Regulation of Mitochondrial Sirtuins Sirt3 and Sirt5 by Small Molecules

REGULATION OF MITOCHONDRIAL SIRTUINS SIRT3 AND SIRT5 BY SMALL MOLECULES A dissertation submitted to The Faculty of Biology, Chemistry and Geosciences University of Bayreuth, Germany To attain the degree Doctor of Natural Sciences (Dr. rer. nat.) Presented by Giang Thi Tuyet Nguyen Bayreuth, 2013 II This doctoral thesis was prepared at the Department of Biochemistry, University of Bayreuth, Germany from October 2010 until September 2013, under the supervision of Prof. Dr. Clemens Steegborn. This work was funded by Elite Network of Bavaria, BioMedTech International graduate School of Science (BIGSS) Ph.D. program, University of Bayreuth, Germany. This is a full reprint of the dissertation submitted to attain the academic degree of Doctor of Natural Sciences (Dr. rer. nat.) and approved by the Faculty of Biology, Chemistry and Geosciences, University of Bayreuth, Germany. Acting dean: Prof. Dr. Rhett Kempe Date of submission: 30th September, 2013 Date of defense: 27th January, 2014 Doctoral Committee: Prof. Dr. Clemens Steegborn 1st reviewer Prof. Dr. Paul Rösch 2nd reviewer Prof. Dr. Stephan Förster Chairman Prof. Dr. Matthias Breuning III Acknowledgements This work is dedicated to my parents, Dau Dinh Nguyen and Tuyet Thi Than for their love, encouragement and support throughout my life, and to my beloved husband, Trieu Nguyen and my little daughter, My Kim Nguyen who have always stood by me. I would like to express my deepest appreciation to my supervisor, Prof. Dr. Clemens Steegborn for his guidance and continuous support throughout this work. His extensive knowledge and broad vision led me to the right way in research. I would also like to extend my appreciation to my doctoral committee members: Prof. Dr. Paul Rösch (Department of Biopolymers, University of Bayreuth, Germany), Prof. Dr. Stephan Förster (Department of Physical Chemistry, University of Bayreuth, Germany) and Prof. Dr. Matthias Breuning (Department of Organic Chemistry, University of Bayreuth, Germany) for their feedback and assistance. I am indebted to all members of the Department of Biochemistry, University of Bayreuth, Germany for their support during my work here. I am very grateful to Dr. Melanie Gertz for detailed and in-depth discussions. I would like to thank Dr. Micheal Weyand and Dr. Sebastien Moniot for their help in crystallography study. Also thanks to Dr. Frank Fischer for his help in mass spectrometry experiments. Special thanks to Ms. Gabriele Kassler and Ms. Renate Crowe for all their help. Thanks to Benjamin Sünkel, Martin Pannek, Ningna Xu, Norbert Grillenbeck, Susanne Schäfer and the other members for their help and encouragement. I would also like to thank to my Master supervisor, Prof. Dr. Kyeong Kyu Kim (Sungkyunkwan University, South Korea) for providing me the plasmid vector pVFT3S. I thank Prof. Dr. Paul Rösch, PD. Dr. Stephan Schwarzinger and Ms. Violaine Zigan for helping me in Elite Network of Bavaria, BioMedTech International graduate School of Science (BIGSS) Ph.D. program. I am thankful to Elite Network of Bavaria, BioMedTech International graduate School of Science (BIGSS) Ph.D. program, University of Bayreuth, Germany for funding this research. IV I would like to thank my aunt, Nguyet Thi Nguyen for her help and other family members and friends for their encouragement. V Publications and presentations relate to this work: Publications: Nguyen, G.T.T.*, Gertz, M.*, and Steegborn, C. (2013). Crystal structures of Sirt3 complexes with the resveratrol derivative 5-(2-(4-bromophenyl)vinyl)-1,3-benzenediol reveal binding sites and inhibition mechanism. Chem Biol 20, 1375-138. Nguyen, G.T.T., Schaefer, S., Gertz, M., Weyand, M., and Steegborn, C. (2013). Structures of human sirtuin 3 complexes with ADP-ribose and with carba-NAD+ and SRT1720: binding details and inhibition mechanism. Acta Crystallogr D 69, 1423-1432. Gertz, M.*, Fischer, F.*, Nguyen, G.T.T., Lakshminarasimhan, M., Schutkowski, M., Weyand, M., and Steegborn, C. (2013). Ex-527 inhibits Sirtuins by exploiting their unique NAD+- dependent deacetylation mechanism. P Natl Acad Sci 110, E2772-E2781. Gertz, M., Nguyen, G.T., Fischer, F., Suenkel, B., Schlicker, C., Franzel, B., Tomaschewski, J., Aladini, F., Becker, C., Wolters, D., and Steegborn, C. (2012). A molecular mechanism for direct sirtuin activation by resveratrol. PLoS One 7, e49761. * = Equal contribution Conferences, courses and symposiums: Poster presentation entitled “Regulation of mitochondrial sirtuins by small molecules” at the Elite Network of Bavaria Structure Days, Thurnau, Germany, July 2012. Protein crystallography practical course entitled “From data to structures”, Oulu, Finland, January 2012. Oral and poster presentation entitled “Regulation of mitochondrial sirtuins by small molecules” at the Elite Network of Bavaria Structure Days symposium, Thurnau, Germany, July 2011. VI Another publication not included in this thesis: Ta, H.M., Nguyen, G.T.T., Jin, H.M., Choi, J., Park, H., Kim, N., Hwang, H.-Y., and Kim, K.K. (2010). Structure-based development of a receptor activator of nuclear factor-κB ligand (RANKL) inhibitor peptide and molecular basis for osteopetrosis. P Natl Acad Sci 107, 2028120286. VII TABLE OF CONTENTS Abbreviations:.......................................................................................... 1 Abstract .................................................................................................... 5 Zusammenfassung ................................................................................... 7 1. Introduction ......................................................................................... 9 1.1. Caloric restriction (CR) and aging ........................................................................................... 9 1.2. Sirtuins and their roles in CR, aging and human age-related diseases ................................... 9 1.3. The mammalian sirtuin enzyme family.................................................................................. 10 1.3.1. Overview of the mammalian sirtuin family: classification, localization and function 10 1.3.2. Mitochondrial sirtuins ...................................................................................................... 12 1.3.3. Structure of sirtuins .......................................................................................................... 13 1.3.4. Enzymatic activity of sirtuins .......................................................................................... 15 1.4. Sirtuin modulators ................................................................................................................... 17 1.4.1. Activators .......................................................................................................................... 17 1.4.1.1. Resveratrol ................................................................................................................. 17 1.4.1.2. Other activators .......................................................................................................... 19 1.4.2. Inhibitors ........................................................................................................................... 20 1.5. Objectives................................................................................................................................. 21 2. Materials and Methods ..................................................................... 23 2.1. Materials ................................................................................................................................... 23 2.1.1. Chemicals, peptides and compounds ............................................................................... 23 2.1.2. Plasmid vectors ................................................................................................................. 23 2.1.3. Oligonucleotide primers ................................................................................................... 24 VIII 2.1.4. Bacterial strains................................................................................................................. 24 2.2. Methods .................................................................................................................................... 25 2.2.1. Agarose gel electrophoresis ............................................................................................. 25 2.2.2. Cloning .............................................................................................................................. 25 2.2.3. Site-directed mutagenesis................................................................................................. 26 2.2.4. Expression ......................................................................................................................... 26 2.2.5. Cell disruption ................................................................................................................... 27 2.2.6. SDS-Polyacrylamide gel electrophoresis (SDS-PAGE) ................................................ 27 2.2.7. Purification ........................................................................................................................ 27 2.2.7.1. Affinity chromatography (AC) ................................................................................. 28 2.2.7.2. Tag cleavage .............................................................................................................. 28 2.2.7.3. Ion exchange chromatography (IEC) ....................................................................... 29 2.2.7.4. Size exclusion chromatography (SEC)..................................................................... 29 2.2.8. Fluorescence-based Flour-de-Lys assay.......................................................................... 29 2.2.9. Enzyme-coupled continuous assay .................................................................................. 30 2.2.10. Mass spectrometry (MS) ................................................................................................ 30 2.2.11. Thermal denaturation shift assay ................................................................................... 31 2.2.12. Binding analysis by microscale thermophoresis (MST) .............................................. 31 2.2.13. Crystallization and structure determination .................................................................. 31 3. Results ................................................................................................. 33 3.1. Sirt3 studies .............................................................................................................................. 33 3.1.1. Sirt3 purification ............................................................................................................... 33 3.1.2. Resveratrol and its related compounds ............................................................................ 35 3.1.2.1. Resveratrol and its related compounds are hSirt3 inhibitors .................................. 35 IX 3.1.2.2. Crystallization trials of hSirt3 in complex with resveratrol related compounds ... 36 3.1.2.3. Crystal structures and inhibition mechanisms of hSirt3 in complex with resveratrol related compounds ................................................................................................ 