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，需要铁辅因子才能发挥活性。铁辅因子在细胞中合成，但参与血红素、铁硫簇和亚铁/三价铁离子向脱辅基蛋白分布的途径仍不完全确定。特别是，胞质单硫醇谷氧还蛋白和BolA样蛋白已被确定为2Fe-2S-配位复合物在体外和铁调节蛋白在真菌中，但目前还不清楚这些蛋白质在哺乳动物系统中的功能，或如何这种复合物可能会影响Fe-S蛋白或胞质Fe-S组装机制。为了探索这些问题，我们使用定量免疫沉淀和活细胞，邻近依赖性生物素化，监测Glrx 3，BolA 2和细胞溶质铁硫簇组装系统的组件之间的相互作用。我们在人类细胞中表征胞质Glrx 3-BolA 2作为2Fe-2S伴侣复合物。与真菌直向同源物形成的复合物不同，人Glrx 3-BolA 2相互作用需要Fe-S簇的配位，而Glrx 3同型二聚体的形成则不需要。细胞Glrx 3-BolA 2复合物响应于增加的铁而增加6-8倍，形成快速膨胀的Fe-S簇池。Glrx 3-BolA 2的Fe-S配位不依赖于Ciapin 1或Ciao 1，这两种蛋白结合Glrx 3并参与细胞溶质Fe-S簇组装和分布。相反，Glrx 3和BolA 2结合并促进Fe-S掺入Ciapin 1，一种在细胞溶质Fe-S组装途径早期起作用的2Fe-2S蛋白。因此，Glrx 3-BolA是能够将2Fe-2S簇转移到人细胞中的脱辅基蛋白的2Fe-2S伴侣复合物。 2)多聚（rC）结合蛋白（PCBP）是介导核酸、铁辅因子和其他蛋白质之间相互作用的多功能衔接子，影响这些相互作用组分的命运和活性。在这里，我们表明，PCBP 2形成了一个复杂的Hippo通路组件Sav 1，Mst 1，Mst 2和Lats 1在人类细胞和小鼠组织。Hippo是一种激酶级联，其功能是磷酸化和磷酸化转录辅激活因子雅普和TAZ，其控制细胞生长和增殖。PCBP 2特异性地与支架蛋白Sav 1相互作用，并阻止Mst 1激酶的蛋白水解切割，导致通过Hippo的信号传导增加并抑制雅普和TAZ的活性。缺乏PCBP 2的人乳腺上皮细胞表现出TAZ的蛋白酶体降解受损。它们在细胞核和细胞质中积累TAZ，增加雅普和TAZ靶基因CTGF和Cyr 61的表达，并表现出锚定非依赖性生长。因此，PCBP 2可以作为Hippo复合物的组分，增强信号传导，抑制雅普和TAZ的活性，并改变细胞的生长特性。 3)调节细胞因子的转录、翻译和分泌对于控制炎症的适当平衡至关重要。 在这里，我们报告的分选受体分拣蛋白与各种细胞因子相互作用，并参与胞吐运输的IFN-类浆细胞树突状细胞（pDC）。 重组蛋白的SPR分析揭示了分拣蛋白与IFN-γ、IL-10、IL-12和IL-17 A以及与两种已知配体IFN-γ和IL-6的相互作用。 pDC中分拣蛋白耗尽导致IFN-分泌减少，显微镜成像显示分拣蛋白与IFN-共定位，而响应TLR刺激的IFNA基因转录不受分拣蛋白敲低的影响。 这些结果表明分拣蛋白在pDC中IFN-γ的胞吐运输中起关键作用。 此外，我们观察到分拣蛋白mRNA在转录后被各种TLR配体刺激后降解。 有趣的是，分拣蛋白mRNA在3个UTR区域具有富含C的元件（CRE），其充当顺式元件并被多聚rC结合蛋白1（PCBP 1）识别。 PCBP 1与siRNA的耗尽增强了分拣蛋白转录物的降解，并且CRE的破坏与转录物的不稳定相关。 这些结果表明，PCBP 1可以作为一个反式作用因子，以稳定分拣蛋白转录。 体外带移分析表明，PCBP 1的核苷酸结合能力受损的锌离子。 分拣蛋白转录降解锌补充剂，锌螯合剂TPEN防止这种降解，表明细胞锌状态分拣蛋白表达的重要性。 因此，PCBP 1可能通过感应细胞内锌水平来控制分拣蛋白转录物的稳定性。 总的来说，我们的研究结果提供了深入了解翻译后调节细胞因子的产生，通过转录后控制分拣蛋白的表达TLR信号。