Aggregation pathways of human γ D crystallin induced by metal ions revealed by time dependent methods

Cataract formation is a slow accumulative process due to protein aggregates promoted by different factors over time. Zinc and copper ions have been reported to induce the formation of aggregates opaque to light in the human gamma D crystallin (HγD) in a concentration and temperature dependent manner. In order to gain insight into the mechanism of metal-induced aggregation of HγD under conditions that mimic more closely the slow, accumulative process of the disease, we have studied the non-equilibrium process with the minimal metal dose that triggers HγD aggregation. Using a wide variety of biophysics techniques such as turbidimetry, dynamic light scattering, fluorescence, nuclear magnetic resonance and computational methods, we obtained information on the molecular mechanisms for the formation of aggregates. Zn(II) ions bind to different regions at the protein, probably with similar affinities. This binding induces a small conformational rearrangement within and between domains and aggregates via the formation of metal bridges without any detectable unfolded intermediates. In contrast, Cu(II)-induced aggregation includes a lag time, in which the N-terminal domain partially unfolds while the C-terminal domain and parts of the N-terminal domain remain in a native-like conformation. This partially unfolded intermediate is prone to form the high-molecular weight aggregates. Our results clearly show that different external factors can promote protein aggregation following different pathways

Zinc and Copper Ions Induce Aggregation of Human β-Crystallins

Cataracts are defined as the clouding of the lens due to the formation of insoluble protein aggregates. Metal ions exposure has been recognized as a risk factor in the cataract formation process. The γ and β crystallins are members of a larger family and share several structural features. Several studies have shown that copper and zinc ions induce the formation of γ-crystallins aggregates. However, the interaction of metal ions with β-crystallins, some of the most abundant crystallins in the lens, has not been explored until now. Here, we evaluate the effect of Cu(II) and Zn(II) ions on the aggregation of HβA1, as a representative of the acidic form, and HβB2, as a representative of the basic β-crystallins. We used several biophysical techniques and computational methods to show that Cu(II) and Zn(II) induce aggregation following different pathways. Both metal ions destabilize the proteins and impact protein folding. Copper induced a small conformational change in HβA1, leading to high-molecular-weight light-scattering aggregates, while zinc is more aggressive towards HβB2 and induces a larger conformational change. Our work provides information on the mechanisms of metal-induced aggregation of β-crystallins

The Common Bean Small Heat Shock Protein Nodulin 22 from <i>Phaseolus vulgaris</i> L. Assembles into Functional High-Molecular-Weight Oligomers

Small heat shock proteins (sHsps) are present in all domains of life. These proteins are responsible for binding unfolded proteins to prevent their aggregation. sHsps form dynamic oligomers of different sizes and constitute transient reservoirs for folding competent proteins that are subsequently refolded by ATP-dependent chaperone systems. In plants, the sHsp family is rather diverse and has been associated with the ability of plants to survive diverse environmental stresses. Nodulin 22 (PvNod22) is an sHsp of the common bean (Phaseolus vulgaris L.) located in the endoplasmic reticulum. This protein is expressed in response to stress (heat or oxidative) or in plant roots during mycorrhizal and rhizobial symbiosis. In this work, we study its oligomeric state using a combination of in silico and experimental approaches. We found that recombinant PvNod22 was able to protect a target protein from heat unfolding in vitro. We also demonstrated that PvNod22 assembles into high-molecular-weight oligomers with diameters of ~15 nm under stress-free conditions. These oligomers can cluster together to form high-weight polydisperse agglomerates with temperature-dependent interactions; in contrast, the oligomers are stable regarding temperature