Final Document for Webpage:
Function of PrPc
Introduction to the function of cellular prion (PrPc)
Cellular Prion Protein binds Copper
The Cellular Role of Copper
History of the research/hypotheses about the function of PrPc
The exact function of PrPc is unknown.
- Quotes:
- “Human cellular prion protein (PrPc) is involved in several neurodegenerative disorders; however, its normal function is unknown.” (Chacon et al. 2003)
- “The exact function of the cellular prion protein (PrPc) remains unknown.” (Lasmezas 2003)
- “Function and regulation of the intrinsic prion protein (PrPc) are largely unknown.” (Sauer et al. 2003)
- “Although the prion protein (PrP) is known to be the causative agent of the neurodegenerative transmissible spongiform encephalopathies, its normal cellular function remains elusive.” (Watt and Hooper 2003)
- “The prion protein (PrPC) is a copper-binding protein of unknown
function that plays an important role in the etiology of transmissible
spongiform encephalopathies.” (Herms et al. 1999)
Introduction to the function of cellular prion (PrPc)
The actual function of PrPc is unknown, but scientists have been able to gather pieces of information about a number of different aspects of prion function. A majority of this information has also come from studies done in vitro that may or may not be applicable to PrPc function in vivo (citation). The location of PrPc being primarily on the cell surface suggests that it is involved in connecting to and communicating with other cells, bringing molecules into the cell vicinity, and sending signals across the membrane (Pauly and Harris 1998). Despite the significant amount of research that has been done, and continues to go on, there has not been a definitive study or article that connects and confirms what is known about the function.
There is one key feature of the PrPc protein that is very important to its function: PrPc binds copper (Cu2+). This ability is tied to the majority of the hypotheses about what PrPc's function is, which may include functions such as acting as a superoxide dismutase, regulating levels of intracellular calcium, or performing a function at the neuronal synapse (Collinge et al. 1994). It is possible that all three of these functions (or more) could be utilized in one organism simultaneously, depending on where each specific PrPc molecule is being expressed.
Cellular prion protein binds copper
A single cellular (normal) prion protein (PrPc) is able to bind copper ions (Cu2+) in the intercellular space (outside of the cell) presumably after PrPc has been bound to the cell membrane (Brown et al. 1997). This binding ability was first discovered in 1995 (Hornshaw et al. 1995). There is an average of 5.6 copper ions bound per human prion protein (Collinge 2001).
The majority of copper binding takes place on the N-terminal end of the protein, between amino acid residues 60 and 91(Aronoff-Spencer et al. 2000, Chacon et al. 2003). This region is unstructured until it binds to copper (Miura et al 1996). Binding alters the conformation of the PrPc (Pauly and Harris 1998). (Click to learn more about the structure of PrPc.) In this N-terminal region of PrPc there are “octarepeats,” which are sections of 8 amino acids arranged in a repeated pattern. In human PrPc there are five tandem octarepeats. This section of PrPc is highly conserved across mamailan species, always in the pattern PHGGGWGQ (or GPHGGGWG) (Hornshaw et al. 1995, Brown et al 1997, Burns et al. 2003, Chacon et al. 2003, Rachidi et al. 2003). A related pattern of hexarepeats is widely conserved among bird species, although the exact amino acids are different in the avian prion sequence (Rachidi et al. 2003). The high conservation of this octarepeat pattern is very significant as evidence that the correct copper binding ability is vital to the function of PrPc, and that the function of PrPc is vital to the life of the organism.
The particular amino acid residues present in the mammalian octarepeat region that are capable of binding to copper are histidine and glycine, although it seems that histidine is the primary source of binding in PrPc (Pauly and Harris 1998, Lasmezas 2003). Additionally some studies show that side chains from multiple amino acids are needed to create the proper binding arrangement (Garnett and Viles 2003).
Researchers have produced varying results regarding the strength of the PrPc-copper bond. Some studies show that the binding affinity of the N-terminal octarepeat is Kd 10 –16 M (Collinge 2001). This is a tight binding affinity compared to previous tests, which suggests that the binding of copper is vital to the function of PrPc (Collinge 2001). Lasmezas (2003), however, describes the binding affinity as “low” and Brown et al. (1997) describe it as “medium.”
In order for copper binding to occur a high pH is required because at lower pHs the binding affinity is lowered because the possible binding sites for copper are reduced by half (Miura et al. 1999). Prpc also has the ability to change conformations at low pHs, so possibly at low pH the conformation changes and therefore PrPc does not bind to Cu (Colligne 2001, Waggoner 2000, Jackson et al. 1999).
The issue of copper binding and its significance on the function of PrPc has been greatly debated due to different research producing conflicting results. There is a summary of the history of those different hypotheses below.
