Molecular insights into amyloid toxicity using model lipid membranes

Varun Prasath, B 2019, Molecular insights into amyloid toxicity using model lipid membranes, Doctor of Philosophy (PhD), Science, RMIT University.


Document type: Thesis
Collection: Theses

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Title Molecular insights into amyloid toxicity using model lipid membranes
Author(s) Varun Prasath, B
Year 2019
Abstract Functional amyloid-forming peptides can be found in nature, including stored in secretory granules as hormones. In contrast to toxic amyloid-forming peptides, the fibrillisation process for functional amyloids is typically "reversible", a mechanism that may be related to the lack of toxicity for these amyloids. The mechanism of fibrillisation for functional amyloids has been reported to be dependent on changes to environmental conditions such as pH, temperature and solvent conditions. Some molecules are known to act as aggregation enhancers for functional amyloids, including, metal ions, heparin and lipid bilayer while others inhibit fibrillisation.

Lipid membranes have been shown to significantly affect fibrillisation, and membrane-mediated fibrillisation has been well characterised for toxic amyloid species such as the amyloid-beta peptide involved in Alzheimer¿s disease.  Depending on the lipid composition and physicochemical properties of the membrane, lipid membranes can both promote or inhibit fibril growth. The following parameters, including a surface charge on the membrane, membrane fluidity, chain packing, and lipid solubility have been shown to drive changes in the electrostatic and hydrophobic interactions between the peptide and the membrane, and hence the rate of fibrillisation. However, essentially nothing is known about the membrane-mediated assembly of functional amyloid-forming peptides.

In this thesis, the effect of the lipid membrane on fibrillisation of five different peptide hormones (Somatostatin, Oxytocin, Vasopressin, Substance P and Deslorelin) was investigated. In general, liposomes were used as model membranes.  In Chapter 1, the effect of liposomes of different lipid composition on the fibrillisation of SST was investigated.  SST reliably self-assembles into amyloid fibrils above a critical concentration (>5% w/w). Interactions of SST with the lipid bilayer were characterised using synchrotron radiation circular dichroism and intrinsic tryptophan fluorescence. The kinetics of fibrillisation was determined using a Thioflavin T assay, while the morphology of fibrils formed was directly visualised using atomic force microscopy (AFM). Reciprocal effects of the peptide and the growing fibril on the lipid bilayer were investigated using synchrotron small-angle x-ray scattering (SAXS).
 
In Chapter 4 a similar suite of techniques was used to determine the effect of the lipid membrane on the fibrillisation of two additional cyclic peptides, the neurohypophysial hormone-like peptides, vasopressin (AVP-9) and oxytocin (OT-9).  These two nine-amino acid peptides are likely to form aggregates due to mutations and are known to form functional amyloids in secretory granules. They are part of a family of structurally and functionally related peptide hormones with a conserved intramolecular ring, allowing us to determine whether substitutions in amino acids can impact the interactions of these peptides with the membrane and subsequent fibrillisation.
 
In Chapter 5 the effect of linear vs cyclic peptides was determined using two linear peptides, substance P (SP), and Deslorelin (Des).  Substance P has been reported to form fibrils and has also been observed to self-assemble into nanotubes.  Deslorelin is an analogue of LHRH, which has also been shown to adopt nanotubes in solution.
 
Finally, in Chapter 6, the anti-amyloidogenic effect of the polyphenol resveratrol, and its derivatives, Trimethoxy stilbene (TMS) and trans-stilbene (TS) was investigated using Somatostatin. Several studies have reported the anti-amyloidogenic effect of resveratrol on toxic amyloids such as Aß and HIAPP. To our knowledge, this is the first study conducted on the effect of stilbenes on functional amyloids (somatostatin). All three stilbenes were shown to inhibit fibril formation in solution.  When doped in a bilayer, this effect changes, particularly for TS, which actually promoted fibril growth.
 
This body of work has significantly added to our understanding of the effect of the lipid bilayer on the fibrillisation of functional amyloids, about which almost nothing is known to date.  The trends observed not only assist in our fundamental understanding of how such peptide hormones are stored in vivo, but also adds to our understanding of fundamental peptide-lipid interactions, and the differences in toxicity between toxic and functional amyloids.
Degree Doctor of Philosophy (PhD)
Institution RMIT University
School, Department or Centre Science
Subjects Colloid and Surface Chemistry
Keyword(s) Functional Amyloids
Neuropeptide Hormones
Non-Toxic Fibrils
Fibril Structure
Amyloid Self-assembly
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Created: Wed, 31 Jul 2019, 15:01:17 EST by Adam Rivett
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