Contributions of ubiquitin- and PCNA-binding domains to the activity of Polymerase η in Saccharomyces cerevisiae

Bypassing of DNA lesions by damage-tolerant DNA polymerases depends on the interaction of these enzymes with the monoubiquitylated form of the replicative clamp protein, PCNA. We have analyzed the contributions of ubiquitin and PCNA binding to damage bypass and damage-induced mutagenesis in Polymerase η (encoded by RAD30) from the budding yeast Saccharomyces cerevisiae. We report here that a ubiquitin-binding domain provides enhanced affinity for the ubiquitylated form of PCNA and is essential for in vivo function of the polymerase, but only in conjunction with a basal affinity for the unmodified clamp, mediated by a conserved PCNA interaction motif. We show that enhancement of the interaction and function in damage tolerance does not depend on the ubiquitin attachment site within PCNA. Like its mammalian homolog, budding yeast Polymerase η itself is ubiquitylated in a manner dependent on its ubiquitin-binding domain

Ubiquitin ligases and beyond

First paragraph (this article has no abstract): In a review published in 2004 [1] and that still repays reading today, Cecile Pickart traced the evolution of research on ubiquitination from its origins in the proteasomal degradation of proteins through the revelation that it has a central role in cell cycle regulation and the recognition of regulatory roles for ubiquitin in intracellular membrane transport, cell signalling, transcription, translation, and DNA repair

Study on Doping Prevention: A map of Legal, Regulatory and Prevention Practice Provisions in EU 28

Historically, anti-doping efforts have focused on the detection and deterrence of doping in elite and competitive sport. There is, however, a growing concern that doping is occurring outside the organised sporting system; giving rise to the belief that the misuse of doping agents in recreational sport has become a societal problem and a public health issue that must be addressed. The EU Commission awarded a contract (EAC/2013/0617) to a Consortium to undertake this Study with the aim of developing the evidence-base for policies designed to combat doping in recreational sport. Fourteen internationally recognised experts shaped the Study which comprised (i) the collection of primary data through a structured survey, and (ii) secondary data through literature searches and website analysis. All 28 Member States participated in the information-gathering process. Specifically, this involved a systematic study of the ethical considerations, legal position, prevention research landscape, and current practise in relation to the prevention of doping in recreational sport. The Study provides a comprehensive overview of current practice and legislation as it applies to the prevention of doping and promotes and supports the sharing of best practices in the EU regarding the fight against doping in recreational sport. It concludes with seven recommendations for future action that focus on the need for a coordinated response in relation to the problems arising from doping in recreational sport

The quest for the ganglioside functions; what did we learn more from «evo-devo» or signaling of long-term maintenance?

