I obtained my PhD from the department of biochemistry
at the Univeristy of Toronto while
working in the laboratory of Dr.
Bibudhendra Sarkar at the research institute of the Hospital
for Sick Children in Toronto Ontario, Canada.
The main interests in Dr. Sarkar's lab are the study of genetic disorders
of copper metabolism. Specifically, I was involved in the expression and
characterization of the copper binding domain from the Wilson disease copper
transporting P-type ATPase.
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Genetic Disorder of Copper Transport in Humans
Although trace amounts of heavy metals are needed to sustain life, a deficiency or excess of heavy metals can be detrimental. In particular, the levels of redox active metals such as copper and iron must be tightly conrolled to prevent increased free radical generation and lipid peroxidation. When the mechanisms which control metal homeostasis fail, serious disorders usually follow. The two major genetic disorders of copper metabolism in humans are Menkes and Wilson disease. Both disorders arise from the disruption of copper homeostasis but have complementary pathologies.
| Menkes Disease | Wilson Disease | |
|---|---|---|
| Laboratory findings | ||
| Defect | ||
| Treatment | ||
| Gene location |
Menkes Disease (Kinky-Hair Syndrome)
Menkes
disease (or Kinky-hair syndrome) is a genetic disorder of copper metabolism
which was first characterized by John Menkes and coworkers in 1962. Menkes
observed that the disorder seemed to be confined to males and was inherited
in a sex-linked recessive manner. Menkes also observed that patients
displayed several gross abnormalities in their hair leading to the disorder
being referred to as kinky-hair syndrome. This observation was later substantiated
by the localization of the Menkes disease gene to the X-chromosome. The
incidence of Menkes disease is approximately 1/300,000 live births. Clinically,
Menkes disease is characterized by a global deficiency of copper which
then leads to a deficiency of cupro-enyzmes such as superoxide dismutase , cytochrome-c oxidase , lysyl
oxidase , and dopamine
beta-hydroxylase. Many of these enzymes are important in early
development. The absence of a functional lysyl oxidase (which is responsible
for crosslinking collagen and elastin) in early development leads to weakened
connective tissue and many untreated Menkes patients usually die of aortic
rupture. The age of onset is very early with untreated patients usually
dying before the age of 3. Menkes disease treatments revolved around trying
to restore normal copper levels in the body by providing copper.
Although several different forms of copper were evaluated, the Cu-histidine
complex was the most successful. This compound was developed in the
laboratory of Dr.
Bibudhendra Sarkar. Using this treatment regime, several patients
have been able to survive into their early twenties.
Wilson Disease (Hepatolenticular Degeneration)
Wilson disease was first characterized by Kinnear Wilson in 1912. The frequency
of the disorder is approximately 1/100,000 but seems to be more prevalent
in Japan, China and Sardinia. Like Menkes disease, Wilson disease is a
recessive genetic disorder of copper metabolism however; unlike Menkes
disease, Wilson disease is characterized by a buildup of copper in various
tissues. Wilson disease also differs from Menkes disease by not being X-linked,
the Wilson disease gene has been localized to chromosome 13. Clinically,
the age of onset for Wilson disease varies from between 8 years to the
mid-50's with patients usually presenting with severe liver cirrhosis.
One hallmark of Wilson disease are Kayser-Fleischer rings which result
from the deposition of copper in the cornea. The appearance of Kayser-Fleischer
rings is frequently accompanied by neurological disorder.
Treatments for Wilson disease are focused on mobilizing and eliminating
excess copper from the body. Several compounds have been evaluated such as D-penicillamine, tetrathio
molybdate, trientine. Of these D-penicillamine is the most widely used, however; in some patients this
leads to a worsening of the neurological symptoms. Oral zinc has also been used to treat Wilson disease.
Zinc induces the expression of metallothionein in the intestines which functions to chelate copper
before it enters the body.
The genes for both Menkes disease (ATP7A) and Wilson disease (ATP7B) code for copper transporting P-type ATPases consisting of about 1500 amino acids. The N-terminal portion of the protein contains six copies of the conserved HMA domain which contains the conserved motif GMT/HCXXC. These conserved cysteine residues have been shown to be directly involved in metal ligation.
