Counterintuitive structural and functional effects due to naturally occurring mutations targeting the active site of the disease-associated NQO1 enzyme

This work was supported by the ERDF/Spanish Ministry of Science, Innovation and Universities-State Research Agency (Grant number RTI2018-096246-B-I00), Consejeria de Economia, Conocimiento, Empresas y Universidad, Junta de Andalucia (Grant number P18-RT-2413), ERDF/Counselling of Economic transformation, Industry, Knowledge and Universities (Grant B-BIO-84-UGR20), MCIN/AEI/10.13039/501100011033 (Grant number PID2019-103901GB-I00), Government of Aragon-FEDER (Grant number E35_20R). Financial support from Horizon 2020 EU_FT-ICR_MS project (Grant number 731077), EU/MEYS projects BioCeV (CZ.1.05/1.1.00/02.0109) and CIISB (Grant number LM2018127) is gratefully acknowledged. The funding sources had no role in study design, collection, analysis and interpretation of data, writing of the report; and in the decision to submit the article for publication. Funding for open access charge: Universidad de Granada/CBUA.Our knowledge on the genetic diversity of the human genome is exponentially growing. However, our capacity to establish genotype-phenotype correlations on a large scale requires a combination of detailed experimental and computational work. This is a remarkable task in human proteins which are typically multifunctional and structurally complex. In addition, mutations often prevent the determination of mutant high-resolution structures by X-ray crystallography. We have characterized here the effects of five mutations in the active site of the disease-associated NQO1 protein, which are found either in cancer cell lines or in massive exome sequencing analysis in human population. Using a combination of H/D exchange, rapid-flow enzyme kinetics, binding energetics and conformational stability, we show that mutations in both sets may cause counterintuitive functional effects that are explained well by their effects on local stability regarding different functional features. Importantly, mutations predicted to be highly deleterious (even those affecting the same protein residue) may cause mild to catastrophic effects on protein function. These functional effects are not well explained by current predictive bioinformatic tools and evolutionary models that account for site conservation and physicochemical changes upon mutation. Our study also reinforces the notion that naturally occurring mutations not identified as disease-associated can be highly deleterious. Our approach, combining protein biophysics and structural biology tools, is readily accessible to broadly increase our understanding of genotype-phenotype correlations and to improve predictive computational tools aimed at distinguishing disease-prone against neutral missense variants in the human genome.ERDF/Spanish Ministry of Science, Innovation and Universities—State Research Agency RTI2018-096246-B-I00Junta de Andalucía P18-RT-2413ERDF/Counselling of Economic transformation, Industry, Knowledge and Universities B-BIO-84-UGR20MCIN/AEI/10.13039/501100011033 PID2019-103901GB-I00Government of Aragón-FEDER E35_20RHorizon 2020 EU_FT-ICR_MS 731077EU/MEYS projects BioCeV CZ.1.05/1.1.00/02.0109CIISB LM2018127Universidad de Granada/CBU

Segregation-induced grain boundary electrical potential in ionic oxide materials: A first principles model

A first principles continuum analytical model for cationic segregation to the grain boundaries in complex ceramic oxides is presented. The model permits one to determine the electric charge density and the segregation-induced electric potential profiles through the grain and can be extrapolated to the range of nanostructured grain sizes. The theoretical predictions are compared with existing data for yttria-stabilized tetragonal zirconia polycrystals. The implications for physical properties (mainly high temperature plasticity and hardening behaviour) are then discussed.Gobierno de España MAT2009-14351-C02-01, MAT2009-14351-C02-0

Effect of naturally-occurring mutations on the stability and function of cancer-associated NQO1: Comparison of experiments and computation

