Identification and characterization of new regulation mechanisms for the mammalian apoptosis inducer p66Shc^{Shc} and the mitochondrial sirtuin isoform 5

In dieser Arbeit wurden neue Regulationsmechanismen für den Apoptoseinduktor p66$^{Shc}$ beschrieben, die dessen apoptotische Aktivität an den zellulären oxidativen Stress koppeln. Demnach ist p66$^{Shc}$ ein redoxbasierter Sensor, der die Apoptose einleitet, sobald die zellulären Redoxsysteme Thioredoxin, Glutathion und Peroxiredoxin dem oxidativen Stress unterliegen. Die Ergebnisse in dieser Arbeit erlauben Einblicke in die $\textit {in vivo}$ Regulation des Apoptoseinduktors, die insbesondere für die biogerontologische Forschung von Interesse sind. Des Weiteren konnte der Aktivierungsmechanismus der Sirtuinisoform 5 durch Resveratrol mit Aktivitätstests und Kristallstrukturen aufgeklärt werden. Resveratrol aktiviert Sirt5, indem es den Substrat/Sirtuin-Komplex stabilisiert und die Interaktionsfläche für das Substrat vergrößert. Die Ergebnisse geben Hinweise auf mögliche $\textit {in vivo}$ Regulationsmechanismen von Sirtuinen durch kleine Moleküle und stellen die Grundlage für eine weitere Wirkstoffentwicklung

Crystal Structures of Sirt3 Complexes with 4′-Bromo-Resveratrol Reveal Binding Sites and Inhibition Mechanism

SummarySirtuins are protein deacetylases regulating aging processes and various physiological functions. Resveratrol, a polyphenol found in red wine, activates human Sirt1 and inhibits Sirt3, and it can mimic calorie restriction effects, such as lifespan extension in lower organisms. The mechanism of Sirtuin modulation by resveratrol is not well understood. We used 4′-bromo-resveratrol (5-(2-(4-hydroxyphenyl)vinyl)-1,3-benzenediol) to study Sirt1 and Sirt3 modulation. Despite its similarity to the Sirt1 activator resveratrol, the compound potently inhibited both, Sirt1 and Sirt3. Crystal structures of Sirt3 in complex with a fluorophore-labeled and with a native substrate peptide, respectively, in presence of 4′-bromo-resveratrol reveal two compound binding sites. Biochemical studies identify the internal site and substrate competition as the mechanism for inhibition, providing a drug target site, and homology modeling suggests that the second, allosteric site might indicate the site for Sirt1 activation

Sirt5 Deacylation Activities Show Differential Sensitivities to Nicotinamide Inhibition

<div><p>Sirtuins are protein deacylases regulating metabolism and aging processes, and the seven human isoforms are considered attractive therapeutic targets. Sirtuins transfer acyl groups from lysine sidechains to ADP-ribose, formed from the cosubstrate NAD⁺ by release of nicotinamide, which in turn is assumed to be a general Sirtuin inhibitor. Studies on Sirtuin regulation have been hampered, however, by shortcomings of available assays. Here, we describe a mass spectrometry–based, quantitative deacylation assay not requiring any substrate labeling. Using this assay, we show that the deacetylation activity of human Sirt5 features an unusual insensitivity to nicotinamide inhibition. In contrast, we find similar values for Sirt5 and Sirt3 for the intrinsic NAD⁺ affinity as well as the apparent NAD⁺ affinity in presence of peptide. Structure comparison and mutagenesis identify an Arg neighboring to the Sirt5 nicotinamide binding pocket as a mediator of nicotinamide resistance, and statistical sequence analyses along with testing further Sirtuins reveal a network of coevolved residues likely defining a nicotinamide-insensitive Sirtuin deacetylase family. The same Arg was recently reported to render Sirt5 a preferential desuccinylase, and we find that this Sirt5 activity is highly sensitive to nicotinamide inhibition. Analysis of Sirt5 structures and activity data suggest that an Arg/succinate interaction is the molecular basis of the differential nicotinamide sensitivities of the two Sirt5 activities. Our results thus indicate a Sirtuin subfamily with nicotinamide-insensitive deacetylase activity and suggest that the molecular features determining nicotinamide sensitivity overlap with those dominating deacylation specificity, possibly suggesting that other subfamily members might also prefer other acylations than acetylations.</p> </div

Closing the Gap: Ensuring Access to and Quality of Contraceptive Implant Removal Services is Essential to Rights-based Contraceptive Care

The use of the subdermal contraceptive implant is increasing globally, and particularly so in lower- and middle-income countries in sub-Saharan Africa. For initiation or discontinuation of the implant, users need to have access to services for insertion and removal by healthcare providers. Providing access to safe and effective contraceptive implant removal services presents both clinical and programmatic challenges. The most challenging implant removal cases, termed ―difficult removals‖, place additional demands upon removal services. In this commentary, we outline challenges for the provision of removal services. Based on our experience in this field, we make  recommendations on how healthcare providers and health services can plan for these challenges. Through  maximising the provision of comprehensive and accessible implant removal services, including those for difficult  removals, implant users can be empowered to discontinue their use of this method of contraception if they choose, thus upholding the principles of rights-based contraceptive care.Keywords: Contraception, contraceptive implant, implant removal, difficult implant remova