LABORATORY OF CONFORMATIONAL DISEASES

Transmissible spongiform encephalopathies (TSEs) are deadly neuro-degenerative disorders among humans and mammals. The infectiousness is the most alarming feature of these diseases. It can be transmitted to humans from animals through the food chain and from humans by blood or organ donation or by infected medical devices. There is no cure or sufficient sensitive early detection for TSEs. The infectious agent, called prion, is believed to be a normal cell protein, the prion protein, with an unknown conformation that is different from its normal conformation. Our aim is to understand the conformational transition of the prion protein to disease-associated forms at the molecular level by combining the results of biochemical and biophysical measurements, cell-free conversion reactions, and experiments in cell and animal model systems.

The group carries out its research activity in both the Biochemistry and the Enzimology Institutes.

An ever-increasing number of diseases have been shown to originate from protein misfolding. Among them, there are more than 20 diseases [including Alzheimer's, Parkinson's, Huntington's and transmissible spongiform encephalopathies (TSEs)] that are characterized by amyloidal protein deposits and thought to share common structural features and mechanism of action. (The amyloidal deposits usually consist of fibres that contain misfolded proteins with a β-sheet conformation.) TSEs, which we chose as a model for the study of these conformational diseases, have drawn special attention due to the outbreak of a new variant of Creutzfeldt-Jakob disease (nvCJD) in the United Kingdom that seemed to be associated with eating beef from TSE-infected cattle.

TSE in humans may result from infection (nvCJD, iatrogenic CJD, kuru); can be inherited when a germ line mutation exists in the PRNP gene that encodes the prion protein [familial CJD, GerstmannStreüssler-Scheinker Syndrome (GSSS), Fatal Familial Insomnia]; and can be sporadic (sporadic CJD) when the origin of the disease is neither determined nor understood. TSEs are also observed in other mammals including sheep, goats, deer, elk, cattle, mink, cats and zoo animals.

The primary symptoms of TSEs usually include progressive dementia and ataxia, associated with spongiform degeneration of the brain and accumulation of an abnormal protease-resistant form of the prion protein (PrP^res) in the central nervous system. (The normal, cellular form of this protein is denoted as PrP^c.) After symptoms are first manifested, the progression of the disease can be dramatically rapid, leading inevitably to the death of the affected individual.

PrP^c is a mainly α-helical, protease-sensitive, soluble monomer protein with a disordered N-terminus (approximate residues 23-127) and a structured C-terminal domain (residues 128-228, Figure 1.).

Figure 1. PrP is a 208 amino-acid residue glycosylated protein, which is connected to the cell surface by a glycosyl phosphatidyl inositol (GPI) anchor. However, only 110 residues (121-231) of the PrP domain are shown here. The two glycosylation sites are Asn 181 and Asn 197, which are both shown in yellow. One disulfide bond is present in the protein (colored gold here) connecting the second and third helices via Cys179-Cys214. The protein is colored by secondary structure. Alpha helices=magenta, beta sheets=blue

By contrast, PrP^res has a very compact structure, which is high in both α and β conformations, exists as oligomers and can be dissolved only by denaturing in GdnHCl or detergents. The disease-associated protein (PrP^res) has partial protease-resistance; its N-terminal ~6 kDa fragment is digested under conditions in which the highly compact C-terminal domain remains intact. Both forms of the protein contain a single disulfide bond and two glycosylation sites. The generally accepted assertion that the difference between the cellular and disease-associated forms of the prion protein is purely conformational with respect to its disulfide bond patterns is based on our earlier experiments.

There is no cure for the disease, and early diagnosis is not available to discriminate between TSE-infected unsymptomatic and uninfected individuals. Such a sensitive, rapid, economical and non-invasive screening test is vital with respect to animals destined for the human food chain, and to humans, who may participate in tissue and blood donation programs. As with any other disease, a detailed mechanistic understanding of pathogenesis is the most effective approach for the development of sensitive predictive diagnostic and efficacious therapeutic regimens.

Our aim is to understand TSEs at molecular and cellular levels as well as at the level of the whole organism as a model for the formation of amyloid fibrils in conformational diseases.