Iron sulfur (Fe-S) clusters, preassembled on the ISCU scaffold, are transferred to target proteins or to intermediate scaffolds by a dedicated chaperone-cochaperone system. However, the molecular mechanisms that underlie substrate discrimination and guide delivery of nascent clusters to specific subsets of Fe-S recipients are poorly understood. Here, we identified interacting partners of the cochaperone HSC20 and discovered that LYR motifs are molecular signatures of specific recipient Fe-S proteins or accessory factors that assist Fe-S cluster delivery. In succinate dehydrogenase B, two LYR motifs engage the ISCU-HSC20-HSPA9 complex to aid incorporation of three Fe-S clusters within the final structure of complex II. Moreover, we show that members of the LYR motif family which assist assembly of complexes II or III, SDHAF1 and LYRM7, respectively, are HSC20 binding partners. Our studies unveil a network of interactions between HSC20 and LYR motif-containing proteins that are key to the assembly and function of complexes I, II, and III.

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SUMMARY Iron regulatory proteins 1 and 2 (Irps) post-transcriptionally control the expression of transcripts that contain iron responsive element (IRE) sequences, including ferritin, ferroportin, transferrin receptor and hypoxia inducible factor 2α (HIF2α). We report here that mice with targeted deletion of Irp1 developed pulmonary hypertension and polycythemia that was exacerbated by a low iron diet. Hematocrits increased to 65% in iron-starved mice, and many polycythemic mice died of abdominal hemorrhages. Irp1 deletion enhanced HIF2α protein expression in kidneys of Irp1−/− mice, which led to increased erythropoietin (EPO) expression, polycythemia and concomitant tissue iron deficiency. Increased HIF2α expression in pulmonary endothelial cells induced high expression of endothelin-1, likely contributing to the pulmonary hypertension of Irp1−/− mice. Our results reveal why anemia is an early physiological consequence of iron deficiency, highlight the physiological significance of Irp1 in regulating erythropoiesis and iron distribution, and provide important insights into the molecular pathogenesis of pulmonary hypertension.

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With the advancement in biomaterial sciences, tissue-engineered scaffolds are developing as a promising strategy for the regeneration of damaged tissues. However, only a few of these scaffolds have been translated into clinical applications. One of the primary drawbacks of the existing scaffolds is the lack of adequate oxygen supply within the scaffolds. Oxygen-producing biomaterials have been developed as an alternate strategy but are faced with two major concerns. One is the control of the rate of oxygen generation, and the other is the production of reactive oxygen species (ROS). To address these concerns, here, we report the development of an oxygen-releasing antioxidant polymeric cryogel scaffold (PUAO-CPO) for sustained oxygen delivery. PUAO-CPO scaffold was fabricated using the cryogelation technique by the incorporation of calcium peroxide (CPO) in the antioxidant polyurethane (PUAO) scaffolds. The PUAO-CPO cryogels attenuated the ROS and showed a sustained release of oxygen over a period of 10 days. An in vitro analysis of the PUAO-CPO cryogels showed their ability to sustain H9C2 cardiomyoblast cells under hypoxic conditions, with cell viability being significantly better than the normal polyurethane (PU) scaffolds. Furthermore, in vivo studies using an ischemic flap model showed the ability of the oxygen-releasing cryogel scaffolds to prevent tissue necrosis upto 9 days. Histological examination indicated the maintenance of tissue architecture and collagen content, whereas immunostaining for proliferating cell nuclear antigen confirmed the viability of the ischemic tissue with oxygen delivery. Our study demonstrated an advanced approach for the development of oxygen-releasing biomaterials with sustained oxygen delivery as well as attenuated production of residual ROS and free radicals because of ischemia or oxygen generation. Hence, the oxygen-releasing PUAO-CPO cryogel scaffolds may be used with cell-based therapeutic approaches for the regeneration of damaged tissue, particularly with ischemic conditions such as myocardial infarction and chronic wound healing.