38 3.1.2.3.1. hSirt3 in complex with FdL-1 peptide and piceatannol/polydatin .................. 38 3.1.2.3.2. hSirt3 in complex with FdL-1 peptide and 4’-bromo-resveratrol.................... 39 3.1.2.3.3. hSirt3 in complex with ACS2 peptide and 4’-bromo-resveratrol .................... 44 3.1.3. Resveratrol unrelated compounds.................................................................................... 49 3.1.3.1. SRT1720..................................................................................................................... 49 3.1.3.2. Ex-527 ........................................................................................................................ 54 3.2. Sirt5 studies .............................................................................................................................. 57 3.2.1. Sirt5 purification ............................................................................................................... 57 3.2.2. Resveratrol and its related compounds are zSirt5 activators on FdL-1 peptide ........... 59 3.2.3. Crystallization trials and crystal structures of zSirt5 in complex with peptide substrates in the presence of resveratrol .................................................................................... 61 4. Discussion ........................................................................................... 66 4.1. Sirt3 studies .............................................................................................................................. 66 4.1.1. Resveratrol and its related compounds ............................................................................ 66 4.1.2. Resveratrol unrelated compounds.................................................................................... 68 4.2. Sirt5 studies .............................................................................................................................. 71 References............................................................................................... 72 Appendix................................................................................................. 83 (Eidesstattliche) Versicherungen und Erklärungen .......................... 92 Zusammenfassung 7 Zusammenfassung Sirtuine sind eine Familie hochkonservierter Proteindeacylasen, die wichtige Regulatoren des Metabolismus, der Lebensdauer und alters-assoziierter Krankheiten sind. Säugetiere besitzen sieben Sirtuinisoformen, die sich in ihrer subzellulären Lokalisation, ihren Substraten und biologischen Funktionen unterscheiden. Drei Sirtuinisoformen, Sirt3-5, sind mitochondrial lokalisiert und spielen eine entscheidende Rolle in allen mitochondrialen Prozessen. Diese Arbeit untersucht die Modulation von niedermolekularen Wirkstoffen auf die mitochondrialen Sirtuine Sirt3 und Sirt5 mittels struktureller Charakterisierung als auch anhand biochemischer und biophysikalischer Analyse. Über Resveratrol, ein in Rotwein vorkommendes Polyphenol, ist bekannt, dass es Sirt1 aktiviert. Die Untersuchung von Resveratrol und dessen verwandten Wirkstoffen wie Piceatannol, Polydatin, 4´-Bromoresveratrol gegen Sirt3 zeigte eine inhibitorische Wirkung. Von diesen Wirkstoffen ist 4´-Bromoresveratrol der stärkste Sirt3-Inhibitor. Kristallstrukturen von Sirt3 komplexiert mit dem FdL-1-Peptid und Piceatannol/Polydatin zeigten eine direkte Interaktion dieser Wirkstoffe mit dem Coumarinring des Peptids. Dies induziert eine unproduktive Substratbindung, die dadurch die Enzymaktivität inhibiert. Kristallstrukturen der Sirt3/FdL-1/4’-bromo-resveratrolund Sirt3/ACS2/4’-bromo-resveratrol-Komplexe offenbarten zwei unterschiedliche Wirkstoffbindestellen. Biochemische Daten und Bindungsdaten deuteten an, dass die 4’-bromo-resveratrol-Bindestelle im FdL-1-Komplex im Inhibitionsmechanismus involviert ist. Die Wirkstoffbindestelle im ACS2-Komplex hingegen könnte die Bindestelle für den Aktivierungsmechanismus von Resveratrol gegenüber Sirt1 implizieren. Wirkstoffe wie SRT1720, ein potenter synthetischer Sirt1-Aktivator, und Ex-527, ein potenter Sirt1-Inhibitor, die beide Resveratrol nicht ähneln, inhibierten auch Sirt3. In der Komplexstruktur von Sirt3/carba-NAD+/SRT1720 kompetierte der Wirkstoff mit dem Substratpeptid, indem er die Bindestelle des Acetyllysins besetzt. SRT1720 interagiert mit NAD+ und Bindestudien deuten an, dass die Nikotinamidgruppe des NAD+ für die SRT1720-Bindung Zusammenfassung 8 essenziell ist. Im Gegensatz zum Inhibitionsmechanismus von SRT1720 stabilisiert Ex-527 die geschlossene Sirtuinkonformation und verhinderte dadurch die Freisetzung des Produkts 2´-Oacetyl-ADP-Ribose. Die Kristallstruktur des Komplexes aus Sirt3/nativem O-alkylamidatIntermediats lieferte mehr Information über die Deacetylierungsreaktion. 1. Introduction 9 1. Introduction 1.1. Caloric restriction (CR) and aging Caloric restriction (CR) is a dietary regimen based on low calorie intake up to 40 percent. Over 75 years ago, Clive McCay and colleagues first found that rats fed a caloric restricted diet live longer than ad libitum (freely fed) (McCay, et al., 1989). Since then, similar observations were reported in a variety of species including yeast, fruit flies, worms, etc. (Lin, et al., 2002; Masoro, 2005). In addition, many studies found that CR without malnutrition can prevent or delay a wide number of chronic diseases, such as cancer, diabetes, autoimmune, respiratory disease, Alzheimer disease and Parkinson disease (Cohen, et al., 2009; Longo and Fontana, 2010; Masoro, 1990) indicating that CR retards aging processes. 1.2. Sirtuins and their roles in CR, aging and human age-related diseases Sirtuins are a family of NAD+-dependent protein deacetylases conserved throughout evolution from archaebacteria to eukaryotes. They are homologs to the yeast Sir2 (silent information regulator 2) (Lin, et al., 2000). Sir2 was proven to be required for lifespan extension in yeast by CR (Kaeberlein, et al., 1999; Lin, et al., 2000). The ability of glucose restriction to extend lifespan was blocked in yeast deleting Sir2 gene (Lin, et al., 2000). Since then, many studies focus on Sir2 homologs and their relation to aging. Sir2 homolog induces lifespan extension in worms (Wang and Tissenbaum, 2006) and flies (Rogina and Helfand, 2004). Sirt1 was described as a key role in regulating the metabolic response to CR (Cantó and Auwerx, 2009). Sirt3 mediates CR to age-related hearing loss, the hallmark of mammalian aging and required for the reduction of oxidative damage (Someya, et al., 2010). Sirt6 prolongs lifespan in male mice (Kanfi, et al., 2012) and can be act as a tumor suppressor (Lombard and Miller, 2012). Many reports suggested the relation of sirtuins to various age-related diseases such as metabolic abnormalities, cancer, neurodegenerative diseases, cardiovascular, etc. (Sebastian, et 1. Introduction 10 al., 2012). Sirtuins can control tumorigenesis due to their ability in regulation of genomic stability such as Sirt1 modulates cellular stress responses and DNA repair, deacetylates the proto-oncogene Myc to prevent transformation (Martinez-Pastor and Mostoslavsky, 2012; Yuan, et al., 2009). Some reports suggested that Sirt3 and Sirt6 seem to be tumor suppressors due to their ability to destabilize HIF-1α (hypoxia-inducible factor 1-α) through down regulation of ROS (reactive oxygen species) and induce apoptosis in cancer cell lines (Sebastian, et al., 2012). Sirt1 was described as a protector against neurodegenerative diseases such as Alzheimer disease, Parkinson disease and Huntington disease (Arima, et al., 1998; Haass and Selkoe, 2007; Jiang, et al., 2012). Sirt2 supports differentiation and migration of some brain cells through deacetylating α-tubulin and Par-3 (protease activated receptor 3) (Beirowski, et al., 2011; Li, et al., 2007). Moreover, Sirt1 and Sirt7 possess cardiovascular protective properties by deacetylating p53 or regulating fatty acid oxidation (Sebastian, et al., 2012). Sirt3 is a regulator of cardiac function by reducing cellular ROS or suppressing Akt phosphorylation via AMPK (AMP-activated kinase) (Pillai, et al., 2010; Sack, 2011). Sirt6 protect against cardiac hypertrophy by inhibiting NF-κB (nuclear factor-κB) or IGF (insulin-like growth factor)-Akt signaling (Sundaresan, et al., 2012; Yu, et al., 2012). 1.3. The mammalian sirtuin enzyme family 1.3.1. Overview of the mammalian sirtuin family: classification, localization and function In mammals, there are seven members of the sirtuin family, Sirt1–7 that differ in their cellular localization and function (Haigis and Sinclair, 2010; Michan and Sinclair, 2007). The seven mammalian sirtuins share a highly conserved catalytic core domain but have differences in their Nand C-terminal ends (Frye, 2000) (Figure 1.1). 1. Introduction 11 Figure 1.1. Schematic illustration of seven mammalian sirtuins. Based on phylogenetic analysis, mammalian sirtuins can be divided into four classes. Sirt1-3 belong to class I, Sirt4 to class II, Sirt5 to class III and Sirt6, Sirt7 to class IV (Frye, 2000). Sirt1 is mainly found in the nucleus but also present in the cytosol (Michan and Sinclair, 2007), Sirt2 in the cytoplasm (North, et al., 2003), Sirt3-5 in mitochondria (Gertz and Steegborn, 2010), and Sirt6 and Sirt7 in the nucleus (Michishita, et al., 2005). Sirt1-3 have robust deacetylation activity, whereas Sirt4 and Sirt6 were reported to be ADP-ribosyltransferases (Haigis, et al., 2006; Mao, et al., 2011). Sirt6 can also act as a deacetylase (Michishita, et al., 2008). Sirt7 was recently confirmed as a deacetylase due to its important role in deacetylation of H3K18Ac (acetylated lysine 18 of histone H3) (Barber, et al., 2012). Sirt5 was initially reported to deacetylate CPS1 (carbamoyl phosphate synthase 1) (Nakagawa, et al., 2009) but recently described as a protein desuccinylase and demalonylase (Du, et al., 2011) indicating that sirtuins are a family of deacylases. Sirt1 is the most studied mammalian sirtuin isoform which was first described as a histone deacetylase (Haigis and Sinclair, 2010) but also has other protein targets such as p53 which is deacetylated upon DNA damage or oxidative stress (Vaziri, et al., 2001) and forkhead transcription factors (FOXO) in lipid and glucose metabolism (Motta, et al., 2004). Sirt2 is a tubulin deacetylase (North, et al., 2003). Sirt3-5 play important roles in metabolism, apoptosis and intracellular signaling (Verdin, et al., 2010). More details of Sirt3-5 are in the Mitochondrial 1. Introduction 12 sirtuins section (see below). Sirt6 is involved in DNA repair (Lombard, 2009), regulates immune response that relates to NF-κB targets (Kawahara, et al., 2009; Michishita, et al., 2008) and controls TNF (tumor necrosis factors) production (Van Gool, et al., 2009). Sirt7 is H3K18Ac deacetylase that functions in chromatin regulation, cellular transformation programs and tumour formation (Barber, et al., 2012). 1.3.2. Mitochondrial sirtuins NAD+-dependent protein deacylase is a major enzymatic activity of sirtuins. Proteomic studies implied that a large number of mitochondrial proteins are acetylated (Verdin, et al., 2010). NAD+ is an essential electron carrier in various metabolic processes such as energy production, fatty acid metabolism, urea cycle, etc., which are integrated by mitochondria. Since sirtuins use NAD+ as a cosubstrate, Sirt3-5 which have been identified in the mitochondrial matrix are directly linked to mitochondrial processes and influence mitochondrial functions (Figure 1.2). Figure 1.2. Mitochondrial sirtuins and their links to mitochondrial processes. Figure is reused with permission from Elsevier Limited (Verdin, et al., 2010). 