Ferritin Contains Less Iron (59Fe) in Cells When the Protein Pores Are Unfolded by Mutation

Ferric minerals in ferritins are protected from cytoplasmic reductants and Fe2+ release by the protein nanocage until iron need is signaled. Deletion of ferritin genes is lethal; two critical ferritin functions are concentrating iron and oxidant protection (consuming cytoplasmic iron and oxygen in the mineral). In solution, opening/closing (gating) of eight ferritin protein pores controls reactions between external reductant and the ferritin mineral; pore gating is altered by mutation, low heat, and physiological urea (1 mm) and monitored by CD spectroscopy, protein crystallography, and Fe2+ release rates. To study the effects of a ferritin pore gating mutation in living cells, we cloned/expressed human ferritin H and H L138P, homologous to the frog open pore model that was unexpressable in human cells. Human ferritin H L138P behaved like the open pore ferritin model in vitro as follows: (i) normal protein cage assembly and mineralization, (ii) increased iron release (t½ decreased 17-fold), and (iii) decreased α-helix (8%). Overexpression (>4-fold), in HeLa cells, showed for ferritin H L138P equal protein expression and total cell 59Fe but increased chelatable iron, 16%, p < 0.01 (59Fe in the deferoxamine-containing medium), and decreased 59Fe in ferritin, 28%, p < 0.01, compared with wild type. The coincidence of decreased 59Fe in open pore ferritin with increased chelatable 59Fe in cells expressing the ferritin open pore mutation suggests that ferritin pore gating influences to the amount of iron (59Fe) in ferritin in vivo. Ferric minerals in ferritins are protected from cytoplasmic reductants and Fe2+ release by the protein nanocage until iron need is signaled. Deletion of ferritin genes is lethal; two critical ferritin functions are concentrating iron and oxidant protection (consuming cytoplasmic iron and oxygen in the mineral). In solution, opening/closing (gating) of eight ferritin protein pores controls reactions between external reductant and the ferritin mineral; pore gating is altered by mutation, low heat, and physiological urea (1 mm) and monitored by CD spectroscopy, protein crystallography, and Fe2+ release rates. To study the effects of a ferritin pore gating mutation in living cells, we cloned/expressed human ferritin H and H L138P, homologous to the frog open pore model that was unexpressable in human cells. Human ferritin H L138P behaved like the open pore ferritin model in vitro as follows: (i) normal protein cage assembly and mineralization, (ii) increased iron release (t½ decreased 17-fold), and (iii) decreased α-helix (8%). Overexpression (>4-fold), in HeLa cells, showed for ferritin H L138P equal protein expression and total cell 59Fe but increased chelatable iron, 16%, p < 0.01 (59Fe in the deferoxamine-containing medium), and decreased 59Fe in ferritin, 28%, p < 0.01, compared with wild type. The coincidence of decreased 59Fe in open pore ferritin with increased chelatable 59Fe in cells expressing the ferritin open pore mutation suggests that ferritin pore gating influences to the amount of iron (59Fe) in ferritin in vivo. Ferritins are cytoplasmic protein cages with an internal ironoxygen mineral. Gene deletion is embryonic lethal in mice (1Ferreira C. Bucchini D. Martin M.E. Levi S. Arosio P. Grandchamp B. Beaumont C. J. Biol. Chem. 2000; 275: 3021-3024Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar); ferritins concentrate iron over the 1014-fold concentration gradient between cell need and iron solubility under physiological conditions (2Liu X. Theil E.C. Acc. Chem. 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The ferritin pores are gated to control localized folding/unfolding and protect the ferric mineral from reductants and Fe2+ exit when they are closed/folded (12Takagi H. Shi D. Ha Y. Allewell N.M. Theil E.C. J. Biol. Chem. 1998; 273: 18685-18688Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 13Jin W. Takagi H. Pancorbo B. Theil E.C. Biochemistry. 2001; 40: 7525-7532Crossref PubMed Scopus (85) Google Scholar, 14Liu X. Jin W. Theil E.C. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 3653-3658Crossref PubMed Scopus (176) Google Scholar, 15Theil E.C. Liu X.S. Tosha T. Inorg. Chim. Acta. 2008; 361: 868-874Crossref PubMed Scopus (65) Google Scholar). How cells regulate the recovery of iron from the ferritin mineral is only partly understood. A recent study in cells rendered iron-deficient by overexpression of the iron export protein ferroportin showed that iron was released from cytoplasmic ferritin followed by protein ubiquitinylation and proteasomal degradation (16De Domenico I. Vaughn M.B. Li L. Bagley D. Musci G. Ward D.M. Kaplan J. EMBO J. 2006; 25: 5396-5404Crossref PubMed Scopus (170) Google Scholar). A number of other studies used cells with iron excess that increased the amount of ferritin protein mediated by increased DNA transcription (6Hintze K.J. Theil E.C. Cell. Mol. Life Sci. 2006; 63: 591-600Crossref PubMed Scopus (151) Google Scholar), increased mRNA translation (7Hentze M.W. Muckenthaler M.U. Andrews N.C. Cell. 2004; 117: 285-297Abstract Full Text Full Text PDF PubMed Scopus (1398) Google Scholar, 8Theil E.C. Eisenstein R.S. J. Biol. Chem. 2000; 275: 40659-40662Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar), and decreased protein turnover (17Harrison P.M. Arosio P. Biochim. Biophys. 