Recent advances in DNA sequencing technologies are revealing a large individual variability of the human genome. Our capacity to establish genotype-phenotype correlations in such large-scale is, however, limited. This task is particularly challenging due to the multifunctional nature of many proteins. Here we describe an extensive analysis of the stability and function of naturally-occurring variants (found in the COSMIC and gnomAD databases) of the cancer-associated human NAD(P)H:quinone oxidoreductase 1 (NQO1). First, we performed in silico saturation mutagenesis studies (>5,000 substitutions) aimed to identify regions in NQO1 important for stability and function. We then experimentally characterized twenty-two naturally-occurring variants in terms of protein levels during bacterial expression, solubility, thermal stability, and coenzyme binding. These studies showed a good overall correlation between experimental analysis and computational predictions; also the magnitude of the effects of the substitutions are similarly distributed in variants from the COSMIC and gnomAD databases. Outliers in these experimental-computational genotype-phenotype correlations remain, and we discuss these on the grounds and limitations of our approaches. Our work represents a further step to characterize the mutational landscape of NQO1 in the human genome and may help to improve high-throughput in silico tools for genotype-phenotype correlations in this multifunctional protein associated with disease.ERDF/Spanish Ministry of Science, Innovation and Universities-State Research AgencyJunta de Andalucia RTI 2018-096246-B-I00ERDF/Counseling of Economic transformation, Industry, Knowledge and Universities P18-RT-2413Comunidad Valenciana B-BIO-84-UGR20Novo Nordisk FoundationNovocure Limited CIAICO/2021/135 NNF18OC003395

A single evolutionarily divergent mutation determines the different FAD-binding affinities of human and rat NQO1 due to site-specific phosphorylation

ALP thanks Professors Jose Manuel Sanchez-Ruiz and Beatriz Ibarra-Molero (both from the University of Granada) for providing access and advice on their home-built software for electrostatic calculations. BR acknowledges kind hospitality and use of computational resources in the European Magnetic Resonance Center (CERM), Sesto Fiorentino (Florence), Italy. This work was supported by Spanish Ministry of Economy and Competitiveness and European ERDF Funds (MCIU/AEI/FEDER, EU) [RTI2018-097991-BI00 to JLN and RTI2018-096246-B-I00 to ALP]; FEDER/Junta de Andalucia-Consejeria de Transformacion Economica, Industria, Conocimiento y Universidades [Grant P18-RT-2413 to ALP]. Financial support from EU Horizon 2020 project EU FT-ICR MS (731077) as well as institutional (CZ.1.05/1.1.00/02.0109) and MS facility support (LM2018127 CIISB) are gratefully acknowledged. Funding for open charge: Universidad de Granada/CBUA.The phosphomimetic mutation S82D in the cancer-associated, FADdependent human NADP(H):quinone oxidoreductase 1 (hNQO1) causes a decrease in flavin-adenine dinucleotide-binding affinity and intracellular stability. We test in this work whether the evolutionarily recent neutral mutation R80H in the vicinity of S82 may alter the strong functional effects of S82 phosphorylation through electrostatic interactions. We show using biophysical and bioinformatic analyses that the reverse mutation H80R prevents the effects of S82D phosphorylation on hNQO1 by modulating the local stability. Consistently, in rat NQO1 (rNQO1) which contains R80, the effects of phosphorylation were milder, resembling the behaviour found in hNQO1 when this residue was humanized in rNQO1 (by the R80H mutation). Thus, apparently neutral and evolutionarily divergent mutations may determine the functional response of mammalian orthologues towards phosphorylation.Spanish GovernmentEuropean ERDF Funds (MCIU/AEI/FEDER, EU) RTI2018-097991-BI00 RTI2018-096246-B-I00FEDER/Junta de Andalucia-Consejeria de Transformacion Economica, Industria, Conocimiento y Universidades P18-RT-2413EU Horizon 2020 project EU FT-ICR MS 731077Universidad de Granada/CBUA CZ.1.05/1.1.00/02.0109 LM2018127 CIIS

Loss of stability and unfolding cooperativity in hPGK1 upon gradual structural perturbation of its N‑terminal domain hydrophobic core

