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Proteinase Inhibitors: An Overview of their Structure and Possible Function in the Acute Phase
Published in Andrzej Mackiewicz, Irving Kushner, Heinz Baumann, Acute Phase Proteins, 2020
The chief characteristic of cystatins, as implied by their name, is the ability to inhibit cysteine proteinases. Cystatins do not inhibit proteinases with other catalytic mechanisms, and they are usually thought to be selective for cysteine proteinases of the papain superfamily, which include the lysosomal proteinases cathepsin B, H, and L, and the cytosolic calpains. Some evidence suggests that other types of cysteine proteinases, including clostripain and polioviral proteinases, may be inhibited, although the interactions have not been studied in detail. Members of this superfamily are unique among the inhibitors considered in this chapter, since some of the members of families 1 and 2 are able to inhibit the exopeptidase known as dipeptidyl peptidase I, an enzyme that sequentially removes dipeptides from the N terminus of proteins.
Reactivities of Amino Acids and Proteins with Iodine
Published in Erwin Regoeczi, Iodine-Labeled Plasma Proteins, 2019
Proteolysis by a broad-spectrum protease provides information on the overall composition of a protein with respect to iodamino acids. Location of these amino acids within the protein structure can be undertaken by sequential digestion with restricted enzymes and/or by fragmentation using agents like cyanogen bromide (see Section I.B.1) followed by peptide mapping.319 One obvious drawback of applying this approach to plasma proteins is their size and the numerous fragments thereby obtained. Clostripain (specific for the carboxy link of arginyl residues) and trypsin (hydrolyzes bonds between the carboxyl group of arginine or lysine and the amino group of another amino acid) are enzymes for consideration in the first place.
Protease-Catalyzed Semisyntheses
Published in Willi Kullmann, Enzymatic Peptide Synthesis, 1987
The preceding studies on the covalent reconstitution of RNase-A55,56 were based on the complex formation between S-peptide and S-protein. In such a complex the arrangement of the reactive groups is such as to ensure both their spatial proximity and their accessibility to the protease. However, in other reports on the preparation of a “deletion mutant” of RNase-S, Komoriya et al.57 and Homandberg et al.58 described the enzymatic condensation of two ex arte subfragments of the S-peptide which were ordinarily unable to associate with each other or to form, either individually or in combination, a complex with the S-protein. Following the formation of a peptide bond between these subfragments, however, the newly formed condensation product could then noncovalently bind to the S-protein (cf. Figure 6). Unlike the protease-catalyzed semisynthesis of the modified trypsin inhibitor, which was mediated by an autonomous “extrinsic” factor, namely by trypsin,41 the present semisynthesis is driven by an “intrinsic” trap, namely by the provision of an integral part of the prospective product. The nonassociating fragments, which corresponded to the sequences (1 to 10) and (11 to 15) of the S-peptide, were enzymatically coupled in the presence of natural S-protein. The peptide bond forming step was catalyzed by clostripain (clostridi-opeptidase B) a protease from Clostridium histolyticum, the primary specificity of which is directed toward an arginine residue at the P1-substrate site.59 This enzyme was chosen as catalyst because of its selective action on the arginine residue occupying the C-terminal position of the S-peptide subfragment (1 to 10). The use of trypsin, for example, would jeopardize not only the Lys7-Phe8 linkage within the S-peptide but also some further peptide bonds within the S-protein. The yield of the novel “deletion mutant” des-(16-20)-RNase-S, which displays the same activity as RNase-S, was 80% with respect to the S-protein which was present at 1/20 of the molar concentration of the synthetic fragments. Thus 4% of the synthetic fragments, which were present in equimolar concentrations, were condensed, whereas only 0.05% of the condensed product would have been expected in the absence of the S-protein. Since the reaction was carried out in an aqueous medium, the 80-fold improvement in yield can be attributed exclusively to the coupling of the energetically unfavorable process of peptide bond formation with the energetically favorable complex formation, rather than to any solvent effect.
Design, synthesis, and in vitro evaluation of aza-peptide aldehydes and ketones as novel and selective protease inhibitors
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2020
Thomas S. Corrigan, Leilani M. Lotti Diaz, Sarah E. Border, Steven C. Ratigan, Kayla Q. Kasper, Daniel Sojka, Pavla Fajtova, Conor R. Caffrey, Guy S. Salvesen, Craig A. McElroy, Christopher M. Hadad, Özlem Doğan Ekici
Incorporation of the aza-peptide motif in protease inhibition was first introduced by Dolle and co-workers3. Several aza-peptidyl inhibitors, including aza-peptide halomethyl ketones and aza-peptide ketones, were designed and synthesised to target the clan CA cysteine proteases cathepsin B and calpain, as well as the serine proteases chymotrypsin and elastase. All of them were found to be ineffective inhibitors of these enzymes. However, to date, several other classes of aza-peptidyl inhibitors have been reported as potent and selective inhibitors of other proteases. Aza-peptide epoxides and aza-peptide Michael acceptors have been shown to potently and selectively inhibit clan CD proteases such as caspases, legumains, gingipains, and clostripain by Powers and co-workers4–7. Aza-peptide nitriles were developed as effective and stable inhibitors for clan CA proteases such as cathepsins L, S, and K by Guetschow8,9.
Evaluation of collagenase gold plus BP protease in isolating islets from human pancreata
Published in Islets, 2018
Bashar Khiatah, Amber Tucker, Kuan-Tsen Chen, Rachel Perez, Shiela Bilbao, Luis Valiente, Leonard Medrano, Jeffrey Rawson, Elena Forouhar, Keiko Omori, Fouad Kandeel, Meirigeng Qi, Ismail H. Al-Abdullah
In this study, VitaCyte Collagenase Gold plus BP protease were found to be effective for pancreas digestion from donors >38 years of age. It is tempting to speculate that such an enzyme may also be efficient to digest pancreata from younger donors, especially when multiple proteases such as Clostripain were used.29 Further investigation is required to substantiate this hypothesis. Roche MTF C/T and Serva NB1/NP enzymes are GMP products and are often the most suitable for clinical use. Comparatively, VitaCyte Collagenase Gold is sterile but is a non-GMP product and therefore used as a research grade enzyme. However, in this study, the Collagenase Gold/BP protease was filtered in the GMP facility prior to use according to our SOP and Quality Assurance regulations at City of Hope. Nevertheless, improving islet isolation outcomes need to be continuously enhanced, so that ultimately every pancreas could be utilized for clinical applications; this can only be achieved with GMP enzymes once the initial results of low cost enzyme(s) show positive outcomes. In fact, the VitaCyte Company has made progress in developing a recombinant collagenase class I and II, in addition to Collagenase Gold, which would show great potential for isolating islets.30,31