Suppression of DTT-induced aggregation of abrin by αA- and αB-crystallins: a model aggregation assay for α-crystallin chaperone activity in vitro1
| Autor: | Ira Surolia, Sriram Narayanan, G. Bhanuprakash Reddy, P. Yadagiri Reddy |
|---|---|
| Rok vydání: | 2002 |
| Předmět: |
biology
Chemistry Ribosome-inactivating protein Biophysics Cell Biology biology.organism_classification Biochemistry eye diseases In vitro Dithiothreitol chemistry.chemical_compound Structural Biology Crystallin Chaperone (protein) Heat shock protein Abrus precatorius Genetics biology.protein sense organs Abrin Molecular Biology |
| Zdroj: | FEBS Letters. 522:59-64 |
| ISSN: | 0014-5793 |
| DOI: | 10.1016/s0014-5793(02)02884-3 |
| Popis: | The eye lens small heat shock proteins (sHSP), alphaA-and alphaB-crystallins, have been shown to function like molecular chaperones, both in vitro and in vivo. It is essential to assess the protective effect of alphaA- and alphaB-crystallins under native conditions to extrapolate the results to in vivo conditions. Insulin and alpha-lactalbumin have widely been used to investigate the chaperone mechanism of alpha-crystallin under native conditions. Due to its smaller size, insulin B-chain may not represent the binding of putative physiological substrate proteins. As it stands, the aggregation of alpha-lactalbumin and binding of alpha-crystallin to it varies under different experimental conditions. Abrin, a ribosome inactivating protein isolated from the seeds of Abrus precatorius, consists of a 30 kDa A-chain and a lectin-like B-chain of 33 kDa joined by a single disulfide bond. Reduction of the disulfide link between the two chains of abrin leads to the aggregation of the B-chain. In this study, we demonstrate that dithiothreitol (DTT)-induced aggregation of abrin B-chain could be monitored by light scattering similar to that of insulin. Moreso, this process could be suppressed by recombinant human alphaA- and alphaB-crystallins in a concentration dependent manner, notably by binding to aggregation prone abrin B-chain. SDS-PAGE and HPLC gel filtration analysis indicate that there is a soluble complex formation between alpha-crystallin and abrin B-chain. Interestingly, in contrast to insulin, there is no significant difference between alphaA- and alphaB-crystallin in suppressing the aggregation of abrin B-chain at two different temperatures (25 and 37degreesC). HSP26, an another small beat shock/alpha-crystallin family protein, was also able to prevent the DTT-induced aggregation of abrin. These results suggest that due to relatively larger size of its B-chain (33 kDa), compared to insulin B-chain (about 3 kDa), abrin may serve as a better model substrate for in vitro chaperone studies of alpha-crystallin and as well as other sHSP. |
| Databáze: | OpenAIRE |
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