Long-term changes in corneal endothelial cell density after repeat penetrating keratoplasty in eyes with endothelial decompensation.

PURPOSE: To compare the longitudinal changes in corneal endothelial cell density (ECD) and the incidence of postoperative complications between eyes with endothelial decompensation after repeat penetrating keratoplasty (RPK) and those after primary penetrating keratoplasty (PPK). METHODS: Fifty-seven eyes with endothelial decompensation scheduled for RPK (RPK group) and 57 eyes with endothelial decompensation scheduled for PPK (PPK group) were enrolled. Corneal ECD was evaluated using a specular microscope at 1, 3, 6, 9, and 12 months, and every 6 months until 60 months postoperatively. Visual acuity (VA) and incidence of graft failure, graft rejection, or marked increase in intraocular pressure were examined. RESULTS: Corneal ECD decreased gradually and percentage of cell loss at 60 months was approximately 73% in both groups; these did not differ significantly between groups throughout the follow-up (P ≥ 0.2209). The incidence of graft failure (52.6% in the RPK group and 36.8% in the PPK group), immune rejection, and marked increase in intraocular pressure did not differ significantly between groups (P ≥ 0.0898), although corrected VA was worse in the RPK group. The most common cause of graft failure in both groups was late endothelial failure. The outcomes were not significantly different between eyes that underwent a first RPK and those that underwent a second or subsequent RPK. CONCLUSIONS: Corneal endothelial cell loss and complications are comparable between eyes with endothelial decompensation after RPK and those after PPK, although VA is worse in eyes after RPK. The outcomes after a first RPK did not differ from those after multiple RPKs.

Redox-dependent domain rearrangement of protein disulfide isomerase from a thermophilic fungus.

Protein disulfide isomerase (PDI) acts as folding catalyst and molecular chaperone for disulfide-containing proteins through the formation, breakage, and rearrangement of disulfide bonds. PDI has a modular structure comprising four thioredoxin domains, a, b, b', and a', followed by a short segment, c. The a and a' domains have an active site cysteine pair for the thiol-disulfide exchange reaction, which alters PDI between the reduced and oxidized forms, and the b' domain provides a primary binding site for substrate proteins. Although the structures and functions of PDI have studied, it is still argued whether the overall conformation of PDI depends on the redox state of the active site cysteine pair. Here, we report redox-dependent conformational and solvation changes of PDI from a thermophilic fungus elucidated by small-angle X-ray scattering (SAXS) analysis. The redox state and secondary structures of PDI were also characterized by nuclear magnetic resonance and circular dichroic spectroscopy, respectively. The oxidized form of PDI showed SAXS differences from the reduced form, and the low-resolution molecular models restored from the SAXS profiles differed between the two forms regarding the positions of the a'-c region relative to the a-b-b' region. The normal mode analysis of the crystal structure of yeast PDI revealed that the inherent motions of the a-b-b' and a'-c regions expose the substrate binding surface of the b' domain. The apparent molecular weight of the oxidized form estimated from SAXS was 1.1 times larger than that of the reduced form, whereas the radius of gyration (ca. 33 A) was nearly independent of the redox state. These results suggest that the conformation of PDI is controlled by the redox state of the active site cysteine residues in the a and a' domains and that the conformational alternation accompanies solvation changes in the active site cleft formed by the a, b, b', and a' domains. On the basis of the results presented here, we propose a mechanism explaining the observed redox-dependent conformational and solvation changes of PDI.

Redox-dependent domain rearrangement of protein disulfide isomerase coupled with exposure of its substrate-binding hydrophobic surface.

Protein disulfide isomerase (PDI) is a major protein in the endoplasmic reticulum, operating as an essential folding catalyst and molecular chaperone for disulfide-containing proteins by catalyzing the formation, rearrangement, and breakage of their disulfide bridges. This enzyme has a modular structure with four thioredoxin-like domains, a, b, b', and a', along with a C-terminal extension. The homologous a and a' domains contain one cysteine pair in their active site directly involved in thiol-disulfide exchange reactions, while the b' domain putatively provides a primary binding site for unstructured regions of the substrate polypeptides. Here, we report a redox-dependent intramolecular rearrangement of the b' and a' domains of PDI from Humicola insolens, a thermophilic fungus, elucidated by combined use of nuclear magnetic resonance (NMR) and small-angle X-ray scattering (SAXS) methods. Our NMR data showed that the substrates bound to a hydrophobic surface spanning these two domains, which became more exposed to the solvent upon oxidation of the active site of the a' domain. The hydrogen-deuterium exchange and relaxation data indicated that the redox state of the a' domain influences the dynamic properties of the b' domain. Moreover, the SAXS profiles revealed that oxidation of the a' active site causes segregation of the two domains. On the basis of these data, we propose a mechanistic model of PDI action; the a' domain transfers its own disulfide bond into the unfolded protein accommodated on the hydrophobic surface of the substrate-binding region, which consequently changes into a "closed" form releasing the oxidized substrate.