Coenzyme A, protein CoAlation and redox regulation in mammalian cells

In a diverse family of cellular cofactors, coenzyme A (CoA) has a unique design to function in various biochemical processes. The presence of a highly reactive thiol group and a nucleotide moiety offers a diversity of chemical reactions and regulatory interactions. CoA employs them to activate carbonyl-containing molecules and to produce various thioester derivatives (e.g. acetyl CoA, malonyl CoA and 3-hydroxy-3-methylglutaryl CoA), which have well-established roles in cellular metabolism, production of neurotransmitters and the regulation of gene expression. A novel unconventional function of CoA in redox regulation, involving covalent attachment of this coenzyme to cellular proteins in response to oxidative and metabolic stress, has been recently discovered and termed protein CoAlation (S-thiolation by CoA or CoAthiolation). A diverse range of proteins was found to be CoAlated in mammalian cells and tissues under various experimental conditions. Protein CoAlation alters the molecular mass, charge and activity of modified proteins, and prevents them from irreversible sulfhydryl overoxidation. This review highlights the role of a key metabolic integrator CoA in redox regulation in mammalian cells and provides a perspective of the current status and future directions of the emerging field of protein CoAlation

Structure and function of epithelial mucins

Mucins are the structural, components of the epithelial mucose that protects the respiratory, gastrointestinal and reproductive tracts from the hostile environments, including microorganisms, toxins and abrasives. Mucins constitute a group of high molecular weight (> 200 kDa), polydisperse and highly glycosytated proteins which are present on the surface of most epithelial tissues. Our understanding of the structure and function of mucins lias advanced significantly in the last decade. This progress was mainly associated with the isolation of the cDNA clones, encoding a family of epithelial mucins. To date, this family includes eight mucin genes (MUC1–MUC8) and more await to be discovered. Based on sequence analysis and studies of subcellular localisation, epithelial mucins could be divided into two classes: membrane-associated (MUC1) and secretory (MUC2–8). This review is focused on our current knowledge of the structure of products of mucin genes and their function in normal tissues and in disease. The regulation of the expression of mucin genes, posttranslational modifications and alterations in secretion and processing will also be discussed.Муцини – це структурні компоненти епітеліального шару мукози, які захищають респіраторний, травний та репродуктивний тракти від негативних впливів зовнішнього оточення (мікроорганізмів, токсинів та хімічних або механічних подразників). Муцини складають групу високо молекулярних (> 200 кДа), полідисперсних та сильноглікозійованих білків, що знаходяться на поверхні більшості епітеліальних тканин. За останнє десятиріччя прогрес в розумінні структури та функцій муцинів, в основному, пов'язаний з одержанням кДНК клонів, кодуючих сімейство епітеліальних муцинів. На сьогодні це сімейство охоплює вісім муцинових генів (MUC1–MUC8) і очікується відкриття нових. Базуючись на первинній структурі та вивченні внутрішньоклітинної локалізації, епітеліальні муцини можна поділити на два класи: мембран-асоційовані (MUC1) та секреторні (MUC2–MUC8). Цей огляд сфокусовано на сучасних уявленнях про структуру продуктів муцинових генів та їхні функції в нормальних тканинах і при патологіях. Обговорюються також регуляція експресії муцинових генів, посттрансляційні модифікації та зміни в секреції і процссингу.Муцины – структурные компоненты эпителиального слоя мукозы – защищают респираторный, пищеварительный и репродуктивный тракты от отрицательного воздействия внешнего окружения, включающего микроорганизмы, токсины и механические раздражители. Муцины составляют группу высокомолекулярных (> 200 кДа) полидисперсных и силыюгликозилированных белков, расположенных на поверхности большинства эпителиальных тканей. За последнее 10-летие прогресс в выяснении структуры и функции муцинов был связан, в основном, с выделением кДНК клонов, кодирующих семейство эпителиальных муцинов. На сегодня это семейство включает уже восемь муциновых генов (MUC1–MUC8) и ожидается открытие новых. На основе анализа первичной структуры и изучения внутриклеточной локализации эпителиальные муцины можно разделить на два класса: мембран-ассоциированные (MUC1) и секреторные (MUC2–MUC8). Данный обзор сфокусирован на современных представлениях о структуре продуктов муциновых генов и их функциях в нормальных тканях и при патологиях. Обсуждаются также регуляция экспрессии муциновых генов, посттрансляционные модификации и изменения в секреции и процессинге муцинов

The Writers, Readers, and Erasers in Redox Regulation of GAPDH.

