Elastin is a structural protein that provides resilience to biological tissues. We examined the contributions of elastin to the quasi-static tensile response of porcine medial collateral ligament through targeted disruption of the elastin network with pancreatic elastase. Elastase concentration and treatment time were varied to determine a dose response. Whereas elastin content decreased with increasing elastase concentration and treatment time, the change in peak stress after cyclic loading reached a plateau above 1 U/ml elastase and 6 hr treatment. For specimens treated with 2 U/ml elastase for 6 hr, elastin content decreased approximately 35%. Mean peak tissue strain after cyclic loading (4.8%, p≥0.300), modulus (275 MPa, p≥0.114) and hysteresis (20%, p≥0.553) were unaffected by elastase digestion, but stress decreased significantly after treatment (up to 2 MPa, p≤0.049). Elastin degradation had no effect on failure properties, but tissue lengthened under the same pre-stress. Stiffness in the linear region was unaffected by elastase digestion, suggesting that enzyme treatment did not disrupt collagen. These results demonstrate that elastin primarily functions in the toe region of the stress-strain curve, yet contributes load support in the linear region. The increase in length after elastase digestion suggests that elastin may pre-stress and stabilize collagen crimp in ligaments.
Caenorhabditis elegans ftn-1 and ftn-2, which encode the iron-storage protein ferritin, are transcriptionally inhibited during iron deficiency in intestine. Intestinal specific transcription is dependent on binding of ELT-2 to GATA binding sites in an iron-dependent enhancer (IDE) located in ftn-1 and ftn-2 promoters, but the mechanism for iron regulation is unknown. Here, we identify HIF-1 (hypoxia-inducible factor -1) as a negative regulator of ferritin transcription. HIF-1 binds to hypoxia-response elements (HREs) in the IDE in vitro and in vivo. Depletion of hif-1 by RNA interference blocks transcriptional inhibition of ftn-1 and ftn-2 reporters, and ftn-1 and ftn-2 mRNAs are not regulated in a hif-1 null strain during iron deficiency. An IDE is also present in smf-3 encoding a protein homologous to mammalian divalent metal transporter-1. Unlike the ftn-1 IDE, the smf-3 IDE is required for HIF-1–dependent transcriptional activation of smf-3 during iron deficiency. We show that hif-1 null worms grown under iron limiting conditions are developmentally delayed and that depletion of FTN-1 and FTN-2 rescues this phenotype. These data show that HIF-1 regulates intestinal iron homeostasis during iron deficiency by activating and inhibiting genes involved in iron uptake and storage.
Ferritin is a ubiquitous protein that sequesters iron and protects cells from iron toxicity. Caenorhabditis elegans express two ferritins, FTN-1 and FTN-2, which are transcriptionally regulated by iron. To identify the cis-acting sequences and proteins required for iron-dependent regulation of ftn-1 and ftn-2 expression, we generated transcriptional GFP reporters corresponding to 5-upstream sequences of the ftn-1 and ftn-2 genes. We identified a conserved 63-bp sequence, the iron-dependent element (IDE), that is required for iron-dependent regulation of a ftn-1 GFP reporter in intestine. The IDE contains two GATAbinding motifs and three octameric direct repeats. Site-directed mutagenesis of the GATA sequences, singly or in combination, reduces ftn-1 GFP reporter expression in the intestine. In vitro DNA mobility shift assays show that the intestine-specific GATA protein ELT-2 binds to both GATA sequences. Inhibition of ELT-2 function by RNA interference blocks ftn-1 GFP reporter expression in vivo. Insertion of the IDE into the promoter region of a heterologous reporter activates iron-dependent transcription in intestine. These data demonstrate that the activation of ftn-1 and ftn-2 transcription by iron requires ELT-2 and that the IDE functions as an iron-dependent enhancer in intestine.Iron is essential in many biological processes, including DNA synthesis, respiration, nitrogen fixation, oxygen transport, heme synthesis, and photosynthesis. At high levels, however, iron can be toxic because of its ability to react with molecular oxygen to generate free radicals that oxidize DNA and proteins and initiate lipid peroxidation, all of which can lead to cell injury and death (1). Consequently, organisms have developed mechanisms to sense, acquire and store this metal within a narrow physiologic range.Ferritin sequesters iron in a form that is biologically available but unable to catalyze free radical formation (2, 3). Mammalian ferritin has a molecular mass of ϳ480,000 and consists of 24 related subunits of two types, a light subunit (L) 2 and a heavy subunit (H). These subunits assemble to form a shell surrounding a cavity that can accommodate up to 4500 iron atoms. The H-subunits oxidize ferrous iron to ferric iron within the cavity; the L-subunits lack ferroxidase activity and function with the H-subunits in iron nucleation. Mutations in either the ferritin H or the ferritin L gene can be deleterious. Ferritin H knock-out mice die early in embryogenesis (4), while mutations in ferritin H or ferritin L genes are associated with hereditary conditions, such as hyperferritinemia cataract syndrome (5, 6), adult onset basal ganglia disease (7), and autosomal dominant iron-overload disease (8).Ferritin induction by iron is regulated by transcriptional and post-transcriptional mechanisms that are organism-specific (9 -11). In vertebrates, ferritin expression is primarily regulated at the translational level by cytosolic proteins that bind to ironresponsive elements (IREs) in the 5Ј-or 3Ј-untranslated regions of mRNAs of...
