Abstract
Clusterin, also known as apolipoprotein J, is expressed from a variety of tissues and implicated in pathological disorders such as neurodegenerative diseases, ischemia and cancer. In contrast to secretory clusterin (sCLU), which acts as an extracellular chaperone, the synthesis, subcellular localization and function(s) of intracellular CLU isoforms is currently a matter of intense discussion. By investigating human CLU mRNAs we here unravel mechanisms leading to the synthesis of distinct CLU protein isoforms and analyze their subcellular localization and their impact on apoptosis and on NF-κB-activity. Quantitative PCR-analyses revealed the expression of four different stress-inducible CLU mRNA variants in non-cancer and cancer cell lines. In all cell lines variant 1 represents the most abundant mRNA, whereas all other variants collectively account for no more than 0.34% of total CLU mRNA, even under stressed conditions. Overexpression of CLU cDNAs combined with in vitro mutagenesis revealed distinct translational start sites including a so far uncharacterized non-canonical CUG start codon. We show that all exon 2-containing mRNAs encode sCLU and at least three non-glycosylated intracellular isoforms, CLU1‑449, CLU21‑449 and CLU34‑449, which all reside in the cytosol of unstressed and stressed HEK‑293 cells. The latter is the only form expressed from an alternatively spliced mRNA variant lacking exon 2. Functional analysis revealed that none of these cytosolic CLU forms modulate caspase-mediated intrinsic apoptosis or significantly affects TNF-α-induced NF-κB-activity. Therefore our data challenge some of the current ideas regarding the physiological functions of CLU isoforms in pathologies.
Highlights
Clusterin (CLU), known as Apolipoprotein J, is a secreted glycoprotein constitutively expressed from a broad spectrum of tissues, especially in brain, neuronal tissue, liver, adrenal glands and testis
In the ER the protein is present as a high mannose single chain precursor that is terminally glycosylated in the Golgi, where it is further cleaved by a furin-like protease in an N-terminal α-chain and a C-terminal β-chain
Mature secretory clusterin (sCLU) is secreted as a heterodimeric protein with an apparent molecular weight (MW) of 75-80 kDa in non-reducing SDS-PAGE analyses
Summary
Clusterin (CLU), known as Apolipoprotein J, is a secreted glycoprotein constitutively expressed from a broad spectrum of tissues, especially in brain, neuronal tissue, liver, adrenal glands and testis. Its expression has been shown to be induced in a broad range of pathologies such as Alzheimer’s disease [1,2], spongiform encephalopathies [3], ischemic injury of the hippocampus and the heart [4,5], myocarditis [6], atherosclerosis [7,8] as well as cancer [9] Common to these diverse pathological disorders is the induction of a cellular stress response due to injury, increased oxidative or proteotoxic stress or dysregulation of particular signal transduction pathways [10]. This in turn poses a severe threat for cells but can be antagonized by upregulation of a “defense machinery” including proteolytic, metabolic and DNA/RNA modifying enzymes, detoxifying proteins and molecular chaperones altogether known as heat shock proteins [11]
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