title: Biochemical and structural characterization of the regulation of human Sirtuin 1 by small molecules and proteins
Creators: name: Adolph, Ramona S.
Publisher: name: University of Bayreuth
Year: 2020
Subjects: subject: Sirtuin
subject: Signaltransduktion
subject: Deacetylase
subject: NAD+
subject: Regulation
subject: HIV1
subject: Human immunodeficiency virus 1
subject: Tat
subject: Transactivator of transcription
subject: Hic1
subject: Hypermethylated in cancer 1
subject: AROS
subject: Active regulator of Sirt1
subject: 540
subject: 570

description: Sirtuins are NAD+-dependent lysine deacylases, which regulate various cell signaling pathways and are associated with lifespan extension through caloric restriction. The human isoforms Sirt1-7 are linked to diverse age-related diseases such as Alzheimer’s, Parkinson’s, but also cancer and AIDS. Sirtuins are therefore emerging targets for therapeutic approaches and regulating sirtuin activity contextually is important. While current pharmacological modulation of sirtuin activity is almost exclusively restricted to inhibitors, which often lack isoform-specificity, potency, or bioavailability, the physiological modulation of sirtuin activity by other proteins is incompletely understood. This study covers important and new aspects for the physiological and pharmacological regulation of human Sirtuin 1. Concerning regulation by small molecules, the activation of hSirt1 by dehydroabietic acid was characterized providing potential for pharmacological modulation with a new nature-derived activator scaffold. In addition, the proposed anti-tumor potential of tranilast was linked to inhibition of hSirt1. Regarding the physiological modulation of hSirt1 activity by other proteins, conflictive results were available for hAROS. Within this thesis, hAROS was validated as hSirt1 inhibitor. Furthermore, a stable core was suggested for the intrinsically disordered protein hAROS, which will be used for future interaction analyses. Conversely, an hSirt1-activity modulating effect for human Hic1 has not been described yet. Focusing on the interaction between hSirt1 and the BTB/POZ domain of Hic1, this study showed that the interaction is limited to the catalytic domain of hSirt1 and does not require phosphorylation of hSirt1, nor the presence of NAD+ or sirtuin substrate. Hic1-BTB might even have a dual and concentration-dependent effect on hSirt1 activity, which will be subject to future studies. In addition, the first crystal structure of Hic1 was solved demonstrating a typical BTB fold and offering an analysis of putative interacting regions with hSirt1. Finally, the region of hSirt1 interacting with HIV1-Tat was also identified as the catalytic domain and an additional in vitro inhibition of the structurally similar catalytic domains of hSirt2 and hSirt3 was observed. Tat binding to hSirt1-3 was competitive to the sirtuin substrate, but not to NAD+. In line with this observation, several complex structures of hSirt3 with Tat peptides could demonstrate that acetylated or deacetylated Tat binds to the sirtuin substrate binding cleft with its basic region. Using structural superpositions and crosslinking, the major contribution to binding was found to be mediated by HIV1-Tat amino acids 49-52, while Tat amino acids 53-59 provide the additional high potency needed for physiological inhibition of hSirt1 through disruption of an important salt bridge between the hSirt1 SBD and catalytic core.