What Is Menkes Disease

Published Date: 02 Nov 2017

Tamina Begum

[Bsc Biological Chemistry]

Both wilsons and Menkes disease are recessive disorders whose occurrence is solely down to a mutation in the copper transporting channels of ATP7A or ATP7B respectively. These channels consequently effect copper levels in the body by either gross deficiency or toxicity .

There are two groups of nutrients required in the human body these are macronutrients and micronutrients. Macronutrients are required in large quantities in the body and there primary function is to provide the body with large amounts of energy. Proteins, Fats and Carbohydrate foods are regarded as macronutrients. Micronutrients on the other hand are required in very small amounts but partake in a very diverse range of physiological functions. Zinc, Copper, Cobalt and Chromium are all micronutrients.

Copper (Cu)

Copper is a very important micronutrient which is vital to all living organisms, it is found in nuts, seeds and seafood.(1,3) Copper has the ability to be easily converted between different oxidation states such as Cu+ and Cu 2+ and it is this behaviour that enables the transitional metal to function as a catalytic cofactor within numerous metalloenzymes in a range of biochemical reactions. These include Cu/Zn superoxide dismutase (antioxidant defense), cellular respiration (cytochrome oxidase), connective tissue formation (lysyl oxidase) and pigmentation (tyronsinase). (2)

In the human body Copper absorption occurs in the large intestine and is then distributed to the copper dependant enzymes. The volume of copper absorbed at the large intestine depends solely on the dietary copper intake of an individual. The World Health Organisation recommends that an adult should have no more than 2-3mg of Copper intake in a day. If an individual’s copper intake is less than 1mg/day then copper absorption will be much higher at around 50% whereas if more than 5mg/day of Copper intake the absorption will be much lower at around 20%. (3) Copper regulation is very important as excess copper levels in the body can lead to toxicity and consequently causes numerous physiological problems.

The reactive nature of ionic copper also makes it a toxic metal if not properly handled by the cell. In the cell, under normal conditions, free copper is virtually nonexistent as the cell has an overcapacity for copper sequestration [2]. However, under conditions of copper overload, free copper ions can accumulate and react to generate hydroxyl radicals that can engage in reactions that can adversely modify proteins, lipids,and nucleic acids [3]. Free copper ions may also exert their toxic property by displacing other essential metal co-factors from metalloenzymes. For example, it has been reported that copper can substitute for Zn(II) in zinc-finger transcription factors that renders the proteins unable to bind their target sequence [4]. Cellular copper concentrations must therefore be maintained at levels where nutritional deficiency and toxicity are avoided.

Linder MC. Biochemistry of Copper. New York: Plenum Press, 1991.

Wang Y, Zhu S, Weisman GA, Gitlin JD, Petris MJ (2012) Conditional Knockout of the Menkes Disease Copper Transporter Demonstrates Its Critical Rolein Embryogenesis. PLoS ONE 7(8): e43039. doi:10.1371/journal.pone.0043039

Turnlund JR. Human whole-body copper metabolism. Am J Clin Nutr 1998;67(5 suppl):960S–4S.

Homeostasis

Copper homeostasis is a very complex and tightly controlled process using membrane transporters and Copper Chaperones at both intracellular and intercellular levels.(1) This is because Copper has to be delivered to a vast number of metalloproteins located within organelles such as the chloroplast, mitochondria and other secretory compartments. To fully understand the cellular trafficking of Copper the yeast Saccharomyces Cerevisiae was studied.

CTR1

In the yeast Saccharomyces Cerevisiae copper is taken up by the transporters Ctr1 and Ctr3 which appear to be functionally redundant high affinity copper transporters. The Ctr family of importers have a distinguishing methionie-rich amino termini and it is this feature which binds extracellular Cu+ hence increasing the local concentration of Copper. Research into the cDNA of Ctr1 and Ctr3 revealed the presence of two already suggested Cu +coordination motives which are MxM and MxxM, and CxxC and CC respectively.(15) The concluding outcome of this research was that Copper which bound to these motive could be transported directly onto intracellular Cu chaperones, such as Atx1,hence demonstrating an efficient Cu transport system.

