Rapid one-step purification of ferritin from cell-free translation systems

A method is described for the one-step affinity purification of ferritin from other proteins synthesized in an in vitro wheat germ protein synthesizing system programmed with rat liver mRNA. The peptide products released from the polysomes were chromatographed on an antiferritin-Sepharose 4B affinity column. The ferritin isolated in this manner was judged pure by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. The recovery of the purified protein from the antibody column ranged from 83 to 88%. Antibody affinity columns can be arranged sequentially to allow the isolation of numerous proteins from one protein synthesis reaction mixture. This method of isolation is applicable to any protein for which antibody is available.     

Reappraisal of the diagnostic and prognostic value of morning stiffness in arthralgia and early arthritis: results from the Groningen EARC,Leiden EARC,ESPOIR, Leiden EAC and REACH

IntroductionMorning stiffness is assessed daily in the diagnostic process of arthralgia and arthritis, but large-scale studies on the discriminative ability are absent. This study explored the diagnostic value of morning stiffness in 5,202 arthralgia and arthritis patients and the prognostic value in early rheumatoid arthritis (RA).MethodsIn arthralgia patients referred to the Early Arthritis Recognition Clinics (EARC) of Leiden (n = 807) and Groningen (n = 481) or included in the Rotterdam Early Arthritis Cohort (REACH) study (n = 353), the associations (cross-sectional analyses) between morning stiffness and presence of arthritis at physical examination were studied. In early arthritis patients, included in the Leiden Early Arthritis Clinic (EAC) (n = 2,748) and Evaluation et Suivi de POlyarthrites Indifférenciées Récentes (ESPOIR) (n = 813), associations with fulfilling the 2010-RA criteria after one year were assessed. In 2010-RA patients included in the EAC (n = 1,140) and ESPOIR (n = 677), association with the long-term outcomes of disease-modifying antirheumatic drug (DMARD)-free sustained remission and radiological progression were determined. Morning stiffness was defined as a duration ≥60 minutes; sensitivity analyses were performed for other definitions.ResultsIn arthralgia, morning stiffness (≥60 minutes) associated with the presence of arthritis; Leiden EARC odds ratio (OR) 1.49 (95% CI 1.001 to 2.20), Groningen EARC OR 2.21 (1.33 to 3.69) and REACH OR 1.55 (0.97 to 2.47) but the areas under the receiver operating characteristic curve (AUCs) were low (0.52, 0.57, 0.54). In early arthritis, morning stiffness was associated with 2010-RA independent of other predictors (Leiden EAC OR 1.72 (95% CI 1.31 to 2.25, AUC 0.68), ESPOIR OR 1.68 (1.03 to 2.74, AUC 0.64)). Duration of ≥30 minutes provided optimal discrimination for RA in early arthritis. Morning stiffness was not associated with radiological progression or DMARD-free sustained remission.ConclusionsMorning stiffness in arthralgia and early arthritis is associated with arthritis and RA respectively. This supports the incorporation of morning stiffness in the diagnostic process.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0616-3) contains supplementary material, which is available to authorized users.     

Novel haem co-ordination variants of flavocytochrome P450BM3

Bacillus megaterium flavocytochrome P450 BM3 is a catalytically self-sufficient fatty acid hydroxylase formed by fusion of soluble NADPH-cytochrome P450 reductase and P450 domains. Selected mutations at residue 264 in the haem (P450) domain of the enzyme lead to novel amino acid sixth (distal) co-ordination ligands to the haem iron. The catalytic, spectroscopic and thermodynamic properties of the A264M, A264Q and A264C variants were determined in both the intact flavocytochromes and haem domains of P450 BM3. Crystal structures of the mutant haem domains demonstrate axial ligation of P450 haem iron by methionine and glutamine ligands trans to the cysteine thiolate, creating novel haem iron ligand sets in the A264M/Q variants. In contrast, the crystal structure of the A264C variant reveals no direct interaction between the introduced cysteine side chain and the haem, although EPR data indicate Cys(264) interactions with haem iron in solution. The A264M haem potential is elevated by comparison with wild-type haem domain, and substrate binding to the A264Q haem domain results in a approximately 360 mV increase in potential. All mutant haem domains occupy the conformation adopted by the substrate-bound form of wild-type BM3, despite the absence of added substrate. The A264M mutant (which has higher dodecanoate affinity than wild-type BM3) co-purifies with a structurally resolved lipid. These data demonstrate that a single mutation at Ala(264) is enough to perturb the conformational equilibrium between substrate-free and substrate-bound P450 BM3, and provide firm structural and spectroscopic data for novel haem iron ligand sets unprecedented in nature.     

