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Prion protein in cell culture

Date:
February 9, 2010
Source:
Norwegian School of Veterinary Science
Summary:
The fatal brain disease Creutzfeldt-Jakob in humans, BSE (bovine spongiform encephalopathy) in cattle and scrapie in sheep are so-called prion diseases, whereby one of the body's normal proteins, the prion protein PrPc misfolds into a pathogenic form: PrPSc. In spite of several years of extensive research, little is still known about what actually happens in this process.

N2a-cells showing a transient expression of PrP in sheep. The prion protein is tagged with green fluorescent protein (GFP). To the left: PrP expressed on the cell membrane. To the right: PrP expressed in the cytoplasm. The picture is taken with a Zeiss confoncal microscope (63x magnification).
Credit: Christel Morζus Olsen

The fatal brain disease Creutzfeldt-Jakob in humans, BSE (bovine spongiform encephalopathy) in cattle and scrapie in sheep are so-called prion diseases, whereby one of the body's normal proteins, the prion protein PrPc misfolds into a pathogenic form: PrPSc. In spite of several years of extensive research, little is still known about what actually happens in this process.

In spite of the fact that PrP is one of most intensely studied proteins in the human genome, its physiological function is still unknown. The pathogenic variant PrPSc arises as a result of changes in the structural folding of PrPc.

We need to know more about how PrPc is expressed and treated in cells in order to understand how the misfolding of PrPc occurs and why cells die as a result. In the course of the work connected with his PhD, Christoffer Lund has therefore carried out detailed studies of cell cultures of normal PrP in sheep. By means of green fluorescent protein (GFP) cloned into PrP, PrP in cell cultures can be studied under a microscope. In addition, genetically manipulated variants of PrP have been made in order to uncover important factors regarding the localisation of PrP in cells and the enzymatic cutting of PrP.

PrP is normally cut into fragments in the course of its cellular lifespan. Lund has studied one of these cutting processes, the α-cut. Where the PrP α-cut occurs in the cell, and to what purpose, is unknown. Through his studies, Lund has shown that PrP is cut in the same place, even when the amino acid composition at the place of cutting is changed. PrP is also cut at the same place, irrespective of whether it is joined to the outside of the cell membrane or whether it is localised in the cell cytoplasm. Lund's findings indicate that the cutting occurs at the same place in PrP, but that the cutting is caused by different mechanisms, depending on where the PrP is localised in the cell.

A phenomenon associated with PrP's localisation in cells that is still poorly understood is that in some types of cells, PrP is positioned in the cell's cytoplasm instead of on the cell membrane, where it most likely fulfils its function. A predominant theory on why proteins may be found in the cytoplasm instead of on the cell membrane is that the cell in question is in a state of stress. Furthermore, PrP has been shown to have an inefficient signal sequence compared to other proteins and may therefore be less efficient at following its natural route out onto the cell membrane, even under normal cellular conditions.

Lund's work reveals that a completely different mechanism related to the actual translation of PrP may also be the reason why a proportion of the PrP molecules end up in the cytoplasm. By studying different mutated variants of PrP, Lund has demonstrated that a cytoplasmic variant of PrP can emerge after PrP molecules have been synthetised from a downstream start codon in the PrP gene. The result of this translation is a shortened form of PrP which lacks large portions of the signal sequence and therefore ends up in the cytoplasm of the cell.

Lund has carried out a detailed characterisation of an antibody (3F4), which is frequently used in prion research. He has shown that the criteria that are necessary for the antibody to bind with PrP are different to those previously assumed necessary. This new knowledge is of great significance when it comes to the interpretation of data where 3F4 has been used. Taken as a whole, the results of this work lead to a greater understanding of fundamental processes related to PrP and new insight into the utility value of fluorescently tagged PrP for studying PrP in cell cultures. Lund's work also highlights other challenges facing studies of proteins in cell cultures.

Christoffer Lund presented his doctoral thesis on 3re December 2009 at The Norwegian School of Veterinary Science. The thesis is entitled: "Studies on fluorescently tagged prion protein in cell culture."


Story Source:

The above story is based on materials provided by Norwegian School of Veterinary Science. Note: Materials may be edited for content and length.


Cite This Page:

Norwegian School of Veterinary Science. "Prion protein in cell culture." ScienceDaily. ScienceDaily, 9 February 2010. <www.sciencedaily.com/releases/2010/02/100205115944.htm>.
Norwegian School of Veterinary Science. (2010, February 9). Prion protein in cell culture. ScienceDaily. Retrieved October 21, 2014 from www.sciencedaily.com/releases/2010/02/100205115944.htm
Norwegian School of Veterinary Science. "Prion protein in cell culture." ScienceDaily. www.sciencedaily.com/releases/2010/02/100205115944.htm (accessed October 21, 2014).

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