Biochemistry

Krishna K. Sharma

Professor of Opthalmology
Joint Professor of Biochemistry

E-mail:sharmak@missouri.edu

Office: 213 Eye Clinic

Mail: Mason Eye Institute
213EC Mason Eye Institute
One Hospital Drive
Columbia, MO 65212

Phone: 573-882-8478

Fax: 573-884-4100

Lab: 573-882-8481

Educational background

Notable honors and service

• Robert E. McCormick Scholar Award from Research to Prevent Blindness, 1999
• Cataract Research Award from National Foundation for Eye Research, 2000
• Lew R. Wasserman Merit Award from Research to Prevent Blindness, 2002
• Program Committee Chair for Lens Section, Annual Meeting of Association for Research in Vision and Ophthalmology, 2007

Research description

The lens of the eye is an excellent model for studying the effects of aging. The lens is primarily composed of long-lived highly stable proteins called crystallins. The normally transparent lens often gradually becomes cloudy with aging, leading to cataract formation. Cataract is a major cause of blindness worldwide. By age 80, more than half of Americans either have a cataract or have had cataract surgery. Cataract primarily develops as a result of extensive modification, aggregation and precipitation of the lens proteins called crystallins. In our studies of the molecular mechanisms of cataract formation, we are investigating the role of cellular enzymes called proteases in cataractogenesis and the structure and function of the major lens crystallin, alpha-crystallin.

Every cataract lens analyzed thus far in laboratories across the world has exhibited evidence of proteolysis (the degradation of crystallin proteins). However, the specific proteases responsible for the proteolysis of lens crystallins and the subsequent alteration in their properties are yet to be characterized. Using specific peptide substrates that mimic the in vivo cleavage sites in crystallin, we have demonstrated in lens extracts the presence of proteases that may be responsible for the breakdown of lens proteins. We are now focusing on the isolation and complete characterization of these proteases. The results of these studies will enable us to develop strategies to control the crystallin degradation in vivo, which may eventually lead to the development of interventions to prevent cataract formation.

The human lens expresses acylpeptidehydrolase, a member of the unique high-molecular weight serine peptidases. We are investigating the role of this protease in lens proteolysis. We have found that cataract develops in mice that overexpress this protease. The lenses from these animals show the accumulation of specific peptides. To understand the role of these peptides in cataract formation, we are now investigating the source of these peptides and the interaction of these peptides with crystallins.

The crystallins account for approximately 95% of lens proteins. There are three types of lens crystallins: alpha, beta and gamma. Alpha crystallin is a protein aggregate with a molecular weight of about 800 kDa. It is formed by the subunits A and B, each with a molecular weight of 20 kDa. These subunits have high sequence homology to small heat shock proteins. They display chaperone-like properties. The chaperone-like properties of alpha-crystallin (and its subunits) are likely to play a significant role in preventing the protein aggregation and light scattering that are associated with clouding of the lens, thereby helping to maintain lens transparency. Studies in our laboratory are directed toward the identification of the chaperone sites in both A and B subunits of alpha-crystallin and the characterization of the chemical modifications that occur at these sites and affect the chaperone activity of alpha-crystallin. We have also chemically synthesized to peptides, called mini-alpha A-crystallin and mini-alpha B-crystallin, and have shown that these peptides possess the antiaggregation properties of molecular chaperones. The availability of mini-alpha A and mini-alpha B gives us an opportunity to investigate the structural requirements of the chaperone site as well as the conformational specificity (structure or shape) of target proteins during chaperone action. This information will help us understand the mechanisms for maintaining lens transparency and the changes that occur to make the lens become cloudy.

While the primary sequence of alpha-crystallin subunits has been known for many years, the tertiary and quaternary organization of alpha-crystallin remains unknown. The structural organization of alpha-crystallin and the interaction of A and B subunits in alpha-crystallin aggregate are other areas of study in our laboratory. We are using site-directed cysteine mutations to study the A and B subunit contact sites in alpha-crystallin. Understanding the structural organization and interaction of A and B subunits will help us learn how cataracts develop and how they can be prevented.

Selected publications

Santhoshkumar P., Murugesan R., Sharma K.K. (2009) Deletion of (54)FLRAPSWF(61) residues decreases the oligomeric size and enhances the chaperone function of alphaB-crystallin. Biochemistry 48(23): 5066-73

Murugesan R., Santhoshkumar P., Sharma K.K. (2008) Role of alphaBI5 and alphaBT162 residues in subunit interaction during oligomerization of alphaB-crystallin. Mol. Vis. 14: 1835-44

Rao G., Santhoshkumar P., Sharma K.K. (2008) Anti-chaperone betaA3/A1(102-117) peptide interacting sites in human alphaB-crystallin. Mol. Vis. 14: 666-74

Santhoshkumar P., Udupa P., Murugesan R., Sharma K.K. (2008) Significance of interactions of low molecular weight crystallin fragments in lens aging and cataract formation. J. Biol. Chem. 283(13): 8477-85

Murugesan R., Santhoshkumar P., Sharma K.K. (2007) Cataract-causing alphaAG98R mutant shows substrate-dependent chaperone activity. Mol. Vis. 13: 2301-9

A. Aziz, P. Santhoshkumar, K. Krishna Sharma, E.C. Abraham. (2007) Cleavage of the C-Terminal Serine of Human alpha A-Crystallin Produces alpha (1-172) with Increased Chaperone Activity and Oligomeric Size. Biochemistry, 46(9):2510-2519