Biology subjects

Raymond, G. J.

Publications and source records attributed to Raymond, G. J..

3 recordsLinked to original sources

bioRxiv2022

Cryo-EM of prion strains from the same genotype of host identifies conformational determinants

Prion strains in a given type of mammalian host are distinguished by differences in clinical presentation, neuropathological lesions, survival time, and characteristics of the infecting prion protein (PrP) assemblies. Near-atomic structures of prions from two host species with different PrP sequences have been determined but comparisons of distinct prion strains of the same amino acid sequence are needed to identify purely conformational determinants of prion strain characteristics. Here we report a 3.2 [A] resolution cryogenic electron microscopy-based structure of the 22L prion strain purified from the brains of mice engineered to express only PrP lacking glycophosphatidylinositol anchors (a22L). Comparison of this near-atomic structure to our recently determined structure of the aRML strain propagated in the same inbred mouse reveals that these two mouse prion strains have distinct conformational templates for growth via incorporation of PrP molecules of the same sequence. Both a22L and aRML are assembled as stacks of PrP molecules forming parallel in-register intermolecular {beta}-sheets and intervening loops, with single monomers spanning the ordered fibril core. Each monomer shares an N-terminal steric zipper, three major {beta}-arches, and an overall V-shape, but the details of these and other conformational features differ markedly. Thus, variations in shared conformational motifs within a parallel in-register {beta}-stack fibril architecture provide a structural basis for prion strain differentiation within a single host genotype.

biophysics↗

bioRxiv2021

Structure of anchorless RML prion reveals motif variation between strains

Little is known about the structural basis of prion strains. Here we provide a high (3.0 [A]) resolution cryo-electron microscopy-based structure of brain-derived fibrils of the mouse anchorless RML scrapie strain which, like the recently determined hamster 263K strain, has a parallel in-register {beta}-sheet-based core. However, detailed comparisons reveal that variations in shared structural motifs provide a basis for prion strain determination. One-sentence summaryCryo-electron microscopy reveals a near-atomic structure of an infectious, brain-derived murine prion fibril and strain differences.

biochemistry↗

bioRxiv2019

Domain-specific quantification of prion protein in cerebrospinal fluid by targeted mass spectrometry

Therapies currently in preclinical development for prion disease seek to lower prion protein (PrP) expression in the brain. Trials of such therapies are likely to rely on quantification of PrP in cerebrospinal fluid (CSF) as a pharmacodynamic biomarker and possibly as a trial endpoint. Studies using PrP ELISA kits have reproducibly shown that CSF PrP is lowered in the symptomatic phase of disease, a potential confounder for reading out the effect of PrP-lowering drugs in symptomatic patients. To date it has been unclear whether the reduced abundance of PrP in CSF results from its incorporation into plaques, retention in intracellular compartments, downregulation as a function of the disease process, or other factors. Because misfolding or proteolytic cleavage could potentially render PrP invisible to ELISA even if its concentration were constant or increasing in disease, we sought to establish an orthogonal method for CSF PrP quantification. We developed a targeted mass spectrometry method based on multiple reaction monitoring (MRM) of nine PrP tryptic peptides quantified relative to known concentrations of isotopically labeled standards. Analytical validation experiments showed process replicate coefficients of variation below 15%, good dilution linearity and recovery, and suitable performance for both CSF and brain homogenate and across humans as well as preclinical species of interest. In N=55 CSF samples from individuals referred to prion surveillance centers with rapidly progressive dementia, all six human PrP peptides, spanning the N- and C-terminal domains of PrP, were uniformly reduced in prion disease cases compared to individuals with non-prion diagnoses. This confirms the findings from ELISA studies, demonstrating that lowered CSF PrP concentration in prion disease is a genuine result of the disease process and not merely an artifact of ELISA-based measurement. We provide a targeted mass spectrometry-based method suitable for preclinical and clinical quantification of CSF PrP as a tool for drug development.

neuroscience↗