Manganese transport and its regulation in bacteria.

Regulation of manganese acquisition by bacteria occurs by both biochemical regulation of the activity of the transporters and transcriptional regulation of gene expression. Structural analysis suggests that calcium ions may regulate the function of an Mn ATP-binding cassette (ABC)-permease in Synechocystis 6803, a cyanobacterium, as well as in a number of other bacteria. The expression of genes encoding the manganese transporter in Synechocystis 6803 is regulated by a two-component signal-transduction mechanism that has not been previously observed for manganese and zinc transport in bacteria.

Pro-collagenase-1 (matrix metalloproteinase-1) binds the alpha(2)beta(1) integrin upon release from keratinocytes migrating on type I collagen.

In injured skin, collagenase-1 (matrix metalloproteinase-1 (MMP-1)) is induced in migrating keratinocytes. This site-specific expression is regulated by binding of the alpha(2)beta(1) integrin with dermal type I collagen, and the catalytic activity of MMP-1 is required for keratinocyte migration. Because of this functional association among substrate/ligand, receptor, and proteinase, we assessed whether the integrin also directs the compartmentalization of MMP-1 to its matrix target. Indeed, pro-MMP-1 co-localized to sites of alpha(2)beta(1) contacts in migrating keratinocytes. Furthermore, pro-MMP-1 co-immunoprecipitated with alpha(2)beta(1) from keratinocytes, and alpha(2)beta(1) co-immunoprecipitated with pro-MMP-1. No other MMPs bound alpha(2)beta(1), and no other integrins interacted with MMP-1. Pro-MMP-1 also provided a substrate for alpha(2)beta(1)-dependent adhesion of platelets. Complex formation on keratinocytes was most efficient on native type I collagen and reduced or ablated on denatured or cleaved collagen. Competition studies suggested that the alpha(2) I domain interacts with the linker and hemopexin domains of pro-MMP-1, not with the pro-domain. These data indicate that the interaction of pro-MMP-1 with alpha(2)beta(1) confines this proteinase to points of cell contact with collagen and that the ternary complex of integrin, enzyme, and substrate function together to drive and regulate keratinocyte migration.

Alpha2beta1 integrin recognition of the carboxyl-terminal propeptide of type I procollagen: integrin recognition and feed-back regulation of matrix biosynthesis are mediated by distinct sequences.

It has recently been established that the carboxyl-terminal propeptide of type I collagen exert a feedback regulatory effect on extracellular matrix biosynthesis and that the propeptide bind to the alpha2beta1 integrin. This raises the intriguing hypothesis that the regulatory propeptide sequences exert their effects as a consequence of binding to the integrin. We show that recombinant alpha1(I) collagen chain C-terminal propeptide contains a binding site for the intact alpha2beta1 integrin and for a recombinant alpha2 integrin I domain, but not for the alpha1beta1 integrin, a structurally and functionally related collagen/laminin receptor. Additional studies employing a series of recombinant N-terminal and C-terminal deletion mutants, internal fragments of the propeptide, synthetic peptides, recombinant alpha2 integrin I domain and inhibitory monoclonal antibodies established that the previously identified sequences within the alpha1(I) C-terminal propeptide that mediate regulation of matrix biosynthesis are neither necessary nor sufficient for alpha2beta1 integrin binding. In contrast, the integrin recognition site is composed of a conformationally complex determinant located within a structurally distinct 115 amino acid region of the propeptide.

Ligand binding results in divalent cation displacement from the alpha 2 beta 1 integrin I domain: evidence from terbium luminescence spectroscopy.

