Integrated genomics and proteomics define huntingtin CAG length-dependent networks in mice.

To gain insight into how mutant huntingtin (mHtt) CAG repeat length modifies Huntington's disease (HD) pathogenesis, we profiled mRNA in over 600 brain and peripheral tissue samples from HD knock-in mice with increasing CAG repeat lengths. We found repeat length-dependent transcriptional signatures to be prominent in the striatum, less so in cortex, and minimal in the liver. Coexpression network analyses revealed 13 striatal and 5 cortical modules that correlated highly with CAG length and age, and that were preserved in HD models and sometimes in patients. Top striatal modules implicated mHtt CAG length and age in graded impairment in the expression of identity genes for striatal medium spiny neurons and in dysregulation of cyclic AMP signaling, cell death and protocadherin genes. We used proteomics to confirm 790 genes and 5 striatal modules with CAG length-dependent dysregulation at the protein level, and validated 22 striatal module genes as modifiers of mHtt toxicities in vivo

Comparative analysis of canine monocyte- and bone-marrow-derived dendritic cells

Dendritic cells (DC) represent a heterogeneous cell family of major importance for innate immune responses against pathogens and antigen presentation during infection, cancer, allergy and autoimmunity. The aim of the present study was to characterize canine DC generated in vitro with respect to their phenotype, responsiveness to toll-like receptor (TLR) ligands and T-cell stimulatory capacity. DC were derived from monocytes (MoDC) and from bone marrow hematopoietic cells cultured with either Flt3-ligand (FL-BMDC) or with GM-CSF (GM-BMDC). All three methods generated cells with typical DC morphology that expressed CD1c, CD11c and CD14, similar to macrophages. However, CD40 was only found on DC, CD206 on MΦ and BMDC, but not on monocytes and MoDC. CD1c was not found on monocytes but on all in vitro differentiated cells. FL-BMDC and GM-BMDC were partially positive for CD4 and CD8. CD45RA was expressed on a subset of FL-BMDC but not on MoDC and GM-BMDC. MoDC and FL-DC responded well to TLR ligands including poly-IC (TLR2), Pam3Cys (TLR3), LPS (TLR4) and imiquimod (TLR7) by up-regulating MHC II and CD86. The generated DC and MΦ showed a stimulatory capacity for lymphocytes, which increased upon maturation with LPS. Taken together, our results are the basis for further characterization of canine DC subsets with respect to their role in inflammation and immune responses

Molecular Insights into Frataxin-Mediated Iron Supply for Heme Biosynthesis in <i>Bacillus subtilis</i>

<div><p>Iron is required as an element to sustain life in all eukaryotes and most bacteria. Although several bacterial iron acquisition strategies have been well explored, little is known about the intracellular trafficking pathways of iron and its entry into the systems for co-factor biogenesis. In this study, we investigated the iron-dependent process of heme maturation in <i>Bacillus subtilis</i> and present, for the first time, structural evidence for the physical interaction of a frataxin homologue (Fra), which is suggested to act as a regulatory component as well as an iron chaperone in different cellular pathways, and a ferrochelatase (HemH), which catalyses the final step of heme <i>b</i> biogenesis. Specific interaction between Fra and HemH was observed upon co-purification from crude cell lysates and, further, by using the recombinant proteins for analytical size-exclusion chromatography. Hydrogen–deuterium exchange experiments identified the landscape of the Fra/HemH interaction interface and revealed Fra as a specific ferrous iron donor for the ferrochelatase HemH. The functional utilisation of the <i>in vitro</i>-generated heme <i>b</i> co-factor upon Fra-mediated iron transfer was confirmed by using the <i>B</i>. <i>subtilis</i> nitric oxide synthase bsNos as a metabolic target enzyme. Complementary mutational analyses confirmed that Fra acts as an essential component for maturation and subsequent targeting of the heme <i>b</i> co-factor, hence representing a key player in the iron-dependent physiology of <i>B</i>. <i>subtilis</i>.</p></div

Influence of Fra on the heme maturation pathway.

