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F. Shang, M. Zetterberg, X. Zhang, E.J. Dudek, A. Taylor; Ubiquitin–Proteasome Pathway Is an Important Protein Quality Control Mechanism in the Lens . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1520.
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© ARVO (1962-2015); The Authors (2016-present)
Accumulation of damaged or modified proteins is associated with cataract. The purpose of this study is to determine roles of ubiquitin–proteasome pathway in removal of damaged or modified proteins.
Recombinant gammaC–crystallin was modified by glutathione or iodoacetamide. To mimic calpain cleavage of alpha A–crystallin, recombinant C–terminal truncated human alpha A–crystallin (1–162) was expressed in E. coli and was purified to homogeneity. Native and modified crystallins were labelled with 125I and were used as substrates for the degradation assay. Lens fiber lysate and reticulocyte lysate were used as source of ubiquitin–proteasome pathway.
Native gamma–crystallin was resistent to proteolysis both in lens fiber lysate and in reticulocyte lysate. In contrast. glutathione–modified or iodoacetamide–modified gammaC–crystallins were readily degraded in lens fiber lysate or in reticulocyte lysate. At least 50% of the degradation is ubiquitin–dependent. Wild–type alpha A–crystallin is a natural substrate for the ubiquitin–proteasome pathway. However, C–terminal cleavage (truncation) enhanced the degradation rate by at least 2–fold. Structural analysis revealed that glutathiolation of gammaC–crystallin results in conformational changes and significant increase in surface hydrophobicity.
These data demonstrate that lens fibers retain an active ubiquitin–proteasome pathway, which discriminates native and damaged proteins, incuding crystallin that was partially cleaved by other proteases. Specifically, exposure of hydrophobic patches of crystallins provides a signal for the ubiquitin–proteasome pathway to distinguish between native and damaged proteins. Dysfunction of this protein quality control mechanism may result in accumulation of damaged proteins in lens cells and may be involved in the pathophysiology of cataract.
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