Sirtuins, NAD(+) -dependent histone deacetylases (HDACs), have recently emerged as potential therapeutic targets for the treatment of a variety of diseases. The discovery of potent and isoform-selective inhibitors of this enzyme family should provide chemical tools to help determine the roles of these targets and validate their therapeutic value. Herein, we report the discovery of a novel class of highly selective SIRT2 inhibitors, identified by pharmacophore screening. We report the identification and validation of 3-((2-methoxynaphthalen-1-yl)methyl)-7-((pyridin-3-ylmethyl)amino)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4(3H)-one (ICL-SIRT078), a substrate-competitive SIRT2 inhibitor with a Ki value of 0.62±0.15??M and more than 50-fold selectivity against SIRT1, 3 and?5. Treatment of MCF-7 breast cancer cells with ICL-SIRT078 results in hyperacetylation of ?-tubulin, an established SIRT2 biomarker, at doses comparable with the biochemical IC50 data, while suppressing MCF-7 proliferation at higher concentrations. In concordance with the recent reports that suggest SIRT2 inhibition is a potential strategy for the treatment of Parkinson's disease, we find that compound ICL-SIRT078 has a significant neuroprotective effect in a lactacystin-induced model of Parkinsonian neuronal cell death in the N27 cell line. These results encourage further investigation into the effects of ICL-SIRT078, or an optimised derivative thereof, as a candidate neuroprotective agent in in vivo models of Parkinson's disease.
The second messenger cyclic adenosine 3',5'-monophosphate (cAMP) regulates a wide range of physiological processes in almost all organisms. cAMP synthesis is catalyzed by adenylyl cyclases (ACs). All ten mammalian AC isoenzymes (AC1-10) belong to AC Class III, which is defined by sequence homologies in the catalytic domains. Nevertheless, the mammalian AC can be separated into two distinct types, nine transmembrane enzymes (tmAC; AC1-9) and one soluble AC (sAC; AC10). tmACs are mainly regulated by heterotrimeric G-proteins as part of the G-protein coupled receptor pathways, while sAC is directly activated by bicarbonate and Ca(2+) and acts as a sensor for ATP, Ca(2+), and bicarbonate/CO2/pH at various intracellular locations. Mammalian sAC has been implicated in processes such as sperm activation, glucose metabolism, and prostate and skin cancer, making it a potential therapeutic target, and first sAC-specific inhibitors have been developed. Mammalian sAC appears evolutionarily closer related to microbial Class III ACs than to tmACs, and sAC-like bicarbonate activated ACs are indeed found in lower organisms and can contribute, e.g., to virulence regulation in microbial pathogens. Here, we review work on the architecture, catalysis, and physiological and pharmacological regulation of sAC-like enzymes, with a main focus on the mammalian enzyme. We further compare the biochemical, regulatory, and structural characteristics of sAC-like enzymes to the evolutionarily and structurally related mammalian tmACs, pointing out common features as well as sAC-specific properties and modulators. This article is part of a Special Issue entitled: The role of soluble adenylyl cyclase in health and disease.
Sirtuins are NAD(+)-dependent deacetylases acting as sensors in metabolic pathways and stress response. In mammals there are seven isoforms. The mitochondrial sirtuin?5 is a weak deacetylase but a very efficient demalonylase and desuccinylase; however, its substrate acyl specificity has not been systematically analyzed. Herein, we investigated a carbamoyl phosphate synthetase?1 derived peptide substrate and modified the lysine side chain systematically to determine the acyl specificity of Sirt5. From that point we designed six potent peptide-based inhibitors that interact with the NAD(+) binding pocket. To characterize the interaction details causing the different substrate and inhibition properties we report several X-ray crystal structures of Sirt5 complexed with these peptides. Our results reveal the Sirt5 acyl selectivity and its molecular basis and enable the design of inhibitors for Sirt5.
Local levels of active thyroid hormone (3,3',5-triiodothyronine) are controlled by the action of activating and inactivating iodothyronine deiodinase enzymes. Deiodinases are selenocysteine-dependent membrane proteins catalyzing the reductive elimination of iodide from iodothyronines through a poorly understood mechanism. We solved the crystal structure of the catalytic domain of mouse deiodinase 3 (Dio3), which reveals a close structural similarity to atypical 2-Cys peroxiredoxin(s) (Prx). The structure suggests a route for proton transfer to the substrate during deiodination and a Prx-related mechanism for subsequent recycling of the transiently oxidized enzyme. The proposed mechanism is supported by biochemical experiments and is consistent with the effects of mutations of conserved amino acids on Dio3 activity. Thioredoxin and glutaredoxin reduce the oxidized Dio3 at physiological concentrations, and dimerization appears to activate the enzyme by displacing an autoinhibitory loop from the iodothyronine binding site. Deiodinases apparently evolved from the ubiquitous Prx scaffold, and their structure and catalytic mechanism reconcile a plethora of partly conflicting data reported for these enzymes.