<p>(A) Kinetics of the conversion of protoporphyrin IX into heme <i>b</i> by HemH. Varying concentrations of Fe²⁺-charged frataxin as the sole iron source were added in a range of 0.1–10 μM, and conversion of protoporphyrin IX into heme <i>b</i> was followed by fluorescence spectroscopy. Data were fitted according to the Michaelis-Menten model, which resulted in a <i>K</i>_m(obs) of about 2.8 ± 0.5 μM and a <i>k</i>_cat(obs) of 0.925 ± 0.059 s^-1 (error bars represent SEM of three independent experiments). (B) <i>In vitro</i> activities of apo-bsNos (1), holo-bsNos (2) and reconstituted apo-bsNos (3). The reconstitution of apo-bsNos with its heme co-factor was carried out by a coupled transfer assay containing Fe²⁺-charged Fra, HemH and protoporphyrin IX, and led to a partial restoration of bsNos activity (error bars represent SEM of three independent experiments). (C) <i>In vivo</i> bsNos activities in equalized amounts of total cellular protein. Control of the assay with <i>B</i>. <i>subtilis</i> WT crude protein extract assayed without the addition of <i>N</i>^ω-hydroxy-l-arginine (1), with <i>B</i>. <i>subtilis</i> WT crude protein extract (2) and with <i>B</i>. <i>subtilis</i> Δ<i>fra</i> crude protein extract (3). The deletion of <i>fra</i> led to a ~12-fold decrease of bsNos activity in the crude mutant cell extract (error bars represent SEM of three independent experiments). (D) Determination of the relative heme contents in <i>B</i>. <i>subtilis</i> WT (red bar) and Δ<i>fra</i> (blue bar) cells by acidic acetone extraction and fluorescence analysis of the heme <i>b</i> soret band emission at 450 nm upon excitation at 380 nm. The amount of cellular heme was found to be ~2.5-fold reduced in the Δ<i>fra</i> mutant cell (error bars represent SEM of three independent experiments). (E) Determination of the relative protoporphyrin IX concentration in <i>B</i>. <i>subtilis</i> WT (red bar) and Δ<i>fra</i> (blue bar) cells by acidic acetone extraction and fluorescence analysis of the protoporphyrin IX soret band emission at 510 nm upon excitation at 410 nm. The amount of cellular protoporphyrin IX was ~1.2-fold elevated in the Δ<i>fra</i> mutant cell (error bars represent SEM of two independent experiments). (F) Fra mediated heme <i>b</i> (protoheme IX) maturation and its delivery to heme-dependent target proteins.</p

HDX epitope mapping of the Fra/HemH interaction surface.

<p>The acidic ridge of Fra (top) binds directly above the HemH (bottom) iron co-ordination site in its catalytic centre (blue), leading to a reduction in HDX (red). Upon binding, both enzymes undergo a conformational change, which leads either to an increased (green) or a reduced (red) HDX. Black areas indicate no change in HDX. The results were mapped to crystal structures of <i>B</i>. <i>subtilis</i> Fra (Protein Databank ID code 2OC6) and HemH (Protein Databank ID code 2HK6) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122538#pone.0122538.ref039" target="_blank">39</a>].</p

Interaction studies of frataxin (Fra) and ferrochelatase (HemH).

<p>(A) Ultraviolet-visible chromatogram of three independent analytical size-exclusion chromatography runs of heterologously expressed and purified Fra-His₆ (gray dotted line), HemH-Strep II (red dotted line) and both proteins together, revealing the formation of a HemH/Fra interaction complex (blue solid line). The interactions were analysed by using a calibrated Superdex 200 10/300 GL gel-filtration column. The inset shows an SDS-PAGE of the Ni²⁺-NTA elution fraction of <i>B</i>. <i>subtilis</i> WT (WT) crude cell extract and <i>B</i>. <i>subtilis</i> AM09 (AM09) crude cell extract with endogenously expressed Fra-His₆, which was co-purified with several proteins, including ferrochelatase HemH from <i>B</i>. <i>subtilis</i> AM09 crude cell extract. Control experiments did not reveal any unspecific interactions. (B) Fra/HemH dimerization was analysed thermophoretically. Unlabelled HemH was titrated to a constant amount of fluorescent-labelled apo-Fra (squares) and holo-Fra (circles). Dimerization caused a significant change in thermophoresis and a <i>K</i>_d of 1.63 μ 0.02 μM was calculated (red line). Error bars represent SEM of three independent experiments.</p

