O-GlcNAcase Fragment Discovery with Fluorescence Polarimetry

The attachment of the sugar N-acetyl-D-glucosamine (GlcNAc) to specific serine and threonine residues on proteins is referred to as protein O-GlcNAcylation. O-GlcNAc transferase (OGT) is the enzyme responsible for carrying out the modification, while O-GlcNAcase (OGA) reverses it. Protein O-GlcNAcylation has been implicated in a wide range of cellular processes including transcription, proteostasis, and stress response. Dysregulation of O-GlcNAc has been linked to diabetes, cancer, and neurodegenerative and cardiovascular disease. OGA has been proposed to be a drug target for the treatment of Alzheimer’s and cardiovascular disease given that increased O-GlcNAc levels appear to exert a protective effect. The search for specific, potent, and drug-like OGA inhibitors with bioavailability in the brain is therefore a field of active research, requiring orthogonal high-throughput assay platforms. Here, we describe the synthesis of a novel probe for use in a fluorescence polarization based assay for the discovery of inhibitors of OGA. We show that the probe is suitable for use with both human OGA, as well as the orthologous bacterial counterpart from <i>Clostridium perfringens</i>, <i>Cp</i>OGA, and the lysosomal hexosaminidases HexA/B. We structurally characterize <i>Cp</i>OGA in complex with a ligand identified from a fragment library screen using this assay. The versatile synthesis procedure could be adapted for making fluorescent probes for the assay of other glycoside hydrolases

Proteolysis of HCF-1 by Ser/Thr glycosylation-incompetent O-GlcNAc transferase:UDP-GlcNAc complexes

In complex with the cosubstrate UDP-N-acetylglucosamine (UDP-GlcNAc),O-linked-GlcNAc transferase (OGT) catalyzes Ser/ThrO-GlcNAcylation of many cellular proteins and proteolysis of the transcriptional coregulator HCF-1. Such a dual glycosyltransferase-protease activity, which occurs in the same active site, is unprecedented and integrates both reversible and irreversible forms of protein post-translational modification within one enzyme. Although occurring within the same active site, we show here that glycosylation and proteolysis occur through separable mechanisms. OGT consists of tetratricopeptide repeat (TPR) and catalytic domains, which, together with UDP-GlcNAc, are required for both glycosylation and proteolysis. Nevertheless, a specific TPR domain contact with the HCF-1 substrate is critical for proteolysis but not Ser/Thr glycosylation. In contrast, key catalytic domain residues and even a UDP-GlcNAc oxygen important for Ser/Thr glycosylation are irrelevant for proteolysis. Thus, from a dual glycosyltransferase-protease, essentially single-activity enzymes can be engineered both in vitro and in vivo. Curiously, whereas OGT-mediated HCF-1 proteolysis is limited to vertebrate species, invertebrate OGTs can cleave human HCF-1. We present a model for the evolution of HCF-1 proteolysis by OGT

Erhöhung der Zuverlässigkeit der Bestimmung der Neutronenbelastung von WWER-Reaktorkomponenten zwecks Ableitung von Vorschlägen für eine sicherere Betriebsführung von WWER-Reaktoren

The results of a project sponsored by the German Bundesministerium fuer Bildung, Wissenschaft, Forschung und Technologie are presented. The Project aimed to improve the safety against embrittlement of VVER-1000 type reactors by a more reliable and accurate determination of the neutron load of reactor pressure vessels. Therefore, six scientist from three Russian research institutions were sponsored to support with their work another BMBF project of the FZR aimed at the same goal. By providing reliable data for the evaluation of ex-vessel neutron activation experiments at two VVER-1000 and formulating the corresponding reactor models a basis has been established for further investigations as well in the FZR as well as in several Russian and Western research institutions. The leading Russian nuclear data library ABBN/MULTIC has been improved and tested. The uncertainties affecting the calculations of the fluence spectrum at the outer boundary of the pressure vessel have been analysed and a spectrum covariance matrix has been derived. The methodologies for the experimental determination of activation rates and for calculations of fluence spectra and activation rates have been further developed and tested by interlaboratoy comparisons. Measurements of different laboratories were compared with each other, as well as the corresponding calculations. Moreover, measurements and calculations were compared against each other, partly with participation of further Russian, Czech and Western institutes. The results of the Intercomparisons have been evaluated by the "International Workshop on the Balakovo-3 Interlaboratory Dosimetry Experiment" in September 1997 in Rossendorf. As a result of these works a better evaluation of the reached accuracies was possible and proposals for an improvement of the used methods could be derived

