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14 pags, 7 figs, 3 tabs ; Enzymes known as bifunctional and bimodular prokaryotic type-I FAD synthetase (FADS) exhibit ATP:riboflavin kinase (RFK) and FMN:ATP adenylyltransferase (FMNAT) activities in their C-Terminal and N-Terminal modules, respectively, and produce flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). These act as cofactors of a plethora of flavoproteins in all organisms. Therefore, regulation of their production maintains the cellular flavoproteome homeostasis. Here, we focus on regulation of the FMN synthesis in Corynebacterium ammoniagenes (Ca) by the inhibition of its RFK activity by substrates and products of the reaction. We use a truncated CaFADS variant consisting in the isolated C-Terminal RFK module, whose RFK activity is similar to that of the full-length enzyme. Inhibition of the RFK activity by the RF substrate is independent of the FMNAT module, and FMN production, in addition to being inhibited by an excess of RF, is also inhibited by both of the reaction products. Pre-steady-state kinetic and thermodynamic studies reveal key aspects to the substrates induced fit to produce the catalytically competent complex. Among them, the role of Mg2+ in the concerted allocation of substrates for catalysis and the ensemble of non-competent complexes that contribute to the regulated inhibition of the RFK activity are particularly relevant. ; This work has been supported by the Spanish Ministry of Economy, Industry and Competitiveness (MINECO) [BIO2013-42978-P and BIO2016-75183-P AEI/FEDER, UE to M.M.], and the Government of Aragon-FEDER [B18]. ; Peer reviewed

18 pags., 8 figs., 6 tabs. -- This article belongs to the Special Issue The Amazing World of IDPs in Human Diseases II ; Intrinsic disorder plays an important functional role in proteins. Disordered regions are linked to posttranslational modifications, conformational switching, extra/intracellular trafficking, and allosteric control, among other phenomena. Disorder provides proteins with enhanced plasticity, resulting in a dynamic protein conformational/functional landscape, with well-structured and disordered regions displaying reciprocal, interdependent features. Although lacking well-defined conformation, disordered regions may affect the intrinsic stability and functional properties of ordered regions. MeCP2, methyl-CpG binding protein 2, is a multifunctional transcriptional regulator associated with neuronal development and maturation. MeCP2 multidomain structure makes it a prototype for multidomain, multifunctional, intrinsically disordered proteins (IDP). The methyl-binding domain (MBD) is one of the key domains in MeCP2, responsible for DNA recognition. It has been reported previously that the two disordered domains flanking MBD, the N-terminal domain (NTD) and the intervening domain (ID), increase the intrinsic stability of MBD against thermal denaturation. In order to prove unequivocally this stabilization effect, ruling out any artifactual result from monitoring the unfolding MBD with a local fluorescence probe (the single tryptophan in MBD) or from driving the protein unfolding by temperature, we have studied the MBD stability by differential scanning calorimetry (reporting on the global unfolding process) and chemical denaturation (altering intramolecular interactions by a different mechanism compared to thermal denaturation). ; This research was funded by the Spanish Ministry of Economy and Competitiveness and European ERDF Funds (MCIU/AEI/FEDER, EU) (BFU2016-78232-P to A.V.C.; BES-2017-080739 to D.O.A.); Miguel Servet Program from Instituto de Salud Carlos III (CPII13/00017 to O.A.); Fondo de Investigaciones Sanitarias from Instituto de Salud Carlos III and European Union (ERDF/ESF, "Investing in your future") (PI15/00663 and PI18/00349 to O.A.); Diputación General de Aragón (Protein Targets and Bioactive Compounds Group E45_20R to A.V.C. and Digestive Pathology Group B25_20R to O.A.); and the Centro de Investigación Biomédica en Red en Enfermedades Hepáticas y Digestivas (CIBERehd).

