Evolutionary trends in RNA base selectivity within the RNase A superfamily

Altres ajuts: Fundació La Marató de TV3 (Marató 20180310)There is a growing interest in the pharmaceutical industry to design novel tailored drugs for RNA targeting. The vertebrate-specific RNase A superfamily is nowadays one of the best characterized family of enzymes and comprises proteins involved in host defense with specific cytotoxic and immune-modulatory properties. We observe within the family a structural variability at the substrate-binding site associated to a diversification of biological properties. In this work, we have analyzed the enzyme specificity at the secondary base binding site. Towards this end, we have performed a kinetic characterization of the canonical RNase types together with a molecular dynamic simulation of selected representative family members. The RNases' catalytic activity and binding interactions have been compared using UpA, UpG and UpI dinucleotides. Our results highlight an evolutionary trend from lower to higher order vertebrates towards an enhanced discrimination power of selectivity for adenine respect to guanine at the secondary base binding site (B2). Interestingly, the shift from guanine to adenine preference is achieved in all the studied family members by equivalent residues through distinct interaction modes. We can identify specific polar and charged side chains that selectively interact with donor or acceptor purine groups. Overall, we observe selective bidentate polar and electrostatic interactions: Asn to N1/N6 and N6/N7 adenine groups in mammals versus Glu/Asp and Arg to N1/N2, N1/O6 and O6/N7 guanine groups in non-mammals. In addition, kinetic and molecular dynamics comparative results on UpG versus UpI emphasize the main contribution of Glu/Asp interactions to N1/N2 group for guanine selectivity in lower order vertebrates. A close inspection at the B2 binding pocket also highlights the principal contribution of the protein β6 and L4 loop regions. Significant differences in the orientation and extension of the L4 loop could explain how the same residues can participate in alternative binding modes. The analysis suggests that within the RNase A superfamily an evolution pressure has taken place at the B2 secondary binding site to provide novel substrate-recognition patterns. We are confident that a better knowledge of the enzymes' nucleotide recognition pattern would contribute to identify their physiological substrate and eventually design applied therapies to modulate their biological functions

Testing a human antimicrobial RNase chimera against bacterial resistance

Altres ajuts: This work was supported by Fundació La Marató de TV3 (Ref. 20180310)The emergence of bacterial resistance to the most commonly used antibiotics encourages the design of novel antimicrobial drugs. Antimicrobial proteins and peptides (AMPs) are the key players in host innate immunity. They exert a rapid and multifaceted action that reduces the development of bacterial adaptation mechanisms. Human antimicrobial RNases belonging to the vertebrate specific RNase A superfamily participate in the maintenance of tissue and body fluid sterility. Among the eight human canonical RNases, RNase 3 stands out as the most cationic and effective bactericidal protein against Gram-negative species. Its enhanced ability to disrupt the bacterial cell wall has evolved in detriment of its catalytic activity. Based on structure-functional studies we have designed an RNase 3/1 hybrid construct that combines the high catalytic activity of RNase 1 with RNase 3 bactericidal properties. Next, we have explored the ability of this hybrid RNase to target the development of bacterial resistance on an Acinetobacter baumannii cell culture. Synergy assays were performed in combination with colistin, a standard antimicrobial peptide used as an antibiotic to treat severe infections. Positive synergism was observed between colistin and the RNase 3/1 hybrid protein. Subsequently, using an in vitro experimental evolution assay, by exposure of a bacterial culture to colistin at incremental doses, we demonstrated the ability of the RNase 3/1 construct to reduce the emergence of bacterial antimicrobial resistance. The results advance the potential applicability of RNase-based drugs as antibiotic adjuvants

