Allosteric Communication in the Multifunctional and Redox NQO1 Protein Studied by Cavity-Making Mutations

Allosterism is a common phenomenon in protein biochemistry that allows rapid regulation of protein stability; dynamics and function. However, the mechanisms by which allosterism occurs (by mutations or post-translational modifications (PTMs)) may be complex, particularly due to long-range propagation of the perturbation across protein structures. In this work, we have investigated allosteric communication in the multifunctional, cancer-related and antioxidant protein NQO1 by mutating several fully buried leucine residues (L7, L10 and L30) to smaller residues (V, A and G) at sites in the N-terminal domain. In almost all cases, mutated residues were not close to the FAD or the active site. Mutations L -> G strongly compromised conformational stability and solubility, and L30A and L30V also notably decreased solubility. The mutation L10A, closer to the FAD binding site, severely decreased FAD binding affinity (approximate to 20 fold vs. WT) through long-range and context-dependent effects. Using a combination of experimental and computational analyses, we show that most of the effects are found in the apo state of the protein, in contrast to other common polymorphisms and PTMs previously characterized in NQO1. The integrated study presented here is a first step towards a detailed structural-functional mapping of the mutational landscape of NQO1, a multifunctional and redox signaling protein of high biomedical relevance.ERDF/Spanish Ministry of Science, Innovation and Universities-State Research Agency RTI2018-096246-B-I00Junta de Andalucia P18-RT-2413ERDF/Counseling of Economic transformation, Industry, Knowledge and Universities B-BIO-84-UGR20Government of Aragon-FEDER E35_20RDepartment of Science & Technology (India)Science Engineering Research Board (SERB), India MTR/2019/000392Horizon 2020 EPIC-XS project 82383EU/MEYS project BioCeV CZ.1.05/1.1.00/02.0109ERDF/Counseling of Economic transformation, Industry, Knowledge and Universities, Junta de Andalucia B-BIO-84-UGR20EU/MEYS project CIISB LM2018127MCIN/AEI PID2019-103901GB-I0

Different phenotypic outcome due to site-specific phosphorylation in the cancer-associated NQO1 enzyme studied by phosphomimetic mutations

Protein phosphorylation is a common phenomenon in human flavoproteins although the functional consequences of this site-specific modification are largely unknown. Here, we evaluated the effects of site-specific phosphorylation (using phosphomimetic mutations at sites S40, S82 and T128) on multiple functional aspects as well as in the structural stability of the antioxidant and disease-associated human flavoprotein NQO1 using biophysical and biochemical methods. In vitro biophysical studies revealed effects of phosphorylation at different sites such as decreased binding affinity for FAD and structural stability of its binding site (S82), conformational stability (S40 and S82) and reduced catalytic efficiency and functional cooperativity (T128). Local stability measurements by H/D exchange in different ligation states provided structural insight into these effects. Transfection of eukaryotic cells showed that phosphorylation at sites S40 and S82 may reduce steady-levels of NQO1 protein by enhanced proteasome-induced degradation. We show that site-specific phosphorylation of human NQO1 may cause pleiotropic and counterintuitive effects on this multifunctional protein with potential implications for its relationships with human disease. Our approach allows to establish relationships between site-specific phosphorylation, functional and structural stability effects in vitro and inside cells paving the way for more detailed analyses of phosphorylation at the flavoproteome scaleDepartamento de Química-Fisica. Financiación: ERDF/Spanish Ministry of Science, Innovation and Universities—State Research Agency (Grant RTI2018-096246-B-I00), Consejería de Economía, Conocimiento, Empresas y Universidad, Junta de Andalucía (Grant P18-RT-2413) and ERDF/Counseling of Economic transformation, Industry, Knowledge and Universities, Junta de Andalucía (Grant B-BIO-84-UGR20), MCIN/AEI/10.13039/501100011033 (Grant PID2019-103901 GB-I00), Government of Aragon-FEDER ´ (Grant E35_20R