The ubiquitous second messenger cAMP regulates a wide array of functions, from bacterial transcription to mammalian memory. It is synthesized by six evolutionarily distinct adenylyl cyclase (AC) families. In mammals, there are two AC types: nine transmembrane ACs (tmACs) and one soluble AC (sAC). Both AC types belong to the widespread cyclase class III, which has members in numerous organisms from archaeons to mammals. Class III also contains all known guanylyl cyclases (GCs), which synthesize the cAMP-related messenger cGMP in many eukaryotes and possibly some prokaryotes. Among mammalian ACs, sAC is uniquely regulated by bicarbonate, and has been proposed to be more closely related to a bacterial AC subfamily than to mammalian ACs, on the basis of sequence comparisons. Here, we used crystal structures of human sAC catalytic domains to analyze its relationships with other class III ACs and GCs, and to study its substrate selection mechanisms. Structural comparisons revealed a similarity within an sAC-like subfamily but no family-specific structure elements, and an unexpected sAC similarity to eukaryotic GCs and a potential bacterial GC. We further solved novel crystal structures of sAC catalytic domains in complex with a substrate analog, unprocessed ATP substrate, and product after soaking with ATP or GTP. The structures show a novel ATP-binding conformation, and suggest mechanisms for substrate association and recognition. Our results could explain the limited substrate specificity of sAC, suggest how specificity is increased in other cyclases, and indicate evolutionary relationships among class III enzymes, with sAC being close to a putative 'ancestor' cyclase.
cAMP is an evolutionary conserved, prototypic second messenger regulating numerous cellular functions. In mammals, cAMP is synthesized by one of 10 homologous adenylyl cyclases (ACs): nine transmembrane enzymes and one soluble AC (sAC). Among these, only sAC is directly activated by bicarbonate (HCO3(-)); it thereby serves as a cellular sensor for HCO3(-), carbon dioxide (CO2), and pH in physiological functions, such as sperm activation, aqueous humor formation, and metabolic regulation. Here, we describe crystal structures of human sAC catalytic domains in the apo state and in complex with substrate analog, products, and regulators. The activator HCO3(-) binds adjacent to Arg176, which acts as a switch that enables formation of the catalytic cation sites. An anionic inhibitor, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, inhibits sAC through binding to the active site entrance, which blocks HCO3(-) activation through steric hindrance and trapping of the Arg176 side chain. Finally, product complexes reveal small, local rearrangements that facilitate catalysis. Our results provide a molecular mechanism for sAC catalysis and cellular HCO3(-) sensing and a basis for targeting this system with drugs.
Blue mussels adhere to surfaces by the byssus, a holdfast structure composed of individual threads representing a collagen fibre reinforced composite. Here, we present the crystal structure and function of one of its matrix proteins, the proximal thread matrix protein 1, which is present in the proximal section of the byssus. The structure reveals two von Willebrand factor type A domains linked by a two-?-stranded linker yielding a novel structural arrangement. In vitro, the protein binds heterologous collagens with high affinity and affects collagen assembly, morphology and arrangement of its fibrils. By providing charged surface clusters as well as insufficiently coordinated metal ions, the proximal thread matrix protein 1 might interconnect other byssal proteins and thereby contribute to the integrity of the byssal threads in vivo. Moreover, the protein could be used for adjusting the mechanical properties of collagen materials, a function likely important in the natural byssus.
Acetylation is a common post-translational modification of proteins but still challenging to analyse. Only few acetylome studies have been performed to tackle this issue. Yet, the detection of acetylated proteins in complex cell lysates remains to be improved. Here, we present a proteomic approach with proteinase K as a suitable protease to identify acetylated peptides quantitatively. We first optimized the digestions conditions using an artificial system of purified bovine histones to find the optimal protease. Subsequently, the capability of proteinase K was demonstrated in complex HEK293 cell lysates. Finally, SILAC in combination with MudPIT was used to show that quantification with proteinase K is possible. In this study, we identified a sheer number of 557 unique acetylated peptides originating from 633 acetylation sites. This article is protected by copyright. All rights reserved.
