Structural Basis for the Recognition of Cellular mRNA Export Factor REF by Herpes Viral Proteins HSV-1 ICP27 and HVS ORF57

The herpesvirus proteins HSV-1 ICP27 and HVS ORF57 promote viral mRNA export by utilizing the cellular mRNA export machinery. This function is triggered by binding to proteins of the transcription-export (TREX) complex, in particular to REF/Aly which directs viral mRNA to the TAP/NFX1 pathway and, subsequently, to the nuclear pore for export to the cytoplasm. Here we have determined the structure of the REF-ICP27 interaction interface at atomic-resolution and provided a detailed comparison of the binding interfaces between ICP27, ORF57 and REF using solution-state NMR. Despite the absence of any obvious sequence similarity, both viral proteins bind on the same site of the folded RRM domain of REF, via short but specific recognition sites. The regions of ICP27 and ORF57 involved in binding by REF have been mapped as residues 104–112 and 103–120, respectively. We have identified the pattern of residues critical for REF/Aly recognition, common to both ICP27 and ORF57. The importance of the key amino acid residues within these binding sites was confirmed by site-directed mutagenesis. The functional significance of the ORF57-REF/Aly interaction was also probed using an ex vivo cytoplasmic viral mRNA accumulation assay and this revealed that mutants that reduce the protein-protein interaction dramatically decrease the ability of ORF57 to mediate the nuclear export of intronless viral mRNA. Together these data precisely map amino acid residues responsible for the direct interactions between viral adaptors and cellular REF/Aly and provide the first molecular details of how herpes viruses access the cellular mRNA export pathway

Competitive and Cooperative Interactions Mediate RNA Transfer from Herpesvirus Saimiri ORF57 to the Mammalian Export Adaptor ALYREF

The essential herpesvirus adaptor protein HVS ORF57, which has homologs in all other herpesviruses, promotes viral mRNA export by utilizing the cellular mRNA export machinery. ORF57 protein specifically recognizes viral mRNA transcripts, and binds to proteins of the cellular transcription-export (TREX) complex, in particular ALYREF. This interaction introduces viral mRNA to the NXF1 pathway, subsequently directing it to the nuclear pore for export to the cytoplasm. Here we have used a range of techniques to reveal the sites for direct contact between RNA and ORF57 in the absence and presence of ALYREF. A binding site within ORF57 was characterized which recognizes specific viral mRNA motifs. When ALYREF is present, part of this ORF57 RNA binding site, composed of an a-helix, binds preferentially to ALYREF. This competitively displaces viral RNA from the a-helix, but contact with RNA is still maintained by a flanking region. At the same time, the flexible N-terminal domain of ALYREF comes into contact with the viral RNA, which becomes engaged in an extensive network of synergistic interactions with both ALYREF and ORF57. Transfer of RNA to ALYREF in the ternary complex, and involvement of individual ORF57 residues in RNA recognition, were confirmed by UV cross-linking and mutagenesis. The atomic-resolution structure of the ORF57-ALYREF interface was determined, which noticeably differed from the homologous ICP27-ALYREF structure. Together, the data provides the first site-specific description of how viral mRNA is locked by a herpes viral adaptor protein in complex with cellular ALYREF, giving herpesvirus access to the cellular mRNA export machinery. The NMR strategy used may be more generally applicable to the study of fuzzy protein-protein-RNA complexes which involve flexible polypeptide regions

Воздействие высокой концентрации оксида азота на оксигенаторы аппаратов искусственного кровообращения (экспериментальное исследование)

