Amyloidogenesis refers to a process of protein misfolding and aggregation that leads to the formation of highly stable amyloid fibers. Amyloidogenesis may lead to loss of physiological protein function and/or formation of toxic intermediates, which are linked to mutliple human diseases. Amyloidogenesis is inhibited by plasma proteins, which function as extracellular chaperones by binding to stressed and misfolded proteins, including amyloidogenic peptides, and preventing their aggregation. This thesis focuses on the ability of human serum albumin (HSA), the main protein in human plasma, to inhibit amyloidogenesis, with emphasis on the molecular nature of the interactions between HSA and the amyloid β peptide (Aβ) associated with Alzhemier’s disease. HSA is as a key amyloidogenic regulator, a novel function for this protein that goes beyond the traditional HSA roles as plasma osmotic pressure regulator and as binder and carrier of endogenous and exogenous low molecular weight ligands. As a first step towards understanding the detailed molecular nature of these interactions, this thesis will focus on defining the key binding determinants in the interaction between HSA and Aβ peptides. Primarily, we will try to answer two main questions. First, which HSA residues are critical for the recognition of Aβ peptides and the prevention of Aβ aggregation? Second, which Aβ residues are mostly affected by HSA binding? Starting form our knowledge about the stoichiometry and affinity of the Aβ interactions at the level of HSA domains, Chapter 2 addresses the first question through successful applications of a reductionist approach, based on a combination of mutational comparative analyses and fatty acid (FA) competition. This strategy allowed us to identify a short HSA derived peptide that specifically recognizes Aβ and prevents its aggregation. In Chapter 3, we examine the effect of HSA on the pseudo-equilibrium state between Aβ monomers and protofibrils. Using Dark state Exchange Saturation Transfer (DEST), Saturation Transfer Difference (STD) and 15N T2 relaxation experiments, we show that Aβ peptides interact with HSA via a dual mechanism. First, selected residues in Aβ (1-40) monomers bind specifically but weakly to HSA (Kd = 0.1 - 1 mM). Second, HSA competes with Aβ monomers for the binding to the protofibrils, as indicated by an HSA-dependent decrease in the direct vs. tethered probabilities for contacts between Aβ monomer residues and the protofibril surface. The effect of HSA mimics that of dilution for the majority of the Aβ (1-40) residues involved in the cross-beta strands of amyloid fibrils. Finally, Chapter 4 will outline future investigations to address currently open questions about HSA dynamics, HSA-Aβ and HSA-FA interactions, for which we acquired preliminary data. / Thesis / Master of Science (MSc)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/15954 |
| Date | 11 1900 |
| Creators | Algamal, Moustafa |
| Contributors | Melacini, Giuseppe, Chemistry and Chemical Biology |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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