The conserved threonine-rich region of the HCF-1PRO repeat activates promiscuous OGT:UDP-GlcNAc glycosylation and proteolysis activities

O-Linked GlcNAc transferase (OGT) possesses dual glycosyltransferase-protease activities. OGT thereby stably glycosylates serines and threonines of numerous proteins and, via a transient glutamate glycosylation, cleaves a single known substrate-the so-called HCF-1 &lt;sub&gt;PRO&lt;/sub&gt; repeat of the transcriptional co-regulator host-cell factor 1 (HCF-1). Here, we probed the relationship between these distinct glycosylation and proteolytic activities. For proteolysis, the HCF-1 &lt;sub&gt;PRO&lt;/sub&gt; repeat possesses an important extended threonine-rich region that is tightly bound by the OGT tetratricopeptide-repeat (TPR) region. We report that linkage of this HCF-1 &lt;sub&gt;PRO&lt;/sub&gt; -repeat, threonine-rich region to heterologous substrate sequences also potentiates robust serine glycosylation with the otherwise poor R &lt;sub&gt;p&lt;/sub&gt; -αS-UDP-GlcNAc diastereomer phosphorothioate and UDP-5S-GlcNAc OGT co-substrates. Furthermore, it potentiated proteolysis of a non-HCF-1 &lt;sub&gt;PRO&lt;/sub&gt; -repeat cleavage sequence, provided it contained an appropriately positioned glutamate residue. Using serine- or glutamate-containing HCF-1 &lt;sub&gt;PRO&lt;/sub&gt; -repeat sequences, we show that proposed OGT-based or UDP-GlcNAc-based serine-acceptor residue activation mechanisms can be circumvented independently, but not when disrupted together. In contrast, disruption of both proposed activation mechanisms even in combination did not inhibit OGT-mediated proteolysis. These results reveal a multiplicity of OGT glycosylation strategies, some leading to proteolysis, which could be targets of alternative molecular regulatory strategies

Thio-linked UDP-peptide conjugates as O-GlcNAc transferase inhibitors

O-GlcNAc transferase (OGT) is an essential glycosyltransferase that installs the O-GlcNAc post-translational modification on the nucleocytoplasmic proteome. We report the development of S-linked UDP–peptide conjugates as potent bisubstrate OGT inhibitors. These compounds were assembled in a modular fashion by photoinitiated thiol–ene conjugation of allyl-UDP and optimal acceptor peptides in which the acceptor serine was replaced with cysteine. The conjugate VTPVC­(S-propyl-UDP)­TA (<i>K</i>_i = 1.3 μM) inhibits the OGT activity in HeLa cell lysates. Linear fusions of this conjugate with cell penetrating peptides were explored as prototypes of cell-penetrant OGT inhibitors. A crystal structure of human OGT with the inhibitor revealed mimicry of the interactions seen in the pseudo-Michaelis complex. Furthermore, a fluorophore-tagged derivative of the inhibitor works as a high affinity probe in a fluorescence polarimetry hOGT assay

Risk-oriented approach to ecological safety management at oil refinery

The article considers place and role of the risk-oriented approach in environmental management at enterprises, describes mechanisms for use of risk assessment in environmental management processes and documents, tools for use of risk characteristics in development and implementation of programmes and plans for oil refineries.The study relevance is based on the need to improve the environmental management system of “Orsknefteorgsintez” in line with the plans to include risk-oriented approach in supervisory work, including state environmental supervision.The main goal of the work was development of approaches to risk-oriented environmental management at “Orsknefteorgsintez” based on the health risk assessment from environment pollution, followed by interpretation of gained data and appropriate management decisions.The research was conducted in two stages. The first stage included health risk levels determination resulted from air pollution by “Orsknefteorgsintez”. The results included hygienic studies of human health risk assessment from negative impact by “Orsknefteorgsintez” considering the implementation of the middle-term enterprise development program; and results of comparative analysis of human health risk levels prior to and after operational commissioning of reconstructed objects within the middle-term enterprise development program. The recommendation on human health risk management from the “Orsknefteorgsintez” production facilities (including control and monitoring) are based on the results of the first stage.Risk-oriented improves environmental management systems at enterprises, helps in interactions with government bodies, enhances management decisions in line with environmental legislation and increases environmental-economic investment efficiency