The cellular role of copper
Copper has many functions within the cell. Copper is so frequently used because it easily accepts and donates electrons (Eide 1998). Like most other metals in the body copper is used primarily as a cofactor to facilitate enzyme function. A common use of copper in particular is to act as a cofactor to enzymes that move iron in and out of cells (citation). Also, copper, along with zinc, is commonly used as a cofactor to various superoxide dismutases, which stop the damage of free radicals (citation). When copper is inside the body it is almost always bound because free copper can be highly toxic (Radisky and Kaplan 1999).
History of the research/hypotheses about the function of PrPc
When the function of PrPc was first under speculation there were many different hypotheses that arose. Since then, a number of these ideas have been disproved, but the evidence for and against many of the remaining ideas is not strong enough to single out one “most likely” function over the others.
One hypothesis was that PrPc’s entire function was to reduce Cu2+ to Cu1+, which facilitates endocytosis (citation). Cu2+ is the form of copper that is most commonly found in the body, but this form is very toxic to the cells, and cannot be easily transported across the cell membrane. Soandso suggested that PrPc was able to either connect with free copper in the intercellular space or receive copper from an intercellular copper transportation protein and the reduce it from Cu2+ to Cu1+. This reduced form of copper can then be moved into the cell by a transmembrane transport protein that can only interact with the reduced copper. This was disproved or ignored, why?
In the early 1990s it was proposed that the function was to act as a signal and start a certain pathway, “identical to acetylcholine receptor-inducing activity” Bueler et al, nature 356, 577 (1992)
Pauly and Harris (1998) suggested that PrPc takes extracellular copper and deposits it in an endocytic compartment where the copper can then be used for other things in the cell. After making this deposition the PrPc protein can go back to the cell surface.
Another, and more widely accepted hypothesis was that the primary function of PrPc was to bind to copper and bring it into the cell (endocytosis). Once safely in the cell the copper could be directed to a specific location where the cell would put it to use (Pauly and Harris 1998). Some current studies have shown that although PrPc binds copper, copper is usually not endocytosed by PrPc when it is present at physiological levels (Waggoner et al. 2000). Other studies, however, advocate that copper endocytosis is the primary function of PrPc (Garnett and Viles 2003).
PrPc is most likely not simply a metallochaperone transporting copper into the cell, but copper is simply a cofactor for the function of PrPc (Radisky and Kaplan 1999, Waggoner et al. 2000).
Current hypotheses
Three of the current hypotheses are that PrPc may act as a superoxide dismutase, it may act as a sensor for copper, signaling an antioxidant pathway, it may play a central role in the regulation of intracellular calcium, or it may perform some as of yet unknown function at the neuronal synapse.
Cellular prion protein functions as a superoxide dismutase (Megan)
There have been many hypotheses about what exactly the copper binding ability has to do with the function of PrPc under the assumption that PrPc function is not limited to transporting the copper into the cell. One theory is that PrPc appears to function as a superoxide dismutase. This function is dependent on copper binding (Brown et al 1999 in Collinge 2001). When the section of the protein that binds the copper ions is removed, then the SOD activity ceases. (Brown et al. 1999 abstract online). Many other proteins with SOD function bind to a metal, specifically Cu or Zn. A popular hypothesis of function right now is that PrPc acts as a sensor for extracellular copper, and can initiate an antioxidative sequence. Studies show that the function of PrPc is to protect the cell from oxidative damage, specifically by acting as a suerpoxide dismutase (citation). Copper is believed to act as a cofactor of this activity. This theory has been supported by a study by Brown et al. (1999) who showed that when the section of the PrPc protein that forms the octarepeats is removed from the protein, there is no superoxide dismutase activity. Additionally many studies have shown that most superoxide dismutases function with a metal ion (Citation).
Cellular prion affects cellular calcium levels
Prion proteins have also been shown to have connections to the amount of calcium in CNS cells. Herms et al. (2001) suggest that prion proteins directly affect the homeostatic levels of calcium in the brain, which disrupts the function of Ca2+ activated K+currents. There is no evidence, however, that these channels are directly affected by PrPc (Herms et al. 2000). The direct connection between prion, copper, and calcium is unknown.
Cellular prion affects the function of the neuronal synapse
Another hypothesis is that PrPc directly controls the amount of copper that is present at neuronal synapses, which might happen for a variety of reasons. When the PrPc function is hindered the amount of copper at the synapses is affected, which in turn affects the function of those synapses. PrPc is likely involved with maintainence of the homeostatic levels of copper, specifically near the pre-synaptic membrane (Vassallo and Kerms 2003). The function of Cu might have to do with synapse firing (citation). A second function at the synapse could be to regulate the levels of Ca around the synapse (citation). PrPc has been shown to regulate Ca levels (is this true?) and Ca is required for synapse firing, so it has been inferred that PrPc affects synapses by regulating Ca levels.
(Kretzschamr et al 2000 in Lasmezas 2003).
