Copper is well known to be an essential element for all forms of life. It is required for a great variety of biological processes. In contrast, when in excess, this necessary element can be very harmfull due to its ability to generate reactive oxygen species (ROS). Production of ROS can lead to damage of DNA, lipids, proteins, and can have several effects on signal transduction pathways. Various diseases characterized by hepatic copper accumulation have been described. These include Wilson disease and Indian- and non-indian childhood cirrhosis in humans.
A well known animal copper overloading disorder is canine copper toxicosis (CT), an autosomal recessive disorder that occurs frequently in Bedlington terriers. CT is characterized by an inefficient excretion of copper through the bile and affected dogs develop chronic hepatitis, eventually leading to cirrhosis. Usually, affected dogs die between 2 and 6 years of age. Employing positional cloning strategies, our group found that CT is caused by a large genomic deletion in the MURR1 gene. MURR1 is a conserved gene and seems to be confined to mammals. The official nomenclature of MURR1 is now COMMD1.
Since affected dogs suffer from copper toxicosis, the role of COMMD1 was expected to involve copper excretion via bile. The precise function however remains to be elucidated. This project, a collaboration between the Department of Endocrine and Metabolic Diseases and the Department of Medical Genetics, aims to do a detailed functional characterization of COMMD1. We have generated specific polyclonal antibodies against COMMD1. Using these antibodies on multiple tissue western-blotting experiments, we have been able to show that COMMD1 is ubiquitously expressed with highest expression levels observed in the liver, kidney, spleen and colon.
The localization of COMMD1 on tissue and subcellular level is currently being investigated using immunofluorescence microscopy and immunohistochemistry methods. Immunofluorescence experiments performed in several cell-lines showed that COMMD1 is probably localised in vesicles.
Recently, remarkable discoveries have been made that could help to characterize the function of COMMD1. In this respect, COMMD1 was found to interact with several partners like ATP7B (Wilson disease protein), XIAP (X-linked inhibitor of Apoptosis), human delta-ENaC (human delta sodium channel), I-kappaB-alpha and Cul1. The protein ATP7B is known to be involved in copper excretion towards the bile. This interaction therefore supports our hypothesis that COMMD1 is involved in copper excretion. The interaction of COMMD1 with XIAP and human delta-ENaC were first shown in a Yeast-two-hybrid to find interaction partners for respectively XIAP (Burstein et al) and human delta-ENaC (Biasio et al). XIAP negatively regulates COMMD1 protein levels by the formation of ubiquitin chains on MURR1 that promote its degradation leading to increased intracellular copper levels. Interaction of COMMD1 with human delta-EnaC results in decreased sodium current across the membrane. The interaction of COMMD1 with I-kappaB-alpha and Cul1 increases NFkappaB activity by facilitating phospho-I-kappaB-alpha degradation by the proteasome. COMMD1 therefore acts as a restriction factor that inhibits HIV-1 replication and might delay the progression of HIV-1 infection to AIDS.
We have investigated additional possible protein interactions. A yeast two-hybrid screen has been performed using a human liver cDNA library. This screen has identified one very promising candidate for interaction with COMMD1. This candidate is a the product of a novel gene which we refer to as MIP1 (MURR1 Interacting Protein 1). The function of MIP1 is not known, but multiple tissue northern blotting experiments showed that MIP1 has a similar expression profile as COMMD1. The interaction between MIP1 and COMMD1 is currently being further investigated with immuno-precipitations, GST-fusion “pull-down” assays and by sub-cellular co-localisation studies.
In another project, we aim to characterize the function of COMMD1 by looking at in vivo or in vitro systems in which COMMD1 has been deleted by using RNAi against COMMD1 or by looking in a COMMD1 knockout system. In a cell line, transfection with RNAi against COMMD1 has already successfully knocked down COMMD1(over 90 %) and we are currently constructing a COMMD1 knockout mouse. In these systems, several experiments will be performed that could help us to understand the role of COMMD1in cellular processes. These experiments include copper measurements, copper excretion experiments and immuno-histochemical studies to investigate the precise role of COMMD1in copper metabolism. In addition, we will collaborate with the Genomics Laboratory to perform cDNA microarrays that will not only be useful to identify new targets of COMMD1in copper metabolism, but will possibly identify targets in other processes as described above.
This project is a collaboration between two departments of the Division of Biomedical Genetics: the Complex Genetics Section of the Department of Medical Genetics, and the Department of Metabolic and Endocrine Diseases.
Bart van de Sluis - Identification of a copper toxicosis gene in Bedlington terriers. 19 November 2002
Diana Klomp - Mechanisms of cellular copper homeostasis. 9 December 2003.
Prim de Bie - Novel insights in the molecular pathogenesis of human copper homeostasis disorders through studies of protein-protein interactions. 4 October 2007.
Patricia Muller - Transcriptomics as a tool to dissect copper homeostasis and COMMD protein function. 25 March 2008.
Willianne Vonk - Understanding COMMD1 function: Novel perspectives from
in vitro and
in vivo studies. 17 March 2011.
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• Burstein E, Ganesh L, Dick RD, van De Sluis B, Wilkinson JC, Klomp LW, Wijmenga C, Brewer GJ, Nabel GJ, Duckett CS. A novel role for XIAP in copper homeostasis through regulation of MURR1. EMBO J. 2004 Jan 14;23(1):244-54. Epub 2003 Dec 18.
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• van de Sluis BJ, Breen M, Nanji M, van Wolferen M, de Jong P, Binns MM, Pearson PL, Kuipers J, Rothuizen J, Cox DW, Wijmenga C, van Oost BA. Genetic mapping of the copper toxicosis locus in Bedlington terriers to dog chromosome 10, in a region syntenic to human chromosome region 2p13-p16. Hum Mol Genet. 1999 Mar;8(3):501-7.
last update: 22 March 2011