Search

У доповіді наведено результати співпраці вчених Інституту молекулярної біології і генетики НАН України та лабораторії клітинної регуляції Університетського коледжу Лондона зі з’ясування механізму біосинтезу та визначення нових функцій коензиму А (КоА), який відіграє ключову роль у багатьох біохімічних клітинних реакціях. Уперше у світі було ідентифіковано ензим КоА-синтазу, який відповідає за два останні етапи біосинтезу КоА, а також визначено всі ензими, задіяні в цьому процесі. Встановлено новий тип посттрансляційної модифікації білків — коалювання, який виявився поширеним і універсальним механізмом антиоксидантного захисту протеїнів клітин та організму в цілому від оксидативного стресу. Наголошено на перспективності подальшого вивчення ролі коалювання в патогенезі нейродегенеративних захворювань з метою розроблення сучасних підходів до їх лікування та профілактики. The report presents the results of cooperation between scientists from the Institute of Molecular Biology and Genetics of the NAS of Ukraine and the Cell Regulation Laboratory of University College London to elucidate the mechanism of biosynthesis and new functions of coenzyme A (CoA), which plays a key role in many biochemical cell reactions. For the first time in the world, the enzyme CoA synthase, which is responsible for the last two stages of CoA biosynthesis, is identified, and all enzymes involved in this process are determined. A new type of post-translational modification of proteins is established: coagulation, which has proved to be a common and universal mechanism of antioxidant protection of cell proteins and the body as a whole from oxidative stress. Emphasis is placed on the prospects of further study of the role of CoAlation in the pathogenesis of neurodegenerative diseases in order to develop modern approaches to their treatment and prevention.

Staphylococcus aureus (S. aureus) is an aggressive opportunistic pathogen of prominent virulence and antibiotic resistance. These characteristics are due in part to the accessory gene regulator (agr) quorum-sensing system, which allows for the rapid adaptation of S. aureus to environmental changes and thus promotes virulence and the development of pathogenesis. AgrA is the agr system response regulator that binds to the P2 and P3 promoters and upregulates agr expression. In this study, we reveal that S. aureus AgrA is modified by covalent binding of CoA (CoAlation) in response to oxidative or metabolic stress. The sites of CoAlation were mapped by liquid chromatography tandem mass spectrometry (LC–MS/MS) and revealed that oxidation-sensing Cys199 is modified by CoA. Surface plasmon resonance (SPR) analysis showed an inhibitory effect of CoAlation on the DNA-binding activity, as CoAlated AgrA had significantly lower affinity towards the P2 and P3 promoters than non-CoAlated AgrA. Overall, this study provides novel insights into the mode of transcriptional regulation in S. aureus and further elucidates the link between the quorum-sensing and oxidation-sensing roles of the agr system.

Spores of Bacillus species have novel properties, which allow them to lie dormant for years and then germinate under favourable conditions. In the current work, the role of a key metabolic integrator, coenzyme A (CoA), in redox regulation of growing cells and during spore formation in Bacillus megaterium and Bacillus subtilis is studied. Exposing these growing cells to oxidising agents or carbon deprivation resulted in extensive covalent protein modification by CoA (termed protein CoAlation), through disulphide bond formation between the CoA thiol group and a protein cysteine. Significant protein CoAlation was observed during sporulation of B. megaterium, and increased largely in parallel with loss of metabolism in spores. Mass spectrometric analysis identified four CoAlated proteins in B. subtilis spores as well as one CoAlated protein in growing B. megaterium cells. All five of these proteins have been identified as moderately abundant in spores. Based on these findings and published studies, protein CoAlation might be involved in facilitating establishment of spores’ metabolic dormancy, and/or protecting sensitive sulfhydryl groups of spore enzymes., Analysis of protein CoAlation during sporulation and in spores of Bacillus species.

The metastasis suppressor protein NME1 is an evolutionarily conserved and multifunctional enzyme that plays an important role in suppressing the invasion and metastasis of tumour cells. The nucleoside diphosphate kinase (NDPK) activity of NME1 is well recognized in balancing the intracellular pools of nucleotide diphosphates and triphosphates to regulate cytoskeletal rearrangement and cell motility, endocytosis, intracellular trafficking, and metastasis. In addition, NME1 was found to function as a protein-histidine kinase, 3′-5′ exonuclease and geranyl/farnesyl pyrophosphate kinase. These diverse cellular functions are regulated at the level of expression, post-translational modifications, and regulatory interactions. The NDPK activity of NME1 has been shown to be inhibited in vitro and in vivo under oxidative stress, and the inhibitory effect mediated via redox-sensitive cysteine residues. In this study, affinity purification followed by mass spectrometric analysis revealed NME1 to be a major coenzyme A (CoA) binding protein in cultured cells and rat tissues. NME1 is also found covalently modified by CoA (CoAlation) at Cys109 in the CoAlome analysis of HEK293/Pank1β cells treated with the disulfide-stress inducer, diamide. Further analysis showed that recombinant NME1 is efficiently CoAlated in vitro and in cellular response to oxidising agents and metabolic stress. In vitro CoAlation of recombinant wild type NME1, but not the C109A mutant, results in the inhibition of its NDPK activity. Moreover, CoA also functions as a competitive inhibitor of the NME1 NDPK activity by binding non-covalently to the nucleotide binding site. Taken together, our data reveal metastasis suppressor protein NME1 as a novel binding partner of the key metabolic regulator CoA, which inhibits its nucleoside diphosphate kinase activity via non-covalent and covalent interactions., Graphical abstract Image 1, Highlights • NME1 is a major CoA-binding protein. • CoA can bind NME1 through covalent and non-covalent interactions. • NME1 CoAlation is induced by oxidative and metabolic stress in mammalian cells. • CoA inhibits the NDPK activity of NME1 in vitro.