In mammalian cells the majority of copper acqusistion is also mediated by the copper transporter Ctr1, which transports the unstable, reduced, cuprous form of copper (Cu+).(1) It is thought that the stable oxidised Cu2+ form of copper is found in the extracellular environment and is reduced to Cu+ by membrane-associated reductases before being transported across the cellular membrane via Ctr1.(2) Before the cuprous copper can be transported across the membrane 3 CTR1 monomers form a channel like pore through which the Cu+ is then transferred across the membrane. It is though that this transfer occur via a series a trans-chelation reactions involving sulphur or nitrogen containing residues which are located on the inside of the pores.(7)

In mammalian cells Ctr1 is found in 2 main locations. These are at the plasma membrane and in intracellular vesicles. The influence of copper on the distribution of Ctr1 across these 2 areas is cell specific. For example CTR1 is present at the basolateral membrane at the liver and kidney. Its primary function here is to remove the copper from circulation and more than likely retrieving the copper from specific carriers.(6)

Once copper has been transported through the cellular membrane and into a cell the Ctr1 then transfers the translocated Cu+ to one of 3 marked intracellular chaperones which in turn deliver the Cu+ ions to Copper-dependant enzymes. (3)

In regards to copper transport through the plasma membrane and into a mammalian cell CTR1 is seen as the main copper transporter however CTR2 also exists and carries out this function however small. CTR2 is a low affinity copper transporter which is located intracellulary usually to release copper from the Lysosomes.(8) However a small portion of CTR2 are present at the plasma membrane. The contribution of CTR2 in the process of copper transport across the membrane is not very significant however this copper transporter is overexpressed when high copper uptake is needed. This fundamentally shows that CTR2 also has some importance in the initial journey of the transport of Copper to cells.(9)

CTR1 Structure

It is fair to say that Ctr1 is a high affinity copper transporter which is pivotal in the Inital transport of Copper and its structure shows how it is adapted for its function. Ctr1 is a membrane protein which is made up of 3 transmembrane domains, an extracellular N-terminus and an intracellular C-terminus. (4) Further analysis of Ctr1using cryoelectron microscopy and several protein structure simulation techniques created an all-atom model of Ctr1 which found that in the central region of the pore 4 sets of methionine traids existed in the intermembranous region. It was further found that the four triads created a structure that promotes and allows step wise transport of metal ions into and then through the intramembranous channel of the transporter via transient thioether bonds to methionine residues. (5) This structure analysis clearly highlights the structural adaptation of Ctr1 to its function.

Figure 1-

Shows the pore which 3 CTR1 monomers form in the lipid bilayer. The CTR1 pore on the right has one monomer unit removed so the inside of the pore can be seen. There are three locations which span the CTR1 pore which Cu+ can bind to for transport. The Cu+ can bind to the conserved methionine residue in the first transmembrane domain, the Met-Xaa-Xaa-Xaa-Met motifs in the second transmembrane domain and finally to the intracellular cysteine or histidine residues.

The one area in which the structural features of CTR1 has no influence is in the rate of uptake of Copper by the Copper transporter. The rate of uptake of Copper by CTR1 is incited by high K (Potassium) concentration and the extracellular PH preferably being acidic. Energy levels also have no influence on copper uptake by CTR1.(10)

Copper Chaperones

We have established that copper enters the cell via CTR1 or CTR2 transporters which are pivotel in the transport of Copper across the plasma membrane of mammalian cells. The understanding of how the translocated copper is then transported within the cell to the different organelles was founded by lin and Culotta in 1995. The researchers identified in yeast Anti-oxidant Protein 1 (ATX1) which lead to the discovery of Copper Chaperones.

Copper Chaperones have the singular function of transporting the Copper to specific cellular compartments and targets in the cell. This process is initiated on the instant entry of the Copper into the cell. There are 3main cytosolic copper chaperones in the cell which are CCS ,ATX1 and Atox1 which transport the copper to different cellular destinations. CCS activates the Cu/Zn-dependent superoxide dismutase (SOD1) by giving It its copper co-factor whilst Atox1 and ATX1 transfer the copper to the copper transporting ATPase’s such as ATP7A and ATP7B. (11) ATP7A and ATP7B are the copper transporting ATPases for Menkes and Wilsons disease respectively.

Recent research into ATOX1 shows that the copper chaperone plays a vital role in moderating the copper dependent movement of the copper transporting ATPase ,ATP7A from the Trans Golgi Netwwork to the cell surface. And hence allows a threshold for copper-dependent trafficking of ATP7A to be determined. This is very important in gaining a greater understanding of Menkes disease and the roots of its cause. We have already established the relationship between ATOX1 and Menkes and Wilsons Disease, however what is startling is that cells which are deficient in ATOX1 have a tendency to amass hugh amounts of Copper due to a default in the cellular copper efflux system. The cause of Wilsons disease is down to an accumulation of copper In the liver (hepatocyte cells) and this could be seen as one of the causes. No definite explanation can be given as to whether low ATOX1 levels in cells is the primary cause of Wilson disease but this disease will be further analysed and conclusions made.(12)

To obtain a greater insight into CCS and its function research was carried out on rats. The rats in question were fed a low-copper diet and it was found that levels of CCS were increased in the various tissues of the rat depending on the level of copper present in the rats diet.(13) It was also found that an increase in CCS levels occurred specifically when low levels of copper were present in the cells and consequently the rate of CCS degradation was decreased by the 26 S proteasome.(14) The theoretical reasoning behind this process is that because the copper levels in the cells is low then increased levels of CCS will increase efficiency regarding the transfer of the copper to the SOD1. This also emphasizes the diverse role of CCS in copper transport as it shows that CCS has some influence in regards to the use of the copper when its levels are low.

