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）细胞包含数百种需要铁辅因子才能活性的蛋白质。铁辅因子是在细胞中合成的，但是分配血红素，铁硫簇和亚硫蛋白的铁/铁离子所涉及的途径仍未完全定义。特别是，在真菌中的体外和铁调节蛋白中，已经确定为2FE-2S配合复合物，但尚不清楚这些蛋白质在哺乳动物系统中的作用或该复合物如何影响Fe-S蛋白或细胞增多Fe-Sphissbly-Sphissbly Machinery。为了探讨这些问题，我们使用定量免疫沉淀和实时细胞，接近性依赖性生物素化来监视GLRX3，BOLA2和胞质铁硫簇组装系统的组件之间的相互作用。我们将胞质GLRX3-BOLA2表征为人类细胞中2FE-2S伴侣复合物。与真菌直系同源物形成的复合物不同，人Glrx3-Bola2相互作用需要Fe-S簇的协调，而GLRX3同二聚体的形成则不是。细胞GLRX3-BOLA2复合物因铁的增加而增加了6-8倍，形成了Fe-S簇的快速膨胀池。 GLRX3-BOLA2的Fe-S配位不取决于结合GLRX3并参与胞质Fe-S簇组装和分布的CIAPIN1或CIAO1。取而代之的是，GLRX3和BOLA2结合并促进Fe-S掺入CIAPIN1，这是一种2FE-2S蛋白在胞质Fe-S组装途径中起作用的2FE-2S蛋白。因此，GLRX3-BOLA是一种2FE-2S伴侣复合体，能够将2FE-2S簇转移到人类细胞中的Apo蛋白质。 2）聚（RC）结合蛋白（PCBP）是多功能适配器，可介导核酸，铁辅因子和其他蛋白质之间的相互作用，影响这些相互作用的成分的命运和活性。在这里，我们表明PCBP2在人类细胞和小鼠组织中与河马途径成分SAV1，MST1，MST2和LATS1形成复合物。 HIPPO是一种激酶级联反应，可在控制细胞生长和增殖的情况下磷酸化和灭活转录共激活剂YAP和TAZ。 PCBP2特异性与支架蛋白SAV1相互作用，并防止MST1激酶的蛋白水解切割，从而通过河马通过河马和抑制YAP和TAZ的活性增加了信号传导。缺乏PCBP2的人乳腺上皮细胞显示TAZ的蛋白酶体降解受损。它们在细胞核和细胞质中累积TAZ，增加YAP和TAZ靶基因CTGF和CYR61的表达，并表现出非锚定的生长。因此，PCBP2可以充当河马复合物的组成部分，增强信号传导，抑制YAP和TAZ的活性以及改变细胞的生长特征。 3）调节细胞因子的转录，翻译和分泌对于控制适当的炎症平衡至关重要。 在这里，我们报告表明，分类受体分类与各种细胞因子相互作用，并参与质细胞类树突状细胞（PDC）中IFN-的外生运输。 重组蛋白的SPR分析揭示了Tortilin与IFN-，IL-10，IL-12和IL-17A的相互作用，以及与两个已知的配体IFN-和IL-6相互作用。 PDC中的Tortilin耗竭导致IFN分泌的降低，微观成像揭示了与IFN-的共定位，而IFN-，而IFNA基因转录对TLR刺激的响应不受Torcililin敲低的影响。 这些结果表明，Tortilin在IFN-在PDC中的外生运输中起关键作用。 此外，我们观察到，在用各种TLR配体刺激后，托林蛋白mRNA在转录后降解。 有趣的是，Tortilin mRNA在3 UTR区中具有富含C的元素（CRE），该元素充当顺式元素，并通过poly-RC结合蛋白1（PCBP1）识别。 用siRNA耗尽PCBP1可增强Tortilin转录本的降解，而CRE的破坏与转录本的不稳定相关。 这些结果表明，PCBP1可以充当稳定Torsilin转录本的跨作用因素。 一种体外带移分析表明，锌离子会损害PCBP1的核苷酸结合能力。 锌补充剂降解，锌螯合剂TPEN阻止了这种降解，这表明细胞锌状态对托硅蛋白表达的重要性。 因此，PCBP1可以通过感测细胞内锌水平来控制Tortilin转录本的稳定性。 总的来说，我们的发现通过TLR信号对TOLILIN表达的转录后控制，对细胞因子产生的翻译后调节提供了见解。