In order to deal with the dynamic ocean environment, blue mussels adhere to various surfaces via their collagenous byssal threads. PTMP1 (proximal thread matrix protein 1) is one identified matrix protein residing in the proximal thread and is capable of collagen binding. Its sequence comprises two von Willebrand factor type A-like repeats. In order to characterize the structure and domain architecture of PTMP1, recombinant protein was crystallized by vapour diffusion. The obtained crystals diffracted to 1.95?Å resolution and belonged to space group P2?, with unit-cell parameters a=62.0, b=62.3, c=122.6?Å, ?=102.2°. The Matthews coefficient suggested the presence of two monomers in the asymmetric unit and 48.3% solvent content.
The second messenger cAMP is synthesized in mammals by ten differently regulated adenylyl cyclases (AC1-10). These ACs are grouped into nucleotidyl cyclase class III based on homologies in their catalytic domains. The catalytic domain of AC10 is unique, however, in being activated through direct interaction with calcium and bicarbonate. Here, the production, crystallization and X-ray diffraction analysis of the catalytic domain of human AC10 are described as a basis for structural studies of regulator binding sites and mechanisms. The recombinant protein had high specific AC activity, and crystals of AC10 in space group P63 diffracted to ?2.0?Å resolution on a synchrotron beamline. A complete diffraction data set revealed unit-cell parameters a = b = 99.65, c = 98.04?Å, indicating one AC10 catalytic domain per asymmetric unit, and confirmed that the obtained crystals are suitable for structure solution and mechanistic studies.
Sirtuins are an evolutionarily conserved family of NAD(+)-dependent protein lysine deacylases. In mammals, 7 Sirtuin isoforms control various functions in metabolism, stress responses and aging processes. Sirtuins are considered attractive therapeutic targets for metabolic and aging-related diseases, such as metabolic syndrome and neurodegenerative disorders. Extensive development efforts on small-molecule Sirtuin inhibitors and activators have yielded few potent and selective compounds, partly due to shortcomings of available assays and a lack of mechanistic characterization of identified compounds.
Sirtuin enzymes regulate metabolism and aging processes through deacetylation of acetyl-lysines in target proteins. More than 6,800 mammalian acetylation sites are known, but few targets have been assigned to most sirtuin isoforms, hampering our understanding of sirtuin function. Here we describe a peptide microarray system displaying 6,802 human acetylation sites for the parallel characterisation of their modification by deacetylases. Deacetylation data for all seven human sirtuins obtained with this system reveal isoform-specific substrate preferences and deacetylation substrate candidates for all sirtuin isoforms, including Sirt4. We confirm malate dehydrogenase protein as a Sirt3 substrate and show that peroxiredoxin 1 and high-mobility group B1 protein are deacetylated by Sirt5 and Sirt1, respectively, at the identified sites, rendering them likely new in vivo substrates. Our microarray platform enables parallel studies on physiological acetylation sites and the deacetylation data presented provide an exciting resource for the identification of novel substrates for all human sirtuins.
Sirtuins are protein deacetylases regulating metabolism and stress responses. The seven human Sirtuins (Sirt1-7) are attractive drug targets, but Sirtuin inhibition mechanisms are mostly unidentified. We report the molecular mechanism of Sirtuin inhibition by 6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxamide (Ex-527). Inhibitor binding to potently inhibited Sirt1 and Thermotoga maritima Sir2 and to moderately inhibited Sirt3 requires NAD(+), alone or together with acetylpeptide. Crystal structures of several Sirtuin inhibitor complexes show that Ex-527 occupies the nicotinamide site and a neighboring pocket and contacts the ribose of NAD(+) or of the coproduct 2-O-acetyl-ADP ribose. Complex structures with native alkylimidate and thio-analog support its catalytic relevance and show, together with biochemical assays, that only the coproduct complex is relevant for inhibition by Ex-527, which stabilizes the closed enzyme conformation preventing product release. Ex-527 inhibition thus exploits Sirtuin catalysis, and kinetic isoform differences explain its selectivity. Our results provide insights in Sirtuin catalysis and inhibition with important implications for drug development.
