve cardio-protection. Epigallocatechin-3-gallate (EGCG), the major polyphenol found in green tea has been extensively studied for its profound health-beneficial activities. Herein, we designed and synthesized a series of mitochondrial-targeting EGCG derivatives, namely MitoEGCGn (n = 4, 6, 8) by incorporating triphenylphosphonium ion onto it using different linkers. MitoEGCGn were found to be non-toxic to H9c2 rat cardiomyocyte cells even at higher doses in comparison to its parent molecule EGCG. Interestingly, MitoEGCG4 and MitoEGCG6 protected the H9c2 cardiomyocyte cells from the oxidative damage induced by H2O2 whereas EGCG was found to be toxic and ineffective in protecting the cells from H2O2 damage. MitoEGCG4 and MitoEGCG6 also protected the cells from the H2O2-induced disruption of mitochondrial membrane potential as well as activation of apoptosis as revealed by pro-caspase 3 expression profile, DNA fragmentation assay, and AO/EtBr staining. Taken together, our study shows that the mitochondria targeting EGCG derivatives were able to effectively combat the H2O2-induced oxidative stress in H9c2 cardiomyocytes. They eventually augmented the mitochondrial health of cardiomyocytes by maintaining the mitochondrial function and attenuating apoptosis. Overall, MitoEGCG4 and MitoEGCG6 could provision a cardioprotective role to H9c2 cardiomyocytes at the time of oxidative insults related to mitochondrial dysfunction-associated injuries.

20 years since the approval of first EGFR-TKI, gefitinib: Insight and foresight.

Epidermal growth factor receptor (EGFR) actively involves in modulation of various cancer progression related mechanisms including angiogenesis, differentiation and migration. Therefore, targeting EGFR has surfaced as a prominent approach for the treatment of several types of cancers, including non-small cell lung cancer (NSCLC), pancreatic cancer, glioblastoma. Various first, second and third generation of EGFR tyrosine kinase inhibitors (EGFR-TKIs) have demonstrated effectiveness as an anti-cancer therapeutics. However, rapid development of drug resistance and mutations still remains a major challenge for the EGFR-TKIs therapy. Overcoming from intrinsic and acquired resistance caused by EGFR mutations warrants the further exploration of alternative strategies and discovery of novel inhibitors. In this review, we delve into the breakthrough discoveries have been made in previous 20 years, and discuss the currently ongoing efforts aimed to circumvent the chemo-resistance. We also highlight the new challenges, limitations and future directions for the development of improved therapeutic approaches such as fourth-generation EGFR-TKIs, peptides, nanobodies, PROTACs etc.

Role of EGFR and FASN in breast cancer progression.

Breast cancer (BC) emerged as one of the life-threatening diseases among females. Despite notable improvements made in cancer detection and treatment worldwide, according to GLOBACAN 2020, BC is the fifth leading cancer, with an estimated 1 in 6 cancer deaths, in a majority of countries. However, the exact cause that leads to BC progression still needs to be determined. Here, we reviewed the role of two novel biomarkers responsible for 50-70% of BC progression. The first one is epidermal growth factor receptor (EGFR) which belongs to the ErbB tyrosine kinases family, signalling pathways associated with it play a significant role in regulating cell proliferation and division. Another one is fatty acid synthase (FASN), a key enzyme responsible for the de novo lipid synthesis required for cancer cell development. This review presents a rationale for the EGFR-mediated pathways, their interaction with FASN, communion of these two biomarkers with BC, and improvements to overcome drug resistance caused by them.

In situ global proteomics profiling of EGCG targets using a cell-permeable and Click-able bioorthogonal probe.