Microclusters of inhibitory killer immunoglobulin-like receptor signaling at natural killer cell immunological synapses

We report the supramolecular organization of killer Ig–like receptor (KIR) phosphorylation using a technique applicable to imaging phosphorylation of any green fluorescent protein–tagged receptor at an intercellular contact or immune synapse. Specifically, we use fluorescence lifetime imaging (FLIM) to report Förster resonance energy transfer (FRET) between GFP-tagged KIR2DL1 and a Cy3-tagged generic anti-phosphotyrosine monoclonal antibody. Visualization of KIR phosphorylation in natural killer (NK) cells contacting target cells expressing cognate major histocompatibility complex class I proteins revealed that inhibitory signaling is spatially restricted to the immune synapse. This explains how NK cells respond appropriately when simultaneously surveying susceptible and resistant target cells. More surprising, phosphorylated KIR was confined to microclusters within the aggregate of KIR, contrary to an expected homogeneous distribution of KIR signaling across the immune synapse. Also, yellow fluorescent protein–tagged Lck, a kinase important for KIR phosphorylation, accumulated in a multifocal distribution at inhibitory synapses. Spatial confinement of receptor phosphorylation within the immune synapse may be critical to how activating and inhibitory signals are integrated in NK cells.     

Characterization of a human orphon 28 S ribosomal DNA

We have isolated clones in which two regions of the human genome are represented, each containing an orphon: a dispersed copy of 28S rDNA. Nucleotide (nt) sequence analysis established that one of these, H28S-O1, corresponds to nt 3627-4105 of human 28S rDNA, but in a mutated form. The orphon was flanked on one side by a portion of the L1Hs long interspersed repeat family of the human genome. Although H25S-O1 is not flanked by the terminal direct repeats characteristic of transposed DNA, it is possible that it is a processed pseudogene.     

Effect of thermal,oxidative, acidic,osmotic, or nutritional stresses on subsequent culturability of Escherichia coli in seawater

Survival of stressed Escherichia coli with or without the rpoS gene was assessed after 2 and 6 days in sterile seawater. Cells were submitted to thermal (48°C), acidic (pH 5.1), oxidative (H₂O₂ 1mm), nutritional (C, N, P starvation), or osmotic (NaCl 0.5m) stresses for periods ranging from 0 to 4 h. We found a stress-mediated cross protection against seawater relative to controls. Viability was higher when cells were acid, oxidatively, nutritionally or osmotically stressed. Survival increased in cells stressed at 37°C as compared with 20°C. With the exception of osmotic stress, we found that this stress-induced cross protection was rpoS dependent.Correspondence to: P.M. Munro.     

Trp(359) regulates flavin thermodynamics and coenzyme selectivity in Mycobacterium tuberculosis FprA

Mtb (Mycobacterium tuberculosis) FprA (flavoprotein reductase A) is an NAD(P)H-dependent FAD-binding reductase that is structurally related to mammalian adrenodoxin reductase, and which supports the catalytic function of Mtb cytochrome P450s. Trp(359), proximal to the FAD, was investigated in light of its potential role in controlling coenzyme interactions, as observed for similarly located aromatic residues in diflavin reductases. Phylogenetic analysis indicated that a tryptophan residue corresponding to Trp(359) is conserved across FprA-type enzymes and in adrenodoxin reductases. W359A/H mutants of Mtb FprA were generated, expressed and the proteins characterized to define the role of Trp(359). W359A/H mutants exhibited perturbed UV-visible absorption/fluorescence properties. The FAD semiquinone formed in wild-type NADPH-reduced FprA was destabilized in the W359A/H mutants, which also had more positive FAD midpoint reduction potentials (-168/-181 mV respectively, versus the standard hydrogen electrode, compared with -230 mV for wild-type FprA). The W359A/H mutants had lower ferricyanide reductase k(cat) and NAD(P)H K(m) values, but this led to improvements in catalytic efficiency (k(cat)/K(m)) with NADH as reducing coenzyme (9.6/18.8 muM(-1).min(-1) respectively, compared with 5.7 muM(-1).min(-1) for wild-type FprA). Stopped-flow spectroscopy revealed NAD(P)H-dependent FAD reduction as rate-limiting in steady-state catalysis, and to be retarded in mutants (e.g. limiting rate constants for NADH-dependent FAD reduction were 25.4 s(-1) for wild-type FprA and 4.8 s(-1)/13.4 s(-1) for W359A/H mutants). Diminished mutant FAD content (particularly in W359H FprA) highlighted the importance of Trp(359) for flavin stability. The results demonstrate that the conserved Trp(359) is critical in regulating FprA FAD binding, thermodynamic properties, catalytic efficiency and coenzyme selectivity.     