Sirtuins are NAD(+)-dependent protein deacetylases that regulate metabolism and aging processes and are considered to be attractive therapeutic targets. Most available sirtuin modulators are little understood mechanistically, hindering their improvement. SRT1720 was initially described as an activator of human Sirt1, but it also potently inhibits human Sirt3. Here, the molecular mechanism of the inhibition of Sirt3 by SRT1720 is described. A crystal structure of Sirt3 in complex with SRT1720 and an NAD(+) analogue reveals that the compound partially occupies the acetyl-Lys binding site, thus explaining the reported competition with the peptide substrate. The compound packs against a hydrophobic protein patch and binds with its opposite surface to the NAD(+) nicotinamide, resulting in an exceptionally tight sandwich-like interaction. The observed arrangement rationalizes the uncompetitive inhibition with NAD(+), and binding measurements confirm that the nicotinamide moiety of NAD(+) supports inhibitor binding. Consistently, no inhibitor is bound in a second crystal structure of Sirt3 that was solved complexed with ADP-ribose and crystallized in the presence of SRT1720. These results reveal a novel sirtuin inhibitor binding site and mechanism, and provide a structural basis for compound improvement.
Sirtuins are NAD+-dependent protein deacetylases regulating metabolism, stress responses and ageing processes. Among the seven mammalian Sirtuins, Sirt1 is the physiologically best-studied isoform. It regulates nuclear functions such as chromatin remodelling and gene transcription, and it appears to mediate beneficial effects of a low calorie diet which can partly be mimicked by the Sirt1 activating polyphenol resveratrol. The molecular details of Sirt1 domain architecture and regulation, however, are little understood. It has a unique N-terminal domain and CTD (C-terminal domain) flanking a conserved Sirtuin catalytic core and these extensions are assumed to mediate Sirt1-specific features such as homo-oligomerization and activation by resveratrol. To analyse the architecture of human Sirt1 and functions of its N- and C-terminal extensions, we recombinantly produced Sirt1 and Sirt1 deletion constructs as well as the AROS (active regulator of Sirt1) protein. We then studied Sirt1 features such as molecular size, secondary structure and stimulation by small molecules and AROS. We find that Sirt1 is monomeric and has extended conformations in its flanking domains, likely disordered especially in the N-terminus, resulting in an increased hydrodynamic radius. Nevertheless, both termini increase Sirt1 deacetylase activity, indicating a regulatory function. We also find an unusual but defined conformation for AROS protein, which fails, however, to stimulate Sirt1. Resveratrol, in contrast, activates the Sirt1 catalytic core independent of the terminal domains, indicating a binding site within the catalytic core and suggesting that small molecule activators for other isoforms might also exist.
Sirtuins are protein deacetylases used as therapeutic targets. Pharmacological Sirt1 activation has been questioned since the in vitro activator resveratrol failed to stimulate deacetylation of several physiological substrates. We tested the influence of substrate sequence by analyzing resveratrol effects on Sirt1-dependent deacetylation of 6802 physiological acetylation sites using peptide microarrays. Resveratrol stimulated deacetylation of a small set of sites and inhibited deacetylation of another set, whereas most substrates were hardly affected. Solution assays confirmed these substrate categories, and statistical analysis revealed their sequence features. Our results reveal substrate sequence dependence for Sirt1 modulation and suggest substrates contributing to resveratrol effects.
Lipid metabolism is tightly controlled by the nutritional state of the organism. Nutrient-rich conditions increase lipogenesis, whereas nutrient deprivation promotes fat oxidation. In this study, we identify the mitochondrial sirtuin, SIRT4, as a regulator of lipid homeostasis. SIRT4 is active in nutrient-replete conditions to repress fatty acid oxidation while promoting lipid anabolism. SIRT4 deacetylates and inhibits malonyl CoA decarboxylase (MCD), an enzyme that produces acetyl CoA from malonyl CoA. Malonyl CoA provides the carbon skeleton for lipogenesis and also inhibits fat oxidation. Mice lacking SIRT4 display elevated MCD activity and decreased malonyl CoA in skeletal muscle and white adipose tissue. Consequently, SIRT4 KO mice display deregulated lipid metabolism, leading to increased exercise tolerance and protection against diet-induced obesity. In sum, this work elucidates SIRT4 as an important regulator of lipid homeostasis, identifies MCD as a SIRT4 target, and deepens our understanding of the malonyl CoA regulatory axis.
Sirtuins are protein deacetylases regulating aging processes and various physiological functions. Resveratrol, a polyphenol found in red wine, activates human Sirt1 and inhibits Sirt3, and it can mimic calorie restriction effects, such as lifespan extension in lower organisms. The mechanism of Sirtuin modulation by resveratrol is not well understood. We used 4-bromo-resveratrol (5-(2-(4-hydroxyphenyl)vinyl)-1,3-benzenediol) to study Sirt1 and Sirt3 modulation. Despite its similarity to the Sirt1 activator resveratrol, the compound potently inhibited both, Sirt1 and Sirt3. Crystal structures of Sirt3 in complex with a fluorophore-labeled and with a native substrate peptide, respectively, in presence of 4-bromo-resveratrol reveal two compound binding sites. Biochemical studies identify the internal site and substrate competition as the mechanism for inhibition, providing a drug target site, and homology modeling suggests that the second, allosteric site might indicate the site for Sirt1 activation.