Tobler et al 1996 (written notes)
Prion affects the circadian rhythm
According to Tobler et al. (1996) and Huber et al. (2002) there is evidence that PrPc is related circadian rhythm and sleep regulation in mice. PrnP knockout mice had significantly altered sleep patterns compared to wild-type mice (Tobler et al 1996). In humans the major evidence for this relationship is the disease Fatal Familial Insomnia (FFI), which is a hereditary prion disease caused by a mutations at codon 178 and codon 129 (Lugaresi et al. 1986, Reder et al. 1995, Montagna et al. 1998). Additionally, at least one case of FFI also had a deletion of one octarepeat segment (which would reduce copper binding ability) (Reder et al. 1995). The major symptom of FFI is an irregular wake-sleep cycle (Lugaresi et al. 1986, Portaluppif et al. 1994). Multiple studies show that patients with FFI have hypometabolism and significant degeneration of the thalamus and cerebral cortex (Montagna et al. 1998, Montagna et al. 2003). The areas that are damaged also show the presence of PrPsc particles (Reder et al. 1995).
One hypothesis about the connection between PrPc function and circadian rhythm is that in cases of FFI the conversion of PrPc to PrPsc causes neurodegeneration in the thalamus via as of yet unknown mechanisms. This damage to the thalamus causes impairment of the endocrine pathways, such as the secretion of melatonin, which is one hormone that has shown reduced secretion in patients with FFI. The reduction of melatonin is what actually causes the sleep changes that are common to FFI (Portaluppif et al. 1994). True to many of the other hypotheses about PrPc function, this theory also has evidence against it. Reder et al. (1995) showed that typically circadian related hormones still functioned despite the presence of FFI.
One particularly interesting aspect of the debate about PrPc function and circadian rhythm is that a number of the experiments have provided evidence to the hypothesis that the thalamus is important for sleep regulation (Montagna et al. 2003). The physiological basis for sleep is in itself a topic of great debate, so the information that can be gleaned from PrPc studies may provide information that is helpful to getting a deeper understanding of sleep and circadian rhythms.
Cellular prion protein represses Doppel
The PRNP gene is located just upstream from its homolog, PRND. The PRND gene codes for the protein doppel, which is expressed primarily in the testes. When Dpl is expressed in the central nervous system, as is the case in some types of knockout mice, it causes severe neurodegeneration.
Why is it important to understand the normal function of prions?
For many years it was assumed that the damage caused by prion diseases was the direct result of the conformational changes undergone by PrPc and the aggregation of amyloid plaques of PrPsc in the brain. Studies have shown, however, that a more likely cause of the pathology of prion diseases is the lack of function of the PrPc protein than by the creation of PrPsc. (Tobler et al. 1986, Collinge et al. 1994)
Whether the loss is of an SOD function, detrimental changes in Ca homeostasis, or interrupted synaptic firing, understanding the function of PrPc will be a significant clue to understanding, preventing, and treating prion diseases. There is likely some effect on the brain by the amyloid plaques, but at this point that effect seems minimal, or maybe the loss of PrPc has such a fast effect on the brain that there is not enough time to observe the detrimental effects of PrPsc aggregation.
The core to this issue of PrPc function and copper binding is that when PrPc becomes PrPsc (changes conformation) it loses its ability to bind to copper. This would mean the loss of all of the functions of PrPc that are related to copper binding.
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Works Cited
Rachidi, W., A. Mange, A. Senatr, P. Guiraud, J. Riondel, M. Benboubetra, A. Favier, S. Lehmann. 2003. Prion infection impairs copper binding of cultured cells. Journal of Biological Chemistry 278:14595-14598.
Radisky, D., J. Kaplan. 1999. Regulation of transition metal transport across the yeast plasma membrane. Journal of Biological Chemistry 274: 4481.
Reder, A. T., A. S. Mednick, P. Brown, J. P. Spire, E Vancauter, R. L. Wollmann, L. Cerenakova, L. G. Goldfab, A. Garay, F. Ovsiew, D. C. Gajdusek, R. P. Roos. 1995. Clinical and genetic-studies of fatal familial insomnia. Neurology 45:1068-1075.
Sauer, H., K. Wefer, V. Vetrugno, M. Pocchiari, C. Gissel, A. Sachinidis, J. Hescheler, M. Wartenberg. 2003. Regulation of intrinsic prion protein by growth factors and TNF-alpha: the role of intracellular reactive oxygen species. Free Radical Biology and Medicine 35:586-94.
Tobler, I., S. Gaus, T. Deboer, P. Achermann, M. Fischer, T. Ruelicke, M. Moser, B. Oesch, P. McBride, J. Manson. 1996. Altered circadian activity rhythms and sleep in mice devoid of prion protein. Nature 380:639-642.
Waggoner, David J., Bettina Drisaldi, Thomas Bartnikas, Ruby Leah Casareno, Joseph Prohaska, Johnathan Gitlin, David Harris. 2000. Brain copper content and cuproenzyme activity do not vary with Prion protein expression level. Journal of Biological Chemistry 275:7455-7458.
Watt NT, N. M. Hooper. 2003. The prion protein and neuronal zinc homeostasis. Trends in Biochemical Sciences 28:406.