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a key glycolytic enzyme, which is crucial for the breakdown of glucose to provide cellular energy. Over the past decade, GAPDH has been reported to be one of the most prominent cellular targets of post-translational modifications (PTMs), which divert GAPDH toward different non-glycolytic functions. Hence, it is termed a moonlighting protein. During metabolic and oxidative stress, GAPDH is a target of different oxidative PTMs (oxPTM), e.g., sulfenylation, S-thiolation, nitrosylation, and sulfhydration. These modifications alter the enzyme's conformation, subcellular localization, and regulatory interactions with downstream partners, which impact its glycolytic and non-glycolytic functions. In this review, we discuss the redox regulation of GAPDH by different redox writers, which introduce the oxPTM code on GAPDH to instruct a redox response; the GAPDH readers, which decipher the oxPTM code through regulatory interactions and coordinate cellular response via the formation of multi-enzyme signaling complexes; and the redox erasers, which are the reducing systems that regenerate the GAPDH catalytic activity. Human pathologies associated with the oxidation-induced dysregulation of GAPDH are also discussed, featuring the importance of the redox regulation of GAPDH in neurodegeneration and metabolic disorders

Development of monoclonal antibodies specific to ribosomal protein S6 kinase 2

Ribosomal protein S6 kinase 2 (S6K2) is a serine/threonine kinase that belongs to the family of AGC kinases, which includes PKB/Akt, PKC, PDK1, and SGK1. Mammalian cells express two isoforms of S6K, termed S6K1 and S6K2. Each of these has nuclear and cytoplasmic spicing variants, which originate from different initiation start codons. Nuclear isoforms of S6K1 and S6K2 are slightly longer, as they possess additional sequences at the N-terminus with nuclear localization signals. Biochemical and genetic studies implicated S6Ks in the regulation of cell size, growth, and energy metabolism. Deregulation of S6K signaling has been linked to various human pathologies, making them excellent targets for drug discovery. The aim of this study was to produce monoclonal antibodies directed at the N-terminal regulatory region of S6K2, which shows very low homology to S6K1 or other members of the AGC family. To achieve this goal, two S6K2 fragments covering 1-64aa and 14-64aa N-terminal sequences were expressed in bacteria as GST/6His fusion proteins. Affinity purified recombinant proteins were used as antigens for immunization, hybridoma screening, and analysis of generated clones. We produced a panel of S6K2-specific antibodies, which recognized recombinant S6K2 proteins in ELISA and Western blot analysis. Further analysis of selected clones revealed that three clones, termed B1, B2, and B4, specifically recognized not only recombinant, but also endogenous S6K2 in Western blot analysis of HEK293 cell lysates. Specificity of B2 clone has been confirmed in additional commonly used immunoassays, including immunoprecipitation and immunocytochemistry. These properties make B2 MAb particularly valuable for elucidating signal transduction pathways involving S6K2 signaling under physiological conditions and in human pathologies

Asymmetric Dimethylation of Ribosomal S6 Kinase 2 Regulates Its Cellular Localisation and Pro-Survival Function