Sirtuins are NAD(+)-dependent protein deacetylases that regulate metabolism and aging-related processes. Sirt2 is the only cytoplasmic isoform among the seven mamalian Sirtuins (Sirt1-7) and structural information concerning this isoform is limited. We crystallized Sirt2 in complex with a product analog, ADP-ribose, and solved this first crystal structure of a Sirt2 ligand complex at 2.3Å resolution. Additionally, we re-refined the structure of the Sirt2 apoform and analyzed the conformational changes associated with ligand binding to derive insights into the dynamics of the enzyme. Our analyses also provide information on Sirt2 peptide substrate binding and structural states of a Sirt2-specific protein region, and our insights and the novel Sirt2 crystal form provide helpful tools for the development of Sirt2 specific inhibitors.
Sirtuins are NAD+-dependent protein deacetylases regulating metabolism, stress responses, and aging processes. Mammalia possess seven Sirtuin isoforms, Sirt1-7, which differ in their subcellular localization and in the substrate proteins they deacetylate. The physiological roles of Sirtuins and their potential use as therapeutic targets for metabolic and aging-related diseases have spurred interest in the development of small-molecule Sirtuin modulators. Here, we describe an approach exploiting the structures available for four human Sirtuins for the development of isoform-specific inhibitors. Virtual docking of a compound library into the peptide binding pockets of crystal structures of Sirt2, 3, 5 and 6 yielded compounds potentially discriminating between these isoforms. Further characterization in activity assays revealed several inhibitory compounds with little isoform specificity, but also two compounds with micromolar potency and high specificity for Sirt2. Structure comparison and the predicted, shared binding mode of the Sirt2-specific compounds indicate a pocket extending from the peptide-binding groove as target side enabling isoform specificity. Our family-wide structure-based approach thus identified potent, Sirt2-specific inhibitors as well as lead structures and a target site for the development of compounds specific for other Sirtuin isoform, constituting an important step toward the identification of a complete panel of isoform-specific Sirtuin inhibitors.
Pseudomonas aeruginosa is an opportunistic bacterial pathogen and a major cause of healthcare-associated infections. While the organisms intrinsic and acquired resistance to most antibiotics hinders treatment of P. aeruginosa infections, the regulatory networks controlling its virulence provide novel targets for drug development. CyaB, a key regulator of P. aeruginosa virulence, belongs to the Class III adenylyl cyclase (AC) family of enzymes that synthesize the second messenger cyclic adenosine 3,5-monophosphate. These enzymes consist of a conserved catalytic domain fused to one or more regulatory domains. We describe here the biochemical and structural characterization of CyaB and its inhibition by small molecules. We show that CyaB belongs to the Class IIIb subfamily, and like other subfamily members, its activity is stimulated by inorganic carbon. CyaB is also regulated by its N-terminal MASE2 (membrane-associated sensor 2) domain, which acts as a membrane anchor. Using a genetic screen, we identified activating mutations in CyaB. By solving the crystal structure of the CyaB catalytic domain, we rationalized the effects of these mutations and propose that CyaB employs regulatory mechanisms similar to other Class III ACs. The CyaB structure further indicates subtle differences compared to other Class III ACs in both the active site and the inhibitor binding pocket. Consistent with these differences, we observed a unique inhibition profile, including identification of a CyaB selective compound. Overall, our results reveal mechanistic details of the physiological and pharmacological regulation of CyaB and provide the basis for its exploitation as a therapeutic drug target.
Mitochondria are central organelles in cellular energy metabolism, apoptosis, and aging processes. A signaling network regulating these functions was recently shown to include soluble adenylyl cyclase as a local source of the second messenger cAMP in the mitochondrial matrix. However, a mitochondrial cAMP-degrading phosphodiesterase (PDE) necessary for switching off this cAMP signal has not yet been identified. Here, we describe the identification and characterization of a PDE2A isoform in mitochondria from rodent liver and brain. We find that mitochondrial PDE2A is located in the matrix and that the unique N terminus of PDE2A isoform 2 specifically leads to mitochondrial localization of this isoform. Functional assays show that mitochondrial PDE2A forms a local signaling system with soluble adenylyl cyclase in the matrix, which regulates the activity of the respiratory chain. Our findings complete a cAMP signaling cascade in mitochondria and have implications for understanding the regulation of mitochondrial processes and for their pharmacological modulation.
