Biochemical and physiological characterization of caspase activity in haloarchaea
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Seth Pasricha, Mansha.
Biochemical and physiological characterization of caspase activity in haloarchaea. Retrieved from
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TitleBiochemical and physiological characterization of caspase activity in haloarchaea
Date Created2015
Other Date2015-01 (degree)
Extent1 online resource (xii, 138 p. : ill.)
DescriptionCaspases, cysteine aspartate-specific proteases, are key initiators and executioners of programmed cell death across a wide array of life. Archaea had been absent from the caspase inheritance discussion due to a notable lack of gene homologues with diagnostic domain signatures. Nonetheless, extremely high, basal caspase-like catalytic activity linked to the cellular stress response was recently demonstrated in the model haloarchaeon, Haloferax volcanii, and shown to be widespread among diverse phyla of archaeal extremophiles. In this dissertation, the catalytic specificity of observed caspase activity in H. volcanii was rigorously tested using hydrolytic assays with a diverse suite of canonical, fluorogenic protease substrates and inhibitors, with model serine and cysteine proteases serving as controls. It was demonstrated that H. volcanii possesses an extremely high level and highly specific caspase-like activity in exponentially growing cells that most closely resembles caspase-4. It is the dominant cellular proteolytic activity, is preferentially inhibited by a pan- caspase inhibitor, and has no cross-reactivity with other known protease families. Biochemical purification and in situ trapping with biotinylated fmk- and AOMK-based inhibitors, combined with genome-enabled proteomics and structural alignments were collectively used to identify the protein(s) that are associated with this caspase activity. These analyses identified a diverse suite of cellular proteins including thermosomes, proteasomes, a cell division protein, an ATPase (recently identified as an activator of proteasomal degradation), a putative nuclease, a putative aminopeptidase, elongation factor αEF-2, and an ornithine cyclodeaminase as key proteins associated with caspase activity. These findings biochemically connected caspase activity in H. volcanii to specific stress-related protein complexes, including those involved in the unfolded protein response (UPR). A subset of these candidate proteins were targeted for gene knockouts to empirically test their relationship to caspase activity, to assess their physiological roles, and link to UPR through incubations with canavanine. We show that loss of this activity or reduction from a critical threshold level has important consequences to organismal fitness, placing caspase activity in a novel cellular context. Given the deep archaeal roots of eukaryotes, we posit that it evolved as part of a cellular protein-quality control system, ensuring proper production, folding, and degradation.
NotePh.D.
NoteIncludes bibliographical references
Noteby Mansha Seth Pasricha
Genretheses, ETD doctoral
Languageeng
CollectionGraduate School - New Brunswick Electronic Theses and Dissertations
Organization NameRutgers, The State University of New Jersey
RightsThe author owns the copyright to this work.
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