1. Introduction 13 Sirt3 seems to be the major mitochondrial deacetylase since mice lacking Sirt3 showed a hyperacetylation of mitochondrial proteins (Lombard, et al., 2007). The mitochondrial protein acetylcoenzyme A synthase 2 (ACS2) which converts acetate to acetyl-CoA in the presence of ATP is the first identified Sirt3 substrate (Hallows, et al., 2006). Glutamate dehydrogenase (GDH) is also Sirt3 substrate since Sirt3 deacetylates and activates GDH activity by 10% (Schlicker, et al., 2008). Sirt3 interacts with Complex I of the electron transport chain and deacetylates various proteins in this complex (Ahn, et al., 2008). The mitochondrial matrix enzyme CPS1 which plays an important role in the rate-limiting step of the urea cycle was identified as a Sirt5 substrate (Nakagawa, et al., 2009). Recently, Sirt5 has been determined as a major demalonylase and desuccinylase since mice lacking Sirt5 showed hypermalonylation and hypersuccinylation (Peng, et al., 2011). Two Sirt5 residues in the catalytic pocket, Tyr102 and Arg105, are mandatory for the demalonylase and desuccinylase activities (Du, et al., 2011). These two residues are conserved in sirtuin class III of different species (Du, et al., 2011). The enzymatic function of Sirt4 remains unclear. So far, Sirt4 has no detectable deacetylase activity and weak ADP-ribosyltransferase activity (Ahuja, et al., 2007; Haigis, et al., 2006). 1.3.3. Structure of sirtuins Crystal structures of different sirtuin homologs including apo protein or in complex with substrates or small molecules have been published. The structure of yeast Hst2 (PDB ID 1Q14) (Zhao, et al., 2003) contains the full-length protein whereas the remaining structures show only the core domain. The full length yHst2 structure implies the role of Nand C-terminal region of sirtuins in the regulation of substrate binding (Zhao, et al., 2003). The conserved catalytic core consists of two domains, a large Rossmann-fold domain for NAD+ binding and a variant small zinc-binding domain that may be involved in substrate binding (Sanders, et al., 2010; Sauve, et al., 2006) (Figure 1.3). The Rossmann-fold domain consists of six parallel β strands forming a β sheet which is sandwiched between several α helices on each side (Min, et al., 2001). The two modules of the zinc-binding domain include three β strands forming an antiparallel β sheet and a helical region with three or four helices 1. Introduction 14 dependent on the sirtuin member. Four cysteine residues coordinate the zinc ion in a tetrahedral conformation to stabilize the structure (Min, et al., 2001). A cleft where catalysis takes place is formed between two domains by four linking loops. This region is the most conserved with high sequence homology among sirtuin family. The largest of these four linking loops, called cosubstrate binding loop, is very dynamic. The cosubstrate binding loop is highly flexible when NAD+ is not bound and becomes wellordered through NAD+ binding (Zhao, et al., 2004) indicating that its conformation is dependent on the presence of NAD+. The NAD+ binding site can be divided in three pockets: an adenine binding pocket (pocket A), a nicotinamide (NAM) ribose binding pocket (pocket B) and a NAM binding pocket (pocket C). Different ligands in NAD+ site induce slightly conformational changes of the cosubstrate binding loop (Avalos, et al., 2005; Sanders, et al., 2007). The acetyl lysine of peptide substrate inserts into a hydrophobic tunnel of the cleft between two domains. When comparing the protein conformation with and without peptide substrate, peptide binding induces a shift in the linking loop between two domains and brings two domains closer together (Cosgrove, et al., 2006). The substrate peptide orientation and interaction with protein residues described in different crystal structures strengthen one mechanism that different sirtuins discriminate among substrates. The NAD+ cosubstrate inserts from opposite site with the acetylated substrate into the cleft between two domains (Sanders, et al., 2010). The conformation of NAM ribose is variable dependent on NAD+ analogs, substrate peptides and sirtuin homologs. The density of the ADPR part including the adenine ribose and NAM ribose is well defined whereas the NAM moiety is almost invisible in different sirtuin structures in the presence of NAD+ (Chang, et al., 2002; Nguyen, et al., 2013; Pan, et al., 2011) indicating the flexibility of this part or the hydrolysis of NAD+ during crystallization. 1. Introduction 15 Figure 1.3. Overall structure of sirtuins. The catalytic core of human Sirt3 (ribbon) in complex with carba-NAD+ (stick, orange) and ACS2 peptide (acetyl lysine in stick, yellow) (PDB ID 4FVT) (Szczepankiewicz, et al., 2012) is shown as a representative. The large Rossmann-fold domain is in purple. The small zinc-binding domain that contains Zn2+ ion (sphere, yellow) is in blue. The loops connecting two domains are in green. The cosubstrate binding loop is highlighted in red. 1.3.4. Enzymatic activity of sirtuins Although the initial activity of sirtuins was reported as NAD+-dependent ADPribosylation (Tanny, et al., 1999), protein deacylation is the most prevalent reaction that sirtuin enzymes catalyze. The deacetylation reaction occurs in two continuous stages to generate deacetylated protein, NAM and 2’-O-acetyl-ADP-ribose (2’-OAADPr) (Sauve, et al., 2006; Tanner, et al., 2000) (Figure 1.4). In the first stage, sirtuins cleave NAD+ to produce NAM and the nucleophilic addition of the acetyl oxygen to C1’ of the ADP-ribose moiety to form C1’-Oalkylamidate intermediate (Sauve, 2010). The nucleophilic attack mechanism has been subject to debate between SN1 and SN2 type for the cleavage of the glycosidic bond between NAM and the 1. Introduction 16 rest of NAD+ (Smith and Denu, 2007). NAM can inhibit sirtuins by rebinding to reverse the reaction through the base-exchange mechanism (Sauve, et al., 2006). In the second stage, the C1’-O-alkylamidate intermediate converts to the bicyclic intermediate by using the conserved Histidine as a general base to induce nucleophilic attack of the 2’-OH group of the ribose onto the iminium carbon of the O-alkylamidate intermediate. The crystal structure of the bicyclic intermediate between thiosuccinyl H3 peptide and NAD+ on Sirt5 was recently solved to provide an evidence for the mechanism (Zhou, et al., 2012). The bicyclic intermediate is disrupted by a base activated water molecule to form deacetylated protein and 2’-O-acetyl-ADP-ribose (Sauve and Youn, 2012). Both 2’-O-acetyl-ADP-ribose and 3’-O-acetyl-ADP-ribose exist in equilibrium as solution products of sirtuins (Jackson and Denu, 2002). Figure 1.4. Mechanism of sirtuin-catalyzed deacetylation. Protein deacetylation reaction with two continuous stages catalyzed by sirtuins. Figure is reused with permission from American Society for Biochemistry and Molecular Biology (Feldman, et al., 2012). Protein Ac-Protein Stage 1 Stage 2 2. Materials and Methods 23 2. Materials and Methods 2.1. Materials 2.1.1. Chemicals, peptides and compounds All chemicals were from Sigma, Roth and Applichem if not stated differently. Fluor-delys 1 (FdL-1) peptide was from Enzo Life Science (New York, USA). The fluorophore-free peptides listed in Table 2.1 were synthesized and HPLC (high performance liquid chromatography) purified by GL Biochem (Shanghai, China). 4’-bromo-resveratrol was from Matrix Scientific (Columbia, USA). SRT1720 was from Cayman Chemical (Ann Arbor, USA). Table 2.1. List of peptides used in this study. Peptide Protein Sequence Acetylated position ACS2 Acetyl-coenzyme A synthetase 2 TRSG(acK)VMRRL Lys642 p53-short p53 RHK(acK)LMFK Lys382 p53-long p53 STSRHK(acK)LMFKTE Lys382 CPS1 Carbamoyl-phosphate synthetase 1 FKRGVL(acK)EYGVKV Lys527 ME Mutant electron-transferring flavoprotein dehydrogenase ILTE(acK)YRI Lys153 ME-long Mutant electron-transferring flavoprotein dehydrogenase FGILTE(acK)YRIPVP Lys153 H3 Histone 3 IHA(acK)RVT Lys116 Lamin-B2 Lamin-B2 YKFTP(acK)YILRA Lys500 AIF Apoptosis-inducing factor 1 DFRSLE(acK)ISREVK Lys295 2.1.2. Plasmid vectors The catalytic core domain gene of zebrafish Sirt5 (zSirt5) (residues 30-298) was cloned into the vector pET151/D-TOPO (Life Technologies, USA) coding for His-tag (hexahistidine tag) and carrying the resistance marker to ampicillin. The catalytic core domain gene of human Sirt3 (hSirt3) (residues 118-399) was cloned into the vector pVFT3S (Sungkyunkwan university, 2. Materials and Methods 24 South Korea) coding for His-Trx-tag (hexahistidine-thioredoxin tag) and carrying the resistance marker to kanamycin. 2.1.3. Oligonucleotide primers All primers using for cloning or site-directed mutagenesis listed in Table 2.2 were HPLC purified or HPSF (high purity salt free) from Sigma, USA or Eurofins MWG Operon, Germany. Table 2.2. List of primers used in this study. Restriction sites or stop codon are underlined. Positions of mutation labeled as bold and italic. Name Sequence zSirt5-5’TOPO 5’-CACCACCAGACCAAGCTCGGATTTA-3’ zSirt5-3’stop 5’-CTATTCCAGCGCGGGGGGCAA-3’ hSirt3-5’BamHI 5’-CTAGCTGGATCCAGTGACAAGGGGAAGCT-3’ hSirt3-3’XhoI 5’-GACCCGCTCGAGTCATTTGTCTGGTCCATC-3’ R139A_F 5’-GCCAGAGCCTGCCAGGCGGTGGTGGTCATGGTG-3’ R139A_R 5’-CACCATGACCACCACCGCCTGGCAGGCTCTGGC-3’ M311A_F 5’-GTGGTTGATTTCCCCGCGGCAGATCTGCTGCTC-3’ M311A_R 5’-GAGCAGCAGATCTGCCGCGGGGAAATCAACCAC-3’ R335A_F 5’-TTGACCGAGGCCGTGGCGAGCTCAGTTCCCCGA-3’ R335A_R 5’-TCGGGGAACTGAGCTCGCCACGGCCTCGGTCAA-3’ R384A_F 5’-TGGACAGAAGAGATGGCGGACCTTGTGCAGCGG-3’ R384A_R 5’-CCGCTGCACAAGGTCCGCCATCTCTTCTGTCCA-3’ 2.1.4. Bacterial strains The E. coli strain TOP10 (Life Technologies, USA) was used for cloning, plasmid propagation and site-directed mutagenesis. The E. coli strain Rosetta (DE3) (Merck, Germany) was used for overexpression of recombinant proteins. 2. Materials and Methods 25 The strains have the following genotypes: TOP10: F– mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 ΔlacX74 recA1 araD139 Δ(ara leu) 7697 galU galK rpsL (StrR) endA1 nupG Rosetta (DE3): F– ompT hsdSB(rB – mB–) gal dcm lacY1(DE3) pRARE (CamR) 2.2. Methods 2.2.1. Agarose gel electrophoresis Bio-Rad Sub-cell horizontal gel electrophoresis system (Bio-Rad, USA) was used to perform nucleic acid electrophoreses with 1x TAE (Tris-acetate-EDTA (ethylenediaminetetraacetic acid)) as the running buffer. Samples and DNA maker (New England Biolabs, USA) were mixed with loading buffer (6 mM EDTA, 6 % glycerol and 0.015 % bromophenol blue) before loading on a 1 % (w/v) agarose matrix (in 1x TAE buffer) containing 1 µg/ml ethidium bromide. After electrophoresis, the gel was placed under UV light for DNA visualization. 2.2.2. Cloning The catalytic core domain genes of zSirt5 and hSirt3 were amplified using PCR (polymerase chain reaction). 