Molecular basis of USP7 inhibition by selective small-molecule inhibitors

Ubiquitination controls the stability of most cellular proteins, and its deregulation contributes to human diseases including cancer. Deubiquitinases remove ubiquitin from proteins, and their inhibition can induce the degradation of selected proteins, potentially including otherwise 'undruggable' targets. For example, the inhibition of ubiquitin-specific protease 7 (USP7) results in the degradation of the oncogenic E3 ligase MDM2, and leads to re-activation of the tumour suppressor p53 in various cancers. Here we report that two compounds, FT671 and FT827, inhibit USP7 with high affinity and specificity in vitro and within human cells. Co-crystal structures reveal that both compounds target a dynamic pocket near the catalytic centre of the auto-inhibited apo form of USP7, which differs from other USP deubiquitinases. Consistent with USP7 target engagement in cells, FT671 destabilizes USP7 substrates including MDM2, increases levels of p53, and results in the transcription of p53 target genes, induction of the tumour suppressor p21, and inhibition of tumour growth in mice

Evidence for STIM1- and Orai1-dependent store-operated calcium influx through ICRAC in vascular smooth muscle cells: role in proliferation and migration

The identity of store-operated calcium (Ca2+) entry (SOCE) channels in vascular smooth muscle cells (VSMCs) remains a highly contentious issue. Whereas previous studies have suggested that SOCE in VSMCs is mediated by the nonselective transient receptor potential canonical (TRPC) 1 protein, the identification of STIM1 and Orai1 as essential components of ICRAC, a highly Ca2+-selective SOCE current in leukocytes, has challenged that view. Here we show that cultured proliferative migratory VSMCs isolated from rat aorta (called “synthetic”) display SOCE with classic features, namely inhibition by 2-aminoethoxydiphenyl borate, ML-9, and low concentrations of lanthanides. On store depletion, synthetic VSMCs and A7r5 cells display currents with characteristics of ICRAC. Protein knockdown of either STIM1 or Orai1 in synthetic VSMCs greatly reduced SOCE, whereas Orai2, Orai3, TRPC1, TRPC4, and TRPC6 knockdown had no effect. Orai1 knockdown reduced ICRAC in synthetic VSMCs and A7r5 cells. Synthetic VSMCs showed up-regulated STIM1/Orai1 proteins and SOCE compared with quiescent freshly isolated VSMC. Knockdown of STIM1 and Orai1 inhibited synthetic VSMC proliferation and migration, whereas STIM2, Orai2, and Orai3 knockdown had no effect. To our knowledge, these results are the first to show ICRAC in VSMCs and resolve a long-standing controversy by identifying CRAC as the elusive VSMC SOCE channel important for proliferation and migration.—Potier, M., Gonzalez, J. C., Motiani, R. K., Abdullaev, I. F., Bisaillon, J. M., Singer, H. A., and Trebak, M. Evidence for STIM1- and Orai1-dependent store-operated calcium influx through ICRAC in vascular smooth muscle cells: role in proliferation and migration

Разработка системы интеллектуального поиска товаров на основе онтологий для интернет-магазина на платформе CS-Cart

The article presents the implementation of a intelligence search system for an CS-Cart platform online store. The system is based on the formation of a domain products ontology. The system assumes the processing and expansion of the user request. This article describes the structure of an OWL ontology. The article describes the algorithm for processing custom queries, as well as the results of experiments. В статье представлены особенности реализации системы интеллектуального поиска для интернет-магазина на платформе CS-Cart. Предлагаемая система основана на формировании онтологии предметной области к которой относятся товары. В рамках исследования рассматриваются процессы системы по обработке и расширению запроса пользователя. В статье представлена структура онтологии в формате OWL. В работе описан алгоритм обработки пользовательских запросов, а также представлены результаты экспериментов

