Every test procedure, scientific and non-scientific, has inherent uncertainties, even when performed according to a standard operating procedure (SOP). In addition, it is prone to errors, defects,... Show moreEvery test procedure, scientific and non-scientific, has inherent uncertainties, even when performed according to a standard operating procedure (SOP). In addition, it is prone to errors, defects, and mistakes introduced by operators, laboratory equipment, or materials used. Adherence to an SOP and comprehensive validation of the test method cannot guarantee that each test run produces data within the acceptable range of variability and with the precision and accuracy determined during the method validation. We illustrate here (part I) why controlling the validity of each test run is an important element of experimental design. The definition and application of acceptance criteria (AC) for the validity of test runs is important for the setup and use of test methods, particularly for the use of new approach methods (NAM) in toxicity testing. AC can be used for decision rules on how to handle data, e.g., to accept the data for further use (AC fulfilled) or to reject the data (AC not fulfilled). The adherence to AC has important requirements and consequences that may seem surprising at first sight: (i) AC depend on a test method's objectives, e.g., on the types/concentrations of chemicals tested, the regulatory context, the desired throughput; (ii) AC are applied and documented at each test run, while validation of a method (including the definition of AC) is only performed once; (iii) if AC are altered, then the set of data produced by a method can change. AC, if missing, are the blind spot of quality assurance: Test results may not be reliable and comparable. The establishment and uses of AC will be further detailed in part II of this series. Show less
Luscher, B.; Ahel, I.; Altmeyer, M.; Ashworth, A.; Bai, P.T.; Chang, P.; ... ; Ziegler, M. 2021
ADP-ribosylation, a modification of proteins, nucleic acids, and metabolites, confers broad functions, including roles in stress responses elicited, for example, by DNA damage and viral infection... Show moreADP-ribosylation, a modification of proteins, nucleic acids, and metabolites, confers broad functions, including roles in stress responses elicited, for example, by DNA damage and viral infection and is involved in intra- and extracellular signaling, chromatin and transcriptional regulation, protein biosynthesis, and cell death. ADP-ribosylation is catalyzed by ADP-ribosyltransferases (ARTs), which transfer ADP-ribose from NAD(+) onto substrates. The modification, which occurs as mono- or poly-ADP-ribosylation, is reversible due to the action of different ADP-ribosylhydrolases. Importantly, inhibitors of ARTs are approved or are being developed for clinical use. Moreover, ADP-ribosylhydrolases are being assessed as therapeutic targets, foremost as antiviral drugs and for oncological indications. Due to the development of novel reagents and major technological advances that allow the study of ADP-ribosylation in unprecedented detail, an increasing number of cellular processes and pathways are being identified that are regulated by ADP-ribosylation. In addition, characterization of biochemical and structural aspects of the ARTs and their catalytic activities have expanded our understanding of this protein family. This increased knowledge requires that a common nomenclature be used to describe the relevant enzymes. Therefore, in this viewpoint, we propose an updated and broadly supported nomenclature for mammalian ARTs that will facilitate future discussions when addressing the biochemistry and biology of ADP-ribosylation. This is combined with a brief description of the main functions of mammalian ARTs to illustrate the increasing diversity of mono- and poly-ADP-ribose mediated cellular processes. Show less
Poly(ADP-ribosyl)ation (PAR) is a versatile and complex posttranslational modification composed of repeating units of ADP-ribose arranged into linear or branched polymers. This scaffold is linked... Show morePoly(ADP-ribosyl)ation (PAR) is a versatile and complex posttranslational modification composed of repeating units of ADP-ribose arranged into linear or branched polymers. This scaffold is linked to the regulation of many of cellular processes including the DNA damage response, alteration of chromatin structure and Wnt signalling. Despite decades of research, the principles and mechanisms underlying all steps of PAR removal remain actively studied. In this work, we synthesise well-defined PAR branch point molecules and demonstrate that PARG, but not ARH3, can resolve this distinct PAR architecture. Structural analysis of ARH3 in complex with dimeric ADP-ribose as well as an ADP-ribosylated peptide reveal the molecular basis for the hydrolysis of linear and terminal ADP-ribose linkages. We find that ARH3-dependent hydrolysis requires both rearrangement of a catalytic glutamate and induction of an unusual, square-pyramidal magnesium coordination geometry. Show less