50 µl of a PCR reaction contains the following: 10-50 ng of template DNA, 0.5 µM of each primer, 2 Units of DNA polymerase (Thermo Scientific, USA) and 1x HF buffer, 0.2 µM of each deoxynucleotide. The PCR program included 1) initial denaturation at 95 ºC for 2 minutes; 2) denaturation at 95 ºC for 1 minute, annealing at 60 ºC for 1 minute, extension at 72 ºC for 1 minute and 3) final elongation at 72 ºC for 10 minutes. Step 2 was repeated 30 times. The PCR products were visualized and purified using agarose gel electrophoresis and gel extraction kit (Qiagen, USA). zSirt5 gene was directly mixed with the vector pET151/D-TOPO without using restriction enzymes. hSirt3 gene and the vector pVFT3S were treated with restriction enzymes 2. Materials and Methods 26 BamHI and XhoI (Thermo Scientific, USA). After visualized and purified using agarose gel electrophoresis, hSirt3 gene was ligated into the vector using a molar ratio of 3:1 (gene : vector) in the presence of T4 DNA ligase (New England Biolabs, USA) and incubation at 20 ºC overnight. 3 µl of the ligated products was used for the transformation of the recombinant plasmids into 50 µl of TOP10 competent cells to amplify the plasmids. The mixture was placed on ice for 30 minutes, heat shock at 42 ºC for 30 seconds, and then put on ice for 5 minutes. 450 µl of LB media was added to recover the cells at 37 ºC for 1 hour followed by plating on LB agar plates containing appropriate antibiotics and incubated at 37 ºC overnight. Subsequently, the plasmids were extracted using plasmid extraction kit (Qiagen, USA). 2.2.3. Site-directed mutagenesis 50 µl of the PCR reaction for site-directed mutagenesis contains the following: 5-50 ng of template DNA, 125 ng of each forward and reverse primers, 0.2 mM deoxynucleotide mix, 1.25 Units of Pfu Turbo DNA polymerase (Agilent Technologies, USA), 1x cloned Pfu DNA polymerase reaction buffer. The PCR program for site-directed mutagenesis was: 1) initial denaturation at 95 ºC for 5 minutes; 2) denaturation at 95 ºC for 1 minute, annealing at 55 ºC for 1 minute, extension at 68 ºC for 10 minutes; 3) final elongation at 68 ºC for 10 minutes. Step 2 was repeated 18 times. Subsequently, the PCR product was treated with 5 Units of DpnI restriction enzyme at 37 ºC for 1 hour to digest the template plasmid vector and 1 µl of the reaction mixture was transformed into 50 µl of TOP10 competent cells using the transformation protocol as in the Cloning section. 2.2.4. Expression The recombinant plasmids were transformed into 50 µl of E. coli Rosetta (DE3) competent cells for expression. The cells were placed on a 1 millimeter electroporation cuvette (Serva, Germany) and pulsed with a voltage of 2.5 kV using the Bio-Rad Gene Pulser electroporation system (Bio-Rad, USA). 450 µl of LB media was added to recover the cells at 37 ºC for 1 hour followed by transferring to LB media containing appropriate antibiotics and 2. Materials and Methods 27 incubation at 37 ºC by shaking. When the OD600 reached 0.6 – 0.8, the temperature was reduced to 15 ºC. IPTG (isopropyl β-D-thiogalactopyranoside) was added into media to induce protein expression. The cells were grown at 15 ºC overnight and harvested by centrifugation at 5,000 RPM for 20 minutes at 4 ºC and stored at -80 ºC. 2.2.5. Cell disruption Frozen cells were resuspended in an appropriate lysis buffer and disrupted using Microfluidizer (Microfluidics, USA) at 4 ºC. The lysed cells were then centrifuged at 18,000 RPM for 45 minutes in a refrigerated Beckman Coulter Avanti J-26XP centrifuge fitted with a JA-30.50 Ti rotor (Beckman Coulter, USA) to remove cell debris. 2.2.6. SDS-Polyacrylamide gel electrophoresis (SDS-PAGE) SDS-PAGE was performed to analyze the purity and size of proteins. The stacking gel, running gel (15% acrylamide), buffers were prepared by following a published protocol (Sambrook and Russell, 2001). Protein samples were denatured for 5 minutes at 95 ºC before loading on the gel. The Mini-PROTEAN Tetra Cell vertical electrophoresis system (Bio-Rad, USA) was used for electrophoresis. After electrophoresis, the gel was rinsed in water, followed by a quick soaking in hot Coomassie blue solution (0.025 % (w/v) Coomassie-Briliant Blue R250 (Applichem, Germany), 50 % (v/v) methanol, 10 % (v/v) acetic acid). Subsequently, the gel was transferred to a destaining solution containing 20 % (v/v) methanol and 12 % (v/v) acetic acid. 2.2.7. Purification The catalytic core domain of zSirt5 and hSirt3 were purified using similar protocols. The fusion proteins were purified by affinity chromatography with TALON resin followed by removing the His-tag of zSirt5 or His-Trx-tag of hSirt3 using tobacco etch virus (TEV) protease. 2. Materials and Methods 28 To separate tag and protease, the tag-cleaved hSirt3 was resubjected to a TALON column while the digested zSirt5 was loaded into a HiTrap SP column. Finally, zSirt5 and hSirt3 were subjected to Superose-12 gel filtration column (GE Healthcare, Waukesha, USA), and the purified proteins were shock frozen and stored at -80 °C. 2.2.7.1. Affinity chromatography (AC) The first step to purify His-tagged hSirt3 and zSirt5 was affinity chromatography. 1 ml bed volume of TALON resin was used for every liter of E. coli culture. The resin was washed with water followed by equilibration in lysis buffer. The supernatant containing the recombinant protein in lysis buffer was incubated with the equilibrated resin at 4 °C for 1 hour. After the incubation, the flow through was collected by gravity flow using a glass column (Bio-Rad, USA). The column was washed twice with 20 bed volumes of two wash buffers and the protein was then eluted with elution buffer. The purity and size of the proteins were analyzed using SDS-PAGE. Lysis buffer: 50 mM Tris, pH 7.8 for hSirt3 and 8.5 for zSirt5, 200 mM NaCl The first wash buffer: 50 mM Tris, pH 7.8 for hSirt3 and 8.5 for zSirt5, 500 mM NaCl The second wash buffer: 50 mM Tris, pH 7.8 for hSirt3 and 8.5 for zSirt5, 200 mM NaCl, 5 mM Imidazole Elution buffer: 50 mM Tris, pH 7.8 for hSirt3 and 8.5 for zSirt5, 200 mM NaCl, 250 mM Imidazole In the second AC of hSirt3 purification, the protein was eluted using gel filtration buffer. 2.2.7.2. Tag cleavage The His-tag of zSirt5 and the His-Trx-tag of hSirt3 were cleaved using TEV protease. The purified proteins after AC step were dialyzed in the buffer containing 30 mM HEPES, pH 2. Materials and Methods 29 6.5, 50 mM NaCl for zSirt5 and gel filtration buffer for hSirt3 at 4 °C. The ratio of protease : protein is 1:20 mg and incubated at 4 °C overnight. 2.2.7.3. Ion exchange chromatography (IEC) IEC was performed using a 1 ml HiTrap SP cation exchange column (GE Healthcare, USA) that was equilibrated with buffer A (30 mM HEPES, pH 6.5). After tag cleavage, zSirt5 was applied on the column. The column was washed with 3 column volumes of buffer A followed by elution of the protein using a linear gradient against buffer B (30 mM HEPES, pH 6.5, 1 M NaCl). Protein fractions were analyzed using SDS-PAGE and then pooled. 2.2.7.4. Size exclusion chromatography (SEC) Elution samples from the second AC of hSirt3 or from the IEC of zSirt5 purification were pooled and concentrated to 1 ml using an Amicon centrifugal concentrator (Millipore, USA) and injected to an equilibrated Superose-12 size exclusion column (GE Healthcare, USA) and eluted with gel filtration buffer containing 20 mM Tris, pH 7.8 for hSirt3 and 8.5 for zSirt5, 150 mM NaCl. Subsequently, the purity of the eluted fractions was assessed using SDS-PAGE before appropriate fractions were pooled and concentrated. 2.2.8. Fluorescence-based Flour-de-Lys assay Deacetylase activity of sirtuins was tested using a commercial FdL assay kit (Enzo Life Sciences, USA) containing the p53-derived FdL-1 substrate peptide RHK(acK) with a Cterminally attached fluorophore. 50 µl of a reaction mixture consisting of 1.5 µg of sirtuin, 100 µM FdL-1, 2.5 mM NAD+ in the appropriate protein buffer was incubated at 37 °C for 30 minutes. Subsequently, a developer mixture containing 2 mM NAM and 10 mg/ml trypsin was added to the reaction mixture and incubated at room temperature for 45 minutes. Trypsin cleaves the coumarin tag from deacetylated FdL-1. Fluorescence was determined at an excitation wavelength of 360 nm and an emission wavelength of 460 nm using a FluoDiaT70 microplate 2. Materials and Methods 30 reader (Photal Otsuka Electronics, Japan). A blank containing all the components of the assay except the enzyme was subtracted. 2.2.9. Enzyme-coupled continuous assay The continuous assay was performed using a published protocol (Smith, et al., 2009). NAM, one of the products of the deacetylation reaction, is first converted to nicotinic acid and ammonia by nicotinamidase. The ammonia is then transferred to α-ketoglutarate via glutamate dehydrogenase yielding glutamate, under consumption of NADPH which is measured spectrophotometrically at 340 nm and thus proportional to sirtuin activity. 100 µl of a reaction mixture contains 2 µM of hSirt3 or 10 µM of zSirt5, 500 µM substrate peptide, 640 µM NAD+, 1 mM DTT, 3.3 mM α-ketoglutarate, 2 µM nicotinamidase, 2 units of bovine GDH and 0.2 mM NADPH in a buffer containing 20 mM Na-PO4, pH 7.5. The reaction was performed at room temperature for 1 hour and continuously measured using a spectrophotometer (Cary 50, Agilent technologies, USA). 2.2.10. Mass spectrometry (MS) 50 µl of a reaction mixture consisting of 10 μM hSirt3 (in 20 mM Tris pH 7.8, 150 mM NaCl) or zSirt5 (in 20 mM Tris pH 8.5, 150 mM NaCl), 0.5 mM ACS2 peptide and 2.5 mM NAD+ in the presence of different compound concentrations in 2% (v/v) DMSO, or with 2% (v/v) DMSO as a control was incubated at 37 °C. The reaction was stopped after different time points by adding 0.25% (v/v) trifluoroacetic acid (TFA) followed by dilution to 1 µM peptide in 0.1% (v/v) formic acid (FA). Subsequently, the solution was filtered to separate the substrate peptide from the reaction mixture using 10 kDa cutoff concentrators (Pall Life Sciences, USA). Finally, 5 µl of each sample containing the filtered substrate peptide was subjected to nano-LCMS/MS analysis as described before (Fischer, et al., 2012). Specific deacetylation activity was determined by linear fitting of the time-series experiments. The results were analyzed using Xcalibur (Thermo Scientific, USA). 2. Materials and Methods 31 2.2.11. Thermal denaturation shift assay Protein thermal denaturation assay measures the thermal stability of a target protein and a subsequent increase in protein melting temperature due to the binding of a ligand to the protein based on the fluorescence change of the dye SYPRO Orange (Life Technologies, USA). 50 µl of a sample mixture contains 0.1 mg/ml of protein, 1 µl of 1:10 diluted SYPRO dye, 500 µM NAD+, compounds or 2% (v/v) DMSO as a control followed by adding 15 µl of mineral oil. The temperature was gradually increased from 25 °C to 73 °C using 2 °C intervals. The change in fluorescence was measured at an excitation wavelength of 465 nm and an emission wavelength of 580 nm using a FluoDiaT70 microplate reader (Photal Otsuka Electronics, Japan). 2.2.12. Binding analysis by microscale thermophoresis (MST) Binding affinities were measured by microscale thermophoresis (Wienken, et al., 2010) with 1 μM hSirt3 in 20 mM Tris pH 7.8, 150 mM NaCl in the presence or absence of different concentrations of compounds or ACS2 peptide. Protein and ligands were mixed at room temperature and transferred to capillaries for scanning before thermophoresis analysis at 25 ºC using the NanoTemper Monolith NT.label-free instrument (NanoTemper Technologies, Germany) with the intrinsic protein fluorescence signal (excitation at 280 nm, emission at 360 nm). The excitation UV-LED power was set to 25% and IR-laser power to 20; 40 and 80%. The Kd values were determined through non-linear fitting (1-site equation) of the measured thermophoresis values using Prism (Graphpad Software, CA, USA). Each experiment was repeated at least twice. 