Membrane Transport
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Lab Head: Assoc Prof V. Nathan Subramaniam
nathanS@qimr.edu.au
The Membrane Transport Laboratory has an interest in both basic and applied studies of membrane biology. Many human diseases are associated with defects in the localisation and trafficking of membrane proteins. By understanding how cells regulate the trafficking, localisation and numerous molecular interactions of proteins implicated in a number of human diseases, we hope to advance our understanding of these disorders with the prospect of potential therapeutic interventions. In particular, our laboratory focuses on the mechanisms and interactions which regulate iron homeostasis. The majority of projects in the laboratory, focus on defining how the liver and a number of liver-expressed molecules regulate the absorption, recycling and distribution of iron in the body. The identification of the molecules involved and defining the way they work has major implications for iron related disorders such as hereditary haemochromatosis and anaemia.
Iron Disorders
Iron is the fourth most abundant element in nature and is indispensable for life. It is invaluable as a cofactor for a number of enzymes and is vital in haem which is the functional group for the binding of oxygen by haemoglobin and myoglobin. The levels of iron in the body are regulated very closely, a deficiency can lead to anaemia whereas an excess of iron results in disorders such as haemochromatosis. Hereditary haemochromatosis is a genetic disorder of iron metabolism which results in the deposition of iron in the major organs, primarily in the liver, pancreas and heart. Untreated, the disorder can result in organ damage, resulting in conditions such as cirrhosis, diabetes, arthritis and heart failure.
The liver plays an important role in the sensing and regulation of body iron levels. Many of the proteins implicated in iron metabolism
are expressed at very high levels in the liver. Alterations in a number of these proteins have been shown to lead to various forms of
hereditary haemochromatosis (HH). The majority of cases of hereditary haemochromatosis are caused by mutations in the HFE gene, located
on chromosome 6. Atypical forms of HH, accounting for 5% of all HH cases and termed non-HFE haemochromatosis, are classified according
to the gene responsible. The study of these molecules has been imperative in defining the various processes involved in maintaining the
appropriate levels of iron within the body.
RESEARCH PROJECTS
Hereditary haemochromatosis caused by HFE mutations The laboratory undertakes molecular analysis of the haemochromatosis gene, HFE, the haemochromatosis gene product, HFE, and the mutation responsible for the majority of haemochromatosis cases. HFE is thought to regulate iron metabolism through its interaction with the transferrin receptor and subsequent regulation of hepcidin. The mutation in HFE results in a protein which is misfolded and is therefore retained in the endoplasmic reticulum. Using cellular model systems we are studying the mechanism by which the mutated HFE protein is retained in the endoplasmic reticulum, the chaperone molecules which it interacts with, and how it is degraded. Studies are also directed at identifying the ultimate localisation of HFE in the cell and understanding the process by which HFE is transported. HFE has been shown to interact with the transferrin receptor at the cell surface. Through its interaction with the transferrin receptor, HFE is endocytosed into the cell. The interaction of HFE with transferrin and its receptor, as well as transporters of iron present on these membranes is also being analysed.
Juvenile haemochromatosis
Juvenile haemochromatosis (JH) is a rare and more severe form of haemochromatosis with an earlier age of onset than HFE-HH.
Generally patients with JH present with clinical symptoms before 30 years of age and often in the teenage years. Reproductive
problems are common and if untreated, death can result from heart failure. Mutations in Hemojuvelin cause Type 2A JH while mutations
in hepcidin cause Type 2B JH. Hemojuvelin, located on chromosome 1, is a member of a previously identified glycoslyphosphatidylinositiol-anchored
gene family, and it's role in iron homeostasis is obscure. Mutational analysis and cellular studies have been undertaken to
determine hemojuvelin's role in iron metabolism. Hepcidin is an important iron regulatory hormone which is produced in the liver and
regulates body iron levels by reducing iron absorption in the gut and recycling in the reticulo-endothelial system. Hepcidin functions
by binding to the iron exporter ferroportin on the cell surface, inducing its internalisation and targeting it for degradation. In this
way, hepcidin is capable of rapidly reducing iron release from cells. We have produced a specific antibody against hepcidin and used it
to determine the hepcidin protein's localisation in hepatocytes and how it is regulated in mouse models of haemochromatosis and anaemia.
Using specific reagents for prohepcidin we have shown that this peptide accumulates in the Golgi compartment of hepatocytes and that this
could be another potential level of regulation.