Iron regulatory protein 2 (IRP2) is an RNA-binding protein that regulates the posttranscriptional expression of proteins required for iron homeostasis such as ferritin and transferrin receptor 1. IRP2 RNA-binding activity is primarily regulated by iron-mediated proteasomal degradation, but studies have suggested that IRP2 RNA binding is also regulated by thiol oxidation. We generated a model of IRP2 bound to RNA and found that two cysteines (C512 and C516) are predicted to lie in the RNA-binding cleft. Site-directed mutagenesis and thiol modification show that, while IRP2 C512 and C516 do not directly interact with RNA, both cysteines are located within the RNA-binding cleft and must be unmodified/reduced for IRP2-RNA interactions. Oxidative stress induced by cellular glucose deprivation reduces the RNA-binding activity of IRP2 but not IRP2-C512S or IRP2-C516S, consistent with the formation of a disulfide bond between IRP2 C512 and C516 during oxidative stress. Decreased IRP2 RNA binding is correlated with reduced transferrin receptor 1 mRNA abundance. These studies provide insight into the structural basis for IRP2-RNA interactions and reveal an iron-independent mechanism for regulating iron homeostasis through the redox regulation of IRP2 cysteines.Iron is an essential nutrient required for a variety of cellular processes, including DNA synthesis, respiration, and heme biosynthesis. However, ferrous iron readily reacts with hydroperoxides to produce hydroxyl radicals that can cause cellular damage. As both iron excess and deficiency are deleterious, cells have developed mechanisms to ensure that iron levels are sufficient for cellular need but at the same time limit iron toxicity.Iron regulatory proteins 1 and 2 (IRP1 and IRP2) are the key iron sensors in mammalian cells (32,48). IRPs are cytosolic proteins that bind RNA stem-loops known as iron-responsive elements (IREs) located in the 5Ј or 3Ј untranslated regions of mRNAs encoding proteins involved in iron homeostasis. IRP binding to a 5Ј IRE present in ferritin (iron storage) or ferroportin (iron exporter) mRNAs represses protein translation. IRP binding to 3Ј IREs, such as those in transferrin receptor 1 (TfR-1) and divalent metal transporter 1 (DMT1) (iron importers), stabilizes the mRNA and increases protein expression. While both IRPs function as RNA-binding proteins when iron content is low, increased cellular iron regulates IRP1 and IRP2 by different mechanisms. Increased cellular iron results in the assembly of an [4Fe-4S] cluster in the RNA-binding cleft of IRP1, which allows IRP1 to function as a cytosolic aconitase. Unlike IRP1, IRP2 does not coordinate an [4Fe-4S] cluster or function as an aconitase. Instead, the RNA-binding activity of IRP2 is reduced by iron-dependent polyubiquitylation and proteasomal degradation. The role of IRP2 as the predominant RNA-binding protein in vivo has been established in murine knockout models in which Irp1 Ϫ/Ϫ mice display no overt phenotype, whereas Irp2 Ϫ/Ϫ mice develop microcytic anemia and locomotor deficits...
Regulation of cellular iron homeostasis is crucial as both iron excess and deficiency cause hematological and neurodegenerative diseases. Here we show that mice lacking ironregulatory protein 2 (Irp2), a regulator of cellular iron homeostasis, develop diabetes. Irp2 post-transcriptionally regulates the iron-uptake protein transferrin receptor 1 (TfR1) and the iron-storage protein ferritin, and dysregulation of these proteins due to Irp2 loss causes functional iron deficiency in β cells. This impairs Fe-S cluster biosynthesis, reducing the function of Cdkal1, an Fe-S cluster enzyme that catalyzes methylthiolation of t 6 A37 in tRNA Lys UUU to ms 2 t 6 A37. As a consequence, lysine codons in proinsulin are misread and proinsulin processing is impaired, reducing insulin content and secretion. Iron normalizes ms 2 t 6 A37 and proinsulin lysine incorporation, restoring insulin content and secretion in Irp2 −/− β cells. These studies reveal a previously unidentified link between insulin processing and cellular iron deficiency that may have relevance to type 2 diabetes in humans.
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