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SHOWCASE ON RESEARCH
Copper: the Jekyll and Hyde Element
Julian F.B. Mercer1 and Jim Camakaris2
1Centre
for Cellular and Molecular Biology, School of Biological and Chemical Sciences, Deakin
University, VIC 3125 and 2Department of Genetics, University of Melbourne, VIC 3010
Copper was discovered as an essential nutrient in
animals in the 1920s and 1930s but it took several
decades after this to establish the importance of copper
for human health (1). Copper is required for the activity
of a range of cuproenzymes, and the most important
clinically are cytochrome c oxidase and lysyl oxidase.
The ability of copper to undergo facile interconversion
between the two oxidation states, Cu (I) and Cu (II), has
been utilised in a range of oxidative enzymes. This
useful property can be regarded as the Dr Jekyll side of
copper which provides functions needed for
maintaining good health.
Unfortunately Dr Jekyll is accompanied by the
dangerous Mr Hyde. If copper is not handled properly
by cells, the activity of Mr Hyde is unleashed in the
form of copper-catalysed free radicals. The most
damaging of these is the hydroxyl radical, which is
formed by copper-catalysed reaction of hydrogen
peroxide in the Fenton reaction. Hydroxyl radicals can
cause substantial damage to all cellular constituents,
including DNA, proteins and membranes. Thus the
problem for all organisms living in an oxidising
environment is how to use Dr Jekyll without releasing
Mr Hyde. This problem was solved by the development
of tightly regulated copper homeostatic mechanisms.
The identification of the genes involved in a genetic
copper deficiency (Menkes disease; Dr Jekyll is out of
action), or copper toxicity (Wilson disease; Mr Hyde has
escaped) has identified two key players in copper
homeostasis, and studies of yeast mutants have
revealed other important members of the cast of
molecules that regulate copper absorption, distribution
and excretion.
Copper Deficiency Diseases
Animal studies revealed the symptoms of copper
deficiency, which include osteoporosis, anaemia, arterial
rupture, neurological defects and, in sheep, wool
abnormalities known as 'steely wool'. Species vary
widely in their susceptibility to copper deficiency (and
toxicity) and the particular symptoms that predominate
(1). Anaemia and neutropenia are found in all cases of
severe nutritional copper deficiency of animals,
including humans. Severe copper deficiency in humans
is usually only reported in premature infants and
reflects the high demand for copper in the early period
of growth (1). The fact that copper deficiency can
induce common conditions such as osteoporosis and
anaemia, however, raises the issue of how many such
cases have chronic marginal copper deficiency as a
contributing factor.
The most severe example of copper deficiency is seen
in the genetic disorder Menkes disease. This fatal XVol 35 No 3 December 2004
linked condition is characterised by growth
retardation, neurological degeneration, connective
tissue abnormalities and peculiar hair (2). Menkes
disease was first shown to be a copper deficiency
disease by David Danks at the Children's Hospital in
Melbourne (3). David's familiarity with the symptoms
of copper deficiency in animals explains why this
seminal discovery in the copper field was made in
Australia. Allelic variants of Menkes disease are
known and one of these, occipital horn syndrome, is
primarily a connective tissue disorder.
The isolation of the Menkes gene by positional
cloning provided an insight into not only the disease
itself, but also the molecular basis of copper
homeostasis (4-6). It led directly to the cloning of the
gene affected in Wilson disease. The Menkes gene
(ATP7A) encodes a transmembrane copper P-type
ATPase, ATP7A protein, the first heavy metal P-type
ATPase to be described in mammals.
Copper Toxicity Disorders
Wilson disease is an autosomal recessive copper
toxicosis disease caused by a mutation in a Menkes
gene paralogue, ATP7B. It is primarily a disease of
copper accumulation in the liver that can lead to liver
failure, or neurological disease when copper escapes
from the liver and deposits in the central nervous
system. The Wilson protein, ATP7B, has a central role in
copper homeostasis, as it eliminates excess copper from
the body in the bile. It is the failure of this excretory
mechanism that results in the slow accumulation of
copper in hepatocytes. The excess copper causes the
death of the hepatocyte and subsequent liver failure.
Wilson disease is readily treated with copper chelators if
diagnosed before liver failure.
Another copper toxicity disorder is known as copperassociated childhood cirrhosis. This disease appears to
be an autosomal recessive condition that requires
exposure to excess copper in early childhood for the
symptoms to occur (7). The sporadic occurrence of this
disease in several countries has caused public health
authorities to review the possible risks to the wider
population of copper in drinking water, primarily
originating in copper pipes. The cases of childhood
deaths suggest that genetic subgroups may be at risk
and further work into possible polymorphic variants of
copper transporters may identify sensitive subgroups.
