Cryo-EM structure of the β3-adrenergic receptor reveals the molecular basis of subtype selectivity

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Exploring sand fly salivary proteins to design multiepitope subunit vaccine to fight against visceral leishmaniasis

Visceral leishmaniasis (VL) is caused by the parasites of Leishmania donovani complex, leads to the death of 20 000 to 40 000 people from 56 affected countries, worldwide. Till date, there is not a single available vaccine candidate to prevent the VL infection, and treatment only relies upon expensive and toxic chemotherapeutic options. Consequently, immunoinformatics approach was applied to design a multiepitope-based subunit vaccine to enhance the humoral as well as cell-mediated immunity. Constructed vaccine candidate was further subjected to evaluation on allergenicity and antigenicity and physiochemical parameters. Later on, disulfide engineering was performed to increase the stability of vaccine construct. Also, molecular docking and molecular dynamics simulation study were performed to check the binding affinity and stability of toll-like receptor-4 to vaccine construct complex. Finally, codon optimization and in silico cloning were performed to ensure the expression of proposed vaccine construct in a microbial expression system.     

Long noncoding RNA LINC00958 regulates cell sensitivity to radiotherapy through RRM2 by binding to microRNA-5095 in cervical cancer

Long noncoding RNAs (lncRNAs) have been implicated in the regulation of resistance to radiotherapy in cervical cancer, which is a type of gynecological disease with high mortality in women around the world. Hence, our purpose is to delineate the involvement of LINC00958 in regulating cell sensitivity to radiotherapy in cervical cancer. LINC00958 expression in cervical cancer was assayed, followed by verification of the relationship among LINC00958, microRNA-5095 (miR-5095) and ribonucleotide reductase subunit M2 (RRM2). Hela cells were transduced with up-/downregulation of miR-5095 or RRM2, or LINC00958 silencing, respectively, and then treated with or without a 6 Gy dose of X-ray irradiation. Then the cell proliferation, apoptosis, survival fraction rate, as well as sensitivity to radiotherapy, were assessed. Finally, xenograft tumor in nude mice was established by transplanting Hela cells transfected with sh-LINC00958 and irradiated with 6 Gy of X-ray. High expression of LINC00958 was revealed in The Cancer Genome Atlas and Gene Expression Profiling Interactive Analysis, as well as in radiation-resistant patients, which was associated with lower sensitivity to radiotherapy in cervical cancer. Moreover, cervical cancer patients with higher LINC00958 expression exhibited a shorter overall survival according to Kaplan–Meier analysis. In addition, LINC00958 could regulate the expression of RRM2 by competing for miR-5095. A combination of radiotherapy with LINC00958 silencing, RRM2 downregulation or miR-5095 overexpression was found to inhibit cervical cancer cell proliferation and tumor growth, while promoting cell apoptosis both in vitro and in vivo. Collectively, our results suggest that LINC00958 could regulate RRM2 by competing to miR-5095, which regulates cell sensitivity to radiotherapy in cervical cancer.     

Ribulose bisphosphate carboxylase of the procaryotic symbiont of a hydrothermal vent tube worm: kinetics, activity and gene hybridization

Abstract The giant tube worm, Riftia pachyptila , which is abundant at deep-sea hydrothermal vents, contains an extremely high density of bacterial symbionts in a specialized 'trophosome' tissue. Although the symbiont has not been cultured, enzymatic studies by others indicate that the symbiont is capable of hydrogen-sulfide- or sulfur-based lithoautotrophy and fixes CO₂ via the Calvin-Benson cycle. Here we report additional findings for a specimen from the Guaymas Basin vent site (Gulf of California, 2000 m). Under assay conditions where activity was proportional to cell-free extract concentration, ribulose bisphosphate carboxylase/oxygenase (RuBisCO) activity was 6.3 nmol CO₂/mg protein per min (30°C). This is within the range observed for non-CO₂ limited cultures of sulfur bacteria. The activity vs. temperature profile suggests that the symbiont is a mesophile and not a thermophile. A substrate saturation curve shows an apparent K _m (with respect to ribulose 1,5-bisphosphate) of 65 μM which is considerably lower than the single previous report for a sulfur bacterial symbiont. Strong hybridization was detected between a gene probe derived from the RuBisCO large subunit gene of Anacystis nidulans and Riftia trophosome DNA. A Rhodospirillum rubrum -derived probe also showed hybridization with the same restriction fragments of symbiont DNA.     

