Structural and Functional Impact of SRP54 Mutations Causing Severe Congenital Neutropenia

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Characterization of four herbicide-resistant mutants of Rhodopseudomonas viridis by genetic analysis, electron paramagnetic resonance, and optical spectroscopy

Herbicides of the triazine class block electron transfer in the photosynthetic reaction centers of purple bacteria and PSII of higher plants. They are thought to act by competing with one of the electron acceptors, the secondary quinone, QB, for its binding site. Several mutants of the purple bacterium Rhodopseudomonas viridis resistant to terbutryn [2-(methylthio)-4-(ethylamino)-6-(tert-butylamino)-s-triazine] have been isolated by their ability to grow photosynthetically in the presence of the herbicide. Sequence analysis of the genes coding for the L and M subunits of the reaction center showed that four different mutants were obtained, two of them being double mutated: T1 (SerL223----Ala and ArgL217----His), T3 (PheL216----Ser and ValM263----Phe), T4 (TyrL222----Phe), and T6 (PheL216----Ser). The residues L223 and L216 are involved in binding of QB, whereas L217 and L222 are not. M263 is part of the binding pocket of the primary quinone, QA. The affinity of the reaction centers for terbutryn and the electron transfer inhibitor o-phenanthroline, determined via the biphasic charge recombination after one flash, is decreased for all mutants. The affinity for ubiquinone 9 is also decreased, except in T1. Characterization by EPR spectroscopy showed that the QB.-Fe2+ signal of T4, having a g = 1.93 peak, is different from the signals obtained with the wild type and the other mutants but very similar to those of Rhodospirillum rubrum and PSII. The results obtained by the combination of these different techniques are discussed with respect to the three-dimensional structure of the wild type and the mode of binding of ubiquinone, terbutryn, and o-phenanthroline as determined by X-ray structure analysis.     

Weight loss and wrestling training: effects on growth-related hormones

Roemmich, James N., and Wayne E. Sinning. Weight lossand wrestling training: effects on growth-related hormones.J. Appl. Physiol. 82(6):1760-1764, 1997.Adolescent wrestlers(n = 9, 15.4 yr) and recreationallyactive control males (n = 7, 15.7 yr)were measured before, at the end of, and 3.5-4 mo after acompetitive wrestling season to assess the influence of dietary restriction on growth-related hormones. Wrestlers had significant elevations preseason to late season for morning serum concentrations (mean of 8 serial samples) of growth hormone (GH; 2.9 ± 0.7 vs. 6.5 ± 1.4 ng/ml) and sex hormone-binding globulin (SHBG; 16.1 ± 2.3 vs. 27.9 ± 6.9 nmol/l) and significant reductions in GH-binding protein (GHBP; 178 ± 19 vs. 109 ± 17 pmol/l), insulin-likegrowth factor I (IGF-I; 332 ± 30 vs. 267 ± 34 ng/ml),testosterone (T; 4.9 ± 0.4 vs. 3.6 ± 0.4 ng/ml), and freetestosterone (Free-T; 22.4 ± 3.6 vs. 15.7 ± 2.8 pg/ml).Wrestlers had significant postseason reductions in GH (3.44 ± 1.30 ng/ml) and SHBG (10.43 ± 4.13 nmol/l) but elevationsin GHBP (66.7 ± 23.8 pmol/l), IGF-I (72.9 ± 25.1 ng/ml),T (2.10 ± 0.46 ng/ml), and Free-T (9.76 ± 3.01 pg/ml). Concentrations of luteinizing hormone (LH), estradiol,prolactin, cortisol, insulin, and thyroid hormones did not differbecause of exercise-dietary practices of wrestlers. In-seasonelevations in GH, with concomitant reductions in GHBP and IGF-I, thatwere reversed during the postseason suggest a reduction in GH receptor number and partial GH resistance during the season. Nonelevated LH withreduced T levels suggests a central hypothalamic-pituitary-gonadal (H-P-G) axis impairment. In conclusion, undernutrition may lead toaltered H-P-G and GH-IGF-I axes function in adolescent wrestlers. However, only the wrestlers' late-season Free-T concentrations wereoutside the normal range, and the hormone axis impairments were quicklyreversed. The present data do not address hormonal axis responses toseveral years of wrestling and weight loss.

     

Immunohistochemistry in the evaluation of neovascularization in tumor xenografts

Angiogenesis, or neovascularization, is known to play an important role in the neoplastic progression leading to metastasis. CD31 or Factor VIII-related antigen (F VIII RAg) immunohistochemistry is widely used in experimental studies for quantifying tumor neovascularization in immunocompromised animal models implanted with transformed human cell lines. Quantification, however, can be affected by variations in the methodology used to measure vascularization including antibody selection, antigen retrieval (AR) pretreatment, and evaluation techniques. To examine this further, we investigated the microvessel density (MVD) and the intensity of microvascular staining among five different human tumor xenografts and a mouse syngeneic tumor using anti-CD31 and F VIII RAg immunohistochemical staining. Different AR methods also were evaluated. Maximal retrieval of CD31 was achieved using 0.5 M Tris (pH 10) buffer, while maximum retrieval of F VIII RAg was achieved using 0.05% pepsin treatment of tissue sections. For each optimized retrieval condition, anti-CD31 highlighted small vessels better than F VIII RAg. Furthermore, the MVD of CD31 was significantly greater than that of F VIII RAg decorated vessels (p<0.001). The choice of antibody and AR method has a significant affect on immunohistochemical findings when studying angiogenesis. One also must use caution when comparing studies in the literature that use different techniques and reagents.     

