Estructura y estabilidad en plastocianinas de cianobacterias

Falta resumenESTRUCTURA Y ESTABILIDAD EN PLASTOCIANINAS DE CIANOBACTERIAS La elucidación de los factores que determinan las diferencias en la termorresistencia en las proteínas de organismos termófilos respecto a sus homólogas de organismos mesófilos es un tema de elevado interés en relación a la ingeniería de proteínas. Cada familia proteica adopta mecanismos propios para adaptarse a las altas temperaturas. Las proteínas de cobre azul o cobre tipo-1 son de especial interés por la utilidad de las mismas en diversos procesos biotecnológicos. Entre ellas, la plastocianina constituye un modelo experimental excelente al estar constituida por un único dominio, que en las enzimas de interés industrial se encuentra duplicado o triplicado. En los últimos años se ha investigado en nuestro grupo las características estructurales que rigen la mayor estabilidad de la plastocianina de la cianobacteria termófila Phormidium laminosum respecto a su homóloga mesófila en la cianobacteria Synechocystis sp. PCC6803, así como la distinta dependencia que los puntos de fusión que las dos proteínas muestran respecto al estado redox del metal. Análisis computacionales realizados indican que la región comprendida entre los residuos 39 y 65 determina las diferencias en la termoestabilidad entre ambas proteínas. Asimismo, sustituciones de aminoácidos en dicha región disminuyen la estabilidad de la proteína termorresistente y alteran la relación entre el estado redox del metal y el punto de fusión de la proteína. Sin embargo, se desconoce por qué los residuos situados en una región flexible (residuos 44 - 56) y distante del centro metálico modulan la estabilidad térmica. En el presente trabajo de Tesis se ha abordado la estabilización de la plastocianina de la cianobacteria mesofílica Synechocystis sp. PCC6803 mediante mutagénesis dirigida, con el fin de comprender cómo la región entre los residuos 39 y 65 modula la estabilidad de la proteína en sus distintos estados redox. Para ello se han aplicado criterios normalmente asumidos para optimizar la termoresistencia de proteínas. El análisis espectroscópico de las distintas proteínas recombinantes ha permitido detectar la existencia de intermediarios en la ruta de desplegamiento de la proteína de Synechocystis. Asimismo, tales análisis de distintas variantes indican una cooperatividad entre el centro cobre y la región flexible que comprende los residuos desde el 44 al 56. Los datos de difracción de rayos-X y los cálculos de dinámica molecular que se presentan indican que las mutaciones afectan a dicha cooperatividad a través del concierto entre los movimientos de las distintas regiones flexibles de la proteína, sin afectar a la estructura global de la misma

Voltammetric study of the adsorbed thermophilic plastocyanin from Phormidium laminosum up to 90 °c

Redox thermodynamics and kinetics of plastocyanin from Phormidium laminosum, and of azurin from Pseudonomas aeruginosa, have been investigated as a function of temperature by protein film voltammetry. To this purpose, both proteins have been physisorbed on a pyrolytic graphite edge electrode. A pronounced negative shift of the plastocyanin standard potential, compared to a slight shift in the case of azurin, has been found upon increasing the temperature. Hence, significant conformational and/or solvation changes accompany the redox conversion of plastocyanin. Lower electron transfer rate constants (by c.a. one order of magnitude) and higher activation enthalpies have been found for plastocyanin as compared to azurin. The voltammetric response of azurin vanishes irreversibly at temperatures close to 60 °C, whereas the redox properties of plastocyanin remain unaltered, except for some loss of electroactive protein, after heating the electrode at temperatures as high as 90 °C.Ministerio de Economía y Competitividad CTQ 2008-00371, BFU2009-07190Junta de Andalucía P07-FQM-02492, P06-CVI-0171

Discovery, optimization, and target identification of novel coumarin derivatives as HIV-1 reverse transcriptase-associated ribonuclease H inhibitors

