Abnormal Aspartyl Residues and Amyloid Formation

              ?Unneeded or damaged proteins are generally removed from tissues by the actions of a variety of specific enzymes. Those that escape these degradative systems can severely disrupt normal metabolic activity. A prime example of this is Alzheimer’s disease (AD), in which a small protein, ß-amyloid (Aß), is deposited as insoluble fibrillar deposits in the spaces between the cells of the brain which function in reasoning and memory, and around the vessels that provide blood to the brain tissues. These fibrils form dense plaques that either directly or indirectly result in the death of the surrounding cells, presumably causing the dementia associated with AD. Aß is generally thought to be an unwanted byproduct of aging brain tissues; while it may in fact play some role in the normal. functioning of the brain, this is as yet unproven. Interestingly, unaggregated AB, the precursor to the stable deposits, is observed in normal individuals as well as in those with AD. Furthermore, aged normal individuals have diffuse amyloid deposits that cause neither cell death nor dementia. Thus, the critical step in the development of AD might be the failure to remove Aß  deposits before they can form dense plaques, rather than simply the presence of the Aß itself.      What causes the greater stability of the Aß deposits found in AD brain? In the majority of AD cases, the basic composition of the Aß is at least initially the same as that produced in normal individuals. Another possibility, however, is that Aß in AD brains is modified after it is synthesized into a form having different properties than the original form. As proteins age, they can be damaged by a variety of reactive molecules that are common in the body, including oxygen and water. The products of these reactions are particularly abundant in the long lived proteins found in human red blood cells and eye lens cells, and within the brain.    Two sites in proteins that are among the most sensitive’to age- related damage by spontaneous reactions are the amino acids aspartate and asparagine. In collaboration with Dr. A. Roher at Wayne State University, we have. recently examined AB isolated from amyloid deposits in AD brains and found that two of the aspartates are extensively damaged, with only 20% of these amino acids remaining in the undamaged form. This is an important finding because it demonstrates that the structure of AB in the brain is different from that which is usually studied in the laboratory. We hypothesize that damaged aspartyl residues arising in AB may in some way contribute to the greater stability observed for the amyloid deposits in AD brains. The presence of these residues has been found in previous studies to alter the structure and function of a number of proteins and to limit the ability of certain degradative enzymes to recognize proteins and peptides containing these residues. To determine the importance of the damaged amino acids in Aß we will measure how quickly .each of the four aspartyl and asparaginyl residues in synthetic ~ are damaged under conditions resembling those found within the brain. Our results will tell us which residues might be particularly prone to these damage reactions, and why, in those afflicted with AD. Knowing these rates will also tell us whether the altered residues arise rapidly prior to aggregation, or whether they originate well after the time expected for stable deposits to already have formed. Finally, we will be able to use these rates to estimate the age of the amyloid deposits taken from AD brains. It is currently not known how long these deposits exist in the brain prior to detection of AD symptoms. We will also investigate the effect of damaged aspartyl residues on the physical properties of the AB. Using techniques that separate single Aß molecules from those in large aggregates, the rate at which damaged forms of Aß aggregate and the stability of the aggregates can be determined, as well as the ability of the damaged forms to resist degradative enzymes. Additionally, it is believed by some researchers that the binding of other proteins (apolipoprotein E, for example) and/or metal ions (zinc, iron, and aluminum) in the brain contributes to the formation of AD, perhaps by stabilizing AB deposits. We will therefore examine the ability of these components to bind damaged ~’ and their effect on the rate at which damaged aspartyl residues are generated in Aß. If the presence of damaged aspartyl residues does in fact contribute to the conversion of the diffuse amyloid deposits found in normal brains into the dense plaques found in AD brains, this will be an important finding. At this time, little is known about what causes dense plaque formation. Determining which conditions favor the damage reactions, and the time during the development of AD at which these reactions are most detrimental, may suggest a method by which dense plaque formation might be limited by inhibiting these reactions. Furthermore, if damaged aspartyl residues are preventing degradative enzymes from removing Aß in the AD brain, it might be possible to modify these enzymes so that they can degrade damaged residues, and thus be used pharmaceutically to remove deposited amyloid.