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

Методика перестроения маршрута полета воздушного судна в процессе его выполнения

   A significant number of aviation incidents is related to loss of control in flight and controlled flight into terrain (LOC-I, CFIT, LALT categories). Investigation of these aviation incidents has revealed that these incidents often occur due to the need for rapid changes in flight routes as a result of detecting obstacles, such as thunderstorms, along the aircraft's path. During the determination of alternative routes to circumvent the encountered obstacle, as well as during the implementation process of the chosen rerouted route, the flight crew makes errors due to increased psycho-physiological workload and time constraints. This article presents an approach to the automatic rerouting of the aircraft's flight route to avoid obstacles detected during flight. The algorithm proposed by the authors allows for evaluating the safety of the original route, calculating alternative route options to bypass the obstacles encountered during flight, verifying their feasibility considering the aircraft's flight technical characteristics and control parameter limitations, and selecting the optimal rerouted route based on specific criteria, such as minimizing the increase in the flight route length, reducing additional fuel consumption, time required for implementing the new flight route, etc. Examples of rerouting the flight route of a hypothetical aircraft with detected obstacles along the flight path are provided in the article to demonstrate the algorithm's functionality. It is shown, in particular, that in the considered example, the shortest route for obstacle avoidance is not optimal in terms of time. It is also demonstrated that the safety of flying along the identified alternative rerouted routes depends, among other factors, on the selected flight speed. Therefore, for each calculated rerouted route, the algorithm determines a range of speeds within which the implementation of the obtained rerouted route is possible. This highlights the complexity and non-triviality of the pilot's task of autonomously finding a safe obstacle avoidance route on board the aircraft.   Большое количество авиационных происшествий связано с потерей управления в полете, а также со столкновением с землей в управляемом полете (категории LOC-I, CFIT, LALT). В результате расследования данных авиационных происшествий выявлено, что часто указанные авиационные происшествия обусловлены необходимостью быстрого изменения маршрута полета вследствие выявления на пути следования воздушного судна препятствий, например, грозового фронта. При определении альтернативных маршрутов облета возникшего препятствия, а также впроцессе реализации выбранного маршрута облета экипаж совершает ошибки ввиду повышенной психофизиологической нагрузки и дефицита времени. В данной статье представлен подход к автоматическому перестроению маршрута полета воздушного судна для облета обнаруженных в процессе полета препятствий. Предлагаемый авторами алгоритм позволяет оценить безопасность исходного маршрута, рассчитать варианты альтернативных маршрутов облета обнаруженных впроцессе полета препятствий, проверить их на реализуемость с учетом летно-технических характеристик воздушного судна, ограничений на управляющие параметры, а также выбрать среди найденных маршрутов облета оптимальный с точки зрения какого-либо критерия, например, исходя из минимизации увеличения протяженности маршрута полета, сокращения дополнительных затрат топлива, времени, необходимого на реализацию нового маршрута полета, и т. д. Для демонстрации работоспособности алгоритма в статье представлены примеры перестроения маршрута полета гипотетического воздушного судна с выявленными на пути следования препятствиями. Показано, в частности, что в рассмотренном примере самый короткий маршрут облета препятствий не является оптимальным с точки зрения временных затрат. Также демонстрируется, что безопасность пролета по найденным альтернативным маршрутам облета препятствий зависит в том числе от выбранной скорости полета. Поэтому для каждого рассчитанного маршрута облета препятствий алгоритм определяет диапазон скоростей, в котором возможна реализация полученного маршрута облетапрепятствий. Последнее указывает на сложность и нетривиальность самостоятельного решения задачи поиска безопасного маршрута облета препятствий пилотом на борту воздушного судна

A structural and biochemical model of processive chitin synthesis

Chitin synthases (CHS) produce chitin, an essential component of the fungal cell wall. The molecular mechanism of processive chitin synthesis is not understood, limiting the discovery of new inhibitors of this enzyme class. We identified the bacterial glycosyltransferase NodC as an appropriate model system to study the general structure and reaction mechanism of CHS. A high throughput screening-compatible novel assay demonstrates that a known inhibitor of fungal CHS also inhibit NodC. A structural model of NodC, on the basis of the recently published BcsA cellulose synthase structure, enabled probing of the catalytic mechanism by mutagenesis, demonstrating the essential roles of the DD and QXXRW catalytic motifs. The NodC membrane topology was mapped, validating the structural model. Together, these approaches give insight into the CHS structure and mechanism and provide a platform for the discovery of inhibitors for this antifungal target

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