2.2.13. Crystallization and structure determination Crystallization trials were performed using a Phoenix robot (Art Robbins, USA) for initial screening with a mixture of 0.15 μl of protein and 0.15 μl of reservoir solution on a 96 well sitting drop plate (Corning, Intelli, Greiner etc. plates) and incubated at 20 °C in a Formulatrix imager (Formulatrix Inc., USA). The selected conditions were further optimized 2. Materials and Methods 32 manually by mixing 1 μl of protein and 1 μl of reservoir solutions on a 24 well sitting drop corning plates and incubation at 20 °C. The X-ray diffraction data were collected at 100 K with an MX-225 CCD detector (Rayonix, Evanston, IL, USA) at beam line MX14.1 of the BESSY II electron storage ring (Berlin, Germany) (Mueller, et al., 2012). The wavelength was 0.92 Å allowing to observe the anomalous diffraction of the Br atom. Diffraction data were processed using XDS (Kabsch, 2010). Crystal structures were solved by Patterson searches with the program MolRep (Vagin and Isupov, 2001) using chain A of the complex hSirt3/FdL-1/PCT (PDB ID 4HD8) (Gertz, et al., 2012) as a search model for hSirt3 structures and the complex hSirt5/suramin (PDB ID 2NYR) (Schuetz, et al., 2007) as a search model for zSirt5 structures. Structure refinement was performed using Refmac (Murshudov, et al., 1997), and manual rebuilding was done in Coot (Emsley and Cowtan, 2004). Parameter files for 4’-bromo-resveratrol, polydatin and SRT1720 were generated using ProDrg (Schuttelkopf and van Aalten, 2004). The quality of the refined structures was evaluated using Coot and MolProbity (Chen, et al., 2010). The structure figures were prepared using Pymol (The PyMOL Molecular Graphics System, Schrödinger, LLC). . 3. Results 39 Figure 3.6. Crystal structure of hSirt3 in complex with PCT/PD and FdL-1 peptide. (A) Overall structures of the complex hSirt3/FdL-1/PCT and hSirt3/FdL-1/PD. PCT is shown in stick-yellow, PD in stick-green. FdL-1 peptides of two complexes are in stick and in the same color as the corresponding compound. (B, C) The interface with the neighboring symmetryrelated monomer: Two FdL-1 peptides form π-stacking interactions and two PCT/PD molecules overlay each other. Omit Fo-Fc difference density is contoured at 3.0σ. The symmetry-related monomer is in grey. 3.1.2.3.2. hSirt3 in complex with FdL-1 peptide and 4’-bromo-resveratrol In the hSirt3/FdL-1/brRESV complex structure, the compound was found in the active site (Figure 3.7) and different from the PCT/PD binding site. 3. Results 40 Figure 3.7. Crystal structure of hSirt3 in complex with brRESV and FdL-1 peptide. (A) Overall structure of hSirt3/FdL-1/brRESV complex. The dashed line indicates a loop not defined by electron density. (B) FdL-1 peptide and brRESV ligands of hSirt3, overlaid with omit Fo-Fc difference density (2.5 σ; green) and anomalous density (5 σ; magenta) showing the positions of Br and Zn2+. (C) 2Fo-Fc electron density (1 σ; blue) of FdL-1 peptide and brRESV in hSirt3/ FdL-1/brRESV complex. A closer look at the compound binding site (Figure 3.8) shows that the A-ring hydroxyl groups of brRESV form hydrogen bonds with Asn229 and Asp231 of hSirt3. Furthermore, residues Ile230, Leu199, and Ile154 form a hydrophobic patch for A-ring binding, and Phe157, Leu195, and Phe180 a hydrophobic cleft for accommodating the B-ring. This cleft extends in a hydrophobic pocket (formed by Ile179, Leu173, and Tyr171) for binding the bromine atom, and Arg158 and Pro176 form a lid shielding this pocket from solvent. 3. Results 41 Figure 3.8. Closer view on the brRESV binding site showing interacting residues. Hydrogen bonds are indicated by dashed lines. Superposition of the crystal structure of the complex hSirt3/FdL-1/brRESV and Sir2Tm in complex with p53 peptide and NAD+ (Hoff, et al., 2006) reveals that brRESV occupies part of the NAD+ binding pocket (Figure 3.9), thus prevents the C-pocket insertion of the NAD+ nicotinamide moiety necessary for catalysis. Figure 3.9. Superposition of the hSirt3/FdL-1/brRESV structure with a Sir2Tm/p53/NAD+ complex (PDB ID 2H4F) (Hoff, et al., 2006). FdL-1 peptide and brRESV are in pink, p53 peptide and NAD+ are in green. The protein part of the Sir2Tm/p53/NAD+ complex is omitted for clarity. 3. Results 42 The overall hSirt3 and FdL1-peptide conformations in the superposition of the brRESV complex with hSirt3 bound to FdL-1 and other resveratrol-related compounds, PCT/PD, are identical (Figure 3.10). However, FdL-1 peptide conformations are different, in particular the fluorophore orientation. PCT/PD interact extensively with the FdL-1 fluorophore to induce nonproductive peptide binding (Gertz, et al., 2012) whereas brRESV does not directly contact this substrate peptide but blocks productive NAD+ binding. Figure 3.10. Superposition of the hSirt3/FdL-1/brRESV with the hSirt3/FdL-1/PCT complex. PCT is shown in stick-yellow, brRESV in stick-pink. FdL-1 peptides of two complexes are in stick and in the same color as the corresponding compound. To test for competition between brRESV and FdL-1 peptide, IC50 values for brRESV inhibition of hSirt3 were determined at three different concentrations of FdL-1 peptide (50, 100, 3. Results 43 and 200 µM). The IC50 value obtained were ~100 µM for all three peptide concentrations (Figure 3.11), indicating that the inhibitor brRESV does not compete against FdL-1. Figure 3.11. IC50 determination for brRESV inhibition of hSirt3 at 50, 100, and 200 M FdL-1 substrate peptide. We also investigated the competition between brRESV and NAD+ for hSirt3 binding. The binding affinity of brRESV to the apo protein was 7.6 ± 0.9 µM, and the Kd increased to higher than 50 µM in the presence of 2 mM NAD+ (Figure 3.12). The results reveal that brRESV competes with NAD+ for binding to hSirt3, supporting the conclusion that the internal brRESV binding site is the one relevant for inhibition of hSirt3 activity. Figure 3.12. Binding affinity of brRESV to hSirt3 in the presence or absence of 2 mM NAD+. 3. Results 44 3.1.2.3.3. hSirt3 in complex with ACS2 peptide and 4’-bromo-resveratrol In the complex structure hSirt3/ACS2/brRESV, the compound molecule was found at the bottom of the Rossmann-fold domain, interacting with Arg139, Met331, and Arg335 (Figure 3.13). In this exposed position, the compound interacts only through its A-ring with this shallow hSirt3 pocket, and the bromo-containing aromatic ring points towards the symmetry-related monomer in the crystal lattice. Figure 3.13. Crystal structure of hSirt3 in complex with brRESV and ACS2 substrate peptide. (A) Overall structure of hSirt3/ACS2/brRESV complex. A missing loop is indicated by a dashed line. (B) 2Fo-Fc electron density (1 σ; blue) of ACS2 peptide in hSirt3/ACS2/brRESV complex. (C) brRESV ligand of hSirt3, overlaid with omit Fo-Fc difference density (2.5 σ; green) and anomalous density (5 σ; magenta) showing the position of Br and surrounding residues important for compound binding. Residues from the symmetry-related monomer are labeled with a star. (D) 2Fo-Fc electron density (1 σ; blue) of brRESV in hSirt3/ACS2/brRESV complex. 3. Results 45 The superposition of the hSirt3/brRESV complexes with FdL-1 and ACS2 peptide, respectively, reveals that the inhibitor cannot bind at the catalytic pocket when the ACS2 peptide is bound, since it would clash with the C-terminal part of this substrate peptide (Figure 3.14). Figure 3.14. Superposition of the hSirt3/ACS2/brRESV structure with the hSirt3/FdL1/brRESV complex. FdL-1 peptide and brRESV are in pink, ACS2 peptide is in cyan. Phe157 of the hSirt3/ACS2/brRESV and hSirt3/FdL-1/brRESV complex are shown in cyan and pink, respectively. brRESV in the complex structure with hSirt3/ACS2-peptide does not show many interactions with hSirt3, rendering it a less likely inhibition site. Figure 3.15 illustrates the hydrogen bonds between brRESV with Arg139, Met331 (backbone), and Arg335 and additionally with Arg384 of the symmetry-related monomer. 3. Results 46 Figure 3. 15. Closer view on the brRESV binding site in the hSirt3/ACS2/brRESV complex showing interacting residues. Hydrogen bonds are indicated by dashed lines and residues of the next symmetry-related monomer are labeled with a star. To test whether the surface site occupied by brRESV in its hSirt3/ACS2 complex is relevant for inhibition, the role of the interacting residues R139, M331, R335, and R384 were tested by site-directed mutagenesis to alanine. Microscale thermophoresis (MST) results indicate that the hSirt3-R335A variant has a slightly reduced binding affinity for the compound compared to wildtype hSirt3 (Figure 3.16A), but the change was not statistically significant. The hSirt3 variants R384A, R139A, and M331A showed no change in the affinity for the compound. Moreover, we performed activity assays with 500 µM ACS2 as peptide substrate and in presence of brRESV (at its IC50 concentration, 140 µM) to examine the effects on the activity of the mutant proteins. The screening indicated that the activity of hSirt3 R335A is slightly higher than for the other mutants and the wildtype protein (data not shown). The IC50 value of brRESV on the R335A variant activity was then determined. Figure 3.16B shows only a small shift for the brRESV inhibition curves between wildtype protein and the R335A variant, resulting in IC50 values of 143.0 ± 3.6 and 179.2 ± 12.3 µM, respectively. Therefore, the position of brRESV in the complex structure hSirt3/ACS2/brRESV seems not to be primarily responsible for the inhibitory effect of the compound on hSirt3. 3. Results 47 Figure 3.16. (A) Affinity measurements for binding of brRESV to hSirt3 wildtype as well as R335A and R384A variants. Error bars represent standard errors of two independent measurements. (B) IC50 determination of brRESV on wildtype hSirt3 or the R335A variant against 500 µM ACS2 peptide using MS. Error bars represent standard errors of linear fits to time series experiments. To test for brRESV competition with ACS2 peptide, MS assays were performed to determine Km and Vmax values for the peptide at different compound concentrations. The result shows that the higher the brRESV concentration, the more the Km for substrate peptide increases, or affinity for substrate decreases, while the Vmax is not altered, indicating competitive inhibition (Figure 3.17). 3. Results 48 Figure 3.17. brRESV inhibition of hSirt3 with ACS2 peptide as a substrate. brRESV concentrations of 0, 50, and 150 µM resulted in Km values for substrate peptide of 31.3 ± 9.0, 48.6 ± 7.5, and 253.3 ± 52.9 µM, respectively, while the Vmax is roughly constant at ~50 nmol mg-1 min-1. Error bars represent standard errors of linear fits to time series experiments. This competition was confirmed through binding data from MST measurements. ACS2 peptide bound to apo hSirt3 with a Kd value of 64.4 ± 9.1 µM. In presence of 50 µM brRESV, the Kd value increased to more than 200 µM (Figure 3.18A). Consistently, binding affinity of brRESV to the apo protein was 7.6 ± 0.9 µM, and the Kd increased to higher than 37 µM in the presence of 500 µM ACS2 peptide (Figure 3.18B). Thus, brRESV inhibits hSirt3 through binding competition with ACS2 peptide substrate. Figure 3.18. (A) Binding affinity of ACS2 peptide to hSirt3 in the presence or absence of 50 µM brRESV. (B) Binding affinity of brRESV to hSirt3 in the presence or absence of 500 µM ACS2 peptide. Error bars represent standard errors of two independent measurements. 