Deregulation of Hepatic Mek1/2–Erk1/2 Signaling Module in Iron Overload Conditions

The liver, through the production of iron hormone hepcidin, controls body iron levels. High liver iron levels and deregulated hepcidin expression are commonly observed in many liver diseases including highly prevalent genetic iron overload disorders. In spite of a number of breakthrough investigations into the signals that control hepcidin expression, little progress has been made towards investigations into intracellular signaling in the liver under excess of iron. This study examined hepatic signaling pathways underlying acquired and genetic iron overload conditions. Our data demonstrate that hepatic iron overload associates with a decline in the activation of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (Erk) kinase (Mek1/2) pathway by selectively affecting the phosphorylation of Erk1/2. We propose that Mek1/2-Erk1/2 signaling is uncoupled from iron-Bmp-Smad-mediated hepcidin induction and that it may contribute to a number of liver pathologies in addition to toxic effects of iron. We believe that our findings will advance the understanding of cellular signaling events in the liver during iron overload of different etiologies

Liposome Reconstitution and Modulation of Recombinant Prenylated Human Rac1 by GEFs, GDI1 and Pak1

Small Rho GTPases are well known to regulate a variety of cellular processes by acting as molecular switches. The regulatory function of Rho GTPases is critically dependent on their posttranslational modification at the carboxyl terminus by isoprenylation and association with proper cellular membranes. Despite numerous studies, the mechanisms of recycling and functional integration of Rho GTPases at the biological membranes are largely unclear. In this study, prenylated human Rac1, a prominent member of the Rho family, was purified in large amount from baculovirus-infected Spodoptera frugiperda insect cells using a systematic detergent screening. In contrast to non-prenylated human Rac1 purified from Escherichia coli, prenylated Rac1 from insect cells was able to associate with synthetic liposomes and to bind Rho-specific guanine nucleotide dissociation inhibitor 1 (GDI1). Subsequent liposome reconstitution experiments revealed that GDI1 efficiently extracts Rac1 from liposomes preferentially in the inactive GDP-bound state. The extraction was prevented when Rac1 was activated to its GTP-bound state by Rac-specific guanine nucleotide exchange factors (GEFs), such as Vav2, Dbl, Tiam1, P-Rex1 and TrioN, and bound by the downstream effector Pak1. We found that dissociation of Rac1-GDP from its complex with GDI1 strongly correlated with two distinct activities of especially Dbl and Tiam1, including liposome association and the GDP/GTP exchange. Taken together, our results provided first detailed insights into the advantages of the in vitro liposome-based reconstitution system to study both the integration of the signal transducing protein complexes and the mechanisms of regulation and signaling of small GTPases at biological membranes

Mechanistic Insights into Specificity, Activity, and Regulatory Elements of the Regulator of G-protein Signaling (RGS)-containing Rho-specific Guanine Nucleotide Exchange Factors (GEFs) p115, PDZ-RhoGEF (PRG), and Leukemia-associated RhoGEF (LARG)*

The multimodular guanine nucleotide exchange factors (GEFs) of the Dbl family mostly share a tandem Dbl homology (DH) and pleckstrin homology (PH) domain organization. The function of these and other domains in the DH-mediated regulation of the GDP/GTP exchange reaction of the Rho proteins is the subject of intensive investigations. This comparative study presents detailed kinetic data on specificity, activity, and regulation of the catalytic DH domains of four GEFs, namely p115, p190, PDZ-RhoGEF (PRG), and leukemia-associated RhoGEF (LARG). We demonstrate that (i) these GEFs are specific guanine nucleotide exchange factors for the Rho isoforms (RhoA, RhoB, and RhoC) and inactive toward other members of the Rho family, including Rac1, Cdc42, and TC10. (ii) The DH domain of LARG exhibits the highest catalytic activity reported for a Dbl protein till now with a maximal acceleration of the nucleotide exchange by 107-fold, which is at least as efficient as reported for GEFs specific for Ran or the bacterial toxin SopE. (iii) A novel regulatory region at the N terminus of the DH domain is involved in its association with GDP-bound RhoA monitored by a fluorescently labeled RhoA. (iv) The tandem PH domains of p115 and PRG efficiently contribute to the DH-mediated nucleotide exchange reaction. (v) In contrast to the isolated DH or DH-PH domains, a p115 fragment encompassing both the regulator of G-protein signaling and the DH domains revealed a significantly reduced GEF activity, supporting the proposed models of an intramolecular autoinhibitory mechanism for p115-like RhoGEFs

