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چکیده
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Apoptosis is an important type of programmed cell death executed by cysteine proteases called caspases. It plays role in many diseases such as cancer, stroke, sepsis and Alzheimer, Parkinson, and Huntington diseases. The activity of these caspases is inhibited by a protein called X-linked inhibitor of apoptosis protein (XIAP). Considering the essential roles of these proteins in apoptosis, inhibition of caspases, its activation as well as XIAP inhibition is of therapeutica value. Caspase-9 as the first enzyme activated in the intrinsic apoptosis pathway, and BIR3 domain of XIAP, which inhibits caspase-9, were the target of this research. Thus, approximately 300 compounds synthesized in the Faculty of Pharmacy as well as extracts from several Iranian soil bacterial isolates were screened for caspase-9 and XIAP-BIR3 inhibition. To do so, caspase-9 and XIAP-BIR3 were produced in E. coli and purified to homogeneity with Ni-NTA affinity chromatography. To obtain cellular extracts, the microorganisms were grown on appropriate media and subjected to methanol extractions. Caspase-9, activity was monitored using the chromogenix substrate (Ac-LEHD-pNA). Upon hydrolysis with the enzyme, pNA dissociates from tetrapeptide moiety and produces yellow color, which can be measured spectrophotometrically at 405nm. For screening XIAP inhibitors, compounds and extracts were preincubated with XIAP-BIR3 protein and the mixture was added to the assay solution containing caspase-9. The control reaction included caspase 9 in the presence of XIAP-BIR3 alone. Neither the synthetic compounds nor the natural extracts from Iranian soil bacteria could antagonize XIAP-BIR3 activity. However, extracts from three Iranian soil bacteria could inhibit caspase-9 activity. Experiments show that the inhibitor is heat stable molecule smaller than 5 kDa. Furthermore, it had no effect on two other enzymes namely thrombin or papain. Efforts are underway to identify the effective ingredient.
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