Research SummaryFor the last twenty years my main research interest has centered on the biochemical and biophysical properties of lens proteins (mainly α-, β- and γ-crystallins) and the changes in their properties during age-related cataractogenesis (ARC). Since lens transparency is a manifestation of the remarkably ordered supramolecular assembly of lens proteins, disruption of this assembly may cause lens opacification. The mechanisms disrupting the assembly therefore are fundamentally important to our understanding of cataractogenesis. To understand these mechanisms, my lab began a study of experimental models of age-related cataract using crystallins isolated from clear and cataractous human lenses along with young and old bovine lenses. We defined the modifications of amino acid residues, the conformational changes and high-molecular-weight (HMW) protein aggregations that occurred with aging. These appeared to have key roles in ARC. α-Crystallin is the major and most abundant crystallin in HMW aggregates as well as in the insoluble protein fraction of the cataractous lens. By using the recombinant proteins, we are able to duplicate the changes observed in the proteins of human lenses, thus confirming our original speculation about the importance of these processes in ARC. We believe that understanding the mechanisms of HMW protein aggregation may lead to therapeutic developments to slow the process of age-related cataract formation. In addition to ARC, there are many human autosomal dominant congenital cataracts in which a single mutation in a specific crystallin or non-crystallin gene is responsible for the cataract. Some examples of such congenital cataract crystallin genes include CRYAA (Rl16C), CRYAB (R120G), CRYBB2 (Q155*), and CRYGC (T5P). My research team has prepared by site-specific mutation the crystallin mutants associated with these congenital cataracts, and we have studied their biophysical properties. We have documented the changes in protein conformation, aggregation size, chaperone activity, and protein-protein interactions. The congenital cataract model provided an excellent model to define the specific relationship between a protein modification (single mutation) and cataract formation. Such a study was not possible in the ARC, in which many post-translational modifications occur concurrently in various crystallins (e.g. such as glycation, photooxidation, deamidation, and disulfide formation). α-Crystallin, the major crystallin in the lens, has two subunits αA and αB that form aggregates with molecular sizes of 600-800 kDa. While αA-crystallin is lens-specific, αB-crystallin has been found in many nonlenticular tissues. Recently they have been extensively studied because of their ability to function as chaperones. However, the three-dimensional structures of these aggregates have not been determined. Also, the mechanisms of their aggregation and subunit interaction are not known, largely because of the inability to crystallize them for X-ray diffraction study. Our recent effort has been to determine the subunit interaction domains using two techniques: the two-hybrid system assay of protein-protein interactions, and site-specific mutagenesis. Once the domains are determined, we will be able to prepare small peptides having the domain sequences that competitively bind to the interaction domain in α-crystallin, thus preventing subunit aggregation. We hope that this will lead to the successful crystallization of each subunit. 1. Biophysical Studies of Age- and Congenital Cataract-related Changes in Lens Proteins. (Role: Principal Investigator) We are interested in studies of the modifications in lens proteins that occur during cataract formation. Cataract is a pathological state in which the lens becomes opaque. There are many types of cataracts and the most prevalent one is age-related cataract (ARC). It is believed that ARC is caused by environmental factors such as UV radiation (sun light damage), free radical oxidation, metabolic disorders (enzyme dysfunction), and nonenzymatic glycation (diabetes mellitus). Another type of cataract is congenital cataracts that are caused by a single missense mutation in a specific gene, such as CRYAA (R116C) in zonular nuclear cataract and CRYGC (T5P) in Coppock-like cataract. Both ARC and congenital cataract involve conformational change, high-molecular weight aggregation, and cellular damage. Our research aim is to determine how protein modifications or mutations cause these changes. To achieve the goal, we have cloned α-, β-, and γ-crystallin wild type and mutant genes and expressed recombinant proteins. Posttranslational modifications are being induced. Spectroscopy and some biophysical techniques are being used to compare the changes in conformation, stability, solubility, and aggregation size between modified or mutated and control crystallins. 