Neurological Diseases Involving Copper
The amyloid precursor protein (APP), which is
involved in Alzheimer's disease, may have a role in
copper homeostasis in neurons (8). Recent findings
demonstrate that mice with a knockout of the APP
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Copper: the Jekyll
and Hyde Element
SHOWCASE ON
RESEARCH
gene have increased copper accumulation in cultured
neurons (9) and that copper regulates the expression of
the APP gene (10). Amyloid plaques contain high levels
of copper and zinc; and copper bound to Aβ can directly
produce hydrogen peroxide, setting up conditions for
Fenton chemistry. Copper chelators have been found to
dissolve these plaques and to produce a significant
improvement in Alzheimer patients (11).
Prion diseases, such as Kuru and mad cow disease,
produce fatal neurodegeneration. The prion protein
contains copper-binding motifs and the toxic peptide
fragment PrP106-126 generates hydroxyl radicals in the
presence of copper, strongly suggesting a role for copper
in these diseases (12). It is vital that copper homeostasis
in the brain is understood in order to clarify the role of
copper in aetiology of neurodegenerative diseases.
Key Molecules in Copper Homeostasis
The cloning of the Menkes and Wilson proteins and
studies of yeast mutants have led to the isolation of a
range of molecules important for copper homeostasis
(summarised in Fig. 1). Copper homeostasis is achieved
by a balance between absorption and excretion, and the
rates of each process are modulated by the dietary and
tissue copper levels. Copper is absorbed from the small
intestine and is passed into the blood via the intestinal
enterocytes. The most likely transporter at the apical
surface of the cell is hCTR1, a trimeric complex that was
first identified in yeast (13). In the cytoplasm a group of
small molecules known as copper chaperones receive
the copper from hCTR1 (14). In Fig. 1 we show only
ATOX1 which delivers copper to the Menkes and
Wilson proteins. In the enterocyte, the Menkes protein
(ATP7A) transports copper across the basolateral
membrane into the circulation, and this explains the
block to copper transport at this level in patients with
Menkes disease (3). Copper uptake is regulated by the
level of dietary copper, and the mechanism possibly
involves the endocytosis of hCTR1 (see below). Copper
is distributed to tissues in the blood, bound to a variety
of molecules and small peptides and the details of this
are still being investigated. The hepatocyte plays a
pivotal role in copper homeostasis. Much of the
absorbed copper is taken up by the hepatocyte, and
excess copper is excreted into the bile by the Wilson
protein (ATP7B). This process is regulated by the
copper-induced trafficking of ATP7B to vesicles which
move to the apical surface of the hepatocyte (see next
section). The critical function of copper delivery to the
brain across the blood brain barrier requires the Menkes
protein and, in a mouse model of Menkes disease,
copper is trapped in the astrocytes and endothelial cells
that form the blood brain barrier (15). The Menkes
protein is also required for efflux of copper out of
various tissues, such as kidney proximal tubules, and
copper accumulates in the kidneys of patients with
Menkes disease. Thus mutations in ATP7A
paradoxically result in overall copper deficiency despite
the accumulation of copper in some tissues. When the
cellular efflux molecules, ATP7A and ATP7B, are
inactivated by mutations as in Menkes and Wilson
diseases, intracellular copper rises and this induces the
synthesis of the metal binding metallothioneins (MTs)
which sequester the excess copper.
Fig. 1. Some molecules involved in the physiological regulation of copper.
The trimeric protein CTR1 is primarily responsible for copper uptake in a variety of cells. The Menkes protein
ATP7A effluxes copper across the basolateral surface of the intestinal enterocyte and is also required for copper
entry into the brain. The Wilson protein ATP7B is the molecule that removes excess copper from the liver in the
bile. Copper exists as Cu (I) [ I ] inside cells and Cu (II) [ II ] in the extracellular environment.
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Copper: the Jekyll
and Hyde Element
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Trafficking of Copper Transporters:
a Central Homeostatic Mechanism
In addition to the efflux of copper across the plasma
membrane, both ATP7A and ATP7B supply copper to
secreted copper-dependent enzymes, a process that
occurs in the trans Golgi network (TGN). In low copper
conditions, the transporters are localised in the TGN.
However, as copper levels rise in the cytoplasm, the
proteins are induced to traffic out of the TGN. This
process was first described in our laboratories in the
case of the Menkes protein, which traffics to the
plasma membrane in response to copper (16).
Subsequently, ATP7B was found to traffic to large
vesicles in response to copper. This trafficking is central
to the maintenance of cellular and whole body copper
levels. The central role of excretion of excess copper in
the bile is achieved by trafficking of ATP7B to vesicles
closely associated with the apical surface of the cell and
thus the biliary canaliculus (17) (Fig. 1). The basolateral
targeting of ATP7A is consistent with its proposed role
in copper absorption at the basolateral surface of the
intestinal enterocyte (18) (Fig. 2).
Fig. 2. Trafficking of copper transporters maintains
cellular copper homeostasis.
CTR1 traffics from the plasma membrane to
endosomal-like vesicles in response to high copper,
thus limiting copper uptake. ATP7A traffics from the
trans Golgi network to the basolateral plasma
membrane when cytoplasmic copper increases, and
ATP7B traffics to subapical vesicles in high copper.