19 pags., 8 figs., 4 tabs. -- This article belongs to the Special Issue The Amazing World of IDPs in Human Diseases ; Methyl-CpG binding protein 2 (MeCP2) is a transcriptional regulator and a chromatin-binding protein involved in neuronal development and maturation. Loss-of-function mutations in MeCP2 result in Rett syndrome (RTT), a neurodevelopmental disorder that is the main cause of mental retardation in females. MeCP2 is an intrinsically disordered protein (IDP) constituted by six domains. Two domains are the main responsible elements for DNA binding (methyl-CpG binding domain, MBD) and recruitment of gene transcription/silencing machinery (transcription repressor domain, TRD). These two domains concentrate most of the RTT-associated mutations. R106W and R133C are associated with severe and mild RTT phenotype, respectively. We have performed a comprehensive characterization of the structural and functional impact of these substitutions at molecular level. Because we have previously shown that the MBD-flanking disordered domains (N-terminal domain, NTD, and intervening domain, ID) exert a considerable influence on the structural and functional features of the MBD (Claveria-Gimeno, R. et al. Sci Rep. 2017, 7, 41635), here we report the biophysical study of the influence of the protein scaffold on the structural and functional effect induced by these two RTT-associated mutations. These results represent an example of how a given mutation may show different effects (sometimes opposing effects) depending on the molecular context. ; This research was funded by the Spanish Ministry of Economy and Competitiveness and European ERDF Funds (MCIU/AEI/FEDER, EU) (BFU2016-78232-P to A.V.C.; BES-2017-080739 to D.O.A.); Miguel Servet Program from Instituto de Salud Carlos III (CPII13/00017 to O.A.); Fondo de Investigaciones Sanitarias from Instituto de Salud Carlos III and European Union (ERDF/ESF, "Investing in your future") (PI15/00663 and PI18/00349 to O.A.); Diputación General de Aragón (Protein Targets and Bioactive Compounds Group E45_17R to A.V.C. and Digestive Pathology Group B25_17R to O.A.); and the Centro de Investigación Biomédica en Red en Enfermedades Hepáticas y Digestivas (CIBERehd). ; Peer reviewed

22 pags., 4 figs., 2 tabs. -- This article belongs to the Special Issue The Amazing World of IDPs in Human Diseases ; Several carrier proteins are involved in protein transport from the cytoplasm to the nucleus in eukaryotic cells. One of those is importin α, of which there are several human isoforms; among them, importin α3 (Impα3) has a high flexibility. The protein NUPR1, a nuclear protein involved in the cell-stress response and cell cycle regulation, is an intrinsically disordered protein (IDP) that has a nuclear localization sequence (NLS) to allow for nuclear translocation. NUPR1 does localize through the whole cell. In this work, we studied the affinity of the isolated wild-type NLS region (residues 54–74) of NUPR1 towards Impα3 and several mutants of the NLS region by using several biophysical techniques and molecular docking approaches. The NLS region of NUPR1 interacted with Impα3, opening the way to model the nuclear translocation of disordered proteins. All the isolated NLS peptides were disordered. They bound to Impα3 with low micromolar affinity (1.7–27 μM). Binding was hampered by removal of either Lys65 or Lys69 residues, indicating that positive charges were important; furthermore, binding decreased when Thr68 was phosphorylated. The peptide phosphorylated at Thr68, as well as four phospho-mimetic peptides (all containing the Thr68Glu mutation), showed the presence of a sequential NN(i,i + 1) nuclear Overhauser effect (NOE) in the 2D-1H-NMR (two-dimensional–proton NMR) spectra, indicating the presence of turn-like conformations. Thus, the phosphorylation of Thr68 modulates the binding of NUPR1 to Impα3 by a conformational, entropy-driven switch from a random-coil conformation to a turn-like structure. ; This research was funded by the Spanish Ministry of Economy and Competitiveness and European ERDF Funds (MCIU/AEI/FEDER, EU) (RTI2018-097991-B-I00 to J.L.N. and BFU2016-78232-P to A.V.C.); La Ligue Contre le Cancer, INCa, Canceropole PACA, and INSERM to J.L.I.; Miguel Servet Program from Instituto de Salud Carlos III (CPII13/00017 to OA); Fondo de Investigaciones Sanitarias from Instituto de Salud Carlos III and European Union (ERDF/ESF, "Investing in your future") (PI15/00663 and PI18/00349 to O.A.); Diputación General de Aragón (Protein Targets and Bioactive Compounds Group E45_17R to A.V.C. and Digestive Pathology Group B25_17R to O.A.); and the Centro de Investigación Biomédica en Red en Enfermedades Hepáticas y Digestivas (CIBERehd). The NMR equipment used in this work was funded by the Generalitat Valenciana and cofinanced with ERDF funds (OP ERDF of Comunitat Valenciana 2014-2020). ; Peer reviewed

