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The study evaluated the effect of inhibitors on chymotrypsin activities. It collected two sets of data with the first set comparing protein concentration to chymotrypsin activity while the second set of data compared the level of chymotrypsin activity without an inhibitor…
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The study evaluated the effect of inhibitors on chymotrypsin activities. Consequently, it collected two sets of data with the first set comparing protein concentration to chymotrypsin activity while the second set of data compared the level of chymotrypsin activity without an inhibitor and the level of chymotrypsin activity with an inhibitor present. This in essence allowed for testing of the effect of the inhibitor on chymotrypsin activity. The results showed that with increase in protein content, chymotrypsin activity is also likely to increase. However, the trend was not very clear in the case of comparison of chymotrypsin activity without and with an inhibitor. While initially, the inhibitor is seen to result into increased activity levels, later, it inhibits activity and chymotrypsin activity in the presence of an inhibitor is seen to be lower than without an inhibitor.
Chymotrypsin activity
Introduction
Proteasome is an enzymatic complex which plays a major role in regulation of cellular processes via selective degradation of proteins (intracellular). Conventionally, there are 3 different enzymatic activities related to proteasome, these are, chymotrypsin-like (CT-L), trypsin-like (T-L) and caspase-like (C-L). A number of regulatory proteins are degraded by proteasome pathway. Proteasome inhibition can result into cellular apoptosis through their effect on levels of short-lived proteins, creating inhibition of NF-κB activity, increased p53 activity and Bax proteins, as well as accumulation of cyclin-dependent kinase inhibitors. Various studies have shown that malignant, transformed, and proliferating cells are more vulnerable to proteasome inhibition than normal cells.
Purpose of the study
The study seeks to establish the effect of inhibitors on chymotrypsin activity. Additionally, it reports how the different levels of protein concentration affect chymotrypsin activity.
Literature review
A number of proteasome inhibitors have been developed and are discussed in various studies. In Bogyo (2008) it was shown that lactacystin; an irreversible inhibitor of catalytic β-sub-unit of proteasome creates an induction of apoptosis of human monoblastic U937 cells. In later studies, Adams & Stein (2010) showed that benzyloxycarbonyl (Z)-Leu-Leu-leucinal; a tripeptide aldehyde inhibitor of proteasome creates a p53-dependent apoptosis induction in leukemic cells. Chu-Ping, Slaughter & DeMartino (2010) also made similar observations with respect to proteasome inhibition. These, alongside other studies are proof of the principle that proteasome is a realistic target for anti-cancer therapy. Nonetheless, most of the available inhibitors lack specificity. As a result, Dick & Cruikshank (2009) designed a number of boronic acid, derived compounds which inhibit proteasome path-way in highly specific manner.
26S proteasome
26S proteasome is a multi-catalytic protease that is responsible for regulation of intracellular protein degradation. Its function is mediated by 3 main catalytic activities, that is, chymotrypsin-like, trypsin-like, and peptidylglutamyl peptide hydrolyzing. Proteasome inhibition is an emergent therapy for cancers and is novel treatment for multiple myeloma.
Synthetic Inhibitors of the Proteasome
Potency and specificity are the two features critical in designing of protease inhibitors. In absence of these traits in inhibitor designs can result into adversely impact on understanding of proteases. This is well illustrated in initial synthetic compounds that are used in blocking proteasomes. The compounds lack specificity, due to their original designs aimed at blocking non-proteasomal proteases. Nonetheless, with proteasome’s catalytic mechanisms established as well as its three-dimensional structure, synthetic chemistry allows for further refining of inhibitory compounds that lead to greater potency and increased selectivity for proteasome. Peptide aldehydes were the first synthetic inhibitors designed to target proteasomes. This was mainly due to their synthesis ease.
Peptide aldehydes like leupeptin and calpain inhibitors I, II, and other closely related compounds, including, MG-132 and MG-115 frequently used in blocking proteasome activity in vitro and in vivo. Further, tetra peptide aldehyde developed by Wilk is amongst commercially available proteasome inhibitors. Most of the compounds inhibit chymotrypsin-like activity of proteasome but can modifying all three basic catalytic b-sub-units at high concentrations. Amongst the drawbacks is their reactivity to serine and cysteine proteases via formation of hemiacetals or hemithioacetals with hydroxyl or thiol nucleophiles.