It has not yet been established how the copper chaperones get a hold of the copper in the cells. Hama et al. have reported however that the copper chaperone HAH1 is not affected by low levels of copper in HeLa cells. This shows that if both CCS and HAH1 obtain their copper form the same cellular area then priority will go to CCS in times of copper deficiency.

Constant research is carried out in the field of copper chaperones with identification of new Copper Chaperones being made. An example is Cox17 which is a copper chaperone which transports Copper to the mitochondria for cellular respiration (cytochrome oxidase).(16,17) Copper chaperones are crucial in maintaining the copper levels needed by mammalian cells.

Maintenance of Copper Homeostasis

Copper Homeostasis is maintaining a constant internal copper environment in cells. This is very important as any slight increase or decrease in copper levels in cells can cause numerous physiological problems. It can be stated generically that excess copper levels in cells which do not require the increase copper leads to toxicity and causes Wilsons Disease. Whereas reduced copper levels in cells which have a greater need for copper leads to deficiency and ultimately Menkes Disease.

Copper-Transporting ATPases (Cu-ATPases) have dual function. They primarily transport Cu+ to the copper dependant enzymes in mammalian cells. They do this by accepting copper from the copper chaperone Atox1 and then using energy from ATP hydrolysis to transport the copper into the secretory pathway where the copper is integrated into the copper-dependant enzymes. The secondary function of Cu-ATPases is to maintain homeostasis intracellulary.

To prevent toxicity in cells when the copper level is raised it is the Cu-ATPases which try to remove the excess copper. They do this by undergoing kinase-mediated phosphorylation and then delocalised to a different region. The favoured delocalised regions are usually the apical (ATP7B) and basolateral (ATP7B) membranes. The copper is subsequently not taken up by the Cu-ATPases and is therefore exported out of the cell via vesicle-mediated fusion. (18) This is the mechanism in place to prevent a build-up of Copper in cells. The mechanism is simple and effective but not completely foul proof. Mutations in the Cu-ATPases ruins the logistics of the cells homeostasis mechanism.

We have already analysed in detail the intricate and complex copper transport system in the Human body however what we will focus on now is uncontrolled errors which occur in the transport system which lead to detrimental and life changing physiological problems.

Summary of Copper Transport In a normal mammalian Cell:

Figure 2-

Shows the Copper transport system in a generic mammalian cell. CRT1 accepts the copper from the extracellual

Copper distribution pathways in a generalized mammalian cell. CTR1 accepts cooper from the extracellular copper carriers and transfers copper into

cytosol; changes in copper levels induce reversible trafficking between the plasma membrane and intracellular vesicles. CTR2 is predominantly

intracellular but can be found at the plasma membrane. Entering copper is either binds directly or is retrieved from CTR1/CTR2 by copper chaperones

that have multiple functions; CCS distributes copper to SOD1 in cytosol and mitochondria, while Atox1 transfers copper to the secretory pathway and

nucleus. An ensemble of proteins regulates copper delivery to cytochrome c oxidase (CCO) in mitochindria. Cu-ATPases transport copper to the

secretory pathways for incorporation into cuproenzumes and mediate copper excretion by sequestering excess copper in vesicles. The trafficking of

Cu-ATPases between these two locations is associated with phosphorylation by a kinase (indicated by stars), which increases in response to copper

elevation.

Current Opinion

Disorders of copper metabolism were instrumental in

forwarding our understanding of how copper is distributed

in the body. Menkes syndrome and Wilson’s disease, disorders

of copper deficiency and overload, respectively, led

to the discovery of the gene products mutated in these

diseases.

1-P.V van de Berghe and L.W Klomp, J.Biol. Inorg.Chem., 2010,15,37-46

2-R.Hassett and D.J Kosman J.Biol.Chem 1995,270 128-134

3-

4-

5-Cell biochemistry and Biophysics july 2012,issue 3 pp223-234

6- Moriya M, Ho YH, Grana A, Nguyen L, Alvarez A, Jamil R,

Ackland ML, Michalczyk A, Hamer P, Ramos D et al.: Copper is

taken up efficiently from albumin and alpha2-macroglobulin

by cultured human cells by more than one mechanism. Am J

Physiol Cell Physiol 2008, 295(3):C708-C721.