Despite possessing a wide spectrum of biological activities, molecular targets of EGCG remain elusive and as a result, its precise mode of action is still unknown. Herein, we have developed a novel cell-permeable and Click-able bioorthogonal probe for EGCG, YnEGCG for in situ detection and identification of its interacting proteins. The strategic structural modification on YnEGCG allowed it to retain innate biological activities of EGCG (IC50 59.52 ± 1.14 µM and 9.07 ± 0.01 µM for cell viability and radical scavenging activity, respectively). Chemoproteomics profiling identified 160 direct EGCG targets, with H:L ratio ≥ 1.10 from the list of 207 proteins, including multiple new proteins that were previously unknown. The targets were broadly distributed in various subcellular compartments suggesting a polypharmacological mode of action of EGCG. GO analysis revealed that the primary targets belonged to the enzymes that regulate key metabolic processes including glycolysis and energy homeostasis, also the cytoplasm (36 %) and mitochondria (15.6 %) contain the majority of EGCG targets. Further, we validated that EGCG interactome was closely associated with apoptosis indicating its role in inducing toxicity in cancer cells. For the first time, this in situ chemoproteomics approach could identify a direct and specific EGCG interactome under physiological conditions in an unbiased manner.

Chemico-biological aspects of (-)-epigallocatechin-3-gallate (EGCG) to improve its stability, bioavailability and membrane permeability: Current status and future prospects.

Natural products have been a bedrock for drug discovery for decades. (-)-Epigallocatechin-3-gallate (EGCG) is one of the widely studied natural polyphenolic compounds derived from green tea. It is the key component believed to be responsible for the medicinal value of green tea. Significant studies implemented in in vitro, in cellulo, and in vivo models have suggested its anti-oxidant, anti-cancer, anti-diabetic, anti-inflammatory, anti-microbial, neuroprotective activities etc. Despite having such a wide array of therapeutic potential and promising results in preclinical studies, its applicability to humans has encountered with rather limited success largely due to the poor bioavailability, poor membrane permeability, rapid metabolic clearance and lack of stability of EGCG. Therefore, novel techniques are warranted to address those limitations so that EGCG or its modified analogs can be used in the clinical setup. This review comprehensively covers different strategies such as structural modifications, nano-carriers as efficient drug delivery systems, synergistic studies with other bioactivities to improve the chemico-biological aspects (e.g., stability, bioavailability, permeability, etc.) of EGCG for its enhanced pharmacokinetics and pharmacological properties, eventually enhancing its therapeutic potentials. We think this review article will serve as a strong platform with comprehensive literature on the development of novel techniques to improve the bioavailability of EGCG so that it can be translated to the clinical applications.

Structure-based design and synthesis of a novel long-chain 4''-alkyl ether derivative of EGCG as potent EGFR inhibitor: in vitro and in silico studies.

Herein, we report the discovery of a novel long-chain ether derivative of (-)-epigallocatechin-3-gallate (EGCG), a major green tea polyphenol as a potent EGFR inhibitor. A series of 4''-alkyl EGCG derivatives have been synthesized via regio-selectively alkylating the 4'' hydroxyl group in the D-ring of EGCG and tested for their antiproliferative activities against high (A431), moderate (HeLa), and low (MCF-7) EGFR-expressing cancer cell lines. The most potent compound, 4''-C14 EGCG showed the lowest IC50 values across all the tested cell lines. 4''-C14 EGCG was also found to be significantly more stable than EGCG under physiological conditions (PBS at pH 7.4). Further western blot analysis and imaging data revealed that 4''-C14 EGCG induced cell death in A431 cells with shrunken nuclei, nuclear fragmentation, membrane blebbing, and increased population of apoptotic cells where BAX upregulation and BCLXL downregulation were observed. In addition, autophosphorylation of EGFR and its downstream signalling proteins Akt and ERK were markedly inhibited by 4''-C14 EGCG. MD simulation and the MM/PBSA analysis disclosed the binding mode of 4''-C14 EGCG in the ATP-binding site of EGFR kinase domain. Taken together, our findings demonstrate that 4''-C14 EGCG can act as a promising potent EGFR inhibitor with enhanced stability.

Pharmacological and genetic perturbation establish SIRT5 as a promising target in breast cancer.