Advances in research on mitochondria have elucidated their importance in cell survival and cell death regulation in addition to their function in energy production. Mitochondria are further implicated in various metabolic and aging-related diseases, which are now assumed to be caused by misregulation of physiological systems rather than pure accumulation of oxidative damage. Thus, the signaling mechanisms within mitochondria and between the organelle and its environment have gained interest as potential drug targets. Emerging mitochondrial signaling systems with potential for exploiting them for therapeutic intervention include, among others, the NAD(+)-dependent protein deacetylases of the Sirtuin family, the redox enzyme p66(Shc), and enzymes of the cyclic adenosine monophosphate (cAMP) signaling pathways. Here, we discuss functions of these signaling systems in mitochondria, their roles in aging processes and disease, and their potential to serve as therapeutic targets.
Mitochondria contribute to various diseases and aging phenotypes. Reactive oxygen species (ROS), mainly formed by the respiratory chain, were long thought to cause these effects by damaging proteins, DNA, and lipids. The emerging understanding that ROS act not only destructively but also as dedicated signaling molecules, and that aging processes are regulated by specific signaling networks has stimulated research on mitochondrial signaling systems and the regulation of mitochondrial ROS metabolism. p66Shc is a lifespan-regulating protein contributing to mitochondrial ROS metabolism and regulating the mitochondrial apoptosis pathway. It was found to participate in aging processes and has been implicated in several pathologies. Considerable progress has been made recently concerning the molecular function of p66Shc. It appears that p66Shc responds to a variety of proapoptotic stimuli by increasing ROS levels in the mitochondrial intermembrane space through an inherent ROS-producing activity, and that this ROS formation might trigger initiation of the mitochondrial apoptosis pathway. In this review, we will discuss the current knowledge on the molecular architecture of the p66Shc protein, its role in ROS metabolism and apoptosis regulation in the mitochondrial intermembrane space, the regulation of its mitochondrial transport, and the molecular mechanisms and interactions involved in these processes.
When colonising host-niches or non-animated medical devices, individual cells of the fungal pathogen Candida albicans expand into significant biomasses. Here we show that within such biomasses, fungal metabolically generated CO(2) acts as a communication molecule promoting the switch from yeast to filamentous growth essential for C. albicans pathology. We find that CO(2)-mediated intra-colony signalling involves the adenylyl cyclase protein (Cyr1p), a multi-sensor recently found to coordinate fungal responses to serum and bacterial peptidoglycan. We further identify Lys 1373 as essential for CO(2)/bicarbonate regulation of Cyr1p. Disruption of the CO(2)/bicarbonate receptor-site interferes selectively with C. albicans filamentation within fungal biomasses. Comparisons between the Drosophila melanogaster infection model and the mouse model of disseminated candidiasis, suggest that metabolic CO(2) sensing may be important for initial colonisation and epithelial invasion. Our results reveal the existence of a gaseous Candida signalling pathway and its molecular mechanism and provide insights into an evolutionary conserved CO(2)-signalling system.
Sirtuins are a family of protein deacetylases that catalyze the nicotinamide adenine dinucleotide (NAD(+))-dependent removal of acetyl groups from modified lysine side chains in various proteins. Sirtuins act as metabolic sensors and influence metabolic adaptation but also many other processes such as stress response mechanisms, gene expression, and organismal aging. Mammals have seven Sirtuin isoforms, three of them - Sirt3, Sirt4, and Sirt5 - located to mitochondria, our centers of energy metabolism and apoptosis initiation. In this review, we shortly introduce the mammalian Sirtuin family, with a focus on the mitochondrial isoforms. We then discuss in detail the current knowledge on the mitochondrial isoform Sirt5. Its physiological role in metabolic regulation has recently been confirmed, whereas an additional function in apoptosis regulation remains speculative. We will discuss the biochemical properties of Sirt5 and how they might contribute to its physiological function. Furthermore, we discuss the potential use of Sirt5 as a drug target, structural features of Sirt5 and of an Sirt5/inhibitor complex as well as their differences to other Sirtuins and the current status of modulating Sirt5 activity with pharmacological compounds.