Aim. To generate HEK-293 cells with disrupted expression of S6K1 isoforms: p85, p70 and p60. Methods. CRISPR/Cas9 gene editing, Western blotting, immunofluorescent analysis, RT-PCR analysis, MTT assay, scratch assay. Results. Several clones of HEK-293 cells with a complete loss of p85/p70/p60-S6K1 protein expression were generated. The effects of p85/p70/p60-S6K1 knockout on Akt/mTORC1/S6K1 signaling and cell proliferation and migration were assessed. Conclusions. The generated cell lines can be used to study a role played by S6K1 in cell physiology and to gain more detailed insight into cellular functions of the S6K1 isoforms. The HEK-293 cells exhibit down-regulation of Akt phosphorylation on Ser473 and subsequent attenuation of cell growth rate, as well as inhibition of cell motility. Мета. Створити клітини HEK293 з порушеною експресією S6K1 ізоформ: p85, p70 і Р60. Методи. CRISPR/cas9 система редагування генома, Вестерн-блот, імунофлуоресцентний аналіз, ПЛР-аналіз, MTT тест, метод «раневої поверхні». Результати. Отримано декілька клонів клітин НЕК-293 з повною втратою експресії білка p85/p70/p60-S6K1. Було оцінено ефект нокауту p85/p70/p60-S6K1 на Akt/mTORC1/S6K1 сигналінг та клітинні проліферацію і міграцію. Висновки. Створені клітинні лінії можуть бути використані для вивчення ролі, яку відіграє S6K1 в фізіології клітини, що дозволяє отримати більш детальне уявлення про клітинні функції ізоформ S6K1. Клітини / / HEK-293 виявляють знижений рівень фосфорилювання Akt по Сер473 і подальше пригнічення швидкості клітинного росту разом з інгібуванням клітинної рухливості. Цель. Создать клетки HEK293 с нарушенной экспрессией S6K1 изоформ: p85, p70 и Р60. Методы. CRISPR/cas9 система редактирования генома, Вестерн-блот, иммунофлуоресцентный анализ, ПЦР-анализ, MTT тест, метод «раневой поверхности». Результаты. Получено несколько клонов клеток НЕК-293 с полной потерей экспрессии белка p85/p70/p60-S6K1. Была проведена оценка влияния нокаута p85/p70/p60-S6K1 на Akt/mTORC1/S6K1 сигналинг и клеточные пролиферацию и миграцию. Выводы. Созданные клеточные линии могут быть использованы для изучения роли, которую играет S6K1 в физиологии клетки и позволить получить более детальное представление о клеточных функциях изоформ S6K1. Клетки / / HEK-293 показывают пониженный уровень фосфорилирования Akt по Сер473 и дальнейшее угнетение скорости клеточного роста вместе с ингибированием клеточной подвижности.

Peroxiredoxins (Prdxs) are antioxidant enzymes that catalyse the breakdown of peroxides and regulate redox activity in the cell. Peroxiredoxin 5 (Prdx5) is a unique member of Prdxs, which displays a wider subcellular distribution and substrate specificity and exhibits a different catalytic mechanism when compared to other members of the family. Here, the role of a key metabolic integrator coenzyme A (CoA) in modulating the activity of Prdx5 was investigated. We report for the first time a novel mode of Prdx5 regulation mediated via covalent and reversible attachment of CoA (CoAlation) in cellular response to oxidative and metabolic stress. The site of CoAlation in endogenous Prdx5 was mapped by mass spectrometry to peroxidatic cysteine 48. By employing an in vitro CoAlation assay, we showed that Prdx5 peroxidase activity is inhibited by covalent interaction with CoA in a dithiothreitol-sensitive manner. Collectively, these results reveal that human Prdx5 is a substrate for CoAlation in vitro and in vivo, and provide new insight into metabolic control of redox status in mammalian cells.