2. The Interaction Domains in the Lens Crystallins. (Role: Principle Investigator) The lens structural protein consists of three major crystallins, designated as α-, β- and γ-crystallins. Each contains many components, e.g., αA and αB in α-crystallin, βA1-βA4 and βB1-βB3 in β-crystallin, and γA-γF in γ-crystallin. It is believed that they interact to form a supramolecular assembly that is fundamentally important in lens transparency. With aging and cataract formation, this normal structure is deteriorated or disrupted. Therefore, the study of structural aspects in the normal and diseased states is important in the understanding of cataractogenesis. Among the three crystallins, the three-dimensional structures for many of β- and γ-crystallin have been elucidated by X-ray diffraction. But no X-ray structure has been determined for α-crystallin, either αA- or αB-crystallin, due to their inability to form crystals, which in turn is caused by their aggregated and polydispersed states. The difficulty to isolate α-crystallin in a monomer native state that may be crystallized has remained to be a challenging work for many decades and has hindered the progress in the study of cataractogenesis. To overcome this difficulty, we aim to determine the nature of subunit interactions that are responsible for oligomerization. Once the domains of subunit interactions are determined, we can use site-specific mutation to alter the domains so oligomerization can be prevented. To identify the domains responsible for oligomerization, we are using a two-hybrid system assay and a site specific-mutation. 3. The Functional Role of the Small Heat Shock Proteins in Cataract Formation. (Role: Principle Investigator) The lens crystallins (α-, β- and γ-crystallin) were thought to serve only structural and refractive purposes, but α-crystallin was found to function as a chaperone. In the lens, α-crystallin is a hetero oligomer composing of two subunits, αA and αB. The two subunits can form homo oligomers in vitro and αB-crystallin is also a homo oligomer in nonlenticular tissues. They belong to the family of small heat shock proteins (sHsps) with the characteristic “α-crystallin domain”. While αA-crystallin is lens-specific, αB-crystallin is widely expressed in other tissues, especially under stress and in neuro degenerative diseases, such as Alzheimer’s disease (AD). In addition to α-crystallin, the lens may have other sHsps, such as Hsp27, Hsp40 and Hsp70, whose functions have not been as widely studied as those of α-crystallin. Though it is not known whether these sHsps function as chaperones, if so, they must be overshadowed by the relatively abundant α-crystallin. However, the amount of soluble α-crystallin decreases with age until none remains in the nuclear region of old lens. For the lens to maintain transparency, other sHsps need to take over the responsibility of protection. Our hypothesis is that sHps besides α-crystallin are expressed in the lens and that their expression increases with age; but at some point they become either inefficient or insufficient for chaperone protection, allowing cataract formation. To test this hypothesis, we will determine non α-crystallin sHsps gene and protein expression levels in young, old, and age-matched cataractous lenses 4. Functional Role of αB-Crystallin in the Nonlenticular Tissues. (Role: Principle Investigator) Recently αB-crystallin was found widely expressed in nonlenticular tissues and the expression was enhanced in some neurodisorder diseases. This has greatly intensified the study of functional role of αB-crystallin in the nonlenticular tissues. We have cloned human αA- and αB-crystallins and some congenital mutants, facilitating study not only in cataract but also in diseases of other tissues, such as Alzheimer’s disease (AD). AD and cataract are two of the protein condensation diseases that involve protein aggregation as an etiology. Many studies report that αB-crystallin colocalizes with Aβ peptides in the AD plaques indicating some functional role of αB-crystallin in the pathology of AD. Since αB-crystallin is a heat shock protein and has chaperone activity, its function may be to protect Aβ peptides from aggregation and thus to maintain the cell viability. This is demonstrated by in vitro study that αB-crystallin suppresses Aβ peptide aggregation. Other studies indicate that expressions of αB-crystallin and Hsp27 increase during heat shock stress. They may provide a protective mechanism. We are studying this possible protective mechanism of αB-crystallin in the aggregation of Aβ peptides. Back to the top |
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