Cristal•lització i difracció de monocristalls al Sincrotró ALBA per a la determinació estructural d’una proteïna Relaxassa

Treballs Finals de Grau de Química, Facultat de Química, Universitat de Barcelona, Any: 2016, Tutor: Daniel Sainz García i Roeland BoerThe aim of the present work is the structure description of two variant (full length and Nterminal) of a protein with Relaxase activity. Both pure variants were crystallized using sitting drop vapor diffusion technique. Crystals obtained were optimized increasing its quality and some of them were treated to derivatize it with metals. Diffraction of crystals and pre-treatment of protein were integrally performed in ALBA Synchrotron (Barcelona) installations. In BL-13 XALOC beamline were collected the data which were processed and evaluated as good quality data. The phase problem solution was not possible by applying Molecular Replacement nor detecting the anomalous signal of derivatives crystals. The lack of phases did not permit to solve the electron density equation, and therefore, it was not possible to complete the model buildin

Construcción, implementación y evaluación de una vía clínica de la disfagia en una unidad de ictus agudo

Introducción: las vías clínicas (VC), son herramientas que desarrollan actividades para evitar la aparición de problemas, se aplican a una clínica previsible, facilitan la atención multidisciplinar a los pacientes, donde describen actividades, secuencias y duración de cualquier proceso asistencial, interviniendo en la coordinación entre sanitarios. El ictus es un problema de salud de primer orden y su asistencia neurológica es urgente. Dicta la buena práctica que deben ser atendidas en unidades de ictus (UI) con monitorización continua y cuidados específicos. Uno de los síntomas que tiene gran importancia en el ictus es la disfagia, que afecta al paciente en múltiples niveles suponiendo la primera causa de aspiraciones y complicaciones respiratorias, siendo necesario realizar su identificación para dar tratamiento oportuno. Objetivos: valorar la calidad de la atención a la disfagia, analizar los resultados de la implantación de una vía para mejorar su identificación y manejo, unificar criterios de actuación diaria de Enfermería, mapear los procesos de detección y tratamientos de la disfagia, definir los indicadores que sirvan para medir la calidad del proceso asistencial e identificar las oportunidades de mejora para los procesos de atención a la disfagia en los pacientes con ictus. Material y métodos: este proyecto constituyó el diseño e implementación de una vía clínica. Incluyó un estudio observacional descriptivo de corte transversal para la validación de indicadores. Además, para su implantación y evaluación se desarrolló un diseño cuasi-experimental con metodología de ciclo de mejora. Se desarrolló en la unidad de ictus del Hospital Clínico Universitario Virgen de la Arrixaca, con una muestra de 43 pacientes para las evaluaciones, y todo el tamaño muestral para indicadores de baja casuística. Se incluyeron todos los pacientes que ingresaron en la UI en los meses de marzo-julio de 2021 para la 1º medición, y de enero a marzo 2022 para la 2º medición. El trabajo se desarrolló gracias al apoyo de un grupo de trabajo multidisciplinar. Resultados: a través del proceso de elaboración de la VC se valoró la calidad de la atención a la disfagia en la UI. Se unificaron criterios de actuación en la práctica enfermera incluidos en la VC, la cual está compuesta por unos documentos como son la matriz final de actividades, flujograma de detección de la disfagia, hoja de variaciones, hoja informativa para familiares/pacientes, y guía rápida sobre la disfagia tras un ictus. Para evaluar la implantación de la VC, se seleccionaron 7 indicadores, extraídos de una búsqueda de artículos, y se crearon 2 indicadores que se consideraban útiles para la evaluación, teniendo un total de 9 indicadores (proceso n=7 y de resultados n=2). Los resultados de 5 de los indicadores demostraron una fiabilidad perfecta, solo dos de ellos una fiabilidad moderada, el Nº 3 “Registro del tipo de test utilizado en la detección de la disfagia” con un valor kappa de 0,47, y el indicador Nº 6 “ITC a nutrición/endocrino al paciente con SNG”, con un valor kappa de 0,50. Los tres indicadores con mayor incumplimiento de la 1º medición fueron: el Nº 3 “Registro de test utilizado para la detección de la disfagia” (IC 95% 0-10,90), Nº 4 “Registro de complicaciones respiratorias” (IC 95% 3,60-24,30), y el indicador Nº 1 “Realización del test de disfagia” (IC 95% 24,90-54,10). Las mejoras conseguidas de los 3 principales indicadores tras la implantación de la VC fueron: el Nº 7 “ITC a RHB disfagia al paciente con disfagia al alta” MR=100% y MA=38%, Nº4 “Registro de complicaciones respiratorias” MR=94% MA=81%, y el Nº 6 “ITC a nutrición/endocrino al paciente con SNG” MR= 76% MA=38. El indicador que no mejoró, pero tampoco empeoró fue el Nº 2 “Registro de los tipos de disfagia” ya que tuvo el mismo cumplimiento tanto en la primera como en la segunda medición. Conclusiones: el diseño de la VC ha logrado unificar algunos criterios