Molecular, cellular and functional characterisation of Transferrin Receptor 2
The membrane receptor most closely identified with iron transport is the transferrin receptor. A homologue of this molecule, transferrin
receptor 2, has recently been identified. It has been shown that mutations in this receptor can also cause haemochromatosis (Type 3).
We are characterising this protein and studying its role in iron transport, its interaction with the transferrin receptor, HFE, and
other components of the iron transport pathway. Our laboratory has generated the first complete TfR2 knockout mouse, which models
Type 3 hereditary haemochromatosis. We have demonstrated that regulation of the iron regulatory peptide hepcidin is impaired in the
knockout mouse. We have also generated the first liver-specific TfR2 knockout mouse model, which duplicates the effect demonstrated
in the complete knockout, suggesting that the liver is the primary site of aberrant iron metabolism causing haemochromatosis. The knockout
models also serve as a tool to further study the effect and relationship of TfR2, HFE, hepcidin and HJV within the liver.
Ferroportin disease or Type 4 haemochromatosis
Ferroportin is an iron exporter and is expressed highly on the basolateral membrane of duodenal enterocytes and the plasma membrane
of reticuloendothelial cells. It plays a key role in the absorption of dietary iron and in the release of iron derived from the
breakdown of erythrocytes. Type 4 haemochromatosis is caused by mutations in the ferroportin gene located on chromosome 2 and is
also termed ferroportin disease. We have identified the first cases of ferroportin disease in the Australian population. Studies
in the lab focus on cellular analysis of ferroportin and the effect of mutations on trafficking and iron transport ability.
Clinical applications and Population studies
The lab has a number of productive and interesting clinical collaborations dealing with both typical and atypical haemochromatosis cases.
By analysing the various genes involved in the regulation of iron metabolism we have been able to identify the molecular basis of many
atypical forms of iron overload and aid in diagnosis of these disorders. We have identified several cases of ferroportin disease in
Australia and the Asia-Pacific region due to novel mutations in ferroportin. We have also identified HJV mutations as the molecular
cause of juvenile haemochromatosis in young Australian patients. The laboratory has a number of active and ongoing projects aimed at
identifying the molecular cause of atypical iron overload disorders, identifying new mutations and potential new genes.
Staff
| Labhead: | Assoc Prof V. Nathan Subramaniam (Gastroenterological Society of Australia Senior Research Fellow) |
| Senior Research Officer: | Dr Daniel Wallace (NHMRC RD Wright Fellow) |
| Research Officer: | Dr Emily Crampton |
| Research Assistant: | Ms Lesa Summerville |
| Students: | Denny Muslim Elizabeth Leddy Nick Barker |
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Funding
NHMRC, Australia
Gastroenterological Society of Australia
National Institutes of Health, USA
Haemochromatosis Society of Australia
Collaborators
Professor Lawrie Powell, Royal Brisbane Hospital, AustraliaProf Greg Anderson, QIMR, Australia
Assoc Prof Grant Ramm, QIMR, Australia
Dr Darrell Crawford, Princess Alexandria Hosital, Brisbane, Australia
Professor Hong Wanjin, IMCB, Singapore
Dr Ann Walker, University College London, UK
Dr Dianne Watters, Griffith University
Prof Peter Browett, University of Auckland
Professor Jim Camakaris, University of Melbourne, Melbourne
Professor Sharad Kumar, IVMS, Adelaide
Dr Paul Sharp, King's College London, UK
Key Publications
Wallace DF, Summerville L, Crampton E, Subramaniam VN. Defective Trafficking and Localization of Mutated Transferrin Receptor 2: Implications for Type 3 Hereditary Hemochromatosis. Am J Physiol Cell Physiol. 2008 Feb;294(2):C383-90. Epub 2007 Dec 19. [pubmed abstract]