Expression, purification and characterization of cytochrome P450 Biol: a novel P450 involved in biotin synthesis in Bacillus subtilis.

The bioI gene has been sub-cloned and over-expressed in Escherichia coli, and the protein purified to homogeneity. The protein is a cytochrome P450, as indicated by its visible spectrum (low-spin haem iron Soret band at 419 nm) and by the characteristic carbon monoxide-induced shift of the Soret band to 448 nm in the reduced form. N-terminal amino acid sequencing and mass spectrometry indicate that the initiator methionine is removed from cytochrome P450 BioI and that the relative molecular mass is 44,732 Da, consistent with that deduced from the gene sequence. SDS-PAGE indicates that the protein is homogeneous after column chromatography on DE-52 and hydroxyapatite, followed by FPLC on a quaternary ammonium ion-exchange column (Q-Sepharose). The purified protein is of mixed spin-state by both electronic spectroscopy and by electron paramagnetic resonance [g values=2.41, 2.24 and 1.97/1.91 (low-spin) and 8.13, 5.92 and 3.47 (high-spin)]. Magnetic circular dichroism and electron paramagnetic resonance studies indicate that P450 BioI has a cysteine-ligated b-type haem iron and the near-IR magnetic circular dichroism band suggests strongly that the sixth ligand bound to the haem iron is water. Resonance Raman spectroscopy identifies vibrational signals typical of cytochrome P450, notably the oxidation state marker v4 at 1,373 cm(-1) (indicating ferric P450 haem) and the splitting of the spin-state marker v3 into two components (1,503 cm(-1) and 1,488 cm(-1)), indicating cytochrome P450 BioI to be a mixture of high- and low-spin forms. Fatty acids were found to bind to cytochrome P450 BioI, with myristic acid (Kd=4.18+/-0.26 microM) and pentadecanoic acid (Kd=3.58+/-0.54 microM) having highest affinity. The fatty acid analogue inhibitor 12-imidazolyldodecanoic acid bound extremely tightly (Kd<1 microM), again indicating strong affinity for fatty acid chains in the P450 active site. Catalytic activity was demonstrated by reconstituting the P450 with either a soluble form of human cytochrome P450 reductase, or a Bacillus subtilis ferredoxin and E. coli ferredoxin reductase. Substrate hydroxylation at the omega-terminal position was demonstrated by turnover of the chromophoric fatty acid para-nitrophenoxydodecanoic acid, and by separation of product from the reaction of P450 BioI with myristic acid.     

High sensitivity of mouse neuronal cells to tetanus toxin requires a GPI-anchored protein.

Tetanus neurotoxin (TeNT) produced by Clostridium tetani specifically cleaves VAMP/synaptobrevin (VAMP) in central neurons, thereby causing inhibition of neurotransmitter release and ensuing spastic paralysis. Although polysialogangliosides act as components of the neurotoxin binding sites on neurons, evidence has accumulated indicating that a protein moiety is implicated as a receptor of TeNT. We have observed that treatment of cultured mouse neuronal cells with the phosphatidylinositol-specific phospholipase C (PIPLC) inhibited TeNT-induced cleavage of VAMP. Also, we have shown that the blocking effects of TeNT on neuroexocytosis can be prevented by incubation of Purkinje cell preparation with PIPLC. In addition, treatment of cultured mouse neuronal cells with cholesterol sequestrating agents such as nystatin and filipin, which disrupt clustering of GPI-anchored proteins in lipid rafts, prevented intraneuronal VAMP cleavage by TeNT. Our results demonstrate that high sensitivity of neurons to TeNT requires rafts and one or more GPI-anchored protein(s) which act(s) as a pivotal receptor for the neurotoxin.