Self-assembling behaviour of the peptide hormone PYY3-36 and its lipidated conjugates for future applications in controlling satiety levels

Obesity is a global healthissue with nations struggling to deal with itstreatmentand economic impacts. Modified peptide hormones have already been shown to be effective at improving the treatment of other chronic conditions such as lymphoma or anaemia. However, peptide hormones, especially those targeted in treating obesity, are known to suffer from short drug lifetimes and thus requiring frequent administration. For the drug to take effect over a longer period requires a gel matrix-based drug carrier or a modification to the peptide itself. This thesis looks at how to use peptide modifications to potentially improve the delivery methods and efficacy of drugs used to control satiety levels. Anovel lipopeptide, based on Peptide Tyrosine-Tyrosine (PYY3-36), was characterised by using circular dichroism spectroscopy (CD), small angle X-ray scattering, critical aggregation concentration experiments, and cryo-TEM. Studies were completed to investigate the secondary structures and the self-assembly processes over a range of physiological pHs and temperatures. While native PYY3-36failedto form gels, PYY lipopeptides formed into gels with elongated fibre-like structures with a secondary structural change from α-helical to β-sheet. Gels formed from natural peptides and water offer the potential for a highly biocompatible gel with a slow release profile. Using the same analytical techniques, the thesis goes on to investigate co-aggregation of PYY3-36with β-cyclodextrin to establish if this common low-cost drug carrier can be used as an alternative delivery method. The results showed that β-cyclodextrin did show aggregation around the twisted sheet structure of the self-assembled PYY3-36butdidnot show co-assembly or encapsulation, rendering it ineffective as a delivery mechanism. Lipidated PYY3-36was mixed with lipopolysaccharide (LPS), providing some insight into how lipidated PYY3-36 could interact with amphiphilic molecules such as bacteria endotoxins. The results show that LPS do co-assemble with lipidated PYY3-36,however, unexpectedly the lipidated PYY3-36 elongated fibre structure remains largely unchanged following the co-assembly. The implications of this observation warrant further analysis. Ultimately, the findings of this thesis could be applied to the development of drug formulations for other conditions, such as lymphoma or anaemia, which would benefit from the lipidation of large peptides that enable slow release mechanisms. Additionally, the results within this thesis add knowledge to the fundamental research of self-assembly and clarifies the effects of lipidation on larger peptides that allow gelation to occur

The effect of lipidation on the self-assembly of the gut-derived peptide hormone PYY3–36

Lipidation is a powerful strategy to improve the stability in vivo of peptide drugs. Attachment of a lipid chain to a hydrophilic peptide leads to amphiphilicity and the potential for surfactant-like self-assembly. Here, the self-assembly and conformation of three lipidated derivatives of the gastrointestinal peptide hormone PYY3–36 is examined using a comprehensive range of spectroscopic, scattering, and electron microscopy methods and compared to those of the parent PYY3–36 peptide. The peptides are lipidated at Ser(11), Arg(17), or Arg(23) in the peptide; the former is within the β-turn domain (based on the published solution NMR structure), and the latter two are both within the α-helical domain. We show that it is possible to access a remarkable diversity of nanostructures ranging from micelles to nanotapes and fibrillar hydrogels by control of assembly conditions (concentration, pH, and temperature). All of the lipopeptides self-assemble above a critical aggregation concentration (cac), determined through pyrene fluorescence probe measurements, and they all have predominantly α-helical secondary structure at their native pH. The pH and temperature dependence of the α-helical conformation were probed via circular dichroism spectroscopy experiments. Lipidation was found to provide enhanced stability against changes in temperature and pH. The self-assembled structures were investigated using small-angle X-ray scattering (SAXS) and cryogenic transmission electron microscopy (cryo-TEM). Distinct differences in nanostructure were observed for lipidated and unlipidated peptides, also depending on the position of lipidation. Remarkably, micelles containing lipopeptides with α-helical peptide conformation were observed. Gelation was observed at higher concentrations in certain pH intervals for the lipidated peptides, but not for unlipidated PYY3–36. Thus, lipidation, in addition to enhancing stability against pH and temperature variation, also provides a route to prepare PYY peptide hydrogels. These findings provide important insights into the control of PYY3–36 conformation and aggregation by lipidation, relevant to the development of future therapeutics based on this peptide hormone, for example, in treatments for obesity

Shear Alignment of Bola-Amphiphilic Arginine-Coated Peptide Nanotubes.

The bola-amphiphilic arginine-capped peptide RFL4RF self-assembles into nanotubes in aqueous solution. The nanostructure and rheology are probed by in situ simultaneous rheology/small-angle scattering experiments including rheo-SAXS, rheo-SANS, and rheo-GISANS (SAXS: small-angle X-ray scattering, SANS: small-angle neutron scattering, GISANS: grazing incidence small-angle neutron scattering). Nematic alignment of peptide nanotubes under shear is observed at sufficiently high shear rates under steady shear in either Couette or cone-and-plate geometry. The extent of alignment increases with shear rate. A shear plateau is observed in a flow curve measured in the Couette geometry, indicating the presence of shear banding above the shear rate at which significant orientation is observed (0.1-1 s-1). The orientation under shear is transient and is lost as soon as shear is stopped. GISANS shows that alignment at the surface of a cone-and-plate cell develops at sufficiently high shear rates, very similar to that observed in the bulk using the Couette geometry. A small isotope effect (comparing H2O/D2O solvents) is noted in the CD spectra indicating increased interpeptide hydrogen bonding in D2O, although this does not influence nanotube self-assembly. These results provide new insights into the controlled alignment of peptide nanotubes for future applications

The Effect of Lipidation on the Self-Assembly of the Gut-Derived Peptide Hormone PYY_3–36

Lipidation is a powerful strategy to improve the stability in vivo of peptide drugs. Attachment of a lipid chain to a hydrophilic peptide leads to amphiphilicity and the potential for surfactant-like self-assembly. Here, the self-assembly and conformation of three lipidated derivatives of the gastrointestinal peptide hormone PYY_3–36 is examined using a comprehensive range of spectroscopic, scattering, and electron microscopy methods and compared to those of the parent PYY_3–36 peptide. The peptides are lipidated at Ser(11), Arg(17), or Arg(23) in the peptide; the former is within the β-turn domain (based on the published solution NMR structure), and the latter two are both within the α-helical domain. We show that it is possible to access a remarkable diversity of nanostructures ranging from micelles to nanotapes and fibrillar hydrogels by control of assembly conditions (concentration, pH, and temperature). All of the lipopeptides self-assemble above a critical aggregation concentration (cac), determined through pyrene fluorescence probe measurements, and they all have predominantly α-helical secondary structure at their native pH. The pH and temperature dependence of the α-helical conformation were probed via circular dichroism spectroscopy experiments. Lipidation was found to provide enhanced stability against changes in temperature and pH. The self-assembled structures were investigated using small-angle X-ray scattering (SAXS) and cryogenic transmission electron microscopy (cryo-TEM). Distinct differences in nanostructure were observed for lipidated and unlipidated peptides, also depending on the position of lipidation. Remarkably, micelles containing lipopeptides with α-helical peptide conformation were observed. Gelation was observed at higher concentrations in certain pH intervals for the lipidated peptides, but not for unlipidated PYY_3–36. Thus, lipidation, in addition to enhancing stability against pH and temperature variation, also provides a route to prepare PYY peptide hydrogels. These findings provide important insights into the control of PYY_3–36 conformation and aggregation by lipidation, relevant to the development of future therapeutics based on this peptide hormone, for example, in treatments for obesity

New insights into the genetic etiology of Alzheimer’s disease and related dementias

Characterization of the genetic landscape of Alzheimer’s disease (AD) and related dementias (ADD) provides a unique opportunity for a better understanding of the associated pathophysiological processes. We performed a two-stage genome-wide association study totaling 111,326 clinically diagnosed/‘proxy’ AD cases and 677,663 controls. We found 75 risk loci, of which 42 were new at the time of analysis. Pathway enrichment analyses confirmed the involvement of amyloid/tau pathways and highlighted microglia implication. Gene prioritization in the new loci identified 31 genes that were suggestive of new genetically associated processes, including the tumor necrosis factor alpha pathway through the linear ubiquitin chain assembly complex. We also built a new genetic risk score associated with the risk of future AD/dementia or progression from mild cognitive impairment to AD/dementia. The improvement in prediction led to a 1.6- to 1.9-fold increase in AD risk from the lowest to the highest decile, in addition to effects of age and the APOE ε4 allele

New insights into the genetic etiology of Alzheimer’s disease and related dementias

Characterization of the genetic landscape of Alzheimer’s disease (AD) and related dementias (ADD) provides a unique opportunity for a better understanding of the associated pathophysiological processes. We performed a two-stage genome-wide association study totaling 111,326 clinically diagnosed/‘proxy’ AD cases and 677,663 controls. We found 75 risk loci, of which 42 were new at the time of analysis. Pathway enrichment analyses confirmed the involvement of amyloid/tau pathways and highlighted microglia implication. Gene prioritization in the new loci identified 31 genes that were suggestive of new genetically associated processes, including the tumor necrosis factor alpha pathway through the linear ubiquitin chain assembly complex. We also built a new genetic risk score associated with the risk of future AD/dementia or progression from mild cognitive impairment to AD/dementia. The improvement in prediction led to a 1.6- to 1.9-fold increase in AD risk from the lowest to the highest decile, in addition to effects of age and the APOE ε4 allele