The alpha 2 beta 1 integrin serves as a cell surface collagen or collagen/laminin receptor. Binding of the integrin to its ligands is largely mediated by the alpha 2 subunit I domain and requires the presence of divalent cations. Terbium ion (Tb3+), a fluorescent trivalent cation that often binds divalent cation-binding sites on proteins, supported binding of the I domain to collagen with half-maximal binding occurring at 5.2 +/- 1.7 microM Tb3+. By fluorescence resonance energy transfer spectroscopy, Tb3+ showed specific and saturable binding to the recombinant I domain with a Kd of 27 +/- 4 microM. Although both Mg2+ and Mn2+ were capable of quenching Tb3+ fluorescence, Mn2+ was much more effective than Mg2+. The alpha 2 beta 1 integrin also binds the pro-alpha 1(I) collagen carboxyl-terminal propeptide in a Mg2+-dependent manner via the I domain. Recombinant propeptide was used to examine the effect of ligand on the Tb3+ binding properties of the alpha 2 integrin I domain. As propeptide bound to the I domain, Tb3+ fluorescence progressively diminished suggesting that as ligand binds to the I domain, either Tb3+ is displaced or its fluorescence is quenched. Consistent with the former possibility, little dissociation of collagen-bound I domain occurred upon the addition of EDTA and subsequent incubation. These data support a model in which (1) the divalent cation is required for initial ligand-binding activity of the I domain and (2) ligand binding results in subsequent metal ion displacement to generate a metal-free I domain-ligand complex.

Irreversible steps in the ferritin synthesis induction pathway.

The ability of cells to re-repress ferritin synthesis after removal of an inducing agent (iron or heme) was investigated. Re-repression was found to be a slow process, requiring approximately 4 (after iron removal) to 10 h (after heme removal) for completion. Desferrioxamine mesylate (Desferal) had only a slight effect on the rate of re-repression, whereas cycloheximide was strongly inhibitory, indicating that new protein synthesis is required for re-repression. Re-repression occurred at a slow but significant rate in the presence of both Desferal and cycloheximide. These results indicate that, in the absence of an iron chelator, the induction of ferritin synthesis is essentially irreversible. The kinetics of the previously reported covalent modification of IRE-binding protein (IRE-BP) were then examined, to see whether this phenomenon might account (at least in part) for the irreversibility of induction. It was found that the heme- or iron-dependent disappearance of 98-kDa IRE-BP occurred rapidly (within 1 h), and was equally rapidly reversed upon removal of heme after a 1-h exposure. By contrast, after a 4-h exposure to heme, little 98-kDa IRE-BP could be regenerated after heme removal. These results suggest that the slow, irreversible covalent modification of IRE-BP correlates closely over time with the induction of ferritin synthesis. The covalent modification of IRE-BP depends on cell growth rate, and is most readily detected in rapidly growing cells.

Specificity of a promoter from the rice tungro bacilliform virus for expression in phloem tissues.

The major promoter region for the transcription of the genome of rice tungro bacilliform virus (RTBV), a newly described badnavirus, has been identified. Fragments of the RTBV genome upstream of the site of transcription initiation were isolated and tested for promoter activity using a beta-glucuronidase receptor gene (gusA). Assays of transient gusA expression were performed following introduction of the chimeric gene into protoplasts via electroporation. The chimeric RTBV-promoter: gusA gene was more active in rice protoplasts than in maize or tobacco protoplasts, but was weaker than gusA controlled by an enhanced 35S promoter from cauliflower mosaic virus. Analysis of gusA gene expression following introduction of chimeric reporter genes into intact leaves via micro-projectile bombardment indicated that the GUS activity is present primarily in vascular tissues. Transgenic rice plants carrying the chimeric gusA gene had GUS activity only in the phloem of the vascular bundles in the leaf. Tissue printing studies demonstrated that RTBV accumulates in the vascular bundles of infected rice leaves. The results of our study indicate that phloem-specific expression from the RTBV promoter is an intrinsic property of the viral promoter.

Enhanced degradation of the ferritin repressor protein during induction of ferritin messenger RNA translation.

Induction of ferritin synthesis in cultured cells by heme or iron is accompanied by degradation of the ferritin repressor protein (FRP). Intermediates in the degradative pathway apparently include FRP covalently linked in larger aggregates. The effect of iron on FRP degradation is enhanced by porphyrin precursors but is decreased by inhibitors of porphyrin synthesis, which implies that heme is an active agent. These results suggest that translational induction in this system may be caused by enhanced repressor degradation. While unique among translational regulatory systems, this process is common to a variety of other biosynthetic control mechanisms.