PREDICTORS OF ADVERSE CLINICAL COURSE OF CORONARY HEART DISEASE: THE RESULTS FROM DYNAMICAL OBSERVATION

Aim. To reveal the molecular genetic predictors of adverse clinical course of coronary heart disease (CHD).Material and methods. A clinical genetic investigation performed, of 567 CHD patients, of those 199 underwent dynamic follow-up. Genotypes Pro12Pro, Pro12Ala, Ala12Ala of the gene PPAR-γ2, genotypes L162L and L162V gene PPAR-α, genotypes A603A, A603G, G603G gene of tissue factor were assessed with polymerase chain reaction and further restrictional analysis.Results. Carriage of the allele V162 gene PPAR-α and allele Ala12 gene PPAR-γ2 is associated with development of the following endpoints in CHD patients: recurrent angina, progression of heart failure, life-threatening arrhythmias, stroke and transient cerebral ischemia, myocardial infarction, fatal outcomes. Diabetes type 2 (DM2) in CHD patients was associated with the risk of adverse outcome 2,55 times. There was relation of DM2 in CHD patients and mortality. In CHD patients that undergone percutaneous coronary intervention and bypass grafting, the combination endpoint was registered rarer than in CHD patients with no interventions, with a decline of adverse CHD prognosis 2 times. The results can be explained by decreased inhibition of NF-kВ pathway in carriers of V162 and Ala12 genes PPAR-α and PPAR-γ2, that facilitates activation of the factors of immune inflammation and atherogenesis with further adverse outcomes of CHD. It is known that DM2 is a risk factor of CHD and its complications.Conclusion. Presence of DM2, carriage of allele V162 gene PPAR-α and allele Ala12 gene PPAR-γ2 is associated with adverse outcomes of CHD. In CHD patients with surgical revascularization of coronary arteries the risk of adverse CHD outcomes declined 2 times

ВЛИЯНИЕ ПОЛИМОРФИЗМА С3435Т ГЕНА MDR1 НА ЭФФЕКТИВНОСТЬ ТЕРАПИИ ЮВЕНИЛЬНОГО ИДИОПАТИЧЕСКОГО АРТРИТА

Juvenile idiopathic arthritis (JIA) is a multifactorial disease: its pathogenesis includes immunological and genetic factors. Gene MDR1 is responsible for resistance to various cytotoxic drugs. Product of gene MDR1 - P-glycoprotein (P-gp) acts as a transmembrane pump, thus affecting action of drugs. The research objective was to determine connection of C3435T polymorphism of gene MDR1 with P-protein’s expression level in children with JIA. P-protein was revealed in all the patents involved in our trial: the protein was revealed on peripheral blood lymphocytes before and after stimulation of interleukin 2. The authors also picked out genome DNA using phenol-chloroform extraction and detected C3435T polymorphism of the gene MDR1 using polymerase chain reaction. Methotrexate concentration in blood serum was determined using a standard method of fluorescent polarization (FPIA) using the Abbott apparatus TDxFLx. Statistical manipulation of the data obtained in the course of the trial was conducted using software Statistica 6.0. C3435T polymorphism of gene MDR1 affects therapy efficacy. Determination of both basal and stimulated (in vitro) P-glycoprotein may be used as additional criterion in the evaluation of disease activity.Ювенильный идиопатический артрит (ЮИА) — многофакторное заболевание; патогенез включает иммунологические и генетические факторы. MDR1-ген отвечает за устойчивость к различным цитотоксическим препаратам. Продукт MDR1-гена — Р-гликопротеин (P-gp) — действует как трансмембранный насос, влияя на действие лекарств. Целью нашего исследования было определить связь полиморфизма С3435Т гена MDR1 c уровнем экспрессии Р-протеинау детей, больных ЮИА. Всем пациентам в ходе нашего исследования был определен Р-протеин: белок определялся на лимфоцитах периферической крови до и после стимуляции интерлейкина 2. Также из крови всех больных была выделена геномная ДНК методом фенолхлороформной экстракции, и определен полиморфизм С3435Т гена MDR1 припомощи полимеразной цепной реакции. Концентрация метотрексата в сыворотке крови определялась стандартным методом флуоресцентной поляризации (ФПИА) на приборе TDxFLx фирмы Abbott. Статистическая обработка полученных в ходе исследования данных проводилась при помощи программы Statistica 6.0. Полиморфизм С3435Т MDR1-генавлияет на эффективность терапии. Определение как базального, так и стимулируемого (in vitro) Р-гликопротеина может быть использовано в качестве дополнительного критерия в оценке активности заболевания