This proposal focuses on how mammals can mitigate the damage resulting from the inevitable accumulation of covalently altered proteins within cells. Two of the major sites of spontaneous covalent damage in proteins are aspartic acid and asparagine residues, where isomerization, racemization, and deamidation reactions can affect protein function as well as the ability of the protein to interact normally with other proteins. Evidence has been presented that cells can limit this damaged by utilizing a repair pathway driven by the intracellular enzyme, L- isoaspartate (D-aspartate) O-methyltransferase, or protein carboxyl methyltransferase(pcmt). Pcmt recognized damaged proteins containing isomerized and racemized aspartyl residues and initiates the conversion of those residues to normal L-aspartyl residues. We have been interested in asking how pcmt prevents the build-up of senescent and potentially toxic proteins, which otherwise might lead to a loss of cellular function in the aging process. To study this issue, we have generated pcmt-deficient mice. The cytosolic proteins of the knockout mice accumulate high levels of D- aspartyl and L-isopartyl residues. The most remarkable phenotypes of the knockout mice are retard growth and a seizure disorder, which results in death at an average of 42 days of age. We now propose to use the knockout mice and their cells and tissue to answer a number of questions that related to the effect of damaged proteins on the aging process. We will use a cell culture system the examine the hypothesis that the accumulation of proteins containing altered aspartyl residues causes cellular dysfunction and results in diminished cell survival. Because homozygous knockout mice die early from seizures, they cannot be used to assess long-term effect of the protein methyltransferase deficiency in various tissues. Thus, we propose to do bone-marrow transplantation experiments to determine if activation of the methyltransferase gene in hematopoietic or immune cells causes age- dependent cellular dysfunction. We will also rescue the knockout mice from the seizure disorder with a human pcmt transgene under the control of a neuron-specific promoter. Using the rescued mice, we will be able to determine if the absence of methyltransferase activity in non-neuronal tissues (such as the heart or liver) results in age-dependent pathology or cellular dysfunction. Finally, we propose to ask whether the expression of the repair methyltransferase in the extracellular compartment would limit the accumulation of spontaneous damage to extracellular proteins.
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