Gangliosides are characteristic extracellular-facing plasma membrane determinants in vertebrate brain. The four major gangliosides (GM1, GD1a, GD1b and GT1b) dominate among more than one hundred glycolipid structures in nervous tissue. During brain development the expression of simple gangliosides shifts toward more complex ones, accompanied by a multiple increase in their total amount. The shift is precisely regulated and some specific structures represent well established neurodevelopmental milestones. From the evolutionary perspective, the ganglioside content in fish and amphibian brain is significantly lower than in mammalian brain, but the general variability is greater. More-polar structures, abundant in Antarctic fishes, are rare in higher vertebrates or expressed only in a narrow developmental frame. Reptiles, birds and mammals share identical common structures expressed in similar patterns with minor interspecies differences. On the contrary, fish and amphibian brains show significant interspecies differences in amount, structure and expression patterns. The initial assumption of evolutionary studies was that the variations in lipid content, particularly the glycolipid content, during temperature adaptations in ectothermic and hibernating heterothermic animals, represent an efficient molecular mechanism of the membrane function preservation. Studies of ordered lipid domains in the last decade verified the ganglioside-mediated regulation of membrane proteins (receptor kinases, neurotransmitter receptors and ion channels) as well as receptor-ligand interaction important for cell signaling.Гангліозиди – характеристичні детермінанти, які локалізовані на зовнішній поверхні мембран клітин мозку хребетних. Чотири основних гангліозиди (GM1, GD1a, GD1b i GT1b) переважають серед сотень інших сполук гліколіпідів нервової тканини. У процесі розвитку мозку експресія простих гангліозидів зміщується у бік синтезу більш складних сполук, що супроводжується багаторазовим зростанням їхньої загальної кількості. Зміщення експресії – строго регульований процес, за якого поява деяких специфічних структур репрезентує добре відомі стадії розвитку нервової тканини. З точки зору еволюції вміст гангліозидів у мозку риб та амфібій значно нижчий, ніж у мозку ссавців, проте загальна їхня варіабельність суттєво вища. Більш полярні сполуки, які широко представлені у антарктичних риб, є рідкісними для ссавців або характерними для певного короткотривалого етапу онтогенезу. Плазуни, птахи і ссавці зберігають ідентичні спільні структури, що мають подібні патерни експресії з незначними міжвидовими відмінностями. Навпаки, для мозку риб та амфібій відзначено істотну міжвидову відмінність щодо кількості, структури та патерну експресії. Першочерговим припущенням еволюційного дослідження стало те, що варіації у вмісті ліпідів, зокрема гліколіпідів, під час температурної адаптації у холоднокровних і гетеротермних тварин, які впадають у сплячку, є високоефективним молекулярним механізмом захисту функціонування мембран. Вивчення впорядкованих доменів ліпідів за останнє десятиліття підтверджує гангліозид-опосередковану регуляцію мембранних білків (рецептори з кіназною активністю, рецептори нейротрансмітерів та іонні канали), так само як і взаємодію рецептор–ліганд, важливу для передачі позаклітинного сигналу.Ганглиозиды – характеристические детерминанты, локализованные на внешней поверхности мембран клеток мозга хребетных. Четыре основных ганглиозида (GM1, GD1a, GD1b и GT1b) преобладают среди сотень других соединений гликолипидов нервной ткани. В процессе развития мозга экспрессия простых ганглиозидов замещается синтезом более сложных соединений, что сопровождается многократным увеличением их общего количества. Смещение экспрессии – строго регулированный процесс, при котором появление некоторых специфических структур представляет хорошо известные стадии развития нервной ткани. Содержание ганглиозидов в мозге рыб и амфибий значительно ниже, чем в мозге млекопитающих, однако их общая вариабельность существенно выше. Более полярные соединения, широко представленные у антарктических рыб, являются редкими для млекопитающих или характерными для определенного кратковременного этапа онтогенеза. Пресмыкающиеся, птицы и млекопитающие сохраняют общие идентичные структуры с подобными паттернами экспрессии, имеющими незначительные межвидовые отличия. Наоборот, для мозга рыб и амфибий отмечены существенные межвидовые отличия, касающиеся количества, структуры и паттерна экспрессии. Первоочередным предположением эволюционного исследования стало то, что вариации в содержании липидов, в частности гликолипидов, во время температурной адаптации у хладнокровных и гетеротермных животных, впадающих в спячку, являются высокоэффективным молекулярным механизмом защиты функционирования мембран. Изучение упорядоченных доменов липидов за последнее десятилетие подтверждает ганглиозид-опосредованную регуляцию мембранных белков (рецепторы с киназной активностью, рецепторы нейротрансмиттеров и ионные каналы), так же как и взаимодействие рецептор–лиганд, важное для передачи внеклеточного сигнала

Left atrial volume changes during exercise stress echocardiography in heart failure and hypertrophic cardiomyopathy