Fish oil supplementation reverses the effect of cholesterol on apoptotic gene expression in smooth muscle cells

<p>Abstract</p> <p>Background</p> <p>Nutritional control of gene regulation guides the transformation of smooth muscle cells (SMC) into foam cells in atherosclerosis. Oxidative stress has been reported in areas of lipid accumulation, activating proliferation genes. Suppression of oxidative stress by antioxidant administration reduces this activation and the progression of lesions. We hypothesized that fish oil consumption may protect against atherosclerotic vascular disease. The study objective was to determine the effects of dietary cholesterol and fish-oil intake on the apoptotic pathways induced by 25-hydroxycholesterol (25-HC) in SMC cultures.</p> <p>Methods</p> <p>An <it>in vivo/in vitro </it>cell model was used, culturing SMC isolated from chicks exposed to an atherogenic cholesterol-rich diet with 5% of cholesterol (SMC-Ch) alone or followed by an anti-atherogenic fish oil-rich diet with 10% of menhaden oil (SMC-Ch-FO) and from chicks on standard diet (SMC-C). Cells were exposed to 25-HC, studying apoptosis levels by flow cytometry (Annexin V) and expressions of caspase-3, c-myc, and p53 genes by quantitative real-time reverse transcriptase-polymerase chain reaction. Results: Exposure to 25-HC produced apoptosis in all three SMC cultures, which was mediated by increases in caspase-3, c-myc, and p53 gene expression. Changes were more marked in SMC-Ch than in SMC-C, indicating that dietary cholesterol makes SMC more susceptible to 25-HC-mediated apoptosis. Expression of p53 gene was elevated in SMC-Ch-FO. This supports the proposition that endogenous levels of p53 protect SMC against apoptosis and possibly against the development of atherosclerosis. Fish oil attenuated the increase in c-myc levels observed in SMC-C and SMC-Ch, possibly through its influence on the expression of antioxidant genes.</p> <p>Conclusion</p> <p>Replacement of a cholesterol-rich diet with a fish oil-rich diet produces some reversal of the cholesterol-induced changes, increasing the resistance of SMC to apoptosis.</p

Genetic Landscape of Nonobstructive Azoospermia and New Perspectives for the Clinic

We thank Alejandro Fernández Sevilla for his valuable help in the development of Figure 2 of this review. The authors were funded by the Spanish Ministry of Economy and Competitiveness through the Spanish National Plan for Scientific and Technical Research and Innovation (ref. SAF2016-78722-R) and the “Ramón y Cajal” program (ref. RYC-2014-16458).Nonobstructive azoospermia (NOA) represents the most severe expression of male infertility, involving around 1% of the male population and 10% of infertile men. This condition is characterised by the inability of the testis to produce sperm cells, and it is considered to have an important genetic component. During the last two decades, di erent genetic anomalies, including microdeletions of the Y chromosome, karyotype defects, and missense mutations in genes involved in the reproductive function, have been described as the primary cause of NOA in many infertile men. However, these alterations only explain around 25% of azoospermic cases, with the remaining patients showing an idiopathic origin. Recent studies clearly suggest that the so-called idiopathic NOA has a complex aetiology with a polygenic inheritance, which may alter the spermatogenic process. Although we are far from a complete understanding of the molecular mechanisms underlying NOA, the use of the new technologies for genetic analysis has enabled a considerable increase in knowledge during the last years. In this review, we will provide a comprehensive and updated overview of the genetic basis of NOA, with a special focus on the possible application of the recent insights in clinical practice.Funded by the Spanish Ministry of Economy and Competitiveness through the Spanish National Plan for Scientific and Technical Research and Innovation (ref. SAF2016-78722-R) and the “Ramón y Cajal” program (ref. RYC-2014-16458)

Effect of Oxysterol-Induced Apoptosis of Vascular Smooth Muscle Cells on Experimental Hypercholesterolemia

