Oxidative folding and structural analyses of a Kunitz-related inhibitor and its disulfide intermediates: functional implications.

Tick-derived protease inhibitor (TdPI) is a tight-binding Kunitz-related inhibitor of human tryptase ß with a unique structure and disulfide-bond pattern. Here we analyzed its oxidative folding and reductive unfolding by chromatographic and disulfide analyses of acid-trapped intermediates. TdPI folds through a stepwise generation of heterogeneous populations of one-disulfide, two-disulfide, and three-disulfide intermediates, with a major accumulation of the nonnative three-disulfide species IIIa. The rate-limiting step of the process is disulfide reshuffling within the three-disulfide population towards a productive intermediate that oxidizes directly into the native four-disulfide protein. TdPI unfolds through a major accumulation of the native three-disulfide species IIIb and the subsequent formation of two-disulfide and one-disulfide intermediates. NMR characterization of the acid-trapped and further isolated IIIa intermediate revealed a highly disordered conformation that is maintained by the presence of the disulfide bonds. Conversely, the NMR structure of IIIb showed a native-like conformation, with three native disulfide bonds and increased flexibility only around the two free cysteines, thus providing a molecular basis for its role as a productive intermediate. Comparison of TdPI with a shortened variant lacking the flexible prehead and posthead segments revealed that these regions do not contribute to the protein conformational stability or the inhibition of trypsin but are important for both the initial steps of the folding reaction and the inhibition of tryptase ß. Taken together, the results provide insights into the mechanism of oxidative folding of Kunitz inhibitors and pave the way for the design of TdPI variants with improved properties for biomedical applications.

Engineered cystine knot miniproteins as potent inhibitors of human mast cell tryptase beta.

Here we report the design, chemical and recombinant synthesis, and functional properties of a series of novel inhibitors of human mast cell tryptase beta, a protease of considerable interest as a therapeutic target for the treatment of allergic asthma and inflammatory disorders. These inhibitors are derived from a linear variant of the cyclic cystine knot miniprotein MCoTI-II, originally isolated from the seeds of Momordica cochinchinensis. A synthetic cyclic miniprotein that bears additional positive charge in the loop connecting the N- and C-termini inhibits all monomers of the tryptase beta tetramer with an overall equilibrium dissociation constant K(i) of 1 nM and thus is one of the most potent proteinaceous inhibitors of tryptase beta described to date. These cystine knot miniproteins may therefore become valuable scaffolds for the design of a new generation of tryptase inhibitors.

Trypsin inhibition by macrocyclic and open-chain variants of the squash inhibitor MCoTI-II.

MCoTI-I and MCoTI-II from the seeds of Momordica cochinchinensis are inhibitors of trypsin-like proteases and the only known members of the large family of squash inhibitors that are cyclic and contain an additional loop connecting the amino- and the carboxy-terminus. To investigate the contribution of macrocycle formation to biological activity, we synthesized a set of open-chain variants of MCoTI-II that lack the cyclization loop and contain various natural and non-natural amino acid substitutions in the reactive-site loop. Upon replacement of P1 lysine residue #10 within the open-chain variant of MCoTI-II by the non-natural isosteric nucleo amino acid AlaG [beta-(guanin-9-yl)-L-alanine], a conformationally restricted arginine mimetic, residual inhibitory activity was detected, albeit reduced by four orders of magnitude. While the cyclic inhibitors MCoTI-I and MCoTI-II were found to be very potent trypsin inhibitors, with picomolar inhibition constants, the open-chain variants displayed an approximately 10-fold lower affinity. These data suggest that the formation of a circular backbone in the MCoTI squash inhibitors results in enhanced affinity and therefore is a determinant of biological activity.

Calpain inhibitor A-558693 in experimental focal cerebral ischemia in rats.