7-S.G.Aller and V.M Unger,Proc. Natl.Acad. Sci.USA 2006,103,3627-3632

8- van den Berghe PV, Folmer DE, Malingre´ HEM, vanBeurden E,

Klomp AEM, vandeSluis B, Merkx M, Berger R, Klomp LWJ:

Human copper transporter 2 is localized in late endosomes

and lysosomes and facilitates cellular copper uptake. Biochem

J 2007, 407(1):49-59

9- Bertinato J, Swist E, Plouffe LJ, Brooks SPJ, L’abbe MR: Ctr2 is

partially localized to the plasma membrane and stimulates

copper uptake in COS-7 cells. Biochem J 2008, 409(3):731-740.

10- Lee J, Pena MM, Nose Y, Thiele DJ. Biochemical characterization of

the human copper transporter Ctr1. J Biol Chem 2002;277:4380–7.

11- Walker JM, Tsivkovskii R, Lutsenko S. Metallochaperone Atox1

transfers copper to the NH2-terminal domain of the Wilson’s disease

protein and regulates its catalytic activity. J Biol Chem 2002;277:

27953–9.

12- Hamza I, Prohaska J, Gitlin JD. Essential role for Atox1 in the

copper-mediated intracellular trafficking of the Menkes ATPase. Proc

Natl Acad Sci USA 2003;100:1215–20.

13- Bertinato J, Iskandar M, L’Abbe´ MR. Copper deficiency induces the

upregulation of the copper chaperone for Cu/Zn superoxide dismutase

in weanling male rats. J Nutr 2003;133:28–31.

14- Bertinato J, L’Abbe´ MR. Copper modulates the degradation of copper

chaperone for Cu,Zn superoxide dismutase by the 26 S proteosome.

J Biol Chem 2003;278:35071–8.

15-Yamaguchi, I. Y., Serpe, M., Haile, D., Yang, W., Kosman, D. J.,

Klausner, R. D., and Dancis, A. (1997) J. Biol. Chem. 272,

17711–17718.

[16] Glerum DM, Shtanko A, Tzagoloff A. Characterization of COX17, a

yeast gene involved in copper metabolism and assembly of cytochrome

oxidase. J Biol Chem 1996;271:14504–9.

[17] Amaravadi R, Glerum DM, Tzagoloff A. Isolation of a cDNA encoding

the human homolog of COX17, a yeast gene essential for

mitochondrial copper recruitment. Hum Genet 1997;99:329–33.

(18)Veldhuis NA, Gaeth AP, Pearson RB, Gabriel K, Camakaris J: The

multi-layered regulation of copper translocating P-type

ATPases. Biometals 2009, 22(1):177-190.

Figure 1- Yasuhiro Nose et al., Structure of the Ctr1 copper trans‘PORE’ter reveals novel architecture, TRENDS in Biochemical Sciences (2006),

doi:10.1016/j.tibs.2006.09.003

What is Wilsons disease?

Wilsons disease is an autosomal recessive disorder caused by copper accumulation in the liver and other extrahepatic tissue including the brain, cornea and kidneys. The disease is caused as a direct result of mutations which occur in the copper transporting p-type ATPase, called ATP7B.(1)

Wilsons disease is easily treated and its effects can be reversed if a diagnosis has been made early.(2)

What is Menkes Disease?

Menkes disease is a very dangerous recessive x-linked neurodegenerative disorder which is most common in females compared to males. Two recessive alleles are required for a female to develop the disease. A mutation occurs in ATP7A which encodes a copper-transporting ATPase as a result copper transport is disrupted in the body and leads to copper deficiency. (2)

The direct cause of Menkes disease is Copper transport is disrupted beacsu

which occurs in the copper transporting ATPase

(2) S.G. Kaler, S. Packman, Inherited disorders of human copper metabolism, In: D.L.

Rimoin, J.M. Connor, R.E. Pyeritz, B.R. Korf (Eds.), Emery and Rimoin's Principles

and Practice of Medical Genetics (6th edition). Churchill Livingstone/Elsevier,

New York (in press).

A variant transcript of the Menkes gene has also

been identified in several human cell lines that encodes for

a protein that is targeted to the nucleus [68]. Thus, this

variant was referred to as nuclear Menkes-like 45 (NML45),

and this protein may function as a copper chaperone that

shuttles copper to the nucleus. Studies examining how the

activities of copper chaperones and their respective enzymes

are influenced by copper deficiency or overload will

provide valuable insight into the distribution and utilization

of cellular copper when it is abundant or scarce.