Copper Stimulates Endocytosis of the Prion Protein

• DOI: 10.1074/jbc.273.50.33107
• Publication Date: 2002-07-26T15:13:03Z
• Authors: Peter C. Pauly; David A. Harris
• Keywords: 0301 basic medicine; Mice; 03 medical and health sciences; Prions; Phosphatidylinositol Diacylglycerol-Lyase; Type C Phospholipases; Tumor Cells, Cultured; Animals; Copper; Endocytosis

Prion diseases result from conformational alteration of PrPC, a cell surface glycoprotein expressed in brain, spinal cord, and several peripheral tissues, into PrPSc, protease-resistant isoform that is the principal component infectious prion particles. Although great deal known about pathogenic role physiological function PrPC has remained mystery. Several lines evidence have recently suggested possibility PrPCmay play metabolism copper. To further investigate interaction copper, we analyzed effect this metal ion on endocytic trafficking cultured neuroblastoma cells. We report here copper rapidly reversibly stimulates endocytosis surface. This may be physiologically relevant suggests hypothesis could serve as recycling receptor for uptake ions extracellular milieu. are neurodegenerative disorders conversion normal (PrPC) 1The abbreviations used are: cellular protein; scrapie PrP, chPrP, chicken moPrP, mouse PAGE, polyacrylamide gel electrophoresis; PIPLC, phosphatidylinositol-specific phospholipase C; SOD, superoxide dismutase. (PrPSc) appears to absence nucleic acid (1Prusiner S.B. Science. 1997; 278: 245-251Crossref PubMed Scopus (852) Google Scholar, 2Prusiner Scott M.R. DeArmond S.J. Cohen F.E. Cell. 1998; 93: 337-348Abstract Full Text PDF (819) Scholar). wealth information now available PrPSc disease process, relatively little normal, PrPC. neurons glia brain cord at lower levels tissues leukocytes (3Bendheim P.E. Brown H.R. Rudelli R.D. Scala L.J. Goller N.L. Wen G.Y. Kascsak R.J. Cashman N.R. Bolton D.C. Neurology. 1992; 42: 149-156Crossref 4Manson J. West J.D. Thomson V. McBride P. Kaufman M.H. 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Commun. 207: 621-629Crossref (321) 16Hornshaw Lakey J.H. 214: 993-999Crossref (294) 17Miura Horii Takeuchi FEBS 396: 248-252Crossref (141) 18Brown D.R. Qin K.F. Herms J.W. Madlung Strome Fraser Kruck Vonbohlen Schulzschaeffer W. Giese Westaway D. Kretzschmar 390: 684-687Crossref (37) 19Stöckel Safar Wallace A.C. Biochemistry. 37: 7185-7193Crossref (491) Copper also facilitates restoration protease resistance infectivity during refolding guanidine-denatured (20McKenzie Bartz Mirwald J.D.O. Marsh Aiken Biol. Chem. 273: 25545-25547Abstract (123) Second, total content other metals only 20% crude membranes, synaptosomes, endosomes derived brains ofPrn-p0/0 mice contain disrupted (18Brown major copper-binding protein brain. Third, neuronal Cu-Zn dismutase (SOD) Prn-p0/0 less enzymatically active incorporates radioactive than enzyme mice; opposite true SOD transgenic over-express (21Brown W.J. Schmidt H.A. Exp. Neurol. 146: 104-112Crossref (390) 22Brown Besinger 334: 423-429Crossref (243) Neurons fromPrn-p0/0 more sensitive oxidative insult, perhaps reduced activity These observations raise involved delivery cuproenzymes. Finally, PC12 selected toxicity increased expression suggesting removal detoxification (23Brown Neurochem. 70: 1686-1693Crossref (144) Scholar).Exactly how functionally related unclear present. One bound serves an essential cofactor yet undetermined enzymatic it does cuproproteins such cytochrome c oxidase (24Linder M.C. Goode C.A. Biochemistry Copper. Plenum Press, New York1991Crossref By virtue its presence surface, sink chelation carrier intracellular targets. An piece shed light functional affects shown previously constitutively endocytosed plasma membrane via clathrin-coated pits transits early endosomal compartment before being recycled back (25Shyng S.L. Huber M.T. Harris 268: 15922-15928Abstract 26Shyng Heuser J.E. Cell 125: 1239-1250Crossref (221) 27Shyng Moulder K.L. Lesko 270: 14793-14800Abstract (148) 28Harris Gorodinsky Lehmann Shyng S.-L. Curr. Top. Microbiol. Immunol. 