entre los profesionales para que los pacientes se beneficien de una atención multidisciplinar en la detección de la disfagia. La inclusión del MECV-V como test de detección de disfagia en la UI, permitirá que todas las unidades del hospital trabajen de manera homogénea con el mismo test. El mapeo de procesos, permite reconocer gráficamente la trayectoria de las funciones que desempeñan los sanitarios. Se han pilotado 9 indicadores factibles y fiables a la hora de medir la calidad de la atención de la disfagia en pacientes con ictus. Se corrigieron ITC a nutrición/endocrino, rehabilitación disfagia, e ITC a ORL, así como también protocolos de glucemia y electrocardiograma.Introduction: clinical pathways (CPWs) are tools that develop activities to avoid the appearance of problems and are applied to a predictable clinic, facilitate multidisciplinary care for patients, where they describe activities, sequences and duration of any care process, intervening in coordination between health workers. Stroke is a health problem of the first order and its neurological assistance is urgent. Good practice dictates that should be treated in stroke units with continuous monitoring and specific care. One of the most important symptoms in stroke is dysphagia, which affects the patient on multiple levels, assuming the first cause of aspirations and respiratory complications, making it necessary to identify it in order to give timely treatment. Objectives: to assess the quality of care for dysphagia, to analyze the results of the implementation of a pathway to improve its identification and management, unify criteria for daily nursing action, map the processes of detection and treatment of dysphagia, define indicators to measure the quality of the care process, and identify opportunities for improvement in the processes of dysphagia care in stroke patients. Material and methods: this project constituted the design and implementation of a CPW. It included a cross-sectional descriptive observational study for the validation of indicators. In addition, for its implementation and evaluation, a quasi-experimental design was developed with an improvement cycle methodology. It was developed in the stroke unit of the Hospital Clinic Universitary Virgen of the Arrixaca, with a sample of 43 patients for evaluations, and the entire sample size for indicators of low casuistry. All patients admitted to the stroke unit between March-July 2021 were included for the 1st measurement, and from January to March 2022 for the 2nd measurement. The work was developed thanks to the support of a multidisciplinary working group. Results: through the process of elaboration of the CPW, the quality of care for dysphagia in the stroke unit was assessed. Criteria for action in nursing practice included in the CPW were unified, which is composed of documents such as the final activity matrix, dysphagia detection flowchart, variation sheet, information sheet for relatives/patients, and quick guide on dysphagia after a stroke. To evaluate the implementation of the CPW, 7 indicators were selected, extracted from a search of articles, and 2 indicators were created that were considered useful for the evaluation, having a total of 9 indicators (process n=7 and results n=2). The results of 5 of the indicators demonstrated perfect reliability, only two of them moderate reliability, No. 3 "Register of the type of test used in the detection of dysphagia" with a kappa value of 0.47, and indicator No. 6 "ITC to nutrition/endocrine in the patient with NGS", with a kappa value of 0.50. The three indicators with the highest non-compliance with the 1st measurement were: No. 3 "Register of the test used to detect dysphagia" (95% CI 0-10.90), No. 4 "Register of respiratory complications" (95% CI 3.60-24.30), and indicator No. 1 "Performance of the dysphagia test" (95% CI 24.90-54.10). The improvements achieved in the 3 main indicators after CPW implantation were: No. 7 "ITC to RHB dysphagia in the patient with dysphagia at discharge" MR=100% and MA=38%, No. 4 "Register of respiratory complications" MR=94% MA=81%, and No. 6 "ITC to nutrition/endocrine in the patient with SNG" MR=76% MA=38. The indicator that did not improve, but did not worsen either, was No. 2 "Register of types of dysphagia" since it had the same compliance both in the first and in the second measurement. Conclusions: the design of the CPW has managed to unify some criteria among professionals so that patients benefit from multidisciplinary care in the detection of dysphagia. The inclusion of the volumen viscosity swallow test as a dysphagia detection test in the stroke unit will allow all hospital units to work homogeneously with the same test. Process mapping allows the trajectory of the functions carried out by the healthcare workers to be recognized graphically. Nine feasible and reliable indicators have been piloted when measuring the quality of care for dysphagia in patients with stroke. Interconsultations to nutrition/endocrine, dysphagia rehabilitation, and interconsultations to Otorhinolaryngology, glycemia and electrocardiogram protocols were corrected

