Nickel and gtp modulate helicobacter pylori ureg structural flexibility

UreG is a P-loop GTP hydrolase involved in the maturation of nickel-containing urease, an essential enzyme found in plants, fungi, bacteria, and archaea. This protein couples the hydrolysis of GTP to the delivery of Ni(II) into the active site of apo-urease, interacting with other urease chaperones in a multi-protein complex necessary for enzyme activation. Whereas the conformation of Helicobacter pylori (Hp) UreG was solved by crystallography when it is in complex with two other chaperones, in solution the protein was found in a disordered and flexible form, defining it as an intrinsically disordered enzyme and indicating that the well-folded structure found in the crystal state does not fully reflect the behavior of the protein in solution. Here, isothermal titration calorimetry and site-directed spin labeling coupled to electron paramagnetic spectroscopy were successfully combined to investigate HpUreG structural dynamics in solution and the effect of Ni(II) and GTP on protein mobility. The results demonstrate that, although the protein maintains a flexible behavior in the metal and nucleotide bound forms, concomitant addition of Ni(II) and GTP exerts a structural change through the crosstalk of different protein regions

Diversification of EPR signatures in site directed spin labeling using a beta-phosphorylated nitroxide

Site Directed Spin Labeling (SDSL) combined with EPR spectroscopy is a very powerful approach to investigate structural transitions in proteins in particular flexible or even disordered ones. Conventional spin labels are based on nitroxide derivatives leading to classical 3-line spectra whose spectral shapes are indicative of the environment of the labels and thus constitute good reporters of structural modifications. However, the similarity of these spectral shapes precludes probing two regions of a protein or two partner proteins simultaneously. To overcome the limitation due to the weak diversity of nitroxide label EPR spectral shapes, we designed a new spin label based on a β-phosphorylated nitroxide giving 6-line spectra. This paper describes the synthesis of this new spin label, its grafting at four different positions of a model disordered protein able to undergo an induced α-helical folding and its characterization by EPR spectroscopy. For comparative purposes, a classical nitroxide has been grafted at the same positions of the model protein. The ability of the new label to report on structural transitions was evaluated by analyzing the spectral shape modifications induced either by the presence of a secondary structure stabilizer (trifluoroethanol) or by the presence of a partner protein. Taken together the results demonstrate that the new phosphorylated label gives a very distinguishable signature which is able to report from subtle to larger structural transitions, as efficiently as the classical spin label. As a complementary approach, molecular dynamics (MD) calculations were performed to gain further insights into the binding process between the labeled NTAIL and PXD. MD calculations revealed that the new label does not disturb the interaction between the two partner proteins and reinforced the conclusion on its ability to probe different local environments in a protein. Taken together this study represents an important step forward in the extension of the panoply of SDSL-EPR approaches. This journal is © 2014 the Owner Societies

Status of coronary disease and results from early endovascular aneurysm repair after preventive percutaneous coronary revascularization

Background: The incidence of coronary artery disease (CAD) is high in patients with an aortic aneurysm but preoperative routine coronary angiography and preventive coronary revascularization are not recommended to reduce cardiac events in patients with severe CAD. Aim: This study evaluated the safeness and efficacy of preventive percutaneous coronary intervention (PCI) in patients with severe CAD scheduled for endovascular aneurysm repair (EVAR). Methods: All patients with descending thoracic aneurysm (DTA) or abdominal aortic aneurysm (AAA) scheduled for EVAR underwent preliminary coronary angiography. Based on coronary angiography results, 917 patients (40.7%) had significant CAD and were treated by percutaneous coronary intervention (PCI; CAD group) and 1337 patients (59.3%) were without or with mild/moderate CAD and were considered as controls (no‐CAD group). To evaluate the safeness and efficacy of preventive PCI in patients with severe CAD undergoing EVAR, groups were compared for hospital and 12‐month cardiac adverse events. Results: CAD was present in 1210 patients (53.6%): significant in 917 patients (38%) and mild to moderate in 293 patients (5.3%). Hospital and 12‐month cardiac events occurred in 15 (1.6%) and 13 (1.4%) CAD group patients and in 9 (0.7%) and 8 (0.4%) no‐CAD group patients (p = .05 and p = .08), respectively. Hospital and 12‐month cardiac deaths occurred in 3 (0.3%) and 2 (0.2%) CAD group patients and in 3 (0.2%) and 2 (0.2%) no‐CAD group patients (p = .9 and p = .9), respectively. Conclusion: The strategy to treat severe CAD preoperatively by PCI and early subsequent EVAR brings a similar outcome to that in patients without or with mild/ moderate CAD

