Using Biophysics to Understand Cataracts

June is Cataract Awareness Month in the US. By age 80, more than half of all Americans either have a cataract or have had cataract surgery, according to the National Eye Institute. To recognize this month, we spoke with BPS member Doug Tobias, University of California, Irvine, about his research on crystallin proteins, the proper functioning of which is key to lens clarity. 


What is the connection between your research and cataracts?

The eye lens, which focuses light on the retina, is composed of bundled fiber cells that lose their nuclei, ribosomes, and organelles during embryonic development. The transparency and refractive properties required for proper lens function results from the liquid-like order of structural proteins called crystallins, which are present in these cells at concentrations exceeding 300 g/L. Because the central nucleus of the lens undergoes very little protein turnover, crystallin proteins must remain stably in solution for a lifetime. A cataract is an opacification of the eye lens caused by the loss of solubility of the crystallin proteins, leading to the formation of aggregates that scatter light.

Tobias image 2

All-atom model of AQP0 tetramer embedded in a hydrated lipid bilayer and complexed with two CaM molecules. Reference: S. L. Reichow, D. Clemens, J. A. Freites, K. L. Németh-Cahalan, M. Heyden, D. J. Tobias, J. E. Hall, and T. Gonen, Allosteric mechanism of water-channel gating by Ca2+–calmodulin, Nat. Struct. Molec. Biol. 20, 1085-1092 (2013).

Our research seeks to understand cataract formation on the molecular level from two different angles, both involving atomically-detailed computer simulations (molecular dynamics, Brownian dynamics, and Monte Carlo), in concert with experiments carried out by our collaborators. On one front, we are attempting to elucidate how minor changes to the chemical structure of the crystallin proteins – such as the single-point mutations that are associated with congenital, early onset cataract, or post-translational modifications (e.g., truncation, disulfide bond formation, UV damage, deamidation, etc.) in the case of the much more common age-related cataract – give rise to altered interprotein interactions, which, in turn, lead to protein aggregation.

On the other front, we are learning how the protein aquaporin 0 (AQP0) is regulated. AQP0 is expressed exclusively in the eye lens, where it comprises roughly half the membrane protein content and functions as a water channel and, hence, is a key player in maintaining lens homeostasis. AQP0’s water permeability is regulated by pH and Ca2+, whose concentrations change dramatically with distance into the lens. Defects in AQP0 can lead to cataract. We are using molecular dynamics simulations, in conjunction with experimental measurements carried out by our collaborators, to determine the mechanism of the alteration of AQP0 permeability by certain mutations, as well as by changes in pH and Ca2+ levels.

Why is your research important to those concerned about cataracts?

There is good reason to be concerned about cataracts. In its latest assessment of priority eye diseases, the World Health Organization estimated that cataract is responsible for more than 50% of world blindness, which amounts to roughly 20 million people. Presently, cataracts are treated by surgical replacement of the opaque lens with an artificial intraocular lens.  Due to lack of access to eye care, millions of people in the developing world remain blind from cataract. The long-term goal of our research is to gain new insights into cataract formation that could ultimately guide the development of new therapies for cataract prevention and treatment.

How did you get into this area of research?

I got into this area of research through interactions with colleagues at UC Irvine, specifically, Jim Hall and Rachel Martin. Jim Hall is a lens physiologist in the Department of Physiology and Biophysics and an expert on AQP0, and Rachel Martin is a biophysical chemist in the Department of Chemistry and an expert on the crystallins. Their enthusiasm for their respective subjects was infectious and irresistible, and I jumped at the opportunity to collaborate with them. Our collaboration with Jim Hall was initiated by a graduate student in his lab who wanted to spend time in my lab learning molecular modeling. Our work with Rachel Martin began as a summer undergraduate research project.

How long have you been working on it?

We started dabbling in the field about five years ago and then increased our effort substantially after receiving funding for the work about three years ago.

Do you receive public funding for this work? If so, from what agency?

Our work on the crystallin proteins is supported by both the National Institutes of Health and the National Science Foundation, and our work on AQP0 is supported by the National Institutes of Health.

Tobias image

Computational model of an aggregate formed by the congenital cataract-related W42R mutant of human gD-crystallin in a solution at 220 g/L concentration. The N-terminal domains are colored red and the C-terminal domains blue. The aggregates formed by the W42R mutant display enhanced interprotein contacts involving the N-terminal domain, where the mutation is located, vs. the wild-type protein, which displays primarily non-specific interactions at the same concentration.

Have you had any surprise findings thus far?

Ca2+ regulation of AQP0 is mediated by the calcium binding protein calmodulin (CaM), which forms a complex with AQP0 of unusual stoichiometry (2 CaM to 1 AQP0 tetramer). We expected that CaM binding would lower AQP0 permeability by simply blocking the water conducting pores. However, we found instead that CaM binding lowers the open probability of a gate deep within the pore through long-range allosteric interactions. We are now learning, also unexpectedly, that pH regulation occurs through a similar (allosteric) mechanism involving a second gate on the other end of the pore.

Crystallin proteins are constantly bumping into one another due to their high concentration in the lens. “Healthy” (wild type) crystallins stay in solution because they don’t stick to each other, i.e., their interactions are non-specific. It is fascinating to me that a single-point mutation in a single class of crystallin proteins can lead to early-onset cataract. The aggregation mechanisms of these mutant proteins are only beginning to be worked out, and it is likely that different mechanisms will be at play in different cataract-related variants. In one case that we are currently studying, we found that the W42R mutation “cracks open” the interface between the two domains in gD-crystallin, exposing a sticky patch that promotes the formation of large-scale protein clusters.

What is particularly interesting about the work from the perspective of other researchers?

Our work produces atomically-detailed descriptions of molecular mechanisms that are often critical to the interpretation of experimental observations. We take great pains to validate our simulations so that their predictions can be trusted.

What is particularly interesting about the work from the perspective of the public?

Cataract is the leading cause of blindness worldwide.  Even in developed countries like the US, where cataract surgery is widely available, and safe and effective in the vast majority of cases, delaying it for just a few years would greatly reduce health care costs. We hope that our research will yield new fundamental insights that will inform non-surgical approaches to cataract treatment.

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