Structure-Based Design of an RNase Chimera for Antimicrobial Therapy

Altres ajuts: Fundació La Marató de TV3/TV3-201803-10Bacterial resistance to antibiotics urges the development of alternative therapies. Based on the structure-function of antimicrobial members of the RNase A superfamily, we have developed a hybrid enzyme. Within this family, RNase 1 exhibits the highest catalytic activity and the lowest cytotoxicity; in contrast, RNase 3 shows the highest bactericidal action, alas with a reduced catalytic activity. Starting from both parental proteins, we designed a first RNase 3/1-v1 chimera. The construct had a catalytic activity much higher than RNase 3, unfortunately without reaching an equivalent antimicrobial activity. Thus, two new versions were created with improved antimicrobial properties. Both of these versions (RNase 3/1-v2 and -v3) incorporated an antimicrobial loop characteristic of RNase 3, while a flexible RNase 1-specific loop was removed in the latest construct. RNase 3/1-v3 acquired both higher antimicrobial and catalytic activities than previous versions, while retaining the structural determinants for interaction with the RNase inhibitor and displaying non-significant cytotoxicity. Following, we tested the constructs' ability to eradicate macrophage intracellular infection and observed an enhanced ability in both RNase 3/1-v2 and v3. Interestingly, the inhibition of intracellular infection correlates with the variants' capacity to induce autophagy. We propose RNase 3/1-v3 chimera as a promising lead for applied therapeutics

Magnetite Nanoparticles Functionalized with RNases against Intracellular Infection of Pseudomonas aeruginosa

Altres ajuts: Fundació La Marató de TV3/20180310Current treatments against bacterial infections have severe limitations, mainly due to the emergence of resistance to conventional antibiotics. In the specific case of Pseudomonas aeruginosa strains, they have shown a number of resistance mechanisms to counter most antibiotics. Human secretory RNases from the RNase A superfamily are proteins involved in a wide variety of biological functions, including antimicrobial activity. The objective of this work was to explore the intracellular antimicrobial action of an RNase 3/1 hybrid protein that combines RNase 1 high catalytic and RNase 3 bactericidal activities. To achieve this, we immobilized the RNase 3/1 hybrid on Polyetheramine (PEA)-modified magnetite nanoparticles (MNPs). The obtained nanobioconjugates were tested in macrophage-derived THP-1 cells infected with Pseudomonas aeruginosa PAO1. The obtained results show high antimicrobial activity of the functionalized hybrid protein (MNP-RNase 3/1) against the intracellular growth of P. aeruginosa of the functionalized hybrid protein. Moreover, the immobilization of RNase 3/1 enhances its antimicrobial and cell-penetrating activities without generating any significant cell damage. Considering the observed antibacterial activity, the immobilization of the RNase A superfamily and derived proteins represents an innovative approach for the development of new strategies using nanoparticles to deliver antimicrobials that counteract P. aeruginosa intracellular infection