Aim. To identify a splicing mRNA of S6K1 kinase specific for the p60-S6K1 isoform alone. Methods. RT-PCR, DNA sequencing, Western blotting. Results. RT-PCR analysis of total RNA extracted from MCF-7 cells and subsequent DNA sequencing revealed a novel S6K1 splice variant that possesses additional exon 1a and encodes the p60 isoform of S6K1 only. Moreover, RT-PCR and western blotting were applied to estimate the expression levels of the p60-S6K1 mRNA and protein. A heterogeneous expression of the p60-S6K1 transcript was observed; it did not correlate with the total S6K1 mRNA expression and with the protein content of p60-S6K1 in the studied cell lines. Conclusions. We provide the evidence for the existence of a novel S6K1 splice variant coding for the p60-S6K1 isoform. The cell can employ two distinct pathways of the p60-S6K1 expression based on alternative translation of mRNA common for the main S6K1 isoforms mRNA and/or translation of the novel p60-S6K1 specific mRNA. Since the levels of the p60-S6K1 transcript expression do not correlate with the expression of total S6K1 mRNA, it is plausible that the p60-S6K1 isoform plays a role distinct from that of other isoforms in cellular physiology, as well as in the development of S6K1-related pathologies. Мета. З'ясувати можливість існування сплайсової мРНК кінази S6К1 специфічної виключно для р60-S6К1 ізоформи. Методи. ЗТ-ПЛР, ДНК секвенування, Вестерн-блоттинг. Результати. ЗТ-ПЛР аналіз тотальної РНК, виділеної із клітин лінії MCF-7, з наступним секвенуванням ДНК виявив новий сплайсовий варіант мРНК S6K1, що містить додатковий екзон 1а і кодує лише p60 ізоформу S6K1. Для оцінки рівня експресії p60-S6K1 мРНК та білку було застосовано ЗТ-ПЛР та вестерн-блот. Результати. вказують на гетерогенну експресію транскрипту p60-S6K1, що не корелює ні з експресією загальної S6K1 мРНК, ні з білковим вмістом p60-S6K1 у досліджуваних клітинних лініях. Висновки. Надано докази існування нової сплайсової мРНК S6K1, яка кодує ізоформу p60-S6K1, що свідчить про існування в клітині двох незалежних шляхів експресії p60-S6K1, а саме альтернативну трансляцію спільної для головних S6K1 ізоформ мРНК та/чи трансляцію нової специфічної лише для р60-S6K1 сплайсової мРНК. Оскільки рівень експресії p60-S6K1 транскрипту в різних клітинних лініях не корелює із експресією загальної мРНК S6K1, цілком можливо, що p60-S6K1 ізоформа може відігравати відмінну від інших S6K1 ізоформ роль у клітинній фізіології, а також при розвитку патологій, які пов’язані із S6K1. Цель. Выяснить возможность существования сплайсинговой мРНК киназы S6К1 специфической исключительно для р60-S6К1 изоформы. Методы. ОТ-ПЦР, ДНК секвенирование, Вестерн-блоттинг. Результаты. ОТ-ПЦР анализ тотальной РНК, выделенной из клеток линии MCF-7, с последующим секвенированием ДНК выявил новый сплайсовый вариант S6K1, содержит дополнительный экзон 1а и кодирует только изоформу p60-S6K1. Далее были использованы ОТ-ПЦР и вестерн-блоттинг для оценки уровней p60-S6K1 мРНК и белка. Результаты показали гетерогенную экспрессию транскрипта p60-S6K1, экспрессия которого не коррелировала ни с тотальной S6K1 мРНК, ни с белковым содержанием p60-S6K1 в исследуемых клеточных линиях. Выводы. Предоставлены доказательства существования нового сплайсового варианта S6K1, который кодирует изоформу p60-S6K1, что указывает на существование в клетке двух независимых путей экспрессии p60-S6K1 изоформы, а именно альтернативную трансляцию общей для основных изоформ мРНК и/или трансляцию новой специфичной лишь для p60-S6K1 сплайсовой мРНК. Поскольку уровень экспрессии p60-S6K1 транскрипта в различных клеточных линиях не коррелируют с экспрессией тотальной S6K1 мРНК, можно предположить, что p60-S6K1 изоформа может играть отличную то других S6K1 изоформ роль в клеточной физиологии, а также при развитии патологий, которые связаны с S6K1.

Coenzyme A (CoA) is an essential cofactor present in all living cells. Under physiological conditions, CoA mainly functions to generate metabolically active CoA thioesters, which are indispensable for cellular metabolism, the regulation of gene expression, and the biosynthesis of neurotransmitters. When cells are exposed to oxidative or metabolic stress, CoA acts as an important cellular antioxidant that protects protein thiols from overoxidation, and this function is mediated by protein CoAlation. CoA and its derivatives are strictly maintained at levels controlled by nutrients, hormones, metabolites, and cellular stresses. Dysregulation of their biosynthesis and homeostasis has deleterious consequences and has been noted in a range of pathological conditions, including cancer, diabetes, Reye&rsquo, s syndrome, cardiac hypertrophy, and neurodegeneration. The biochemistry of CoA biosynthesis, which involves five enzymatic steps, has been extensively studied. However, the existence of a CoA biosynthetic complex and the mode of its regulation in mammalian cells are unknown. In this study, we report the assembly of all five enzymes that drive CoA biosynthesis, in HEK293/Pank1&beta, and A549 cells, using the in situ proximity ligation assay. Furthermore, we show that the association of CoA biosynthetic enzymes is strongly upregulated in response to serum starvation and oxidative stress, whereas insulin and growth factor signaling downregulate their assembly.

Treatment of class III is a current problem in orthodontics that requires constant improvement of its methods, development of new or modifications of known methods and techniques. We have developed and studied the modification of removable functionally-directing orthodontic appliances for treatment of Class III, which consists of a plastic base, vestibular arc, retaining clasps, ramp, which is connected with the base by means of two torsion springs. Its usage ensures a prolonged contact of ramp with the teeth. We studied two types of club-shaped springs (torsion springs): one spring, which create an amortization effect during the action of the ramp, but do not change its inclination angle and second one – spring that seek to increase the angle of the ramp inclination due to the disclosure of its curl.

Aurora A kinase is a master mitotic regulator whose functions are controlled by several regulatory interactions and post-translational modifications. It is frequently dysregulated in cancer, making Aurora A inhibition a very attractive antitumor target. However, recently uncovered links between Aurora A, cellular metabolism and redox regulation are not well understood. In this study, we report a novel mechanism of Aurora A regulation in the cellular response to oxidative stress through CoAlation. A combination of biochemical, biophysical, crystallographic and cell biology approaches revealed a new and, to our knowledge, unique mode of Aurora A inhibition by CoA, involving selective binding of the ADP moiety of CoA to the ATP binding pocket and covalent modification of Cys290 in the activation loop by the thiol group of the pantetheine tail. We provide evidence that covalent CoA modification (CoAlation) of Aurora A is specific, and that it can be induced by oxidative stress in human cells. Oxidising agents, such as diamide, hydrogen peroxide and menadione were found to induce Thr 288 phosphorylation and DTT-dependent dimerization of Aurora A. Moreover, microinjection of CoA into fertilized mouse embryos disrupts bipolar spindle formation and the alignment of chromosomes, consistent with Aurora A inhibition. Altogether, our data reveal CoA as a new, rather selective, inhibitor of Aurora A, which locks this kinase in an inactive state via a “dual anchor” mechanism of inhibition that might also operate in cellular response to oxidative stress. Finally and most importantly, we believe that these novel findings provide a new rationale for developing effective and irreversible inhibitors of Aurora A, and perhaps other protein kinases containing appropriately conserved Cys residues.

