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Suresh

This study aimed to understand the function of ATP7B, the copper transporting ATPase defective in Wilson's disease, by overexpressing ATP7B in mammalian cells. HEp2 laryngeal epithelial cells that do not naturally express ATP7B were transfected with ATP7B plasmids. Cells overexpressing ATP7B showed increased resistance to copper toxicity compared to non-transfected cells, with 40% vs 5% survival at lethal copper concentrations. This suggests ATP7B overexpression increased copper transport and detoxification, demonstrating its role in protecting against copper toxicity. Further studies are needed to fully characterize ATP7B function.

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15 views

Suresh

This study aimed to understand the function of ATP7B, the copper transporting ATPase defective in Wilson's disease, by overexpressing ATP7B in mammalian cells. HEp2 laryngeal epithelial cells that do not naturally express ATP7B were transfected with ATP7B plasmids. Cells overexpressing ATP7B showed increased resistance to copper toxicity compared to non-transfected cells, with 40% vs 5% survival at lethal copper concentrations. This suggests ATP7B overexpression increased copper transport and detoxification, demonstrating its role in protecting against copper toxicity. Further studies are needed to fully characterize ATP7B function.

Uploaded by

cgeethaedwin
Copyright
© Attribution Non-Commercial (BY-NC)
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Download as PPT, PDF, TXT or read online on Scribd
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In vitro study of ATP7B (Wilson s disease protein) function.

S Suresh Kumar, Pramod Yadav#, K A Balasubramanian, A Oommen*, G Kurian, G Chandy, GR Jayandharan**, CE Eapen, S Mazumdar#. Departments of Gastrointestinal Sciences; Neurochemistry* and Hematology**, Christian Medical College, Vellore; Department of Chemical Sciences#, Tata Institute of Fundamental Research, Mumbai.

Background : Defective copper transporting ATPase called ATP7B, leads to copper accumulation in Wilson s disease This study is a preliminary step to understand ATP7B in Indian patients with Wilson s disease. Understanding the WD protein better may improve diagnostic methods. We selected laryngeal epithelial (HEp2) cells for our experiment, as these cells do not express ATP7B. Aim : To study the function of ATP7B by over expressing these proteins in mammalian cells.
ATP7b protein and copper transport

Step 3: Transfection HEp2 cells were transfected with the green fluorescent (GFP) plasmid (control), GFP + ATP7B and ATP7B (non-fluorescent) plasmids using lipofectamine 2000. From the 2nd day the cells were incubated with Geneticin (G4181800g/ml).
Plasmid with GFP GFP +ATP7B ATP7B in UV ATP7B in Visible light

Hep2 cells transfected with GFP , GFP+ATP7B & ATP7B


Day 2 Day 5 Day 7 Day 14

Hep2 cells transfected with GFP (green flourescent protein) Study design Result: After 7 days, cells with GFP+ATP7B were giving fluorescence whereas the cells with out ATP7B lost its fluorescence

Step 4: Copper toxicity assay HEp2 cells transfected with ATP7B and non-transfected cells (controls) were exposed to varying concentrations of copper sulphate for 24 hours.

Results :

Step 1: Transformation
Undigested Digested with SmaI

At lethal dosage (300M) of copper, 5% of non-transfected HEp2 cells (ATP7B absent) survived whereas 40% of ATP7B transfected HEp2 cells survived (p value = 0.00015)

Conclusion : Transfection of ATP7B gene into laryngeal epithelial cells increased the resistance of these cells to copper toxicity.
Plasmids with ATP7B were inserted into E.coli and selected using ampicillin (1mg/l) containing agar medium. Reconfirmed by (restriction) Sma 1 digestion.

Further steps to do
ATP7B over expression has to be confirmed by Immunoblotting Change in Amino acid and Molecular weight of mutated ATP7B has to be determined. 3D structure of these protein should be analysed. Copper binding activity of ATP7B has to be checked.

Step 2: Selection of cells for transfection


Liver samples show WD protein * patient with cirrhosis, not Wilson s disease (Lane 1) * patient with hepatic Wilson s disease (Lane2) * Hep2 (laryngeal epithelial) cells do not express WD protein (Lanes 3, 4)

Acknowledgement This work is supported by Department of Science and Technology Govt . of India

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