A novel in vitro assay to study chondrocyte-to-osteoblast transdifferentiation

Purpose!#!Endochondral ossification, which involves transdifferentiation of chondrocytes into osteoblasts, is an important process involved in the development and postnatal growth of most vertebrate bones as well as in bone fracture healing. To study the basic molecular mechanisms of this process, a robust and easy-to-use in vitro model is desirable. Therefore, we aimed to develop a standardized in vitro assay for the transdifferentiation of chondrogenic cells towards the osteogenic lineage.!##!Methods!#!Murine chondrogenic ATDC5 cells were differentiated into the chondrogenic lineage for seven days and subsequently differentiated towards the osteogenic direction. Gene expression analysis of pluripotency, as well as chondrogenic and osteogenic markers, cell-matrix staining, and immunofluorescent staining, were performed to assess the differentiation. In addition, the effects of Wnt3a and lipopolysaccharides (LPS) on the transdifferentiation were tested by their addition to the osteogenic differentiation medium.!##!Results!#!Following osteogenic differentiation, chondrogenically pe-differentiated cells displayed the expression of pluripotency and osteogenic marker genes as well as alkaline phosphatase activity and a mineralized matrix. Co-expression of Col2a1 and Col1a1 after one day of osteogenic differentiation indicated that osteogenic cells had differentiated from chondrogenic cells. Wnt3a increased and LPS decreased transdifferentiation towards the osteogenic lineage.!##!Conclusion!#!We successfully established a rapid, standardized in vitro assay for the transdifferentiation of chondrogenic cells into osteogenic cells, which is suitable for testing the effects of different compounds on this cellular process

Nucleotide-independent extraction of Rac1^Ic from the liposomes by GDI1.

<p>(<b>A</b>) GST-GDI1 pull-down of Rac1^Ic but not of Rac1<i>^Ec</i>. Input is the total mixture of beads and proteins, and output is the pull-down (PD). (<b>B</b>) Liposome binding of Rac1^Ic but not of Rac1<i>^Ec</i>. In the liposome sedimentation assay, Rac1^Ic efficiently binds to liposomes in the absence of GDI1 and independent of whether it was loaded with GDP or GppNHp, a non-hydrolysable GTP analog. Rac1<i>^Ec</i> failed to bind to liposomes under the same conditions. (<b>C</b>) Preferential binding Rac1^Ic to GDI1 than to liposomes. GDI1 binds to both GDP-bound and GppNHp-bound Rac1^Ic proteins and prevents their association with the liposomes. (<b>D</b>, <b>E</b>) GDI1 efficiently extracted GDP-bound Rac1^Ic from the liposomes and to a lower extend also Rac1^Ic-GppNHp. Same amount of GDP-bound and GppNHp-bound forms of Rac1^Ic associated with the liposomes were prepared before incubation with 5-fold molar excess of GDI1 and sedimentation at 20,000x<i>g</i> (<b>D</b>). Using increasing molar excess of GDI1 (2-, 5-, 10-, 15- and 20-fold) showed that higher concentrations of GDI1 are required to extract Rac1^Ic-GppNHp from the liposomes to supernatants in comparison to Rac1^Ic-GDP (<b>E</b>). CBB, coomassie brilliant blue; <i>Ec</i>, <i>E. coli</i>; Ic, insect cells; P, liposome pellet; S, supernatant.</p

Detergent screening for optimal extraction of GDP-bound Rac1 from the insect cell membrane.

<p>(<b>A</b>) Schematic workflow for the isolation of insect cell membrane fraction, detergent extraction and pull-down assay using GST-GDI1. (<b>B</b>) Effects of eighteen various detergents on Rac1 extraction from the membrane fraction of insect cells (upper panel) and inspection of Rac1 prenylation <i>via</i> pull-down with GST-GDI1 (lower panel). Membrane fractions mixed with two different concentrations (0.5% and 1%) of the respective detergents (Table S1 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102425#pone.0102425.s001" target="_blank">File S1</a>) were incubated at room temperature for 30 min, separated in supernatants (S1) and pellets (P1) by centrifugation and immunoblotted using anti-Rac1 antibody. The Supernatants S1 were used in pull-down assays (PD) by using GST-GDI1, which selectively binds to the intact, nucleotide-bound Rac1. Resulted pellets (P2, corresponding to the GSH beads) and supernatant (S2) were visualized by anti-Rac1 antibody in immunoblots. Underlined detergents, especially CHAPS, showed the best properties in the extraction of GDP-bound Rac1 from the insect cell membranes.</p

A restricted spectrum of NRAS mutations causes Noonan syndrome

Noonan syndrome, a developmental disorder characterized by congenital heart defects, reduced growth, facial dysmorphism and variable cognitive deficits, is caused by constitutional dysregulation of the RAS-MAPK signaling pathway. Here we report that germline NRAS mutations conferring enhanced stimulus-dependent MAPK activation account for some cases of this disorder. These findings provide evidence for an obligate dependency on proper NRAS function in human development and growth