Our laboratory work s in multidisciplinary research environment focused on understanding how small heat shock proteins function in vivo.
We investigate the role of the protein aggregate formation in eye lens cataract initiation and growth.
To study the intricate relationship between protein aggregation, cell adhesion and cell cytoskeleton proteins and small heat shock proteins in hereditary cataracts, we have generated several genetically-engineered mouse models.
Our laboratory is specifically interested in understanding how lens epithelial cells control cataract development and progression in vivo.
We are also applying state-of-the art proteomic, imaging and genetic resources at Washington University to develop specific markers to study protein aggregation and insolubilization in lens epithelial and fiber cells and to identify anti-cataract strategies that specifically target protein aggregation.
Knock-in mice expressing mutations associated with human hereditary cataracts are the focus of our present research. The influence of cataract on autophagy and the unfolded protein response will be important to understand the role of protein degradation of misfolded proteins in human cataract formation.
These studies will enhance the understanding of cataract development and progression in animal models, which can be correlated with human cataract development. Using knock-in and knock-out mouse models for cataracts, we have recently identified the likely in vivo substrates of alpha crystallin in the lens using proteomic techniques. In a major new endeavor, we are testing novel small molecule pharmaceutical compounds to reverse and delay cataract development using animal models generated in our laboratory. View the article in science magazine.
Alpha-crystallin and cataracts:
The vertebrate lens expresses two α-crystallin proteins, αA and αB, at a high concentration in lens fiber cells and at lower levels in the lens epithelium. In lens fiber cells, α-crystallin is a heteroaggregate of αA- and αB-crystallin in a 3:1 ratio. αA- and αB-crystallins are members of the small heat shock protein family of molecular chaperones.
Elegant work has shown that αA-crystallin, αB-crystallin, and the α-crystallin heteroaggregate possess chaperone-like activity, binding to partially unfolded or denatured proteins to suppress non-specific protein aggregation. This suggests that α-crystallin is likely to have considerable functional importance in vivo and may help preserve unfolded proteins in a folding-competent conformation, delaying protein aggregation and cataract formation.
Our research is focused on the in vivo substrates of α-crystallin to gain insight into the relationship between chaperone function, substrate binding, and human cataract formation.
Mechanism of alpha-crystallin function:
Targeted disruption of the mouse aA gene demonstrated a critical role of this protein in the maintenance of lens transparency. Lenses of aA knockout mice are 35% smaller in size and develop opacities by 4-6 weeks of age as a result of the presence of dense inclusion bodies containing aB-crystallin, HSP25, and γ-crystallin in the fiber cells, suggesting that αA may be necessary for solubility of other crystallins in vivo.
Using αA-/- mouse lenses, we demonstrated that aA-crystallin expression is necessary for optimal growth of lens epithelial cells in vivo, and ensures the successful completion of mitosis and cytokinesis. αA-/- cells demonstrate abnormal microtubule cytoskeleton during anaphase and cytokinesis. The reduced size of the aA-/- lens may be due, in part, to the reduced survival of aA-/- lens epithelial cells in vivo. Our lab is collaborating with Dr. Sue Menko to investigate expression and/or phosphorylation of specific cell survival and cell death-related signaling proteins in αA-/- mouse lens epithelial and fiber cell fractions.
Models for congenital and age-related cataracts:
The molecular mechanisms of hereditary human cataract formation by point mutations in α-, β- and γ-crystallin genes are not yet completely understood. These point mutations result in an altered structure, higher aggregate molecular mass, abnormal membrane association and reduced ability to prevent aggregation of several target proteins in vitro. Our studies aim to increase our understanding of the mechanisms underlying congenital cataracts caused by αA-crystallin mutations and provide crucial information concerning the mechanisms of age-related cataract.
Makley LN, McMenimen KA, DeVree BT, Goldman JW, McGlasson BN, Rajagopal P, Dunyak BM, McQuade TJ,Thompson AD, Sunahara R, Klevit RE, Andley UP, Gestwicki JE. Pharmacological chaperone for α-crystallin partially restores transparency in cataract models. Science 2015; 350 (6261): 674-677.
Andley UP, Malone J, Hamilton PD, Ravi N, Townsend RR. Comparative proteomic analysis identifies increases in the abundance of βB2- crystallin and vimentin after the deletion of the small heat shock proteins αA- and αB-crystallins. Biochemistry. 2013; 52, 2933-2948.