Identification of Proteins That Interact with Podocin Using the Yeast 2-hybrid System

Overview

Journal Yonsei Med J

Specialty General Medicine

Date 2009 May 12

PMID 19430563

Citations 5

Authors

Soo Jin Park

Byung Ha Lee

Dae Joong Kim

Affiliations

Soon will be listed here.

Abstract

Purpose: As a membrane protein at the insertion site of the slit diaphragm (SD) complex in podocyte foot processes, podocin has been reported to act as a scaffolding protein required to maintain or regulate the structural integrity of the SD. In order to identify proteins that associate or interact with podocin, we screened a mouse kidney complementary DNA (cDNA) library using a yeast 2-hybrid system.

Materials And Methods: 1) The full-length cDNA of podocin from the mouse kidney was amplified by Polymerase Chain Reaction (PCR), 2) The PCR product was cloned into a pGBKT7 vector, pGBKT7-podocin, 3) After the pGBKT7-podocin was transformed into AH109, the AH109/pGBKT7-podocin product was obtained, 4) The mouse kidney cDNA library was transformed into the AH109/pGBKT7-podocin and screened by selection steps, 5) Next, twelve clones were cultured and isolated, 6) The yeast-purified plasmids were transformed into Escherichia coli (E. coli) by heat shock, and 7) To identify the activation domain (AD)/library inserts, we digested them with Him III, and the fragments were then sequenced.

Results: 12 positive clones that interacted with podocin were obtained by screening a mouse kidney cDNA library using pGBKT7-podocin. Among them, only 4 clones were found to function at the podocyte where podocin is present.

Conclusion: Additional studies are needed to clarify the role and interaction with podocin and candidates.

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References

Baricos W, Shah S . Proteolytic enzymes as mediators of glomerular injury. Kidney Int. 1991; 40(2):161-73. DOI: 10.1038/ki.1991.196. View

Sharp D, Yu W, Ferhat L, Kuriyama R, Rueger D, Baas P . Identification of a microtubule-associated motor protein essential for dendritic differentiation. J Cell Biol. 1997; 138(4):833-43. PMC: 2138050. DOI: 10.1083/jcb.138.4.833. View

FIEGELSON E, Drake J, RECANT L . Experimental aminonucleoside nephrosis in rats. J Lab Clin Med. 1957; 50(3):437-46. View

Ito K, Ger Y, Kawamura S . Actin filament alterations in glomerular epithelial cells of adriamycin-induced nephrotic rats. Acta Pathol Jpn. 1986; 36(2):253-60. DOI: 10.1111/j.1440-1827.1986.tb01477.x. View

Uetz P, Giot L, Cagney G, Mansfield T, Judson R, Knight J . A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae. Nature. 2000; 403(6770):623-7. DOI: 10.1038/35001009. View

Donoviel D, Freed D, Vogel H, Potter D, Hawkins E, Barrish J . Proteinuria and perinatal lethality in mice lacking NEPH1, a novel protein with homology to NEPHRIN. Mol Cell Biol. 2001; 21(14):4829-36. PMC: 87176. DOI: 10.1128/MCB.21.14.4829-4836.2001. View

Yu W, Cook C, Sauter C, Kuriyama R, KAPLAN P, Baas P . Depletion of a microtubule-associated motor protein induces the loss of dendritic identity. J Neurosci. 2000; 20(15):5782-91. PMC: 6772545. View

Smoyer W, Mundel P . Regulation of podocyte structure during the development of nephrotic syndrome. J Mol Med (Berl). 1998; 76(3-4):172-83. DOI: 10.1007/s001090050206. View

Schwarz K, Simons M, Reiser J, Saleem M, Faul C, Kriz W . Podocin, a raft-associated component of the glomerular slit diaphragm, interacts with CD2AP and nephrin. J Clin Invest. 2001; 108(11):1621-9. PMC: 200981. DOI: 10.1172/JCI12849. View

10.

Boute N, Gribouval O, Roselli S, Benessy F, Lee H, Fuchshuber A . NPHS2, encoding the glomerular protein podocin, is mutated in autosomal recessive steroid-resistant nephrotic syndrome. Nat Genet. 2000; 24(4):349-54. DOI: 10.1038/74166. View

11.

Putaala H, Soininen R, Kilpelainen P, WARTIOVAARA J, Tryggvason K . The murine nephrin gene is specifically expressed in kidney, brain and pancreas: inactivation of the gene leads to massive proteinuria and neonatal death. Hum Mol Genet. 2001; 10(1):1-8. DOI: 10.1093/hmg/10.1.1. View

12.

Fields S, Song O . A novel genetic system to detect protein-protein interactions. Nature. 1989; 340(6230):245-6. DOI: 10.1038/340245a0. View

13.

Yu W, Sharp D, Kuriyama R, Mallik P, Baas P . Inhibition of a mitotic motor compromises the formation of dendrite-like processes from neuroblastoma cells. J Cell Biol. 1997; 136(3):659-68. PMC: 2134303. DOI: 10.1083/jcb.136.3.659. View

14.

Schmitz P, Kasiske B, ODonnell M, Keane W . Lipids and progressive renal injury. Semin Nephrol. 1989; 9(4):354-69. View

15.

Kriz W, Hahnel B, Rosener S, Elger M . Long-term treatment of rats with FGF-2 results in focal segmental glomerulosclerosis. Kidney Int. 1995; 48(5):1435-50. DOI: 10.1038/ki.1995.433. View

16.

Farquhar M, Vernier R, GOOD R . An electron microscope study of the glomerulus in nephrosis, glomerulonephritis, and lupus erythematosus. J Exp Med. 1957; 106(5):649-60. PMC: 2136823. DOI: 10.1084/jem.106.5.649. View

17.

Daniels B, Deen W, Mayer G, Meyer T, Hostetter T . Glomerular permeability barrier in the rat. Functional assessment by in vitro methods. J Clin Invest. 1993; 92(2):929-36. PMC: 294932. DOI: 10.1172/JCI116668. View

18.

Holthofer H, Ahola H, Solin M, Wang S, Palmen T, Luimula P . Nephrin localizes at the podocyte filtration slit area and is characteristically spliced in the human kidney. Am J Pathol. 1999; 155(5):1681-7. PMC: 1866978. DOI: 10.1016/S0002-9440(10)65483-1. View

19.

Gesualdo L, Pinzani M, Floriano J, Hassan M, Nagy N, Schena F . Platelet-derived growth factor expression in mesangial proliferative glomerulonephritis. Lab Invest. 1991; 65(2):160-7. View

20.

Gwinner W, Landmesser U, Brandes R, Kubat B, Plasger J, Eberhard O . Reactive oxygen species and antioxidant defense in puromycin aminonucleoside glomerulopathy. J Am Soc Nephrol. 1997; 8(11):1722-31. DOI: 10.1681/ASN.V8111722. View