IDENTIFICAZIONE E CARATTERIZZAZIONE DI PARTNERS MOLECOLARI DELLE PROTEINE HMGA

2005/2006XIX Ciclo1973Versione digitalizzata della tesi di dottorato cartacea

The second AT-hook of the architectural transcription factor HMGA2 is determinant for nuclear localization and function

High Mobility Group A (HMGA) is a family of architectural nuclear factors which play an important role in neoplastic transformation. HMGA proteins are multifunctional factors that associate both with DNA and nuclear proteins that have been involved in several nuclear processes including transcription. HMGA localization is exclusively nuclear but, to date, the mechanism of nuclear import for these proteins remains unknown. Here, we report the identification and characterization of a nuclear localization signal (NLS) for HMGA2, a member of the HMGA family. The NLS overlaps with the second of the three AT-hooks, the DNA-binding domains characteristic for this group of proteins. The functionality of this NLS was demonstrated by its ability to target a heterologous β-galactosidase/green fluorescent protein fusion protein to the nucleus. Mutations to alanine of basic residues within the second AT-hook resulted in inhibition of HMGA2 nuclear localization and impairment of its function in activating the cyclin A promoter. In addition, HMGA2 was shown to directly interact with the nuclear import receptor importin-α2 via the second AT-hook. HMGA proteins are overexpressed and rearranged in a variety of tumors; our findings can thus help elucidating their role in neoplastic transformation

The AT-hook of the Chromatin Architectural Transcription Factor High Mobility Group A1a Is Arginine-methylated by Protein Arginine Methyltransferase 6

The HMGA1a protein belongs to the high mobility group A (HMGA) family of architectural nuclear factors, a group of proteins that plays an important role in chromatin dynamics. HMGA proteins are multifunctional factors that associate both with DNA and nuclear proteins that have been involved in several nuclear processes, such as transcriptional regulation, viral integration, DNA repair, RNA processing, and chromatin remodeling. The activity of HMGA proteins is finely modulated by a variety of post-translational modifications. Arginine methylation was recently demonstrated to occur on HMGA1a protein, and it correlates with the apoptotic process and neoplastic progression. Methyltransferases responsible for these modifications are unknown. Here we show that the protein arginine methyltransferase PRMT6 specifically methylates HMGA1a protein both in vitro and in vivo. By mass spectrometry, the sites of methylation were unambiguously mapped to Arg(57) and Arg(59), two residues which are embedded in the second AT-hook, a region critical for both protein-DNA and protein-protein interactions and whose modification may cause profound alterations in the HMGA network. The in vivo association of HMGA and PRMT6 place this yet functionally uncharacterized methyltransferase in the well established functional context of the chromatin structure organization

A novel TMPRSS6 mutation that prevents protease auto-activation causes IRIDA

IRIDA (iron-refractory iron-deficiency anaemia) is a rare autosomal-recessive disorder hallmarked by hypochromic microcytic anaemia, low transferrin saturation and high levels of the iron-regulated hormone hepcidin. The disease is caused by mutations in the transmembrane serine protease TMPRSS6 (transmembrane protease serine 6) that prevent inactivation of HJV (haemojuvelin), an activator of hepcidin transcription. In the present paper, we describe a patient with IRIDA who carries a novel mutation (Y141C) in the SEA domain of the TMPRSS6 gene. Functional characterization of the TMPRSS6(Y141C) mutant protein in cultured cells showed that it localizes to similar subcellular compartments as wild-type TMPRSS6 and binds HJV, but fails to auto-catalytically activate itself. As a consequence, hepcidin mRNA expression is increased, causing the clinical symptoms observed in this IRIDA patient. The present study provides important mechanistic insight into how TMPRSS6 is activated

Iron- and erythropoietin-resistant anemia in a spontaneous breast cancer mouse model

