Iron regulatory proteins (IRP1 and IRP2) are two cytoplasmic RNA-binding proteins that control iron metabolism in mammalian cells. Both IRPs bind to specific sequences called iron-responsive elements (IREs) located in the 3 or 5 untranslated regions of several mRNAs, in particular mRNA encoding ferritin and transferrin receptor. In this study, we followed in parallel the in vivo regulation of the two IRPs in physiologically stimulated macrophages. We show that stimulation of mouse RAW 264.7 macrophage-like cells increased IRP1 IRE binding activity 4-fold, whereas IRP2 activity decreased 2-fold 8 h after interferon-␥/lipopolysaccharide treatment. Decrease in IRP2 was not due to nitric oxide (NO) production and did not require de novo protein synthesis. Our data therefore indicate that the two IRPs can be conversely regulated in response to the same stimulus. In addition, the effect of endogenously produced NO on IRP1 was further characterized in an activated macrophage/ target cell system. We show that NO acts as an intercellular signal to increase IRP1 activity in adjacent cells. As the effect was detectable within 1 h and did not require de novo protein synthesis, this result supports a direct action of NO on IRP1.Iron regulatory proteins (IRP1 and IRP2) 1 are cytoplasmic trans-regulators that modulate expression of several mRNA containing one or several regulatory sequences in their untranslated regions termed iron-responsive elements (IREs) (1, 2). An IRE has been located at the 5Ј end of the mRNA of ferritin H-and L-chain and erythroid 5-aminolevulinate synthase and, as revealed more recently, on mitochondrial aconitase and subunit b of Drosophila melanogaster succinate dehydrogenase mRNA (3-8). IRE/IRP interaction in the 5Ј untranslated region inhibits mRNA translation (9 -14). Five IRE sequences have also been located in the 3Ј untranslated region of transferrin receptor mRNA and, in that case, IRE/IRP interaction confers stability against endonucleolytic cleavage (15, 16). Thus, IRPs control uptake, storage, and intracellular metabolism of iron through their IRE binding activity.Two IRPs called IRP1 and IRP2 have been characterized and cloned in several cell types (12,17,18). IRP1 exhibits considerable sequence homology with mitochondrial aconitase and has been identified as the cytoplasmic aconitase (19 -21). The two activities of this protein are mutually exclusive. The form that presents aconitase activity converting citrate into isocitrate in the cytosol possesses an intact 4Fe-4S cluster, whereas the IRE-binding form lacks it (22, 23). Thus, the status of the Fe-S cluster is crucial to determination of IRP1 function. In iron-repleted cells, holoIRP1 predominates and exhibits aconitase activity. Conversely, apoIRP1, which binds IRE with high affinity, is the major form in iron-depleted cells. This first discovered regulation of IRP1 by iron led to the suggestion that a switch between the holo-and apoprotein without any change in IRP1 protein levels explains the regulation (2). In vitro, IRP2 binds IRE...

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Transport of DNA into preformed procapsids is a general strategy for genome packing inside virus particles. In most viruses, this task is accomplished by a complex of the viral packaging ATPase with the portal protein assembled at a specialized vertex of the procapsid. Such molecular motor translocates DNA through the central tunnel of the portal protein. A central question to understand this mechanism is whether the portal is a mere conduit for DNA or whether it participates actively on DNA translocation. The most constricted part of the bacteriophage SPP1 portal tunnel is formed by twelve loops, each contributed from one individual subunit. The position of each loop is stabilized by interactions with helix ␣-5, which extends into the portal putative ATPase docking interface. Here, we have engineered intersubunit disulfide bridges between ␣-5s of adjacent portal ring subunits. Such covalent constraint blocked DNA packaging, whereas reduction of the disulfide bridges restored normal packaging activity. DNA exit through the portal in SPP1 virions was unaffected. The data demonstrate that mobility between ␣-5 helices is essential for the mechanism of viral DNA translocation. We propose that the ␣-5 structural rearrangements serve to coordinate ATPase activity with the positions of portal tunnel loops relative to the DNA double helix.Portal proteins are hollow oligomers localized asymmetrically at a single vertex of icosahedral capsids of tailed bacteriophages and herpesviruses (1, 2) providing a tunnel for doublestranded DNA entry and exit (see Fig. 1, A-C) (5-7). The viral genome is packaged into procapsids by a DNA translocation motor that assembles at the portal vertex of the procapsid structure (step from states I to II of Fig. 1A). The motor is composed of the portal protein, an ATPase (large terminase subunit), and a third component (small terminase subunit or a pRNA) (8). How this large complex uses ATP hydrolysis (9 -13) to pump double-stranded DNA against a steep concentration gradient remains a mystery. Although it is well established that the ATPase energetically fuels DNA translocation, biochemical and genetic evidence shows also that the portal protein regulates ATPase activity (13) and that portal mutations impair DNA packaging (13-16). However, a mechanistic role of the portal protein and the function of its cross-talk with the terminase in the DNA packaging reaction remain to be demonstrated. X-ray structures of portal proteins from bacteriophages phi29 (p10) (5) and SPP1 (gp6) (17) show that they share a very similar structure. Their most unusual feature is the presence of a prominent distortion in helix ␣6. Helix ␣6 of the SPP1 portal presents a 136°kink stabilized by hydrogen bonding with crown residues and by van der Waals interactions with the carboxyl terminus of helix ␣5 (see Fig. 1C) (17). In isolated gp6, a 13-mer, helices ␣5 and ␣6 are connected by a loop that protrudes to the portal tunnel interior (17). This region is disorganized in the phi29 portal structure (5).The gp6 form found in ...