1. Structure and Function
UNSW ANATOMY 2004
Prion Proteins
Andy Clare, Brian S. Borbe, David V. Le, Joel A. Jukes, Mitchell Clare.
ANAT3231 – Cell Biology
Introduction
Memory and Prions 4 1
Most of the kits exploit the protease resistance of the PrPsc protein in brain samples to distinguish between the PrPc and PrPsc molecules. The most common proteinase used is called proteinase K, which will break down all proteins present in the sample, except the resistant PrPsc form. In combination with a proteinase, an antibody is then use to detect the presence of the protease resistant PrPsc by binding to it. After this various techniques are used to display the results. For example running a gel to separate molecules, or using fluorecent markers.
Future Research
The need for the development of techniques that can be used to diagnose living organisms is very important and as such extensive research has begun to both help develop a better understanding, and to help improve diagnostic and treatments.
A promising area is the use of peripheral tissues as samples. Two of these are blood and urine. Techniques testing blood have shown to have tremendous sensitivity and are promising since only small samples are needed. Using urine for detection of the prion protein firstly depends on the protein being small enough to be filtered through the kidneys. Then an antibody is used to quantify the presence of the prion. So far this technique has detected infected hamsters within 60 days of being infected
The word Prions (pronounced Pree-Ons) is short for proteinaceous infectious particles and has been found to be small mutated protein molecules related to infectious diseases like that of mad cow and scrapies in animals including kuru and Creutzfeldt-Jakob disease in humans.
The term prions itself was first coined by Stanley B. Prusiner an American scientist whom for two decades struggled to convince his peers that certain proteins were possibly responsible for various unusual brain diseases, which in medical terms is called spongiform encephalopathies. Prusiner was later in 1997 presented with the Nobel prize in medicine.
Prion proteins are as stated in their name protein molecules and as such have no genetic information. Instead the proteins are made up of several amino acids, which due to a defect subsequently direct the prion protein to fold in a dysfunctional manner. This abnormal folding occurs when the normal PrPc protein mutates to form an abnormal PrPsc protein.
It is speculated that prion proteins are involved in memory formation. A protein within neurons called cytoplasmic polyadenylation element binding protein (CPEB) has prion like properties and is linked to “long term synaptic changes”(1) that occur with memory storage.
CPEB is a protein that binds to the 3’ tail of mRNA that codes for structural proteins to “promote polyadenylation and translation”(2). MRNA that undergoes poly(A) elongation is stabilized and translated, whereas those with shortening are deactivated.
Figure: Illustration of neurons.
When synapses are stimulated, CPEB is transformed to self perpetuate, causing other CPEB proteins to assimilate and stabilize the mRNA involved, a higher level of structural proteins is produced and the integrity of the synapse is maintained, forming memory.
This proposed process is a highly efficient form of memory storage, because the protein is induced by a neurotransmitter, then “once the protein achieves its prion like state it is self perpetuating and no longer requires for maintenance continued signaling either by kinases or phosphatases”(3).
Prion Diseases 5
Prion related diseases are often classified as transmissible spongiform encephalopathic disease, or TSE, and are found in both humans and mammals. The most well known TSE disease is Bovine Spongiform Encephalopathy, commonly known as Mad Cow Disease. Several forms of TSE that manifest themselves in
humans are Creutzfeldt-Jakob disease (CJD), fatal familial
insomnia, Gerstmann Straussler Scheinker syndrome, and
variant Creutzfeldt-Jakob disease (vCJD).
It is believed that vCJD, the human manifestation of bovine
spongiform encephalopathy, is the direct result of ingesting
infected bovine tissues. As a result of this ingestion of
infected tissue, there begins to be a build up of abnormal prion
proteins however, unlike the other human TSE, where the
abnormal prion protein is largely confined to the central nerv-
ous system (CNS), vCJD illustrates a widespread distribution
of the abnormal protein in peripheral lymphoid tissue. In
addition to the CNS, the abnormal protein is detectable in the
spleen, lymph nodes, tonsils, optic nerve and retina of vCJD
cases[1]. Currently, diagnosis during life is confined to the
visible symptoms, and characteristic appearances on magnetic
resonance imaging (MRI) of the brain, while also
excluding other causes[2].
Compared to other infectious diseases, TSEs have very
long incubation periods hence their description as 'slow viruses and the incubation period of vCJD could be anything from 1 year to more than 30 years’[4].
Current prion research is yet to discover a key in preventing TSE or even curing TSE. Hopefully it will not be too long before we see this trend change. As long as research continues, it may be possible to detect prion diseases more rapidly and possibly slow down the onset of the various diseases even further.