Ribosomal S6 kinases (S6Ks) are critical regulators of cell growth, homeostasis, and survival, with dysregulation of these kinases found to be associated with various malignancies. While S6K1 has been extensively studied, S6K2 has been neglected despite its clear involvement in cancer progression. Protein arginine methylation is a widespread post-translational modification regulating many biological processes in mammalian cells. Here, we report that p54-S6K2 is asymmetrically dimethylated at Arg-475 and Arg-477, two residues conserved amongst mammalian S6K2s and several AT-hook-containing proteins. We demonstrate that this methylation event results from the association of S6K2 with the methyltransferases PRMT1, PRMT3, and PRMT6 in vitro and in vivo and leads to nuclear the localisation of S6K2 that is essential to the pro-survival effects of this kinase to starvation-induced cell death. Taken together, our findings highlight a novel post-translational modification regulating the function of p54-S6K2 that may be particularly relevant to cancer progression where general Arg-methylation is often elevated

Regulation of the CoA Biosynthetic Complex Assembly in Mammalian Cells

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’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/Pank1β 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

Диференціація генотипів сосни звичайної за поліморфізмом довжини інтронів генів дефензинів

The search for highly informative DNA markers to support the breeding programs aimed at increasing the productivity and biological stability of forest stands is an urgent task, especially in the context of global climate change. In this paper, we first investigate a new type of genetic markers based on the intron length polymorphism (ILP) of defensin genes to determine their informativeness and promising use for the estimation of the potential resistance of pine genotypes to biological damage. In the course of our study we applied the following methods: total DNA isolation by STAB method, PCR amplification with specific primers to pine defensin genes and Heterobasidion annosum s. s., electrophoretic separation of PCR fragments on a polyacrylamide gel under nondenaturing conditions, and also data analysis using software GenAlEx 6.053 and Statistica 10. Having conducted the research, we can present the following results. To investigate the defensin genes polymorphism in pine trees, we used ILP markers. These markers were developed based on the nucleotide sequences of the exons of PsDef1-4 genes. Analysis of PCR fragments obtained after amplification of each pair of primers with genomic DNA of pine showed that only one pair of primers, which is specific to defensin 2 (IPL-PsDef2), forms a wide range of amplified products, indicating their promising use for genetic characterization of genotypes of Scots pine. To clarify the use of IPL-PsDef2 markers for the study of genetic polymorphism we analyzed 196 trees and revealed that the average PIC was 0.287. IPL-PsDef2 markers were used to analyze the different genotypes of Scots pine on the areas affected by root rot disease. Based on the results of cluster analysis, the samples were divided into two groups, which differ in resistance to the root sponge. In addition, we identified PCR fragments that are specific to each of these groups and can serve as markers for the evaluation of the resistance of pine genotypes to Heterobasidion annosum. Thus, our conclusion is as follows: genotyping of defensin genes loci of pine breeding material is promising for the development of environmentally friendly technologies in order to enhance the sustainability of improved genetic material of pine trees to biotic stress.Наведено результати дослідження генотипів сосни звичайної (Pinus sylvestris L.) із використанням молекулярних маркерів на підстаі генетичного поліморфізму довжини інтронів генів дефензинів PsDef1-4. Виявлено високу інформативність маркерів на підстаі інтрону гена PsDef2 (IPL-PsDef2) для дослідження внутрішньовидового поліморфізму в сосни звичайної. Встановлено межі осередка інфекції кореневої губки (Heterobasidion annosum (Fr.) Bref.) в сосновому насадженні за допомогою полімеразно-ланцюгової реакції (ПЛР) із використанням видоспецифічних праймерів. Проведено ДНК-профілювання модельних дерев способом електрофоретичного розділення ампліконів IPL-PsDef2 маркерів. За результатами поліморфізму довжини інтронів генів дефензинів здійснено кластеризацію генотипів сосни звичайної із використанням алгоритму UPGMA. на підстаі дендрограми виділено два кластери генотипів зі 100 % бутстреп підтримкою, які відрізняються за стійкістю до кореневої губки. Методом К-середніх визначено значущість кожного з ампліконів для диференціації генотипів сосни на групи. Виявлено амплікони, які можуть бути генетичними маркерами для оцінки потенційної стійкості генотипів сосни звичайної до кореневої губки. Отримані дані щодо поліморфізму локусів генів дефензинів, а також результати кластерного аналізу вказують на перспективність використання IPL-PsDef2 маркерів для генотипування сосни звичайної

