Characterization of Spontaneous Bone Marrow Recovery after Sublethal Total Body Irradiation: Importance of the Osteoblastic/Adipocytic Balance

Many studies have already examined the hematopoietic recovery after irradiation but paid with very little attention to the bone marrow microenvironment. Nonetheless previous studies in a murine model of reversible radio-induced bone marrow aplasia have shown a significant increase in alkaline phosphatase activity (ALP) prior to hematopoietic regeneration. This increase in ALP activity was not due to cell proliferation but could be attributed to modifications of the properties of mesenchymal stem cells (MSC). We thus undertook a study to assess the kinetics of the evolution of MSC correlated to their hematopoietic supportive capacities in mice treated with sub lethal total body irradiation. In our study, colony-forming units – fibroblasts (CFU-Fs) assay showed a significant MSC rate increase in irradiated bone marrows. CFU-Fs colonies still possessed differentiation capacities of MSC but colonies from mice sacrificed 3 days after irradiation displayed high rates of ALP activity and a transient increase in osteoblastic markers expression while pparγ and neuropilin-1 decreased. Hematopoietic supportive capacities of CFU-Fs were also modified: as compared to controls, irradiated CFU-Fs significantly increased the proliferation rate of hematopoietic precursors and accelerated the differentiation toward the granulocytic lineage. Our data provide the first evidence of the key role exerted by the balance between osteoblasts and adipocytes in spontaneous bone marrow regeneration. First, (pre)osteoblast differentiation from MSC stimulated hematopoietic precursor's proliferation and granulopoietic regeneration. Then, in a second time (pre)osteoblasts progressively disappeared in favour of adipocytic cells which down regulated the proliferation and granulocytic differentiation and then contributed to a return to pre-irradiation conditions

Brain expressed and X-linked (Bex) proteins are intrinsically disordered proteins (IDPs) and form new signaling hubs

Intrinsically disordered proteins (IDPs) are abundant in complex organisms. Due to their promiscuous nature and their ability to adopt several conformations IDPs constitute important points of network regulation. The family of Brain Expressed and X-linked (Bex) proteins consists of 5 members in humans (Bex1-5). Recent reports have implicated Bex proteins in transcriptional regulation and signaling pathways involved in neurodegeneration, cancer, cell cycle and tumor growth. However, structural and biophysical data for this protein family is almost non-existent. We used bioinformatics analyses to show that Bex proteins contain long regions of intrinsic disorder which are conserved across all members. Moreover, we confirmed the intrinsic disorder by circular dichroism spectroscopy of Bex1 after expression and purification in E. coli. These observations strongly suggest that Bex proteins constitute a new group of IDPs. Based on these findings, together with the demonstrated promiscuity of Bex proteins and their involvement in different signaling pathways, we propose that Bex family members play important roles in the formation of protein network hubs.Ministry of Economy and Competitiveness (MINECO) from the Government of Spain. Projects: BFU2010-15276 and BFU2013-42746-P to MV. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscriptS

Disorder predictions for the Bex protein family in human: A) Bex1 and Bex2; B) Bex3 and Bex5; C) Bex4.

<p>Disorder predictions are sorted from top to bottom according to decreasing average disorder tendency in the region of the coiled coil as predicted using COIL (green box). The location of the α-helix predicted by PSIPRED is shown in red. Disordered regions have scores >0.5.</p

Brain Expressed and X-Linked (Bex) Proteins Are Intrinsically Disordered Proteins (IDPs) and Form New Signaling Hubs

<div><p>Intrinsically disordered proteins (IDPs) are abundant in complex organisms. Due to their promiscuous nature and their ability to adopt several conformations IDPs constitute important points of network regulation. The family of Brain Expressed and X-linked (Bex) proteins consists of 5 members in humans (Bex1-5). Recent reports have implicated Bex proteins in transcriptional regulation and signaling pathways involved in neurodegeneration, cancer, cell cycle and tumor growth. However, structural and biophysical data for this protein family is almost non-existent. We used bioinformatics analyses to show that Bex proteins contain long regions of intrinsic disorder which are conserved across all members. Moreover, we confirmed the intrinsic disorder by circular dichroism spectroscopy of Bex1 after expression and purification in <i>E. coli</i>. These observations strongly suggest that Bex proteins constitute a new group of IDPs. Based on these findings, together with the demonstrated promiscuity of Bex proteins and their involvement in different signaling pathways, we propose that Bex family members play important roles in the formation of protein network hubs.</p></div

Analysis of mBex1 by far-UV circular dichroism (CD).

<p>A) Schematic showing the mBex1 purification process. After inducing protein expression with IPTG, pelleted bacteria were boiled. NaCl was added to the supernatant at a final concentration of 3 M and the solution was subjected to hydrophobic chromatography. B) SDS-PAGE of purified from <i>E. coli</i> by boiling and hydrophobic chromatography. Lane 0: no induction; lane 1: after incubation for 3 h with IPTG; lane 3: soluble fraction after boiling bacteria for 5 minutes; lane 4: eluent from hydrophobic chromatography. See text and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117206#sec002" target="_blank">Materials and Methods</a> for details. Molecular weight standards are shown on the left. C) Far-UV CD spectra of mBex1 in 20 mM acetate buffer pH 5.1 containing increasing concentrations of trifluorethanol (TFE). The CD spectra shown represent the average of five independently determined spectra.</p

The Bex protein network.

<p>Bex proteins are promiscuous, interacting with a variety of proteins and participating in several signaling pathways. Bex1–4 are represented by green circles. Direct interactions described between Bex members and protein interactors are indicated with blue circles. Bex1–4 have been shown to modulate NF-kB signaling pathways, as indicated by the yellow square. Orange circles represent proteins whose expression or activation is modulated by the indicated Bex proteins.</p

HCA analysis of all human Bex proteins. Bex1 (A), Bex2 (B), Bex3 (C), Bex5 (D) and Bex4 (E) were analyzed using the HCA server (http://bioserv.impmc.jussieu.fr/).

<p>The form of the clusters is generally indicative of the type of secondary structure formed (vertical clusters often correspond to β-strands while horizontal clusters correspond to α-helices) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117206#pone.0117206.ref030" target="_blank">30</a>]. Symbols are used to denote specific amino acids: star for proline; square for threonine; dotted square for serine; diamond for glycine.</p