Aggregation, Inhibition, Degradation: The Cystatin C-Beta-Amyloid-Cathepsin B System

University Of Wisconsin-Madison, Madison WI

Investigators

Abstract

Degenerative neurological disorders such as Alzheimer's or Parkinson's disease are caused by abnormal clumping of proteins into deposits called "amyloid". Amyloid can be prevented by enzymes that chop up the protein into nontoxic fragments, or by molecules that attach to the protein and prevent clumping. In this project, three brain proteins will be studied: (1) beta-amyloid (BA), the protein that forms deposits related to Alzheimer's disease, (2) cathepsin B, an enzyme that chops up BA, and (3) cystatin C, which can form amyloid deposits, prevents cathepsin B from degrading BA, and attaches to BA and prevents clumping. The interactions among these three proteins will be explored, providing new knowledge on protein clumping and degradation in healthy and diseased brain cells, and potentially providing some clues as to more effective treatment strategies for these diseases. Research opportunities will be provided to students from underrepresented groups, and an undergraduate engineering text will be revised to incorporate more examples related to protein folding diseases. Amyloid fibrils are protein aggregates that share physicochemical properties such as cross-beta-sheet structure and fibrillar morphology. Much research has focused on aggregation of individual amyloidogenic proteins because of their importance in neurodegenerative disorders. However, protein aggregation occurs not in isolation but in a complex biological milieu of competing interactions. The triad of cystatin C, beta-amyloid, and cathepsin B constitute a network in which protein aggregation, binding, and degradation are intertwined. Cystatin C is a constituent of cerebrospinal fluid, where it serves as an inhibitor of proteases such as cathepsin B. Cystatin C amyloid deposits are found in patients with cerebral amyloid angiopathy. Beta-amyloid is a peptide of unknown biological function that aggregates into amyloid fibrils in Alzheimer's disease. Cathepsin B degrades beta-amyloid but is inhibited by cystatin C. Furthermore, binding of cystatin C to beta-amyloid inhibits beta-amyloid fibrillogenesis. Thus, cystatin C has two opposing roles: sequestering beta-amyloid and preventing fibril formation, while inhibiting cathepsin B-mediated proteolysis of beta-amyloid. The objectives of this project are to (1) characterize the structure and formation of cystatin C dimers, oligomers, and fibrils, (2) measure the effect of cystatin C on beta-amyloid aggregation, and (3) determine the role of cystatin C- beta-amyloid interactions in regulation of cathepsin B proteolytic activity. The experimental data will be used to build a mathematical model of an "amyloid regulatory network". Identification of such a network, and exploration of its interactions, will open up new lines of inquiry in protein misfolding, aggregation, and degradation in healthy and diseased tissues.