EndoPDI, a Novel Protein-disulfide Isomerase-like Protein That Is Preferentially Expressed in Endothelial Cells Acts as a Stress Survival Factor

Abstract

We have identified a novel protein-disulfide isomerase and named it endothelial protein-disulfide isomerase (EndoPDI) because of its high expression in endothelial cells. Isolation of the full-length cDNA showed EndoPDI to be a 48 kDa protein that has three APWCGHC thioredoxin motifs in contrast to the two present in archetypal PDI. Ribonuclease protection and Western analysis has shown that hypoxia induces EndoPDI mRNA and protein expression. In situ hybridization analysis showed that EndoPDI expression is rare in normal tissues, except for keratinocytes of the hair bulb and syncytiotrophoblasts of the placenta, but was present in the endothelium of tumors and in other hypoxic lesions such as atherosclerotic plaques. We have compared the function of EndoPDI to that of PDI in endothelial cells using specific siRNA. PDI was shown to have a protective effect on endothelial cells under both normoxia and hypoxia. In contrast, EndoPDI has a protective effect only in endothelial cells exposed to hypoxia. The loss of EndoPDI expression under hypoxia caused a significant decrease in the secretion of adrenomedullin, endothelin-1, and CD105; molecules that protect endothelial cells from hypoxia-initiated apoptosis. The identification of an endothelial PDI further extends this increasing multigene family and EndoPDI, unlike archetypal PDI, may be a molecule with which to target tumor endothelium.Protein-disulfide isomerase (PDI) 1 is a ubiquitously expressed multifunctional protein found in the endoplasmic reticulum (ER). It constitutes around 0.8% of total cellular protein and can reach near millimolar concentrations in the ER lumen of some tissues. PDI plays a role in protein folding because of its ability to catalyze the formation of native disulfide bonds and disulfide bond rearrangement (1). Proteins targeted for secretion by the cell are inserted into and translocated across the ER membrane and enter the ER lumen in an unfolded state. PDI, together with a variety of other folding factors and molecular chaperones resident in the ER correctly fold the proteins ready for secretion (2). The accumulation of misfolded proteins in the ER, known as the Unfolded Protein Response, results in increased transcription of chaperones and folding catalysts. Proteins that fail to fold correctly are relocated to the cytosol for proteasomal degradation.PDI is a modular protein consisting of a, b, bЈ, aЈ, and c domains (3). The a and aЈ domains show sequence and structural homology to thioredoxin (Trx) and both contain the active site WCGHCK motif, constituting two independent catalytic sites for thiol-disulfide bond exchange reactions (4 -7). A ratelimiting step in the folding of many newly synthesized proteins is the formation of disulfide bridges (1) and the presence of WCGHCK in PDI is essential for this process, as confirmed by the loss of PDI activity following mutation of the cysteine residues within these motifs (5, 8). The b and bЈ domains also have the thioredoxin structural fold but lack the active site motif. Thus, PDI conta...

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“…It has been shown that PDI protects cells against apoptosis/death under both normoxic and hypoxic conditions. 38,39 In the present study, we found that P4HB was expressed in the Leydig cells and in all types of germ cells ( Figure 5). TU + LNG treatment resulted in the down-regulation of the expression of P4HB; this might have weakened the protective effect of P4HB against apoptosis that is triggered by exogenous hormone administration.…”

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