GRK2-Dependent HuR Phosphorylation Regulates HIF1α Activation under Hypoxia or Adrenergic Stress.

Adaptation to hypoxia is a common feature in solid tumors orchestrated by oxygen-dependent and independent upregulation of the hypoxia-inducible factor-1α (HIF-1α). We unveiled that G protein-coupled receptor kinase (GRK2), known to be overexpressed in certain tumors, fosters this hypoxic pathway via phosphorylation of the mRNA-binding protein HuR, a central HIF-1α modulator. GRK2-mediated HuR phosphorylation increases the total levels and cytoplasmic shuttling of HuR in response to hypoxia, and GRK2-phosphodefective HuR mutants show defective cytosolic accumulation and lower binding to HIF-1α mRNA in hypoxic Hela cells. Interestingly, enhanced GRK2 and HuR expression correlate in luminal breast cancer patients. GRK2 also promotes the HuR/HIF-1α axis and VEGF-C accumulation in normoxic MCF7 breast luminal cancer cells and is required for the induction of HuR/HIF1-α in response to adrenergic stress. Our results point to a relevant role of the GRK2/HuR/HIF-1α module in the adaptation of malignant cells to tumor microenvironment-related stresses.This research was funded by the Instituto de Salud Carlos III: PI17-00576; Ministerio de Economia, Industria y Competitividad, Gobierno de Espana: SAF2017-84125-R; Ministerio de Economia, Industria y Competitividad, Gobierno de Espana: SAF2017-87301-R; Instituto de Salud Carlos III: CIBERCV CB16/11/00278; Instituto de Salud Carlos III: PI14-00435; Fundacion Ramon Areces and the Comunidad de Madrid: B2017/BMD-3671-INFLAMUNE.S

HuR biological function involves RRM3-mediated dimerization and RNA binding by all three RRMs

HuR/ELAVL1 is an RNA-binding protein involved in differentiation and stress response that acts primarily by stabilizing messenger RNA (mRNA) targets. HuR comprises three RNA recognition motifs (RRMs) where the structure and RNA binding of RRM3 and of full-length HuR remain poorly understood. Here, we report crystal structures of RRM3 free and bound to cognate RNAs. Our structural, NMR and biochemical data show that RRM3 mediates canonical RNA interactions and reveal molecular details of a dimerization interface localized on the -helical face of RRM3. NMR and SAXS analyses indicate that the three RRMs in full-length HuR are flexibly connected in the absence of RNA, while they adopt a more compact arrangement when bound to RNA. Based on these data and crystal structures of tandem RRM1,2- RNA and our RRM3-RNA complexes, we present a structural model of RNA recognition involving all three RRM domains of full-length HuR. Mutational analysis demonstrates that RRM3 dimerization and RNA binding is required for functional activity of fulllength HuR in vitro and to regulate target mRNAs levels in human cells, thus providing a fine-tuning for HuR activity in vivo.Peer reviewe

S-adenosylmethionine Levels Regulate the Schwann Cell DNA Methylome

SummaryAxonal myelination is essential for rapid saltatory impulse conduction in the nervous system, and malformation or destruction of myelin sheaths leads to motor and sensory disabilities. DNA methylation is an essential epigenetic modification during mammalian development, yet its role in myelination remains obscure. Here, using high-resolution methylome maps, we show that DNA methylation could play a key gene regulatory role in peripheral nerve myelination and that S-adenosylmethionine (SAMe), the principal methyl donor in cytosine methylation, regulates the methylome dynamics during this process. Our studies also point to a possible role of SAMe in establishing the aberrant DNA methylation patterns in a mouse model of diabetic neuropathy, implicating SAMe in the pathogenesis of this disease. These critical observations establish a link between SAMe and DNA methylation status in a defined biological system, providing a mechanism that could direct methylation changes during cellular differentiation and in diverse pathological situations

Stabilization of LKB1 and Akt by neddylation regulates energy metabolism in liver cancer

The current view of cancer progression highlights that cancer cells must undergo through a post-translational regulation and metabolic reprogramming to progress in an unfriendly environment. In here, the importance of neddylation modification in liver cancer was investigated. We found that hepatic neddylation was specifically enriched in liver cancer patients with bad prognosis. In addition, the treatment with the neddylation inhibitor MLN4924 in Phb1-KO mice, an animal model of hepatocellular carcinoma showing elevated neddylation, reverted the malignant phenotype. Tumor cell death in vivo translating into liver tumor regression was associated with augmented phosphatidylcholine synthesis by the PEMT pathway, known as a liver-specific tumor suppressor, and restored mitochondrial function and TCA cycle flux. Otherwise, in protumoral hepatocytes, neddylation inhibition resulted in metabolic reprogramming rendering a decrease in oxidative phosphorylation and concomitant tumor cell apoptosis. Moreover, Akt and LKB1, hallmarks of proliferative metabolism, were altered in liver cancer being new targets of neddylation. Importantly, we show that neddylation-induced metabolic reprogramming and apoptosis were dependent on LKB1 and Akt stabilization. Overall, our results implicate neddylation/signaling/metabolism, partly mediated by LKB1 and Akt, in the development of liver cancer, paving the way for novel therapeutic approaches targeting neddylation in hepatocellular carcinoma