CAD‐score: A new contact area difference‐based function for evaluation of protein structural models

Evaluation of protein models against the native structure is essential for the development and benchmarking of protein structure prediction methods. Although a number of evaluation scores have been proposed to date, many aspects of model assessment still lack desired robustness. In this study we present CAD‐score, a new evaluation function quantifying differences between physical contacts in a model and the reference structure. The new score uses the concept of residue–residue contact area difference (CAD) introduced by Abagyan and Totrov (J Mol Biol 1997; 268:678–685). Contact areas, the underlying basis of the score, are derived using the Voronoi tessellation of protein structure. The newly introduced CAD‐score is a continuous function, confined within fixed limits, free of any arbitrary thresholds or parameters. The built‐in logic for treatment of missing residues allows consistent ranking of models of any degree of completeness. We tested CAD‐score on a large set of diverse models and compared it to GDT‐TS, a widely accepted measure of model accuracy. Similarly to GDT‐TS, CAD‐score showed a robust performance on single‐domain proteins, but displayed a stronger preference for physically more realistic models. Unlike GDT‐TS, the new score revealed a balanced assessment of domain rearrangement, removing the necessity for different treatment of single‐domain, multi‐domain, and multi‐subunit structures. Moreover, CAD‐score makes it possible to assess the accuracy of inter‐domain or inter‐subunit interfaces directly. In addition, the approach offers an alternative to the superposition‐based model clustering. The CAD‐score implementation is available both as a web server and a standalone software package at http://www.ibt.lt/bioinformatics/cad‐score/ . Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Discovery of allostery in PKA signaling

Cyclic AMP (cAMP)-dependent protein kinase (PKA) was the second protein kinase to be identified, and the PKA catalytic (C)-subunit serves as a prototype for the large protein kinase superfamily that contains over 500 gene products. The protein kinases regulate many biological functions in eukaryotic cells and are now also a major therapeutic target. The discovery of PKA nearly 50 years ago was quickly followed by the identification of the regulatory subunits that bind cAMP and release the catalytic activity from the holoenzyme. Thus in PKA we see the convergence of two major signaling mechanisms—protein phosphorylation and second messenger signaling through cAMP. Crystallography provides a foundation for understanding function, and detailed knowledge of the structure of the isolated regulatory (R)- and catalytic (C)-subunits has been extremely informative. Yet it is the R₂C₂ holoenzyme that predominates in cells, and the allosteric features of PKA signaling can only be fully appreciated by seeing the full-length protein. The symmetry and the quaternary constraints that one R:C heterodimer exerts on the other in the holoenzyme simply are not present in the isolated subunits or even in the R:C heterodimer.     

Heterotrimeric G protein mediates ethylene‐induced stomatal closure via hydrogen peroxide synthesis in Arabidopsis

Heterotrimeric G proteins function as key players in hydrogen peroxide (H₂O₂) production in plant cells, but whether G proteins mediate ethylene‐induced H₂O₂ production and stomatal closure are not clear. Here, evidences are provided to show the Gα subunit GPA1 as a missing link between ethylene and H₂O₂ in guard cell ethylene signalling. In wild‐type leaves, ethylene‐triggered H₂O₂ synthesis and stomatal closure were dependent on activation of Gα. GPA1 mutants showed the defect of ethylene‐induced H₂O₂ production and stomatal closure, whereas wGα and cGα overexpression lines showed faster stomatal closure and H₂O₂ production in response to ethylene. Ethylene‐triggered H₂O₂ generation and stomatal closure were impaired in RAN1, ETR1, ERS1 and EIN4 mutants but not impaired in ETR2 and ERS2 mutants. Gα activator and H₂O₂ rescued the defect of RAN1 and EIN4 mutants or etr1‐3 in ethylene‐induced H₂O₂ production and stomatal closure, but only rescued the defect of ERS1 mutants or etr1‐1 and etr1‐9 in ethylene‐induced H₂O₂ production. Stomata of CTR1 mutants showed constitutive H₂O₂ production and stomatal closure, but which could be abolished by Gα inhibitor. Stomata of EIN2, EIN3 and ARR2 mutants did not close in responses to ethylene, Gα activator or H₂O₂, but do generate H₂O₂ following challenge of ethylene or Gα activator. The data indicate that Gα mediates ethylene‐induced stomatal closure via H₂O₂ production, and acts downstream of RAN1, ETR1, ERS1, EIN4 and CTR1 and upstream of EIN2, EIN3 and ARR2. The data also show that ETR1 and ERS1 mediate both ethylene and H₂O₂ signalling in guard cells.     

Evidence for an unusual transmembrane configuration of AGG3, a class C Gγ subunit of Arabidopsis

Heterotrimeric G proteins are crucial for the perception of external signals and subsequent signal transduction in animal and plant cells. In both model systems, the complex comprises one Gα, one Gβ, and one Gγ subunit. However, in addition to the canonical Gγ subunits (class A), plants also possess two unusual, plant‐specific classes of Gγ subunits (classes B and C) that have not yet been found in animals. These include Gγ subunits lacking the C–terminal CaaX motif (class B), which is important for membrane anchoring of the protein; the presence of such subunits gives rise to a flexible sub‐population of Gβ/γ heterodimers that are not necessarily restricted to the plasma membrane. Plants also contain class C Gγ subunits, which are twice the size of canonical Gγ subunits, with a predicted transmembrane domain and a large cysteine‐rich extracellular C–terminus. However, neither the presence of the transmembrane domain nor the membrane topology have been unequivocally demonstrated. Here, we provide compelling evidence that AGG3, a class C Gγ subunit of Arabidopsis, contains a functional transmembrane domain, which is sufficient but not essential for plasma membrane localization, and that the cysteine‐rich C–terminus is extracellular.