3. Results 55 Figure 3.27. Active site of the hSirt3/ NAD+/Ex-527 complex. 2Fo-Fc electron density of NAD+ and Ex-527 is contoured at 1.0σ. To examine whether Ex-527 can bind to hSirt3 during the step of forming Oalkylamidate intermediate, the crystal of hSirt3/ACS2 was soaked with NAD+ and Ex-527 in 80 minutes. The obtained structure showed very clear native O-alkylamidate intermediate state of the reaction but density for Ex-527 was not found (Figure 3.28). In comparison to the Salkylamidate intermediate, the ribose moiety of the native O-alkylamidate intermediate has a different conformation (Figure 3.29). In combination with inhibition kinetics and binding analysis, the inhibition mechanism of Ex-527 on sirtuins was revealed to be that the compound stabilizes the closed enzyme conformation of the complex with 2’-O-acetyl-ADP-ribose, thus prevents product release (Gertz, et al., 2013). 3. Results 56 Figure 3.28. Active site of the hSirt3/ O-alkylamidate intermediate complex. 2Fo-Fc electron density of the intermediate is contoured at 1.0σ. Figure 3.29. Superposition of the hSirt3/ O-alkylamidate intermediate complex with hSirt3/Salkylamidate intermediate complex (pink, PDB ID 3GLT) (Jin, et al., 2009). 3. Results 57 3.2. Sirt5 studies 3.2.1. Sirt5 purification In a previous study, human Sirt5 (hSirt5) purification yielded high amount of the purified protein (Gertz, et al., 2012). However, crystallization trials resulted in twinned crystals and low occupancy of ligands. Therefore, zSirt5, an orthologue of hSirt5 was used in this study to overcome the issue. The zSirt5 purification protocol in this study was illustrated by a diagram in figure 3.30A. After the AC step, the highly expressed protein with the size of ~32 kDa was collected. The zSirt5 construct (30-298) is ~31 kDa and the His-tag is ~1 kDa. After TEV protease incubation and cation exchange, the His-tag was removed and zSirt5 was eluted in the fractions of the first peak with a small remaining contamination. After SEC, the last step of purification, the purified zSirt5 was obtained in the fractions B5 – B12 with the purity ≥ 95% (Figure 3.30B). The yield of purification was 6 mg of the purified protein per 12 liters of the expressed media. 3. Results 58 Figure 3.30. Zebrafish Sirt5 purification. (A) Four steps of zSirt5 purification. (B) Step 4: SEC, contamination was separated, purified zSirt5 including fractions B5-B12 (green box) was pooled for further studies. B.L, before loading. 3. Results 59 3.2.2. Resveratrol and its related compounds are zSirt5 activators on FdL-1 peptide Similar to Sirt3 study, the FdL assay was performed to investigate the effects of RESV and its related compounds including PCT, PD and brRESV on zSirt5. Among these compounds, 1 mM RESV can activates zSirt5 up to 12-fold, thus becomes the most potent activator of the enzyme (Figue 3.31). PCT, PD and brRESV slightly activate zSirt5 with nearly 3-fold in the presence of 0.2 mM and 6-fold in the presence of 1 mM compound concentration, except brRESV. Figure 3.31. Activation of zSirt5 activity on FdL-1 peptide by resveratrol-related compounds at 0.2 mM and 1 mM compound concentration. Activities were normalized to the control in the absence of compound. Error bars represent standard errors of two independent measurements. To prevent the artificial effect of FdL-1 peptide as mentioned in the Sirt3 study section, several fluorophore-free acetylated peptides were screened to find potent peptide substrates for zSirt5 using continuous assay. Among chosen peptides, p53 peptide is the most potent substrate of zSirt5 but not CPS1, the acetylated peptide derived from the physiological mammalian Sirt5 substrate (Figure 3.32). Lamin_B2 peptide is also a potent zSirt5 peptide substrate but has low solubility due to its rich hydrophobic residues. ME peptide has the similar linear of NADPH 3. Results 60 consumption as CPS1 peptide indicating that it is also a zSirt5 substrate (Figure 3.32). ME and p53 peptide were chosen for further study. The regulations of resveratrol related compounds on these peptides were examined using MS. However, these compounds did not show significant effects (weak or unclear activation or inhibition) on the peptides (Figure 3.33). Figure 3.32. zSirt5 substrates were identified using continuous assays Figure 3.33. Insignificant effects of resveratrol related compounds on zSirt5 using MS. (A) p53 peptide. (B) ME peptide with 0.2 mM of each compound. Error bars represent standard errors of linear fits to time series experiments. 3. Results 61 3.2.3. Crystallization trials and crystal structures of zSirt5 in complex with peptide substrates in the presence of resveratrol Since RESV is the most potent activator of zSirt5 in comparison to its related compounds, it was used for crystallization study to determine the activation mechanism on the enzyme. Different crystallization trials including zSirt5 in the presence or absence of peptide substrate and RESV were setup. The diamond crystals were obtained from the condition containing the mixture of zSirt5 and RESV in comparison with no crystal when using DMSO as a control (Figure 3.34). The compound thus seems to be important for crystal growing. The crystals of the complex zSirt5/FdL-1 in the presence of RESV are in rod shape with a nice packing (Figure 3.34). However, the diffractions of these crystals are quite weak and the best data set is only 3.2 Å. The complex zSirt5/ME in the presence of RESV has very big rod crystals (Figure 3.34) and their diffractions are up to 2 Å. The complex zSirt5/p53 in the presence of RESV formed long stick crystals (Figure 3.34). Figure 3.34. Crystals of different zSirt5 complexes with FdL-1 or p53 or ME peptide in the presence of RESV. Since the compound was not found in the structures, it was labeled as italic. 3. Results 62 Similar to other sirtuin/peptide complexes, ME or p53 peptide binds to the cleft between the Rossmann-fold and zinc-binding domain of zSirt5 and the acetyl lysine binds into a hydrophobic tunnel pointing toward the catalytic residue His158 (Figure 3.35). No density fits to RESV implying the compound could not bind or bound with very low occupancy that could not be observed, consistent with the lack of an effect in activity assays with these peptides. Superposition of the complex zSirt5/ME/RESV and zSirt5/p53/RESV indicates that the protein has the same conformation in both structures (data not shown). Figure 3.35. Active site of complex structures. (A) zSirt5/p53/RESV complex and (B) zSirt5/ME/RESV complex. Omit Fo-Fc difference density is contoured at 3.0σ. zSirt5 was crystallized in the presence of RESV but the obtained structure only showed the apo enzyme. The asymmetric unit of the apo-zSirt5 has four monomers whereas the complex zSirt5/FdL-1/RESV contains five zSirt5 monomers and the electron density of FdL-1 was found in only one monomer (Figure 3.36). RESV was included in the solution but not present in the structures. The protein packing may be caused by the unspecific binding between monomers including disulfide bonds formed by the residues Cys278. 3. Results 63 Figure 3.36. (A) Superposition of apo-zSirt5 (salmon pink) and the zSirt5/FdL-1/RESV complex (cyan), the density of the compound was not found. The asymmetric unit of apo structure is tetramer and of the zSirt5/FdL-1/RESV is pentamer but only one monomer contains FdL-1 peptide. zSirt5 is shown in cartoon, Zn2+ as a sphere and the peptide is in stick representation. (B) Active site of the monomer containing FdL-1 peptide. 2Fo-Fc electron density of FdL-1 peptide is contoured at 1.0σ. Superposition of the FdL-1-containing monomer and apo monomer reveals two positions of conformation change: the loop 250 – 260 and the loop 277 – 284 (Figure 3.37). The loop 250 – 260 is peptide-binding loop, thus it moves closer to the peptide when the peptides bind to the protein. The loop 277 – 284 is on the surface of monomer contacts. Figure 3.38 shows the crystal contact of FdL-1 containing monomer of the zSirt5/FdL-1/RESV complex. The peptide is close to the loop 277 – 284 of the next symmetry-related monomer indicating that the loop conformation is also influenced by the presence of the peptide. The overall conformations of the complex zSirt5/FdL-1/RESV and zSirt5/ME/RESV are almost identical except a slight difference in the loop 277-284 (Figure 3.39). This may cause by different sequences and lengths of the peptides. Therefore, the loop 277-284 conformation is very flexible and depends on the presence, sequence and length of peptides. 3. Results 64 Figure 3.37. Superposition of apo-zSirt5 (salmon pink) and the FdL-1 containing zSirt5 monomer (cyan) with the positions of conformation changes showed in black boxes: loop 250-260 and loop 277-284. Figure 3.38. Crystal contact of the zSirt5/FdL-1/RESV complex. The symmetry-related monomer is shown in grey. The FdL-1 peptide and the loop 277-284 of the complex is in dark blue and of the symmetry-related monomer is in grey-black. 4. Discussion 71 4.2. Sirt5 studies An effort to investigate the activation mechanism of RESV on zSirt5 has not been successful. The compound was not found in crystal structures of zSirt5 complex that might due to its low solubility. However, the study obtained different zSirt5 structures including the apo structure and the protein in complex with different peptide substrates. Therefore, it provides structural models for further study on screening and investigating the regulation of other small molecule compounds with higher solubility, potency and selectivity on zSirt5. References 72 References Ahn, B.H., Kim, H.S., Song, S., Lee, I.H., Liu, J., Vassilopoulos, A., Deng, C.X., and Finkel, T. (2008). A role for the mitochondrial deacetylase Sirt3 in regulating energy homeostasis. Proc Natl Acad Sci USA 105, 14447-14452. Ahuja, N., Schwer, B., Carobbio, S., Waltregny, D., North, B.J., Castronovo, V., Maechler, P., and Verdin, E. (2007). Regulation of Insulin Secretion by SIRT4, a Mitochondrial ADPribosyltransferase. J Biol Chem 282, 33583-33592. Alcaín, F.J., and Villalba, J.M. (2009). Sirtuin activators. Expert Opin Ther Pat 19, 403-414. Arima, K., Uéda, K., Sunohara, N., Hirai, S., Izumiyama, Y., Tonozuka-Uehara, H., and Kawai, M. (1998). Immunoelectron-microscopic demonstration of NACP/α-synuclein-epitopes on the filamentous component of Lewy bodies in Parkinson's disease and in dementia with Lewy bodies. Brain Res 808, 93-100. Avalos, J.L., Bever, K.M., and Wolberger, C. (2005). Mechanism of sirtuin inhibition by nicotinamide: altering the NAD(+) cosubstrate specificity of a Sir2 enzyme. Mol Cell 17, 855868. Barber, M.F., Michishita-Kioi, E., Xi, Y., Tasselli, L., Kioi, M., Moqtaderi, Z., Tennen, R.I., Paredes, S., Young, N.L., Chen, K., et al. (2012). SIRT7 links H3K18 deacetylation to maintenance of oncogenic transformation. Nature 487, 114-118. Bedalov, A., Gatbonton, T., Irvine, W.P., Gottschling, D.E., and Simon, J.A. (2001). Identification of a small molecule inhibitor of Sir2p. Proc Natl Acad Sci U S A 98, 15113-15118. Beher, D., Wu, J., Cumine, S., Kim, K.W., Lu, S.