The beta-carbonic anhydrase (CA, EC 18.104.22.168) from the fungal pathogen Candida albicans (Nce103) is involved in a CO(2) sensing pathway critical for the pathogen life cycle and amenable to drug design studies. Herein we report an inhibition study of Nce103 with a library of sulfonamides and one sulfamate, showing that Nce103, similarly to the related enzyme from Cryptococcus neoformans Can2, is inhibited by these compounds with K(I)s in the range of 132 nM-7.6 microM. The best Nce103 inhibitors were acetazolamide, methazolamide, bromosulfanilamide, and 4-hydroxymethylbenzenesulfonamide (K(I)s<500 nM). A homology model was generated for Nce103 based on the crystal structure of Can2. The model shows that compounds with zinc-binding groups incorporating less polar moieties and compact scaffolds generate stronger Nce103 inhibitors, whereas highly polar zinc-binding groups and bulkier compounds appear more promising for the specific inhibition of Can2. Such compounds may be useful for the design of antifungal agents possessing a new mechanism of action.
In the pathogenic fungus Cryptococcus neoformans, a CO(2)-sensing system is essential for survival in the natural environment (approximately 0.03% CO(2)) and mediates the switch to virulent growth in the human host (approximately 5% CO(2)). This system is composed of the carbonic anhydrase (CA) Can2, which catalyzes formation of bicarbonate, and the fungal, bicarbonate-stimulated adenylyl cyclase Cac1. The critical role of these enzymes for fungal metabolism and pathogenesis identifies them as targets for antifungal drugs. Here, we prove functional similarity of Can2 to the CA Nce103 from Candida albicans and describe its biochemical and structural characterization. The crystal structure of Can2 reveals that the enzyme belongs to the "plant-type" beta-CAs but carries a unique N-terminal extension that can interact with the active-site entrance of the dimer. We further tested a panel of compounds, identifying nanomolar Can2 inhibitors, and present the structure of a Can2 complex with the inhibitor and product analog acetate, revealing insights into interactions with physiological ligands and inhibitors.
Damage caused by reactive oxygen species (ROS) contributes to many aging processes and accompanying diseases. ROS are toxic side products of cellular respiration, but also function as signal, e.g. in the mitochondrial apoptosis pathway. The protein p66Shc, which has been implicated in life-span regulation and aging-related diseases, is a central player in stress-induced apoptosis and the associated ROS burst. Stress signals, such as UV radiation or ROS themselves, activate p66Shc, which was proposed to stimulate its H(2)O(2) forming activity, ultimately triggering mitochondrial disintegration. However, mechanistic details of H(2)O(2) formation and apoptosis induction by p66Shc and regulation of these activities remain to be revealed. Here, we describe the effects of Ser36 phosphorylation and Pin1 binding on p66Shc activity, and the identification of Peroxiredoxin 1 (Prx1) as a novel interaction partner for the unique p66Shc N-terminal domain. Prx1 was identified in affinity experiments as dominant interaction partner. Complex formation leads to disassembly of Prx1 decamers, which is known to increase its peroxidase activity. The interaction leads to reduction of the p66CH2CB tetramer, which reduces its ability to induce mitochondrial rupture. Our results indicate that p66CH2CB and Prx1 form a stress-sensing complex that keeps p66Shc inactive at moderate stress levels.
Sirtuins are NAD(+) consuming protein deacylases involved in many cellular processes from DNA-repair to metabolism. Their contribution to age-related and metabolic diseases makes them attractive pharmaceutical targets. Few pharmacological inhibitors have been reported yet for human Sirt5 since substrates and assays for reliable testing of its activity were unavailable until recently, and most modulators of other Sirtuins were not tested against Sirt5 and therefore have only partially characterized isoform selectivities. We used here improved substrates and assays for testing of known Sirtuin inhibitors for their effects on two activities of human Sirt5, the generic Sirtuin activity deacetylation and the more pronounced Sirt5 activity desuccinylation. Our tests show that most of the compounds have no significant effect on either Sirt5 activity. The indole GW5074, however, was found to be a potent inhibitor for Sirt5s desuccinylation activity, identifying a first pharmacological scaffold for development into Sirt5-specific inhibitors. Interestingly, the compound showed weaker effects in Sirt5 deacetylation assays and also varying potencies against different peptide sequences, indicating a substrate-specific effect of GW5074.
