Proteolysis, protease inhibitors

Griese M ci al. Reduced proteolysis of surfactant protein A and changes of the bronchoalveolar lavage fluid proteome by inhaled alpha 1-protease inhibitor in cystic fibrosis. Electrophoresis 2001 22 165-171. 

Contrary to the previous case, using strategies with low MOTs, cell infection will not be synchronised and the cell population will be distributed over different cell status and different specific infection times (T]) [102], leading to a non-obvious definition of the optimal harvest time, extension of cell lysis and the increase in the proteolytic activity is not easy to predict and account. Although this could be considered a drawback in low MOI strategies, it is possible to control the extension of proteolysis [33,83] by the addition of protease inhibitors. 

A second example of protease inhibitor design that properly illustrates the peptide scaffold-based approach is that of thrombin inhibitors. Work with these compounds led to the identification of highly potent, selective, and in vivo-effective lead compounds. A member of the serine protease family, thrombin cleaves a number of substrates (e.g., fibrinogen) and activates its platelet receptor (a G-protein-coupled receptor) by proteolysis of the extracellular N-terminal domain which results in self-activation (for a review see Reference 66). Initial lead inhibitors of thrombin were substrate-based, including the fibrinogen P3-Pi Phe-Pro-Arg sequence [67] and simple Arg derivatives such as Tos-Arg-OMe [68]. 

Previously, both in our laboratory and elsewhere, cellulases subjected to purification procedures were obtained from commercial sources (5,6, 8,9,10,13,39,46). Three cellobiases and several endoglucanases and cellobiohydrolases from commercial preparations were purified in our laboratory. While use of protease inhibitors in the fractionation procedures minimized proteolysis during enzyme purification, the existence of enzymes proteolytically modified, presumedly during prolonged fermentation (required for obtaining high titres for commercial production), was a source of confusion, as previously explained. Therefore, we prepared T. reesei cellulase harvested from young culture broth. This was used to carry out the enzyme purification procedures described below. 

Okagawa, T., et al. 1994. Susceptibility of ebiratide to proteolysis in rat intestinal fluid and homogenates and its protection by various protease inhibitors. Life Sci 55 677. 

Certain precautions have to be taken in order to prevent proteins being denatured or inactivated during purification by physical or biological factors. These include buffering the pH of the solutions, undertaking the procedures at a low temperature and including protease inhibitors to prevent unwanted proteolysis. 

Since a major component of all cells is protein, proteases could be very destructive if they were not carefully controlled or compartmentalized. The potential seriousness of uncontrolled proteolysis can be recognized by the fact that ca. 10% of the proteins by weight found in human plasma are protease inhibitors. The currently recognized plasma protease inhibitors are listed in Table I (3,4). In addition to the plasma inhibitors, there are other inhibitors that are more localized and have not been as well characterized. 

As with all complex biological systems we should not forget the close interplay between oxidative and proteolytic systems (see Fig. 2). For example, it has been shown that at a localised inflammatory site, oxidative inactivation of protease inhibitors may lead to a proteolytic cascade resulting in down-stream MMP activation through the localised action of serine proteinases activating previously latent MMPs (see Fig. 2). Equally, the generation of active MMPs (post-oxidant exposure) may be involved in the site-specific catalytic inactivation of serine-protease inhibitors [59] at an inflammatory site with the consequent generation of an elevated serine protease load and connective tissue proteolysis (see Fig. 2). 

Beside proteolysis during phage production, one should keep in mind that purified phages could also lose their displayed proteins because of contaminating proteases. Cocktails of protease inhibitors should be added to prevent proteolysis, especially for long-term storage of phage stocks. 

Fig. 10.1. Schematic of CaaX protein biosynthesis. A precursor containing a CaaX motif is sequentially modified to yield a product with an isoprenylated and carboxylmethylated C-ter-minus. Rcelp is specific for Ras substrates and partially redundant with Ste24p in mediating proteolysis of certain other CaaX proteins (step 2). FTI, farnesyl transferase inhibitor RPI, Rcelp protease inhibitor.

Cystatin refers to a diverse family of protein cysteine protease inhibitors. There are three general types of cystatins Type 1 (stefens), which are primarily found in the cytoplasm but can appear in extracellular fluids Type 2, which are secreted and found in most extracellular fluids and Type 3, which are multidomain protease inhibitors containing carbohydrates and that include the kininogens. Cystatin 3 is used to measure renal function in clinical chemistry. See Barrett, A.J., The cystatins a diverse superfamily of cysteine peptidase inhibitors, Biomed. Biochim. Acta 45,1363-1374,1986 Katunuma, N., Mechanisms and regulation of lysosomal proteolysis, Revis. Biol. Cellular 20, 35-61, 1989 Gauthier, F., Lalmanach, G., Moeau, T. et al., Cystatin mimicry by synthetic peptides, Biol Chem. Hoppe Seyler 373, 465-470, 1992 Bobek, L.A. and Levine,... 

Protein contaminants can occur as natural isoforms or can arise during purification. Adventitious proteolysis and cysteine oxidation are probably the most common sources of microheterogeneity that occur during isolation (Lorber et al, 1987). This has frequently motivated the inclusion of protease inhibitors and/or reducing agents in crystallization solutions, as well as during purification. In many cases modifications that produce molecular heterogeneity are reflected in enzyme activity. For... 

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