Anemia of cancer (AoC) with its multifactorial etiology and complex pathology is a poor prognostic indicator for cancer patients. One of the main causes of AoC is cancer-associated inflammation that activates mechanisms, commonly observed in anemia of inflammation, where functional iron deficiency and iron-restricted erythropoiesis is induced by increased hepcidin levels in response to IL-6 elevation. So far only a few AoC mouse models have been described, and most of them did not fully recapitulate the interplay of anemia, increased hepcidin levels and functional iron deficiency in human patients. To test if the selection and the complexity of AoC mouse models dictates the pathology or if AoC in mice per se develops independently of iron deficiency, we characterized AoC in Trp53floxWapCre mice that spontaneously develop breast cancer. These mice developed AoC associated with high IL-6 levels and iron deficiency. However, hepcidin levels were not increased and hypoferremia coincided with anemia rather than causing it. Instead, an early shift in the commitment of common myeloid progenitors from the erythroid to the myeloid lineage resulted in increased myelopoiesis and in the excessive production of neutrophils that accumulate in necrotic tumor regions. This process could neither be prevented by iron nor erythropoietin (EPO) treatment. Trp53floxWapCre mice are the first mouse model where EPO-resistant anemia is described and may serve as a disease model to test therapeutic approaches for a subpopulation of human cancer patients with normal or corrected iron levels that do not respond to EPO

Elevated hepcidin serum level in response to inflammatory and iron signals in exercising athletes is independent of moderate supplementation with vitamin C and E

Iron deficiency among endurance athletes is of major concern for coaches, physicians, and nutritionists. Recently, it has been observed that hepcidin, the master regulator of iron metabolism, was upregulated after exercise and was found to be related to interleukin-6 (IL-6) elevation. In this study performed on noniron deficient and well-trained runners, we observed that hepcidin concentrations remain elevated in response to inflammatory and iron signals despite a 28-days supplementation period with vitamins C (500 mg/day) and E (400 IU/day)

Dietary stearic acid regulates mitochondria in vivo in humans.

Since modern foods are unnaturally enriched in single metabolites, it is important to understand which metabolites are sensed by the human body and which are not. We previously showed that the fatty acid stearic acid (C18:0) signals via a dedicated pathway to regulate mitofusin activity and thereby mitochondrial morphology and function in cell culture. Whether this pathway is poised to sense changes in dietary intake of C18:0 in humans is not known. We show here that C18:0 ingestion rapidly and robustly causes mitochondrial fusion in people within 3 h after ingestion. C18:0 intake also causes a drop in circulating long-chain acylcarnitines, suggesting increased fatty acid beta-oxidation in vivo. This work thereby identifies C18:0 as a dietary metabolite that is sensed by our bodies to control our mitochondria. This could explain part of the epidemiological differences between C16:0 and C18:0, whereby C16:0 increases cardiovascular and cancer risk whereas C18:0 decreases both

Transferrin receptor 2 controls bone mass and pathological bone formation via BMP and Wnt signalling

Transferrin receptor 2 (Tfr2) is mainly expressed in the liver and controls iron homeostasis. Here, we identify Tfr2 as a regulator of bone homeostasis that inhibits bone formation. Mice lacking Tfr2 display increased bone mass and mineralization independent of iron homeostasis and hepatic Tfr2. Bone marrow transplantation experiments and studies of cell-specific Tfr2 knockout mice demonstrate that Tfr2 impairs BMP-p38MAPK signaling and decreases expression of the Wnt inhibitor sclerostin specifically in osteoblasts. Reactivation of MAPK or overexpression of sclerostin rescues skeletal abnormalities in Tfr2 knockout mice. We further show that the extracellular domain of Tfr2 binds BMPs and inhibits BMP-2-induced heterotopic ossification by acting as a decoy receptor. These data indicate that Tfr2 limits bone formation by modulating BMP signaling, possibly through direct interaction with BMP either as a receptor or as a co-receptor in a complex with other BMP receptors. Finally, the Tfr2 extracellular domain may be effective in the treatment of conditions associated with pathological bone formation

Hfe Is Highly Expressed in Liver Sinusoidal Endothelial Cells But Is Not Needed to Maintain Systemic Iron Homeostasis In Vivo

Supplemental Digital Content is available in the text