SummaryAurora A is a cell cycle protein kinase implicated in multiple human cancers, and several Aurora A-specific kinase inhibitors have progressed into clinical trials. In this study, we report structural and cellular analysis of a novel biochemical mode of Aurora A inhibition, which occurs through reversible covalent interaction with the universal metabolic integrator coenzyme A (CoA). Mechanistically, the CoA 3’-phospho ADP moiety interacts with Thr 217, an Aurora A selectivity filter, which permits the formation of an unprecedented covalent bond with Cys 290 in the kinase activation segment, lying some 15 Å away. CoA modification (CoAlation) of endogenous Aurora A is rapidly induced by oxidative stresses at Cys 290 in human cells, and microinjection of CoA into mouse embryos perturbs meitoic spindle formation and chromosome alignment. Aurora A regulation by CoA reveals how targeting of Aurora A might be accomplished in the future by development of a ‘double-anchored’ covalent inhibitor.

In all living organisms, coenzyme A (CoA) is an essential cofactor with a unique design allowing it to function as an acyl group carrier and a carbonyl-activating group in diverse biochemical reactions. It is synthesized in a highly conserved process in prokaryotes and eukaryotes that requires pantothenic acid (vitamin B5), cysteine and ATP. CoA and its thioester derivatives are involved in major metabolic pathways, allosteric interactions and the regulation of gene expression. A novel unconventional function of CoA in redox regulation has been recently discovered in mammalian cells and termed protein CoAlation. Here, we report for the first time that protein CoAlation occurs at a background level in exponentially growing bacteria and is strongly induced in response to oxidizing agents and metabolic stress. Over 12% of Staphylococcus aureus gene products were shown to be CoAlated in response to diamide-induced stress. In vitro CoAlation of S. aureus glyceraldehyde-3-phosphate dehydrogenase was found to inhibit its enzymatic activity and to protect the catalytic cysteine 151 from overoxidation by hydrogen peroxide. These findings suggest that in exponentially growing bacteria, CoA functions to generate metabolically active thioesters, while it also has the potential to act as a low-molecular-weight antioxidant in response to oxidative and metabolic stress.

Purpose. To investigate a frequency of antibody response to SEREX-identified medullary breast carcinoma autoantigens ZRF1 and KRR1 in sera of breast cancer patients taking into account clinical and molecular characteristics of tumors for opening of new perspectives in creation of minimally invasive immunological tests for cancer diagnostics.Methods. Enzyme-linked immunosorbent assay and bioinformatics analysis.Results. Increased frequency of antibody response was found in sera of breast cancer patients to ZRF and KRR1 antigens. The antibody response to these antigens was higher in sera of patients with invasive ductal carcinoma than in sera of patients with other histological types of breast tumors. Moreover, more frequent antibody response to ZRF antigen was found in sera of patients with less aggressive tumors. The sequence analysis of ZRF1 antigen SEREX clones obtained from cDNA libraries of different tumors demonstrates that they encode different protein isoforms.Conclusion. Tumor-associated antigens KRR1 and ZRF1 and their cognate autoantibodies could be considered as potential molecular markers of breast cancer which need to be further investigated.

© 2016 Informa UK Limited, trading as Taylor & Francis Group.Content: Identification of panel of SEREX-defined antigens for breast cancer autoantibodies profile detection. Objective: To create panel of antigens that can differentiate breast cancer patients and healthy individuals. Methods: SEREX (serological analysis of cDNA expression libraries) method, ELISA (enzyme-linked immunosorbent assay), qPCR (quantitative polymerase chain reaction). Results: In large-scale screening of 16 SEREX-antigens by sera of breast cancer patients and healthy donors, a combination of six antigens (RAD50, PARD3, SPP1, SAP30BP, NY-BR-62 and NY-CO-58) was identified, which can differentiate breast cancer patients and healthy donors with 70% sensitivity and 91% specificity. Elevated mRNA expression of SPP1 gene was revealed in breast tumors (2–7-fold) that correlated with SPP1 antigen immunoreactivity in autologous patients’ sera. Conclusions: The new panel of six SEREX-antigens was proposed, which enables creation of serological assay for breast cancer diagnostics and/or prognosis.

The aim of this study was to produce monoclonal antibodies directed to the N-terminal regulatory region of S6K1, which shows very low homology to S6K2. Methods. Hybridoma technology, ELISA, Western blot, Immunoprecipitation. Results. Three hybridoma clones (A1, A2 and A3) producing mAbs specific to ribosomal protein S6 kinase 1 have been generated. Specificity of mAbs has been confirmed by Western blot and immunoprecipitation technique. Conclusions. The obtained antibodies are suitable for elucidating signal transduction pathways involving S6K1. Keywords: ribosomal protein S6 kinase, mAbs. Мета. Отримати моноклональні антитіла проти N-кінцевої регуляторної ділянки кінази рибосомного білка S6-S6K1, який має низький рівень гомології з ізоформою S6K2. Методи. Гібридомна технологія, ІФА, Вестерн-блот, імунопреципітація. Результати. Створено тригібридомних клони (A1, A2 і A3), які продукують моноклональні антитіла, специфічні до кінази S6K1. Висновки. Одержані моноклональні антитіла за своїми характеристикам можна використовувати для дослідження S6K1-залежних сигнальних шляхів. Ключові слова: кіназа рибосомного білка S6, МкАТ. Цель. Получить моноклональные антитела против N-концевого регуляторного участка киназы рибосомного белка S6 – S6K1, имеющего низкий уровень гомологии с S6K2 изоформой. Методы. Гибридомная технология, ИФА, Вестерн-блот, иммунопреципитация. Результаты. Созданы три гибридомных клона (A1, A2 и A3), продуцирующих моноклональные антитела, специфичные к киназе S6K1. Специфичность антител подтверждена методами Вестерн блота и иммунопреципитации. Выводы. Полученные моноклональные антитела по своим характеристикам можно использовать для изучения S6K1-зависимых сигнальных путей. Ключевые слова: киназа рибосомного белка S6, МкАТ.