Exploring the pharmacological properties of human antimicrobial ribonucleases

Aquesta tesi s’enfoca en la caracterització estructural i funcional de les propietats biològiques de les RNases antimicrobianes de la superfamília de la RNasa A. Concretament, s’han assolit els següents objectius a curt termini: La caracterització estructural i funcional de la RNasa 6 per cristal·lografia de raigs X, dinàmica molecular, mutagènesi dirigida i anàlisis enzimàtics destaca el paper clau de les regions remotes d’unió al substrat. A part, hem identificat un possible segon centre actiu en la RNasa 6. Finalment, un estudi evolutiu dels diversos membres de la superfamília de la RNasa A ha revelat una tendència clara, al llarg de l’evolució en vertebrats, des de la preferència de guanina cap a la d’adenina en l’arquitectura de la regió secundària d’unió a bases B2. Al llarg del treball experimental realitzat en aquesta tesi, hem buscat la caracterització del mecanisme d’acció bactericida de les RNases, una de les principals línies de recerca del nostre grup de recerca. En aquest treball, ens hem enfocat específicament en l’optimització del pèptid antimicrobià derivat de l‘N-terminal de la RNasa 3, ECP(5-17P24-36), i en el disseny d’una RNasa quimèrica antimicrobiana (RNasa 3/1). Respecte el pèptid ECP(5-17P24-36), s’ha optimitzat mitjançant diverses metodologies, arribant a la conclusió que el millor candidat antimicrobià és el seu enantiòmer total D-ECP(5–17P24–36). Pel que fa a la RNasa 3/1, aquesta incorpora les característiques estructurals de les RNases 1 i 3, combinant així la seva elevada activitat catalítica i bactericida, respectivament. Es va dissenyar un primer constructe amb èxit, malgrat que no presentava els mateixos nivells d’activitat bactericida que la RNasa 3. Aleshores, vam dissenyar dues versions noves de la RNasa 3/1 que incorporaven el loop C-terminal de la RNasa 3, en el qual es va identificar un motiu estructural específic associat al reclutament de l’autofagosoma. És interessant destacar la capacitat de la primera versió de la quimera RNasa 3/1 d’endarrerir l’adquisició de resistència a la colistina en un assaig evolutiu in vitro d’exposició a la colistina en cultius d’Acinetobacter baumannii. En global, aquests resultats ajudaran a elucidar el mode d’unió a l’RNA de les ribonucleases i el seu mecanisme antimicrobià, així com la seva contribució en el sistema immunitari innat, amb prometedores aplicacions farmacològiques.Esta tesis se enfoca en la caracterización estructural y funcional de las propiedades biológicas de las RNasas antimicrobianas de la superfamilia de la RNasa A. Concretamente, se han alcanzado los siguientes objetivos en el corto plazo: La caracterización estructural y funcional de la RNasa 6 por cristalografía de rayos X, dinámica molecular, mutagénesis dirigida y análisis enzimáticos destaca el papel clave de las regiones remotas de unión al sustrato. A parte, hemos identificado un posible segundo centro activo en la RNasa 6. Finalmente, un estudio evolutivo de los distintos miembros de la superfamilia de la RNasa A ha revelado una tendencia clara, a lo largo de la evolución en vertebrados, desde la preferencia de la guanina hacia adenina en la arquitectura de la región secundaria de unión a bases B2. A lo largo del trabajo experimental realizado en esta tesis, hemos buscado la caracterización del mecanismo de acción bactericida de las RNasas, una de las principales líneas de investigación de nuestro grupo de investigación. En este trabajo, nos hemos enfocado específicamente en la optimización del péptido derivado del N-terminal de la RNasa 3, ECP(5-17P24-36), y en el diseño de una RNasa quimérica antimicrobiana (RNasa 3/1). Respecto al péptido ECP(5-17P24-36), se ha optimizado mediante varias metodologías, llegando a la conclusión que el mejor candidato antimicrobiano es su enantiómero total D-ECP(5-17P24-36). Por lo que respecta a la RNasa 3/1, esta incorpora las características estructurales de las RNasas 1 y 3, combinando así su elevada actividad catalítica y bactericida, respectivamente. Se diseñó un primer constructo con éxito, pese a que no presentaba los mismos niveles de actividad bactericida que la RNasa 3. Entonces, diseñamos dos nuevas versiones de la RNasa 3/1 que incorporaban el loop C-terminal de la RNasa 3, en el que se identificó un motivo estructural específico asociado al reclutamiento del autofagosoma. Es interesante destacar la capacidad de la primera versión de la quimera RNasa 3/1 de retrasar la adquisición de resistencia a la colistina en un ensayo evolutivo in vitro de exposición a la colistina en cultivos de Acinetobacter baumannii. En global, estos resultados ayudarán a elucidar el modo de unión al RNA de las ribonucleasas y su mecanismo antimicrobiano, así como su contribución en el sistema inmunitario innato, con prometedoras aplicaciones farmacológicas.This thesis project focuses on the structural-functional characterization of the biological properties of antimicrobial RNases from the RNase A superfamily. Specifically, the following short-term goals have been achieved: Structural and functional characterization of RNase 6 by X-ray crystallography, molecular dynamics, site-directed mutagenesis and enzymatic analysis have highlighted the key role of remote binding subsites. Besides, we have identified in RNase 6 a putative novel secondary active site. In addition, an evolutionary study of several members of the RNase A superfamily have revealed a clear drift from guanine to adenine preference at the secondary base binding site (B2) architecture along vertebrate evolution. During this thesis’ experimental work, we have pursued the characterization of RNases’ bactericidal mechanism of action, a long-term object of study in our research group. Here, we have specifically focused on the optimisation of the antimicrobial peptide derived from RNase 3, ECP(5-17P24-36), and the design of a chimeric antimicrobial RNase (RNase 3/1). Regarding the N-terminus peptide ECP(5-17P24-36), it has been optimised by several methodologies. We have concluded the best antimicrobial candidate to be its total enantiomer, D-ECP(5-17P24-36). As for RNase 3/1, this chimera encompasses structural features from RNases 1 and 3 parental proteins to combine both high catalytic and bactericidal activities. A first construct was successfully achieved, albeit not reaching the bactericidal activity levels of RNase 3. Therefore, we designed two more versions of RNase 3/1 that incorporate the RNase 3 C-terminus loop. A specific tag motif was identified in that region associated to autophagosome recruitment. Interestingly, the hybrid chimera RNase 3/1 was able to delay the acquisition of bacterial resistance to colistin using an in vitro evolutionary exposure assay in Acinetobacter baumannii cultures. Overall, the results shed light on the elucidation of substrate binding architecture and antimicrobial mechanism of action of RNases and their contribution to the innate immune system, with promising pharmacological applications.Universitat Autònoma de Barcelona. Programa de Doctorat en Bioquímica, Biologia Molecular i Biomedicin