We assessed feasibility and functional correlates of LAVI (left atrial volume index) changes during exercise stress echocardiography (ESE).ESE on bike or treadmill was performed in 363 patients with heart failure with preserved ejection fraction (HFpEF, n = 173), reduced ejection fraction (HFrEF, n = 59) or hypertrophic cardiomyopathy (HCM, n=131). LAVI stress-rest increase ≥ 6.8 ml/m2 was defined as dilation.LAVI measurements were feasible in 100%. LAVI did not change in HFrEF being at rest 32 (25-45) vs. at stress 36 (24 - 54) ml/m2, P = NS and in HCM at rest 35 (26 - 48) vs. at stress 38 (28 - 48) ml/m2, P = NS whereas it decreased in HFpEF from 30 (24 -40) to 29 (21 - 37) ml/m2 at stress, P = 0.007. LA dilation occurred in 107 (30%) patients (27% with treadmill vs. 33% with bike ESE, P = NS): 26 with HFpEF (15%), 26 with HFrEF (44%) and 55 with HCM (42%) with P 14 at rest with OR 4.4, LVEF < 50% with OR 2.9, and LAVI at rest < 35 ml/m2 with OR 2.7.LAVI assessment during ESE was highly feasible and dilation equally frequent with treadmill or bike. LA dilation was threefold more frequent in HCM and HFrEF and could be predicted by increased resting E/e' and impaired EF as well as smaller baseline LAVI

Influence of Higenamine on Exercise Performance of Recreational Female Athletes: A Randomized Double-Blinded Placebo-Controlled Trial

The aim of this study was to determine the ergogenic effects and the safety profile of a one-component higenamine supplement in female recreational athletes. Twelve recreational female basketball players (age 29–41 years, oxygen consumption (VO2max) > 30 ml⋅kg–1⋅min–1, with training > 5 h wk–1) were randomized either to the higenamine group, or to the placebo group for 3 weeks. In order to determine ergogenic effects and safety profile of higenamine administration, we assessed the following variables before and after 3 weeks of supplementation: anthropometric parameters, resting metabolic rate (RMR), exercise testing variables, serum free fatty acids (FFAs), blood pressure, enzyme activity, urea, lipid profile, and complete blood count. There were no differences between groups in anthropometric parameters, including basal metabolic rate (BMR), RMR and body fat [p = 0.706 (Cohen’s d 0.223), p = 0.169 (Cohen’s d 0.857), and p = 0.223 (Cohen’s d 0.750), respectively], FFAs [0.43 ± 0.03 vs. 0.54 ± 0.23, p = 0.206 (Cohen’s d 0.540)], neither significant differences in cardiopulmonary parameters after the intervention period. Furthermore, all measured outcome variables in the safety assessment were not significant, with values remaining stable during the intervention period for participants in both groups. This is the first study to document the effects and the safety profile of higenamine-based dietary supplements at a specified dose in female recreational athletes. Our data indicate that 21-day of supplementation with 75 mg higenamine would not result in improving cardiopulmonary exercise fitness and weight loss in female recreational athletes. Moreover, supplementation with 75 mg higenamine is safe and well-tolerated in younger recreational female athletes

The degradation of p53 and its major E3 ligase Mdm2 is differentially dependent on the proteasomal ubiquitin receptor S5a.

p53 and its major E3 ligase Mdm2 are both ubiquitinated and targeted to the proteasome for degradation. Despite the importance of this in regulating the p53 pathway, little is known about the mechanisms of proteasomal recognition of ubiquitinated p53 and Mdm2. In this study, we show that knockdown of the proteasomal ubiquitin receptor S5a/PSMD4/Rpn10 inhibits p53 protein degradation and results in the accumulation of ubiquitinated p53. Overexpression of a dominant-negative deletion of S5a lacking its ubiquitin-interacting motifs (UIM)s, but which can be incorporated into the proteasome, also causes the stabilization of p53. Furthermore, small-interferring RNA (siRNA) rescue experiments confirm that the UIMs of S5a are required for the maintenance of low p53 levels. These observations indicate that S5a participates in the recognition of ubiquitinated p53 by the proteasome. In contrast, targeting S5a has no effect on the rate of degradation of Mdm2, indicating that proteasomal recognition of Mdm2 can be mediated by an S5a-independent pathway. S5a knockdown results in an increase in the transcriptional activity of p53. The selective stabilization of p53 and not Mdm2 provides a mechanism for p53 activation. Depletion of S5a causes a p53-dependent decrease in cell proliferation, demonstrating that p53 can have a dominant role in the response to targeting S5a. This study provides evidence for alternative pathways of proteasomal recognition of p53 and Mdm2. Differences in recognition by the proteasome could provide a means to modulate the relative stability of p53 and Mdm2 in response to cellular signals. In addition, they could be exploited for p53-activating therapies. This work shows that the degradation of proteins by the proteasome can be selectively dependent on S5a in human cells, and that this selectivity can extend to an E3 ubiquitin ligase and its substrate

