5 research outputs found
Biochemical characterization of prostasin, a channel activating protease.
Human prostasin was recently identified as a potential regulator of epithelial sodium channel (ENaC) function. Through the use of positional scanning combinatorial substrate libraries, prostasin was shown to have a preference for poly-basic substrates: in position P4 preference was for arginine or lysine; in P3 preference was for histidine, lysine or arginine; in P2 preference was for basic or large hydrophobic amino acids; and in P1 preference was for arginine and lysine. P1', P2', and P3' displayed broad selectivity with the exception of a lack of activity for isoleucine, and P4' had a preference for small, unbranched, amino acids such as alanine and serine. A prostasin-preferred poly-basic cleavage site was found in the extracellular domains of the ENaC alpha- and beta-subunits, and may present a mechanism for prostasin activation. The absence of activity seen with substrates containing isoleucine in position P1' explains the inability of prostasin to autoactivate and suggests that prostasin proteolytic activity is regulated by an upstream protease. Prostasin activity was highly influenced by mono- and divalent metal ions which were potent inhibitors and substrate specific modulators of enzymatic activity. In the presence of sub-inhibitory concentrations of zinc, the activity of prostasin increased several-fold and its substrate specificity was significantly altered in favor of a strong preference for histidine in positions P3 or P4 of the substrate
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Diastereoselective SelfâAssembly of [2]Catenanes
Two chiral Ïâelectronârich crown ethers incorporating either a binaphthol or two Dâmannitol units have been synthesized and their abilities to bind bipyridinium guests demonstrated. Both crown ethers could be interlocked mechanically with cyclobis(paraquatâpâphenylene) to afford two chiral [2]catenanes. Furthermore, these crown ethers were also mechanically interlocked with a tetracationic cyclophane incorporating a 2,2âČâdihydroxyâ1,1âČâbinaphthyl spacer to afford mixtures of diastereoisomeric [2]catenanes. The composition of these mixtures was determined by 1HâNMRâspectroscopic and HPLC analyses which revealed that modest diastereoselection (56:44â67:33) occurs during the kinetically controlled selfâassembly of the catenanes. The free energy barriers (12.8â16.8 kcal molâ1) associated with the circumrotation of one macrocyclic component through the cavity of the other and vice versa were determined by variableâtemperature 1HâNMR spectroscopy. In addition, another dynamic process involving the ârockingâ of the mean planes of the mechanically interlocked macrocycles with respect to each other was also identified and the associated free energy barriers (10.3â10.4 kcal molâ1) determined
Activity Profile of Dust Mite Allergen Extract Using Substrate Libraries and Functional Proteomic Microarrays
Enzymatic activity in the fecal droppings from the house dust mite has been postulated to contribute to the elicited allergic response. Screening dust mite extracts through 137,180 tetrapeptide fluorogenic substrates allowed for the characterization of proteolytic substrate specificity from the potential cysteine and serine proteases in the extract. The extract was further screened against a 4000 member peptide nucleic acid (PNA) encoded inhibitor library designed to target cysteine proteases using microarray detection. Affinity chromatography coupled with mass spectrometry identified Der p 1 as one of the proteases targeted by the PNA inhibitors in the dust mite lysate. A phenotypic readout of Der p 1 function in allergy progression was demonstrated by the inhibition of CD25 cleavage from T cells by dust mite extract that had been treated with the Der p 1 inhibitor identified from the PNA-encoded inhibitor library