A Common Polymorphism in RNASE6 Impacts Its Antimicrobial Activity toward Uropathogenic Escherichia coli

Human Ribonuclease (RNase) 6 is a monocyte and macrophage-derived protein with potent antimicrobial activity toward uropathogenic bacteria. The RNASE6 gene is heterogeneous in humans due to the presence of single nucleotide polymorphisms (SNPs). RNASE6 rs1045922 is the most common non-synonymous SNP, resulting in a G to A substitution that determines an arginine (R) to glutamine (Q) transversion at position 66 in the protein sequence. By structural analysis we observed that R66Q substitution significantly reduces the positive electrostatic charge at the protein surface. Here, we generated both recombinant RNase 6-R66 and -Q66 protein variants and determined their antimicrobial activity toward uropathogenic Escherichia coli (UPEC), the most common cause of UTI. We found that the R66 variant, encoded by the major SNP rs1045922 allele, exhibited superior bactericidal activity in comparison to the Q66 variant. The higher bactericidal activity of R66 variant correlated with an increase in the protein lipopolysaccharide binding and bacterial agglutination abilities, while retaining the same enzymatic efficiency. These findings encourage further work to evaluate RNASE6 SNP distribution and its impact in UTI susceptibility

Exploring the RNase A scaffold to combine catalytic and antimicrobial activities. Structural characterization of RNase 3/1 chimeras

Design of novel antibiotics to fight antimicrobial resistance is one of the first global health priorities. Novel protein-based strategies come out as alternative therapies. Based on the structure-function knowledge of the RNase A superfamily we have engineered a chimera that combines RNase 1 highest catalytic activity with RNase 3 unique antipathogen properties. A first construct (RNase 3/1-v1) was successfully designed with a catalytic activity 40-fold higher than RNase 3, but alas in detriment of its anti-pathogenic activity. Next, two new versions of the original chimeric protein were created showing improvement in the antimicrobial activity. Both second generation versions (RNases 3/1-v2 and -v3) incorporated a loop characteristic of RNase 3 (L7), associated to antimicrobial activity. Last, removal of an RNase 1 flexible loop (L1) in the third version enhanced its antimicrobial properties and catalytic efficiency. Here we solved the 3D structures of the three chimeras at atomic resolution by X-ray crystallography. Structural analysis outlined the key functional regions. Prediction by molecular docking of the protein chimera in complex with dinucleotides highlighted the contribution of the C-terminal region to shape the substrate binding cavity and determine the base selectivity and catalytic efficiency. Nonetheless, the structures that incorporated the key features related to RNase 3 antimicrobial activity retained the overall RNase 1 active site conformation together with the essential structural elements for binding to the human ribonuclease inhibitor (RNHI), ensuring non-cytotoxicity. Results will guide us in the design of the best RNase pharmacophore for anti-infective therapies

Human RNase3 immune modulation by catalytic-dependent and independent modes in a macrophage-cell line infection model

Altres ajuts: Fundació Marató TV3 [20180310]The human RNase3 is a member of the RNaseA superfamily involved in host immunity. RNase3 is expressed by leukocytes and shows broad-spectrum antimicrobial activity. Together with a direct antimicrobial action, RNase3 exhibits immunomodulatory properties. Here, we have analysed the transcriptome of macrophages exposed to the wild-type protein and a catalytic-defective mutant (RNase3-H15A). The analysis of differently expressed genes (DEGs) in treated THP1-derived macrophages highlighted a common pro-inflammatory "core-response" independent of the protein ribonucleolytic activity. Network analysis identified the epidermal growth factor receptor (EGFR) as the main central regulatory protein. Expression of selected DEGs and MAPK phosphorylation were inhibited by an anti-EGFR antibody. Structural analysis suggested that RNase3 activates the EGFR pathway by direct interaction with the receptor. Besides, we identified a subset of DEGs related to the protein ribonucleolytic activity, characteristic of virus infection response. Transcriptome analysis revealed an early pro-inflammatory response, not associated to the protein catalytic activity, followed by a late activation in a ribonucleolyticdependent manner. Next, we demonstrated that overexpression of macrophage endogenous RNase3 protects the cells against infection by Mycobacterium aurum and the human respiratory syncytial virus. Comparison of cell infection profiles in the presence of Erlotinib, an EGFR inhibitor, revealed that the receptor activation is required for the antibacterial but not for the antiviral protein action. Moreover, the DEGs related and unrelated to the protein catalytic activity are associated to the immune response to bacterial and viral infection, respectively. We conclude that RNase3 modulates the macrophage defence against infection in both catalytic-dependent and independent manners