Long non-coding RNA PCAT19 safeguards DNA in quiescent endothelial cells by preventing uncontrolled phosphorylation of replication protein A2

In healthy vessels, endothelial cells maintain a stable, differentiated, and growth-arrested phenotype for years. Upon injury, a rapid phenotypic switch facilitates proliferation to restore tissue perfusion. Here we report the identification of the endothelial cell-enriched long non-coding RNA (lncRNA) PCAT19, which contributes to the proliferative switch and acts as a safeguard for the endothelial genome. PCAT19 is enriched in confluent, quiescent endothelial cells and binds to the full replication protein A (RPA) complex in a DNA damage- and cell-cycle-related manner. Our results suggest that PCAT19 limits the phosphorylation of RPA2, primarily on the serine 33 (S33) residue, and thereby facilitates an appropriate DNA damage response while slowing cell cycle progression. Reduction in PCAT19 levels in response to either loss of cell contacts or knockdown promotes endothelial proliferation and angiogenesis. Collectively, PCAT19 acts as a dynamic guardian of the endothelial genome and facilitates rapid switching from quiescence to proliferation

UTP and ATP increase extracellular signal-regulated kinase 1/2 phosphorylation in bovine chromaffin cells through epidermal growth factor receptor transactivation

Adenosine triphosphate (ATP) is coreleased with catecholamines from adrenal medullary chromaffin cells in response to sympathetic nervous system stimulation and may regulate these cells in an autocrine or paracrine manner. Increases in extracellular signal-regulated kinase (ERK) 1/2 phosphorylation were observed in response to ATP stimulation of bovine chromaffin cells. The signaling pathway involved in ATP-mediated ERK1/2 phosphorylation was investigated via Western blot analysis. ATP and uridine 5′-triphosphate (UTP) increased ERK1/2 phosphorylation potently, peaking between 5 and 15 min. The mitogen-activated protein kinase (MAPK/ERK)-activating kinase (MEK) inhibitor PD98059 blocked this response. UTP, which is selective for G-protein-coupled P2Y receptors, was the most potent agonist among several nucleotides tested. Adenosine 5′-O-(3-thio) triphosphate (ATPγS) and ATP were also potent agonists, characteristic of the P2Y2 or P2Y4 receptor subtypes, whereas agonists selective for P2X receptors or other P2Y receptor subtypes were weakly effective. The receptor involved was further characterized by the nonspecific P2 antagonists suramin and reactive blue 2, which each partially inhibited ATP-mediated ERK1/2 phosphorylation. Inhibitors of protein kinase C (PKC), protein kinase A (PKA), Ca2+/calmodulin-dependent protein kinase II (CaMKII), and phosphoinositide-3 kinase (PI3K) had no effect on ATP-mediated ERK1/2 phosphorylation. The Src inhibitor PP2, epidermal growth factor receptor (EGFR) inhibitor AG1478, and metalloproteinase inhibitor GM6001 decreased ATP-mediated ERK1/2 phosphorylation. These results suggest nucleotide-mediated ERK1/2 phosphorylation is mediated by a P2Y2 or P2Y4 receptor, which stimulates metalloproteinase-dependent transactivation of the EGFR