Think You Know Pathological Protein Mutations? Think Again!
Using NMR to Reveal the Mechanism of Misfolding in The HuPrP Protein
Background
The human prion protein, also known as huPrP, is a protein that is expressed most predominantly in our nervous system. Normally, it plays an important role in the creation and function of neurons, with the highest concentration in pre-synaptic neurons, where it is essential for cell signaling. However, most people are far more familiar with the mutated version of huPrP, and the fatal diseases it can cause. Transmissible spongiform encephalopathies, or TSEs, are deadly neurodegenerative disorders caused by huPrP misfolding and its eventual aggregation within the neural tissue. Some of the more well-known transmissible spongiform encephalopathies include Kuru, Fatal Familial Insomnia, and variant Creutzfeldt-Jakob disease (and, of course, Mad Cow Disease — its bovine counterpart), which are all incredibly destructive to the brain’s functioning and presently have no cure.
While scientists have known for years that these diseases are transmitted through the consumption of infected tissue, the mechanism triggering its unfolding has remained unknown…that is until now! A technique called nuclear magnetic resonance spectroscopy (NMR), which allows scientists to observe the magnetic fields of atomic nuclei combined with computational analysis, has revealed the molecular mechanism at work when the human prion protein misfolds.
Within the cell, huPrP is a glycosylated protein bound to the plasma membrane. Glycosylated is a term that means that a carbohydrate is bound to a protein at one of its functional groups. The basic structure of this protein can be observed using NMR spectroscopy and X-ray crystallography. The C-terminus of huPrP binds to the plasma membrane though a glycosylphosphatidylinositol anchor and is composed of three α-helical segments (labeled H1, H2, and H3), and two β-strands (labeled S1 and S2) that form an antiparallel β-sheet. The opposite end of the protein, or the N-terminus, lacks an ordered structure and contains five copper binding chains of eight amino acids each.
Summary: The Misbehaving Helix
In transmissible spongiform encephalopathies, the mutations that lead to illness primarily affect the C-terminal, with one study (Minikel et al. 2016) identifying approximately 40 single point mutations associated with TSEs. These mutations can either arise sporadically or be inherited mutations of the prion gene. One mutation in a gene known as T183A seems to be the most destabilizing to the protein’s normal structure and is linked to the phenotype of early-onset dementia and spongiform encephalopathies. The mutation eliminates a hydrogen bond between the β-strand S2 and the α-helix H3.
In this research, scientists from Imperial College London and the University of Zurich measured the NMR resonances of the C-terminus of mutated huPrP and compared that to the wild type protein. The wild type huPrP spectroscopy readings were consistent with previously published data. However, when mutations to T183A were induced, instability could be visualized from immediate precipitation of the protein within the NMR tube at low concentrations, as well from lower signal intensities. For those unfamiliar with how NMR works and what these results mean, more information can be found here. Despite this instability, the mutated protein could still be analyzed by NMR, but with significant signal reduction. This signal reduction became most apparent in a loop-shaped portion of the protein connecting the β-strand S2 and the α-helix H2. The destabilization of the α-helix H2, in particular, leads to instability in more distal portions of the protein, suggesting that the mutation of T183 has downstream effects of structural destabilization.
With this new data on the structure of mutated huPrP, Monoclonal antibodies were produced that were able to target the mechanism of transition from normal to pathogenic forms. These antibodies are known as POM and suppressed transition by binding to and locking the protein in its normal structure (more specifically, preventing the α-helix H1 from detaching). One particular antibody, named POM1, completely suppressed transition of huPrP. Other notable antibodies included POM7, which prevents detachment of the helices H1 and H2 while also preventing aggregation of mutated proteins. POM 17 only stabilizes the H1 helix but is incredibly useful at suppressing aggregation.
Using NMR spectroscopy, this study confirmed exactly which portion of the human prion protein mutates to lead to transmissible spongiform encephalopathies. Targeted antibodies were created that successfully blocked the transition to the pathogenic form. Although it is far from a cure to these fatal diseases, this study outlines the mechanism of misfolding and identifies one gene, known as T183A, which is particularly vulnerable to pathogenic mutation. The information collected from this research will be valuable in the creation of future treatments to save the lives of those inflicted with these devastating illnesses.