Structure of the catalytic domain of the Tannerella forsythia matrix metallopeptidase karilysin in complex with a tetrapeptidic inhibitor

5 páginas, 1 figura, 1 tabla.-- et al.Karilysin is the only metallopeptidase identified as a virulence factor in the odontopathogen Tannerella forsythia owing to its deleterious effect on the host immune response during bacterial infection. The very close structural and sequence-based similarity of its catalytic domain (Kly18) to matrix metalloproteinases suggests that karilysin was acquired by horizontal gene transfer from an animal host. Previous studies by phage display identified peptides with the consensus sequence XWFPXXXGGG (single-letter amino-acid codes; X represents any residue) as karilysin inhibitors with low-micromolar binding affinities. Subsequent refinement revealed that inhibition comparable to that of longer peptides could be achieved using the tetrapeptide SWFP. To analyze its binding, the high-resolution crystal structure of the complex between Kly18 and SWFP was determined and it was found that the peptide binds to the primed side of the active-site cleft in a substrate-like manner. The catalytic zinc ion is clamped by the α-amino group and the carbonyl O atom of the serine, thus distantly mimicking the general manner of binding of hydroxamate inhibitors to metallopeptidases and contributing, together with three zinc-binding histidines from the protein scaffold, to an octahedral-minus-one metal-coordination sphere. The tryptophan side chain penetrates the deep partially water-filled specificity pocket of Kly18. Together with previous serendipitous product complexes of Kly18, the present results provide the structural determinants of inhibition of karilysin and open the field for the design of novel inhibitory strategies aimed at the treatment of human periodontal disease based on a peptidic hit molecule. © 2013.This study was supported in part by grants from European, American, Polish, Spanish, Danish and Catalan agencies (2012/04/A/NZ1/00051, 2011/03/N/NZ1/00586, 2137/7.PR-EU/2011/2, DE09761, FP7-HEALTH-F3-2009-223101 ‘AntiPathoGN’, FP7-HEALTH-2010-261460 ‘Gums&Joints’, FP7-PEOPLE-2011-ITN-290246 ‘RAPID’, BIO2009-10334, BFU2012-32862, CSD2006-00015, Lundbeck Foundation grant R54-A5291 and Fundació ‘La Marató de TV3’ grants 2009-100732 and 2009SGR1036). The Faculty of Biochemistry, Biophysics and Biotechnology of the Jagiellonian University in Kraków (Poland) is a beneficiary of structural funds from the European Union (grant No POIG.02.01.00-12-064/08 ‘Molecular Biotechnology for Health’).Peer Reviewe

Structure and mechanism of a bacterial host-protein citrullinating virulence factor, Porphyromonas gingivalis peptidylarginine deiminase