Background: Autoantibodies, which are produced against tumor-associated antigens, are potential tumor markers and attract a growing interest for cancer detection, differential diagnostics and prognosis.Objective: To evaluate the diagnostic significance of 40 antigens identified by immunoscreening of cDNA libraries from thyroid and colon cancers by allogenic screening with different tumor types patients’ sera.Method: Plaque-spot serological assay.Results: Increased frequency of antibody response in sera of cancer patients compared with that of healthy donors was shown toward 14 antigens, 8 of which (CG016, BTN3A3, FKBP4, XRCC4, TSGA2, ACTR1A, FXYD3 and CTSH) have revealed exclusively cancer-related serological profile.Conclusion: Allogenic screening of 40 SEREX-antigens with sera from cancer patients and healthy donors allowed us to reveal 14 antigens with potential diagnostic significance. These antigens and their cognate autoantibodies could be considered as valuable targets for further analysis as poten...

Chitinase 3-like 2 (CHI3L2) is one of the most overexpressed genes in glioblastoma. Despite this, both the CHI3L2 gene and its protein product CHI3L2 are poorly characterized. Here we report the generation and characterization of monoclonal antibodies to CHI3L2 protein (CHI3L2 MAbs). Bacterially expressed 6 His-tagged full-length CHI3L2 was used as antigen. Spleen cells from immunized mice were collected and fused with SP2/0 myeloma cells. Hybridoma clones 2D3 and 4D2 producing high titer CHI3L2 MAbs were identified by enzyme-linked immunosorbent assay (ELISA) and further examined for their activity with the CHI3L2 protein by Western blot analysis and immunoprecipitation. The 2D3 clone was chosen for mouse inoculation and ascites formation. Antibodies derived from the ascitic fluid specifically recognized the recombinant CHI3L2 protein and strongly interacted with CHI3L2 in glioblastoma tissue lysate, as determined by Western blot analysis. The antibodies generated may be useful as a tool in various aspects of CHI3L2 investigation.

Tumor-associated antigen MX35, which is overexpressed in 70-90% of epithelial ovarian cancers, has been recently identified as phosphate transporter NaPi2b. This finding has raised significant interest in understanding NaPi2b function under physiological conditions and its deregulation in human pathologies, such as cancer. As a member of the sodium-dependent phosphate transporter family, NaPi2b is primarily involved in the maintenance of phosphate homeostasis in the human body. The role of NaPi2b in oncogenic transformation and malignant growth is not well understood. To date, several monoclonal antibodies specific to NaPi2b have been reported. However, available monoclonal antibodies are not very efficient in recognizing endogenous NaPi2b under reducing conditions. In addition, these antibodies could not recognize the mutant form of transporter (NaPi2b-T330V). In this study we describe the production of monoclonal antibodies raised against the N-terminal region of NaPi2b. One of them, designated N-NaPi2b(15/1), possesses very useful immunological characteristics. We found that N-NaPi2b(15/1) specifically recognizes NaPi2b protein in immunohistochemical analysis and immunoprecipitation assay. Importantly, N-NaPi2b(15/1) antibody detects very efficiently endogenous and expressed wild-type and mutant forms of NaPi2b under both reducing and non-reducing conditions in Western blot analysis. These features make N-NaPi2b(15/1) antibody a very useful tool for studying the pattern of NaPi2b expression in health and pathologies.

Detection of cell proliferation index is widely used in experimental and clinical research. Earlier it was shown that nuclear Ki-67 protein expression is strictly related to cell proliferation. It was revealed during all active phases of the cell cycle in mammals but was absent in G0 phase, so Ki-67 presence in cell nuclei reflects a potential growth fraction of whole cell population. The main area of Ki-67 antibody application is in immunocytochemical and immunohistochemical analyses. The aim of our work was to generate mouse monoclonal antibodies for Ki-67 antigen detection in mammalian tissues and in cultured cells. His-tagged fragment of Ki-67 expressed in bacteria was used as an antigen. Antibody-producing hybridoma cells were generated by standard procedure by fusing SP2/0 myeloma cells with splenocytes of immunized mice. Monoclonal antibodies were analyzed using paraffin-embedded human melanoma tissue samples and breast cancer cell line MCF-7. It was shown that generated anti-Ki-67 antibodies revealed proliferating cells in MCF-7 culture and after heat-induced epitope retrieval on paraffin sections of human melanoma tissue. In summary, generated antibodies could be useful for detection of proliferating cells in immunohistochemical and immunofluorescence studies of mammalian cells and tissues.

Medullary breast carcinoma (MBC) is a relatively rare malignancy with heavy lymphocytic infiltration that despite cytologically anaplastic features and high mitotic index has more favorable prognosis than other types of breast cancer. Lymphocytic infiltration of tumors reflects ongoing immune response against tumor antigens which could represent a great interest as potential targets for cancer immunotherapy. The search for MBC antigens by SEREX methodology has not been successful due to a very high titer of false positive clones, representing immunoglobulin genes. Here, we describe a novel approach for generating cDNA expression libraries from MBC tumor samples which are depleted of IgG cDNA clones and, therefore, are suitable for the identification of novel tumor-associated antigens (TAA) by SEREX approach. Modified methodology allowed us to isolate a panel of known and novel TAA which are currently under further investigation.

PTEN is a tumor suppressor with dual protein and lipid-phosphatase activity, which is frequently deleted or mutated in many human advanced cancers. Recent studies have also demonstrated that PTEN is a promising target in type II diabetes and obesity treatment. Using C-terminal PTEN sequence in pEG202-NLS as bait, yeast two-hybrid screening on Mouse Embryo, Colon Cancer, and HeLa cDNA libraries was carried out. Isolated positive clones were validated by mating assay and identified through automated DNA sequencing and BLAST database searches. Sequence analysis revealed a number of PTEN-binding proteins linking this phosphatase to a number of different signaling cascades, suggesting that PTEN may perform other functions besides tumor-suppressing activity in different cell types. In particular, the interplay between PTEN function and adipocyte-specific fatty-acid-binding protein FABP4 is of notable interest. The demonstrable tautology of PTEN to FABP4 suggested a role for this phosphatase in the regulation of lipid metabolism and adipocyte differentiation. This interaction was further studied using coimmunoprecipitation and gel-filtration assays. Finally, based on Biacore assay, we have calculated the K(D) of PTEN-FABP4 complex, which is around 2.8 microM.