Application of serex-analysis for identification of human colon cancer antigens

Copyright © Experimental Oncology, 2015. Background: Colorectal, lung and breast tumors are the most devastating and frequent malignances in clinical oncology. SEREX-analysis of colon cancer leads to identification of more than hundred antigens which are potential tumor markers. With idea that immunoscreening with pool of allogeneic sera is more productive for antigen isolation, SEREX-analysis was applied to four cases of stages II-IV primary colon tumor and 22 new antigens were isolated. Objective: To characterize 22 primary colon cancer antigens isolated by SEREXtechnique. Materials and Methods: Allogenic screening, real-time PCR analysis. Results: After allogeneic immunoscreening, for 5 of 22 (22%) isolated antigens were confirmed colon cancer restricted serological profile solely positive for 14% of tested colon cancer sera. Through these five antigens, KY-CC-17/β-actin has cytoskeleton function; KY-CC-14/ACTR1A and KY-CC-19/TSGA2 participate in chromosome segregation; KY-CC-12/FKBP4 regulates steroid receptor function and KY-CC-15/PLRG1 is a component of spliceosome complex. For the last four antigens tested were found aberrant mRNA expression in some cases of colon tumor. Conclusion: The exploration of identified antigens may define suitable targets for immunotherapy or diagnostic of colon cancer

Asymmetric dimethylation of ribosomal S6 kinase 2 regulates its cellular localisation and pro-survival function

Ribosomal S6 kinases (S6Ks) are critical regulators of cell growth, homeostasis, and survival, with dysregulation of these kinases found to be associated with various malignancies. While S6K1 has been extensively studied, S6K2 has been neglected despite its clear involvement in cancer progression. Protein arginine methylation is a widespread post-translational modification regulating many biological processes in mammalian cells. Here, we report that p54-S6K2 is asymmetrically dimethylated at Arg-475 and Arg-477, two residues conserved amongst mammalian S6K2s and several AT-hook-containing proteins. We demonstrate that this methylation event results from the association of S6K2 with the methyltransferases PRMT1, PRMT3, and PRMT6 in vitro and in vivo and leads to nuclear the localisation of S6K2 that is essential to the pro-survival effects of this kinase to starvation-induced cell death. Taken together, our findings highlight a novel post-translational modification regulating the function of p54-S6K2 that may be particularly relevant to cancer progression where general Arg-methylation is often elevated

Human HMGN1 and HMGN2 are not required for transcription-coupled DNA repair

Transcription-coupled repair (TCR) removes DNA lesions from the transcribed strand of active genes. Stalling of RNA polymerase II (RNAPII) at DNA lesions initiates TCR through the recruitment of the CSB and CSA proteins. The full repertoire of proteins required for human TCR - particularly in a chromatin context - remains to be determined. Studies in mice have revealed that the nucleosome-binding protein HMGN1 is required to enhance the repair of UV-induced lesions in transcribed genes. However, whether HMGN1 is required for human TCR remains unaddressed. Here, we show that knockout or knockdown of HMGN1, either alone or in combination with HMGN2, does not render human cells sensitive to UV light or Illudin S-induced transcription-blocking DNA lesions. Moreover, transcription restart after UV irradiation was not impaired in HMGN-deficient cells. In contrast, TCR-deficient cells were highly sensitive to DNA damage and failed to restart transcription. Furthermore, GFP-tagged HMGN1 was not recruited to sites of UV-induced DNA damage under conditions where GFP-CSB readily accumulated. In line with this, HMGN1 did not associate with the TCR complex, nor did TCR proteins require HMGN1 to associate with DNA damage-stalled RNAPII. Together, our findings suggest that HMGN1 and HMGN2 are not required for human TCR.Genome Instability and Cance