The importance of mitochondrial iron-sulfur cluster (ISC) biogenesis for human health has been well established, but the roles of some components of this critical pathway still remain uncharacterized in mammals. Among them is human heat shock cognate protein 20 (hHSC20), the putative human homolog of the specialized DnaJ type co-chaperones, which are crucial for bacterial and fungal ISC assembly. Here, we show that the human HSC20 protein can complement for its counterpart in yeast, Jac1p, and interacts with its proposed human partners, hISCU and hHSPA9. hHSC20 is expressed in various human tissues and localizes mainly to the mitochondria in HeLa cells. However, small amounts were also detected extra-mitochondrially. RNA interference-mediated depletion of hHSC20 specifically reduced the activities of both mitochondrial and cytosolic ISC-containing enzymes. The recovery of inactivated ISC enzymes was markedly delayed after an oxidative insult of hHSC20-deficient cells. Conversely, overexpression of hHSC20 substantially protected cells from oxidative insults. These results imply that hHSC20 is an integral component of the human ISC biosynthetic machinery that is particularly important in the assembly of ISCs under conditions of oxidative stress. A cysteine-rich N-terminal domain, which clearly distinguishes hHSC20 from the specialized DnaJ type III proteins of fungi and most bacteria, was found to be important for the integrity and function of the human co-chaperone.

In mammals, two homologous cytosolic regulatory proteins, iron regulatory protein 1 (also known as IRP1 and Aco1) and iron regulatory protein 2 (also known as IRP2 and Ireb2), sense cytosolic iron levels and posttranscriptionally regulate iron metabolism genes, including transferrin receptor 1 (TfR1) and ferritin H and L subunits, by binding to iron-responsive elements (IREs) within target transcripts. Mice that lack IRP2 develop microcytic anemia and neurodegeneration associated with functional cellular iron depletion caused by low TfR1 and high ferritin expression. IRP1 knockout (IRP1 ؊/؊ ) animals do not significantly misregulate iron metabolism, partly because IRP1 is an iron-sulfur protein that functions mainly as a cytosolic aconitase in mammalian tissues and IRP2 activity increases to compensate for loss of the IRE binding form of IRP1. The neurodegenerative disease of IRP2 ؊/؊ animals progresses slowly as the animals age. In this study, we fed IRP2 ؊/؊ mice a diet supplemented with a stable nitroxide, Tempol, and showed that the progression of neuromuscular impairment was markedly attenuated. In cell lines derived from IRP2 ؊/؊ animals, and in the cerebellum, brainstem, and forebrain of animals maintained on the Tempol diet, IRP1 was converted from a cytosolic aconitase to an IRE binding protein that stabilized the TfR1 transcript and repressed ferritin synthesis. We suggest that Tempol protected IRP2 ؊/؊ mice by disassembling the cytosolic iron-sulfur cluster of IRP1 and activating IRE binding activity, which stabilized the TfR1 transcript, repressed ferritin synthesis, and partially restored normal cellular iron homeostasis in the brain.anemia ͉ axonopathy ͉ ferritin ͉ iron-sulfur cluster ͉ transferrin receptor

Oxidative stress plays an important role in various pathological conditions, such as wound healing, inflammation, myocardial infarction, and biocompatibility of the materials. Antioxidant polymers to attenuate oxidative stress is an emerging field of biomaterial research with a huge impact in the field of tissue engineering and regenerative medicine. We describe here the fabrication and evaluation of an elastomeric antioxidant polyurethane (PUAO) for tissue engineering applications. Uniaxial and cyclic tensile testing, thermal analysis, degradation, cytotoxicity and antioxidant analysis was carried out. An in vitro oxidative stress model demonstrated that PUAO reduced intracellular oxidative stress in H9C2 cardiomyocytes (p < 0.05) and attenuated reactive oxygen species (ROS) induced cell death (p < 0.001). Under simulated ischemic reperfusion, PUAO could rescue hypoxia induced cell death. Further as a proof of concept, neonatal rat cardiomyocytes cultured on PUAO film displayed synchronous beating with mature phenotype showing expression of cardiac specific α-actinin, troponin-T, and connexin-43 proteins. Intracellular calcium transients established the functionality of cultured cardiomyocytes on PUAO film. Our study demonstrated the potential of this biomaterial to be developed into tissue engineered scaffold to attenuate oxidative stress for treatment of diseased conditions with increased oxidative stress, such as cardiovascular diseases, chronic wound healing, and myocardial infarction.