SIRT5 is a member of the sirtuin family of NAD+-dependent protein lysine deacylases implicated in a variety of physiological processes. SIRT5 removes negatively charged malonyl, succinyl, and glutaryl groups from lysine residues and thereby regulates multiple enzymes involved in cellular metabolism and other biological processes. SIRT5 is overexpressed in human breast cancers and other malignancies, but little is known about the therapeutic potential of SIRT5 inhibition for treating cancer. Here we report that genetic SIRT5 disruption in breast cancer cell lines and mouse models caused increased succinylation of IDH2 and other metabolic enzymes, increased oxidative stress, and impaired transformation and tumorigenesis. We, therefore, developed potent, selective, and cell-permeable small-molecule SIRT5 inhibitors. SIRT5 inhibition suppressed the transformed properties of cultured breast cancer cells and significantly reduced mammary tumor growth in vivo, in both genetically engineered and xenotransplant mouse models. Considering that Sirt5 knockout mice are generally normal, with only mild phenotypes observed, these data establish SIRT5 as a promising target for treating breast cancer. The new SIRT5 inhibitors provide useful probes for future investigations of SIRT5 and an avenue for targeting SIRT5 as a therapeutic strategy.

Plant-derived natural polyphenols as potential antiviral drugs against SARS-CoV-2 via RNA-dependent RNA polymerase (RdRp) inhibition: an in-silico analysis.

The sudden outburst of Coronavirus disease (COVID-19) caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) poses a massive threat to global public health. Currently, no therapeutic drug or vaccine exists to treat COVID-19. Due to the time taking process of new drug development, drug repurposing might be the only viable solution to tackle COVID-19. RNA-dependent RNA polymerase (RdRp) catalyzes SARS-CoV-2 RNA replication and hence, is an obvious target for antiviral drug design. Interestingly, several plant-derived polyphenols effectively inhibit the RdRp of other RNA viruses. More importantly, polyphenols have been used as dietary supplementations for a long time and played beneficial roles in immune homeostasis. We were curious to study the binding of polyphenols with SARS-CoV-2 RdRp and assess their potential to treat COVID-19. Herein, we made a library of polyphenols that have shown substantial therapeutic effects against various diseases. They were successfully docked in the catalytic pocket of RdRp. The investigation reveals that EGCG, theaflavin (TF1), theaflavin-3'-O-gallate (TF2a), theaflavin-3'-gallate (TF2b), theaflavin 3,3'-digallate (TF3), hesperidin, quercetagetin, and myricetin strongly bind to the active site of RdRp. Further, a 150-ns molecular dynamic simulation revealed that EGCG, TF2a, TF2b, TF3 result in highly stable bound conformations with RdRp. The binding free energy components calculated by the MM-PBSA also confirm the stability of the complexes. We also performed a detailed analysis of ADME prediction, toxicity prediction, and target analysis for their druggability. Overall, our results suggest that EGCG, TF2a, TF2b, TF3 can inhibit RdRp and represent an effective therapy for COVID-19.Communicated by Ramaswamy H. Sarma.

An in-silico study on selected organosulfur compounds as potential drugs for SARS-CoV-2 infection via binding multiple drug targets.

The emerging paradigm shift from 'one molecule, one target, for one disease' towards 'multi-targeted small molecules' has paved an ingenious pathway in drug discovery in recent years. We extracted this idea for the investigation of drugs for COVID-19. Perceiving the importance of organosulfur compounds, seventy-six known organosulfur compounds were screened and studied for the interaction with multiple SARS-CoV-2 target proteins by molecular dynamics simulation. Lurasidone and its derivatives displayed substantial binding affinity against five proteins (Mpro, PLpro, Spro, helicase and RdRp). The pharmacokinetics, ADMET properties and target prediction studies performed in this work further potentiates the effectiveness against SARS-CoV-2.

Direct Comparison of SIRT2 Inhibitors: Potency, Specificity, Activity-Dependent Inhibition, and On-Target Anticancer Activities.

