310 research outputs found

    Structures, Substrates, and Regulators of Mammalian Sirtuins – Opportunities and Challenges for Drug Development

    Get PDF
    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

    Sirt1 activation by resveratrol is substrate sequence-selective

    Get PDF
    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. ONE SENTENCE SUMMARY: Testing 6802 acetylation sites reveals that resveratrol effects on Sirt1-dependent deacetylation depend on substrate sequence and suggests substrates relevant for in vivo effects

    Molecular architecture of the human protein deacetylase Sirt1 and its regulation by AROS and resveratrol

    Get PDF
    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

    Molecular, Enzymatic, and Cellular Characterization of Soluble Adenylyl Cyclase From Aquatic Animals.

    Get PDF
    The enzyme soluble adenylyl cyclase (sAC) is the most recently identified source of the messenger molecule cyclic adenosine monophosphate. sAC is evolutionarily conserved from cyanobacteria to human, is directly stimulated by [Formula: see text] ions, and can act as a sensor of environmental and metabolic CO2, pH, and [Formula: see text] levels. sAC genes tend to have multiple alternative promoters, undergo extensive alternative splicing, be translated into low mRNA levels, and the numerous sAC protein isoforms may be present in various subcellular localizations. In aquatic organisms, sAC has been shown to mediate various functions including intracellular pH regulation in coral, blood acid/base regulation in shark, heart beat rate in hagfish, and NaCl absorption in fish intestine. Furthermore, sAC is present in multiple other species and tissues, and sAC protein and enzymatic activity have been reported in the cytoplasm, the nucleus, and other subcellular compartments, suggesting even more diverse physiological roles. Although the methods and experimental tools used to study sAC are conventional, the complexity of sAC genes and proteins requires special considerations that are discussed in this chapter

    Pharmacological activation of SIRT6 triggers lethal autophagy in human cancer cells

    Get PDF
    Sirtuin 6 (SIRT6) is a member of the NAD+-dependent class III deacetylase sirtuin family, which plays a key role in cancer by controlling transcription, genome stability, telomere integrity, DNA repair, and autophagy. Here we analyzed the molecular and biological effects of UBCS039, the first synthetic SIRT6 activator. Our data demonstrated that UBCS039 induced a time-dependent activation of autophagy in several human tumor cell lines, as evaluated by increased content of the lipidated form of LC3B by western blot and of autophagosomal puncta by microscopy analysis of GFP-LC3. UBCS039-mediated activation of autophagy was strictly dependent on SIRT6 deacetylating activity since the catalytic mutant H133Y failed to activate autophagy. At the molecular level, SIRT6-mediated autophagy was triggered by an increase of ROS levels, which, in turn, resulted in the activation of the AMPK-ULK1-mTOR signaling pathway. Interestingly, antioxidants were able to completely counteract UBCS039-induced autophagy, suggesting that ROS burst had a key role in upstream events leading to autophagy commitment. Finally, sustained activation of SIRT6 resulted in autophagy-related cell death, a process that was markedly attenuated using either a pan caspases inhibitor (zVAD-fmk) or an autophagy inhibitor (CQ). Overall, our results identified UBCS039 as an efficient SIRT6 activator, thereby providing a proof of principle that modulation of the enzyme can influence therapeutic strategy by enhancing autophagy-dependent cell death

    Inelastic J/psi Photoproduction

    Full text link
    Inelastic photoproduction of J/ψJ/\psi particles at high energies is one of the processes to determine the gluon distribution in the nucleon. We have calculated the QCD radiative corrections to the color-singlet model of this reaction. They are large at moderate photon energies, but decrease with increasing energies. The cross section and the J/ψJ/\psi energy spectrum are compared with the available fixed-target photoproduction data and predictions are given for the HERA energy range.Comment: 14 pages, latex, 7 uuencoded figure

    Fragmentation production of doubly heavy baryons

    Get PDF
    Baryons with a single heavy quark are being studied experimentally at present. Baryons with two units of heavy flavor will be abundantly produced not only at future colliders, but also at existing facilities. In this paper we study the production via heavy quark fragmentation of baryons containing two heavy quarks at the Tevatron, the LHC, HERA, and the NLC. The production rate is woefully small at HERA and at the NLC, but significant at pppp and ppˉp\bar{p} machines. We present distributions in various kinematical variables in addition to the integrated cross sections at hadron colliders.Comment: 13 pages, macro package epsfig needed, 6 .eps figure files in a separate uuencoded, compressed and tarred file; complete paper available at http://www.physics.carleton.ca/~mad/papers/paper.p
    • 

    corecore