Smooth muscle cells (SMCs) undergo changes related to proliferation and apoptosis in the physiological remodeling of vessels and in diseases such as atherosclerosis and restenosis. Recent studies also have demonstrated the vascular cell proliferation and programmed cell death contribute to changes in vascular architecture in normal development and in disease. The present study was designed to investigate the apoptotic pathways induced by 25-hydroxycholesterol in SMCs cultures, using an in vivo/in vitro cell model in which SMCs were isolated and culture from chicken exposed to an atherogenic cholesterol-rich diet (SMC-Ch) and/or an antiatherogenic fish oil-rich diet (SMC-Ch-FO). Cells were exposed in vitro to 25-hydroxycholesterol to study levels of apoptosis and apoptotic proteins Bcl-2, Bcl- and Bax and the expression of bcl-2 and bcl- , genes. The quantitative real-time reverse transcriptase-polymerase chain reaction and the Immunoblotting western blot analysis showed that 25-hydroxycholesterol produces apoptosis in SMCs, mediated by a high increase in Bax protein and Bax gene expression. These changes were more marked in SMC-Ch than in SMC-Ch-FO, indicating that dietary cholesterol produces changes in SMCs that make them more susceptible to 25-hydroxycholesterol-mediated apoptosis. Our results suggest that the replacement of a cholesterol-rich diet with a fish oil-rich diet produces some reversal of cholesterol-induced changes in the apoptotic pathways induced by 25-hydroxycholesterol in SMCs cultures, making SMCs more resistant to apoptosis.This work was supported partly by Junta de Andalucía (Group code CTS 168) and also by the research grants from Instituto de Salud Carlos III, ISCIII (Grant no. PI030829)

Sertoli cell-specific ablation of miR-17-92 cluster significantly alters whole testis transcriptome without apparent phenotypic effects

MicroRNAs are frequently organized into polycistronic clusters whose transcription is controlled by a single promoter. The miR-17-92 cluster is expressed in most embryonic and postnatal organs. It is a potent oncogene associated to several types of cancer and it is involved in several important developmental processes. In the testis, expression of the miR- 17-92 cluster in the germ cells is necessary to maintain normal spermatogenesis. This cluster is also expressed in Sertoli cells (the somatic cells of the seminiferous tubules), which require miRNAs for correct cell development and survival. To study the possible role of miR- 17-92 in Sertoli cell development and function and, in order to overcome the postnatal lethality of miR-17-92-/ mice, we conditionally deleted it in embryonic Sertoli cells shortly after the sex determination stage using an Amh-Cre allele. Mutant mice developed apparently normal testes and were fertile, but their testis transcriptomes contained hundreds of moderately deregulated genes, indicating that testis homeostasis is tightly controlled in mammals and that miR-17-92 expression in Sertoli cells contribute to maintain normal gene expression levels, but is unnecessary for testis development and function. Our results show that significant deregulation of hundreds of genes might have no functional consequences.This work was supported by grants from the Andalusian Government, Junta de Andalucía, BIO-109 to R. Jiménez and P11-CVI-7291 to M. Burgos and grants from the Spanish Ministry of Science and Innovation (CGL2011-23368 and CGL2015-67108-P) to R. Jiménez and F.J. Barrionuevo. The authors would like to thank the Spanish Ministry of Science and Innovation for the 'Ramón y Cajal' fellowship granted to F.D. Carmona (RYC-2014-16458) and the 'FPU' PhD fellowship granted to A. Hurtado

Different phenotypic outcome due to site-specific phosphorylation in the cancer-associated NQO1 enzyme studied by phosphomimetic mutations