Современные методы предотвращения выкатываний воздушных судов за пределы взлетно-посадочной полосы

The landing of the aircraft has always been the most challenging and dangerous stage of the flight. In order to make a safe landing, the aircraft (A/C) requires reducing the vertical (at the stage of flare-out) and horizontal (prior to touchdown) components of the aircraft's flight speed vector, which in turn reduces the capabilities to increase lift and limits the crew's ability to perform maneuvers. At the same time, during landing the crew must align the aircraft with the runway (RW) and make a touchdown, subsequent A/C landing roll and stop within a rather limited area, which eventually and particularly, under the effect of contributing adverse factors (piloting errors, wind shear, icing, engine failure, aquaplaning, etc.) can cause the aircraft to overshoot and overrun the RW. Currently, as the analysis of aviation accidents statistics shows, the issue of preventing and alerting aircraft overrun is quite relevant. The search for a solution, in terms of preventing aircraft overrunning the runway (RW), is conducted as at the level of aviation authorities as among aircraft manufacturers, operators. Within the framework of this review, an attempt is made to identify and analyze the key factors affecting the dynamics of aircraft motion during landing, using information about aviation accidents that have occurred over the past few years. Notably, such aspects as a human factor and technical features of the operation of modern jet aircraft, influencing the A/C landing roll, are considered. In addition, special attention is paid to consider the methods of prevention and warning of A/C overrun with highlighting the approaches of passive and active protection. Within the framework of the analysis of active protection techniques, the principles of on-board avionic systems operation of the most major aircraft manufacturers, such as Boeing and Airbus, are considered. As an example of the passive protection, the experience of using special energy-absorbing destructible blocks installed next to the runway threshold, is analyzed.Посадка воздушного судна была и остается наиболее сложным и опасным этапом полета. Для совершения безопасной посадки воздушному судну (ВС) необходимо уменьшить вертикальную (на этапе выравнивания)и горизонтальную (на этапе выдерживания) составляющие вектора скорости полета ВС, что в свою очередь уменьшает возможности по увеличению подъемной силы и ограничивает экипаж в возможностях совершения маневров. Также экипаж во время посадки должен подвести ВС к взлетно-посадочной полосе (ВПП) и совершить касание, последующие пробег и остановку ВС в пределах довольно ограниченной по своим размерам площадки, что в конечном итоге, в частности при воздействии сопутствующих неблагоприятных факторов (ошибки пилотирования, сдвиг ветра, обледенение, отказ двигателя, гидроглиссирование и др.), может привести к перелету и выкатыванию самолетов за пределы ВПП. В настоящее время вопрос возможности предотвращения и предупреждения выкатываний ВС, как показывает анализ статистики авиационных происшествий, достаточно актуален. Поиск решения в части предотвращения выкатываний ВС за пределы ВПП ведется как на уровне авиационных властей, так и на уровнях производителей и эксплуатантов ВС. В рамках данной обзорной статьи предпринята попытка выделить и проанализировать ключевые факторы, влияющие на динамику движения ВС при посадке, используя информацию об авиационных происшествиях, произошедших за последние несколько лет. В частности, рассмотрены такие аспекты, как человеческий фактор и технические особенности работы современных реактивных ВС, влияющие на пробег самолета по полосе. Кроме того, особое внимание в статье уделено рассмотрению методов предотвращения и предупреждения выкатываний ВС с выделением методов пассивной и методов активной защиты. В рамках анализа методов активной защиты рассмотрены принципы работы бортовых электронных систем крупнейших авиапроизводителей, таких как Boeing и Airbus. В качестве примера пассивной защиты проанализирован опыт использования специальных энергопоглощающих разрушаемых блоков, размещаемых после торца ВПП