Despite significant advances in antiretroviral therapy, acquired immunodeficiency syndrome remains as one of the leading causes of death worldwide. New antiretroviral drugs combined with updated treatment strategies are needed to improve convenience, tolerability, safety, and antiviral efficacy of available therapies. In this work, a focused library of coumarin derivatives was exploited by cell phenotypic screening to discover novel inhibitors of HIV-1 replication. Five compounds (DW-3, DW-4, DW-11, DW-25 and DW-31) showed moderate activity against wild-type and drug-resistant strains of HIV-1 (IIIB and RES056). Four of those molecules were identified as inhibitors of the viral RT-associated RNase H. Structural modification of the most potent DW-3 and DW-4 led to the discovery of compound 8a. This molecule showed increased potency against wild-type HIV-1 strain (EC = 3.94 ± 0.22 μM) and retained activity against a panel of mutant strains, showing EC values ranging from 5.62 μM to 202 μM. In enzymatic assays, 8a was found to inhibit the viral RNase H with an IC of 12.3 μM. Molecular docking studies revealed that 8a could adopt a binding mode similar to that previously reported for other active site HIV-1 RNase H inhibitors.Natural Science Foundation of China (NSFC Nos. 81973181, 81903453), Shandong Provincial Key research and development project (Nos. 2019JZZY021011), Shandong Provincial Natural Science Foundation (ZR2019BH011, ZR2020YQ61, ZR2020JQ31), Foreign cultural and educational experts Project (GXL20200015001), Qilu Young Scholars Program of Shandong University, the Taishan Scholar Program at Shandong Province, and KU Leuven (GOA 10/014). Work in Madrid was supported by the Spanish Ministry of Science and Innovation (grant PID2019-104176RB-I00/AEI/10.13039/501100011033), and an institutional grant of Fundación Ramón Areces (Madrid, Spain)

Novel indolylarylsulfone derivatives as covalent HIV-1 reverse transcriptase inhibitors specifically targeting the drug-resistant mutant Y181C

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are widely used in combination therapies against HIV-1. However, emergent and transmitted drug resistance compromise their efficacy in the clinical setting. Y181C is selected in patients receiving nevirapine, etravirine and rilpivirine, and together with K103N is the most prevalent NNRTI-associated mutation in HIV-infected patients. Herein, we report on the design, synthesis and biological evaluation of a novel series of indolylarylsulfones bearing acrylamide or ethylene sulfonamide reactive groups as warheads to inactivate Cys181-containing HIV-1 RT via a Michael addition reaction. Compounds I-7 and I-9 demonstrated higher selectivity towards the Y181C mutant than against the wild-type RT, in nucleotide incorporation inhibition assays. The larger size of the NNRTI binding pocket in the mutant enzyme facilitates a better fit for the active compounds, while stacking interactions with Phe227 and Pro236 contribute to inhibitor binding. Mass spectrometry data were consistent with the covalent modification of the RT, although off-target reactivity constitutes a major limitation for further development of the described inhibitors.by grants PID2019-104176RB-I00/AEI/10.13039/501100011033) (Spanish Ministry of Science and Innovation) and 2019AEP001 (CSIC), as well as an institutional grant of Fundación Ramón Areces (awarded to the CBMSO)

Reverse Transcriptase: From Transcriptomics to Genome Editing

Reverse transcriptases (RTs) are enzymes that can generate a complementary strand of DNA (cDNA) from RNA. Coupled with PCR, RTs have been widely used to detect RNAs and to clone expressed genes. Classical retroviral RTs have been improved by protein engineering. These enzymes and newly characterized RTs are key elements in the development of next-generation sequencing techniques that are now being applied to the study of transcriptomics. In addition, engineered RTs fused to a CRISPR/Cas9 nickase have recently shown great potential as tools to manipulate eukaryotic genomes. In this review, we discuss the properties and uses of wild type and engineered RTs in biotechnological applications, from conventional RT-PCR to recently introduced prime editing.Ministry of Science and Innovation of Spain (grant BIO2016‐76716‐R (AEI/FEDER, UE)), and Consejo Superior de Investigaciones Científicas (2019AEP001). S.M.‐A. is a predoctoral fellow of the Ministry of Science and Innovation of Spain (BES‐2017‐079836). An institutional grant to CBMSO of Fundación Ramón Arece