20 pags, 5 figs, 7 tabs ; Risk population screening programs are instrumental for advancing cancer management and reducing economic costs of therapeutic interventions and the burden of the disease, as well as increasing the survival rate and improving the quality of life for cancer patients. Lung cancer, with high incidence and mortality rates, is not excluded from this situation. The success of screening programs relies on many factors, with some of them being the appropriate definition of the risk population and the implementation of detection techniques with an optimal discrimination power and strong patient adherence. Liquid biopsy based on serum or plasma detection of circulating tumor cells or DNA/RNA is increasingly employed nowadays, but certain limitations constrain its wide application. In this work, we present a new implementation of thermal liquid biopsy (TLB) for lung cancer patients. TLB provides a prediction score based on the ability to detect plasma/serum proteome alterations through calorimetric thermograms that strongly correlates with the presence of lung cancer disease (91% accuracy rate, 90% sensitivity, 92% specificity, diagnostic odds ratio 104). TLB is a quick, minimally-invasive, low-risk technique that can be applied in clinical practice for evidencing lung cancer, and it can be used in screening and monitoring actions. ; This research was funded by Miguel Servet Program from Instituto de Salud Carlos III (CPII13/00017 to OA); Fondo de Investigaciones Sanitarias from Instituto de Salud Carlos III, and European Union (ERDF/ESF, 'Investing in your future') (PI15/00663 and PI18/00349 to OA, and PI17/01109 to AL); Spanish Ministry of Economy and Competitiveness (BFU2016-78232-P to AVC); Diputación General de Aragón (Protein Targets and Bioactive Compounds Group – E45_17R to AVC, and Digestive Pathology Group – E25_17R to OA and AL); and Centro de Investigación Biomédica en Red en Enfermedades Hepáticas y Digestivas (CIBERehd). ; Peer reviewed

13 p.-5 fig.-3 tab. ; Bifunctional FAD synthetases (FADSs) fold in two independent modules; The C-terminal riboflavin kinase (RFK) catalyzes the RFK activity, while the N-terminal FMN-adenylyltransferase (FMNAT) exhibits the FMNAT activity. The search for macromolecular interfaces in the Corynebacterium ammoniagenes FADS (CaFADS) crystal structure predicts a dimer of trimers organization. Within each trimer, a head-to-tail arrangement causes the RFK and FMNAT catalytic sites of the two neighboring protomers to approach, in agreement with active site residues of one module influencing the activity at the other. We analyze the relevance of the CaFADS head-to-tail macromolecular interfaces to stabilization of assemblies, catalysis and ligand binding. With this aim, we evaluate the effect of point mutations in loop L1c-FlapI, loop L6c, and helix α1c of the RFK module (positions K202, E203,F206, D298, V300, E301 and L304), regions at the macromolecular interface between two protomers within the trimer. Although none of the studied residues is critical in the formation and dissociation of assemblies, residues at L1c-FlapI and helix α1c particularly modulate quaternary architecture, as well as ligand binding and kinetic parameters involved with RFK and FMNAT activities. These data support the influence of transient oligomeric structures on substrate accommodation and catalysis at both CaFADS active sites. ; This work has been supported by MINECO, Spain (BIO2013-42978-P and BIO2016-75183-P AEI/FEDER, UE to M.M.) and the Government of Aragon DGA-FEDER (B-18). ; Peer reviewed

3 pags, 1 fig ; Intrinsically disordered proteins (IDPs) do not have a well-defined structure, but they have key biological tasks in cancer development. By using the disordered cancer-related protein NUPR1 as a proof-of-concept, we have developed a new multidisciplinary approach to tackle drug-design against IDPs, using it to repurpose drugs for treating pancreatic adenocarcinoma (PDAC). ; This work in the authors' laboratories was supported by [La Ligue Contre le Cancer], INCa, [Canceropole PACA] and [INSERM] to JLI and LP; [Miguel Servet Program from Instituto de Salud Carlos III] under grant [CPII13/00017] to OA; Fondo de Investigaciones Sanitarias from Instituto de Salud Carlos III, and European Union (ERDF/ESF, 'Investing in your future') under grants [PI15/00663 and PI18/00349] to OA; Ministerio de Economia, Industria y Competitividad, Gobierno de Espana under grants [BFU2016-78232-P to AVC, CTQ2015-64445-R to JLN]; [Diputacion General de Aragon] under grants [Protein Targets and Bioactive Compound Group to AVC, and Digestive Pathology Group to OA]; [Centro de Investigacion Biomedica en Red en Enfermedades Hepaticas y Digestivas] (CIBERehd) toOAand AVC; [Programme XUGUANGQI] to YX and JLI; and [National Natural Science Foundation of China (81502920), the Fundamental Research Funds for the Central Universities] under grant [106112017CDJQJ468823] to YX. The Fundacion Alfonso Martin-Escudero and Fondation de France supported to PSC. ; Peer reviewed

