HαB-Crystallin Suppresses The Aggregation Upon Refolding Of Its Physiological Substrates HγD-, HγC- And HγS-Crystallin

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The passive chaperone α-crystallin, a small heat shock protein, is one of the ubiquitous crystallins in vertebrate lenses, along with the βγ-crystallins. It is composed of two subunits (∼ 20 kDa) αA- and αB-crystallin (αA- and αB-crys), which form an hetero-oligomeric, polydisperse complex of ∼ 800 kDa in the lens. Aggregates isolated from mature-onset cataracts, the major cause of sight loss worldwide, contain damaged and misfolded forms of βγ-crystallins, as well as α-crystallins. We have studied the chaperone function of Human αB-crystallin interacting with its physiological Human γ-crystallin substrates. Human γD-crystallin (HγD-crys) and γC-crystallin (HγC-crys) are stable and long-lived mammalian γ-crystallins localized in the lens nucleus. Human γS-crystallin (HγS-crys) is abundant in the lens outer cortex. All three γ-crystallins can refold in vitro to their native state after unfolding in high concentrations of GdnHCl. However, at very low denaturant concentrations (< 1 M GdnHCl) aggregation of refolding HγC- and HγD-Crys intermediates competes with productive refolding. Diluting unfolded HγC-, HγD-, or HγS-crys to low GdnHCl concentrations (100 μg/ml, 37°C) resulted in the protein population partitioning between productive refolding and aggregation pathways. HγD-, HγC- or HγS-Crys protein was allowed to refold and aggregate in the presence of HαB-Crys homo-oligomers at different monomer-to-monomer ratios of γ-Crys to αB-Crys. HγD- and HγC-Crys aggregation was suppressed to similar levels, whereas HγS-Crys aggregation was not suppressed as strongly in assays measuring solution turbidity at 350 nm. SEC chromatograms of the products of suppression reactions showed the presence of a high molecular weight complex containing the chaperone-substrate complex in ratios of 1γC:5αB and 1γD:5αB chains. This complex was still present 4 days after the suppression reaction was initiated. These results provide a model for how α-crystallin interacts with aggregation-prone substrates in vivo.