Sirtuin inhibitors have attracted much interest due to the involvement of sirtuins in various biological processes. Several SIRT2-selective inhibitors have been developed, and some exhibit anticancer activities. To facilitate the choice of inhibitors in future studies and the development of better inhibitors, we directly compared several reported SIRT2-selective inhibitors: AGK2, SirReal2, Tenovin-6, and TM. In vitro, TM is the most potent and selective inhibitor, and only TM could inhibit the demyristoylation activity of SIRT2. SirReal2, Tenovin-6, and TM all showed cytotoxicity in cancer cell lines, with Tenovin-6 being the most potent, but only TM showed cancer-cell-specific toxicity. All four compounds inhibited the anchorage-independent growth of HCT116 cells, but the effect of TM was most significantly affected by SIRT2 overexpression, suggesting that the anticancer effect of TM depends more on SIRT2 inhibition. These results not only provide useful guidance about choosing the right SIRT2 inhibitor in future studies, but also suggest general practices that should be followed for small-molecule inhibitor development activities.

Emerging Roles of DHHC-mediated Protein S-palmitoylation in Physiological and Pathophysiological Context.

Protein S-palmitoylation refers to a post-translational modification (PTM) wherein palmitic acid, a 16-carbon long saturated fatty acid gets covalently attached to Cys sidechain of a protein. It has been known to the literature for almost 50 years and in general, this PTM is believed to facilitate membrane attachments of proteins for the obvious hydrophobicity of the palmitoyl group. But after the discovery of the protein palmitoyl acyltransferases (PATs, also known as DHHC-PATs), a major paradigm shift has been observed in the field of protein S-palmitoylation. A family of 23 mammalian DHHC-PATs has been identified and the majority of them are associated with many human diseases spanning from neuropsychiatric diseases to cancers. Novel unique and essential role of DHHC-mediated protein S-palmitoylation has been revealed apart from its membrane trafficking role. Biomedical importance of DHHCs has also been reiterated with small molecule inhibitors for DHHCs as well as in DHHC-knockout mice or mouse Xenograft models. In this review, we present recent advances in the field of protein S-palmitoylation and the involvement of individual DHHC isoforms in human diseases. In addition, the recent development of the analytical tools to study S-palmitoylation and their inhibitors are discussed in detail. We also highlight the issues that need to be addressed in detail to further develop our understanding on protein S-palmitoylation and strongly believe that pharmacological modulation of DHHC-mediated protein S-palmitoylation has a massive potential to emerge as a novel therapeutic strategy for human diseases. It will not be surprising if reversible protein S-palmitoylation prove to be an indispensable PTM that regulates a host of cellular processes, just like protein phosphorylation or ubiquitination.

SIRT7 Is an RNA-Activated Protein Lysine Deacylase.

Mammalian SIRT7 is a member of the sirtuin family that regulates multiple biological processes including genome stability, metabolic pathways, stress responses, and tumorigenesis. SIRT7 has been shown to be important for ribosome biogenesis and transcriptional regulation. SIRT7 knockout mice exhibit complications associated with fatty liver and increased aging in hematopoietic stem cells. However, the molecular basis for its biological function remains unclear, in part due to the lack of efficient enzymatic activity in vitro. Previously, we have demonstrated that double-stranded DNA could activate SIRT7's deacetylase activity in vitro, allowing it to deacetylate H3K18 in the context of chromatin. Here, we show that RNA can increase the catalytic efficiency of SIRT7 even better and that SIRT7 can remove long chain fatty acyl groups more efficiently than removing acetyl groups. Truncation and mutagenesis studies revealed residues at both the amino and carboxyl termini of SIRT7 that are involved in RNA-binding and important for activity. RNA immunoprecipitation-sequencing (RIP-seq) identified ribosomal RNA (rRNA) as the predominant RNA binding partner of SIRT7. The associated RNA was able to effectively activate the deacetylase and defatty-acylase activities of SIRT7. Knockdown of SIRT7 increased the lysine fatty acylation of several nuclear proteins based on metabolic labeling with an alkyne-tagged fatty acid analog, supporting that the defatty-acylase activity of SIRT7 is physiologically relevant. These findings provide important insights into the biological functions of SIRT7, as well as an improved platform to develop SIRT7 modulators.

