Prolyl oligopeptidase (PREP) is a serine protease that has been studied particularly in the context of neurodegenerative diseases for decades but its physiological function has remained unclear. We have previously found that PREP negatively regulates beclin1-mediated macroautophagy (autophagy), and that PREP inhibition by a small-molecule inhibitor induces clearance of protein aggregates in Parkinson's disease models. Since autophagy induction has been suggested as a potential therapy for several diseases, we wanted to further characterize how PREP regulates autophagy. We measured the levels of various kinases and proteins regulating beclin1-autophagy in HEK-293 and SH-SY5Y cell cultures after PREP inhibition, PREP deletion, and PREP overexpression and restoration, and verified the results in vivo by using PREP knockout and wild-type mouse tissue where PREP was restored or overexpressed, respectively. We found that PREP regulates autophagy by interacting with protein phosphatase 2A (PP2A) and its endogenous inhibitor, protein phosphatase methylesterase 1 (PME1), and activator (protein phosphatase 2 phosphatase activator, PTPA), thus adjusting its activity and the levels of PP2A in the intracellular pool. PREP inhibition and deletion increased PP2A activity, leading to activation of deathassociated protein kinase 1 (DAPK1), beclin1 phosphorylation and induced autophagy while PREP overexpression reduced this. Lowered activity of PP2A is connected to several neurodegenerative disorders and cancers, and PP2A activators would have enormous potential as drug therapy but development of such compounds has been a challenge. The concept of PREP inhibition has been proved safe, and therefore, our study supports the further development of PREP inhibitors as PP2A activators.

The chemokine system is a key player in the functioning of the immune system and a sought-after target for drug candidates. The number of experimental structures of chemokines in complex with chemokine receptors has increased rapidly over the past few years, providing essential information for rational development of chemokine receptor ligands. Here, we perform a comparative analysis of all chemokine−chemokine receptor structures, with the aim of characterizing the molecular recognition processes and highlighting the relationships between chemokine structures and functional processes. The structures show conserved interaction patterns between the chemokine core and the receptor N-terminus, while interactions near ECL2 display subfamily-specific features. Detailed analyses of the interactions of the chemokine N-terminal domain in the 7TM cavities reveal activation mechanisms for CCR5, CCR2, and CXCR2 and a mechanism for biased agonism in CCR1.

This work describes a new nanotechnology‐based immobilization strategy for cytochrome P450s (CYPs), the major class of drug metabolizing enzymes. Immobilization of CYPs on solid supports provides a significant leap forward compared with soluble enzyme assays by enabling the implementation of through‐flow microreactors for, for example, determination of time‐dependent inhibition. Immobilization of the complex CYP membrane‐protein system is however particularly challenging as the preservation of the authentic enzyme kinetic parameters requires the full complexity of the lipid environment. The developed strategy is based on the spontaneous fusion of biotinylated fusogenic liposomes with lipid bilayers to facilitate the gentle biotinylation of human liver microsomes that incorporate all main natural CYP isoforms. The same process is also feasible for the biotinylation of recombinant CYPs expressed in insect cells, same as any membrane‐bound enzymes in principle. As a result, CYPs could be immobilized on streptavidin‐functionalized surfaces, both those of commercial magnetic beads and customized microfluidic arrays, so that the enzyme kinetic parameters remain unchanged, unlike in previously reported immobilization approaches that often suffer from restricted substrate diffusion to the enzyme's active site and steric hindrances. The specificity and robustness of the functionalization method of customized microfluidic CYP assays are also carefully examined.

We introduce rapid replica molding of ordered, high-aspect-ratio, thiol-ene micropillar arrays for implementation of microfluidic immobilized enzyme reactors (IMERs). By exploiting the abundance of free surface thiols of off-stoichiometric thiol-ene compositions, we were able to functionalize the native thiol-ene micropillars with gold nanoparticles (GNPs) and these with proteolytic α-chymotrypsin (CHT) via thiol-gold interaction. The micropillar arrays were replicated via PDMS soft lithography, which facilitated thiol-ene curing without the photoinitiators, and thus straightforward bonding and good control over the surface chemistry (number of free surface thiols). The specificity of thiol-gold interaction was demonstrated over allyl-rich thiol-ene surfaces and the robustness of the CHT-IMERs at different flow rates and reaction temperatures using bradykinin hydrolysis as the model reaction. The product conversion rate was shown to increase as a function of decreasing flow rate (increasing residence time) and upon heating of the IMER to physiological temperature. Owing to the effective enzyme immobilization onto the micropillar array by GNPs, no further purification of the reaction solution was required prior to mass spectrometric detection of the bradykinin hydrolysis products and no clogging problems, commonly associated with conventional capillary packings, were observed. The activity of the IMER remained stable for at least 1.5 h (continuous use), suggesting that the developed protocol may provide a robust, new approach to implementation of IMER technology for proteomics research. Graphical abstract Electronic supplementary material The online version of this article (10.1007/s00216-019-01674-9) contains supplementary material, which is available to authorized users.