Investigation of Proteasome Chymotryptic Activities and Effects on their Inhibition in Rat and Human Natural Killer Cells

Lu, Min, Investigation of Proteasome Chymotryptic Activites and Effects of Their Inhibition in Rat and Human Natural Killer Cells. Doctor of Philosophy (Biochemistry and Molecular Biology), April, 2003, 185 pp., 3 tables, 32 illustrations, bibliography, 158 titles. The proteasome is a multicatalytic proteinase complex that is involved in the major extralysosomal pathway responsible for intracellular protein degradation in mammalian cells. This dissertation focuses on investigating proteasome chymotryptic activities and the effects of selective inhibitors of these activities on the function of natural killer (NK) cells. In this dissertation, 20S proteasomes derived from rat RNK16 cells were purified and some of their biochemical and biophysical properties were investigated extensively. The results indicated that RNK16 cell-derived proteasome differ from the proteasome of other origins in many aspects including substrate selectivity, inhibitor specificity, and kinetic regulation, although they may share some common biochemical properties with others. To investigate the effects of proteasomal inhibition on the function of NK cells, several proteasome inhibitors were used including MG115, MG132, clasto-lactacystin-β-lactone, EGCG and LLnL. MG115 and MG 132 were shown to induce apoptosis of RNK16 cells, as evidenced by DNA fragmentation, caspase-3 activation and the appearance of sub-G1 cell populations. Activation of multiple caspases and increased expression of cell surface Fas (CD95) protein were also observed following the treatment of RNK16 cells by these two inhibitors. This dissertation also tested the hypothesis that different cell types could respond differentially to proteasome inhibitors. The effects of several proteasome inhibitors were determined on the purified 20S proteasomal and 26S proteasomal chymotrypsin-like activity in whole cell extracts and intact YT and Jurkat cells, human NK and T cell lines respectively. Following such treatment, caspase-3 activation occurred much earlier in Jurkat cells than YT cells; cell cycle analysis indicated a sub-G1 apoptotic cell population in Jurkat cells and G2/M arrest in YT cells. In addition, accumulation of p27 and IκB-α was detected only in Jurkat cells, but not YT cells. Therefore, proteasome inhibitors appear to act differentially in cell cycle progression and apoptosis signaling pathways between human NK and T cells. These studies indicate that the generation of ideal proteasome inhibitors for the treatment of malignancies could be screened or designed to specifically induce cancer cells to undergo programmed cell death, while having little or no apoptosis-inducing abilities for natural killer cells and other cells of the immune response, thus enhancing the selectivity and specificity of the anti-cancer, apoptosis-inducing capabilities of proteasome inhibitors.