Date of Award

1-1-2012

Language

English

Document Type

Master's Thesis

Degree Name

Master of Science (MS)

College/School/Department

Department of Biomedical Sciences

Content Description

1 online resource (vii, 62 pages) : illustrations (some color)

Dissertation/Thesis Chair

Nicholas J Mantis

Committee Members

April D Burch, Douglas S Conklin

Keywords

Ricin, Microbial toxins, Ribosomes, Apoptosis

Subject Categories

Biology

Abstract

Ribotoxins such as ricin and T-2 toxin bind to ribosomes and trigger cleavage of 28S rRNA and inhibition of protein synthesis. Upon detection of ribosomal alterations, the cell initiates a ribotoxic stress response (RSR), resulting in activation of the stress-activated protein kinase and mitogen-activated protein kinase (SAPK/MAPK) signaling cascade, upregulated expression of both pro-and anti-apoptotic stress-inducible genes, increased secretion of inflammatory cytokines, and ultimately culminating in apoptosis. While it is not immediately clear the exact mechanism by which ribosomes induce the RSR, nor the exact cellular components involved upstream of MAPK activation, it has recently been proposed, in the case of the fungal ribotoxin, deoxynivalenol (DON), that suppressing PKR activation attenuates MAPK activation, cytokine secretion, and cell death. This suggests that PKR may be an important component in sensing alterations in ribosomes and transducing the ribotoxic stress response, and thus a potentially useful therapeutic target against ribotoxin-induced inflammation and cell death. The purpose of this study was to use an established experimental model of ricin and T-2 toxin-induced PKR and RSR in J774 murine macrophages to determine whether PKR may represent a common host cellular protein induced by a wide array of ribotoxins. Using western blot analysis of p~PKR, p~eIF2á, and p~JNK, cascades in response to low and high toxin doses, and the use of a chemical inhibitor of PKR phosphorylation, it was determined that ricin and T-2 toxin induction of JNK is largely PKR-independent and that additional eIF2á kinases may be activated in response to both toxins. As well, it was also shown that a small molecule, PW66, which has previously been shown to suppress ricin induced SAPK/MAPK activation, also suppressed both PKR and eIF2á phosphorylation. This study thus reveals that a PKR-independent pathway is largely responsible for p~eIF2á and p~JNK in response to ricin and T-2 toxin and that PW 66 may be a useful tool to uncover the crucial signaling components involved in initiating the RSR.

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