The mTOR (mammalian target of rapamycin) promotes growth in response to nutrients and growth factors and is deregulated in numerous pathologies, including cancer. The mechanisms by which mTOR senses and regulates energy metabolism and cell growth are relatively well understood, whereas the molecular events underlining how it mediates survival and proliferation remain to be elucidated. Here, we describe the existence of the mTOR splicing isoform, TOR beta, which, in contrast to the full-length protein (mTOR alpha), has the potential to regulate the G(1) phase of the cell cycle and to stimulate cell proliferation. mTOR beta is an active protein kinase that mediates downstream signaling through complexing with Rictor and Raptor proteins. Remarkably, overexpression of mTOR beta transforms immortal cells and is tumorigenic in nude mice and therefore could be a proto-oncogene.

CoA Synthase (CoASy, 4'-phosphopantetheine adenylyltransferase/dephospho-CoA kinase) mediates two final stages of de novo coenzyme A (CoA) biosynthesis in higher eukaryotes. Unfortunately very little is known about regulation of this important metabolic pathway. In this study, we demonstrate that CoASy interacts in vitro with Src homology-2 (SH2) domains of a number of signaling proteins, including Src homology-2 domains containing protein tyrosine phosphatase (Shp2PTP). Complexes between CoASy and Shp2PTP exist in vivo in mammalian cells and this interaction is regulated in a growth-factor-dependent manner. We have also demonstrated that endogenous CoASy is phosphorylated on tyrosine residues in vivo, and that cytoplasmic protein tyrosine kinases can mediate this phosphorylation in vitro and in vivo. Importantly, Shp2PTP-mediated CoASy in vitro dephosphorylation leads to an increase in CoASy enzymatic phosphopantetheine adenylyltransferase (PPAT) activity. We therefore argue that CoASy is a novel potential substrate of Shp2PTP and phosphorylation of CoASy at tyrosine residue(s) could represent unrecognized before mechanism of modulation intracellular CoA level in response to hormonal and (or) other extracellular stimuli.

The complex interplay between cellular signaling and metabolism in eukaryotic cells just start to emerge. Coenzyme A (CoA) and its derivatives play a key role in cell metabolism and also participate in regulatory processes. CoA synthase (CoASy) is a mitochondria-associated enzyme which mediates two final stages of de novo CoA biosynthesis. Here, we report that CoASy is involved in signaling events in the cell and forms a functional complex with p85alphaPI3K in vivo. Importantly, observed interaction of endogenous CoASy and p85alphaPI3K is regulated in a growth factor dependent manner. Surprisingly, both catalytic p110alpha and regulatory p85alpha subunits of PI3K were detected in mitochondrial fraction where mitochondria-localized p85alphaPI3K was found in complex with CoASy. Unexpectedly, significant changes of PI3K signaling pathway activity were observed in experiments with siRNA-mediated CoASy knockdown pointing on the role of CoA biosynthetic pathway in signal transduction.

Mammalian target of rapamycin (mTOR) is a serine/threonine kinase that plays a critical role in the regulation of basic cellular functions, including cellular growth and proliferation. In this study we describe the generation and characterization of novel monoclonal antibodies directed against mTOR protein kinase. A GST-tagged fragment of mTOR expressed in bacteria was used as an antigen. Antibody-producing hybridoma cells were obtained by fusing SP2/0 myeloma cells with splenocytes from immunized mice. Anti-mTOR antibody-producing hybridoma cell lines were first identified by enzyme-linked immunosorbent assay and then subcloned by limiting dilution. Antibodies produced by selected clones were further tested for their reactivity towards the GST/mTOR 1334-1504 recombinant protein. Furthermore, antibody produced by F11 clone was shown to recognize specifically mTOR in different tissues and cell lines in Western blotting, immunoprecipitation, and immunohistochemistry. In addition, mTOR F11 antibody was suitable for immunoprecipitating and testing mTOR activity in in vitro kinase assay. In summary, generated antibodies will be useful for investigating mTOR signaling complexes in normal and pathological states.

Homeostasis of inorganic phosphate in the human body is maintained by regulated absorption, metabolism, and excretion. Sodium-dependent phosphate transporters (NaPi) mediate the transport of inorganic phosphate (P(i)) in cells in response to dietary phosphate consumption, hormones, and growth factors. NaPi2b is a member of the sodium-dependent phosphate transporter family, with a distinct pattern of expression and regulation. Signaling pathways activated by mitogens, glucocorticoids, and metabolic factors have been implicated in regulating P(i) transport via NaPi2b. Inactivation of NaPi2b function by mutations has been linked to human pathologies, such as pulmonary alveolar microlithiasis. In this study, we describe the generation and characterization of monoclonal antibodies against human NaPi2b. The monoclonal antibodies were shown to recognize specifically transiently overexpressed and endogenous NaPi2b in commonly used immunoassays, including Western blotting, immunoprecipitation, and immunohistochemistry. These properties make them particularly valuable reagents for elucidating NaPi2b function in health and disease.