The aim of the study. To study the effect of high nitric oxide concentrations on hollow polypropylene fibers of oxygenators.Materials and methods. The study was conducted in two stages. At the first stage, we evaluated the stability of oxygenator membrane made of hollow polypropylene fibers after six hours of exposure to air-oxygen mixture containing NO at 500 parts per million, or 500 pro pro mille (ppm) concentration, using mass spectrometry and infrared spectroscopy. At the second stage, an experiment with cardiopulmonary bypass (CPB) was conducted on 10 pigs. In the study group (n=5) animals sweep gas was supplied to the oxygenator as an air-oxygen mixture with NO at 100 ppm. In the control group animals (n=5) an air-oxygen mixture was used without NO. The CPB lasted for 4 hours, followed by observation for 12 hours. NO, NO2 (at the inlet and outlet of the oxygenator), and the dynamics of methemoglobin were evaluated. After weaning of animals from CPB, the oxygenators were tested for leakproofness, and scanning electron microscopy (SEM) was performed.Results. The oxygenator made of polypropylene hollow fibers retained its gas transfer parameters after six hours of exposure to air-oxygen mixture containing NO at 500 ppm. Based on IR-Fourier spectroscopy findings, NO did not affect structural integrity of polypropylene membranes. NO added to gas mixture at 100 ppm did not increase NO2 to toxic level of 2 ppm in 91% of control tests during 4 hours CPB in pigs; mean value was 1.58 ± 0.28 ppm. Methemoglobin concentration did not exceed the upper limit of permissible level (3%), and there were no statistically significant differences with the control group. All tested oxygenators have passed the leakproofness test. According to SEM findings, larger amounts of fibrin deposits were found in the control group oxygenators vs study group.Conclusion. There were no negative effects of NO at 500 ppm concentration on the oxygenator membrane made of hollow polypropylene fibers. NO at 100 ppm in a gas-mixture supplied to oxygenators did not lead to an exceedance of safe NO2 and methemoglobin concentrations in an animal model. Reduced fibrin deposits on hollow fibers of polypropylene oxygenator membranes were observed when with NO at a level of 100 ppm was added to a gas mixture.  Цель исследования. Изучить воздействие высоких концентраций оксида азота на полипропиленовые полые волокна оксигенаторов.Материалы и методы. Исследование провели в два этапа. На первом этапе с помощью масс-спектрометрии и инфракрасной спектроскопии выполнили оценку стабильности мембраны оксигенатора из полых волокон полипропилена после шестичасового воздействия воздушно-кислородной смеси, содержащей NO в концентрации 500 пропромилле, или 500 частей на миллион – parts per million (ppm). На втором этапе провели эксперимент на 10 свиньях с подключением аппарата искусственного кровообращения (ИК). Животным основной группы (n=5) в оксигенатор подавали воздушно-кислородную смесь, содержащую NO в концентрации 100 ppm. Животным контрольной группы (n=5) в оксигенатор подавали воздушно-кислородную смесь без NO. Процедура ИК длилась 4 часа, затем следовало наблюдение в течение 12 часов. Оценивали NO, NO2 (на входе и выходе из оксигенатора), динамику метгемоглобина. После отключения от ИК оксигенаторы тестировали на герметичность, а также выполняли сканирующую электронную микроскопию (СЭМ).Результаты. Оксигенатор из полипропиленовых полых волокон сохранял свои газотранспортные характеристики после шестичасового воздействия воздушно-кислородной смеси с добавлением NO в концентрации 500 ppm. По данным ИК-Фурье спектроскопии показали, что NO не влияет на структуру мембран из полипропилена. Добавление NO в дозировке 100 ppm во время 4 часов ИК у свиней не сопровождалось повышением концентрации NO2 до токсичного уровня 2 ppm в 91% измерений: среднее значение составило 1,58 ± 0,28 ppm. Концентрация метгемоглобина не превышала верхнего  предела  допустимых  значений  (3%),  не  обнаружили  каких-либо статистически значимых различий при сравнении с группой контроля. Все исследуемые оксигенаторы выдержали тестирование на герметичность. По результатам СЭМ оксигенаторы группы контроля характеризовались большим количеством отложений фибрина, чем оксигенаторы основной группы.Заключение. Негативного воздействия NO в концентрации 500 ppm на мембраны оксигенаторов из полых волокон полипропилена не обнаружили. Подача в оксигенатор NO в концентрации 100 ppm NO2 не приводила к превышению безопасного содержания NO2 и метгемоглобина в эксперименте на животных. Выявили снижение образования отложений фибрина на полых волокнах мембран оксигенаторов из полипропилена при подаче NO в концентрации 100 ppm

Stability of a high-concentration monoclonal antibody solution produced by liquid-liquid phase separation

Subcutaneous injection of a low volume (100 mg/mL) formulation is an attractive administration strategy for monoclonal antibodies (mAbs) and other biopharmaceutical proteins. Using concentrated solutions may also be beneficial at various stages of bioprocessing. However, concentrating proteins by conventional techniques, such as ultrafiltration, can be time consuming and challenging. Isolation of the dense fraction produced by macroscopic liquid–liquid phase separation (LLPS) has been suggested as a means to produce high-concentration solutions, but practicality of this method, and the stability of the resulting protein solution have not previously been demonstrated. In this proof-of-concept study, we demonstrate that LLPS can be used to concentrate a mAb solution to >170 mg/mL. We show that the structure of the mAb is not altered by LLPS, and unperturbed mAb is recoverable following dilution of the dense fraction, as judged by (1)H nuclear magnetic resonance spectroscopy. Finally, we show that the physical properties and stability of a model high concentration protein formulation obtained from the dense fraction can be improved, for example through the addition of the excipient arginine·glutamate. This results in a stable high-concentration protein formulation with reduced viscosity and no further macroscopic LLPS. Concentrating mAb solutions by LLPS represents a simple and effective technique to progress toward producing high-concentration protein formulations for bioprocessing or administration. Abbreviations Arginine·glutamate (Arg·Glu), Carr-Purcell-Meiboom-Gill (CPMG), critical temperature (T(C)), high-performance size-exclusion chromatography (HPSEC), liquid–liquid phase separation (LLPS), monoclonal antibody (mAb), nuclear magnetic resonance (NMR), transverse relaxation rate (R(2)

Michael Addition of 3-Oxo-3-phenylpropanenitrile to Linear Conjugated Enynones: Approach to Polyfunctional δ-Diketones as Precursors for Heterocycle Synthesis

Reaction of linear conjugated enynones, 1,5-diarylpent-2-en-4-yn-1-ones [Ar1C≡CCH=CHC(=O)Ar2], with 3-oxo-3-phenylpropanenitrile (NCCH2COPh) in the presence of sodium methoxide MeONa as a base in MeOH at room temperature for 4–26 h affords polyfunctional δ-diketones as a product of regioselective Michael addition to the double carbon–carbon bond of starting enynones. The δ-diketones have been formed as mixtures of two diastereomers in a ratio of 2.5:1 in good general yields of 53–98%. A synthetic potential of the obtained δ-diketones has been demonstrated by heterocyclization with hydrazine into substututed 5,6-dihydro-4H-1,2-diazepine