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Obada Shamaa Dissertation Final.pdf (2.51 MB)

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Intracellular and extracellular regulation of the inflammatory protease caspase-1

Shamaa, Obada
http://rave.ohiolink.edu/etdc/view?acc_num=osu1399024292

Abstract Details

2014, Doctor of Philosophy, Ohio State University, Biomedical Sciences.
Caspase-1 is an inflammatory cysteine protease which cleaves the pro-inflammatory cytokines, interleukin-1beta (IL-1beta) and IL-18, into their mature, bioactive forms. In addition, caspase-1 plays a role in an inflammatory form of programmed cell death termed pyroptosis. Caspase-1 is constitutively expressed in monocytes as a zymogen which requires the assembly of a multi-protein complex, termed the inflammasome. Assembly of the inflammasome occurs in response to pathogen or danger associated molecular patterns (PAMP/DAMPs) that are sensed by pattern recognition receptors (PRR) which facilitates caspase-1 dimerization and cleavage into the mature enzyme. Where and how caspase-1 activation occurs is poorly understood. Our central hypothesis is that in response to inflammasome activation, caspase-1 is released from the monocyte in an active, functional form. Intracellularly, caspase-1 is tightly regulated and rapidly inhibited. To address this we compared the activation of caspase-1 in a cell-extract model, in which concentrated monocyte lysates endogenously activate capsase-1, to an in vitro model in which monocytes release mature caspase-1 from the cell upon inflammasome activation. We also assessed the role of mitochondria which have been identified as playing a role in the localization and assembly of the inflammasome. In the cell-extract model, concentrated monocytic lysates incubated at 37 C spontaneously activate caspase-1 which cleaves endogenous proIL-18, but rapidly lose function, with a t(1/2)=10 min. Addition of mitochondria to non-concentrated monocytic lysates also induced caspase-1 activation. We found that the inflammasome adaptor protein, apoptosis-associated speck-like protein containing a CARD (ASC), was enriched on the mitochondria and this was critical for caspase-1 activation. High concentrations of Ca+2, which are tightly regulated by the mitochondria, induced the formation of a stable caspase-1 intermediate. At more physiologic concentrations, Ca+2 enhanced the activity of caspase-1 in the cell-extract. Intact monocytic cells release mature caspase-1 in response to inflammatory stimuli. We used the classic inflammasome activators, endotoxin (LPS) and adenosine triphosphate (ATP) and found that in contrast to the cell-extract activated caspase-1 the released caspase-1 has stable activity over 12h and is inhibited by the tetra-peptide caspase-1 specific inhibitor, YVAD-cmk. This released caspase-1 activity existed in a high molecular weight complex distinct in size from inactive lower molecular weight forms. The active caspase-1 was not immunodepleteable nor able to cleave exogenous proIL-1beta. These results suggest that in the intracellular environment, caspase-1 is rapidly activated and that Ca+2 and the mitochondria play a role in this process. However, in this cytosolic environment caspase-1 rapidly loses its activity. Monocytes release mature caspase-1 extracellularly. An active fraction of caspase-1 exists in a unique complex that stabilizes its ability to cleave low molecular weight substrates but is sequestered in its interaction with target cytokines after release. The inactive fraction of mature caspase-1 in the supernatant is readily immunodepleteable and exists in a low molecular weight fraction by gel chromatography. Understanding the significance of the unique release of caspase-1, especially in the context of inflammation and disease, will yield new insights into the function of this protease and the efficacy of therapeutic inhibition of caspase-1.
Mark Wewers, MD (Advisor)
Larry Schlesinger, MD (Committee Chair)
Elliott Crouser, MD (Committee Member)
Peter Mohler, PhD (Committee Member)
164 p.
Biochemistry; Biomedical Research; Immunology; Medicine
caspase-1; inflammasome; IL-1beta; IL-18; mitochondria; calcium; monocyte; THP1

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Citations

  • Shamaa, O. (2014). Intracellular and extracellular regulation of the inflammatory protease caspase-1 [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1399024292

    APA Style (7th edition)

  • Shamaa, Obada. Intracellular and extracellular regulation of the inflammatory protease caspase-1. 2014. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1399024292.

    MLA Style (8th edition)

  • Shamaa, Obada. "Intracellular and extracellular regulation of the inflammatory protease caspase-1." Doctoral dissertation, Ohio State University, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=osu1399024292

    Chicago Manual of Style (17th edition)

osu1399024292
860
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This open access ETD is published by The Ohio State University and OhioLINK.