An Enzymatic Pathway for the Biosynthesis of the Formylhydroxyornithine Required for Rhodochelin Iron Coordination

Rhodochelin, a mixed catecholate–hydroxamate type siderophore isolated from <i>Rhodococcus jostii</i> RHA1, holds two l-δ-<i>N</i>-formyl-δ-<i>N</i>-hydroxyornithine (l-fhOrn) moieties essential for proper iron coordination. Previously, bioinformatic and genetic analysis proposed <i>rmo</i> and <i>rft</i> as the genes required for the tailoring of the l-ornithine (l-Orn) precursor [Bosello, M. (2011) <i>J. Am. Chem. Soc.</i> <i>133</i>, 4587–4595]. In order to investigate if both Rmo and Rft constitute a pathway for l-fhOrn biosynthesis, the enzymes were heterologously produced and assayed <i>in vitro</i>. In the presence of molecular oxygen, NADPH and FAD, Rmo monooxygenase was able to convert l-Orn into l-δ-<i>N</i>-hydroxyornithine (l-hOrn). As confirmed in a coupled reaction assay, this hydroxylated intermediate serves as a substrate for the subsequent <i>N</i>¹⁰-formyl-tetrahydrofolate-dependent (<i>N</i>¹⁰-fH₄F) Rtf-catalyzed formylation reaction, establishing a route for the l-fhOrn biosynthesis, prior to its incorporation by the NRPS assembly line. It is of particular interest that a major improvement to this study has been reached with the use of an alternative approach to the chemoenzymatic FolD-dependent <i>N</i>¹⁰-fH₄F conversion, also rescuing the previously inactive CchA, the Rft-homologue in coelichelin assembly line [Buchenau, B. (2004) <i>Arch. Microbiol.</i> <i>182</i>, 313–325; Pohlmann, V. (2008) <i>Org. Biomol. Chem.</i> <i>6</i>, 1843–1848]

Scheme of the proposed iron channelling pathway.

<p>Ferrous iron is bound to the iron chaperone Fra, which transfers it by physical interaction to the ferrochelatase HemH. Upon incorporation of ferrous iron into the protoporphyrin IX scaffold by HemH, heme <i>b</i> is generated and can serve as a co-factor for heme-dependent target enzymes, such as nitric oxide synthase bsNos.</p

Crystal Structure of Bacillus subtilis Cysteine Desulfurase SufS and Its Dynamic Interaction with Frataxin and Scaffold Protein SufU.

The biosynthesis of iron sulfur (Fe-S) clusters in Bacillus subtilis is mediated by a SUF-type gene cluster, consisting of the cysteine desulfurase SufS, the scaffold protein SufU, and the putative chaperone complex SufB/SufC/SufD. Here, we present the high-resolution crystal structure of the SufS homodimer in its product-bound state (i.e., in complex with pyrodoxal-5'-phosphate, alanine, Cys361-persulfide). By performing hydrogen/deuterium exchange (H/DX) experiments, we characterized the interaction of SufS with SufU and demonstrate that SufU induces an opening of the active site pocket of SufS. Recent data indicate that frataxin could be involved in Fe-S cluster biosynthesis by facilitating iron incorporation. H/DX experiments show that frataxin indeed interacts with the SufS/SufU complex at the active site. Our findings deepen the current understanding of Fe-S cluster biosynthesis, a complex yet essential process, in the model organism B. subtilis