Iron and Copper Transport via DMT-1 in HEK-293 Cells Alex Jackson, York College of Pennsylvania...
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Iron and Copper Transport via DMT-1 in HEK-293 Cells Alex Jackson, York College of Pennsylvania Biology Department Introduction • Neurodegenerative diseases are currently incurable fatal conditions. They are associated with the accumulation of proteins and metals in brain tissue leading to inflammation and eventual loss of structure and function. • Copper is both an essential and potentially toxic metal. Copper is utilized by most structures involved in energy transfer, including mitochondria and chloroplast. While important, excess copper can lead to oxidative damage and cell death. Copper has also been associated with a pre-cursor protein associated with AD. In the body copper levels result from the mediation of absorption and secretion; however the mechanisms that govern intestinal copper absorption remain largely a mystery. • Divalent Metal Transporter Protein 1 (DMT-1) is a nonspecific transport protein known to transport a substantial number of divalent metal ions, including Fe, Zn, Mn, Cd, and Co. It is very highly expressed in the kidney and small intestine. Methods HEK-293 cells in culture Exposed to Uptake buffer for 30 min. Wash cells, lyse cells Treat cellswith metal(s) for 24 hrs. Analyze cell lysate for Fe Review of Literature • The most prevalent neurodegenerative diseases; Alzheimer’s disease (AD), Parkinson’s disease (PD), and Wilson’s disease (WD) have been shown to be associated with the imbalanced transport of one or more divalent metals (Cu, Fe, Mn and Zn). AD and PD have an association with both nonfunctional Cu accumulation and nonfunctioning metal transporters (Yokel 2006). Objectives • Determine DMT-1’s role, if any, in Cu uptake. • Characterize the interaction of Fe and Cu with regards to uptake. • Determine if additional transport proteins are utilized to mediate Cu uptake. Figure 2: Copper transported by DMT-1 from the mucosal to serosal side of the gut in a Belgrade rat model. Figure from Jiang et al. (2013). Expected Results • Copper should be observed at a noticeable concentration but at a lower concentration than iron in single metal treatments. • Copper concentrations in + Cu 2+ /Fe 2 treated cells should be lower than only Cu 2+ cells. • Copper concentration in cells in CB/Cu 2+ treatment is expected to be reduced but not completely eliminated since copper is also mediated by other transporters such as ATP7B, the transport protein associated with WD. • Iron concentration in CB/Fe 2+ treated cells should be nearly entirely reduced. Work Cited • Bannon, D.I., Abounader, R., Lees, P.S.J., Bressler, J.P., 2003 Effect of DMT1 Knockdown on iron, cadmium and lead uptake in caco-2 cells. American Journal of Physiology – Cell Physiology . 284:44-50. • Byrne, S. L., Buckett, P.D., Kim, J., Luo, F., Sanford, J., Chen, J., Enns, C., Wessling- Resnick, M., 2013. Ferristatin II Promotes Degradation of Transferrin Receptor-1 In Vitro and In Vivo. PLOS One . 8: 1-12. • Cerpa, W., Varela-Nallar, L., Reyes, A.E., Minniti, A.N., Inestrosa, N.C., 2005. Is there a role for Copper in Neurodegenerative Diseases? Molecular Aspects of Medicine . 26: 405-420 • Criponi, G., Nurchi, V.M., Fanni, D., Gerosa, C., Nemolato, S., Faa, G., 2010. Copper-related diseases: From chemistry to molecular pathology. Coordination Chemistry Reviews. 254 (2010) 876–889. • Illing, A.C., Shawki, A., Cunningham, C.L., Mackenzie, B. 2012. Substrate Profile and Metal-ion Selectivity of Human Divalent Metal- ion Transporter-1. Journal of Biological Chemistry. 287:30485-30496. • Jiang, L., Garrick, M.D., Garrick, L.M., Zhao, L., Collins., J.F. 2013. Divalent metal transporter 1 (DMT1) Mediates Copper Transport in the Duodenum of Iron-Deficient Rats and When Overexpressed in Iron-Deprived HEK-293 Cells. Journal of Nutrition. 143(12):1927-1933. Acknowledegments I would like to thank Dr. Kaltreider for his guidance and advice. • DMT-1’s role in the transport of Cu remains contested (Illing et al. 2012). • There have been several proposed conditions in which DMT-1 may mediate Cu uptake but only in Fe2+ deficient diets or when intentionally overexpressed (Jiang et al. 2013). Figure 1: Mechanism for how Chlorazol Black inhibits DMT-1. Figure from Byrne et al.2013. Figure 3: Uptake of Cu2+ and Fe2+ by metal treatment. Cu2+ is inhibited by Fe2+. Cu Fe C u/Fe CB/Cu CB/Fe CB/Cu/Fe 0.0 0.5 1.0 1.5 Treatm ent M etalC oncentration (pm ol/mL) C opper Iron
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Transcript of Iron and Copper Transport via DMT-1 in HEK-293 Cells Alex Jackson, York College of Pennsylvania...