Additionally, specificity for proteasome is achievable by designing of peptide sequences which are optimized for proteasome binding. As a matter of fact, few examples of selective peptide aldehydes are documented as they often exhibit broad specificity. The other drawback in use of the aldehyde electrophiles is their reactivity with free thiols as well as their instability in aqueous solution. Nonetheless, irrespective of the shortcomings, initial contributions of peptide aldehyde inhibitors lay foundation for generations of proteasome inhibitors.
Proteasome inhibition
Proteasome inhibition has displayed great promise in treatment of multiple myeloma, a malignancy which remains incurable using conventional therapies. Typically, there are 3 distinct proteolytic activities in relation to proteasome. The CT-L activity is the main target of most of the proteasome inhibitors. The roles of these 3 activities in functioning of proteasome are under vast investigations, and there exists conflicting evidence on how the activities contribute to overall biological processes. Although Coux, Tanaka & Goldberg (2007) suggested existence of a hierarchy among different sites, with CT-L activity determining rate of protein breakdown, he further indicated a redundancy degree for active sub-units.
Results
Figure 1: Protein concentration vs. chymotrypsin activity
The graph plots protein concentration against chymotrypsin activity. It allows for study of the relationship between the two. A plot is generated based on the resulting points in order to establish a relationship between the variables. The results derived from this graph suggest an increase in chymotrypsin activity each time protein concentration is increased although not linearly.
The figure compares chymotrypsin activity in the absence of an inhibitor and in the presence of an inhibitor. Interestingly the reported results are mixed in nature. While chymotrypsin activity with an inhibitor is higher, later, from 10-22, chymotrypsin activity with an inhibitor is reduced.
Discussion
The dominant protease dedicated to protein turnover, proteasome shapes cellular protein repertoire. The existing knowledge on proteasome regulation as well as activity has immensely improved considerably over the last decade. Conventional inhibitors, in particularly, have assisted in advancement of proteasome biology comprehension. A range of small peptide-based structures which can be modified to vary specificity to large macro-molecular inhibitors, proteins inclusive. While reagents play have important role in establishment of current knowledge of proteasomes catalytic mechanism, rapid advances in synthesis as well as identification of new proteasome inhibitor classes over the past 10 years serve as positive indicators. Figure 1 shows the relationship between protein concentration and chymotrypsin activity. The results reveal a definite relationship between the two and it can be concluded that as protein concentration increases, chymotrypsin activity also increases. Figure 2 further compares chymotrypsin activity without an inhibitor and chymotrypsin activity with an inhibitor. In this case, mixed findings are recorded. Initially, activities in the presence of an inhibitor are high but gradually, this changes and the effect of an inhibitor are felt with the activity with an inhibitor immensely decreases as compared to without an inhibitor. The future lies in design of compounds which can function as drugs to target processes in disease progression. It might only be a short while before products of these researches find safe application in practical settings.
References
Adams, J. & Stein, R. (2010). Novel Inhibitors of the proteasome and their therapeutic use in inflammation. Annu Rep Med Chem., 31, 279 – 288.
Bogyo, M. (2008). Covalent modification of the active site threonine of proteasomal b subunits and the Escherichia coli homolog HslV by a new class of inhibitors. Proc. Natl. Acad. Sci., 94, 6629 – 663.
Chu-Ping, M., Slaughter, C. A. & DeMartino, G. N. (2002). Purification and characterization of a protein inhibitor of the 20S proteasome (macropain). Biochim Biophys Acta, 1119, 303 - 311
Coux, O., Tanaka, K., & Goldberg, A. L. (2007). Structure and functions of the 20S and 26S proteasomes. Annu Rev Biochem, 65, 801 – 847
Dick, L. R. & Cruikshank, A. A. (2009). Mechanistic studies on the inactivation of the proteasome by lactacystin in cultured cells. J Biol. Chem., 272, 182 - 188
Dick, T. P, Nussbaum, A. K. (1998) Contribution of proteasomal beta-subunits to the cleavage of peptide substrates analyzed with yeast mutants. J Biol Chem., 273, 25637 – 25646.
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