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Future studies are had a need to addresses the timing and spatial contexts of the phosphorylation occasions as well as the sign transduction

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Matriptase-1, a TTSP of about 855 amino acids, belongs to the family of S1 trypsin-like proteases. It combines an amino terminal hydrophobic transmembrane region with an extracellular section of several domains, among them a trypsin-like catalytic and a low-density lipoprotein region. Autocatalytic activation of the zymogen is assisted by its cognate inhibitor HAI-1 and does not depend on other proteases. To date, the mechanism of autocatalytic activation has not been fully understood. Interestingly, matriptase-1 is also activated via acidification of the enzyme, therefore indicating its role in cellular acidosis. Studies on knock-out mice have shown that matriptase-1 is essential for epidermal barrier functions, hence postnatal survival, as well as growth of hair follicles, and thymic homeostasis. Moreover, matriptase- 1 has been reported to be expressed not only in epithelial cells, but also in mast cells, B-cells, and blood monocytes. Among its numerous substrates of which most are important for cell adhesion and tissue remodeling, processing of pro-uPA and pro-HGF have been shown to be significantly involved in tumor growth and metastasis. Expression rates of matriptase- 1 were reported to reflect the degree of tumor progression in several types of cancerous cells, thus indicating a crucial role of this protease in tumor metastasis. This was evidenced through various experiments, both in vitro and in vivo, in which the enzyme was inhibited. The ratio of matriptase-1 and HAI-1, which is shifted towards matriptase-1 in cancer cells, is of major importance for tumor invasiveness. Moreover, matriptase- 1 has been reported to be implicated in a number of other diseases, among them osteoarthritis and atherosclerosis, and to induce cancer itself. In conclusion, matriptase-1 has become a promising target for drug development. To date, only one peptide-based inhibitor of matriptase-1 with a picomolar Ki has been reported. Despite its excellent inhibition constants against matriptase-1, this four-amino-acid peptide with the sequence H-R-Q-A-R-Bt displays a low selectivity. Since for in vivo experiments a high selectivity and serum half-life are indispensable, this inhibitor presumably is not suitable for experiments towards tumor targeting in vivo. Here we describe the isolation of selective cystine-knot peptides of high affinity from knowledge-based combinatorial miniprotein libraries and their functional characterization in vitro and in cell culture. Since the overall structure of matriptase-1 is similar to trypsin, the preference for cleavage at basic residues at the P1 position is maintained. Hence, we considered trypsin-inhibiting miniproteins as a starting point for functional combinatory library design to isolate inhibitors of matriptase-1. From the plethora of miniproteins that are characterized to date, scaffolds were selected matching the following criteria: inhibitor of a trypsin-like protease, known three-dimensional structure, tolerance to variation of loop lengths and sequence, known mechanism of folding and disulfide bond formation, as well as availability through chemical and recombinant routes of synthesis. Two different scaffold proteins have been selected based on the aforementioned requirements. The first selected scaffold was based on the spinachderived inhibitor SOTI-III. The structure of this protease inhibitor has been recently elucidated by X-ray crystallography. Since the inhibitor loop of SOTI-III is located between CysV and CysVI, this miniprotein is structurally and sequentially very distinct to MCoTI-II, which was chosen as second scaffold. This scaffold is based on miniproteins from the seeds of the squash plant M. cochinchinensis. This plant produces a number of miniprotein-based trypsin inhibitors, both backbone-cyclized macrolactams and variants lacking this motif, which are slightly different in their sequences. To evaluate which of the natural MCoTI variants could serve as a scaffold for the generation of matriptase-1 inhibitors, natural inhibitors were isolated form the M. cochinchinensis seeds using known extraction procedures followed by HPLC separation. Miniproteins from various fractions were identified by ESI-MS and examined for inhibition of matriptase-1. MCoTI-II, a macrolactam-cyclized miniprotein consisting only of natural amino acids, was found to be the most efficient natural inhibitor of matriptase-1 and therefore chosen as starting scaffold. Synthetic open-chain MCoTI-II displayed a Ki app similar to that of its cyclic counterpart. Interestingly, SOTI-III is a less potent inhibitor of trypsin and did not display measurable inhibitory activity against matriptase-1.
asked 5 years ago in English by mondayitaly57 (280 points)

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