Identification of human genes of iron homeostasis

人类铁稳态基因的鉴定

• 批准号: 9361476
• 负责人: Caroline Philpott
• 金额: $ 149.36万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9361476
• 关键词: Affect; Anchorage-Independent Growth; Beryllium; Binding; Binding Proteins; Biochemical; Biological; Biological Assay; Biotinylation; Breast Epithelial Cells; Cell Nucleus; Cell Proliferation; Cells; Characteristics; Chelating Agents; Complex; Cytosol; Dendritic Cells; Elements; Equilibrium; Exhibits; Friedreich Ataxia; Gene Targeting; Genes; Genetic; Genetic Transcription; Growth; Heme Iron; Hereditary Disease; Hereditary hemochromatosis; Homeostasis; Human; Immunoprecipitation; In Vitro; Indium; Inflammation; Interferons; Interleukin-10; Interleukin-12; Interleukin-17; Interleukin-6; Intracellular Transport; Ions; Iron; Iron Overload; Iron-Regulatory Proteins; Life; Ligands; Mediating; Messenger RNA; Molecular Chaperones; Monitor; Mus; Nucleic Acids; Nucleotides; Nutrient; Organism; Orthologous Gene; Pathway interactions; Phosphotransferases; Play; Post-Translational Regulation; Process; Production; Protein S; Proteins; Recombinant Proteins; Regulation; Reporting; Role; Scaffolding Protein; Signal Transduction; Small Interfering RNA; Sorting - Cell Movement; Sulfur; System; Tissues; Toxin; Trans-Activators; Transcript; Transcription Coactivator; Translations; Untranslated Regions; Zinc; Zinc supplementation; cell growth; cofactor; connective tissue growth factor; cytokine; fungus; glutaredoxin; human disease; insight; iron metabolism; knock-down; microscopic imaging; prevent; protein function; receptor; response; sortilin; trafficking; uptake

1) Cells contain hundreds of proteins that require iron cofactors for activity. Iron cofactors are synthesized in the cell, but the pathways involved in distributing heme, iron-sulfur clusters, and ferrous/ferric ions to apo-proteins remain incompletely defined. In particular, cytosolic monothiol glutaredoxins and BolA-like proteins have been identified as 2Fe-2S-coordinating complexes in vitro and iron-regulatory proteins in fungi, but it is not clear how these proteins function in mammalian systems or how this complex might affect Fe-S proteins or the cytosolic Fe-S assembly machinery. To explore these questions, we use quantitative immunoprecipitation and live-cell, proximity-dependent biotinylation, to monitor interactions between Glrx3, BolA2, and components of the cytosolic iron-sulfur cluster assembly system. We characterize cytosolic Glrx3-BolA2 as a 2Fe-2S chaperone complex in human cells. Unlike complexes formed by fungal orthologs, human Glrx3-BolA2 interaction required the coordination of Fe-S clusters, while Glrx3 homodimer formation did not. Cellular Glrx3-BolA2 complexes increased 6-8-fold in response to increasing iron, forming a rapidly-expandable pool of Fe-S clusters. Fe-S coordination by Glrx3-BolA2 did not depend on Ciapin1 or Ciao1, proteins that bind Glrx3 and are involved in cytosolic Fe-S cluster assembly and distribution. Instead, Glrx3 and BolA2 bound and facilitated Fe-S incorporation into Ciapin1, a 2Fe-2S protein functioning early in the cytosolic Fe-S assembly pathway. Thus, Glrx3-BolA is a 2Fe-2S chaperone complex capable of transferring 2Fe-2S clusters to apo-proteins in human cells. 2) Poly (rC)-binding proteins (PCBPs) are multifunctional adapters that mediate interactions between nucleic acids, iron cofactors, and other proteins, affecting the fate and activity of the components of these interactions. Here we show that PCBP2 forms a complex with the Hippo pathway components Sav1, Mst1, Mst2 and Lats1 in human cells and mouse tissues. Hippo is a kinase cascade that functions to phosphorylate and inactivate the transcriptional coactivators YAP and TAZ, which control cell growth and proliferation. PCBP2 specifically interacts with the scaffold protein, Sav1, and prevents proteolytic cleavage of the Mst1 kinase, resulting in increased signaling through Hippo and suppressed activity of YAP and TAZ. Human breast epithelial cells lacking PCBP2 exhibit impaired proteasomal degradation of TAZ. They accumulate TAZ in both the nucleus and the cytosol, increase expression of YAP and TAZ target genes CTGF and Cyr61, and exhibit anchorage-independent growth. Thus, PCBP2 can function as a component of the Hippo complex, enhancing signaling, suppressing activity of YAP and TAZ, and altering growth characteristics of cells. 3) Regulating the transcription, translation and secretion of cytokines is crucial for controlling the appropriate balance of inflammation. Here we report that the sorting receptor sortilin interacts with various cytokines and is involved in the exocytic trafficking of IFN- in plasmacytoid dendritic cells (pDCs). SPR analysis with recombinant proteins revealed interactions of sortilin with IFN-, IL-10, IL-12 and IL-17A as well as with two known ligands, IFN- and IL-6. Sortilin depletion in pDCs led to a reduction of IFN- secretion, and microscopic imaging revealed the co-localization of sortilin with IFN-, whereas IFNA gene transcription in response to TLR stimulation was unaffected by sortilin knockdown. These results suggest that sortilin plays a pivotal role in the exocytic trafficking of IFN- in pDCs. Moreover, we observed that sortilin mRNA was degraded posttranscriptionally upon stimulation with various TLR ligands. Interestingly, sortilin mRNA possessed a C-rich element (CRE) in the 3 UTR region, which acted as a cis-element and was recognized by poly-rC-binding protein 1 (PCBP1). Depletion of PCBP1 with siRNA enhanced the degradation of sortilin transcripts, and disruption of the CRE correlated with destabilization of transcripts. These results suggest that PCBP1 can act as a trans-acting factor to stabilize sortilin transcripts. An in vitro band shift assay revealed that the nucleotide-binding ability of PCBP1 was impaired by zinc ions. Sortilin transcripts were degraded by zinc supplementation, and the zinc chelator TPEN prevented this degradation, suggesting the importance of cellular zinc status for sortilin expression. PCBP1 may therefore control the stability of sortilin transcripts by sensing intracellular zinc levels. Collectively, our findings provide insights into the posttranslational regulation of cytokine production through the posttranscriptional control of sortilin expression by TLR signals.