Choline head groups stabilize the matrix loop regions of the ATP/ADP carrier ScAAC2

ATP/ADP carriers (AACs) are essential to the cell as they exchange ATP produced in mitochondria for cytosolic ADP. Monoclonal antibodies against the isoform 2 of Saccharomyces cerevisiae AAC (ScAAC2) were used to probe the accessibility of the matrix loops 1 and 3 depending on the environment of the carrier. In mitochondrial membranes ScAAC2 was not recognized, whereas in dodecylmaltoside the antibodies bound to the carrier, suggesting that the epitopes are hidden in the native environment. Exposure of the epitopes by detergents was reversed by reconstitution of the carrier in phospholipids or by exchanging with detergents having a choline or a trimethylammonium head group. Circular dichroism spectroscopy on peptides representing the C-terminal regions of all three matrix loops showed that only phosphocholine detergents induced a structural reorganization. Since in addition phosphatidylcholine was found to be tightly associated with the purified carrier, the matrix loop regions are likely to be associated to the membrane by phosphatidylcholine.     

Functional reconstitution of purified metabotropic glutamate receptor expressed in the fly eye

G-protein-coupled receptors (GPCRs) form one of the largest superfamilies of membrane proteins. Obtaining high yields of GPCRs remains one of the major factors limiting a detailed understanding of their structure and function. Photoreceptor cells (PRCs) contain extensive stacks of specialized membranes where high levels of rhodopsins are naturally present, which makes them ideal for the overexpression of GPCRs. We have generated transgenic flies expressing a number of GPCRs in the PRCs. Drosophila melanogaster metabotropic glutamate receptor (DmGluRA) expressed by this novel strategy was purified to homogeneity, giving at least 3-fold higher yields than conventional baculovirus expression systems due to the higher membrane content of the PRCs. Pure DmGluRA was then reconstituted into liposomes of varying composition. Interestingly, glutamate binding was strictly dependent on the presence of ergosterol.     

Anionic phospholipids are involved in membrane association of FtsY and stimulate its GTPase activity

FtsY, the Escherichia coli homologue of the eukaryotic signal recognition particle (SRP) receptor alpha-subunit, is located in both the cytoplasm and inner membrane. It has been proposed that FtsY has a direct targeting function, but the mechanism of its association with the membrane is unclear. FtsY is composed of two hydrophilic domains: a highly charged N-terminal domain (the A-domain) and a C-terminal GTP-binding domain (the NG-domain). FtsY does not contain any hydrophobic sequence that might explain its affinity for the inner membrane, and a membrane-anchoring protein has not been detected. In this study, we provide evidence that FtsY interacts directly with E.coli phospholipids, with a preference for anionic phospholipids. The interaction involves at least two lipid-binding sites, one of which is present in the NG-domain. Lipid association induced a conformational change in FtsY and greatly enhanced its GTPase activity. We propose that lipid binding of FtsY is important for the regulation of SRP-mediated protein targeting.     

Dietary input of microbes and host genetic variation shape among-population differences in stickleback gut microbiota

To explain differences in gut microbial communities we must determine how processes regulating microbial community assembly (colonization, persistence) differ among hosts and affect microbiota composition. We surveyed the gut microbiota of threespine stickleback (Gasterosteus aculeatus) from 10 geographically clustered populations and sequenced environmental samples to track potential colonizing microbes and quantify the effects of host environment and genotype. Gut microbiota composition and diversity varied among populations. These among-population differences were associated with multiple covarying ecological variables: habitat type (lake, stream, estuary), lake geomorphology and food- (but not water-) associated microbiota. Fish genotype also covaried with gut microbiota composition; more genetically divergent populations exhibited more divergent gut microbiota. Our results suggest that population level differences in stickleback gut microbiota may depend more on internal sorting processes (host genotype) than on colonization processes (transient environmental effects).     

The structure of the regulatory domain of the adenylyl cyclase Rv1264 from Mycobacterium tuberculosis with bound oleic acid

The universal secondary messenger cAMP is produced by adenylyl cyclases (ACs). Most bacterial and all eukaryotic ACs belong to class III of six divergent classes. A class III characteristic is formation of the catalytic pocket at a dimer interface and the presence of additional regulatory domains. Mycobacterium tuberculosis possesses 15 class III ACs, including Rv1264, which is activated at acidic pH due to pH-dependent structural transitions of the Rv1264 dimer. It has been shown by X-ray crystallography that the N-terminal regulatory and C-terminal catalytic domains of Rv1264 interact in completely different ways in the active and inhibited states. Here, we report an in-depth structural and functional analysis of the regulatory domain of Rv1264. The 1.6 A resolution crystal structure shows the protein in a tight, disk-shaped dimer, formed around a helical bundle, and involving a protein chain crossover. To understand pH regulation, we determined structures at acidic and basic pH values and employed structure-based mutagenesis in the holoenzyme to elucidate regulation using an AC activity assay. It has been shown that regulatory and catalytic domains must be linked in a single protein chain. The new studies demonstrate that the length of the linker segment is decisive for regulation. Several amino acids on the surface of the regulatory domain, when exchanged, altered the pH-dependence of AC activity. However, these residues are not conserved amongst a number of related ACs. The closely related mycobacterial Rv2212, but not Rv1264, is strongly activated by the addition of fatty acids. The structure resolved the presence of a deeply embedded fatty acid, characterised as oleic acid by mass spectrometry, which may serve as a hinge. From these data, we conclude that the regulatory domain is a structural scaffold used for distinct regulatory purposes.     

Protein translocation: checkpoint role for SRP GTPase activation

Co-translational protein targeting by the signal recognition particle (SRP) relies on a complex series of structural rearrangements in the SRP and its receptor (SR). In order to precisely coordinate the individual steps, the GTPases of the SRP and the SR form a unique complex in which GTP hydrolysis is activated in a composite active site. A recent study provides new insights on the link between the GTPases and protein translocation.