Office of Biological and Environmental Research

DOE Lowdose Radiation Program Workshop III

Abstract

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Title: Synthesis of Pro-Apoptotic Forms of Clusterin, an Ionizing Radiation-Induced Protein.

Authors: Konstantin S. Leskov, Jing Li, Dmitri V. Klokov, David A. Boothman.

Clusterin (CLU) is an ionizing radiation (IR)-induced protein that is reported to have both pro- and anti-apoptotic functions. The major form of the CLU protein is secreted (sCLU), and is induced by low doses of IR (>2 cGy). Several groups have shown that sCLU acts as a protector against apoptosis.

However, the existence of another, nuclear form of CLU (nCLU) was proposed and reported by us (Yang et al., PNAS, 2000). We demonstrated that nCLU induced caspase-3-independent cell death upon forced over-expression in MCF7:WS8 and SV-40-immortalized mouse embryonic fibroblasts. We also demonstrated that the C-terminal coiled-coil domain of nCLU was responsible for cell death. Our recent data indicate that the N-terminal coiled-coil domain of nCLU bound to the C-terminal coiled-coil domain, promoting either intra-molecular folding or oligomerization. We showed that endogenous nCLU protein was induced in the nuclei of IR-treated cells by higher doses of IR (³1 Gy), and we hypothesized that nCLU protein induction may be necessary for the elimination of severely damaged cells. We recently determined the mechanisms for nCLU synthesis. We isolated a cDNA for nCLU from IR-treated MCF7:WS8 cells. This nCLU mRNA was produced by splicing together exons I and III, and eliminating exon II, which contained the first AUG codon and the endoplasmic reticulum (ER)-targeting signal of sCLU. In the result of this alternative splicing, translation started from the second in-frame AUG codon positioned in exon III leading to the production of the nCLU protein. Surprisingly, in addition to nCLU message, we found that another form of nCLU (tCLU) could be produced using alternative translation start site on the sCLU message. This alternative translation initiation appeared to be cap-independent. The resulted tCLU protein lacked N-terminal coiled-coil domain, which made it a good candidate for the apoptotic form of CLU. We are currently investigating the expression of nCLU and tCLU in mammalian cells, their functions regarding apoptosis, and their responses to IR, including regulation of splicing and translation initiation. We propose that CLU RNA message undergoes alternative splicing and internal translation initiation resulting in two pro- and one anti-apoptotic form of the protein. This work was supported DOE grant #DE-FG02-99EQ62724 to D.A.B.

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