NEI Award #EY015834, "Human Gamma-D-Crystallin Folding, Misfolding and Fibril Forms"
Summary: Lens cataracts,
interfering with normal vision, affect 50% of the American population over
age 75. Human g D-crystallin (H g D-Crys) and Human g S-crystallin (H g S-Crys)
are monomeric two-domain major proteins of the human eye lens and significant
components of mature-onset cataracts.
Mature-onset cataracts are thought to be an aggregated state of modified,
damaged, or partially unfolded states of lens crystallins. This proposal
treats mature-onset cataract as a protein deposition disease, and focuses
on in vitro unfolding,
refolding and aggregation experiments with purified proteins, to find in
vitro models for the precursors to lens protein aggregation and/or chaperone
recognition, in situ. For other protein deposition diseases such systems have
been key steps toward targeted searches for therapeutic agents inhibiting pathological
deposition.
Purified recombinant H g D-Crys can be refolded in vitro at 37 ° C and neutral pH in the absence of cellular factors. Formation of high molecular weight fibrils competes with productive refolding. A partially unfolded species with the C-terminal domain folded, and the N-terminal domain unfolded, has been identified as an intermediate in the unfolding and refolding pathway, and is a candidate for the precursor in fibril formation. H g S-Crys also refolds in vitro. Though H g S molecules do not self-aggregate during refolding, they are recruited into the H g D fibrils when refolding with both proteins present. These co-aggregation reactions of partially folded intermediates - under conditions of productive refolding - provide in vitro models for lens opacity.
In this proposal, we use a set of H g D mutant proteins, including triple-tryptophan replacements, to a) deepen understanding of the molecular basis of the extraordinary stability of the crystallins in their solution state, b) to map the pathway of the partially folded H g D and H g S crystallins polymerizing into fibrillar and other aggregated states, with emphasis on identification of putative nucleating and propagating species, and c) to determine whether partially folded species or their fibrillar state formed in vitro are substrates of the a -crystallins, and whether they are generated in vitro by photo-oxidative stress, thought to be acting on the crystallins within the lens.
Publications
Chen, J., Toptygin, D., Brand, L. and King, J. (2008) Mechanism of the efficient tryptophan fluorescence quenching in human gD-crystallin studied by time-resolved fluorescence.Biochemistry, 47, 10705-10721.
Wang, Y. and King, J. (2008) Cataract as a protein aggregation disease. In, Protein Misfolding Diseases: Current and Emerging Principles and Therapies (Marina Ramirez-Alvarado, Jeffrey W. Kelly, and Christopher M. Dobson, eds.) John Wiley and Sons, Hoboken, NJ. In press.
Papanikolopoulou, K., Mills, I.A., Flaugh, S.L.., Wang, Y., Gross, A.A.R., Kirschner, D.A., Decatur, S.M. and King, J.A. (2008) Formation of amyloid fribrils in vitro by human gamma D-crystallin and its isolated domains. Molecular Vision, 14, 81-89. http://www.molvis.org/molvis/v14/a10
Mills, I.A., Flaugh, S.L., Kosinski-Collins, M.S. and King, J. (2007) Folding and stability of the isolated greek key domains of the long-lived human lens proteins, gamma D-crystallin and gamma S-crystallin.Protein Science, 16, 2427-2444 .
Flaugh, S.L., Mills, I.A. and King, J. (2006) Glutamine deamidation destabilizes human gammaD-crystallin and lowers the kinetic barrier to unfolding. J.Biol.Chem., 281, 30782-30793.
Chen, J., Flaugh, S.L., Callis, P.R. and King, J. (2006) Mechanism of the highly efficient quenching of tryptophan fluorescence in human gammaD-crystallin. Biochemsitry, 45, 11552-11563.
Flaugh, S.L., Kosinski-Collins, M.S. and King, J (2005) Interdomain side-chain interactions in human gammaD crystallin influencing folding and stability. Protein Science, 14, 2030-2043.
Flaugh, S.L., Kosinski-Collins, M.S. and King, J (2005) Contributions of hydrophobic domain interface interactions to the folding and stability of human gammaD-crystallin. Protein Science, 14, 569-581 .
Kosinski-Collins, M., Flaugh, S. & King, J. (2004) Probing folding and fluorescence quenching in Human gamma-D crystallin Greek key domains using Triple Tryptophan mutant proteins. Protein Science, 13, 2223-2235.
Kosinski-Collins,
M. & King, J. (2003) In
vitro unfolding and refolding of human gamma-D crystallin, a
protein involved in cataract formation. Protein Science, 12, 480-490.
Asherie, N., Pande, J., Pande, A., Zarutskie, J.A., Lomakin, J., Lomakin, A., Ogun, O., Stern, L.J., King, J., Benedek, G.B. (2001) Enhanced Crystallization of the Cys18 to Ser Mutant of Bovine yB Crystallin. J. Mol.Biol., 314, 663-669.
Pande. A., Pande, J., Asherie, N., Lomakin, A., Ogun, O., King, J., and Benedek, G.B. (2001) Crystal cataracts: Human genetic cataract caused by protein crystallization. PNAS,98, 6116-6120.
Pande, A., Pande, J., Aserie, N., Lomakin, A., Ogun, O., King, J., Lubsen, N., Walton, D. & Benedek, G. (2000) Molecular basis of a progresssive juvenile-onset hereditary cataract. PNAS, 97, 1993-1998.