Year: 2014
Type: article
Abstract: The prion protein (PrP) is currently one of the most studied molecules in the neurosciences as it causes a group of neurological diseases collectively named transmissible spongiform encephalophaties (TSEs). The TSEs are characterized by a variety of motor and/or cognitive symptoms distinct from those of Parkinson and Alzheimer diseases and severely affect both humans and a variety of mammals. A great effort has thus been made to understand the molecular basis of PrP activity, both in physiological and pathological terms. In this context, the identification of neuronally-relevant interactors of PrP, capable of governing or interfering with its activity, cellular localization and/or expression, plays a crucial role. Through the expression of the proteins of interest in recombinant form in E.coli cells and the analysis of their interaction by Western blot and dot-blot, we identified two specific and neurologically relevant interactions involving the prion protein and on the one hand tyrosine hydroxylase, the enzyme that catalyzes the initial and rate-limiting reaction of the biosynthesis of catecholamines dopamine, norepinephrine and epinephrine; on the other hand the heat shock protein B8, member of the small heat shock protein family that appears to play an important role in those diseases that, like transmissible spongiform encephalopathies, are characterized by the accumulation of misfolded proteins. The association/dissociation constants of the complexes have been calculated using surface plasmon resonance and interactions were confirmed by immunohistochemistry. The data obtained show a specific and high affinity interaction (KD in the nano molar range) between the TH N-terminal regulatory domain (1-152) and the C-terminal structured domain (90-230) of PrP. The co-expression of the two proteins causes a shift in prion protein expression from a prevalent membrane-associated expression to a greater cytoplasmic localization, and also a down-regulation of the levels of expression of the prion protein without, however, affecting the topology and expression of tyrosine hydroxylase. The link between PrP and HSPB8 involves the C-terminal domain of the prion protein and the co-expression of the two proteins leads to a down-regulation of both prion protein and HSPB8. The latter, moreover, protects the prion protein from degradation by Proteinase K, while it seems to accelerate the thermal denaturation. The mutants K141E and K141N of the heat shock protein, associated with the development of neuropathy, show minor binding affinity with respect to the wild type protein and they are less effective in regulating the levels of expression, localization and degradation of the prion protein. Our results, in particular if confirmed in pathological patterns, can help to understand the physiological and pathological mechanisms of action of the prion protein and suggest tyrosine hydroxylase and heat shock protein B8 as prion protein modulators, thus as potential targets for therapeutic applications.
Author: Mattia Vicario
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W96326872