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|Title:||Novel mechanisms of programmed cell death in the protozoan parasite blastocystis||Authors:||YIN JING||Keywords:||programmed cell death; protozoan; Blastocystis; proteomics; legumain; cysteine proteases||Issue Date:||21-Aug-2009||Citation:||YIN JING (2009-08-21). Novel mechanisms of programmed cell death in the protozoan parasite blastocystis. ScholarBank@NUS Repository.||Abstract:||Programmed cell death (PCD) is crucial for cellular growth and development in multicellular organisms. Although distinct PCD features have been described for unicellular eukaryotes, homology searches have failed to reveal clear PCD-related orthologs among these organisms.Previous studies revealed that a surface-reactive monoclonal antibody MAb 1D5 could induce apoptosis-like PCD in the protozoan parasite Blastocystis. In the present study, through two-dimensional gel electrophoresis and mass spectrometry, the cellular target of MAb 1D5 was identified as a cell surface-localized legumain, an asparagine endopeptidase that is usually found in lysosomes of other organisms. Recombinant Blastocystis legumain displayed biphasic pH optima in substrate assays, with peaks at pH 4 and 7.4. Activity of Blastocystis legumain was greatly inhibited by legumain specific inhibitor Cbz-Ala-Ala-AAsn-EPCOOEt, and moderately inhibited by MAb 1D5, cystatin and caspase-1 inhibitor. It was found that inhibition of legumain activity induced apoptosis-like PCD in Blastocystis. In contrast to plants, in which legumains have been shown to play a pro-death role, legumain appears to display a pro-survival role in Blastocystis. The data strongly suggest that legumain has a key role in the regulation of Blastocystis cell death. Previous studies demonstrated that besides apoptosis, MAb 1D5 could elicit a PCD response in Blastocystis independent of caspases-like activity, mitochondria, or both, suggesting the existence of an alternative cell death pathway. In this study, the use of autophagic marker monodansylcadaverine (MDC) and autophagic inhibitors 3-methyladenine and wortmannin showed the existence of autophagic cell death in MAb 1D5-treated Blastocystis. MAb 1D5-triggered autophagy was intensified in the presence of the caspase inhibitor zVAD.fmk and appeared to be dependent on mitochondrial outer membrane permeabilization (MOMP) since the MOMP inhibitor cyclosporine A could abolish MDC incorporation in MAb 1D5-treated cells, even in the presence of zVAD.fmk. This study is the first to report the occurrence of autophagy in Blastocystis through induction by a cytotoxic antibody. MDC staining of Blastocystis colony forms revealed that autophagy also occurs naturally in this organism. Amino acid starvation and rapamycin treatment are two common triggers of autophagy in mammalian cells and Blastocystis was found to rapidly up-regulate MDC-labeled autophagic vacuoles upon these inductions. Confocal microscopic and transmission electron microscopic studies also showed morphological changes suggestive of autophagy. The unusually large size of the autophagic compartments within the parasite central vacuole was found to be unique in Blastocystis. These results suggest that the core machinery for autophagy is conserved in Blastocystis and plays an important role in starvation response and cell death of the parasite.The last part of this study reports that staurosporine, a common apoptosis-inducer in mammalian cells, also induces cytoplasmic and nuclear features of apoptosis in Blastocystis, including cell shrinkage, PS externalization, maintenance of plasma membrane integrity, extensive cytoplasmic vacuolation, nuclear condensation and DNA fragmentation. Staurosporine-induced PS exposure and DNA fragmentation was abolished by the MOMP inhibitor cyclosporin A and significantly inhibited by the broad cysteine protease inhibitor iodoacetamide. Interestingly, the apoptosis phenotype was insensitive to inhibitors of caspases and cathepsins B and L while calpain-specific inhibitors augmented staurosporine-induced apoptosis response. While the identities of the proteases responsible for staurosporine-induced apoptosis warrants further investigation, these findings demonstrate that PCD in Blastocystis is complex and regulated by multiple mediators.||URI:||http://scholarbank.nus.edu.sg/handle/10635/16829|
|Appears in Collections:||Master's Theses (Open)|
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