Isoform-Dependent Differences in Apolipoprotein E Mercury Binding

نویسنده

  • Erinn Jochimsen
چکیده

The relationship between apolipoprotein E4 (Apo E4) and the occurrence of Alzheimer’s Disease is well known; however, the biochemical mechanism behind this association is not well characterized. This research project examines the potential differences in mercury binding between free Apo E2 and Apo E4 isoforms. An equilibrium dialysis chamber is used to monitor the movement and binding of mercury through a membrane. Samples are then run through a cold vapor atomic absorption spectrometry (CVAAS) to measure the amounts of metal bound to each of the Apo E isoforms. The detection limit of the CVAAS technique is 3.3 ppt Hg. Results of these experiments showed that the Apo E2 isoform bound a statistically greater amount of mercury metal than did the Apo E4 isoform. Introduction Alzheimer’s disease (AD) is affecting an increasing number of people each year. The cost of the disease is both emotional and financial, estimating over 70 billion dollars spent per year. Though the exact cause is unknown, many believe that Alzheimer’s disease is the death of cells in the cerebral cortex caused by senile plaques (SP) and nerofibrilary tangles (NT). SP are lesions surrounded by protein fragments that accumulate between nerve cells in the brain to form hard, insoluble barriers (See Fig 1). In a normal brain, the protein fragments are degraded and removed. NT, on the other hand, are found inside the cells (See Fig 2). In Alzheimer’s patients, NT causes the tau protein to function abnormally or collapse by blocking transport signals. Apolipoprotein E Apolipoprotein E (Apo E) is a protein that is not attached to any lipids. Apo E has three isoforms, which are proteins with similar functions: Apo E2, Apo E3, and Apo E4. Previous studies have shown that individuals homozygous for Apo E4 are at a greater risk for AD than those homozygous for Apo E2 (Meyers and Goate, 2001). The structural differences between the three isoforms are found on the amino acids of 112 and 158. Apo E2 has a cysteine on both, while Apo E3 has an arginine on 112 and a cysteine of 158, and Apo E4 has an arginine on both amino acids. The difference between cysteine and arginine is that cysteine incorporates a sulfur atom, and heavy metals such as Hg have a high affinity for sulfur binding. Apo E is an important protein needed for both the regulation and maintenance of cholesterol and other lipids, besides being a ligand for lipoproteins like LDL and chylomicons. The ability to transport is important because Apo E is able to take the harmful lipoproteins to the appropriate disposal areas of the body. Recent research suggests that cholesterol transport may be involved in AD. Metals in AD It is believed that metals (zinc, iron, etc.) may play a role in the effects of binding or formation of plaques. When copper and zinc are present, Beta amyloid has increased oxidative activities. When zinc and copper were located with Apo E, the metal aggregation of Beta Amyloid increased, suggesting a 1 to 1 ratio. The increase in metal aggregation means that more plaques are formed. Though extremely toxic, mercury has also been found at elevated levels in Alzheimer’s disease patients. A report by Thompson et al (1988) found statistically significant mercury elevations in the limbic systems of AD patients. It was suggested that amalgam from dental fillings and other dental work released mercury vapors into the olfactory during the process of chewing and normal wear and tear (Stortbecker, 1986). The Hg was then inhaled into the limbic system. Recent unpublished research suggests that AD patients have higher levels of Hg in their systems than non-AD affected persons (Cornett, 2004). Hypothesis The presence of cysteine residues at 112 and 158 in the Apo E2 compared to arginine residues at those sides in Apo E4 will lead to a higher heavy metal binding capacity with the Apo E2 isoform. Experiment Materials and Instrumentation Cold Vapor Atomic Absorption System (CVAAS) – model number 01000073-1 Stannous chloride (SnCl2) – used as a reductant Nitric acid 2% used as a cleansing rinse 2% HCl – rinse solution during procedure 10,000 ppb Hg in 5 % HNO3 – to prepare 0.2 – 50 ppb Hg standards in 2 % HCl for instrument calibration 0.7 M (NH4)2CO3 – used as buffer during equilibrium dialysis Semi-permeable membranes – used in dialysis chamber Apo E 2 and Apo E 4 isoforms Methods This research project uses an equilibrium dialysis chamber, was used to monitor the movement of the metal across a membrane. Equilibrium dialysis consists of two chambers separated by a semi-permeable membrane. The mercury is put on one side, and the protein is put into the other (See Fig 3). The Hg side required the addition of an approximately 20ppb Hg solution. The semi permeable membrane was place in the middle of the dialysis chamber, and then 20 μl of protein solution and 230 μl of buffer was added to the other side. The chamber was then placed in a shaker for a specific amount of time. Results The samples were analyzed using a cold vapor atomic absorption spectrometry (CVAAS) to measure the amounts of metal bound to each of the Apo E isoforms (See Fig 4). The calibration curve of the CVAAS has an R value = 1, meaning good accuracy and precision of the instrument (See Fig 5). Equilibrium dialysis experiments to date resulted in free metal cell Hg concentrations of 60 + 11 ppt for the Apo E2 (n = 8) and 210 + 20 ppt for the Apo E4 (n = 5) isoforms. The ANOVA comparison demonstrated that this difference in Hg binding was statistically significant (p < 0.05). Conclusion The major success of this experiment was adopting Hg protocols and the new CVAAS to this analysis. Also, the data lead to the acceptance of the experimental hypothesis that the cysteine-rich Apo E2 isoform binds more Hg than Apo E4. Future experiments will alter equilibrium dialysis conditions to better examine the metal binding with the protein. Additional experiments will examine the applicability of using intrinsic fluorescence to monitor the actual protein conformation changes that occur as the metal binds to the Apo E.

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تاریخ انتشار 2004