Protein phosphorylation is a common phenomenon in human flavoproteins although the functional consequences of this site-specific modification are largely unknown. Here, we evaluated the effects of site-specific phosphorylation (using phosphomimetic mutations at sites S40, S82 and T128) on multiple functional aspects as well as in the structural stability of the antioxidant and disease-associated human flavoprotein NQO1 using biophysical and biochemical methods. In vitro biophysical studies revealed effects of phosphorylation at different sites such as decreased binding affinity for FAD and structural stability of its binding site (S82), conformational stability (S40 and S82) and reduced catalytic efficiency and functional cooperativity (T128). Local stability measurements by H/D exchange in different ligation states provided structural insight into these effects. Transfection of eukaryotic cells showed that phosphorylation at sites S40 and S82 may reduce steady-levels of NQO1 protein by enhanced proteasome-induced degradation. We show that site-specific phosphorylation of human NQO1 may cause pleiotropic and counterintuitive effects on this multifunctional protein with potential implications for its relationships with human disease. Our approach allows to establish relationships between site-specific phosphorylation, functional and structural stability effects in vitro and inside cells paving the way for more detailed analyses of phosphorylation at the flavoproteome scale

A1298C polymorphism in the MTHFR gene predisposes to cardiovascular risk in rheumatoid arthritis

8 páginas, 1 figura, 3 tablas.-- et al.[Introduction]: We determined the contribution of the methylene tetrahydrofolate reductase (MTHFR) 677 C>T and 1298 A>C gene polymorphisms to the susceptibility to rheumatoid arthritis (RA). We also assessed whether these two MTHFR gene polymorphisms may be implicated in the development of cardiovascular (CV) events and subclinical atherosclerosis manifested by the presence of endothelial dysfunction, in a series of Spanish patients with RA. [Methods]: Six hundred and twelve patients fulfilling the 1987 American College of Rheumatology classification criteria for RA, seen at the rheumatology outpatient clinics of Hospital Xeral-Calde, Lugo and Hospital San Carlos, Madrid, were studied. Patients and controls (n = 865) were genotyped using predesigned TaqMan SNP genotyping assays. [Results]: No significant differences in allele or genotype frequencies for the MTHFR gene polymorphisms between RA patients and controls were found. Also, no association between the MTHFR 677 C>T polymorphism and CV events or endothelial dysfunction was observed. However, the MTHFR 1298 allele C frequency was increased in patients with CV events after 5 years (38.7% versus 30.3%; odds ratio = 1.45; 95% confidence interval = 1.00 to 2.10; P = 0.04) and 10 years (42.2% versus 31.0%; odds ratio = 1.62; 95% confidence interval = 1.08 to 2.43; P = 0.01) follow up. Moreover, patients carrying the MTHFR 1298 AC and CC genotypes had a significantly decreased flow-mediated endothelium-dependent vasodilatation (4.3 ± 3.9%) compared with those carrying the MTHFR 1298 AA genotype (6.5 ± 4.4%) (P = 0.005). [Conclusions]: Our results show that the MTHFR 1298 A>C gene polymorphism confers an increased risk for subclinical atherosclerosis and CV events in patients with RA.The present study was supported by two grants from Fondo de Investigaciones Sanitarias PI06-0024 and PS09/00748 (Spain). This work was partially supported by RETICS Program RD08/0075 (RIER) from the Instituto de Salud Carlos III.Peer reviewe

Immune and spermatogenesis-related loci are involved in the development of extreme patterns of male infertility