Inhibiting the main protease 3CLpro is the most common strategy in the search for antiviral drugs to fight the infection from SARS-CoV-2. We report that the natural compound eugenol is able to hamper in vitro the enzymatic activity of 3CLpro, the SARS-CoV-2 main protease, with an inhibition constant in the sub-micromolar range (Ki = 0.81 μM). Two phenylpropene analogs were also tested: the same effect was observed for estragole with a lower potency (Ki = 4.1 μM), whereas anethole was less active. The binding efficiency index of these compounds is remarkably favorable due also to their small molecular mass (MW < 165 Da). We envision that nanomolar inhibition of 3CLpro is widely accessible within the chemical space of simple natural compounds ; This work was supported by Fundación hna (grant to A.V.-C. and O.A.); Miguel Servet Program from Instituto de Salud Carlos III (CPII13/00017 to O.A.); Fondo de Investigaciones Sanitarias from Instituto de Salud Carlos III, and European Union (ERDF/ESF, 'Investing in your future') (PI18/00349 to O.A. and a FIS Research Contract to L.C.-L.); Spanish Ministry of Economy and Competitiveness (BFU2016-78232-P to A.V.-C.); Spanish Ministry of Science, Innovation and Universities (FPI Predoctoral Research Contract BES-2017-080739 to D.O.-A.); Diputación General de Aragón (Predoctoral Research Contract 2019 to A.J.-A., 'Protein Targets and Bioactive Compounds Group' E45_20R to A.V.-C., 'Digestive Pathology Group' B25_20R to O.A.); Centro de Investigación Biomédica en Red en Enfermedades Hepáticas y Digestivas (CIBERehd). ; Peer reviewed

Intrinsically disordered proteins (IDPs) are emerging as attractive drug targets by virtue of their physiological ubiquity and their prevalence in various diseases, including cancer. NUPR1 is an IDP that localizes throughout the whole cell, and is involved in the development and progression of several tumors. We have previously repurposed trifluoperazine (TFP) as a drug targeting NUPR1 and, by using a ligand-based approach, designed the drug ZZW-115 starting from the TFP scaffold. Such derivative compound hinders the development of pancreatic ductal adenocarcinoma (PDAC) in mice, by hampering nuclear translocation of NUPR1. Aiming to further improve the activity of ZZW-115, here we have used an indirect drug design approach to modify its chemical features, by changing the substituent attached to the piperazine ring. As a result, we have synthesized a series of compounds based on the same chemical scaffold. Isothermal titration calorimetry (ITC) showed that, with the exception of the compound preserving the same chemical moiety at the end of the alkyl chain as ZZW-115, an increase of the length by a single methylene group (i.e., ethyl to propyl) significantly decreased the affinity towards NUPR1 measured in vitro, whereas maintaining the same length of the alkyl chain and adding heterocycles favored the binding affinity. However, small improvements of the compound affinity towards NUPR1, as measured by ITC, did not result in a corresponding improvement in their inhibitory properties and in cellulo functions, as proved by measuring three different biological effects: hindrance of the nuclear translocation of the protein, sensitization of cells against DNA damage mediated by NUPR1, and prevention of cancer cell growth. Our findings suggest that a delicate compromise between favoring ligand affinity and controlling protein function may be required to successfully design drugs against NUPR1, and likely other IDPs. ; This work was supported by Spanish Ministry of Economy and Competitiveness and European ERDF Funds (MCIU/AEI/FEDER, EU) [RTI2018-097991-B-I00 to J.L.N.]; La Ligue Contre le Cancer, INCa, Canceropole PACA and INSERM to JLI; Fondo de Investigaciones Sanitarias from Instituto de Salud Carlos III, and European Union (ERDF/ESF, 'Investing in your future') (PI18/00349 to O.A.); Diputacion General de Aragon ('Protein Targets and Bioactive Compounds Group' E45_20R to A.V.-C., 'Digestive Pathology Group' B25_20R to O.A.); Centro de Investigacion Biomedica en Red en Enfermedades Hepaticas y Digestivas (CIBERehd) (O.A. and A.V.-C.); CAI YUANPEI Scholarship (201906050187) to Y.X.; and Jeunes Talents France-Chine Program (JTFC) to Y.X. P.S.-C. was supported by Fondation de France; and W.L. by China Scholarship Council. ; Peer reviewed