The main aim of this work was to determine subcellular localization of S6K1 and S6K2 forms of ribosomal protein S6 kinase in the primary monolayer culture of thyrocytes obtained from undamaged follicles. In the thyroid follicles S6K1, S6K2 have been detected predominantly in the cytoplasm of the cells, however, in the monolayer culture of thyrocytes in the course of follicles outspreading S6K1 and S6K2 have been observed in nuclei as well. Such redistribution of S6K was not directly related to the appearance of proliferating Ki-67 positive cells. At the same time there was a correlation between the appearance of S6K1, S6K2 positive nuclei in monolayer thyrocyte culture and decrease in thyroglobulin content in cultured cells. Thus, the obtained results indicated that the down regulation of thyrocyte functional activity caused by the loss of follicle organization was accompanied by subcellular redistribution of S6K1 and S6K2. мунохімічно визначено субклітинну локалізацію S6K1 і S6K2 у тканині та моношаровій культурі щитовидної залози щурів. Встановлено, що в тканині щитовидної залози S6K1 і S6K2 локалізуються переважно в цитоплазмі клітин. При розпластуванні фолікулів з’являлася позитивна реакція в ядрах тиреоцитів. Такий перерозподіл не пов’язаний прямо з появою проліферуючих Ki-67 позитивних клітин. У той же час відмічено кореляцію між появою S6K1, S6K2 позитивних ядер і зменшенням вмісту тиреоглобулину у культивованх клітинах. Таким чином, отримані результати свідчать про те, що зниження функціональної активності тиреоцитів внаслідок втрати фолікулярної організації супроводжується зміною субклітинної локалізації S6K1 і S6K2. Иммунохимически определена субклеточная локализация S6K1, S6K2 в ткани и монослойной культуре клеток щитовидной железы крыс. В исходной ткани S6K1 и S6K2 локализовались преимущественно в цитоплазме. При распластывании фолликулов положительная реакция появлялась и в ядрах тиреоцитов. Такое перераспределение напрямую не связано с появлением пролиферирующих Ki-67 положительных клеток. При этом отмечалась корреляция между появлением S6K1, S6K2 положительных ядер и снижением содержания тиреоглобулина в культивируемых клетках. Полученные нами результаты свидетельствуют о том, что снижение функциональной активности тиреоцитов вследствие потери фолликулярной организации сопровождается изменением субклеточной локализации S6K1 и S6K2.

The eEF1A1 and eEF1A2 isoforms of translation elongation factor 1A have 98% similarity and perform the same protein synthesis function catalyzing codon-dependent binding of aminoacyl-tRNA to 80S ribosome. However, the isoforms apparently play different non-canonical roles in apoptosis and cancer development which are awaiting further investigations. We hypothesize that the difference in non-translational functions could be caused, in particular, by differential ability of the isoforms to be involved in phosphotyrosine-mediated signalling. The ability of eEF1A1 and eEF1A2 to interact with SH2 and SH3 domains of different signalling molecules in vitro was compared. Indeed, contrary to eEF1A1, eEF1A2 was able to interact with SH2 domains of Grb2, RasGAP, Shc and C-terminal part of Shp2 as well as with SH3 domains of Crk, Fgr, Fyn and phospholipase C-gamma1. Interestingly, the interaction of both isoforms with Shp2 in vivo was found using stable cell lines expressing eEF1A1-His or eEF1A2-His. The formation of a complex between endogenous eEF1A and Shp2 was also shown. Importantly, a higher level of tyrosine phosphorylation of eEF1A2 as compared to eEF1A1 was demonstrated in several independent experiments and its importance for interaction of eEF1A2 with Shp2 in vitro was revealed. Thus, despite the fact that both isoforms of eEF1A could be involved in the phosphotyrosine-mediated processes, eEF1A2 apparently has greater potential to participate in such signalling pathways. Since tyrosine kinases/phosphatases play a prominent role in human cancerogenesis, our observations may gave a basis for recently found oncogenicity of the eEF1A2 isoform.

TSC1 and TSC2 are two recently identified tumor suppressor genes encoding hamartin and tuberin, respectively. They have been implicated in the pathogenesis of tuberous sclerosis, a neurological disorder linked with the development of hamartomas in numerous organs, including the brain, kidneys, heart, and liver. Both protein products of TSC1 and TSC2 form an intracellular complex exerting GTPase-activating (GAP) activity towards a small G protein Rheb (Ras homologue enriched in brain). Inhibition of Rheb is important for the positive regulation of mTOR pathway, while mutations of hamartin or tuberin result in uncontrolled cell cycle progression. Although the precise role for the TSC1/2 complex in tumor suppression is not clear, many studies have established a link with the regulation of transcription and protein biosynthesis, increasing susceptibility to apoptosis, cell differentiation, and cell cycle control. We describe the development of a monoclonal antibody specific towards TSC2/tuberin and characterize the suitability for Western blotting, immunoprecipitation, and immunofluorescent applications. The C-terminal region of TSC2 was expressed as a His-tag fusion protein in bacteria, affinity purified and used as an immunogen. Hybrid myelomas were produced from the spleenocytes of immunized mice and SP2/0 myeloma cells. Testing the specificity of cell culture supernatants from generated hybridomas towards recombinant His-TSC2C in ELISA assay allowed us to isolate a panel of positive clones. Further analysis of selected clones by Western blotting and immunoprecipitation revealed one clone, termed D6, which specifically recognized recombinant and endogenous TSC2. The specificity of generated antibody was also confirmed in TSC2(/) and TSC2(+/+) mouse embryo fibroblasts. In summary, the produced antibody is a useful tool in our research program and will be available for researchers investigating signal transduction pathways involving TSC1/2 signaling under physiological conditions and in human pathologies.