OBJECTIVES: Calpains are intracellular proteases, which are activated in various cerebral injuries. We studied the expression of mu-calpain in a model of focal cerebral ischemia/reperfusion and the efficacy of the calpain inhibitor A-558693. METHODS: A transient occlusion of the middle cerebral artery was produced in male Wistar rats by using the suture model with 3 hours of ischemia and 24 hours of reperfusion. Six animals were given the calpain inhibitor and six animals were treated with placebo. The infarct size was determined by the loss of the calpain substrate microtubule-associated protein-2 (MAP-2) immunohistochemistry using volumetry in serial slices of the brains. Furthermore mu-calpain positive-stained cells were detected by immunohistochemistry and western blotting. RESULTS: In placebo-treated animals the mu-calpain expression was significantly increased in the ischemic hemisphere compared with the contralateral non-ischemic hemisphere (88.6 versus 10.5% in the basal ganglia, 60.7 versus 10.7% in the cortex, p < 0.001, respectively) with a subsequent loss its substrate MAP-2. However, the use of the calpain inhibitor A-558693 did not significantly change the mu-calpain expression, nor significantly reduce the infarct volume. DISCUSSION: The present data indicate that mu-calpain proteolysis plays an important role in the chain of events following cerebral ischemia. However, the calpain inhibitor A-558693 failed to prevent these changes.

Affinity chromatography of tryptases: design, synthesis and characterization of a novel matrix-bound bivalent inhibitor.

beta-Tryptases are mast cell-derived serine proteases that are enzymatically active in the form of an oligomer consisting of four subunits each with trypsin-like activity. The active-site clefts, which are directed toward the central pore of the tetramer, form spatial arrays of four negatively charged S1 binding pockets. Therefore, dibasic inhibitors of appropriate geometry can bind in a bivalent fashion to neighboring subunits. We have recently identified a potent bivalent inhibitor (K(i)=18 nM), based on the bifunctional scaffold cyclo-(-D-Asp-L-Asp-) and the arginine mimetic dl-3-aminomethyl-phenylalanine methyl ester as a ligand for S1 pockets that takes advantage of the this unique tetrameric geometry. To generate an affinity matrix, the bivalent ligand was modified and immobilized on a Sepharose matrix by use of the PEG derivative Jeffamine ED 900 as spacer. This matrix selectively recognizes and binds beta-tryptase from crude protein mixtures and thus is useful as a geometry-driven means of isolating and purifying human mast cell tryptases.

Assessing experimental models in myocardial injury: lack of activation of the proteases TACE and calpain in brief ischaemia and reperfusion.

BACKGROUND: Calpain inhibitors are reportedly cardioprotective. Furthermore, oxidative stress may acutely activate the sheddase tumour necrosis factor (TNF)-alpha-cleaving enzyme (TACE). The aim of this study was to examine whether myocardial reperfusion leads to activation of the proteases mu- and m-calpain, and to evaluate which cardiac cells act as a source of TNF-alpha. METHODS: Isolated hearts (guinea pig) were subjected to global ischaemia (15 min) and reperfused. Calpain activity was determined by zymography. Calpastatin (inhibitor) and troponin I (substrate) were quantified by western blotting. Immunohistology of hearts and a human mast cell line (HMC-1) was used to localise expression of TNF-alpha and TACE. Shedding of TNF-alpha was assessed in Mono Mach, Jurkat-T, HMC-1 and peripheral blood leucocytes with and without oxidative stress. RESULTS: Neither of the ubiquitous calpains (mu- and m-calpain) was significantly activated by brief ischaemia/reperfusion, nor were calpastatin and troponin degraded more than in extracts of control hearts. Cardiac TNF-alpha immunoreactivity was localised to mast cells. None of the tested cell lines shed TNF-alpha in response to non-toxic amounts of oxidants. However, HMC-1 cells showed poor expression of proTNF-alpha, while TACE was abundant. CONCLUSIONS: Although the severity of ischaemia in the current model may have been insufficient, activation of calpain by ischaemia/reperfusion cannot be demonstrated simply in the Langendorff-mode perfused isolated heart. Mast cells are the prime source of myocardial TNF-alpha. A suitable whole-cell model remains to be found to demonstrate acute oxidative activation of TACE.