The primary objective of the proposed research is to define the functional mechanism for the protective actions of the stress protein, human ?B crystallin, a small heat shock protein (sHSP) and molecular chaperone. HYPOTHESIS 1: The activity of multiple interactive sequences in human??B crystallin provides protection against protein unfolding, aggregation, and toxicity in aging disorders that include cataract, neurodegeneration, AMD, and possibly, neuromuscular disorders. Synthetic peptides based on the individual interactive domains in ?B crystallin mediate the formation of amyloid fibrils and will be used to characterize the collective mechanism(s) of the interactive domains in the action of ?B crystallin.
AIM 1 will optimize the function of human ?B crystallin, the archetype of all small heat shock proteins (sHSP). Functional assays will be used to characterize the effects of new mutants of ?B crystallin on the stress response and protection against protein unfolding and aggregation in vitro.
AIM 2 will use slit lamp imaging to conduct phenotype analyses of lenses in transgenic mouse models for neurodegenerative and neuromuscular disorders.
This aim addresses HYPOTHESIS 2: The loss of lens transparency is a sensitive indicator of altered protein interactions in neurodegenerative and neuromuscular disorders. The unique accessibility of the lens for non-invasive optical examinations makes lens cells excellent for the study of basic mechanisms of aggregation and amyloid formation in living animals. HYPOTHESIS 3: The proteins responsible for the lens phenotype in neurodegeneration may also be responsible for the lens phenotype in neuromuscular disorders, and AIM 3 will identify critical protein constituents responsible for the structural phenotype in transgenic mouse models for neurodegenerative and neuromuscular disorders. Mass spectrometry of samples obtained using laser capture microdissection (LCM) will be combined with immunocytochemistry, and light and electron microscopy to correlate protein constituents with structural modifications accounting for the phenotype observed in lenses of mouse models for neurodegenerative and neuromuscular disorders. SIGNIFICANCE FOR PUBLIC HEALTH: Protein unfolding and amyloid/aggregate formation characterize disorders of aging that include cataract, age related macular degeneration (AMD), neurodegeneration, cardiomyopathy and muscular dystrophy. With the increase in human longevity, the impact of aging diseases is increasing dramatically. Defining the basic mechanism for the protective activity of the stress protein ?B crystallin will be a major advance in biomedical research and has the potential to provide novel therapeutic targets for aging disorders. The lens is ideal for innovative functional studies of ?B crystallin function in vivo and may have significance as a biomarker for the initiation and progression of neurodegenerative and neuromuscular disease.
This application is a study of the interactions of the stress protein human ?B crystallin responsible for the fundamental protective mechanism(s) against protein unfolding and aggregation. The approach is to characterize the activity of the interactive domains and quantify their affinities in vitro while studying their influence on the transparent structure of the lens in vivo using the slit lamp. The results will advance biomedical research in understanding the endogenous function of sHSP in diseases of aging that include neurodegeneration, cardiovascular diseases, muscular dystrophy, AMD (age related macular dystrophy) and cataract. This research has the potential for translational applications to the development of novel therapeutics against major diseases of aging. The lens is used for these studies because of its unique accessibility for the in vivo investigation of protein unfolding and aggregation diseases in aging and the well known protective effects of ?B crystallin.
|Clark, John I (2013) Self-assembly of protein aggregates in ageing disorders: the lens and cataract model. Philos Trans R Soc Lond B Biol Sci 368:20120104|
|Qu, Bo; Landsbury, Andrew; Schönthaler, Helia Berrit et al. (2012) Evolution of the vertebrate beaded filament protein, Bfsp2; comparing the in vitro assembly properties of a "tailed" zebrafish Bfsp2 to its "tailless" human orthologue. Exp Eye Res 94:192-202|
|Houck, Scott A; Landsbury, Andrew; Clark, John I et al. (2011) Multiple sites in ýýB-crystallin modulate its interactions with desmin filaments assembled in vitro. PLoS One 6:e25859|
|Houck, Scott A; Clark, John I (2010) Dynamic subunit exchange and the regulation of microtubule assembly by the stress response protein human alphaB crystallin. PLoS One 5:e11795|
|Greiling, Teri M S; Aose, Masamoto; Clark, John I (2010) Cell fate and differentiation of the developing ocular lens. Invest Ophthalmol Vis Sci 51:1540-6|
|Greiling, Teri M S; Stone, Brad; Clark, John I (2009) Absence of SPARC leads to impaired lens circulation. Exp Eye Res 89:416-25|
|Greiling, Teri M S; Clark, John I (2009) Early lens development in the zebrafish: a three-dimensional time-lapse analysis. Dev Dyn 238:2254-65|
|Greiling, Teri M S; Houck, Scott A; Clark, John I (2009) The zebrafish lens proteome during development and aging. Mol Vis 15:2313-25|
|Ghosh, Joy G; Houck, Scott A; Clark, John I (2008) Interactive sequences in the molecular chaperone, human alphaB crystallin modulate the fibrillation of amyloidogenic proteins. Int J Biochem Cell Biol 40:954-67|
|Greiling, Teri M S; Clark, John I (2008) The transparent lens and cornea in the mouse and zebra fish eye. Semin Cell Dev Biol 19:94-9|
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