In vivo evaluation of ECP peptide analogues for the treatment of Acinetobacter baumannii infection

Antimicrobial peptides (AMPs) are alternative therapeutics to traditional antibiotics against bacterial resistance. Our previous work identified an antimicrobial region at the N-terminus of the eosinophil cationic protein (ECP). Following structure-based analysis, a 30mer peptide (ECPep-L) was designed that combines antimicrobial action against Gram-negative species with lipopolysaccharides (LPS) binding and endotoxin-neutralization activities. Next, analogues that contain non-natural amino acids were designed to increase serum stability. Here, two analogues were selected for in vivo assays: the all-D version (ECPep-D) and the Arg to Orn version that incorporates a D-amino acid at position 2 (ECPep-2D-Orn). The peptide analogues retained high LPS-binding and anti-endotoxin activities. The peptides efficacy was tested in a murine acute infection model of Acinetobacter baumannii. Results highlighted a survival rate above 70% following a 3-day supervision with a single administration of ECPep-D. Moreover, in both ECPep-D and ECPep-2D-Orn peptide-treated groups, clinical symptoms improved significantly and the tissue infection was reduced to equivalent levels to mice treated with colistin, used as a last resort in the clinics. Moreover, treatment drastically reduced serum levels of TNF-α inflammation marker within the first 8 h. The present results support ECP-derived peptides as alternative candidates for the treatment of acute infections caused by Gram-negative bacteria.Research work was supported by Fundació La Marató de TV3 (TV3-201803-10), the Ministerio de Economía y Competitividad (PID2019-106123GB-I00) and by AGAUR, Generalitat de Catalunya (2016PROD00060; 2019 LLAV 00002), co-financed by FEDER funds. P.F.-M. was a recipient of Juan de la Cierva postdoctoral fellowship, G.P.-E. was recipient of a PIF-UAB predoctoral fellowship and J.L. was a recipient of a CSC predoctoral fellowship

Insight into the antifungal mechanism of action of human RNase N-terminus derived peptides

Candida albicans is a polymorphic fungus responsible for mucosal and skin infections. Candida cells establish themselves into biofilm communities resistant to most currently available antifungal agents. An increase of severe infections ensuing in fungal septic shock in elderly or immunosuppressed patients, along with the emergence of drug-resistant strains, urge the need for the development of alternative antifungal agents. In the search for novel antifungal drugs our laboratory demonstrated that two human ribonucleases from the vertebrate-specific RNaseA superfamily, hRNase3 and hRNase7, display a high anticandidal activity. In a previous work, we proved that the N-terminal region of the RNases was sufficient to reproduce most of the parental protein bactericidal activity. Next, we explored their potency against a fungal pathogen. Here, we have tested the N-terminal derived peptides that correspond to the eight human canonical RNases (RN1-8) against planktonic cells and biofilms of C. albicans. RN3 and RN7 peptides displayed the most potent inhibitory effect with a mechanism of action characterized by cell-wall binding, membrane permeabilization and biofilm eradication activities. Both peptides are able to eradicate planktonic and sessile cells, and to alter their gene expression, reinforcing its role as a lead candidate to develop novel antifungal and antibiofilm therapies.This research was funded by the Ministerio de Economía y Competitividad (SAF2017-82158-R and SAF2015-66007P, Fundació La Marató de TV3 (20180310) and Generalitat de Catalunya (2016-PROD-00060). M.T. would like to acknowledge support from the Programa Ramon y Cajal (RYC-2012-09999) and the European Society of Clinical Microbiology and Infectious Diseases though ans ESCMID-2016 grant