The Presence of Essential and Non-Essential Stratum Corneum Proteases: The Vital Need for Protease Inhibitors

Dry skin is one of the most important concerns of consumers worldwide. Despite huge efforts over several decades, the personal care industry still does not offer complete solutions that satisfy the unmet needs of consumers for moisturizing treatments. The paucity of data for the underlying biochemical problems in and the effects of moisturizers on facial skin biology and physiology may partly explain this. Our recent color mapping studies based on bio-instrumental evaluations of skin capacitance and transepidermal water loss have revealed the complexity of facial skin. However, the biomolecular reasons for these subtle differences in the different zones of the face are unknown so far. As the maturation of the stratum corneum is vital for skin moisturization and optimal barrier function, we believe that the protease / proteaseinhibitor balance particularly of the plasminogen system may be key in these processes. Thus, our aim was to develop a specific dual plasmin and urokinase inhibitor for topical application to barrier-impaired skin and demonstrate its efficac

Nonlinear Protein Degradation and the Function of Genetic Circuits

The functions of most genetic circuits require sufficient degrees of cooperativity in the circuit components. While mechanisms of cooperativity have been studied most extensively in the context of transcriptional initiation control, cooperativity from other processes involved in the operation of the circuits can also play important roles. In this study, we examine a simple kinetic source of cooperativity stemming from the nonlinear degradation of multimeric proteins. Ample experimental evidence suggests that protein subunits can degrade less rapidly when associated in multimeric complexes, an effect we refer to as cooperative stability. For dimeric transcription factors, this effect leads to a concentration-dependence in the degradation rate because monomers, which are predominant at low concentrations, will be more rapidly degraded. Thus cooperative stability can effectively widen the accessible range of protein levels in vivo. Through theoretical analysis of two exemplary genetic circuits in bacteria, we show that such an increased range is important for the robust operation of genetic circuits as well as their evolvability. Our calculations demonstrate that a few-fold difference between the degradation rate of monomers and dimers can already enhance the function of these circuits substantially. These results suggest that cooperative stability needs to be considered explicitly and characterized quantitatively in any systematic experimental or theoretical study of gene circuits.Comment: 42 pages, 10 figure

Imaging an isolated water molecule using a single electron wave packet

Observing changes in molecular structure requires atomic-scale Ångstrom and femtosecond spatio-temporal resolution. We use the Fourier transform (FT) variant of laser-induced electron diffraction (LIED), FT-LIED, to directly retrieve the molecular structure of H2O+ with picometer and femtosecond resolution without a priori knowledge of the molecular structure nor the use of retrieval algorithms or ab initio calculations. We identify a symmetrically stretched H2O+ field-dressed structure that is most likely in the ground electronic state. We subsequently study the nuclear response of an isolated water molecule to an external laser field at four different field strengths. We show that upon increasing the laser field strength from 2.5 to 3.8 V/Å, the O–H bond is further stretched and the molecule slightly bends. The observed ultrafast structural changes lead to an increase in the dipole moment of water and, in turn, a stronger dipole interaction between the nuclear framework of the molecule and the intense laser field. Our results provide important insights into the coupling of the nuclear framework to a laser field as the molecular geometry of H2O+ is altered in the presence of an external field

X ray Structures of the Proprotein Convertase Furin Bound with Substrate Analogue Inhibitors Reveal Substrate Specificity Determinants beyond the S4 Pocket

The proprotein convertase furin is a highly specific serine protease modifying and thereby activating proteins in the secretory pathway by proteolytic cleavage. Its substrates are involved in many diseases, including cancer and infections caused by bacteria and viruses. Understanding furin’s substrate specificity is crucially important for the development of pharmacologically applicable inhibitors. Using protein X-ray crystallography, we investigated the extended substrate binding site of furin in complex with three peptide-derived inhibitors at up to 1.9 Å resolution. The structure of the protease bound with a hexapeptide inhibitor revealed molecular details of its S6 pocket, which remained completely unknown so far. The arginine residue at P6 induced an unexpected turnlike conformation of the inhibitor backbone, which is stabilized by intra- and intermolecular H-bonds. In addition, we confirmed the binding of arginine to the previously proposed S5 pocket (S5₁). An alternative S5 site (S5₂) could be utilized by shorter side chains as demonstrated for a 4-aminomethyl-phenylacetyl residue, which shows steric properties similar to those of a lysine side chain. Interestingly, we also observed binding of a peptide with citrulline at P4 substituting for the highly conserved arginine. The structural data might indicate an unusual protonation state of Asp264 maintaining the interaction with uncharged citrulline. The herein identified molecular interaction sites at P5 and P6 can be utilized to improve next-generation furin inhibitors. Our data will also help to predict furin substrates more precisely on the basis of the additional specificity determinants observed for P5 and P6

OFF State Specific Inhibition of the Proprotein Convertase Furin

The pro-protein convertase furin is a highly specific serine protease involved in the proteolytic maturation of many proteins in the secretory pathway. It also activates surface proteins of many viruses including the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Furin inhibitors effectively suppress viral replication and thus are promising antiviral therapeutics with broad application potential. Polybasic substrate-like ligands typically trigger conformational changes shifting furin’s active site cleft from the OFF-state to the ON-state. Here, we solved the X-ray structures of furin in complex with four different arginine mimetic compounds with reduced basicity. These guanylhydrazone-based inhibitor complexes showed for the first time an active site-directed binding mode to furin’s OFF-state conformation. The compounds undergo unique interactions within the S1 pocket, largely different compared to substrate-like ligands. A second binding site was identified at the S4/S5 pocket of furin. Crystallography-based titration experiments confirmed the S1 site as the primary binding pocket. We also tested the proprotein convertases PC5/6 and PC7 for inhibition by guanylhydrazones and found an up to 7-fold lower potency for PC7. Interestingly, the observed differences in the Ki values correlated with the sequence conservation of the PCs at the allosteric sodium binding site. Therefore, OFF-state-specific targeting of furin can serve as a valuable strategy for structure-based development of PC-selective small-molecule inhibitors

J. Mol. Biol.

Bivalent peptidic thrombin inhibitors consisting of an N- terminal D-cyclohexylalanine-Pro-N-alpha(Me)Arg active-site fragment, a flexible polyglycine linker, and a C-terminal hirugen-like segment directed towards the fibrinogen recognition exosite inhibit thrombin with K-i values in the pico-molar range, remaining stable in buffered solution at pH 7.8 for at least 15 hours. In order to investigate the structural basis of this increased, stability, the most potent of these inhibitors, I-11 (K-i = 37pM), containing an N- alpha(Me)Arg-Thr bond, was crystallized in complex with human a-thrombin. X-ray data were collected to 1.8 Angstrom resolution and the crystal structure of this complex was determined. The Fourier map displays clear electron density for the N-terminal fragment and for the exosite binding segment. It indicates, however, that in agreement with Edman sequencing, the peptide had been cleaved in the crystal, presumably due to the long incubation time of 14 days needed for crystallization and data collection. The N-alpha(Me) group is directed toward the carbonyl oxygen atom of Ser214, pushing the Ser195 O-gamma atom out of its normal site. This structure suggests that upon thrombin binding, the scissile peptide bond of the intact peptide and the Ser195 O-gamma are separated from each other, impairing the nucleophilic attack of the Ser195 07 toward the N-chi(Me)Arg carbonyl group. In the time-scale of two weeks, however, cleavage geometries favoured by the crystal allow catalysis at a slow rate. (C) 2002 Elsevier Science Ltd