77-93PubMed dramatic cycle.RESULTSWe characterized using N2a stably transfected express either moPrP chPrP (29Harris van Dijken Chait B.T. Wang 32: 1009-1016Crossref (187) 25Shyng therefore examine effects distribution determine whether altered amount treated 90 min CuSO4 then biotinylated them membrane-impermeable reagent. The was determined by immunoprecipitating lysates, running immunoprecipitates SDS-PAGE, developing blots horseradish peroxidase-streptavidin ECL. found concentrations 200 μm above, CuSO4dramatically amounts both (Fig. 1). Similar results were obtained assayed sensitivity externally applied trypsin (data shown).Because tested explained stimulatory endocytosis. measure endocytosis, surface-iodinated 4 °C, warmed 37 °C initiate internalization, scored iodinated became inaccessible C (PIPLC), bacterial cleaves glycolipid anchor responsible attaching membrane. Using assay, 500 μmCuSO4 stimulated internalization 2, A B) (not shown). increase internalized seen after 5 incubation, 30 65% internalized, compared 15% untreated Stimulation demonstrated biotinylation rather iodination label molecules. procedure, minimum concentration CuSO4required produce observable ∼100 D). At concentration, ZnSO4 no CoCl2, MnCl2, FeSO4 due generalized proteins, did significantly alter [125I]-transferrin shown).Figure 2Copper chPrP. A, expressing indicated periods time μmCuSO4. Cells PIPLC immunoprecipitated incubation media (surface,S lanes) lysates (internal, I SDS-PAGE autoradiography. B, quantitated PhosphorImager analysis lanes inpanel internal percentage (surface + internal) each point. (The course slower experiments published work probably differences growth conditions cells).C, surface-biotinylated CuSO4. (surface, S lanes), separated visualized development D, film densitometry panel C, concentration. figure representative least three similar ones.View Large Image Figure ViewerDownload (PPT)We reversible. Within 15 metal, began redistribute inside outside cell, accessibility (Fig.3, B). 60 min, steady-state reached 40% internal, same readjustment presumably documented ability molecules recycle they directly first incubated 20 stimulate stripped treatment finally re-warmed release medium. procedure 3, andD), absent re-warming medium, almost all t 12 10 Fig. 3 Interestingly, present returned although somewhat slowly (t = min). implies markedly rate much recycling.Previous implicated mammalian region efficient (27Shyng N-terminally deleted forms assay 4). normally eight hexapeptide (31Harris Falls D.L. Johnson F.A. Fischbach G.D. 1991; 88: 7664-7668Crossref (112) Deletion one-third molecule (Δ25–91), including repeats, completely abolished underwent degradation experiment four (Δ42–65) last (Δ66–91) attenuated eliminate copper-induced remaining retain residual coordinating activity.Figure 4N-terminally efficiently response copper.Internalization wild-type biotinylation, described legend 2 internal).Bars represent mean ± two six independent experiments.View (PPT)DISCUSSIONOur previous passes through demonstrate arm cycle.It attractive speculate attributable purified, ions, most likely Zinc binds tightly (32Pan K.M. Stahl Protein 1: 1343-1352Crossref (172) Consistent direct find missing full-length altering conformation increases affinity putative localizes pits. Because attached glycosyl-phosphatidylinositol anchor, lacks cytoplasmic domain components coated postulated existence whose signals interacting adapter clathrin Scholar).Enhanced requires range 100–500 10–50-fold greater estimated synthetic synthesized bacteria discrepancy due, part, amino acids Opti-MEM medium our experiments, phenomenon free It possible occurs sites cell-expressed distinct ones bacterially PrPCinternalization. In case, average even higher specific regions (19Stöckel 33Smith R.M. Dreosti I.E. Neurobiology Trace Elements. 1. Human Clifton, NJ1983: 1-40Google Therefore, potentially standpoint.One might deliver within dissociate transferred copper-carrier proteins move cytosol. return begin another cycle. proposed analogous transferrin iron (34Ciechanover Schwartz A.L. Dautry-Varsat Lodish H.F. 1983; 258: 9681-9689Abstract exception case comparable transferrin. transporters chaperones identified yeast humans (35Valentine J.S. Gralla E.B. 817-818Crossref (189) molecular mechanism still poorly understood (36Vulpe C.D. Packman Annu. Rev. Nutr. 15: 293-322Crossref (241) Further exploration between level subject. Pr