Deamidation is the most prevalent post-translational modification in the lens, and increases with age. All of the major crystallins are deamidated, with the beta-crystallins the most heavily deamidated. Our laboratory has recently identified several specific in vivo sites of deamidation in crystallins allowing us to investigate the functional consequences of this modification. Several deamidation sites are in highly conserved regions between beta-subunits, suggesting that these deamidations occur in critical structural regions. However, the effects of deamidation on crystallin structure and stability are not known. The stability of crystallins is fundamental to the transparency of the lens. The formation of cataracts will only be understood when we begin to elucidate factors that alter crystallin stability. The long-term goal of our research is to determine the effects of deamidation on beta-crystallins in the human lens. The objective of this grant application is to characterize several specific in vivo sites of deamidation of beta-crystallins. We hypothesize that deamidation in critical regions disrupt structure and stability promoting insolubilization, while deamidations in non-critical regions foster solubility. Our hypothesis is derived from our previous studies showing that a deamidation at the interface between domains decreased stability, while deamidations at other sites increased stability, or had little effect. To test our hypothesis, we will use site directed mutagenesis to introduce in vivo deamidations in regions predicted to be structurally significantly. We will then characterize their structure, stability, and tendency to aggregate using advanced biophysical techniques, such as multi-angle laser light scattering, and dynamic light scattering. Only after we understand how deamidation alters crystallin structure can we correlate specific deamidations to cataract formation. We can then begin to investigate methods to prevent deamidation or design chaperones that prevent structural changes at these sites.

National Institute of Health (NIH)
National Eye Institute (NEI)
Research Project (R01)
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Special Emphasis Panel (ZRG1-AED (01))
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Liberman, Ellen S
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Oregon Health and Science University
Schools of Dentistry
United States
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Lampi, Kirsten J; Murray, Matthew R; Peterson, Matthew P et al. (2016) Differences in solution dynamics between lens ?-crystallin homodimers and heterodimers probed by hydrogen-deuterium exchange and deamidation. Biochim Biophys Acta 1860:304-14
Lampi, Kirsten J; Wilmarth, Phillip A; Murray, Matthew R et al. (2014) Lens ?-crystallins: the role of deamidation and related modifications in aging and cataract. Prog Biophys Mol Biol 115:21-31
Lampi, Kirsten J; Fox, Cade B; David, Larry L (2012) Changes in solvent accessibility of wild-type and deamidated ýýB2-crystallin following complex formation with ýýA-crystallin. Exp Eye Res 104:48-58
Fort, Patrice E; Lampi, Kirsten J (2011) New focus on alpha-crystallins in retinal neurodegenerative diseases. Exp Eye Res 92:98-103
Mellies, Jay L; Benison, Gregory; McNitt, William et al. (2011) Ler of pathogenic Escherichia coli forms toroidal protein-DNA complexes. Microbiology 157:1123-33
Takata, Takumi; Smith, Joshua P; Arbogast, Brian et al. (2010) Solvent accessibility of betaB2-crystallin and local structural changes due to deamidation at the dimer interface. Exp Eye Res 91:336-46
Michiel, Magalie; Duprat, Elodie; Skouri-Panet, Feriel et al. (2010) Aggregation of deamidated human betaB2-crystallin and incomplete rescue by alpha-crystallin chaperone. Exp Eye Res 90:688-98
Dudek, Edward J; Lampi, Kirsten J; Lampi, Jason A et al. (2010) Ubiquitin proteasome pathway-mediated degradation of proteins: effects due to site-specific substrate deamidation. Invest Ophthalmol Vis Sci 51:4164-73
Takata, Takumi; Woodbury, Luke G; Lampi, Kirsten J (2009) Deamidation alters interactions of beta-crystallins in hetero-oligomers. Mol Vis 15:241-9
Takata, Takumi; Oxford, Julie T; Demeler, Borries et al. (2008) Deamidation destabilizes and triggers aggregation of a lens protein, betaA3-crystallin. Protein Sci 17:1565-75

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