The pandemic, due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has stimulated the search for antivirals to tackle COVID-19 infection. Molecules with known pharmacokinetics and already approved for human use have been demonstrated or predicted to be suitable to be used either directly or as a base for a scaffold-based drug design. Among these substances, quercetin is known to be a potent in vitro inhibitor of 3CLpro, the SARS-CoV-2 main protease. However, its low in vivo bioavailability calls for modifications to its molecular structure. In this work, this issue is addressed by using rutin, a natural flavonoid that is the most common glycosylated conjugate of quercetin, as a model. Combining experimental (spectroscopy and calorimetry) and simulation techniques (docking and molecular dynamics simulations), we demonstrate that the sugar adduct does not hamper rutin binding to 3CLpro, and the conjugated compound preserves a high potency (inhibition constant in the low micromolar range, Ki = 11 μM). Although showing a disruption of the pseudo-symmetry in the chemical structure, a larger steric volume and molecular weight, and a higher solubility compared to quercetin, rutin is able to associate in the active site of 3CLpro, interacting with the catalytic dyad (His41/Cys145). The overall results have implications in the drug-design of quercetin analogs, and possibly other antivirals, to target the catalytic site of the SARS-CoV-2 3CLpro ; This work was supported by Fundación hna (to A.V.C. and O.A.); Miguel Servet Program from Instituto de Salud Carlos III (CPII13/00017 to O.A.); Fondo de Investigaciones Sanitarias from Instituto de Salud Carlos III, and European Union (ERDF/ESF, 'Investing in your future') (PI18/00349 to O.A. and a FIS Research Contract to L.C.-L.); Spanish Ministry of Economy and Competitiveness (BFU2016-78232-P to A.V.-C. and SAF2017-83265-R to H.T.R.); Spanish Ministry of Science, Innovation and Universities (FPI Predoctoral Research Contract BES-2017-080739 to D.O.-A.); the Spanish National Research Council (CSIC, project 202020E079); Diputación General de Aragón (Predoctoral Research Contract 2019 to A.J.-A., 'Protein Targets and Bioactive Compounds Group' E45_20R to A.V.-C., 'Digestive Pathology Group' B25_20R to O.A.); and Centro de Investigación Biomédica en Red en Enfermedades Hepáticas y Digestivas (CIBERehd). ; Peer reviewed

12 páginas, 6 figuras ; Thioredoxin reductases control the redox state of thioredoxins (Trxs)—ubiquitous proteins that regulate a spectrum of enzymes by dithiol–disulfide exchange reactions. In most organisms, Trx is reduced by NADPH via a thioredoxin reductase flavoenzyme (NTR), but in oxygenic photosynthetic organisms, this function can also be performed by an iron-sulfur ferredoxin (Fdx)-dependent thioredoxin reductase (FTR) that links light to metabolic regulation. We have recently found that some cyanobacteria, such as the thylakoid-less Gloeobacter and the ocean-dwelling green oxyphotobacterium Prochlorococcus, lack NTR and FTR but contain a thioredoxin reductase flavoenzyme (formerly tentatively called deeplyrooted thioredoxin reductase or DTR), whose electron donor remained undefined. Here, we demonstrate that Fdx functions in this capacity and report the crystallographic structure of the transient complex between the plant-type Fdx1 and the thioredoxin reductase flavoenzyme from Gloeobacter violaceus. Thereby, our data demonstrate that this cyanobacterial enzyme belongs to the Fdx flavin-thioredoxin reductase (FFTR) family, originally described in the anaerobic bacterium Clostridium pasteurianum. Accordingly, the enzyme hitherto termed DTR is renamed FFTR. Our experiments further show that the redox-sensitive peptide CP12 is modulated in vitro by the FFTR/Trx system, demonstrating that FFTR functionally substitutes for FTR in light-linked enzyme regulation in Gloeobacter. Altogether, we demonstrate the FFTR is spread within the cyanobacteria phylum and propose that, by substituting for FTR, it connects the reduction of target proteins to photosynthesis. Besides, the results indicate that FFTR acquisition constitutes a mechanism of evolutionary adaptation in marine phytoplankton such as Prochlorococcus that live in low-iron environments. ; This work was supported by the Spanish Ministry of Science and Innovation—State Research Agency (MICINN) Grants [PID2019-110900GB-I00 to M.B., PID2019-109671GB-I00 to R.M.B., BFU-2016-78232-P to A.V.-C., and PID2019-103901GB-I00 to M.M.], and the Government of Aragón FEDER [Grupo de Referencia Biología Estructural (E35_20R to M.M., and E45_17R to A.V.-C.)]. D.F.J. was supported by a predoctoral contract from the Junta de Castilla y León. A.G.- L. acknowledges support from CSIC Jae-Intro program. ; Peer reviewed

