The α-crystallin chaperones undergo a quasi-ordered co-aggregation process in response to saturating client interaction

Small heat shock proteins (sHSPs) are ATP-independent chaperones vital to cellular proteostasis, preventing protein aggregation events linked to various human diseases including cataract. The α-crystallins, αA-crystallin (αAc) and αB-crystallin (αBc), represent archetypal sHSPs that exhibit complex polydispersed oligomeric assemblies and rapid subunit exchange dynamics. Yet, our understanding of how this plasticity contributes to chaperone function remains poorly understood. This study investigates structural changes in αAc and αBc during client sequestration under varying degree of chaperone saturation. Using biochemical and biophysical analyses combined with single-particle electron microscopy (EM), we examined αAc and αBc in their apo-states and at various stages of client-induced co-aggregation, using lysozyme as a model client. Quantitative single-particle analysis unveiled a continuous spectrum of oligomeric states formed during the co-aggregation process, marked by significant client-triggered expansion and quasi-ordered elongation of the sHSP scaffold. These structural modifications culminated in an apparent amorphous collapse of chaperone-client complexes, resulting in the creation of co-aggregates capable of scattering visible light. Intriguingly, these co-aggregates maintain internal morphological features of highly elongated sHSP scaffolding with striking resemblance to polymeric α-crystallin species isolated from aged lens tissue. This mechanism appears consistent across both αAc and αBc, albeit with varying degrees of susceptibility to client-induced co-aggregation. Importantly, our findings suggest that client-induced co-aggregation follows a distinctive mechanistic and quasi-ordered trajectory, distinct from a purely amorphous process. These insights reshape our understanding of the physiological and pathophysiological co-aggregation processes of sHSPs, carrying potential implications for a pathway toward cataract formation

Eye lens β-crystallins are predicted by native ion mobility-mass spectrometry and computations to form compact higher-ordered heterooligomers

Eye lens crystallin proteins maintain the refractive properties of the lens but are not replaced after denucleation. Rolland et al. use native ion mobility-mass spectrometry, kinetics experiments, and computations to reveal that b-crystallins form heterodimers. These likely assemble into compact heterooligomers that enable the very high protein concentrations found in lens tissue

Deamidation Alters the Structure and Decreases the Stability of Human Lens βΑ3-Crystallin

According to the World Health Organization, cataracts account for half of the blindness in the world, with the majority occurring in developing countries. A cataract is a clouding of the lens of the eye due to light scattering of precipitated lens proteins or aberrant cellular debris. The major proteins in the lens are crystallins, and they are extensively deamidated during aging and cataracts. Deamidation has been detected at the domain and monomer interfaces of several crystallins during aging. The purpose of this study was to determine the effects of two potential deamidation sites at the predicted interface of the βA3-crystallin dimer on its structure and stability. The glutamine residues at the reported in vivodeamidation sites of Q180 in the C-terminal domain and at the homologous site Q85 in the N-terminal domain were substituted with glutamic acid residues by site-directed mutagenesis. Far-UV and near-UV circular dichroism spectroscopy indicated that there were subtle differences in the secondary structure and more notable differences in the tertiary structure of the mutant proteins compared to that of the wild type βA3-crystallin. The Q85E/Q180E mutant also was more susceptible to enzymatic digestion, suggesting increased solvent accessibility. These structural changes in the deamidated mutants led to decreased stability during unfolding in urea and increased precipitation during heat denaturation. When simulating deamidation at both residues, there was a further decrease in stability and loss of cooperativity. However, multiangle-light scattering and quasi-elastic light scattering experiments showed that dimer formation was not disrupted, nor did higher-order oligomers form. These results suggest that introducing charges at the predicted domain interface in the βA3 homodimer may contribute to the insolubilization of lens crystallins or favor other, more stable, crystallin subunit interactions

Client-Induced Elongation, Expansion, and Co-Aggregation of the Lens Alpha-Crystallins

The long-lived nature of the eye lens presents unique challenges to the maintenance of protein stability and function. Age-related accumulation of chemical modifications to proteins in the lens promote the formation of light-scattering aggregates that disrupt vision, leading to cataract: a leading cause of blindness worldwide. To counteract these effects, ∼40% of the lens cytosol is composed of α-crystallins (αAc and αBc isoforms): ATP-independent chaperone “holdases” that work to prevent formation of protein aggregates capable of scattering light. Like other members of the small heat shock protein (sHSP) family of chaperones, the α-crystallins form large and polydisperse oligomeric assemblies that recognize and sequester destabilized proteins, through the formation of soluble chaperone/client complexes. Over time, saturating conditions of unfolding clientele overwhelm lens chaperone capacity, leading to light-scattering co-aggregates. A mechanistic basis for the chaperone/client co-aggregation pathway is not well understood. Here, we applied single-particle electron microscopy and other biophysical techniques to define the morphological transitions associated with chaperone/client co-aggregation, using the model client lysozyme. We observe a mechanism where αAc and αBc chaperone/client sequestration progresses through an “initiation complex” (∼15–20 nm diameter, akin to apo-state α-crystallins), and proceeds through an intermediate elongation/expansion stage where co-aggregates reach dimensions of 50–200 nm. Ultimately, under saturating client conditions, the elongation/expansion complexes appear to cluster (or collapse) to form large light-scattering aggregates (microns in diameter). Ensemble and single-particle analysis techniques show αAc and αBc adopt a similar overall mechanism of expansion/elongation, while some unique isoform-specific features may be attributed to characteristic differences in activity toward lysozyme client. Overall, this work provides a mechanistic basis for understanding how α-crystallins (and perhaps other sHSP\u27s) accommodate destabilized clients and depicts a potential client-induced co-aggregation pathway leading to lens opacification and age-related vision loss

An ATCUN-like copper site in B2-crystallin plays a protective role in cataract-associated aggregation

Cataracts is the leading cause of blindness worldwide and it is caused by crystallin damage and aggregation. Senile cataractous lenses have relatively high levels of metals, while some metal ions can directly induce aggregation of human -crystallins. Here we evaluated the impact of divalent metal ions in the aggregation of human B2-crystallin, one of the most abundant crystallins in the lens. Turbidity assays showed that Pb2+, Hg2+, Cu2+, and Zn2+ ions induce the aggregation of B2-crystallin. Metal-induced aggregation is partially reverted by a chelating agent, indicating formation of metal-bridged species. Our study focused on the mechanism of copper-induced aggregation of B2-crystallin, finding that it involves metal-bridging, disulfide-bridging, and loss of protein stability. Circular dichroism (CD) and electron paramagnetic resonance (EPR) revealed the presence of at least three Cu2+ binding sites in B2-crystallin; one of them with spectroscopic features typical of Cu2+ bound to an amino-terminal copper and nickel binding motif (ATCUN), a motif found in Cu transport proteins. The ATCUN-like Cu binding site is located at the unstructured N-terminus of B2-crystallin, and it could be modeled by a peptide with the first six residues in the protein sequence (NH2-ASDHQF-). Removal of the N-terminus yields an N-truncated form of B2-crystallin that is more susceptible to Cu-induced aggregation and loss of thermal stability, indicating a protective role for the ATCUN-like site. EPR and X-ray absorption spectroscopy (XAS) studies reveal the presence of a copper redox active site in B2-crystallin that is associated to metal-induced aggregation and formation of disulfide-bridged oligomers. Our study demonstrates metal-induced aggregation of cataract-related B2-crystallin and the presence of putative copper binding sites in the protein. Whether the copper-transport ATCUN-like site in B2-crystallin plays a functional/protective role or constitute a vestige from its evolution as a lens structural protein, remains to be elucidated