Molecular mechanisms of physiological Sirtuin 1 regulation and targeting these mechanisms with drugs

• Applicant: Professor Dr. Clemens Steegborn
• Subject Area: Biochemistry ; Structural Biology
• Term: from 2015 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 277644855

• Final Report Year: 2024

Final Report Abstract

Reversible acylation of proteins at lysine side chains is a widespread regulatory protein modification. Sirtuins are protein lysine deacylases with a unique, NAD+-dependent mechanism that renders them metabolic sensors. The seven mammalian sirtuin isoforms regulate, e.g., energy metabolism and stress responses and contribute to aging processes and aging-related diseases. Sirt1 is a nuclear isoform that deacetylates histones, transcription factors and DNA repair proteins and contributes, e.g., to liver function and brain development. Sirt1 is exploited as a drug target for cancer and inflammatory, metabolic and neurodegenerative diseases. However, physiological regulation and function of Sirt1 are complex and only partially understood, and only few potent and/or specific small molecule Sirt1 modulators are available. We investigated molecular details of physiological Sirt1 regulatory mechanisms and pharmacological Sirt1 modulators. We analyzed the Sirt1 inhibition by the proteins Dbc1 (deleted in breast cancer 1; including the inhibition of poly(ADP-ribose) polymerase 1), Tat (trans-activator of transcription) and HIC1 (hypermethylated in cancer 1) as well as Sirt1 activation by the AROS protein (active regulator of Sirt1) and the interaction with 14-3-3 adapter proteins, and analogously the inhibition and activation by small molecules. As a first step, we established the recombinant production of Sirt1, the regulator proteins and specifically acetylated substrate proteins. These materials allowed us to test the effects of pharmacological modulators against Sirt1 and to verify them with real substrate proteins as well as to reconstitute and characterize the regulatory complexes of Sirt1. We were able to crystallographically solve structures of two Sirt1 states (with and without peptide ligand) as well as the Sirt1-binding regions of Dbc1 and HIC1. Through activity, binding and cross-linking experiments, we identified the affinities and co-ligand dependencies of the interactions as well as involved domains and surfaces. Our results showed that Tat binds to the catalytic domain of Sirt1, but also of Sirt2 and Sirt3. Therefore, we solved and analyzed the structures of Sirt3 complexes with Tat fragments by protein crystallography: Tat utilizes its intrinsic elongated conformation and a charge complementarity for a substrate-like, strong interaction. We were also able to solve structures of Dbc1 and HIC1, which provided important insights into the architecture of Dbc1 and allowed us to propose models for Sirt1 binding and regulation using the biochemical data. This work has provided detailed information on the interaction of Sirt1 with its regulators and improved our understanding of the physiological regulation and function of Sirt1.

Publications

• 1,4-Dihydropyridines Active on the SIRT1/AMPK Pathway Ameliorate Skin Repair and Mitochondrial Function and Exhibit Inhibition of Proliferation in Cancer Cells. Journal of Medicinal Chemistry, 59(4), 1471-1491.
  Valente, Sergio; Mellini, Paolo; Spallotta, Francesco; Carafa, Vincenzo; Nebbioso, Angela; Polletta, Lucia; Carnevale, Ilaria; Saladini, Serena; Trisciuoglio, Daniela; Gabellini, Chiara; Tardugno, Maria; Zwergel, Clemens; Cencioni, Chiara; Atlante, Sandra; Moniot, Sébastien; Steegborn, Clemens; Budriesi, Roberta; Tafani, Marco; Del Bufalo, Donatella ... & Mai, Antonello
  
• Recombinant Preparation, Biochemical Analysis, and Structure Determination of Sirtuin Family Histone/Protein Deacylases. Methods in Enzymology, 183-208.
  Suenkel, B. & Steegborn, C.
  
• Structural Basis of Sirtuin 6 Activation by Synthetic Small Molecules. Angewandte Chemie, 129(4), 1027-1031.
  You, Weijie; Rotili, Dante; Li, Tie‐Mei; Kambach, Christian; Meleshin, Marat; Schutkowski, Mike; Chua, Katrin F.; Mai, Antonello & Steegborn, Clemens
  
• A conserved NAD+binding pocket that regulates protein-protein interactions during aging. Science, 355(6331), 1312-1317.
  Li, Jun; Bonkowski, Michael S.; Moniot, Sébastien; Zhang, Dapeng; Hubbard, Basil P.; Ling, Alvin J. Y.; Rajman, Luis A.; Qin, Bo; Lou, Zhenkun; Gorbunova, Vera; Aravind, L.; Steegborn, Clemens & Sinclair, David A.
  
• Sirtuin activators and inhibitors: Promises, achievements, and challenges. Pharmacology & Therapeutics, 188, 140-154.
  Dai, Han; Sinclair, David A.; Ellis, James L. & Steegborn, Clemens
  
• Structural basis for the activation and inhibition of Sirtuin 6 by quercetin and its derivatives. Scientific Reports, 9(1).
  You, Weijie; Zheng, Wei; Weiss, Sandra; Chua, Katrin F. & Steegborn, Clemens
  
• Molecular Mechanism of Sirtuin 1 Modulation by the AROS Protein. International Journal of Molecular Sciences, 23(21), 12764.
  Weiss, Sandra; Adolph, Ramona S.; Schweimer, Kristian; DiFonzo, Andrea; Meleshin, Marat; Schutkowski, Mike & Steegborn, Clemens
  
• Molecular Mechanism of Sirtuin 1 Inhibition by Human Immunodeficiency Virus 1 Tat Protein. Life, 13(4), 949.
  Adolph, Ramona S.; Beck, Eileen; Schweimer, Kristian; Di Fonzo, Andrea; Weyand, Michael; Rösch, Paul; Wöhrl, Birgitta M. & Steegborn, Clemens