Identification of human genes of iron homeostasis

细胞中含有数百种需要铁辅助因子才能发挥活性的蛋白质。铁辅因子是在细胞内合成的，但将血红素、铁硫簇和铁/铁离子分配给载脂蛋白的途径仍不完全明确。特别是，胞质单硫醇glutaredoxins和bola样蛋白在体外被鉴定为2fe - 2s协调复合物，在真菌中被鉴定为铁调节蛋白，但尚不清楚这些蛋白在哺乳动物系统中的功能，也不清楚这种复合物如何影响铁- s蛋白或胞质铁- 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，邻近依赖的生物素化，来监测Glrx3， BolA2和细胞质铁硫簇组装系统组分之间的相互作用。我们将细胞质Glrx3-BolA2描述为人类细胞中的2Fe-2S伴侣复合物。与真菌同源物形成的复合物不同，人类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需要。细胞Glrx3-BolA2复合物随着铁的增加而增加6-8倍，形成一个快速扩展的Fe-S簇池。Glrx3- bola2的Fe-S配位不依赖于与Glrx3结合并参与胞质Fe-S簇组装和分布的蛋白Ciapin1或Ciao1。相反，Glrx3和BolA2结合并促进Fe-S结合到Ciapin1中，Ciapin1是一种2Fe-2S蛋白，在细胞内Fe-S组装途径的早期起作用。因此，Glrx3-BolA是一种2Fe-2S伴侣复合物，能够将2Fe-2S簇转移到人类细胞的载脂蛋白上。

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.