The ferritins: molecular properties, iron storage function and cellular regulation☆

Formation of the chromophore of green fluorescent protein (GFP) depends on the correct folding of the protein. We constructed a "folding reporter" vector, in which a test protein is expressed as an N-terminal fusion with GFP. Using a test panel of 20 proteins, we demonstrated that the fluorescence of Escherichia coli cells expressing such GFP fusions is related to the productive folding of the upstream protein domains expressed alone. We used this fluorescent indicator of protein folding to evolve proteins that are normally prone to aggregation during expression in E. coli into closely related proteins that fold robustly and are fully soluble and functional. This approach to improving protein folding does not require functional assays for the protein of interest and provides a simple route to improving protein folding and expression by directed evolution.

Rapid protein-folding assay using green fluorescent protein.

FERRITINis important in iron homeostasis. Its twenty-four chains of two types, H and L, assemble as a hollow shell providing an iron-storage cavity1–3. Ferritin molecules in cells containing high levels of iron tend to be rich in L chains, and may have a long-term storage function, whereas H-rich ferritins are more active in iron metabolism3–7. The molecular basis for the greater activity of H-rich ferritins has until now been obscure, largely because the structure of H-chain ferritin has remained unknown owing to the difficulties in obtaining crystals ordered enough for X-ray crys-tallographic analysis. Here we report the three-dimensional structure of a human ferritin H-chain homopolymer. By genetically engineering a change in the sequence of the intermolecular contact region, we obtained crystals isomorphous with the homologous rat L ferritin8–9 and of high enough quality for X-ray diffraction analysis. The X-ray structure of human H ferritin shows a novel metal site embedded within each of its four-helix bundles and we suggest that ferroxidase activity associated with this site accounts for its rapid uptake of iron10.

Solving the structure of human H ferritin by genetically engineering intermolecular crystal contacts.

It is commonly accepted that the reverse-transcribed cellular RNA molecules, called retroposons, integrate at staggered breaks in mammalian chromosomes. However, unlike what was previously thought, most of the staggered breaks are not generated by random nicking. One of the two nicks involved is primarily associated with the 5′-TTAAAA hexanucleotide and its variants derived by a single base substitution, particularly A → G and T → C. It is probably generated in the antisense strand between the consensus bases 3′-AA and TTTT complementary to 5′-TTAAAA. The sense strand is nicked at variable distances from the TTAAAA consensus site toward the 3′ end, preferably within 15–16 base pairs. The base composition near the second nicking site is also nonrandom at positions preceding the nick. On the basis of the observed sequence patterns it is proposed that integration of mammalian retroposons is mediated by an enzyme with endonucleolytic activity. The best candidate for such enzyme may be the reverse transcriptase encoded by the L1 non-long-terminal-repeat retrotransposon, which contains a freshly reported domain homologous to the apurinic/apyrimidinic (AP) endonuclease family [Martin, F., Olivares, M., Lopez, M. C. & Alonso, C. (1996) Trends Biochem. Sci. 21, 283–285; Feng, Q., Moran, J. V., Kazazian, H. H. & Boeke, J. D. (1996) Cell 87, 905–916] and shows nicking in vitro with preference for targets similar to 5′-TTAAAA/3′-AATTTT consensus sequence [Feng, Q., Moran, J. V., Kazazian, H. H. & Boeke, J. D. (1996) Cell 87, 905–916]. A model for integration of mammalian retroposons based on the presented data is discussed.

Sequence patterns indicate an enzymatic involvement in integration of mammalian retroposons

Regulated translation of messenger RNA offers an important mechanism for the control of gene expression. The biosynthesis of the intracellular iron storage protein ferritin is translationally regulated by iron. A cis-acting element that is both necessary and sufficient for this translational regulation is present within the 5' nontranslated leader region of the human ferritin H-chain messenger RNA. In this report the iron-responsive element (IRE) was identified by deletional analysis. Moreover, a synthetic oligodeoxynucleotide was shown to be able to transfer iron regulation to a construct that would otherwise not be able to respond to iron. The IRE has been highly conserved and predates the evolutionary segregation between amphibians, birds, and man. The IRE may prove to be useful for the design of translationally regulated expression systems.

/pdf/identification-of-the-iron-responsive-element-for-the-1dq8vqk7xb.pdf

Identification of the iron-responsive element for the translational regulation of human ferritin mRNA

Differences in the regulation of messenger RNA for housekeeping and specialized-cell ferritin. A comparison of three distinct ferritin complementary DNAs, the corresponding subunits, and identification of the first processed in amphibia.

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Differences in the regulation of messenger RNA for housekeeping and specialized-cell ferritin. A comparison of three distinct ferritin complementary DNAs, the corresponding subunits, and identification of the first processed in amphibia.