-C., Atangan, L., and Wang, M. (2009). Resveratrol is Not a Direct Activator of SIRT1 Enzyme Activity. Chem Biol Drug Des 74, 619624. Beirowski, B., Gustin, J., Armour, S.M., Yamamoto, H., Viader, A., North, B.J., Michán, S., Baloh, R.H., Golden, J.P., Schmidt, R.E., et al. (2011). Sir-two-homolog 2 (Sirt2) modulates peripheral myelination through polarity protein Par-3/atypical protein kinase C (aPKC) signaling. Proc Natl Acad Sci USA 108, E952-E961. Blum, C.A., Ellis, J.L., Loh, C., Ng, P.Y., Perni, R.B., and Stein, R.L. (2011). SIRT1 modulation as a novel approach to the treatment of diseases of aging. J Med Chem 54, 417-432. References 73 Borra, M.T., Langer, M.R., Slama, J.T., and Denu, J.M. (2004). Substrate specificity and kinetic mechanism of the Sir2 family of NAD+-dependent histone/protein deacetylases. Biochemistry 43, 9877-9887. Borra, M.T., Smith, B.C., and Denu, J.M. (2005). Mechanism of human SIRT1 activation by resveratrol. J Biol Chem 280, 17187-17195. Cantó, C., and Auwerx, J. (2009). Caloric restriction, SIRT1 and longevity. Trends Endocrinol Metab 20, 325-331. Cen, Y. (2010). Sirtuins inhibitors: The approach to affinity and selectivity. Biochim Biophys Acta 1804, 1635-1644. Chang, J.H., Kim, H.C., Hwang, K.Y., Lee, J.W., Jackson, S.P., Bell, S.D., and Cho, Y. (2002). Structural basis for the NAD-dependent deacetylase mechanism of Sir2. J Biol Chem 277, 34489-34498. Chen, L. (2011). Medicinal chemistry of sirtuin inhibitors. Curr Med Chem 18, 1936-1946. Chen, V.B., Arendall, W.B., 3rd, Headd, J.J., Keedy, D.A., Immormino, R.M., Kapral, G.J., Murray, L.W., Richardson, J.S., and Richardson, D.C. (2010). MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr D Biol Crystallogr 66, 12-21. Cohen, E., Paulsson, J.F., Blinder, P., Burstyn-Cohen, T., Du, D., Estepa, G., Adame, A., Pham, H.M., Holzenberger, M., Kelly, J.W., et al. (2009). Reduced IGF-1 Signaling Delays AgeAssociated Proteotoxicity in Mice. Cell 139, 1157-1169. Cosgrove, M.S., Bever, K., Avalos, J.L., Muhammad, S., Zhang, X., and Wolberger, C. (2006). The structural basis of sirtuin substrate affinity. Biochemistry 45, 7511-7521. Du, J., Zhou, Y., Su, X., Yu, J.J., Khan, S., Jiang, H., Kim, J., Woo, J., Kim, J.H., Choi, B.H., et al. (2011). Sirt5 is a NAD-dependent protein lysine demalonylase and desuccinylase. Science 334, 806-809. Emsley, P., and Cowtan, K. (2004). Coot: Model-Building Tools for Molecular Graphics. Acta Cryst Section D 60, 2126-2132. Feldman, J.L., Dittenhafer-Reed, K.E., and Denu, J.M. (2012). Sirtuin Catalysis and Regulation. J Biol Chem 287, 42419-42427. References 74 Fischer, F., Gertz, M., Suenkel, B., Lakshminarasimhan, M., Schutkowski, M., and Steegborn, C. (2012). Sirt5 deacylation activities show differential sensitivities to nicotinamide inhibition. PLoS One 7, e45098. Frye, R.A. (2000). Phylogenetic classification of prokaryotic and eukaryotic Sir2-like proteins. Biochem Biophys Res Commun 273, 793-798. Gertz, M., Fischer, F., Nguyen, G.T.T., Lakshminarasimhan, M., Schutkowski, M., Weyand, M., and Steegborn, C. (2013). Ex-527 inhibits Sirtuins by exploiting their unique NAD+-dependent deacetylation mechanism. P Natl Acad Sci 110, E2772-E2781. Gertz, M., Nguyen, G.T., Fischer, F., Suenkel, B., Schlicker, C., Franzel, B., Tomaschewski, J., Aladini, F., Becker, C., Wolters, D., et al. (2012). A molecular mechanism for direct sirtuin activation by resveratrol. PLoS One 7, e49761. Gertz, M., and Steegborn, C. (2010). Function and regulation of the mitochondrial Sirtuin isoform Sirt5 in Mammalia. Biochim Biophys Acta 1804, 1658-1665. Grozinger, C.M., Chao, E.D., Blackwell, H.E., Moazed, D., and Schreiber, S.L. (2001). Identification of a class of small molecule inhibitors of the sirtuin family of NAD-dependent deacetylases by phenotypic screening. J Biol Chem 276, 38837-38843. Haass, C., and Selkoe, D.J. (2007). Soluble protein oligomers in neurodegeneration: lessons from the Alzheimer’s amyloid beta-peptide. Nat Rev Mol Cell Bio 8, 101-112. Haigis, M.C., Mostoslavsky, R., Haigis, K.M., Fahie, K., Christodoulou, D.C., Murphy, A.J., Valenzuela, D.M., Yancopoulos, G.D., Karow, M., Blander, G., et al. (2006). SIRT4 inhibits glutamate dehydrogenase and opposes the effects of calorie restriction in pancreatic beta cells. Cell 126, 941-954. Haigis, M.C., and Sinclair, D.A. (2010). Mammalian sirtuins: biological insights and disease relevance. Annu Rev Pathol 5, 253-295. Hallows, W.C., Lee, S., and Denu, J.M. (2006). Sirtuins deacetylate and activate mammalian acetyl-CoA synthetases. Proc Natl Acad Sci U S A 103, 10230-10235. Heltweg, B., Gatbonton, T., Schuler, A.D., Posakony, J., Li, H., Goehle, S., Kollipara, R., Depinho, R.A., Gu, Y., Simon, J.A., et al. (2006). Antitumor activity of a small-molecule inhibitor of human silent information regulator 2 enzymes. Cancer Res 66, 4368-4377. References 75 Hoff, K.G., Avalos, J.L., Sens, K., and Wolberger, C. (2006). Insights into the sirtuin mechanism from ternary complexes containing NAD+ and acetylated peptide. Structure 14, 1231-1240. Howitz, K.T., Bitterman, K.J., Cohen, H.Y., Lamming, D.W., Lavu, S., Wood, J.G., Zipkin, R.E., Chung, P., Kisielewski, A., Zhang, L.L., et al. (2003). Small molecule activators of sirtuins extend Saccharomyces cerevisiae lifespan. Nature 425, 191-196. Hubbard, B.P., Gomes, A.P., Dai, H., Li, J., Case, A.W., Considine, T., Riera, T.V., Lee, J.E., E, S.Y., Lamming, D.W., et al. (2013). Evidence for a Common Mechanism of SIRT1 Regulation by Allosteric Activators. Science 339, 1216-1219. Jackson, M.D., and Denu, J.M. (2002). Structural Identification of 2′- and 3′-O-Acetyl-ADPribose as Novel Metabolites Derived from the Sir2 Family of β-NAD+-dependent Histone/Protein Deacetylases. J Biol Chem 277, 18535-18544. Jiang, M., Wang, J., Fu, J., Du, L., Jeong, H., West, T., Xiang, L., Peng, Q., Hou, Z., Cai, H., et al. (2012). Neuroprotective role of Sirt1 in mammalian models of Huntington’s disease through activation of multiple Sirt1 targets. Nat Med 18, 153-158. Jin, L., Galonek, H., Israelian, K., Choy, W., Morrison, M., Xia, Y., Wang, X., Xu, Y., Yang, Y., Smith, J.J., et al. (2009). Biochemical characterization, localization, and tissue distribution of the longer form of mouse SIRT3. Protein Sci 18, 514-525. Jin, L., Wei, W., Jiang, Y., Peng, H., Cai, J., Mao, C., Dai, H., Choy, W., Bemis, J.E., Jirousek, M.R., et al. (2009). Crystal structures of human SIRT3 displaying substrate-induced conformational changes. J Biol Chem 284, 24394-24405. Kabsch, W. (2010). Xds. Acta Crystallogr D Biol Crystallogr 66, 125-132. Kaeberlein, M., McDonagh, T., Heltweg, B., Hixon, J., Westman, E.A., Caldwell, S.D., Napper, A., Curtis, R., DiStefano, P.S., Fields, S., et al. (2005). Substrate-specific activation of sirtuins by resveratrol. J Biol Chem 280, 17038-17045. Kaeberlein, M., McVey, M., and Guarente, L. (1999). The SIR2/3/4 complex and SIR2 alone promote longevity in Saccharomyces cerevisiae by two different mechanisms. Genes Dev 13, 2570-2580. Kanfi, Y., Naiman, S., Amir, G., Peshti, V., Zinman, G., Nahum, L., Bar-Joseph, Z., and Cohen, H.Y. (2012). The sirtuin SIRT6 regulates lifespan in male mice. Nature 483, 218-221. References 76 Kawahara, T.L.A., Michishita, E., Adler, A.S., Damian, M., Berber, E., Lin, M., McCord, R.A., Ongaigui, K.C.L., Boxer, L.D., Chang, H.Y., et al. (2009). SIRT6 Links Histone H3 Lysine 9 Deacetylation to NF-ºB-Dependent Gene Expression and Organismal Life Span. Cell 136, 62-74. Lakshminarasimhan, M., Rauh, D., Schutkowski, M., and Steegborn, C. (2013). Sirt1 activation by resveratrol is substrate sequence-selective. Aging. Landry, J., Slama, J.T., and Sternglanz, R. (2000). Role of NAD+ in the Deacetylase Activity of the SIR2-like Proteins. Biochem Bioph Res Co 278, 685-690. Li, W., Zhang, B., Tang, J., Cao, Q., Wu, Y., Wu, C., Guo, J., Ling, E.A., and Liang, F. (2007). Sirtuin 2, a Mammalian Homolog of Yeast Silent Information Regulator-2 Longevity Regulator, Is an Oligodendroglial Protein That Decelerates Cell Differentiation through Deacetylating - Tubulin. J Neurosci 27, 2606-2616. Lin, S.-J., Kaeberlein, M., Andalis, A., Sturtz, L., Defossez, P.-A., Culotta, V., Fink, G., and Guarente, L. (2002). Calorie restriction extends Saccharomyces cerevisiae lifespan by increasing respiration. Nature 418, 344-348. Lin, S.J., Defossez, P.A., and Guarente, L. (2000). Requirement of NAD and SIR2 for life-span extension by calorie restriction in Saccharomyces cerevisiae. Science 289, 2126-2128. Lombard, D.B. (2009). Sirtuins at the Breaking Point: SIRT6 in DNA Repair. Aging 1, 12-16. Lombard, D.B., Alt, F.W., Cheng, H.L., Bunkenborg, J., Streeper, R.S., Mostoslavsky, R., Kim, J., Yancopoulos, G., Valenzuela, D., Murphy, A., et al. (2007). Mammalian Sir2 homolog SIRT3 regulates global mitochondrial lysine acetylation. Mol Cell Biol 27, 8807-8814. Lombard, D.B., and Miller, R.A. (2012). Ageing: sorting out the sirtuins. Nature 483, 166-167. Longo, V.D., and Fontana, L. (2010). Calorie restriction and cancer prevention: metabolic and molecular mechanisms. Trends Pharmacol Sci 31, 89-98. Mai, A., Massa, S., Lavu, S., Pezzi, R., Simeoni, S., Ragno, R., Mariotti, F.R., Chiani, F., Camilloni, G., and Sinclair, D.A. (2005). Design, synthesis, and biological evaluation of sirtinol analogues as class III histone/protein deacetylase (Sirtuin) inhibitors. J Med Chem 48, 77897795. Mao, Z., Hine, C., Tian, X., Van Meter, M., Au, M., Vaidya, A., Seluanov, A., and Gorbunova, V. (2011). SIRT6 Promotes DNA Repair Under Stress by Activating PARP1. Science 332, 14431446. References 77 Martinez-Pastor, B., and Mostoslavsky, R. (2012). Sirtuins, Metabolism, and Cancer. Front Pharm 3. Masoro, E.J. (2005). Overview of caloric restriction and ageing. Mech Ageing Dev 126, 913922. Masoro, E.J. (1990). The Retardation of Aging and Disease by Dietary Restriction. J Nutr 120, 139-139. McCay, C.M., Crowell, M.F., and Maynard, L.A. (1989). The effect of retarded growth upon the length of life span and upon the ultimate body size. 1935. Nutrition 5, 155-171; discussion 172. Medda, F., Russell, R.J.M., Higgins, M., McCarthy, A.R., Campbell, J., Slawin, A.M.Z., Lane, D.P., Lain, S., and Westwood, N.J. (2009). Novel Cambinol Analogs as Sirtuin Inhibitors: Synthesis, Biological Evaluation, and Rationalization of Activity. J Med Chem 52, 2673-2682. Michan, S., and Sinclair, D. (2007). Sirtuins in mammals: insights into their biological function. Biochem J 404, 1-13. Michishita, E., McCord, R.A., Berber, E., Kioi, M., Padilla-Nash, H., Damian, M., Cheung, P., Kusumoto, R., Kawahara, T.L., Barrett, J.C., et al. (2008). SIRT6 is a histone H3 lysine 9 deacetylase that modulates telomeric chromatin. Nature 452, 492-496. Michishita, E., Park, J.Y., Burneskis, J.M., Barrett, J.C., and Horikawa, I. (2005). Evolutionarily conserved and nonconserved cellular localizations and functions of human SIRT proteins. Mol Biol Cell 16, 4623-4635. Milne, J.C., Lambert, P.D., Schenk, S., Carney, D.P., Smith, J.J., Gagne, D.J., Jin, L., Boss, O., Perni, R.B., Vu, C.B., et al. (2007). Small molecule activators of SIRT1 as therapeutics for the treatment of type 2 diabetes. Nature 450, 712-716. Min, J., Landry, J., Sternglanz, R., and Xu, R.