Programmed cell death (PCD) is crucial for development and homeostasis of all multicellular organisms. In human cells, the double role of extra-mitochondrial cytochrome c in triggering apoptosis and inhibiting survival pathways is well reported. In plants, however, the specific role of cytochrome c upon release from the mitochondria remains in part veiled yet death stimuli do trigger cytochrome c translocation as well. Here, we identify an Arabidopsis thaliana 14-3-3ι isoform as a cytosolic cytochrome c target and inhibitor of caspase-like activity. This finding establishes the 14-3-3ι protein as a relevant factor at the onset of plant HO-induced PCD. The in vivo and in vitro studies herein reported reveal that the interaction between cytochrome c and 14-3-3ι exhibits noticeable similarities with the complex formed by their human orthologues. Further analysis of the heterologous complexes between human and plant cytochrome c with plant 14-3-3ι and human 14-3-3ε isoforms corroborated common features. These results suggest that cytochrome c blocks p14-3-3ι so as to inhibit caspase-like proteases, which in turn promote cell death upon HO treatment. Besides establishing common biochemical features between human and plant PCD, this work sheds light onto the signaling networks of plant cell death. ; Spanish Ministry of Economy and Competitiveness (BFU2015-71017/BMC MINECO/FEDER, PGC2018-096049-B-I00 BIO/BMC MICINN/FEDER, and RTI2018-094793-B-I00 MICIU), the Andalusian Government (BIO198, US-1254317, US-1257019, P18-FR-3487 and P18-HO-4091), and the Ramón Areces Foundation. CAER was recipient of a PhD fellowship from the Fundación Cámara – University of Seville

22 pags., 14 figs., 9 tabs. 1 sch. -- This article belongs to the Special Issue Nanostructures Applied to Drug Delivery and Diagnosis ; (1) Background: Biophysical techniques applied to serum samples characterization could promote the development of new diagnostic tools. Fluorescence spectroscopy has been previously applied to biological samples from cancer patients and differences from healthy individuals were observed. Dendronized hyperbranched polymers (DHP) based on bis(hydroxymethyl)propionic acid (bis-MPA) were developed in our group and their potential biomedical applications explored. (2) Methods: A total of 94 serum samples from diagnosed cancer patients and healthy individuals were studied (20 pancreatic ductal adenocarcinoma, 25 blood donor, 24 ovarian cancer, and 25 benign ovarian cyst samples). (3) Results: Fluorescence spectra of serum samples (fluorescence liquid biopsy, FLB) in the presence and the absence of DHP-bMPA were recorded and two parameters from the signal curves obtained. A secondary parameter, the fluorescence spectrum score (FSscore), was calculated, and the diagnostic model assessed. For pancreatic ductal adenocarcinoma (PDAC) and ovarian cancer, the classification performance was improved when including DHP-bMPA, achieving high values of statistical sensitivity and specificity (over 85% for both pathologies). (4) Conclusions: We have applied FLB as a quick, simple, and minimally invasive promising technique in cancer diagnosis. The classification performance of the diagnostic method was further improved by using DHP-bMPA, which interacted differentially with serum samples from healthy and diseased subjects. These preliminary results set the basis for a larger study and move FLB closer to its clinical application, providing useful information for the oncologist during patient diagnosis. ; This research was funded by the Spanish Ministry of Economy and Competitiveness and European ERDF Funds (MCIU/AEI/FEDER, EU) (BFU2016-78232-P to A.V.C.); Projects funded by Instituto de Salud Carlos III and co-funded by European Union (ESF, "Investing in your future"): "PI15/00663 (FIS project to O.A.)", "PI18/00349 (FIS project to O.A.)", "FI19/00146 (PFIS contract for SHD)"; Gobierno de Aragón-ESF (Predoctoral Research Contract FEDER 2014-2020 "Construyendo Europa desde Aragón") to V.M.-V., Protein Targets and Bioactive Compounds Group E45_17R to A.V.C., Digestive Pathology Group B25_17R to S.H.-D., O.A. and CLIP group E47_20R, to V.M.-V. and T.S.); and the Centro de Investigación Biomédica en Red en Enfermedades Hepáticas y Digestivas (CIBERehd)

