Abstract

A large body of evidence supports the hypothesis that lens opacity is due to crystallin aggregation. To determine possible causes of this aggregation, our current research program has been directed towards identifying all of the major covalent modifications occurring in vivo to human lens crystallins with emphasis on distinguishing between clear and cataractous lenses. More broadly, the goal of this research has been to elucidate the chemistry that initiates or propagates cataract, which includes the chemistry of aging. Our use of mass spectrometry has led to major advances in identifying these covalent modifications. For example, it has been possible to unequivocally distinguish among a variety of age-related modifications that cause the proteins to be more acidic, such as phosphorylation, deamidation and lysine modification, and to readily identify multiple degradation products. All the major sites of modification in all the major gene products of the water-soluble portion of the lens have been identified. The renewal application includes four Specific Aims.
Aim 1 will extend our present studies to include identification of covalent modifications to crystallins present in the water-insoluble fractions of clear and cataractous human lenses. Effective pursuit of this goal includes collaboration with Prof. Larry David (Oregon Health Sciences University). Dr. David requests a subcontract for this collaboration in lieu of direct competition with renewal of his R01 grant.
In Aims 2 -4, we shall explore linkages between known in vivo modifications of human lens crystallins and their propensity to aggregate.
Specific Aim 2 will determine the effect of modifications of beta- and gamma- crystallins on their solubility.
In Aim 3, new and highly detailed structural information will be obtained about the mechanisms through which alpha-crystallins solubilize gamma- crystallins.
Aim 4 will address the effects of specific modifications to alpha-crystallins that inhibit their ability to perform this chaperone function. New analytical methods based on amide hydrogen exchange and lysine reactivity will be used to accomplish Aims 3 and 4.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY007609-14
Application #
6384588
Study Section
Visual Sciences A Study Section (VISA)
Program Officer
Liberman, Ellen S
Project Start
1989-04-01
Project End
2004-03-31
Budget Start
2001-04-01
Budget End
2002-03-31
Support Year
14
Fiscal Year
2001
Total Cost
$301,033
Indirect Cost

  Institution

Name
University of Nebraska Lincoln
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
555456995
City
Lincoln
State
NE
Country
United States
Zip Code
68588

  Publications

Lapko, Veniamin N; Cerny, Ronald L; Smith, David L et al. (2005) Modifications of human betaA1/betaA3-crystallins include S-methylation, glutathiolation, and truncation. Protein Sci 14:45-54
Swaim, Catherine L; Smith, Jean B; Smith, David L (2004) Unexpected products from the reaction of the synthetic cross-linker 3,3'-dithiobis(sulfosuccinimidyl propionate), DTSSP with peptides. J Am Soc Mass Spectrom 15:736-49
Hasan, Azeem; Yu, Jiong; Smith, David L et al. (2004) Thermal stability of human alpha-crystallins sensed by amide hydrogen exchange. Protein Sci 13:332-41
Wintrode, Patrick L; Friedrich, Kenneth L; Vierling, Elizabeth et al. (2003) Solution structure and dynamics of a heat shock protein assembly probed by hydrogen exchange and mass spectrometry. Biochemistry 42:10667-73
Lapko, Veniamin N; Smith, David L; Smith, Jean B (2003) Expression of betaA2-crystallin in human lenses. Exp Eye Res 77:383-5
Zhang, Zhongli; Smith, David L; Smith, Jean B (2003) Human beta-crystallins modified by backbone cleavage, deamidation and oxidation are prone to associate. Exp Eye Res 77:259-72
Lapko, Veniamin N; Smith, David L; Smith, Jean B (2003) Methylation and carbamylation of human gamma-crystallins. Protein Sci 12:1762-74
Hasan, Azeem; Smith, David L; Smith, Jean B (2002) Alpha-crystallin regions affected by adenosine 5'-triphosphate identified by hydrogen-deuterium exchange. Biochemistry 41:15876-82
Lapko, Veniamin N; Purkiss, Andrew G; Smith, David L et al. (2002) Deamidation in human gamma S-crystallin from cataractous lenses is influenced by surface exposure. Biochemistry 41:8638-48
Thampi, Prajitha; Hassan, Azeem; Smith, Jean B et al. (2002) Enhanced C-terminal truncation of alphaA- and alphaB-crystallins in diabetic lenses. Invest Ophthalmol Vis Sci 43:3265-72

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