Voltammetric study of the adsorbed thermophilic plastocyanin from Phormidium laminosum up to 90 °c

Redox thermodynamics and kinetics of plastocyanin from Phormidium laminosum, and of azurin from Pseudonomas aeruginosa, have been investigated as a function of temperature by protein film voltammetry. To this purpose, both proteins have been physisorbed on a pyrolytic graphite edge electrode. A pronounced negative shift of the plastocyanin standard potential, compared to a slight shift in the case of azurin, has been found upon increasing the temperature. Hence, significant conformational and/or solvation changes accompany the redox conversion of plastocyanin. Lower electron transfer rate constants (by c.a. one order of magnitude) and higher activation enthalpies have been found for plastocyanin as compared to azurin. The voltammetric response of azurin vanishes irreversibly at temperatures close to 60 °C, whereas the redox properties of plastocyanin remain unaltered, except for some loss of electroactive protein, after heating the electrode at temperatures as high as 90 °C. © 2012 Elsevier B.V. All rights reserved.Peer Reviewe

Electrostatic strain and concerted motions in the transient complex between plastocyanin and cytochrome f from the cyanobacterium Phormidium laminosum

10 páginas, 9 figuras, 1 tablaMany fleeting macromolecular interactions, like those being involved in electron transport, are essential in biology. However, little is known about the behaviour of the partners and their dynamics within their short-lived complex. To tackle such issue, we have performed molecular dynamics simulations on an electron transfer complex formed by plastocyanin and cytochrome f from the cyanobacterium Phormidium laminosum. Besides simulations of the isolated partners, two independent trajectories of the complex were calculated, starting from the two different conformations in the NMR ensemble. The first one leads to a more stable ensemble with a shorter distance between the metal sites of the two partners. The second experiences a significant drift of the complex conformation. Analyses of the distinct calculations show that the conformation of cytochrome f is strained upon binding of its partner, and relaxes upon its release. Interestingly, the principal component analysis of the trajectories indicates that plastocyanin displays a concerted motion with the small domain of cytochrome f that can be attributed to electrostatic interactions between the two proteins.This work was supported in part by research grants from the Spanish Ministry of Science and Innovation (BFU2006-01361) and the Andalusian Government (BIO-198 and P06-CVI-01713). FJML is the recipient of a FPI fellowship (BES-2005-10404) from the Spanish Ministry of Science and Innovation.Peer reviewe

Medicinal chemistry strategies for discovering antivirals effective against drug-resistant viruses

During the last forty years we have witnessed impressive advances in the field of antiviral drug discovery culminating with the introduction of therapies able to stop human immunodeficiency virus (HIV) replication, or cure hepatitis C virus infections in people suffering from liver disease. However, there are important viral diseases without effective treatments, and the emergence of drug resistance threatens the efficacy of successful therapies used today. In this review, we discuss strategies to discover antiviral compounds specifically designed to combat drug resistance. Currently, efforts in this field are focused on targeted proteins (e.g. multi-target drug design strategies), but also on drug conformation (either improving drug positioning in the binding pocket or introducing conformational constraints), in the introduction or exploitation of new binding sites, or in strengthening interaction forces through the introduction of multiple hydrogen bonds, covalent binding, halogen bonds, additional van der Waals forces or multivalent binding. Among the new developments, proteolysis targeting chimeras (PROTACs) have emerged as a valid approach taking advantage of intracellular mechanisms involving protein degradation by the ubiquitin-proteasome system. Finally, several molecules targeting host factors (e.g. human dihydroorotate dehydrogenase and DEAD-box polypeptide 3) have been identified as broad-spectrum antiviral compounds. Implementation of herein described medicinal chemistry strategies are expected to contribute to the discovery of new drugs effective against current and future threats due to emerging and re-emerging viral pandemics. This journal isNational Natural Science Foundation of China; Ministry of Science and Innovation of Spain (BIO2016-76716-R (AEI/FEDER, UE)) and (PID2019-104176RB-I00/AEI/10.13039/501100011033) and CSIC (2019AEP001), as well as an institutional grant of Fundació Ramón Areces awarded to the CBMS

Key Role of the Local Hydrophobicity in the East Patch of Plastocyanins on Their Thermal Stability and Redox Properties

Understanding the molecular basis of the thermal stability and functionality of redox proteins has important practical applications. Here, we show a distinct thermal dependence of the spectroscopic and electrochemical properties of two plastocyanins from the thermophilic cyanobacterium Phormidium laminosum and their mesophilic counterpart from Synechocystis sp. PCC 6803, despite the similarity of their molecular structures. To explore the origin of these differences, we have mimicked the local hydrophobicity in the east patch of the thermophilic protein by replacing a valine of the mesophilic plastocyanin by isoleucine. Interestingly, the resulting mutant approaches the thermal stability, redox thermodynamics, and dynamic coupling of the flexible site motions of the thermophilic protein, indicating the existence of a close connection between the hydrophobic packing of the east patch region of plastocyanin and the functional control and stability of the oxidized and reduced forms of the protein.Peer Reviewe

Novel RNase H Inhibitors Blocking RNA-directed Strand Displacement DNA Synthesis by HIV-1 Reverse Transcriptase

In retroviruses, strand displacement DNA-dependent DNA polymerization catalyzed by the viral reverse transcriptase (RT) is required to synthesize double-stranded proviral DNA. In addition, strand displacement during RNA-dependent DNA synthesis is critical to generate high-quality cDNA for use in molecular biology and biotechnology. In this work, we show that the loss of RNase H activity due to inactivating mutations in HIV-1 RT (e.g. D443N or E478Q) has no significant effect on strand displacement while copying DNA templates, but has a large impact on DNA polymerization in reactions carried out with RNA templates. Similar effects were observed with β-thujaplicinol and other RNase H active site inhibitors, including compounds with dual activity (i.e., characterized also as inhibitors of HIV-1 integrase and/or the RT DNA polymerase). Among them, dual inhibitors of HIV-1 RT DNA polymerase/RNase H activities, containing a 7-hydroxy-6-nitro-2H-chromen-2-one pharmacophore were found to be very potent and effective strand displacement inhibitors in RNA-dependent DNA polymerization reactions. These findings might be helpful in the development of transcriptomics technologies to obtain more uniform read coverages when copying long RNAs and for the construction of more representative libraries avoiding biases towards 5' and 3' ends, while providing valuable information for the development of novel antiretroviral agents.This work was supported in part by the Ministry of Science and Innovation of Spain through grant PID2019-104176RB-I00/AEI/10.13039/501100011033 awarded to L.M.-A. S.M.-A. is a predoctoral fellow of the Ministry of Science and Innovation of Spain (BES-2017-079836). J.M.R. is a predoctoral fellow of the Spanish Ministry of Universities (Formación Personal Universitario, FPU19/01653). An institutional grant of the Fundación Ramón Areces to the CBMSO is also acknowledged. The team at CBMSO is affiliated to the Global Virus Network. Work in Jinan was supported by the National Natural Science Foundation of China (81973181, 81903453), Science Foundation for Outstanding Young Scholars of Shandong Province (ZR2020JQ31), Science Foundation for Excellent Young Scholars of Shandong Province (ZR2020YQ61), Foreign cultural and educational experts project GXL20200015001, and the Shandong Provincial Key research and development project 2019JZZY021011.Peer reviewe