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In normal cells then, mechanisms exist for enhancing the removal of iron from the ferritin nanocages when cell need increases, as in growth or iron deficiency (16De Domenico I. Vaughn M.B. Li L. Bagley D. Musci G. Ward D.M. Kaplan J. EMBO J. 2006; 25: 5396-5404Crossref PubMed Scopus (170) Google Scholar, 20Lipschitz D.A. Simon M.O. Lynch S.R. Dugard J. Bothwell T.H. Charlton R.W. Br. J. Haematol. 1971; 21: 289-303Crossref PubMed Scopus (50) Google Scholar, 21Bothwell T.H. Charlton R.W. Cook J.D. Finch C.A. Jacobs A. Worwood M. Iron in Biochemistry and Medicine. Blackwell Scientific Publications, Oxford1979: Scholar, M. F. M. Proc. Natl. Acad. Sci. U. S. A. 2005; PubMed Scopus Google Scholar). iron from ferritin in is when reductants are that cytoplasmic reductants (12Takagi H. Shi D. Ha Y. Allewell N.M. Theil E.C. J. Biol. Chem. 1998; 273: 18685-18688Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, T. C. Biochemistry. 17: PubMed Scopus Google Scholar, F. F. J. Inorg. Biochem. PubMed Scopus Google Scholar, M.C. A. Harrison P.M. J. Inorg. Biochem. PubMed Scopus Google Scholar), well the of iron removal in Am. J. 2007; PubMed Scopus Google Scholar). mutation of of a of the of that the pores the of iron from ferritin when a reductant such as is (12Takagi H. Shi D. Ha Y. Allewell N.M. Theil E.C. J. Biol. Chem. 1998; 273: 18685-18688Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 13Jin W. Takagi H. Pancorbo B. Theil E.C. Biochemistry. 2001; 40: 7525-7532Crossref PubMed Scopus (85) Google Scholar). of the ferritin protein cage the pores in wild proteins is by or low of urea or in proteins in and CD of the protein cage is to of the ferric mineral to Fe2+ (12Takagi H. Shi D. Ha Y. Allewell N.M. Theil E.C. J. Biol. Chem. 1998; 273: 18685-18688Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 14Liu X. Jin W. Theil E.C. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 3653-3658Crossref PubMed Scopus (176) Google Scholar). now the of a pore mutation the amount of 59Fe in the protein compared with wild to the concentration in HeLa cells. to in human H ferritin, the well frog ferritin H mutation the homologous be in human cells, a protein with to the frog model as follows: increased iron removal in and decreased α-helix the of ferritin pore The amount of iron (59Fe) in the human ferritin H was < wild ferritin when to the in HeLa cells. the 59Fe in cells expressing the ferritin was < to compared with cells expressing wild ferritin H. The a of pore to the recovery of iron from ferritin minerals in vivo. of and DNA for the of human ferritin H used human ferritin human wild ferritin L138P, human ferritin H with pores by of a in the from HeLa cell and in was the of for in the two and A expression for was expression for from as follows: and The was to the of to The for in was with the for by to the in both the and protein used as follows: and expression of DNA to DNA The wild and the pore ferritins in E. and by the Theil E.C. Biochemistry. PubMed Scopus Google Scholar), Iron and Fe2+ was monitored as the in Ferritins by a with to a concentration of protein iron and as (12Takagi H. Shi D. Ha Y. Allewell N.M. Theil E.C. J. Biol. Chem. 1998; 273: 18685-18688Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Iron release was by reductant T. C. Biochemistry. 17: PubMed Scopus Google Scholar) with as a and as (12Takagi H. Shi D. Ha Y. Allewell N.M. Theil E.C. J. Biol. Chem. 1998; 273: 18685-18688Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 14Liu X. Jin W. Theil E.C. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 3653-3658Crossref PubMed Scopus (176) Google Scholar). ferritin, and Fe2+ release was monitored as of the the protein as the in the for T. C. Biochemistry. 17: PubMed Scopus Google Scholar), from the of the from to as we X. Jin W. Theil E.C. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 3653-3658Crossref PubMed Scopus (176) Google Scholar). The are as the of the from protein for of ferritin with for of and the is the CD a The protein nanocages in from to with in a cell with a from The was from the as follows: Biochemistry. PubMed Scopus Google Scholar). in Life was with an of a of as in J. Biol. Chem. Full Text PDF PubMed Google Scholar, to iron as was in and to the 59Fe was to for and the was for a the was and protein concentration by the the 59Fe was in by and Ferritin with of cells in with mm) and (1 mm) cells a of cells well in in of cells with with or ferritin a DNA to of of HeLa cells was used to the the of the ferritin pore by the to the frog H pore ferritin and by the expression we of human wild and L138P protein in cells. was the and with and The cells for and the concentration was the and with 59Fe cells in by and the cells by 59Fe was the cell in a In with was to cells. The was for followed by and of cell and 59Fe in cell and from cell in of by and by 59Fe was by and protein by the of total with and with Ferritin with with a human ferritin for ferritin concentration and in cell in of cells iron or in cell with cell The are and the is as the protein wild and was in the HeLa cell with of used and in and with ferritin was ferritin as the and as the was with an was the by ferritin with of over and with over followed by the addition of for and with Human ferritin was to the over the concentration of ferritin in the cell as To ferritin from cell of protein was from of cells from in of and as ferritin was used with a protein to Ferritin was in and both 59Fe and ferritin protein and human ferritin from by ferritin with in a to the in Human Ferritin ferritin H was to study the effects of ferritin pores by mutation the (12Takagi H. Shi D. Ha Y. Allewell N.M. Theil E.C. J. Biol. Chem. 1998; 273: 18685-18688Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 13Jin W. Takagi H. Pancorbo B. Theil E.C. Biochemistry. 2001; 40: 7525-7532Crossref PubMed Scopus (85) Google Scholar, 14Liu X. Jin W. Theil E.C. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 3653-3658Crossref PubMed Scopus (176) Google Scholar, X.S. Theil E.C. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar) frog ferritin H pore and frog H wild be in or cells. of cells with the expression frog ferritin H and H the pore cell and the expression of proteins was in the cells. to human ferritin H the of compared with or frog ferritins P.M. T.H. Iron and Iron Scholar), by Thus, we human ferritin H L138P, is homologous to a mutation in of the pore gating ferritin as ferritin pore folding/unfolding are only in human and frog ferritins but other known the of frog and human ferritin H is (17Harrison P.M. Arosio P. Biochim. Biophys. Acta. 1996; 1275: 161-203Crossref PubMed Scopus (2252) Google Scholar). of the human ferritin H L138P ferritin protein cage was normal as in the homologous frog ferritin (12Takagi H. Shi D. Ha Y. Allewell N.M. Theil E.C. J. Biol. Chem. 1998; 273: 18685-18688Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). the protein was for wild and H L138P as W. Takagi H. Pancorbo B. Theil E.C. Biochemistry. 2001; 40: 7525-7532Crossref PubMed Scopus (85) Google Scholar). the iron release was increased to the frog protein (12Takagi H. Shi D. Ha Y. Allewell N.M. Theil E.C. J. Biol. Chem. 1998; 273: 18685-18688Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar), the of the The of iron release from human ferritin L138P was the wild protein the to and release of the mineral to the protein cage (12Takagi H. Shi D. Ha Y. Allewell N.M. Theil E.C. J. Biol. Chem. 1998; 273: 18685-18688Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar) was for human ferritin H L138P for the wild protein and A of pore in ferritin is a of by CD X. Jin W. Theil E.C. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 3653-3658Crossref PubMed Scopus (176) Google Scholar); the α-helix of ferritin is A of in the of the human H L138P ferritin was to that the eight pores The are with in the frog H Thus, the of decreased and iron by a are for both the human H L138P and the homologous frog H the of of the pore to iron release from ferritins with by stability the and in HeLa 59Fe the of gated pores in by 59Fe and in HeLa cells or pore human ferritin H to 59Fe was as HeLa cells as the cell expression from of and wild human ferritins was with cells expression of the human ferritin was wild type. The of wild and human ferritins in cells was to human ferritin in both and both normal and normal assembly of the proteins in the HeLa cells. Overexpression increased ferritin by over and and by of compared with the control with The to the that for the of ferritin in are with human ferritin in ferritin we are expressing ferritin in from ferritin by by both ferritin H and ferritin H L138P the ferritin and, for to ferritin pores by mutation ferritin 59Fe in HeLa of 59Fe in HeLa cells are from and the are the control is with is human wild ferritin L138P is human ferritin H with pores by of a in the control is with is human wild ferritin L138P is human ferritin H with pores by of a in the pore control is with is human wild ferritin L138P is human ferritin H with pores by of a in the cell 59Fe concentration of of total 59Fe total are from pore or p < and p < are from pore or p < and p < in the cell of are from pore or p < and p < are from pore or p < and p < The are from and the are the control is with is human wild ferritin L138P is human ferritin H with pores by of a in the are from pore or p < and p < in a To the of ferritin pores 59Fe in we HeLa cells with for was of the protein total cell of 59Fe proteins in the cell by in of the 59Fe in the with ferritin protein 59Fe in was of total The the of cell 59Fe that is in ferritin compared with and the iron the amount of 59Fe in total protein be used to the of protein compared in cells expressing the wild and pore the was < in cells wild ferritin The that the pore ferritin 59Fe in in iron and normal in the human ferritin H L138P of 59Fe in ferritin iron by the of the of Ferritin with pores in HeLa cells and of with only 59Fe of the wild ferritin to the Fe2+ exit in vitro To of the cells the protein in cells ferritin H with the pore mutation L138P, cells for the removal of with 59Fe was of the and for cells with wild or ferritin compared with the of and for cells with wild and ferritin and In the of for with the 59Fe and and In the proteins was < for cells expressing open pore In the of protein for cells with and wild protein was was of 59Fe and was with or the of pore gating to the iron (59Fe) in ferritin pores by mutation chelatable 59Fe in HeLa of 59Fe in HeLa an of from and the are the with the human wild ferritin L138P, human ferritin H with pores by of a in the with the human wild ferritin L138P, human ferritin H with pores by of a in the pore only was with the human wild ferritin L138P, human ferritin H with pores by of a in the cell 59Fe only was 59Fe total are from pore or p < p < are from pore or p < p < 59Fe in the of only was Ferritin concentration of of total cell 59Fe 59Fe total are from pore or p < p < are from pore or p < p < 59Fe in the of are from pore or p < p < are from pore or p < p < Ferritin concentration of of total an of from and the are the with the human wild ferritin L138P, human ferritin H with pores by of a in the only was are from pore or p < p < in a 59Fe to in HeLa Ferritin H L138P ferritin with the pore 59Fe wild ferritin and and we that iron be to iron in the HeLa cells expressing ferritin with pores in cells expressing wild ferritin, as in (2Liu X. Theil E.C. Acc. Chem. Res. 2005; 38: 167-175Crossref PubMed Scopus (431) Google Scholar, X.S. Theil E.C. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). the of protein and in the of a the that iron in the of cells Am. J. 2007; PubMed Scopus Google Scholar) was to the HeLa cell is used to iron deficiency in cells (16De Domenico I. Vaughn M.B. Li L. Bagley D. Musci G. Ward D.M. Kaplan J. EMBO J. 2006; 25: 5396-5404Crossref PubMed Scopus (170) Google Scholar, 18Kidane T.Z. Sauble E. Linder M.C. Am. J. Physiol. 2006; 291: C445-C455Crossref PubMed Scopus (189) Google Scholar). The concentration used decreased ferritin protein in the controls and in the cells ferritin in a in the cytoplasmic 59Fe from cells with the open pore ferritin protein compared with wild In a of 59Fe was in the for cells with the open pore ferritin of with the that of the 59Fe was to the amount of 59Fe in the cytoplasmic of cells in the was in cells expressing the L138P ferritin 59Fe in of in the amount of 59Fe in the was for cells expressing open pore ferritin, that ferritin pore gating is only that influences the amount of iron (59Fe) in Iron is from the ferritin mineral the protein cage in by two During iron iron is from ferritin followed by ubiquitinylation and proteasomal degradation of the protein cage (16De Domenico I. Vaughn M.B. Li L. Bagley D. Musci G. Ward D.M. Kaplan J. EMBO J. 2006; 25: 5396-5404Crossref PubMed Scopus (170) Google Scholar), in iron ferritin is by lysosomal and the ferritin iron is to the T.Z. Sauble E. Linder M.C. Am. J. 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A. 2003; 100: 3653-3658Crossref PubMed Scopus (176) Google Scholar, E.C. J. 2003; PubMed Google Scholar, I. G. S. Biochemistry. PubMed Scopus Google Scholar). Ferritin pores are by by the of the protein pore of the eight of a the protein of the pore gating in a ferritin are the in the other two that the pore the of a mutation in gating is by the of the homologous in (12Takagi H. Shi D. Ha Y. Allewell N.M. Theil E.C. J. Biol. Chem. 1998; 273: 18685-18688Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 13Jin W. Takagi H. Pancorbo B. Theil E.C. Biochemistry. 2001; 40: 7525-7532Crossref PubMed Scopus (85) Google Scholar, 14Liu X. Jin W. Theil E.C. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 3653-3658Crossref PubMed Scopus (176) Google Scholar). The of ferritin pore gating with between mineral and reductants removal as well as the and of ferritin pores by a from in a X.S. Theil E.C. J. Biol. 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