1)细胞含有数百种蛋白质，需要铁辅助因子才能发挥作用。铁辅助因子是在细胞中合成的，但涉及将血红素、铁硫簇和亚铁/铁离子分配到脱辅蛋白的途径仍未完全确定。特别是细胞质中的单硫醇谷氧还蛋白和BoLA样蛋白，在体外被鉴定为2Fe-2S配位蛋白，在真菌中被鉴定为铁调节蛋白，但这些蛋白在哺乳动物系统中如何发挥作用，或者这种复合体如何影响Fe-S蛋白或细胞质中的Fe-S组装机制尚不清楚。为了探索这些问题，我们使用定量免疫沉淀和活细胞，邻近依赖的生物素化，以监测Glrx3，BolA2，和细胞质铁-硫簇组装系统的组件之间的相互作用。我们将Glrx3-BolA2定性为人细胞中的2Fe-2S伴侣复合体。与真菌直系物形成的络合物不同，人Glrx3-BolA2相互作用需要Fe-S簇的配位，而Glrx3同源二聚体的形成不需要。细胞内Glrx3-BolA2复合体随着铁含量的增加而增加6-8倍，形成一个可快速扩展的Fe-S团簇。Glrx3-BolA2对Fe-S的配位作用不依赖于Ciapin1或Ciao1，这两种蛋白与Glrx3结合，参与胞内Fe-S簇的组装和分布。相反，Glrx3和BolA2结合并促进Fe-S整合到Ciapin1中，Ciapin1是一种2Fe-2S蛋白，在细胞质Fe-S组装途径中起早期作用。因此，Glrx3-BoLA是一种2Fe-2S伴侣复合体，能够将2Fe-2S簇转移到人体细胞中的载脂蛋白上。 2)多聚(RC)结合蛋白(PCBP)是调节核酸、铁辅因子和其他蛋白质之间相互作用的多功能适配器，影响这些相互作用成分的命运和活性。在这里，我们证明了PCBP2在人类细胞和小鼠组织中与河马通路成分Sav1、Mst1、Mst2和Lats1形成了一个复合体。河马是一个激酶级联，其功能是磷酸化和失活转录共激活因子YAP和TAZ，后者控制细胞的生长和增殖。PCBP2与支架蛋白Sav1特异性地相互作用，阻止Mst1激酶的蛋白水解性切割，导致通过河马的信号增加，并抑制YAP和TAZ的活性。缺乏PCBP2的人乳腺上皮细胞显示出TAZ的蛋白酶体降解受损。它们在细胞核和胞浆中积聚TAZ，增加YAP和TAZ靶基因CTGF和Cyr61的表达，并表现出锚定无关的生长。因此，PCBP2可以作为河马复合体的一个组成部分，增强信号传递，抑制YAP和TAZ的活性，并改变细胞的生长特性。 3)调节细胞因子的转录、翻译和分泌是控制炎症平衡的关键。在这里，我们报道了分选受体sortilin与多种细胞因子相互作用，并参与血浆细胞样树突状细胞(PDCs)中干扰素的胞外转运。重组蛋白的SPR分析表明，山梨素与干扰素、IL-10、IL-12和IL-17A以及两个已知的配体-干扰素和IL-6相互作用。PDC中索的林素缺乏导致干扰素的分泌减少，显微成像显示索替林素与干扰素共定位，而干扰素基因转录在TLR刺激下的转录不受索替林素基因敲除的影响。这些结果表明，山梨素在干扰素在pDC胞外转运中起着关键作用。此外，我们还观察到在不同的TLR配体刺激下，sortilin mRNA在转录后被降解。有趣的是，sortilin mRNA在3个非编码区含有一个富含C的元件(Cre)，它作为顺式元件被多聚RC结合蛋白1(PCBP1)识别。用siRNA耗尽PCBP1增强了山梨素转录本的降解，Cre的破坏与转录本的不稳定有关。这些结果表明，PCBP1可以作为一个反式作用因子来稳定山梨素转录本。体外带移实验表明，锌离子对PCBP1的核苷酸结合能力有一定的影响。补锌后，山梨素转录本被降解，而锌螯合剂TPEN阻止了这种降解，这表明细胞锌状态对山梨素表达的重要性。因此，PCBP1可能通过检测细胞内锌水平来控制山梨素转录本的稳定性。总而言之，我们的发现为通过TLR信号转录后控制sortilin表达而对细胞因子产生的翻译后调控提供了洞察力。