Metabolomics-assisted proteomics identifies succinylation and SIRT5 as important regulators of cardiac function.

Cellular metabolites, such as acyl-CoA, can modify proteins, leading to protein posttranslational modifications (PTMs). One such PTM is lysine succinylation, which is regulated by sirtuin 5 (SIRT5). Although numerous proteins are modified by lysine succinylation, the physiological significance of lysine succinylation and SIRT5 remains elusive. Here, by profiling acyl-CoA molecules in various mouse tissues, we have discovered that different tissues have different acyl-CoA profiles and that succinyl-CoA is the most abundant acyl-CoA molecule in the heart. This interesting observation has prompted us to examine protein lysine succinylation in different mouse tissues in the presence and absence of SIRT5. Protein lysine succinylation predominantly accumulates in the heart whenSirt5is deleted. Using proteomic studies, we have identified many cardiac proteins regulated by SIRT5. Our data suggest that ECHA, a protein involved in fatty acid oxidation, is a major enzyme that is regulated by SIRT5 and affects heart function.Sirt5knockout (KO) mice have lower ECHA activity, increased long-chain acyl-CoAs, and decreased ATP in the heart under fasting conditions.Sirt5KO mice develop hypertrophic cardiomyopathy, as evident from the increased heart weight relative to body weight, as well as reduced shortening and ejection fractions. These findings establish that regulating heart metabolism and function is a major physiological function of lysine succinylation and SIRT5.

SIRT7 Is Activated by DNA and Deacetylates Histone H3 in the Chromatin Context.

Mammalian sirtuins (SIRT1-7) are members of a highly conserved family of nicotinamide adenine dinucleotide (NAD(+))-dependent protein deacetylases that regulate many biological processes including metabolism, genome stability, and transcription. Among the seven human sirtuins, SIRT7 is the least understood, to a large extent due to the lack of enzymatic activity in vitro. Here, we reported that SIRT7 can be activated by DNA to hydrolyze the acetyl group from lysine residues in vitro on histone peptides and histones in the chromatin context. Both N- and C- termini of SIRT7 are important for the DNA-activated deacetylase activity. The regulatory mechanism of SIRT7 is different from that of SIRT6, which also showed increased activity on chromatin substrates, but the deacetylase activity of SIRT6 on a peptide substrate cannot be activated by DNA. This finding provides an improved enzymatic activity assay of SIRT7 that will promote the development of SIRT7 modulators. Further investigation into the activation mechanism of SIRT7 by DNA could provide new insights into its biological function and help the development of sirtuin activators.

High-Resolution Metabolomics with Acyl-CoA Profiling Reveals Widespread Remodeling in Response to Diet.

The availability of acyl-Coenzyme A (acyl-CoA) thioester compounds affects numerous cellular functions including autophagy, lipid oxidation and synthesis, and post-translational modifications. Consequently, the acyl-CoA level changes tend to be associated with other metabolic alterations that regulate these critical cellular functions. Despite their biological importance, this class of metabolites remains difficult to detect and quantify using current analytical methods. Here we show a universal method for metabolomics that allows for the detection of an expansive set of acyl-CoA compounds and hundreds of other cellular metabolites. We apply this method to profile the dynamics of acyl-CoA compounds and corresponding alterations in metabolism across the metabolic network in response to high fat feeding in mice. We identified targeted metabolites (>50) and untargeted features (>1000) with significant changes (FDR < 0.05) in response to diet. A substantial extent of this metabolic remodeling exhibited correlated changes in acyl-CoA metabolism with acyl-carnitine metabolism and other features of the metabolic network that together can lead to the discovery of biomarkers of acyl-CoA metabolism. These findings show a robust acyl-CoA profiling method and identify coordinated changes of acyl-CoA metabolism in response to nutritional stress.