Wallace DF, Subramaniam VN. Non-HFE haemochromatosis. World J Gastroenterol. 2007 Sep 21;13(35):4690-8. [pubmed abstract]
Wallace DF, Dixon JL, Ramm GA, Anderson GJ, Powell LW, Subramaniam VN. A novel mutation in ferroportin implicated in iron overload. J Hepatol. 2007 May;46(5):921-6. [pubmed abstract]
Wallace DF, Summerville L, Subramaniam VN. Targeted disruption of the hepatic transferrin receptor 2 gene in mice leads to iron overload. Gastroenterology. 2007 Jan;132(1):301-10. [pubmed abstract]
Walsh A, Dixon JL, Ramm GA, Hewett DG, Lincoln DJ, Anderson GJ, Subramaniam VN, Dodemaide J, Cavanaugh JA, Bassett ML, Powell LW. The clinical relevance of compound heterozygosity for the C282Y and H63Dsubstitutions in hemochromatosis. Clin Gastroenterol Hepatol. 2006 Nov;4(11):1403-10. Epub 2006 Sep 18. [pubmed abstract]
Wallace DF, Jones MD, Pedersen P, Rivas L, Sly LI, Subramaniam VN. Purification and partial characterisation of recombinant human hepcidin. Biochimie. 2006 Jan;88(1):31-7. [pubmed abstract]
Wallace DF, Summerville L, Lusby PE, Subramaniam VN. Prohepcidin localises to the Golgi compartment and secretory pathway in hepatocytes. J Hepatol. 2005 Oct;43(4):720-8. [pubmed abstract]
Subramaniam VN, Wallace DF, Dixon JL, Fletcher LM, Crawford DH. Ferroportin disease due to the A77D mutation in Australia. Gut. 2005 Jul;54(7):1048-9. [pubmed abstract]
Wallace DF, Summerville L, Lusby PE, Subramaniam VN. First phenotypic description of transferrin receptor 2 knockout mouse, and the role of hepcidin.Gut. 2005 Jul;54(7):980-6. [pubmed abstract]
Wallace DF, Browett P, Wong P, Kua H, Ameratunga R, Subramaniam VN. Identification of ferroportin disease in the Indian subcontinent. Gut. 2005 Apr;54(4):567-8. [pubmed abstract]
Loh E, Peter F, Subramaniam VN, Hong W. Mammalian Bet3 functions as a cytosolic factor participating in transport from the ER to the Golgi apparatus. J Cell Sci. 2005 Mar 15;118(Pt 6):1209-22. [pubmed abstract]
Wallace DF, Clark RM, Harley HA, Subramaniam VN. Autosomal dominant iron overload due to a novel mutation of ferroportin1 associated with parenchymal iron loading and cirrhosis. J Hepatol. 2004 Apr;40(4):710-3. [pubmed abstract]
Arden KE, Wallace DF, Dixon JL, Summerville L, Searle JW, Anderson GJ, Ramm GA, Powell LW, Subramaniam VN. A novel mutation in ferroportin1 is associated with haemochromatosis in a Solomon Islands patient. Gut. 2003 Aug;52(8):1215-7. [pubmed abstract]
Bridle KR, Frazer DM, Wilkins SJ, Dixon JL, Purdie DM, Crawford DH, Subramaniam VN, Powell LW, Anderson GJ, Ramm GA. Disrupted hepcidin regulation in HFE-associated haemochromatosis and the liver as a regulator of body iron homoeostasis. Lancet. 2003 Feb 22;361(9358):669-73. [pubmed abstract]
Wallace DF, Pedersen P, Dixon JL, Stephenson P, Searle JW, Powell LW, Subramaniam VN. Novel mutation in ferroportin1 is associated with autosomal dominant hemochromatosis. Blood. 2002 Jul 15;100(2):692-4. [pubmed abstract] PMID: 12091366
Subramaniam VN, Loh E, Horstmann H, Habermann A, Xu Y, Coe J, Griffiths G, Hong W. Preferential association of syntaxin 8 with the early endosome.J. Cell Sci. 2000 Mar; 113 ( Pt 6): 997-1008. [pubmed abstract]
Lowe SL, Peter F, Subramaniam VN, Wong SH, Hong W. A SNARE involved in protein transport through the Golgi apparatus. Nature 1997 Oct 23; 389(6653): 881-4. [pubmed abstract]
Subramaniam VN, Loh E, Hong W. N-Ethylmaleimide-sensitive factor (NSF) and alpha-soluble NSF attachment proteins (SNAP) mediate dissociation of GS28-syntaxin 5 Golgi SNAP receptors (SNARE) complex. J. Biol. Chem. 1997 Oct 10; 272(41): 25441-4. [pubmed abstract]
Subramaniam VN, Peter F, Philp R, Wong SH, Hong W. GS28, a 28-kilodalton Golgi SNARE that participates in ER-Golgi transport. Science 1996 May 24; 272(5265): 1161-1163. [pubmed abstract]