Expression pattern and immunogenicity are critical issues that define tumor antigens as diagnostic markers and potential targets for immunotherapy. The development of SEREX (serological analysis of recombinant expression libraries) has provided substantial progress in the identification of tumor antigens eliciting both cellular and humoral immune responses in cancer patients. By SEREX, we have previously identified RAB38/NY-MEL-1 as a melanocyte differentiation antigen that is highly expressed in normal melanocytes and melanoma tissues but not in other normal tissues or cancer types. In this study, we further demonstrate that RAB38/NY-MEL-1 is strongly immunogenic, leading to spontaneous antibody responses in a significant proportion of melanoma patients. The immune response occurs solely in malignant melanoma patients and was not detected in patients with other diseases, such as vitiligo, affecting melanocytes. Fine analysis of the spontaneous anti-RAB38/NY-MEL-1 antibody response reveals a polyclonal B cell recognition targeting various epitopes, although a dominant immunogenic region was preferentially recognized. Interestingly, our data indicate that this recognition is not rigid in the course of a patient's response, as the dominant epitope changes during the disease evolution. Implications for the understanding of spontaneous humoral immune responses are discussed.

Fatty acid binding protein 4 (FABP4) is a key mediator of intracellular transport and metabolism of fatty acids in adipose tissues. FABP4 binds fatty acids with high affinity and transports them to various compartments in the cell. When in complex with fatty acids, FABP4 interacts with and modulates the activity of two important regulators of metabolism: hormone-sensitive lipase and peroxisome proliferator-activated receptor gamma. Genetic studies in mice clearly indicated that deregulation of FABP4 function may lead to the development of severe diseases such as diabetes II type and atherosclerosis. In this study, we report the production and detailed characterization of monoclonal antibodies (MAbs) against FABP4. Recombinant glutathione S-transferase (GST)-FABP4 or His-FABP4 was expressed in bacteria, affinity purified, and used for immunization of mice, enzyme-linked immunosorbent assay (ELISA) screening, and characterization of selected clones. We have isolated two hybridoma clones that produced antibodies specific for recombinant and native FABP4, as shown by Western blotting and immunoprecipitation. The specificity of generated antibodies was further tested in a cell-based model of adipogenesis. In this analysis, the accumulation of FABP4 during NIH 3T3-L1 differentiation into adipocytes was detected by generated antibodies, which correlates well with previously published data. Taken together, we produced MAbs that will be useful for the scientific community working on fatty acid-binding proteins and lipid metabolism.

CoA and its derivatives Acetyl-CoA and Acyl-CoA are important players in cellular metabolism and signal transduction. CoA synthase is a bifunctional enzyme which mediates the final stages of CoA biosynthesis. In previous studies, we have reported molecular cloning, biochemical characterization, and subcellular localization of CoA synthase (CoASy). Here, we describe the existence of a novel CoA synthase isoform, which is the product of alternative splicing and possesses a 29aa extension at the N-terminus. We termed it CoASy beta and originally identified CoA synthase, CoASy alpha. The transcript specific for CoASy beta was identified by electronic screening and by RT-PCR analysis of various rat tissues. The existence of this novel isoform was further confirmed by immunoblot analysis with antibodies directed to the N-terminal peptide of CoASy beta. In contrast to CoASy alpha, which shows ubiquitous expression, CoASy beta is primarily expressed in the brain. Using confocal microscopy, we demonstrated that both isoforms are localized on mitochondria. The N-terminal extension does not affect the activity of CoA synthase, but possesses a proline-rich sequence which can bring the enzyme into complexes with signalling proteins containing SH3 or WW domains. The role of this novel isoform in CoA biosynthesis, especially in the brain, requires further elucidation.

Protein kinase 2 (CK2) is a ubiquitous and constitutively active serine/threonine protein kinase with various cell functions. It typically forms tetrameric complexes consisting of two catalytic (alpha and/or (alpha') and two regulatory (beta) subunits. The aim of this study was to produce monoclonal antibodies (MAbs) against the CK2beta subunit and to characterize their suitability for Western blotting, immunoprecipitation, and immunohistochemical applications. Bacterially expressed CK2beta-6His-GST recombinant protein has been used as an antigen. Balb/c mice were immunized and given a final boost, and their spleen cells were collected and fused with SP2/0 myeloma cells using PEG 2000. The fused cells were then selected in the HAT-RPMI medium. Anti- CK2beta high-titer antibody-producing hybridoma cell lines were identified by enzyme-linked immunosorbent assay (ELISA) and then subcloned by limiting dilution in HT-RPMI medium supplemented with 20% fetal bovine serum (FBS). A total of 10 IgG-producing cell lines were selected and further tested for their reactivity with the CK2beta subunit using ELISA, Western blots, immunoprecipitation, and immunostaining of formaldehyde-fixed paraffin-embedded tissue sections. The results obtained clearly indicate that several clones produce antibodies that recognize specifically recombinant and endogenous CK2beta subunit in Western blotting and immunoprecipitation, and are suitable for immunohistochemical analysis. In summary, the produced antibodies will be useful for researchers investigating signaling pathways involving CK2 kinase and their deregulation in human pathologies.

The ribosomal protein S6 kinase (S6K) belongs to the AGC family of Ser/Thr kinases and is known to be involved in the regulation of protein synthesis and the G(1)/S transition of the cell cycle. There are two forms of S6K, termed S6Kalpha and S6Kbeta, which have cytoplasmic and nuclear splice variants. Nucleocytoplasmic shuttling has been recently proposed for S6Kalpha, based on the use of the nuclear export inhibitor, leptomycin B. However, the molecular mechanisms regulating subcellular localization of S6Ks in response to mitogenic stimuli remain to be elucidated. Here we present data on the in vitro and in vivo phosphorylation of S6Kbeta, but not S6Kalpha, by protein kinase C (PKC). The site of phosphorylation was identified as S486, which is located within the C-terminal nuclear localization signal. Mutational analysis and the use of phosphospecific antibodies provided evidence that PKC-mediated phosphorylation at S486 does not affect S6K activity but eliminates the function of its nuclear localization signal and causes retention of an activated form of the kinase in the cytoplasm. Taken together, this study uncovers a novel mechanism for the regulation of nucleocytoplasmic shuttling of S6KbetaII by PKC-mediated phosphorylation.