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:
o “Human cellular prion protein (PrPc) is involved in several
neurodegenerative disorders; however, its normal function is unknown.”
(Chacon et al. 2003)
o “The exact function of the cellular prion protein (PrPc) remains
unknown.” (Lasmezas 2003)
o “Function and regulation of the intrinsic prion protein (PrPc) are largely
unknown.” (Sauer et al. 2003)
o “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)
o “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 PrPccopper 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 TNFalpha: 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.