Conformational selection underlies recognition of a molybdoenzyme by its dedicated chaperone

Molecular recognition is central to all biological processes. Understanding the key role played by dedicated chaperones in metalloprotein folding and assembly requires the knowledge of their conformational ensembles. In this study, the NarJ chaperone dedicated to the assembly of the membrane-bound respiratory nitrate reductase complex NarGHI, a molybdenum-iron containing metalloprotein, was taken as a model of dedicated chaperone. The combination of two techniques ie site-directed spin labeling followed by EPR spectroscopy and ion mobility mass spectrometry, was used to get information about the structure and conformational dynamics of the NarJ chaperone upon binding the N-terminus of the NarG metalloprotein partner. By the study of singly spin-labeled proteins, the E119 residue present in a conserved elongated hydrophobic groove of NarJ was shown to be part of the interaction site. Moreover, doubly spin-labeled proteins studied by pulsed double electron-electron resonance (DEER) spectroscopy revealed a large and composite distribution of inter-label distances that evolves into a single preexisting one upon complex formation. Additionally, ion mobility mass spectrometry experiments fully support these findings by revealing the existence of several conformers in equilibrium through the distinction of different drift time curves and the selection of one of them upon complex formation. Taken together our work provides a detailed view of the structural flexibility of a dedicated chaperone and suggests that the exquisite recognition and binding of the N-terminus of the metalloprotein is governed by a conformational selection mechanism

Transcriptional Profile Associated with Clinical Outcomes in Metastatic Hormone-Sensitive Prostate Cancer Treated with Androgen Deprivation and Docetaxel

(1) Background: Androgen deprivation therapy (ADT) and docetaxel (DX) combination is a standard therapy for metastatic hormone-sensitive prostate cancer (mHSPC) patients. (2) Methods: We investigate if tumor transcriptomic analysis predicts mHSPC evolution in a multicenter retrospective biomarker study. A customized panel of 184 genes was tested in mRNA from tumor samples by the nCounter platform in 125 mHSPC patients treated with ADT+DX. Gene expression was correlated with castration-resistant prostate cancer-free survival (CRPC-FS) and overall survival (OS). (3) Results: High expression of androgen receptor (AR) signature was independently associated with longer CRPC-FS (hazard ratio (HR) 0.6, 95% confidence interval (CI) 0.3-0.9; p = 0.015), high expression of estrogen receptor (ESR) signature with longer CRPC-FS (HR 0.6, 95% CI 0.4-0.9; p = 0.019) and OS (HR 0.5, 95% CI 0.2-0.9, p = 0.024), and lower expression of tumor suppressor genes (TSG) (RB1, PTEN and TP53) with shorter OS (HR 2, 95% CI 1-3.8; p = 0.044). ARV7 expression was independently associated with shorter CRPC-FS (HR 1.5, 95% CI 1.1-2.1, p = 0.008) and OS (HR 1.8, 95% CI 1.2-2.6, p = 0.004), high ESR2 was associated with longer OS (HR 0.5, 95% CI 0.2-1, p = 0.048) and low expression of RB1 was independently associated with shorter OS (HR 1.9, 95% CI 1.1-3.2, p = 0.014). (4) Conclusions: AR, ESR, and TSG expression signatures, as well as ARV7, RB1, and ESR2 expression, have a prognostic value in mHSPC patients treated with ADT+DX

Morphological evaluation of cardoon (Cynara cardunculus L.): assessing biodiversity for applications based on tradition, innovation and sustainability

Selected cardoon cultivars established in the “Serra da Estrela” region were analysed to evaluate morphological characteristics related principally to the production of cardoon flowers, which are a compulsory ingredient for particular Mediterranean PDO cheese regions. The biodiversity of twelve cardoon cultivars installed in an experimental field were evaluated over three growing seasons using thirty-four morphological descriptors. Statistically significant differences were found between cultivars for twenty-four morphological characteristics which indicate a wide genetic diversity. The relationship among the cultivars and characteristics was analysed using principal component analysis. A three dimensional template was found to be very significant and explained 71% of the total variation. The first component is dominated positively by plant height, diameter of stalk, inflorescence characteristics and flower production, while the second component is positively dominated by leaf characteristics. Cultivars A26, D32 and D33, present a plant architecture simultaneously well adapted for flower production, ease of harvesting and plant biomass. These characterizations and understandings can be useful for a plant breeding programme to develop cultivars for innovative potential applications besides flowers, and also for application by other cheese producers in Mediterranean regions.info:eu-repo/semantics/publishedVersio