Sirtuins are protein deacetylases regulating metabolism, stress responses, and aging processes, and they were suggested to mediate the lifespan extending effect of a low calorie diet. Sirtuin activation by the polyphenol resveratrol can mimic such lifespan extending effects and alleviate metabolic diseases. The mechanism of Sirtuin stimulation is unknown, hindering the development of improved activators. Here we show that resveratrol inhibits human Sirt3 and stimulates Sirt5, in addition to Sirt1, against fluorophore-labeled peptide substrates but also against peptides and proteins lacking the non-physiological fluorophore modification. We further present crystal structures of Sirt3 and Sirt5 in complex with fluorogenic substrate peptide and modulator. The compound acts as a top cover, closing the Sirtuins polypeptide binding pocket and influencing details of peptide binding by directly interacting with this substrate. Our results provide a mechanism for the direct activation of Sirtuins by small molecules and suggest that activators have to be tailored to a specific Sirtuin/substrate pair.
Sirtuins are protein deacylases regulating metabolism and aging processes, and the seven human isoforms are considered attractive therapeutic targets. Sirtuins transfer acyl groups from lysine sidechains to ADP-ribose, formed from the cosubstrate NAD(+) by release of nicotinamide, which in turn is assumed to be a general Sirtuin inhibitor. Studies on Sirtuin regulation have been hampered, however, by shortcomings of available assays. Here, we describe a mass spectrometry-based, quantitative deacylation assay not requiring any substrate labeling. Using this assay, we show that the deacetylation activity of human Sirt5 features an unusual insensitivity to nicotinamide inhibition. In contrast, we find similar values for Sirt5 and Sirt3 for the intrinsic NAD(+) affinity as well as the apparent NAD(+) affinity in presence of peptide. Structure comparison and mutagenesis identify an Arg neighboring to the Sirt5 nicotinamide binding pocket as a mediator of nicotinamide resistance, and statistical sequence analyses along with testing further Sirtuins reveal a network of coevolved residues likely defining a nicotinamide-insensitive Sirtuin deacetylase family. The same Arg was recently reported to render Sirt5 a preferential desuccinylase, and we find that this Sirt5 activity is highly sensitive to nicotinamide inhibition. Analysis of Sirt5 structures and activity data suggest that an Arg/succinate interaction is the molecular basis of the differential nicotinamide sensitivities of the two Sirt5 activities. Our results thus indicate a Sirtuin subfamily with nicotinamide-insensitive deacetylase activity and suggest that the molecular features determining nicotinamide sensitivity overlap with those dominating deacylation specificity, possibly suggesting that other subfamily members might also prefer other acylations than acetylations.
The second messengers cAMP and cGMP mediate a multitude of physiological processes. In mammals, these cyclic nucleotides are formed by related Class III nucleotidyl cyclases, and both ACs (adenylate cyclases) and GCs (guanylate cyclases) comprise transmembrane receptors as well as soluble isoforms. Whereas sGC (soluble GC) has a well-characterized regulatory HD (haem domain) that acts as a receptor for the activator NO (nitric oxide), very little is known about the regulatory domains of the ubiquitous signalling enzyme sAC (soluble AC). In the present study, we identify a unique type of HD as a regulatory domain in sAC. The sAC-HD (sAC haem domain) forms a larger oligomer and binds, non-covalently, one haem cofactor per monomer. Spectral analyses and mutagenesis reveal a 6-fold co-ordinated haem iron atom, probably with non-typical axial ligands, which can bind both NO and CO (carbon monoxide). Splice variants of sAC comprising this domain are expressed in testis and skeletal muscle, and the HD displays an activating effect on the sAC catalytic core. Our results reveal a novel mechanism for regulation of cAMP signalling and suggest a need for reanalysis of previous studies on mechanisms of haem ligand effects on cyclic nucleotide signalling, particularly in testis and skeletal muscle.
Sirtuins are NAD(+)-dependent protein deacetylases regulating metabolism, stress responses, and aging processes. Mammalia have seven Sirtuin isoforms, Sirt1-7, which differ in their substrate specificities and subcellular localizations. The physiological functions of Sirtuins make them interesting therapeutic targets, which has stimulated extensive efforts on development of small molecule Sirtuin modulators. Yet, most Sirtuin inhibitors show limited potency and/or isoform specificity, and the mechanism of Sirtuin activation by small molecules remains obscure. Accumulating information on Sirtuin substrates, structures, and regulation mechanisms offer new opportunities for the challenging task to develop potent and specific small molecule modulators for mammalian Sirtuins for in vivo studies and therapeutic applications. We therefore recapitulate advances in structural and mechanistic studies on substrate recognition and deacetylation by Sirtuins, and in the characterization of compounds and molecular mechanisms regulating their activity. We then discuss challenges and opportunities from these findings for Sirtuin-targeted drug development efforts.
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