AbstractThe Apoptosis-Inducing Factor (AIF) is a moonlighting flavoenzyme involved in the assembly of mitochondrial respiratory complexes in healthy cells, but also able to trigger DNA cleavage and parthanatos. Upon apoptotic-stimuli, AIF redistributes from the mitochondria to the nucleus, where upon association with other proteins such as endonuclease CypA and histone H2AX, it is proposed to organize a DNA–degradosome complex. In this work, we provide evidence for the molecular assembly of this complex as well as for the cooperative effects among its protein components to degrade genomic DNA into large fragments. We have also uncovered that AIF has nuclease activity that is stimulated in the presence of either Mg2+ or Ca2+. Such activity allows AIF by itself and in cooperation with CypA to efficiently degrade genomic DNA. Finally, we have identified TopIB and DEK motifs in AIF as responsible for its nuclease activity. These new findings point, for the first time, to AIF as a nuclease able to digest nuclear dsDNA in dying cells, improving our understanding of its role in promoting apoptosis and opening paths for the development of new therapeutic strategies.

17 pags., 9 figs., 1 tab. ; Repair of injured DNA relies on nucleosome dismantling by histone chaperones and de-phosphorylation events carried out by Protein Phosphatase 2A (PP2A). Typical histone chaperones are the Acidic leucine-rich Nuclear Phosphoprotein 32 family (ANP32) members, e.g. ANP32A, which is also a well-known PP2A inhibitor (a.k.a. IPP2A). Here we report the novel interaction between the endogenous family member B—so-called ANP32B—and endogenous cytochrome c in cells undergoing camptothecin-induced DNA damage. Soon after DNA lesions but prior to caspase cascade activation, the hemeprotein translocates to the nucleus to target the Low Complexity Acidic Region (LCAR) of ANP32B; in a similar way, our group recently reported that the hemeprotein targets the acidic domain of SET/Template Activating Factor-Iβ (SET/TAF-Iβ), which is another histone chaperone and PP2A inhibitor (a.k.a. IPP2A). The nucleosome assembly activity of ANP32B is indeed unaffected by cytochrome c binding. Like ANP32A, ANP32B inhibits PP2A activity and is thus herein referred to as IPP2A. Our data demonstrates that ANP32B-dependent inhibition of PP2A is regulated by respiratory cytochrome c, which induces long-distance allosteric changes in the structured N-terminal domain of ANP32B upon binding to the C-terminal LCAR. In agreement with the reported role of PP2A in the DNA damage response, we propose a model wherein cytochrome c is translocated from the mitochondria into the nucleus upon DNA damage to modulate PP2A activity via its interaction with ANP32B. ; This work was supported by the Spanish Ministry of Science, Innovation and Universities (BFU2012-31670, BFU2015-71017, PGC2018-096049- BI00), Spanish Ministry of Education and Professional Training (FPU013/04373 to FRR, FPU016/01513 to AVC), Regional Government of Andalusia (BIO198, US-1254317 US/JUNTA/FEDER,UE, US- 1257019 US/JUNTA/FEDER,UE, P18-FR-3487 and P18–HO-4091), Ramon Areces Foundation, Biointeractomics Platform (cicCartuja, Seville) and the Microscopy and NMR Services at CITIUS (University of Seville). MPG is funded by a postdoctoral grant from the Spanish Association Against Cancer Scientific Foundation (FC AECC)