A flow cytometry-based neutralization assay for simultaneous evaluation of blocking antibodies against SARS-CoV-2 variants

Vaccines against SARS-CoV-2 have alleviated infection rates, hospitalization and deaths associated with COVID-19. In order to monitor humoral immunity, several serology tests have been developed, but the recent emergence of variants of concern has revealed the need for assays that predict the neutralizing capacity of antibodies in a fast and adaptable manner. Sensitive and fast neutralization assays would allow a timely evaluation of immunity against emerging variants and support drug and vaccine discovery efforts. Here we describe a simple, fast, and cell-free multiplexed flow cytometry assay to interrogate the ability of antibodies to prevent the interaction of Angiotensin-converting enzyme 2 (ACE2) and the receptor binding domain (RBD) of the original Wuhan-1 SARS-CoV-2 strain and emerging variants simultaneously, as a surrogate neutralization assay. Using this method, we demonstrate that serum antibodies collected from representative individuals at different time-points during the pandemic present variable neutralizing activity against emerging variants, such as Omicron BA.1 and South African B.1.351. Importantly, antibodies present in samples collected during 2021, before the third dose of the vaccine was administered, do not confer complete neutralization against Omicron BA.1, as opposed to samples collected in 2022 which show significant neutralizing activity. The proposed approach has a comparable performance to other established surrogate methods such as cell-based assays using pseudotyped lentiviral particles expressing the spike of SARS-CoV-2, as demonstrated by the assessment of the blocking activity of therapeutic antibodies (i.e. Imdevimab) and serum samples. This method offers a scalable, cost effective and adaptable platform for the dynamic evaluation of antibody protection in affected populations against variants of SARS-CoV-2.Funding: This research was supported by the SPRI I+D COVID-19 fund (Basque Government, bG-COVID-19), BIOEF EITB Maratoia (BIO21/COV/037 to AP), the European Research Council (ERC) (ERC-2018-StG 804236-NEXTGEN-IO to AP), the Instituto de Salud Carlos iii (ISCiii, DTS21/00094 to AP and DTS20/00138 to MM-C), Ministerio de Ciencia, Innovación y Universidades (MICINN, PID2019-107956RA-I00 and TED2021-129433B-C21 to AP; PID2020-117116RB-I00 and RTC2019-007125-1 to MM-C) and the FERO Foundation to AP. Personal fellowships: EP-F (Juan de la Cierva-Formación, FJC2018-035449-I), ABo (AECC Bizkaia Scientific Foundation, PRDVZ19003BOSC), AG (Programa Bikaintek from the Basque Government, 48-AF-W1-2019-00012), AA-V (La Caixa Inphinit, LCF/BQ/DR20/11790022), BJ-L (Basque Government, PRE_2019_1_0320), ABl (AECC Bizkaia Scientific Foundation, PRDVZ21640DEBL), PV-B (Proyectos I +D+I, PRE2020-092342) and AP (Ramón y Cajal, RYC2018- 024183-I; and Ikerbasque Research Associate). Acknowledgments: The plasmids for the generation of pseudotyped lentiviral particles were kindly provided by Dr Jesse D. Bloom (Fred Hutchinson Cancer Research Center) and Dr Jean-Philippe Julien (The Hospital for Sick Children). HEK293T-ACE2 cells were kindly provided by Dr. June Ereño-Orbea (CIC bioGUNE) and Dr. Jean-Philippe Julien (The Hospital for Sick Children Research Institute, Toronto)

A Shortcut from Metabolic-Associated Fatty Liver Disease (MAFLD) to Hepatocellular Carcinoma (HCC): c-MYC a Promising Target for Preventative Strategies and Individualized Therapy