Acknowledgements We thank the National DNA Bank Carlos III (University of Salamanca, Spain) for supplying part of the control DNA samples from Spain and all the participants for their essential collaboration. This work was supported by the Spanish Ministry of Science through the Spanish National Plan for Scientific and Technical Research and Innovation (refs. SAF2016-78722-R and PID2020-120157RB-I00), the Andalusian Plan for Research and Innovation (PAIDI 2020) (ref. PY20_00212), and the R+D+i Projects of the FEDER Operational Programme 2020 (ref. B-CTS-584-UGR20). F.D.C. was supported by the “Ramón y Cajal” programme (ref. RYC-2014-16458), and L.B.C. was supported by the Spanish Ministry of Economy and Competitiveness through the “Juan de la Cierva Incorporación” programme (ref. IJC2018-038026-I, funded by MCIN/AEI /10.13039/ 501100011033), all of them including FEDER funds. A.G.J. was funded by MCIN/AEI /10.13039/501100011033 and FSE “El FSE invierte en tu futuro” (ref. FPU20/02926). IPATIMUP integrates the i3S Research Unit, which is partially supported by the Portuguese Foundation for Science and Technology (FCT), financed by the European Social Funds (COMPETE-FEDER) and National Funds (projects PEstC/SAU/LA0003/2013 and POCI-01-0145-FEDER-007274). A.M.L. is funded by the Portuguese Government through FCT (IF/01262/2014). P.I.M. is supported by the FCT post-doctoral fellowship (SFRH/BPD/120777/2016), financed from the Portuguese State Budget of the Ministry for Science, Technology and High Education and from the European Social Fund, available through the Programa Operacional do Capital Humano. ToxOmics—Centre for Toxicogenomics and Human Health, Genetics, Oncology and Human Toxicology, Nova Medical School, Lisbon, is also partially supported by FCT (Projects: UID/BIM/00009/ 2013 and UIDB/UIDP/00009/2020). SLarriba received support from “Instituto de Salud Carlos III” (grant DTS18/00101], co-funded by FEDER funds/European Regional Development Fund (ERDF)—a way to build Europe), and from “Generalitat de Catalunya” (grant 2017SGR191). SLarriba is sponsored by the “Researchers Consolidation Programme” from the SNS-Departament de Salut Generalitat de Catalunya (Exp. CES09/ 020). The German cohort was recruited within the Male Reproductive Genomics (MERGE) study and supported by the German Research Foundation Clinical Research Unit ‘Male Germ Cells’ (DFG CRU326, grants to F.T. and J.G.). This article is related to the Ph.D. Doctoral Thesis of Miriam Cerván-Martín (grant ref. BES-2017-081222 funded by MCIN/AEI/10.13039/501100011033 and FSE “El FSE invierte en tu futuro”).We conducted a genome-wide association study in a large population of infertile men due to unexplained spermatogenic failure (SPGF). More than seven million genetic variants were analysed in 1,274 SPGF cases and 1,951 unaffected controls from two independent European cohorts. Two genomic regions were associated with the most severe histological pattern of SPGF, defined by Sertoli cell-only (SCO) phenotype, namely the MHC class II gene HLA-DRB1 (rs1136759, P = 1.32E-08, OR = 1.80) and an upstream locus of VRK1 (rs115054029, P = 4.24E-08, OR = 3.14), which encodes a protein kinase involved in the regulation of spermatogenesis. The SCO-associated rs1136759 allele (G) determines a serine in the position 13 of the HLA-DRβ1 molecule located in the antigen-binding pocket. Overall, our data support the notion of unexplained SPGF as a complex trait influenced by common variation in the genome, with the SCO phenotype likely representing an immune-mediated condition.Andalusian Plan for Research and InnovationJuan de la Cierva Incorporación IJC2018-038026-IMinistry for Science, Technology and High EducationBES-2017-081222, MCIN/AEI/10.13039/501100011033National Funds IF/01262/2014, PEstC/SAU/LA0003/2013, POCI-01-0145-FEDER-007274, SFRH/BPD/120777/2016PAIDI 2020 PY20_00212R+D+i Projects B-CTS-584-UGR20, RYC-2014-16458Faculty of Science and Engineering, University of Manchester FPU20/02926Deutsche Forschungsgemeinschaft DFG CRU326Fundação para a Ciência e a TecnologiaGeneralitat de Catalunya 2017SGR191Ministerio de Economía y CompetitividadInstituto de Salud Carlos III DTS18/00101Ministerio de Ciencia e InnovaciónEuropean Social Fund UIDB/UIDP/00009/2020European Regional Development FundFundació Catalana de TrasplantamentDepartament de Salut, Generalitat de Catalunya CES09/020Programa Operacional Temático Factores de CompetitividadeSpanish National Plan for Scientific and Technical Research and Innovation PID2020-120157RB-I00, SAF2016-78722-