measuring-interactions-globular-filamentous-proteins-nuclear-magnetic

Measuring Interactions of Globular and Filamentous Proteins by Nuclear Magnetic Resonance Spectroscopy (NMR) and Microscale Thermophoresis (MST)

Filamentous proteins such as vimentin provide organization within cells by providing a structural scaffold with sites that bind proteins containing plakin ...

University of Alberta

Prion diseases are fatal neurodegenerative disorders associated with the polymerization of the cellular form of prion protein (PrP(C)) into an amyloidogenic β-sheet infectious form (PrP(Sc)). The sequence of host PrP is the major determinant of host prion disease susceptibility. In mice, the presence of allele a (Prnp(a), encoding the polymorphism Leu-108/Thr-189) or b (Prnp(b), Phe-108/Val-189) is associated with short or long incubation times, respectively, following infection with PrP(Sc). The molecular bases linking PrP sequence, infection susceptibility, and convertibility of PrP(C) into PrP(Sc) remain unclear. Here we show that recombinant PrP(a) and PrP(b) aggregate and respond to seeding differently in vitro. Our kinetic studies reveal differences during the nucleation phase of the aggregation process, where PrP(b) exhibits a longer lag phase that cannot be completely eliminated by seeding the reaction with preformed fibrils. Additionally, PrP(b) is more prone to propagate features of the seeds, as demonstrated by conformational stability and electron microscopy studies of the formed fibrils. We propose a model of polymerization to explain how the polymorphisms at positions 108 and 189 produce the phenotypes seen in vivo. This model also provides insight into phenomena such as species barrier and prion strain generation, two phenomena also influenced by the primary structure of PrP.

Mouse prion protein polymorphism Phe-108/Val-189 affects the kinetics of fibril formation and the response to seeding: evidence for a two-step nucleation polymerization mechanism.

The deposition of Aβ peptide in the brain is the key event in Alzheimer disease progression. Therefore, the prevention of Aβ self assembly into disease-associated oligomers is a logical strategy for treatment. π stacking is known to provide structural stability to many amyloids; two phenylalanine residues within the Aβ 14-23 self recognition element are in such an arrangement in many solved structures. Therefore, we targeted this structural stacking by substituting these two phenylalanine residues with their D-enantiomers. The resulting peptides were able to modulate Aβ aggregation in vitro and reduce Aβ cytotoxicity in primary neuronal cultures. Using kinetic analysis of fibril formation, electron microscopy and dynamic light scattering characterization of oligomer size distributions, we demonstrate that, in addition to altering fibril structural characteristics, these peptides can induce the formation of larger amorphous aggregates which are protective against toxic oligomers, possibly because they are able to sequester the toxic oligomers during co-incubation. Alternatively, they may alter the surface structure of the oligomers such that they can no longer interact with cells to induce toxic pathways. 

Structure-Based Peptide Design to Modulate Amyloid Beta Aggregation and Reduce Cytotoxicity.

Human amyloid β1-42 (hAβ1-42) peptides are known to self-aggregate into oligomers that contribute to the degeneration of neurons and development of Alzheimer's disease (AD) pathology. Unlike humans, rodents do not develop AD, possibly due to differences in three amino acids (R5G, Y10F and H13R) within the hydrophilic N-terminal domain of Aβ1-42. This is partly supported by evidence that hAβ1-42 is more prone to fibrillization and has a higher cellular toxicity than rodent Aβ1-42 (rAβ1-42). Mutagenesis studies, however, have shown that correlation between fibrillization potential and toxicity is not always direct. Thus, to understand better how N-terminal mutations can affect hAβ1-42 toxicity through oligomerization, we evaluated fibrillization kinetics, oligomer sizes and toxicity profiles of double mutant (human toward rodent) Aβ1-42. Additionally, we tested the mutant peptides in combination with hAβ1-42, to assess effects on hAβ1-42 aggregation/toxicity. Our results clearly show that double mutations to humanize rAβ1-42 result in a significantly reduced efficiency of fibril formation, as determined by Thioflavin-T aggregation assays and confirmed with electron micrographic studies. Interestingly, the mutants are still able to aggregate into oligomers, which are predominantly larger than those comprised of hAβ1-42. Our cell viability experiments further showed a rank order of oligomer toxicity of hAβ1-42 > rAβ1-42 ≫ mutant Aβ1-42, suggesting that toxicity can be influenced by N-terminal Aβ1-42 mutations via reduction of fibril formation and/or alteration of oligomer size. These results, taken together, confirm that N-terminal mutations can affect Aβ fibril and oligomer formation with reduced toxicity despite lying outside the core amyloid region of Aβ peptide.

Article	Year
Mouse prion protein polymorphism Phe-108/Val-189 affects the kinetics of fibril formation and the response to seeding: evidence for a two-step nucleation polymerization mechanism. The Journal of biological chemistry\| PubMed ID: 23283973	2013
Structure-Based Peptide Design to Modulate Amyloid Beta Aggregation and Reduce Cytotoxicity. PloS one\| PubMed ID: 26070139	2015
The Effects of N-terminal Mutations on β-amyloid Peptide Aggregation and Toxicity. Neuroscience\| PubMed ID: 29572166	2018

Jitendra Kumar

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