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A model system for smoking-related damage to proteins

Institution: J. David Gladstone Institutes
Investigator(s): Johan Bjorkegren, M.D., Ph.D.
Award Cycle: 1999 (Cycle 8) Grant #: 8FT-0022 Award: $75,375
Subject Area: Cardiovascular Disease
Award Type: Postdoctoral Fellowship Awards

Initial Award Abstract
Cigarette smoke contains numerous chemicals that can damage proteins, both in the blood and in cells. Proteins consist of many small building blocks called amino acids. Cigarette smoke has been shown to damage many different types of amino acids, including tyrosine, methionine, histidine, lysine, tryptophan, phenylalanine, and aspartic acid. Damage to these amino acids undoubtedly affects the shape and the function of proteins and may contribute to the development of a number of different smoking-related diseases, including chronic lung diseases, heart disease, and cancer.

Smoking related damage to proteins is extremely complex because it involves damage to numerous amino acid residues by many different small molecules, and involves many different types of chemical reactions. As a result of this complexity, smoking related damage to proteins has been difficult to study. In particular, it has been difficult to sort out the relationship between smoking-related damage to proteins and the development of specific smoking-related diseases. In this project, we have taken a step back from studying cigarette smoke-related damage to proteins and instead have tried to identify a much simpler system for analyzing how such damaged proteins might affect the overall health and vitality of an organism. We have decided to investigate a well-defined form of protein damage: spontaneous damage to aspartyl residues within intracellular proteins. Two of the major sites of spontaneous damage in proteins are aspartic acid and asparagine residues. Normal cells can limit this damage by utilizing a repair pathway driven by the intracellular enzyme, protein carboxyl methyltransferase, or pcmt. Pcmt recognizes and initiates the repair of damaged amino acid residues. We are using an animal model in which the pcmt repair pathway is lacking and therefore the organism cannot repair the damage to their proteins. We now propose to use cells lacking pcmt to test the hypothesis that damage to proteins leads to cellular dysfunction and diminished cell survival. I plan to use several experimental strategies to generate viable pcmt-deficient models, so that I will be able to study the consequences of the build-up of damaged proteins in many different tissues.

Final Report
The primary objective of this fellowship was to define the consequences of the accumulation of damaged proteins in a mammalian system. My sponsor’s laboratory created mice (Pcmt1 knockout mice) that accumulated large amounts of damaged proteins in a variety of tissues. Our goal was to use this model system to understand the consequences of the damaged proteins that occur as a result of cigarette smoking.

I decided to use a new approach to understand the consequences of the accumulation of damaged proteins in the Pcmt1-deficient mice. The Pcmt1-deficient mice had a dramatic central nervous system phenotype associated with the accumulation of damaged proteins. We therefore chose to use microarray technology to gain clues regarding how the damaged proteins affected gene expression in the brain.

First, I established DNA microarray technology in our laboratory. Initially, I found that the RNA isolation for microarray experiments requires a more stringent quality control than conventional RNA isolation procedures. I also found that variability in gene-expression data posed a significant potential problem. After overcoming these initial problems, I performed eight successful microarray experiments of brains from four newborn Pcmt1 knockout mice and four littermate controls. We found a total of 15 genes and expression sequence tags (ESTs) that had highly significant changes of expression (P < 0.0002) in brains of Pcmt1 knockout mice compared to controls. Eight of these were known genes and seven were ESTs. Among these most significantly changed genes was, reassuringly, Pcmt. Among the upregulated genes were ATP-binding cassette-1 and superoxide dismutase 3. We find these two genes particularly interesting given their involvement in intracellular lipid metabolism and inflammation and in radical defense, respectively. Among the downregulated genes, we found that Pcmt1-deficient mice completely lack any expression of the neuronal acetylcholine receptor protein, alpha-7 chain gene involved in behavioral effects of nicotine. In addition to these 15 genes with significant altered expression, we also found a total of 272 genes and ESTs with altered expression but at lower statistical significance (P < 0.05, down or upregulated > 1.5–fold).

Based on our first microarray studies, we suspect that some of the identified genes are involved in the development of the central nervous system phenotype in Pcmt1 knockout mice. To further confirm these genes, we are now in the process of performing additional microarray experiments in adult, affected, Pcmt1 knockout mice and unaffected controls. I am confident that these last studies will allow us to ascertain the identity of the genes central to the process of protein accumulation in the brain and in the development of the central nervous system phenotype in Pcmt1 knockout mice.