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1-antitrypsin transgenic mice
Semmelweis University, Department of Medical Chemistry, Puskin str. 9, H-1088 Budapest, Hungary
2Correspondence: Department of Medical Chemistry Department of Medical Chemistry, Semmelweis University, P.O. Box 260. H-1444 Budapest 8, Hungary. E-mail: Csermely{at}puskin.sote.hu
We wanted to elucidate how stress proteins of the endoplasmic reticulum (ER), their complexes, and function change during chronic stress, such as protein aggregation using a transgenic mouse model, which overexpresses the mutant human 
1-antitrypsin (PiZ) protein forming insoluble aggregates in the ER of the liver cells.
PRINCIPAL FINDINGS
1. Examining the stress response in the ER, we found that calnexin, Grp78, Grp94, and PDI were at the same concentration in PiZ transgenic and in age- and gender-matched control mice, which had the same genetic background but did not express the mutant protein.
2. We found an elevated concentration of the cytoplasmic chaperones, Hsp70 and Hsp90, and the antioxidant enzyme, thioredoxin in PiZ animals compared to control mice.
3. Immunoprecipitation with AAT antibodies showed that most of the ER chaperones (such as Grp78, Grp94, calnexin, and the protein disulfide isomerase family member ERp72) did not bind to the PiZ variant of AAT.
4. The same set of experiments showed that the 58 kDa protein disulfide isomerase protein (PDI), the most abundant disulfide isomerase and chaperone of the ER coprecipitated with the PiZ protein even after the reduction of the samples. During PDI immunoprecipitation, a high amount PDI remained unbound to the immunoprecipitational construct in PiZ transgenic mice, and remained in the supernatant.
5. PDI and Grp94 immunoprecipitation experiments showed that the Grp94-PDI complex, which is a typical component of the protein folding machinery, is formed in lower amount in PiZ transgenic mice.
6. Because oxidative stress often prevails in folding diseases, we checked the redox parameters of both the ER and the cytoplasm. We found more reduced proteins in the ER of the PiZ transgenic mice, which was accompanied with a higher glutathione/oxidized glutathione (GSH/GSSG) ratio, and an increased total GSH amount.
7. In contrast, the cytoplasmic redox parameters did not show the signs of abnormality in the concentration of protein thiols. The GSH/GSSG balance showed a slight, but not significant shift toward the more oxidizing state, despite the elevated concentration of total GSH.
8. With the help of a redox-sensitive dye, AMS, we proved that PDI is in a more reduced state in PiZ transgenic mice than in control animals, while the redox state of other, disulfide containing chaperones of the ER (such as ERp72, Grp94, and calnexin) remained unchanged.
9. To get functional data about the efficiency of the microsomal redox folding machinery, we prepared a fluorescent substrate for PDIs, difluorescrein-thiocarbamyl-insulin (di-FiTZ insulin). Our measurements showed that the protein disulfide reductase activity (PDR) of the ER is significantly reduced in PiZ transgenic mice than in control animals (Fig. 2
).
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CONCLUSIONS AND SIGNIFICANCE
The results of our investigations show, that chronic protein aggregation does not provoke the elevation of the major stress proteins participating in unfolded protein response (UPR) in PiZ transgenic mice. This is in good correlation with previous findings on PiZ mice, cell lines, and also on human patients suffering from PiZ aggregation. In cell lines, Grp94 and Grp78, as well as calnexin were found to bind PiZ, but these complexes gradually disappeared with passage of time after PiZ transfection. Because in our model, the aggregation is more chronic, it is not surprising, that the detachment of these early-phase foldases from PiZ became already complete in the approximately 5-mo-old transgenic animals that we examined. PDI, which was not investigated earlier, was found attached to the PiZ complexes. This is in good agreement with the appearance of PDI in lysosyme aggregates. Because PDI is the chaperone, which holds misfolded proteins until their reverse transport and degradation, it is not surprising, that this is the protein, which remained bound to the PiZ complexes, probably helping their unfolding and reverse transport toward the proteasomal degradation system. Because the protein disulfide reductase (PDR) activity is provided predominantly by PDI in the ER, its decrease in PiZ transgenic mice may reflect a decreased availability of PDI, probably due to the sequestration of PDI by the protein aggregates. In addition, a redox-dependent switch of PDI activity has been proposed, that is, PDI acts as a chaperone rather than a disulfide isomerase in its reduced state. Thus the shift of ER redox potential toward a reduced state and the reduction of PDI disulfides, as well as the decreased availability of PDI, may explain the PDR deficiency of PiZ transgenic mice. Whether the higher amount of reduced proteins in the ER and the shift in the GSH/GSSG ratio found in PiZ transgenic mice is a prerequisite to keep PDI in reduced form or whether it is a consequence of the decreased oxidizing power of the redox foldase machinery remains an open question. The fact that in chronic diseases, in which the accumulation of proteins does not occur, such as diabetes, where similar redox shift was found in the ER of the liver cells, supports the idea that the onset of the more reducing environment is a beneficial, protecting mechanism rather than a simple consequence. This is supported further by the finding, that cells with hypo-oxidizing ER can avoid apoptosis better than normal ones.
Hsp90 and Hsp70 are both usually induced during the ERAD process, to protect the proteasome and avoid oxidative stress and aggregation of denatured proteins. The proteasome plays an important role in PiZ degradation. This may explain the elevation of Hsp90 and Hsp70 in PiZ transgenic mice. Both the lower efficiency of the ER-folding machinery and PiZ protein degradation can lead to an increased cytoplasmic oxidative stress. This may explain the increase in the amount of the antioxidant thioredoxin in the cytoplasm.
As a conclusion, our data suggest that in chronic ER stress, such as in PiZ transgenic mice, different protective pathways are activated than in short-term ER stress. The redox changes, the decreased PDR activity and the differences in chaperone complexes in the ER, as well as chaperone and antioxidant enzyme induction in the cytoplasm suggest a long-term adaptive response, which sacrifices efficient protein folding for the sake of long-term survival (Fig. 3
). Our results shed a novel light in the molecular background of the pathophysiology of folding diseases in a complex model of transgenic animals.
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FOOTNOTES
1 E. P. and P. S. contributed equally to this paper and, therefore, both should be considered as first authors of the paper. 
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5065fje
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