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A4. Oxidative Modification of Proteins

Oxidative Modification of Proteins:

Figure:  Oxidative Modification of Proteins

Oxidized levels of proteins (as evidenced by increased levels of aldehydes) increase dramatically with age (especially after age 40 ). The reactions seem to be catalyzed by metals and may proceed by generation of hydroxy free radicals.  Diseases associated with premature aging (Werner's Syndrome, another link to Werner's Syndrome, Progeria) show very high levels of oxidized proteins at an early age. Fibroblasts from 10 yr. old children with progeria have levels of oxidized proteins usually not seen until the age of 70. Beta amyloid protein deposits (found in Alzheimer's and Down's Syndrome) cause neurotoxicity and death, partly by increasing superoxide production by endothelial cells, causing vasoconstriction/dilation, and ultimately disease progression. Beta amyloid aggreagates appear to increase H2O2 levels, in a process facilitated by Fe2+ and Cu+. Free radical scavengers (antioxidants) help to prevent this damage. Recent studies suggest that vitamin E may delay the symptoms of Alzheimers.  

Lou Gehrigs Disease (Amyotrophic Lateral Schlerosis) is a disease of progressive motor neuron degeneration, which affects 1/100,000 people, and is 10-15% familial. Of the familial cases, about 25% have a mutation in superoxide dismutase I, a copper-zinc enzyme.  About 2-3% of ALS patients carry 1 of 60 different dominant mutations in this enzyme.  Mutations often decrease the stability of the protein which decreases Zn2+ affinity 5-50 fold.  The A4V mutation (valine at amino acid 4 substituted for Ala) has the weakest Zn affinity and causes rapid disease progression.   In the absence of Zn2+, the apoprotein somehow seems to induce cell death in neurons.  This superoxide dismutase also expresses a second activity. It also acts as a peroxidase which takes ROH + H2O2 to form an RHO (an aldehyde) plus water.   In some cases, the enzyme retains normal activity against superoxide but altered peroxidase activity.

Is your hair going white?:  Wood et al have shown that millimolar concentrations of hydrogren peroxide build up in hairs that have grayed and whitened.  This was associated with a decrease in catalase and in increases in Met oxidation (to Met-sulfoxide) in proteins, also associated with a decrease in the repair enzyme Met-sulfoxide reductase,  Met 374 in the active site of tyrosinase, an enzyme required for production of melanin in hair follicles, is also damaged, leading to lack of melanin, a pigment necessary for hair coloration and "senile hair graying".

ROS and Protein Folding

As discussed in Chapter 2D:  Protein Folding in Vitro and in Vivo,  the cytoplasm has sufficient concentrations of "β-mercaptoethanol"-like molecules (used to reduce disulfide bonds in proteins in vitro) such as glutathione (γ-Glu-Cys-Gly) and reduced thioredoxin (with an active site Cys) to prevent disulfide bond formation in cytoplasmic proteins.  Disulfide bonds in proteins are typically found in extracellular proteins, where they serve to keep multisubunit proteins together as they become diluted in the extracellular milieu.  These proteins destined for secretion are cotranslationally inserted into the endoplasmic reticulum (see below) which presents an oxidizing environment to the folding protein and where sugars are covalently attached to the folding protein and disulfide bonds are formed (see Chapter 3D:  Glycoproteins - Biosynthesis and Function).   Protein enzymes involved in disulfide bond formation contain free Cys which form mixed disulfides with their target substrate proteins.   The enzymes (thiol-disulfide oxidoreductases, protein disulfide isomerases) have a Cys-XY-Cys motif and can promote disulfide bond formation or their reduction to free sulfhydryls.  They are especially redox sensitive since their Cys side chains must cycle between and free disulfide forms. 

Reactive oxygen species (ROS) can significantly affect redox chemistry, and if present in excess can place the cell in a condition of "oxidative" stress.  ROS can indiscriminately oxidize lipids, nucleic acids, and proteins, but more specifically, they may also oxidize proteins involved in creating and maintaining the normal disulfide bond formation in proteins.  As the concentration of ROS increase, the concentration of cytoplasmic proteins with incorrect disulfides should increase. Using a two dimension PAGE system (first dimension run under nonreducing and the second reducing conditions) of neural cell proteins derived from cells exposed to normal and differing oxidative conditions (hydrogen peroxide or decreased intracellular glutathione levels, Cumming et al showed that oxidizing stress increased the levels of disulfide bonds in redox sensitive enzymes and, unexpectedly, among other cytoplasmic proteins involved in many aspects of life, effecting the activity of many cellular processes, suggesting that disulfide bond formation may have not only a structural but regulatory role.