Perturbations in maturation of secretory proteins and their association with endoplasmic reticulum chaperones in a cell culture model for epithelial ischemia.

Abstract

The effects of ischemia on the maturation of secretory proteins are not well understood. Among several events that occur during ischemia-reperfusion are a rapid and extensive decrease in ATP levels and an alteration of cellular oxidative state. Since the normal folding and assembly of secretory proteins are mediated by endoplasmic reticulum (ER) molecular chaperones, the function of which depends on ATP and maintenance of an appropriate redox environment, ischemia might be expected to perturb folding of secretory proteins. In this study, whole animal and cultured cell models for the epithelial ischemic state were used to examine this possibility. After Ischemia is a major cause of epithelial and other tissue injury and leads to a variety of cellular lesions. These include disruption of tight junctions, the subcortical actin cytoskeleton, cell-substratum interactions mediated by integrins, and possibly of other cellular structures and processes involved in the generation and maintenance of apical-basolateral protein polarity (1-3). On a biochemical level, ischemia and subsequent reperfusion are manifested by a variety of changes that include a rapid and severe decline in cellular ATP levels (4-7), alterations of cellular redox state, and perturbations of Na+/K+ ratio, intracellular pH, and intracellular Ca2+ homeostasis (7-9). Ischemia also leads to a cytosolic stress response manifested by impressive induction of the heat shock proteins (Hsps), especially the members of the Hsp7O family (6, 10, 11). Preinduction of Hsp70 appears to protect from future ischemic insult (12-15).Hsp7O, like a number of other proteins believed to function as molecular chaperones, participates in the ATP-dependent folding and assembly of a variety of newly synthesized cytosolic proteins (16). Proteins belonging to the Hsp7O family are also involved in the detection of misfolded or misassembled proteins and may play a role in targeting them for degradation (17). In ischemia, it is believed that the resulting ATP depletion leads to misfolding and aggregation of proteins (e.g., the actin cytoskeleton) and that Hsp7O participates in the refolding of these proteins and/or their degradation (18).Secretory (membrane and secreted) proteins are translocated into the lumen of the endoplasmic reticulum (ER) shortly after initiation of their synthesis. The ER lumen serves as a site for folding, assembly and degradation of secretory proteins. As in the case of Hsp7O-catalyzed folding of cytosolic proteins, the maturation of secretory proteins in the ER is dependent on ATP and sensitive to the local redox environment; folding reactions are mediated by a set of specific molecular chaperones, most of which are resident ER lumenal proteins (19,20). The best studied of these are immunoglobulin-binding protein [BiP; also known as glucose-regulated protein (grp)78], which is an ER homolog of Hsp7O, and grp94, an ER homolog of Hsp9O (19). BiP and grp94 have been shown to bind . At least in the case of BiP, the role of ATP in its chaperone funct...

The protein disulphide-isomerase family: unravelling a string of folds

“…ERp72 interacts in itro with denatured proteins in association with molecular chaperones, including PDI, BiP and GRp94 [167,168]. Similar findings have been made in i o for thyroglobulin and another secretory protein, thrombospondin [103,169].…”

Preconditioning with Endoplasmic Reticulum Stress Ameliorates Mesangioproliferative Glomerulonephritis

“…Previous reports by our and other groups have emphasized a pathogenic role of ER stress in glomerular and tubular epithelial injury. 14,17,20,21 Immunohistochemical analysis in this study showed upregulation of the ER chaperones GRP78 and ORP150 in the glomeruli of rats with antiThy1 nephritis. Furthermore, activation of the PERK pathway, a UPR pathway involved in the downregulation of translation, was also detected in the glomeruli of these rats.…”

Tight Junction Proteins Form Large Complexes and Associate with the Cytoskeleton in an ATP Depletion Model for Reversible Junction Assembly

“…ATP Depletion-Depletion of ATP was achieved rapidly in MDCK cells by using a combination of glycolytic (2-deoxy-D-glucose; Sigma) and oxidative (antimycin A; Sigma) inhibitors, as described previously (15,22,23). In brief, confluent monolayers were washed with PBS three times, then exposed to Dulbecco's PBS containing 1.5 mM CaCl 2 , 2 mM MgCl 2 , 2 mM deoxy-D-glucose, and 10 M antimycin A for various times.…”

“…After ATP Depletion, TJ Proteins Fractionate into a Large Macromolecular Complex-The marked insolubility of TJ proteins after ATP depletion suggested that they might associate with other proteins (possibly cytoskeletal), leading us to examine whether large complexes containing TJ proteins could be identified (23,25). When analyzed by rate zonal centrifugation through 5-20% sucrose gradients, after ATP depletion, the amount of detergent (1% Triton X-100, 0.5% deoxycholate, and 0.2% SDS)-extractable ZO-1 as well as ZO-2 present in the high density (fractions 13-17) fractions increased 1.5ϳ2-fold by densitometric analysis (Fig.…”