-M. (2001). Crystal Structure of a SIR2 Homolog NAD Complex. Cell 105, 269-279. Motta, M.C., Divecha, N., Lemieux, M., Kamel, C., Chen, D., Gu, W., Bultsma, Y., McBurney, M., and Guarente, L. (2004). Mammalian SIRT1 Represses Forkhead Transcription Factors. Cell 116, 551-563. Mueller, U., Darowski, N., Fuchs, M.R., Forster, R., Hellmig, M., Paithankar, K.S., Puhringer, S., Steffien, M., Zocher, G., and Weiss, M.S. (2012). Facilities for macromolecular crystallography at the Helmholtz-Zentrum Berlin. J Synchrotron Radiat 19, 442-449. References 78 Murshudov, G.N., Vagin, A.A., and Dodson, E.J. (1997). Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr D Biol Crystallogr 53, 240-255. Nakagawa, T., Lomb, D.J., Haigis, M.C., and Guarente, L. (2009). SIRT5 Deacetylates carbamoyl phosphate synthetase 1 and regulates the urea cycle. Cell 137, 560-570. Napper, A.D., Hixon, J., McDonagh, T., Keavey, K., Pons, J.F., Barker, J., Yau, W.T., Amouzegh, P., Flegg, A., Hamelin, E., et al. (2005). Discovery of indoles as potent and selective inhibitors of the deacetylase SIRT1. J Med Chem 48, 8045-8054. Nguyen, G.T.T., Schaefer, S., Gertz, M., Weyand, M., and Steegborn, C. (2013). Structures of human sirtuin 3 complexes with ADP-ribose and with carba-NAD+ and SRT1720: binding details and inhibition mechanism. Acta Crystallogr D 69, 1423-1432. North, B.J., Marshall, B.L., Borra, M.T., Denu, J.M., and Verdin, E. (2003). The human Sir2 ortholog, SIRT2, is an NAD+-dependent tubulin deacetylase. Mol Cell 11, 437-444. Pacholec, M., Bleasdale, J.E., Chrunyk, B., Cunningham, D., Flynn, D., Garofalo, R.S., Griffith, D., Griffor, M., Loulakis, P., Pabst, B., et al. (2010). SRT1720, SRT2183, SRT1460, and resveratrol are not direct activators of SIRT1. J Biol Chem 285, 8340-8351. Pagans, S., Pedal, A., North, B.J., Kaehlcke, K., Marshall, B.L., Dorr, A., Hetzer-Egger, C., Henklein, P., Frye, R., McBurney, M.W., et al. (2005). SIRT1 Regulates HIV Transcription via Tat Deacetylation. PLoS Biol 3, e41. Pan, P.W., Feldman, J.L., Devries, M.K., Dong, A., Edwards, A.M., and Denu, J.M. (2011). Structure and Biochemical Functions of SIRT6. J Biol Chem 286, 14575-14587. Park, S.-J., Ahmad, F., Philp, A., Baar, K., Williams, T., Luo, H., Ke, H., Rehmann, H., Taussig, R., Brown, Alexandra L., et al. (2012). Resveratrol Ameliorates Aging-Related Metabolic Phenotypes by Inhibiting cAMP Phosphodiesterases. Cell 148, 421-433. Peng, C., Lu, Z., Xie, Z., Cheng, Z., Chen, Y., Tan, M., Luo, H., Zhang, Y., He, W., Yang, K., et al. (2011). The First Identification of Lysine Malonylation Substrates and Its Regulatory Enzyme. Mol Cell Proteomics 10. Pillai, V.B., Sundaresan, N.R., Kim, G., Gupta, M., Rajamohan, S.B., Pillai, J.B., Samant, S., Ravindra, P.V., Isbatan, A., and Gupta, M.P. (2010). Exogenous NAD Blocks Cardiac Hypertrophic Response via Activation of the SIRT3-LKB1-AMP-activated Kinase Pathway. J Biol Chem 285, 3133-3144. References 79 Pirola, L., and Frojdo, S. (2008). Resveratrol: one molecule, many targets. IUBMB Life 60, 323332. Rogina, B., and Helfand, S.L. (2004). Sir2 mediates longevity in the fly through a pathway related to calorie restriction. Proc Natl Acad Sci USA 101, 15998-16003. S. Mohar, D. (2012). The Sirtuin System: The Holy Grail of Resveratrol? J Clin Exp Cardiolog 03. Sack, M.N. (2011). Emerging characterization of the role of SIRT3-mediated mitochondrial protein deacetylation in the heart. Am J Physiol Heart Circ Physiol 301, H2191-H2197. Sambrook, J., and Russell, D.W. (2001). Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory; 3rd edition. Sanders, B.D., Jackson, B., and Marmorstein, R. (2010). Structural basis for sirtuin function: What we know and what we don't. Biochim Biophys Acta 1804, 1604-1616. Sanders, B.D., Zhao, K., Slama, J.T., and Marmorstein, R. (2007). Structural Basis for Nicotinamide Inhibition and Base Exchange in Sir2 Enzymes. Mol Cell 25, 463-472. Sauve, A.A. (2010). Sirtuin chemical mechanisms. Biochim Biophys Acta 1804, 1591-1603. Sauve, A.A., and Schramm, V.L. (2003). Sir2 regulation by nicotinamide results from switching between base exchange and deacetylation chemistry. Biochemistry 42, 9249-9256. Sauve, A.A., Wolberger, C., Schramm, V.L., and Boeke, J.D. (2006). The biochemistry of sirtuins. Annu Rev Biochem 75, 435-465. Sauve, A.A., and Youn, D.Y. (2012). Sirtuins: NAD+-dependent deacetylase mechanism and regulation. Curr Opin Chem Biol 16, 535-543. Schlicker, C., Gertz, M., Papatheodorou, P., Kachholz, B., Becker, C.F., and Steegborn, C. (2008). Substrates and regulation mechanisms for the human mitochondrial sirtuins Sirt3 and Sirt5. J Mol Biol 382, 790-801. Schuetz, A., Min, J., Antoshenko, T., Wang, C.L., Allali-Hassani, A., Dong, A., Loppnau, P., Vedadi, M., Bochkarev, A., Sternglanz, R., et al. (2007). Structural basis of inhibition of the human NAD+-dependent deacetylase SIRT5 by suramin. Structure 15, 377-389. Schuttelkopf, A.W., and van Aalten, D.M. (2004). PRODRG: a tool for high-throughput crystallography of protein-ligand complexes. Acta Crystallogr D Biol Crystallogr 60, 1355-1363. References 80 Sebastian, C., Satterstrom, F.K., Haigis, M.C., and Mostoslavsky, R. (2012). From Sirtuin Biology to Human Diseases: An Update. J Biol Chem 287, 42444-42452. Smith, B.C., and Denu, J.M. (2007). Mechanism-Based Inhibition of Sir2 Deacetylases by Thioacetyl-Lysine Peptide†. Biochemistry 46, 14478-14486. Smith, B.C., and Denu, J.M. (2007). Sir2 Deacetylases Exhibit Nucleophilic Participation of Acetyl-Lysine in NAD+ Cleavage. J Am Chem Soc 129, 5802-5803. Smith, B.C., Hallows, W.C., and Denu, J.M. (2009). A continuous microplate assay for sirtuins and nicotinamide-producing enzymes. Anal Biochem 394, 101-109. Solomon, J.M., Pasupuleti, R., Xu, L., McDonagh, T., Curtis, R., DiStefano, P.S., and Huber, L.J. (2006). Inhibition of SIRT1 catalytic activity increases p53 acetylation but does not alter cell survival following DNA damage. Mol Cell Biol 26, 28-38. Someya, S., Yu, W., Hallows, W.C., Xu, J., Vann, J.M., Leeuwenburgh, C., Tanokura, M., Denu, J.M., and Prolla, T.A. (2010). Sirt3 Mediates Reduction of Oxidative Damage and Prevention of Age-Related Hearing Loss under Caloric Restriction. Cell 143, 802-812. Sundaresan, N.R., Vasudevan, P., Zhong, L., Kim, G., Samant, S., Ravindra, P.V., Pillai, V.B., Gupta, M., Cunningham, J.M., Deng, C.-X., et al. (2012). The sirtuin SIRT6 blocks IGF-Akt signaling and development of cardiac hypertrophy by targeting c-Jun. Nat Med 8, 1643-1650. Szczepankiewicz, B.G., Dai, H., Koppetsch, K.J., Qian, D., Jiang, F., Mao, C., and Perni, R.B. (2012). Synthesis of Carba-NAD and the Structures of Its Ternary Complexes with SIRT3 and SIRT5. J Org Chem 77, 7319-7329. Tanner, K.G., Landry, J., Sternglanz, R., and Denu, J.M. (2000). Silent information regulator 2 family of NADdependent histone/protein deacetylases generates a unique product, 1-O-acetylADP-ribose. P Natl Acad Sci 97, 14178-14182. Tanny, J.C., Dowd, G.J., Huang, J., Hilz, H., and Moazed, D. (1999). An Enzymatic Activity in the Yeast Sir2 Protein that Is Essential for Gene Silencing. Cell 99, 735-745. Trapp, J., Meier, R., Hongwiset, D., Kassack, M.U., Sippl, W., and Jung, M. (2007). Structureactivity studies on suramin analogues as inhibitors of NAD+-dependent histone deacetylases (sirtuins). ChemMedChem 2, 1419-1431. Appendix 87 hSirt3/ADPR hSirt3/carba-NAD+/SRT1720 Space group P21212 C2 Unit cell constants a = 63.9 Å, b= 66.7 Å, c = 66.9 Å β = 90º a = 227.8 Å, b = 246.1 Å, c = 127.3 Å, β = 123.9º Resolution (Å) 47.2 – 1.3 45.8 – 3.25 Unique reflections 69702 91148 <I / σ> (outermost shell) 15.1 (1.2) 9.3 (1.1) Completeness (outermost shell) (%) 98.4 (96.8) 99.4 (99.6) Rmeas(a) (outermost shell) (%) 8.8 (136.2) 14.4 (144.0) Protein chains in asymmetric unit No. of amino acids No. of protein atoms No. of ligand atoms No. of waters No. of solvent atoms (except water) No. of metals 1 272 2489 41 266 20 2 12 3275 25548 936 0 24 12 R.m.s. deviations: Bond length (Å) Bond angles (º) 0.02 2.3 0.02 2.4 Average B factor (Å2) Protein Ligands Water 20.4 15.0 37.9 20.0 86.2 --- Final Rcryst/Rfree (b)(c) (%) 13.8/17.9 22.7/24.7 Appendix 88 hSirt3/NAD+/Ex-527 hSirt3/native intermediate Space group P21212 C2221 Unit cell constants a = 60.9 Å, b= 63.4 Å, c = 66.4 Å a = 78.0 Å, b = 131.3 Å, c = 76.5 Å Resolution (Å) 45.8 – 2.0 38.2 – 2.5 Unique reflections 17814 12915 <I / σ> (outermost shell) 15.6 (2.9) 20.8 (3.8) Completeness (outermost shell) (%) 99.3 (95.4) 92.4 (93.9) Rmeas(a) (outermost shell) (%) 11.0 (64.7) 7.2 (52.3) No. of amino acids No. of protein atoms No. of intermediate atoms No. of ligand atoms No. of waters No. of solvent atoms (except water) No. of metals 270 2183 0 97 123 18 1 273 2144 101 0 57 63 1 R.m.s. deviations: Bond length (Å) Bond angles (º) 0.02 2.0 0.02 1.9 Average B factor (Å2) Protein Intermediate Ligands include Zn Water 20.0 16.7 25.0 41.2 53.5 28.3 39.6 Final Rcryst/Rfree (b)(c) (%) 16.3/20.9 18.2/24.8 Appendix 89 zSirt5/ME/Resveratrol zSirt5/p53/Resveratrol Space group I222 C222 Unit cell constants a = 56.9 Å, b = 77.6 Å c = 139.4 Å a = 84.5 Å, b = 105.4 Å, c = 65.9 Å Resolution (Å) 19.93 – 2.0 46.6 – 2.45 Unique reflections 21218 11155 <I / σ> 20.70 (4.20) 15.75 (3.6) Completeness (outermost shell) (%) 99.7 (100) 99.9 (99.9) Rmeas(a) (outermost shell) (%) 4.8 (33.9) 9.7 (53.2) Total reflections Used Current Rcryst/Rfree (b)(c) (%) 20157 20.6/27.5 41463 20.2/27.7 R.m.s. deviations: bond length (Å) bond angles (°) 0.018 1.93 0.013 1.60 Appendix 90 zSirt5/FdL-1/Resveratrol zSirt5/Resveratrol Space group I222 R32 Unit cell constants a = 146.6 Å, b = 148.1 Å c = 248.8 Å a = b= 230.8 Å, c = 167.0 Å Resolution (Å) 19.9 – 3.2 46.2 - 2.6 Unique reflections 44932 52309 <I / σ> 13.35 (3.38) 22.62 (4.18) Completeness (outermost shell) (%) 99.8 (99.8) 100 (100) Rmeas(a) (outermost shell) (%) 12.0 (52.8) 7.5 (51.1) Total reflections Used Current Rcryst/Rfree (b)(c) (%) 42685 18.3/24.2 49693 18.5/24.6 R.m.s. deviations: bond length (Å) bond angles (°) 0.016 2.13 0.016 1.90 Appendix 91 (a) Rmeas =     h h i ih n i ihh hh h nI II n nh , , ˆ 1 with   hn i ih h hI n I, 1 ˆ (b) Rcryst =   obs calcobs F FF ; obs F is the observed and calc F the calculated structure factor amplitude. (c) Rfree was calculated from 5% of measured reflections omitted from refinement. Erklärung/Declaration 92 (Eidesstattliche) Versicherungen und Erklärungen (§ 5 Nr. 4 PromO) Hiermit erkläre ich, dass keine Tatsachen vorliegen, die mich nach den gesetzlichen Bestimmungen über die Führung akademischer Grade zur Führung eines Doktorgrades unwürdig erscheinen lassen. (§ 8 S. 2 Nr. 5 PromO) Hiermit erkläre ich mich damit einverstanden, dass die elektronische Fassung meiner Dissertation unter Wahrung meiner Urheberrechte und des Datenschutzes einer gesonderten Überprüfung hinsichtlich der eigenständigen Anfertigung der Dissertation unterzogen werden kann. (§ 8 S. 2 Nr. 7 PromO) Hiermit erkläre ich eidesstattlich, dass ich die Dissertation selbständig verfasst und keine anderen als die von mir angegebenen Quellen und Hilfsmittel benutzt habe. Ich habe die Dissertation nicht bereits zur Erlangung eines akademischen Grades anderweitig eingereicht und habe auch nicht bereits diese oder eine gleichartige Doktorprüfung endgültig nicht bestanden. (§ 8 S. 2 Nr. 9 PromO) Hiermit erkläre ich, dass ich keine Hilfe von gewerbliche Promotionsberatern bzw. - vermittlern in Anspruch genommen habe und auch künftig nicht nehmen werde. ………………………………………………………………………………………. Ort, Datum, Unterschrift