Although they code for similar proteins, their patterns of expression are quite different. The Menkes disease ATPase is expressed in all tissues except the liver while the Wilson disease ATPase is expressed mainly in the liver, brain and placenta. These patterns of gene exspression correlate well with the clinical manifestations observed. Both proteins are localized to the trans-golgi membrane where they are thought to transport copper from the cytoplasm into the golgi lumen where it is incorporated into newly synthesized copper containing proteins. Both proteins have also been shown to undergo a copper induced translocation from the trans-golgi membrane to both plasma membrane and vesicular compartments. The trans-golgi membrane localization of the proteins also explains the pathology of the diseases. If the Menkes protein is non-functional uptake of copper from the intestines will not be possible and a state of copper deficiency will result. If the Wilson disease protein is non-functional, copper cannot be transported into the golgi and out of the cell leading to the accumulation of copper in the liver and other organs.
My PhD research focused on the characterization of the N-terminal copper binding domain (~70 kDa) from
the Wilson disease copper-transport P-type ATPase (WCBD). As it turned out, this protein was extremely difficult to work with. It
was fairly large with about 650 amino acids and loaded with cysteine residues, 18 in total. As such much of the recombinant
protein had to be refolded from inclusion bodies even when fused to GST, although fusing the domain to GST did pull some into the soluble fraction. Eventually I was able to produce enough protein to work with. I will refer you to my publications for details on the experiments I did and will just provide an overview of my results here.
In our first set of experiments we use Immobilized metal ion affinity chromatography (IMAC) and a competitive 65Zn blotting technique to determine the metal specificity and relative affinity. Both techniques gave comparable results and we determined that the WCBD is able to bind other transition metals besides copper with varying affinity. The relative affinity of these metals was as follows Cu>>Zn>Ni>Co>>Fe. Using neutron activation analysis (NAA) we also determined that the WCBD is able to bind 6 copper atoms, presumably 1 for every HMA domain. A UV/Vis spectroscopic analysis of the WCBD did not reveal any features commonly associated with copper containing proteins suggesting that copper was bound in the Cu(I) oxidation state. Since Cu(I) is a d10 system it is spectroscopically silent explaining the lack of any UV/Vis features.
The finding that copper was bound in the +1 oxidation state was somewhat disappointing in that it meant we had to turn to more exotic spectroscopic methods to continue our analysis. Our next set of experiments utilized X-ray Absorption Spectroscopy (XAS) and Circular Dichroism (CD) to characterize the metal binding sites and any conformational changes taking place upon metal binding. The XAS studies were carried out in collaboration with Dr. Lawrence Que, Jr.. The CD results indicated that significant and progressive secondary and tertiary structure changes were taking place upon copper binding. The XAS results showed that overall (XAS is an averaging technique in proteins with multiple metal centers) copper was bound by two sulfur atoms (from the conserved cysteine residues in the HMA domains) in a distorted linear conformation in the +1 oxidation state. Using these findings and those of other researchers working on the Menkes protein, we proposed the following hypothetical model.
Copper Induced Trafficking of the Wilson Disease ATPase
In this model, copper binds to the "Apo" form of the protein inducing a conformational change that helps initiate the copper transport cycle. As the cytosolic concentration of copper rises, more copper becomes bound to the ATPase inducing additional conformational changes which cause the ATPase to traffic between the trans-golgi network (TGN) and the plasma membrane (PM) where it pumps copper out of the cell. Once the cytosolic concentration of copper drops, the majority of the ATPase recycles back to the TGN. Athough consistent with our results and those of others, some recent work has shown that not all the copper binding domains are necessary for function.
The final set of experiments involved characterizing the binding of Zn to the WCBD again using both XAS and CD. The results of these experiments were somewhat unexpected in that they indicated that zinc was not binding to the same sites as copper. This was puzzling since we had shown earlier that zinc was able to bind to the WCBD and that the addition of copper was able to displace it (DiDonato et al., 1997). The conformational changes observed by CD were also very different from those induced by copper. Our current hypothesis is that the differences between zinc and copper binding are the basis for regulation of ATPase activity in vivo. Copper induced conformational changes stimulate the phosphorylation of the ATPase (hence initiating the transport cycle), while those induced by zinc do not. This hypothesis has been strengthened by recent results which have shown that copper and not zinc specifically activates the ATPase (See references in DiDonato et al., 2002). The mechanism by which increasing concentrations of cytosolic copper trigger the translocation of the ATPase to the plasma membrane has yet to be elucidated.
Research Papers
Book Chapters
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©Michael DiDonato, PhD., The Scripps Research Institute
http://www.scripps.edu/~didonato/Phd.html
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Last updated: Friday, 04-Jun-2004 14:31:24 PDT