Prions : How they work 2
Each and every protein when activated folds in a specific way, prion proteins are no different.
When an abnormal PrPsc protein folds, it folds in an unusual way which adversely affects the cell (neuronal cells). The PrPsc protein then
attaches to other healthy PrPc proteins, building a template
that changes the normal conformation of the healthy prion
protein to that of the disease causing proteins. This continual
cycle occurs until the neuronal Cell dies and disperses the
infectious prion protein to Surrounding brain cells, which
ultimately leads to brain damage.
Fig1.The cortex of sporadic CJD showing Spongiform changes disrupting architecture
Prion Cycle
The normal prion protein (PrPc) found in the secretory pathway of protein expressing cells. (1) The prion moves to the plasma membrane where it binds to an extracellular ligand (3) until it is cycled from the membrane into endocytic vesicles(4).
At its destination it unloads its cargo and the protein either passes to the lysozome for degradation or back to surface for another round of ligand binding. Its activities resemble the typical membrane-resident proteins, however the pathogenic form (PrPSc) also finds its way to endocytic vesicles where it co-opts some of the normal prions to become pathogenic (5).
The Abnormal PrPSc form is quite resistant to degradation therefore accumulates causing neurotoxicity. Jones, I. (2002)
Fig2. An example of plaque found in cerebral cortex of vCJD 3
Structure and Function
As with all proteins prion structure is directly related to its function. The normal protein (PrP) gene product PrPc is kd and is protease sensitive and soluble in nondenaturing detergents (Rosenberg et al; 1998). The prion protein is 253 amino acids long in humans. PrPsc is the infectious agent that causes disease and is a cellular isoform of PrPc. PrPc is made up of 45% alpha
helix and has no beta sheet.
The change into PrPsc occurs via the intermediate
complex PrPc-PrPsc. PrPsc then is made up of 30%
alpha helix and 45% beta sheet. There are however,
many different strains of PrPsc with each having similar
tertiary structure but unique sections of base pairing.
So far we have not been able to deduce the function of
PrP and only know the effects of PrPsc not the
mechanism of action.
Prion Diagnosis
Diagnosis of the prion protein has proven quite difficult with conventional methods due to the unique nature of the disease. Being protein, it contains no nucleic acids and is chemically indistinguishable from the normal cellular form. Thus the pathological form is not detected within the body as being foreign, so inflammatory or immunological are not presented by the host.
The only definite form of diagnosing Transmissible Spongiform Encephalopies (TSE’s) has been via post mortem analysis. This is achieved by histopathological, immunohistochemical and neuropathological means. These tests show prominent features such as spongiform changes (vacuoation), astrocyte gliosis and amyloid plaques.
Currently five kits are available for the testing of the protease resistant PrPsc prion protein. These kits all test samples of brain, brainstem or cervical brain stem tissue. As mentioned before these samples are obtained from deceased animals.
Reference
Introduction & How they work
[1]Hunt, M. Slow virus diseases of the nervous system. April [online] [Article]
[2]Heaphy, S. Prion Diseases [online] [Article]
[3]Image from: Medical Images [online]
Memory and Prions
[1]Darnell RB. Memory, synaptic translation, and...prions?[comment]. [Comment. News] Cell. 115(7):767-8, 2003 Dec 26.
[2]Thomas B. Brody, 1996, CPEB binding RNA and Polyadenylation, [online], available: [2004, May]
[3]Si K. Lindquist S. Kandel ER. A neuronal isoform of the aplysia CPEB has prion-like properties.[see comment]. [Journal Article] Cell. 115(7):879-91, 2003 Dec 26.
Image: Transparencies to Educate, 2002, ‘Science and Research on Visual Learning’, [online], available: [2004, May]
Structure and Function
[1]Rosenberg, R.N., Prusiner, S.B., DiMauro, S. and Barchi, R.L The Molecular and Genetic Basis of Neurological Disease 2nd Edition. Butterworth and Heinemann. Boston.
[2]Baker, H.F Molecular Pathology of the Prion. Humana Press. New Jersey.
Image from: Prion Structure [online]
Prion cycle Image.
Image from: Jones,I. Prions show their metal The. Royal Society of Chemistry 2004.[Online] [Article]
Prion Diseases
Image Fig1 & Fig2: Pathology of Creutzfeldt-Jakob disease. [online]
[1] Horby, P., Variant Creutzfeldt-Jakob disease: An unfolding epidemic of misfolded proteins, Journal of Paediatrics & Child Health. Vol 38(6) Dec 2002,
[2],[3],[4],[5]. Ibid
[1]PrPc structure consists of alpha helices [2]Abnormal PrPsc structure and consists of beta sheets.