Background: Metabolic-associated fatty liver disease (MAFLD) has risen as one of the leading etiologies for hepatocellular carcinoma (HCC). Oncogenes have been suggested to be responsible for the high risk of MAFLD-related HCC. We analyzed the impact of the proto-oncogene c-MYC in the development of human and murine MAFLD and MAFLD-associated HCC. Methods: alb-myctg mice were studied at baseline conditions and after administration of Western diet (WD) in comparison to WT littermates. c-MYC expression was analyzed in biopsies of patients with MAFLD and MAFLD-associated HCC by immunohistochemistry. Results: Mild obesity, spontaneous hyperlipidaemia, glucose intolerance and insulin resistance were characteristic of 36-week-old alb-myctg mice. Middle-aged alb-myctg exhibited liver steatosis and increased triglyceride content. Liver injury and inflammation were associated with elevated ALT, an upregulation of ER-stress response and increased ROS production, collagen deposition and compensatory proliferation. At 52 weeks, 20% of transgenic mice developed HCC. WD feeding exacerbated metabolic abnormalities, steatohepatitis, fibrogenesis and tumor prevalence. Therapeutic use of metformin partly attenuated the spontaneous MAFLD phenotype of alb-myctg mice. Importantly, upregulation and nuclear localization of c-MYC were characteristic of patients with MAFLD and MAFLD-related HCC. Conclusions: A novel function of c-MYC in MAFLD progression was identified opening new avenues for preventative strategies

Assignment of a single disulfide bridge in rat liver methionine adenosyltransferase

Journal now known as FEBS Journal.-- Open Access: content older than 1 year.Rat liver methionine adenosyltransferase incorporated 8 mol of N- ethylmaleimide per mol of subunit upon denaturation in the presence of 8 M urea, whereas 10 such groups were labelled when dithiothreitol was also included. This observation prompted a re-examination of the state of the thiol groups, which was carried out using peptide mapping, amino acid analysis and N-terminal sequencing. The results obtained revealed a disulfide bridge between Cys35 and Cys61. This disulfide did not appear to be conserved because cysteines homologous to residue 61 do not exist in methionine adenosyltransferases of other origins, therefore suggesting its importance for the differential aspects of the liver-specific enzyme.This work was supported by a grant of the Dirección General de Investigación Científica y Técnica (PM 97/0064).Peer Reviewe

Role of thioltransferases on the modulation of rat liver S-adenosylmethionine synthetase activity by glutathione

Rat liver S-adenosylmethionine synthetase, high- and low-M(r) forms, are regulated in vitro by the GSH/GSSG ratio at pH 8. The inhibition and oxidation constants for both forms have been calculated in the presence of thioltransferases. The mechanism of the reaction appeared to involve the formation of intramolecular disulfides. Increases of 3- to 4-fold in the oxidation constants for both S-adenosylmethionine synthetase isoenzymes in the presence of protein disulfide isomerase suggested the possibility of a thiol-disulfide exchange regulatory mechanism for this enzyme in vivo. The significance of these results is discussed on the light of the data available relating glutathione changes and modulation of enzyme activities, either in vivo and in vitro.This work was supported by grants from the Fondo de Investigaciones Sanitarias (94/ 0231) Direcciónn General de Investigación Científica y Técnica (PB 94/0087) the Science Programme of the European Community (SCI*- CT92-0780) and Europharma S.A.Peer Reviewe

The N-terminal domain of the enzyme I is a monomeric well-folded protein with a low conformational stability and residual structure in the unfolded state.

The bacterial phosphoenolpyruvate-dependent sugar phosphotransferase system is a multiprotein complex that phosphorylates and, concomitantly, transports carbohydrates across the membrane into the cell. The first protein of the cascade is a multidomain protein so-called enzyme I (EI). The N-terminal domain of EI from Streptomyces coelicolor, EIN(sc), responsible for the binding to the second protein in the cascade (the histidine phosphocarrier, HPr), was cloned and successfully expressed and purified. We have previously shown that EI(sc) binds to HPr(sc) with smaller affinity than other members of the EI and HPr families [Hurtado-Gómez et al. (2008) Biophys. J. 95, 1336-1348]. We think that the study of the isolated binding HPr(sc) domain, that is EIN(sc), could shed light on the small affinity value measured. Therefore, in this work we present a detailed description of the structural features of the EIN domain, as a first step towards a complete characterization of the molecular recognition process between the two proteins. We show that EIN(sc) is a folded protein, with alpha-helix and beta-sheet structures and also random-coil conformations, as shown by circular dichroism (CD), FTIR and NMR spectroscopies. The acquisition of secondary and tertiary structures, and the burial of hydrophobic regions, occurred concomitantly at acidic pHs, but at very low pH, the domain acquired a molten-globule conformation. The EIN(sc) protein was not very stable, with an apparent conformational free energy change upon unfolding, DeltaG, of 4.1 +/- 0.4 kcal mol(-1), which was pH independent in the range explored (from pH 6.0 to 8.5). The thermal denaturation midpoint, which was also pH invariant, was similar to that measured in the isolated intact EI(sc). Although EIN(sc) shows thermal- and chemical denaturations that seems to follow a two-state mechanism, there is evidence of residual structure in the chemical and thermally unfolded states, as indicated by differential scanning calorimetry and CD measurements.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe