Rational stabilization of complex proteins: A divide and combine approach

© 2015, Macmillan Publishers Limited. All rights reserved. Increasing the thermostability of proteins is often crucial for their successful use as analytic, synthetic or therapeutic tools. Most rational thermostabilization strategies were developed on small two-state proteins and, unsurprisingly, they tend to fail when applied to the much more abundant, larger, non-fully cooperative proteins. We show that the key to stabilize the latter is to know the regions of lower stability. To prove it, we have engineered apoflavodoxin, a non-fully cooperative protein on which previous thermostabilizing attempts had failed. We use a step-wise combination of structure-based, rationally-designed, stabilizing mutations confined to the less stable structural region, and obtain variants that, according to their van't Hoff to calorimetric enthalpy ratios, exhibit fully-cooperative thermal unfolding with a melting temperature of 75°C, 32 degrees above the lower melting temperature of the non-cooperative wild type protein. The ideas introduced here may also be useful for the thermostabilization of complex proteins through formulation or using specific stabilizing ligands (e.g. pharmacological chaperones).Peer Reviewe

Tratamientos actuales para el Síndrome de Duplicación de MECP2

Methyl-CpG-binding protein 2 (MeCP2) is a small, intrinsically disordered protein which has a role in gene activation and repression, and chromatin compaction. When MECP2 gene, located in the X chromosome, suffers a duplication, MeCP2 expression increases, leading to the outbreak of seizures, hypotonia, autistic features and respiratory infections. This disorder is known as MECP2 Duplication Syndrome (M2DS).The aim of this Master’s thesis is to gather all the existing drug trials and auxiliar treatments for M2DS.The search returned 10 cases of drug trials (6 performed on humans, 1 on human iPSCs and 3 on M2DS mouse models). Anticonvulsants are the most frequently prescribed drugs on young patients, although antisense oligonucleotides (ASOs) and CRISPR-Cas9 gene editing are being developed in preclinic studies. The main auxiliar therapies found for M2DS were occupational, physical and speech therapy.Valproic acid (VPA) is the most used anticonvulsant for the treatment of seizures in children with M2DS, but other drugs have also proven to be effective, such as lamotrigine (LTG) and topiramate (TPM). The screening of small compounds able to destabilize the MeCP2-DNA union could also be a fruitful source of long-term drugs against M2DS. Auxiliar therapies will continue to be imperative until a full phenotype reversal can be achieved in human patients via gene editing.<br /

Riboflavin kinase and pyridoxine 5'-phosphate oxidase complex formation envisages transient interactions for FMN cofactor delivery

Enzymes catalysing sequential reactions have developed different mechanisms to control the transport and flux of reactants and intermediates along metabolic pathways, which usually involve direct transfer of metabolites from an enzyme to the next one in a cascade reaction. Despite the fact that metabolite or substrate channelling has been widely studied for reactant molecules, such information is seldom available for cofactors in general, and for flavins in particular. Flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) act as cofactors in flavoproteins and flavoenzymes involved in a wide range of physiologically relevant processes in all type of organisms. Homo sapiens riboflavin kinase (RFK) catalyses the biosynthesis of the flavin mononucleotide cofactor, and might directly interplay with its flavin client apo-proteins prior to the cofactor transfer. Non-etheless, none of such complexes has been characterized at molecular or atomic level so far. Here, we particularly evaluate the interaction of riboflavin kinase with one of its potential FMN clients, pyridoxine-5′-phosphate oxidase (PNPOx). The interaction capacity of both proteins is assessed by using isothermal titration calorimetry, a methodology that allows to determine dissociation constants for interaction in the micromolar range (in agreement with the expected transient nature of the interaction). Moreover, we show that; i) both proteins become thermally stabilized upon mutual interaction, ii) the tightly bound FMN product can be transferred from RFK to the apo-form of PNPOx producing an efficient enzyme, and iii) the presence of the apo-form of PNPOx slightly enhances RFK catalytic efficiency. Finally, we also show a computational study to predict likely RFK-PNPOx binding modes that can envisage coupling between the FMN binding cavities of both proteins for the potential transfer of FMN

Vehiculización de compuestos efectivos frente al Virus de la Hepatitis C (VHC)

En este proyecto, “Vehiculización de compuestos efectivos frente al Virus de la Hepatitis C (VHC)”, se ha pretendido por una parte estudiar el comportamiento de las nanopartículas de oro modificadas y no modificadas con galactosa en su superficie (AuNPGal y AuNPTris respectivamente). Los estudios realizados ofreciendo los dos tipos de AuNPs a tres tipos celulares diferentes, nos han permitido validar un método de detección de la cantidad de AuNPs que son incorporadas a las células tras realizar experimentos paralelos de citometría de flujo y microscopía. Así se ha determinado que la modificación con galactosa es necesaria para la correcta entrada de nanopartículas a la célula, ya que en caso contrario las nanopartículas quedan absorbidas en la superficie celular. Aunque parece que los datos indican que las AuNPGal entrarían a la célula utilizando los receptores de galactosa, no se ha podido concluir de forma clara el mecanismo de entrada y se están llevando a cabo estudios que lo determinen. Por otra parte se ha pretendido validar la estrategia de vehiculización de los compuestos antivirales previamente identificados en el grupo de investigación, (OAVx) utilizando ciclodextrinas. Aunque la formación de complejos OAVx-Ciclodextrinas no ha sido posible de cuantificar mediante absorbancia, utilizando calorimetría isotérmica de titulación de determinaron las constantes de unión y los parámetros termodinámicos de la formación d complejos. Mediante el uso de un algoritmo gratuito en la red, se ha obtenido una simulación que nos permite tanto visualizar los grupos funcionales a través de los cuales se produce la interacción como cuantificar la superficie del compuesto que interacciona. Una vez formados los complejos, se ha determinado la actividad antiviral de los mismos, utilizando líneas celulares que portan un sistema en el que se mimetiza el ciclo de replicación del virus de la hepatitis C. Ha sido posible rescatar compuestos que aunque habían sido seleccionados por su demostrada eficacia in vitro, no lo hacían a nivel celular. Este proyecto máster ha contribuido de forma importante al desarrollo del proyecto global en el que se enmarca. Los datos obtenidos con las AuNPs permiten seleccionar el tipo de nanopartícula al que se le unirá la CDx (tras su modificación química correspondiente) y posteriormente se formará el complejo con el compuesto OAV de interés

Identificación de compuestos inhibidores dirigidos contra la flavodoxina de Streptococcus pneumoniae

Streptococcus pneumoniae, causante de neumonía, otitis media, meningitis y bacteremia provoca más de un millón de muertes anuales y en un 25% de los casos, secuelas a nivel neurológico. Actualmente, la terapia contra este patógeno se basa en la administración de antibióticos y vacunas polivalentes dirigidas contra los serotipos más comunes, conocidos como “serotipos pediátricos”. La elevada diversidad genotípica y fenotípica de Streptococcus pneumoniae impide su erradicación, por ello es necesaria la búsqueda de una nueva diana terapeútica para combatir las infecciones causadas por este patógeno. En este proyecto de investigación, se realiza un cribado de 1120 compuestos dirigidos contra una proteína implicada en el metabolismo de metionina de Streptococcus, flavodoxina. La expresión del gen de flavodoxina se ha reportado como esencial en la supervivencia e invasividad de la bacteria a través de estudios de expresión génica durante la fase infectiva de Streptococcus. Su función esencial en el metabolismo bacteriano, la inexistencia de un homólogo proteico en humanos y la ausencia de variabilidad genotípica conducen a la elección de flavodoxina como posible diana terapeútica. A través del estudio de interacción proteína-ligando efectuado mediante el análisis de sus curvas de desnaturalización térmica (thermal-shift assay) se consigue identificar un compuesto que aumenta la estabilidad proteica de flavodoxina (Tm), L-Tiroxina. Los estudios de ITC (isothermal titration calorimetry) realizados para confirmar la interacción entre L-Tiroxina – SpFld fueron favorables, obteniendo valores de KD razonablemente buenos

Streptococcus pneumoniae TIGR4 flavodoxin: Structural and biophysical characterization of a novel drug target

Streptococcus pneumoniae (Sp) strain TIGR4 is a virulent, encapsulated serotype that causes bacteremia, otitis media, meningitis and pneumonia. Increased bacterial resistance and limited efficacy of the available vaccine to some serotypes complicate the treatment of diseases associated to this microorganism. Flavodoxins are bacterial proteins involved in several important metabolic pathways. The Sp flavodoxin (Spfld) gene was recently reported to be essential for the establishment of meningitis in a rat model, which makes SpFld a potential drug target. To facilitate future pharmacological studies, we have cloned and expressed SpFld in E. coli and we have performed an extensive structural and biochemical characterization of both the apo form and its active complex with the FMN cofactor. SpFld is a short-chain flavodoxin containing 146 residues. Unlike the well-characterized long-chain apoflavodoxins, the Sp apoprotein displays a simple two-state thermal unfolding equilibrium and binds FMN with moderate affinity. The X-ray structures of the apo and holo forms of SpFld differ at the FMN binding site, where substantial rearrangement of residues at the 91-100 loop occurs to permit cofactor binding. This work will set up the basis for future studies aiming at discovering new potential drugs to treat S. pneumoniae diseases through the inhibition of SpFld.We acknowledge financial support from BFU2010-16297 and BFU2010-19504 [Ministerio de Ciencia e Innovación Spain], BFU2013-47064-P, BIO2014-57314-REDT and CTQ2013-44367-C2-2-P [Ministerio de Economía y Competitividad, Spain], and DGA (Protein Targets B89). We also thank synchrotron radiation sources DLS (Oxford), and in particular beamline I04-1 (experiment number MX8035-3 and MX8035-11). The research leading to these results has also received funding from the FP7 (2007–2013) under BIOSTRUCTX-7687. A.R.C. was funded by a Banco Santander Central Hispano/Universidad de Zaragoza predoctoral fellowship. M. C-G was recipient of a predoctoral fellowship from the Government of Aragón.Peer Reviewe

Small molecule inhibitors of the response regulator ArsR exhibit bactericidal activity against Helicobacter pylori

Helicobacter pylori is considered the most prevalent bacterial pathogen in humans. The increasing antibiotic resistance evolved by this microorganism has raised alarm bells worldwide due to the significant reduction in the eradication rates of traditional standard therapies. A major challenge in this antibiotic resistance crisis is the identification of novel microbial targets whose inhibitors can overcome the currently circulating resistome. In the present study, we have validated the use of the essential response regulator ArsR as a novel and promising therapeutic target against H. pylori infections. A high-throughput screening of a repurposing chemical library using a fluorescence-based thermal shift assay identified several ArsR binders. At least four of these low-molecular weight compounds noticeably inhibited the DNA binding activity of ArsR and showed bactericidal effects against antibiotic-resistant strains of H. pylori. Among the ArsR inhibitors, a human secondary bile acid, lithocholic acid, quickly destroyed H. pylori cells and exhibited partial synergistic action in combination with clarithromycin or levofloxacin, while the antimicrobial effect of this compound against representative members of the normal human microbiota such as Escherichia coli and Staphylococcus epidermidis appeared irrelevant. Our results enhance the battery of novel therapeutic tools against refractory infections caused by multidrug-resistant H. pylori strains

Structural and functional characterization of phosphomimetic mutants of cytochrome c at threonine 28 and serine 47

Protein function is frequently modulated by post-translational modifications of specific residues. Cytochrome c, in particular, is phosphorylated in vivo at threonine 28 and serine 47. However, the effect of such modifications on the physiological functions of cytochrome c – namely, the transfer of electrons in the respiratory electron transport chain and the triggering of programmed cell death – is still unknown. Here we replace each of these two residues by aspartate, in order to mimic phosphorylation, and report the structural and functional changes in the resulting cytochrome c variants. We find that the T28D mutant causes a 30-mV decrease on the midpoint redox potential and lowers the affinity for the distal site of Arabidopsis thaliana cytochrome c1 in complex III. Both the T28D and S47D variants display a higher efficiency as electron donors for the cytochrome c oxidase activity of complex IV. In both protein mutants, the peroxidase activity is significantly higher, which is related to the ability of cytochrome c to leave the mitochondria and reach the cytoplasm. We also find that both mutations at serine 47 (S47D and S47A) impair the ability of cytoplasmic cytochrome c to activate the caspases cascade, which is essential for triggering programmed cell death.Peer reviewe

Identifying potential novel drugs against Helicobacter pylori by targeting the essential response regulator HsrA

The increasing antibiotic resistance evolved by Helicobacter pylori has alarmingly reduced the eradication rates of first-line therapies. To overcome the current circulating resistome, we selected a novel potential therapeutic target in order to identify new candidate drugs for treating H. pylori infection. We screened 1120 FDA-approved drugs for molecules that bind to the essential response regulator HsrA and potentially inhibit its biological function. Seven natural flavonoids were identified as HsrA binders. All of these compounds noticeably inhibited the in vitro DNA binding activity of HsrA, but only four of them, apigenin, chrysin, kaempferol and hesperetin, exhibited high bactericidal activities against H. pylori. Chrysin showed the most potent bactericidal activity and the most synergistic effect in combination with clarithromycin or metronidazole. Flavonoid binding to HsrA occurs preferably at its C-terminal effector domain, interacting with amino acid residues specifically involved in forming the helix-turn-helix DNA binding motif. Our results validate the use of HsrA as a novel and effective therapeutic target in H. pylori infection and provide molecular evidence of a novel antibacterial mechanism of some natural flavonoids against H. pylori. The results further support the valuable potential of natural flavonoids as candidate drugs for novel antibacterial strategies

2-oxoglutarate modulates the affinity of FurA for the ntcA promoter in Anabaena sp. PCC 7120

2-oxoglutarate (2-OG) is a central metabolite that acts as a signaling molecule informing about the status of the carbon/nitrogen balance of the cell. In recent years, some transcriptional regulators and even two-component systems have been described as 2-OG sensors. In the nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120, two master regulators, NtcA and FurA, are deeply involved in the regulation of nitrogen metabolism. Both of them show a complex intertwined regulatory circuit to achieve a suitable regulation of nitrogen fixation. In this work, 2-OG is found to bind FurA, modulating the specific binding of FurA to the ntcA promoter. This study provides evidence of a new additional control point in the complex network controlled by the NtcA and FurA proteins