Multiple stable conformations account for reversible concentration-dependent oligomerization and autoinhibition of a metamorphic metallopeptidase

© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Molecular plasticity controls enzymatic activity: the native fold of a protein in a given environment is normally unique and at a global free-energy minimum. Some proteins, however, spontaneously undergo substantial fold switching to reversibly transit between defined conformers, the >metamorphic> proteins. Here, we present a minimal metamorphic, selective, and specific caseinolytic metallopeptidase, selecase, which reversibly transits between several different states of defined three-dimensional structure, which are associated with loss of enzymatic activity due to autoinhibition. The latter is triggered by sequestering the competent conformation in incompetent but structured dimers, tetramers, and octamers. This system, which is compatible with a discrete multifunnel energy landscape, affords a switch that provides a reversible mechanism of control of catalytic activity unique in nature. Shape shifting: A minimal metamorphic, selective, and specific caseinolytic metallopeptidase, selecase, reversibly transits between several different states of defined three-dimensional structure (monomer and tetramer represented in picture). The competent conformation is sequestered in incompetent but structured dimers, tetramers, and octamers, which are associated with loss of enzymatic activity due to autoinhibition.This study was supported in part by grants from European, Spanish, and Catalan agencies (FP7-HEALTH-2010-261460 “Gums&Joints”; FP7-PEOPLE-2011-ITN-290246 “RAPID”; FP7-HEALTH-2012-306029-2 “TRIGGER”; BFU2012-32862; CSD2006-00015; Fundació “La Marató de TV3” grant 2009-100732; 2009SGR1036; and “Pot d’Idees” FGB301793) and FPI Ph.D. fellowships from the former Spanish Ministry for Science and Technology, currently of Economy and Competitiveness, to M.L.-P. and A.C.-P. P.B. acknowledges funds from ANR-CHEX (project SPIN-HD) and ATIP-AvenirPeer Reviewe

Active site-directed inhibitors of prolyl oligopeptidase abolishes its conformational dynamics

Deciphering conformational dynamics is crucial for understanding the biological functions of proteins and for designing compounds targeting them. In particular, providing an accurate description of microsecond-millisecond motions opens the opportunity for regulating protein-protein interactions (PPIs) by modulating the dynamics of one interacting partner. Here we analyzed the conformational dynamics of prolyl oligopeptidase (POP) and the effects of active-site-directed inhibitors on the dynamics. We used an integrated structural biology approach based on NMR spectroscopy and SAXS experiments complemented by MD simulations. We found that POP is in a slow equilibrium in solution between open and closed conformations, and that inhibitors effectively abolished this equilibrium by stabilizing the enzyme in the closed conformation

A multi-stakeholder multicriteria decision analysis for the reimbursement of orphan drugs (FinMHU-MCDA study)

Background: Patient access to orphan medicinal products (OMPs) is limited and varies between countries, reimbursement decisions on OMPs are complex, and there is a need for more transparent processes to know which criteria should be considered to inform these decisions. This study aimed to determine the most relevant criteria for the reimbursement of OMPs in Spain, from a multi-stakeholder perspective, and using multicriteria decision analysis (MCDA). Methods: An MCDA was developed in 3 phases and included 28 stakeholders closely related to the field of rare diseases (6 physicians, 5 hospital pharmacists, 7 health economists, 4 patient representatives and 6 members from national and regional health authorities). Initially [phase A], a bibliographic review was conducted to identify the potential reimbursement criteria. Then, a reduced advisory board (8 members) proposed, selected, and defined the final list of criteria that could be relevant for reimbursement. A discrete choice experiment (DCE) [phase B] was developed to determine the relevance and relative importance weight of such criteria according to the stakeholders’ preferences by choosing between pairs of hypothetical financing scenarios. A multinomial logit model was fitted to analyze the DCE responses. Finally [phase C], the advisory board review the results using a deliberative process. Results: Thirteen criteria were selected, related to 4 dimensions: patient population, disease, treatment, and economic evaluation. Nine criteria were deemed relevant for decision-making and associated with a higher relative importance: Health-related quality of life (HRQL) (23.53%), treatment efficacy (14.64%), availability of treatment alternatives (13.51%), disease severity (12.62%), avoided costs (11.21%), age of target population (7.75%), safety (seriousness of adverse events) (4.72%), quality of evidence (3.82%) and size of target population (3.12%). The remaining criteria had a < 3% relative importance: economic burden of disease (2.50%), cost of treatment (1.73%), cost-effectiveness (0.83%) and safety (frequency of adverse events) (0.03%). Conclusion: The reimbursement of OMPs in Spain should be determined by its effect on patient’s HRQL, the extent of its therapeutic benefit from efficacy and the availability of other therapeutic options. Furthermore, the severity of the rare disease should also influence the decision along with the potential of the treatment to avoid associated costs

Active site-directed inhibitors of prolyl oligopeptidase abolishes its conformational dynamics

Deciphering conformational dynamics is crucial for understanding the biological functions of proteins and for designing compounds targeting them. In particular, providing an accurate description of microsecond-millisecond motions opens the opportunity for regulating protein-protein interactions (PPIs) by modulating the dynamics of one interacting partner. Here we analyzed the conformational dynamics of prolyl oligopeptidase (POP) and the effects of active-site-directed inhibitors on the dynamics. We used an integrated structural biology approach based on NMR spectroscopy and SAXS experiments complemented by MD simulations. We found that POP is in a slow equilibrium in solution between open and closed conformations, and that inhibitors effectively abolished this equilibrium by stabilizing the enzyme in the closed conformation

Noninvasive Structural Analysis of Intermediate Species During Fibrillation: An Application of Small-Angle X-Ray Scattering

Structural investigation of intermediately formed oligomers and pre-fibrillar species is of tremendous importance in order to elucidate the structural principles of fibrillation, and because intermediate species have been suggested as the pathogenic agents in several amyloid diseases. Structural investigations are however greatly complicated by the dynamic changes between structural states of very different sizes and life-times. Small angle X-ray scattering (SAXS) is an ideal method to handle this challenge. The method provides information about the fibrillation process (number of species present and their volume fractions) and low-resolution 3-dimensional structural models of individual species, notably also of the intermediately formed, in-process species from undisturbed fibrillation equilibria. Here, we provide a detailed description of the methods used for the measurement and analysis of SAXS data from fibrillating samples, exemplified using data from our own research