Promoting Protein Folding with N-glycosylation and Amyloid Disaggregation

• Main Author: Murray, Amber N
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-2012
• Subjects: Biochemistry; Biophysics
• ISBN: 9781267789853; 1267789859

Proteins must fold into complex three-dimensional structures to function. Considerable cellular resources are spent to facilitate protein folding. Enzymes catalyze covalent modifications that can destabilize unfolded protein conformations and stabilize folded conformations. Chaperones bind unfolded portions of proteins to prevent misfolding and aggregation. Disaggregases pull apart aggregated species such as amyloid fibrils, allowing them to engage chaperones and fold. The studies presented here focus on N-glycosylation, which can promote folding by conferring intrinsic stability and by modulating interactions with chaperones, and disaggregation, which can shuttle proteins out of aggregated conformations and toward folded conformations. The N-glycoslyation studies demonstrate that aromatic residues at the -2 position relative to a glycosylated asparagine, “enhanced aromatic sequons”, alter glycosylation efficiency by oligosaccharyltransferase (OST) and glycan elaboration. OST preferentially glycosylates enhanced aromatic sequons. Glycoform heterogeneity is decreased by enhanced aromatic sequons, which prevent glycan elaboration in the Golgi apparatus. We hypothesize that N-acetylglucosaminyltransferase-I, which is a gateway in from high-mannose to complex N-glycans, prefers non-aromatic sequons, because aromatic sequons confer greater local stability and render glycans less accessible to catalysis. Thus, enhanced aromatic sequons are tools for influencing glycosylation efficiency and glycoform homogeneity, effecting protein stability, targeting, and many other properties conveyed by N-glycans. The identification and characterization of mammalian amyloid disaggregases, which would better equip us to battle neurodegenerative amyloid diseases, is an important and elusive goal. Studies toward the identification of a mammalian amyloid disaggregase presented here highlight the difficulties inherent in working with amyloidogenic proteins. Because of exposed hydrophobic surfaces, amyloid adsorbs to containers routinely used in in vitro experiments. This adsorption is facilitated by other denatured proteins, which in effect increase hydrophobic surface area to sequester amyloid away from aqueous solution. For this reason, we misinterpreted our initial studies toward the identification of C. elegans and mammalian amyloid disaggregases, believing that a decrease in amyloid was due to amyloid disaggregation rather than sequestration. Strict accounting for all amyloidogenic protein in in vitro studies is therefore crucial, and we delineate useful techniques to this end, in hopes of preventing future misinterpretations and identifying authentic amyloid disaggregases.