Free chaperones reutilization o o o >
Dissociation of chaperones
Protein folding Peptidyl-prolyl isomerization N-linked glycosylation Disulfide bond formation reutilization o •
Dissociation of chaperones
Protein folding Peptidyl-prolyl isomerization N-linked glycosylation Disulfide bond formation
Protein processing in the endoplasmic reticu-lum (ER). To recover from serious injury, cells must synthesize and assemble new membrane (tight junction proteins) and secreted (growth factors) proteins. The ER is the initial site of synthesis of all membrane and secreted proteins. As a protein is translocated into the lumen of the ER it begins to interact with a group of resident ER proteins called molecular chaperones [20, 54-57]. Molecular chaperones bind transiently to and interact with these nascent polypeptides as they fold, assemble, and oligomerize [20, 54, 58]. Upon successful completion of folding or assembly, the molecular chaperones and the secretion-competent protein part company via a reaction that requires ATP hydrolysis, and the chaperones are ready for another round of protein folding [20, 59-61]. If a protein is recognized as being misfolded or misassem-bled it is retained within the ER via stable association with the molecular chaperones and is ultimately targeted for degradation . Interestingly, some of the more characteristic features of epithelial ischemia include loss of cellular functions mediated by proteins that are folded and assembled in the ER (ie, cell adhesion molecules, integrins, tight junctional proteins, transporters). This suggests that proper functioning of the protein-folding machinery of the ER could be critically important to the ability of epithelial cells to withstand and recover from ischemic insult. ADP—adenosine diphosphate.
Kidney Cell Line
Thyroid Cell Line
28 S rRNA
Ischemia upregulates endoplasmic reticulum (ER) molecular chaperones. Molecular chaperones of the ER are believed to function normally to prevent inappropriate intra- or intermolecular interactions during the folding and assembly of proteins [20, 54]. However, ER molecular chaperones are also part of the "quality control" apparatus involved in the recognition, retention, and degradation of proteins that fail to fold or assemble properly as they transit the ER [20, 54]. In fact, the messages encoding the ER molecular chaperones are known to increase in response to intraorganelle accumulation of such malfolded proteins [11, 20, 54, 55]. Here, Northern blot analysis of total RNA from either whole kidney or cultured epithelial cells demonstrates that ischemia or ATP depletion induces the mRNAs that encode the ER molecular chaperones, including immunoglobulin binding protein (BiP), 94 kDa glucose regulated protein (grp94), and 72 kDa endoplasmic reticulum protein (Erp72) . This suggests not only that ischemia or ATP depletion causes the accumulation of mal-folded proteins in the ER but that a major effect of ischemia and ATP depletion could be perturbation of the "folding environment" of the ER and disruption of protein processing. GAPDH—glyceraldehyde-3-phosphate dehydrogenase; Hsp70—70 kDa heat-shock protein. (From Kuznetsov et al. ; with permission.)
ATP depletion perturbs normal endoplasmic reticulum (ER) function. Because ATP and a proper redox environment are necessary for folding and assembly [20, 54, 63, 64] and ATP depletion alters ATP levels and the redox environment, the secretion of proteins is perturbed under these conditions. Here, Western blot analysis of the culture media from thyroid epithelial cells subjected to ATP depletion (ie, treatment with antimycin A, an inhibitor of oxidative phosphorylation) illustrates this point. A, Treatment with as little as 1|xM antimycin A for 1 hour completely blocks the secretion of thyroglobulin (Tg) from these cells.
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B-D, Moreover, indirect immunofluorescence with antithyroglobulin antibody demonstrates that the nonsecreted protein is trapped almost entirely in the ER. Together with data from Northern blot analysis, this suggests that perturbation of ER function and disruption of the secretory pathway is likely to be a key cellular lesion in ischemia . MED—control media; PBS—phos-phate-buffered saline. (From Kuznetsov et al. ; with permission.)
ATP depletion increases the stability of chaperone-folding polypeptide interactions in the endoplasmic reticulum (ER). Immunoglobulin binding protein (BiP), and perhaps other ER molecular chaperones, associate with nascent polypeptides as they are folded and assembled in ER [20, 54, 56, 57, 65-73]. The dissociation of these proteins requires hydrolysis of ATP . Thus, when levels of ATP drop, BiP should not dissociate from the secretory proteins and the normally transient interaction should become more stable. Here, the associations of ER molecular chap-erones with a model ER secretory protein is examined by Western blot analysis of thyroglobulin (Tg) immunoprecipitates from thyroid cells subjected to ATP depletion. After treatment with antimycin A, there is an increase in the amounts of ER molecular chaperones (BiP, grp94 and ERP72) which co-immunoprecipitate with antithyroglobulin antibody , suggesting that ATP depletion causes stabilization of the interactions between molecular chaperones and secretory proteins folded and assembled in the ER. Moreover, because a number of proteins critical to the proper functioning of polarized epithelial cells (ie, occludin, E-cadherin, Na-K-ATPase) are folded and assembled in the ER, this suggests that recovery from ischemic injury is likely to depend, at least in part, on the ability of the cell to rescue the protein-folding and -assembly apparatus of the ER. Control media (MED) and phosphate buffered saline (PBS)—no ATP depletion; 1, 5, 10|xM antimycin A—ATP-depleting conditions. (From Kuznetsov et al. ; with permission.)
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