Ferritin, a metalloprotein which stores iron in a bioavailable form, overcomes the low solubility (10-18M) of iron in living systems. Small amounts of ferritin occur in all cells of higher organisms, providing iron for proteins of DNA synthesis, electron transport, and oxygen activation and transport, while large amounts occur in specialized cells of iron storage, e.g. liver, spleen, and red cells of embryo. Pathological conditions occur when iron stores are low or are overloaded. Superimposed on a highly conserved sequence are cell-specific features of ferritin sequence and structure which may relate to function and/or regulation. Ferritin structure could be further modified by cytoplasmic components leading to functional changes. We have recently identified such a change: ferritin subunit dimer crosslinks, a posttranslational modification of ferritin, appear to regulate the iron content of ferritin in vivo. The observation is the first identified, natural structural modification related to the iron storage function of ferritin, to our knowledge. Crosslinking with F2DNB replicates the effect. The structural requirements for crosslinks (which are not -S-S) will be examined in terms of the identity of the linked amino acids, the sequence and subunit restrictions (by sequence analysis and comparison to known subunit sequences), the sequences recognized by monoclonal antibodies sensitive to the presence of crosslinks, the biosynthetic pathway of crosslink formation (H-3-leucine pulse labeling) and cytoplasmic components which may influence crosslinks (transpeptidases? ascorbate?). Cloned ferritin cDNA from lamb spleen or other sources will be prepared and used to gain information about the sequence and regulation of ferritin mRNA related to crosslinks. The functional effect of crosslinks will be measured as the effect of crosslink-sensitive monoclonal antibodies on iron uptake and release in vitro and as the effect of crosslinks on Fe-protein interactions, phosphate-Fe-protein interactions, and iron core structure, using X-ray absorption spectroscopy (EXAFS and XANES). The results will be important in understanding the molecular basis for pathologically altered iron storage, e.g. iron deficiency anemia, hemochromatosis, thalassemia, and cirrhosis, as well as understanding the relationship among cell-specific features of protein structure, cytoplasmic modifying agents, and regulation of function.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM034675-02
Application #
3286062
Study Section
Biophysics and Biophysical Chemistry B Study Section (BBCB)
Project Start
1985-08-01
Project End
1989-07-31
Budget Start
1986-08-01
Budget End
1987-07-31
Support Year
2
Fiscal Year
1986
Total Cost
Indirect Cost
Name
North Carolina State University Raleigh
Department
Type
Schools of Arts and Sciences
DUNS #
City
Raleigh
State
NC
Country
United States
Zip Code
27695
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Rohrer, J S; Islam, Q T; Watt, G D et al. (1990) Iron environment in ferritin with large amounts of phosphate, from Azotobacter vinelandii and horse spleen, analyzed using extended X-ray absorption fine structure (EXAFS). Biochemistry 29:259-64
Islam, Q T; Sayers, D E; Gorun, S M et al. (1989) A comparison of an undecairon(III) complex with the ferritin iron core. J Inorg Biochem 36:51-62
Theil, E C; Sayers, D E (1989) Iron core formation in ferritins. Basic Life Sci 51:161-7
McKenzie, R A; Yablonski, M J; Gillespie, G Y et al. (1989) Crosslinks between intramolecular pairs of ferritin subunits: effects on both H and L subunits and on immunoreactivity of sheep spleen ferritin. Arch Biochem Biophys 272:88-96
Theil, E C (1987) Ferritin: structure, gene regulation, and cellular function in animals, plants, and microorganisms. Annu Rev Biochem 56:289-315
Rohrer, J S; Joo, M S; Dartyge, E et al. (1987) Stabilization of iron in a ferrous form by ferritin. A study using dispersive and conventional x-ray absorption spectroscopy. J Biol Chem 262:13385-7