Iron and Copper Transport via DMT-1 in HEK-293 Cells
Alex Jackson, York College of Pennsylvania Biology Department
Introduction• Neurodegenerative diseases are currently incurable fatal conditions.
They are associated with the accumulation of proteins and metals in brain tissue leading to inflammation and eventual loss of structure and function.
• Copper is both an essential and potentially toxic metal. Copper is utilized by most structures involved in energy transfer, including mitochondria and chloroplast. While important, excess copper can lead to oxidative damage and cell death. Copper has also been associated with a pre-cursor protein associated with AD. In the body copper levels result from the mediation of absorption and secretion; however the mechanisms that govern intestinal copper absorption remain largely a mystery.
• Divalent Metal Transporter Protein 1 (DMT-1) is a nonspecific transport protein known to transport a substantial number of divalent metal ions, including Fe, Zn, Mn, Cd, and Co. It is very highly expressed in the kidney and small intestine.
MethodsHEK-293 cells in culture
Exposed to Uptake buffer for 30 min.Wash cells, lyse cells
Treat cellswith metal(s) for 24 hrs.
Analyze cell lysate for Fe and Cu levels
Review of Literature• The most prevalent neurodegenerative diseases; Alzheimer’s
disease (AD), Parkinson’s disease (PD), and Wilson’s disease (WD) have been shown to be associated with the imbalanced transport of one or more divalent metals (Cu, Fe, Mn and Zn). AD and PD have an association with both nonfunctional Cu accumulation and nonfunctioning metal transporters (Yokel 2006).
Objectives• Determine DMT-1’s role, if any, in Cu uptake. • Characterize the interaction of Fe and Cu with regards to uptake.• Determine if additional transport proteins are utilized to mediate
Cu uptake.
Figure 2: Copper transported by DMT-1 from the mucosal to serosal side of the gut in a Belgrade rat model. Figure from Jiang et al. (2013).
Expected Results• Copper should be observed at a noticeable
concentration but at a lower concentration than iron in single metal treatments.
• Copper concentrations in +Cu2+/Fe2 treated cells should be lower than only Cu2+ cells.
• Copper concentration in cells in CB/Cu2+ treatment is expected to be reduced but not completely eliminated since copper is also mediated by other transporters such as ATP7B, the transport protein associated with WD.
• Iron concentration in CB/Fe2+ treated cells should be nearly entirely reduced.
Work Cited• Bannon, D.I., Abounader, R., Lees, P.S.J., Bressler, J.P., 2003 Effect of DMT1 Knockdown on iron,
cadmium and lead uptake in caco-2 cells. American Journal of Physiology – Cell Physiology. 284:44-50.• Byrne, S. L., Buckett, P.D., Kim, J., Luo, F., Sanford, J., Chen, J., Enns, C., Wessling- Resnick, M., 2013.
Ferristatin II Promotes Degradation of Transferrin Receptor-1 In Vitro and In Vivo. PLOS One. 8: 1-12.• Cerpa, W., Varela-Nallar, L., Reyes, A.E., Minniti, A.N., Inestrosa, N.C., 2005. Is there a role for Copper
in Neurodegenerative Diseases? Molecular Aspects of Medicine. 26: 405-420• Criponi, G., Nurchi, V.M., Fanni, D., Gerosa, C., Nemolato, S., Faa, G., 2010. Copper-related diseases:
From chemistry to molecular pathology. Coordination Chemistry Reviews. 254 (2010) 876–889.• Illing, A.C., Shawki, A., Cunningham, C.L., Mackenzie, B. 2012. Substrate Profile and Metal-ion
Selectivity of Human Divalent Metal-ion Transporter-1. Journal of Biological Chemistry. 287:30485-30496.
• Jiang, L., Garrick, M.D., Garrick, L.M., Zhao, L., Collins., J.F. 2013. Divalent metal transporter 1 (DMT1) Mediates Copper Transport in the Duodenum of Iron-Deficient Rats and When Overexpressed in Iron-Deprived HEK-293 Cells. Journal of Nutrition. 143(12):1927-1933.
AcknowledegmentsI would like to thank Dr. Kaltreider for his guidance and advice.
• DMT-1’s role in the transport of Cu remains contested (Illing et al. 2012).
• There have been several proposed conditions in which DMT-1 may mediate Cu uptake but only in Fe2+ deficient diets or when intentionally overexpressed (Jiang et al. 2013).
Figure 1: Mechanism for how Chlorazol Black inhibits DMT-1. Figure from Byrne et al.2013.
Cu Fe Cu/Fe CB/Cu CB/Fe CB/Cu/Fe0.0
0.5
1.0
1.5
Treatment
Met
al C
on
cen
trat
ion
(p
mo
l/mL
)
CopperIron
Figure 3: Uptake of Cu2+ and Fe2+ by metal treatment. Cu2+ is inhibited by Fe2+.