Phosphoglycerate kinase has been a model for the stability, folding cooperativity and catalysis of a two-domain protein. The human isoform 1 (hPGK1) is associated with cancer development and rare genetic diseases that affect several of its features. To investigate how mutations affect hPGK1 folding landscape and interaction networks, we have introduced mutations at a buried site in the N-terminal domain (F25 mutants) that either created cavities (F25L, F25V, F25A), enhanced conformational entropy (F25G) or introduced structural strain (F25W) and evaluated their effects using biophysical experimental and theoretical methods. All F25 mutants folded well, but showed reduced unfolding cooperativity, kinetic stability and altered activation energetics according to the results from thermal and chemical denaturation analyses. These alterations correlated well with the structural perturbation caused by mutations in the N-terminal domain and the destabilization caused in the interdomain interface as revealed by H/D exchange under native conditions. Importantly, experimental and theoretical analyses showed that these effects are significant even when the perturbation is mild and local. Our approach will be useful to establish the molecular basis of hPGK1 genotype–phenotype correlations due to phosphorylation events and single amino acid substitutions associated with disease.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-UGR20Department of Science & Technology (India)Science Engineering Research Board (SERB), India MTR/2019/000392Horizon 2020 EU_FT-ICR_MS project 731077EU/MEYS projects BioCeV CZ.1.05/1.1.00/02.0109CIISB LM201812

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

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

Uncovering the adult morphology of the forearm bones from the Sima de los Huesos Site in Atapuerca (Spain), with comments on biomechanical features

[EN] The forearm skeleton is composed of two bones: the radius and the ulna. This is closely related to manipulative movements. The ulna is part of the elbow joint, whereas the radius and ulna together with the scaphoid and lunate bones, form the wrist joints. Thus, morphofunctional analysis of the adult Sima de los Huesos (SH) forearm bones, provides clues about manipulative activities in one Pleistocene population. From 1976 to the present, over 7000 human fossils have been recovered from the SH site. The radial sample comprised 98 labeled fragments, of which 49 belonged to adult individuals, representing at least 7 individuals. The ulnar sample included 31 labeled adult fossils representing at least nine individuals. In this study, we describe the SH radii and ulnae and analyze their functional implications for manipulative and forearm movements. We confirmed that the SH radii are long and curved, with variations in robusticity and radial tuberosity orientation. The SH ulnae are characterized by an anteriorly oriented trochlear notch, a massive olecranon process, an obliquely oriented radial notch, a blunt and short supinator crest, a gracile and curved diaphysis, and a round and anteriorly oriented pronator crests. In general, they exhibit Neanderthal morphology. The SH collection provides a unique opportunity to conduct morphological analyses of these bones in the Middle Pleistocene population.SIPublicación en abierto financiada por el Consorcio de Bibliotecas Universitarias de Castilla y León (BUCLE), con cargo al Programa Operativo 2014ES16RFOP009 FEDER 2014-2020 DE CASTILLA Y LEÓN, Actuación:20007-CL - Apoyo Consorcio BUCL

The use of IRT to investigate the relationships between the g factor and cognitive tasks

Las relaciones entre el factor g y tareas de procesamiento de la información han abierto la puerta a la comprensión de la naturaleza de g ( Jensen, 1998). No obstante, es necesario superar determinados problemas metodológicos para poder comprender hasta qué punto las diferencias en g son ocasionadas por diferencias en procesos cognitivos. En este sentido, la falta de fiabilidad de las puntuaciones en las tareas de procesos puede llevar a conclusiones equivocadas sobre la naturaleza de dichas relaciones. Un medio de abordar este problema es seleccionar a las personas con un menor error de estimación del nivel de habilidad. Los resultados muestran un aumento de las correlaciones entre g y memoria de trabajo en el grupo de sujetos mejor medidos frente al grupo de personas con un error de estimación mayorMany authors have looked for the relations between the g factor and cognitive tasks in order to find out the nurture of the g factor (Jensen, 1998). Nevertheless, some methodological problems must be solved to understand if the individual differences in g are differences in cognitive processes. One of them is the low reliability of the scores in this kind of tasks. On the grounds of the standard error of the ability level estimation, we hypothesized that the correlations between the g factor and a working memory task would be larger in the g roup of the subjects better measured (less standard error). The results support this hypothesi