Identification of human genes of iron homeostasis

National Institute Of Diabetes And Digestive And Kidney Diseases

Investigators

Linked publications & trials

Abstract

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.