E = endosomal-like vesicles, N = nucleus, SC =
subapical compartment, TGN = trans Golgi network.
Regulation of copper uptake by CTR1 also involves
trafficking (19). As shown in Fig. 2, in low copper, the
protein is closely associated with the plasma
membrane, but after a period of time in high copper it
is found inside the cell in large vesicular structures.
Thus, in response to high copper, the transporters
display distinct trafficking responses: CTR1 is removed
from the plasma membrane region reducing uptake,
and ATP7A/B move to the plasma membrane region
to facilitate efflux of excess copper.
We have been studying the regulation of the
trafficking of ATP7A and AT7B using in vitro
mutagenesis to alter various regions of the proteins.
Vol 35 No 3 December 2004
Fig. 3. Proposed reaction cycle of the Menkes
copper-translocating P-type ATPase (ATP7A).
The enzyme has two basic E1 and E2
conformation states and forms a high energy Cudependent acyl-phosphate* intermediate. The
cycle results in translocation of Cu to the lumen
(e.g. of the TGN) or to the outside of the cell.
For example, both proteins have dileucine motifs close
to their C-termini and these motifs are required for the
return of the protein from the plasma membrane (or
vesicles) to the trans Golgi (20). The six metal binding
sites in the N-terminal region of these molecules are
also important for the trafficking process, as mutation
of this region blocks trafficking (21). The trigger for
trafficking appears to be the formation of a high energy
aspartyl-phosphate intermediate (Fig. 3) that is
characteristic of P-type ATPases. It is likely that the
formation of the acyl phosphate induces a
conformation that exposes a trafficking signal.
Interestingly, if ATP7A has a mutation in the domain
that removes the acyl phosphate (the phosphatase
domain), the protein is constitutively located on the
plasma membrane (22). It also appears likely that
kinase phosphorylation of ATP7A and ATP7B is
associated with trafficking (23). Recently we have
identified a putative PDZ target motif at the Cterminus of ATP7A which appears to be involved in
targeting of ATP7A to the basolateral membrane (18).
Functional Effect of Mutations that Cause
Menkes Disease and its Variants
Mutations of the Menkes gene result in three distinct
clinical phenotypes: classical Menkes disease, mild
Menkes disease and occipital horn syndrome. Classical
Menkes disease causes death in early childhood, mild
Menkes disease is characterised by less severe
neurological defects, and occipital horn syndrome is a
connective tissue disorder (2, 24). Classical Menkes
disease results when there is little, if any, active
ATP7A formed from the mutant gene. Mild Menkes
disease appears to result from missense mutations that
allow some residual Cu transport activity. The protein
has lost its ability to traffic in response to copper, but
as it is located in the TGN, it supplies sufficient
copper to lysyl oxidase, thus explaining why the
connective tissue defects are not pronounced. Occipital
horn syndrome is caused by splice site mutations that
allow a small amount of normal splicing and therefore
a small amount of normal ATP7A to be formed.
However, this protein is constitutively localised on the
plasma membrane and insufficient copper is supplied
to lysyl oxidase, thus explaining the pronounced
connective tissue defects (24).
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and Hyde Element
SHOWCASE ON
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Copper Homeostasis in Other Organisms −
Model Systems
Elegant studies in yeast have identified a number of
cytosolic 'Cu chaperones', which are involved in
transporting Cu to various subcellular compartments
(14). The yeast copper-translocating P-type ATPase
CCC2 functions in delivering Cu to the multicopper
ferroxidase, Fet3 (analogous to ATP7B delivering Cu to
caeruloplasmin in the case of mammals), in the Golgi
compartment.
The bacterium Enterococcus hirae has two coppertranslocating P-type ATPases, CopA, which is involved
in Cu uptake, and CopB, which is involved in Cu efflux
(25). These are regulated at the transcription level. This
is in contrast to the mammalian copper-transporting
ATPases which do not appear to be regulated at the
transcriptional level, but which exhibit Cu-regulated
trafficking as described above.
Recently the fruitfly Drosophila melanogaster has been
investigated as a model system for understanding
copper homeostasis. It provides a potentially powerful
metazoan model which is amenable to sophisticated
genetic analysis and has a well annotated genome.
Using mutants, the Ctr1B Cu transporter was
characterised as being vital for normal growth and at
particular stages of development (26). We have recently
demonstrated that cultured Drosophila cells express a
number of putative orthologues of human Cu
homeostasis genes (27). Using double stranded RNA
interference, we have characterised genes involved in
copper detoxification (27). These approaches should lead
to discovery of novel candidate genes involved in Cu
homeostasis.
The copper-regulated trafficking of the copper
transporters provide a fascinating example of a tightly
coordinated homeostatic mechanism that helps keep the
Mr Hyde of copper under control while allowing Dr
Jekyll to carry out useful functions. Further analysis of
these mechanisms and other components will lead to
clarification of the importance of copper toxicity and
deficiency in common diseases, and perhaps lead to the
identification of genetic variants of the transporters that
result in sensitivity to copper deficiency or toxicity.
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