Proteins--Chemical modification

The central premise of this dissertation is that mitochondrial antioxidant enzymes
are essential to lens cell viability by preserving lens cell mitochondria and protecting
and/or repairing lens cell proteins, and two mitochondrial-specific antioxidant enzymes,
Peroxiredoxin 3 (PRDX3) and Methionine sulfoxide reductase A (MsrA), are explored.
In this dissertation, we will examine the expression ofPRDX3 in the human lens, its colocalization
to the lens cell mitochondria, its ability to be induced by H20 2-oxidative
stress, and speculate how PRDX3 function/sf could affect the lens. We will also examine
the reduced levels of MsrA by targeted gene silencing and its effect on reactive oxygen
species production and mitochondrial membrane potential in human lens cells to
determine its role in mitochondrial function in the lens. Lastly, we will examine the
ability of MsrA to repair and restore function to a critical mitochondrial protein,
Cytochrome c. The collective evidence strongly indicates that the loss of mitochondrial-specific enzymes, such as PRDX3 and MsrA, are responsible for increased reactive
oxygen species levels, decreased mitochondrial membrane potential, protein aggregation
and lens cell death, and further indicates that mitochondrial repair, protective, and
reducing systems play key roles in the progression of age-related cataract and other agerelated
diseases.

Oxidative damage is an inevitable consequence of aerobic respiration. Methionine
sulfoxide reductases (Msr) are a group of enzymes that function to repair oxidized
methionine residues in both free methionine and methionine in proteins. MsrA was the
first of these enzymes to be discovered and is the most thoroughly studied. It is thought to
play a role in both the aging process and probably several neurodegenerative diseases. I
recently obtained a strain of Drosophila that was reported to have a P-element transposon
located within Exon 2 (part of the open reading frame) of the eip71cd gene, which is the
Drosophila homolog of MsrA. Thus, the transposon insertion should disrupt expression
of the msrA gene. I did a series of experiments to "jump out" the P-element in an effort
to recover two types of isogenic strains. The first would be a null mutation of the MsrA
gene created by deletion of flanking genomic DNA when the P-element excised from the
chromosome. The second would be a precise excision of the P-element, which would
restore the genetic locus to its original structure. This study looks at the effect of a null
mutant of the MsrA gene on aging and resistance to oxidative stress.

One methionine sulfoxide reductase A (TMSRA) and two methionine sulfoxide reductase
Bs (TMSRB 1 and TMSRB2) were isolated from tobacco plants. TMSRA showed
specificity for the reduction of Met-(S)-SO and both TMSRBs were specific for the
reduction of Met-(R)-SO. TMSRA was the cytosolic form and both TMSRBs were
plastid forms based on sequence comparison and expression tests. TMSRA and TMSRB2
could use either thioredoxin (TRX) or dithiothreitol (DTT) as reducing system, while
TMSRB 1 showed little activity with TRX but much more activity with DTT, which was
similar to the mitochondrial MSRB2 from mammals. Ferredoxin (FD) is not the reducing
system for Msrs, but might reflect the redox status in the cell and control the activity of
Msrs indirectly.