Theodoros Goulas et al.Citrullination is a post-translational modification of higher organisms that deiminates arginines in proteins and peptides. It occurs in physiological processes but also pathologies such as multiple sclerosis, fibrosis, Alzheimer's disease and rheumatoid arthritis (RA). The reaction is catalyzed by peptidylarginine deiminases (PADs), which are found in vertebrates but not in lower organisms. RA has been epidemiologically associated with periodontal disease, whose main infective agent is Porphyromonas gingivalis. Uniquely among microbes, P. gingivalis secretes a PAD, termed PPAD (Porphyromonas peptidylarginine deiminase), which is genetically unrelated to eukaryotic PADs. Here, we studied function of PPAD and its substrate-free, substrate-complex, and substrate-mimic-complex structures. It comprises a flat cylindrical catalytic domain with five-fold α/β-propeller architecture and a C-terminal immunoglobulin-like domain. The PPAD active site is a funnel located on one of the cylinder bases. It accommodates arginines from peptide substrates after major rearrangement of a >Michaelis loop> that closes the cleft. The guanidinium and carboxylate groups of substrates are tightly bound, which explains activity of PPAD against arginines at C-termini but not within peptides. Catalysis is based on a cysteinehistidine-asparagine triad, which is shared with human PAD1-PAD4 and other guanidino-group modifying enzymes. We provide a working mechanism hypothesis based on 18 structure-derived point mutants.This study was financially supported in part by grants from European, US American, Polish, Spanish, and Catalan agencies (UMO-2012/04/A/NZ1/00051, UMO-2012/05/B/NZ6/00581, UMO-2013/08/W/NZ1/00696, 2137/7.PR-EU/2011/2, 2975/7.PR/13/2014/2, NIH NIDCR DE09761; FP7-PEOPLE-2011-ITN-290246 “RAPID”; FP7-HEALTH-2012-306029-2 “TRIGGER”; FP7-HEALTH-2010-261460 “Gums&Joints”; BFU2012-32862; BFU2012-33516; BFU2012-35367; BIO2013-49320-EXP; MDM-2014-0435; 2014SGR9 and 2014SGR997). IGF acknowledges an FPU-fellowship (AP2010-3799) from the former Spanish Ministry for Education, Culture and Sport. TG acknowledges a “Juan de la Cierva” research contract (JCI-2012-13573) from the Spanish Ministry for Economy and Competitiveness. The Department of Structural Biology of IBMB is a “María de Maeztu” Unit of Excellence from the Ministry of Economy and Competitiveness. Funding for data collection was provided in part by ESRFPeer Reviewe

Structural determinants of inhibition of Porphyromonas gingivalis gingipain K by KYT-36, a potent, selective, and bioavailable peptidase inhibitor

Abstract Porphyromonas gingivalis is a member of the dysbiotic oral microbiome and a “keystone pathogen” that causes severe periodontal disease, which is among the most prevalent infectious diseases. Part of the virulence factors secreted by P. gingivalis are the essential cysteine peptidases gingipain K (Kgp) and R (RgpA and RgpB), which account for 85% of the extracellular proteolytic activity of the pathogen and are thus prime targets for inhibition. We report the high-resolution (1.20 Å) complex structure of Kgp with KYT-36, a peptide-derived, potent, bioavailable and highly selective inhibitor, which is widely used for studies in vitro, in cells and in vivo. Sub-nanomolar inhibition of Kgp is achieved by tight binding to the active-site cleft, which is covered for its sub-sites S3 through S1’ under establishment of nine hydrophobic interactions, 14 hydrogen bonds and one salt bridge. In addition, an inhibitor carbonyl carbon that mimics the scissile carbonyl of substrates is pyramidalized and just 2.02 Å away from the catalytic nucleophile of Kgp, C477Sγ. Thus, the crystal structure emulates a reaction intermediate of the first nucleophilic attack during catalysis of cysteine peptidases. The present study sets the pace for the development of tailored next-generation drugs to tackle P. gingivalis

Repositioning small molecule drugs as allosteric inhibitors of the BFT-3 toxin from enterotoxigenic Bacteroides fragilis

Bacteroides fragilis is an abundant commensal component of the healthy human colon. However, under dysbiotic conditions, enterotoxigenic B. fragilis (ETBF) may arise and elicit diarrhea, anaerobic bacteremia, inflammatory bowel disease, and colorectal cancer. Most worrisome, ETBF is resistant to many disparate antibiotics. ETBF's only recognized specific virulence factor is a zinc-dependent metallopeptidase (MP) called B. fragilis toxin (BFT) or fragilysin, which damages the intestinal mucosa and triggers disease-related signaling mechanisms. Thus, therapeutic targeting of BFT is expected to limit ETBF pathogenicity and improve the prognosis for patients. We focused on one of the naturally occurring BFT isoforms, BFT-3, and managed to repurpose several approved drugs as BFT-3 inhibitors through a combination of biophysical, biochemical, structural, and cellular techniques. In contrast to canonical MP inhibitors, which target the active site of mature enzymes, these effectors bind to a distal allosteric site in the proBFT-3 zymogen structure, which stabilizes a partially unstructured, zinc-free enzyme conformation by shifting a zinc-dependent disorder-to-order equilibrium. This yields proBTF-3 incompetent for autoactivation, thus ablating hydrolytic activity of the mature toxin. Additionally, a similar destabilizing effect is observed for the activated protease according to biophysical and biochemical data. Our strategy paves a novel way for the development of highly specific inhibitors of ETBF-mediated enteropathogenic conditions

Molecular and in vivo studies of a glutamate-class prolyl-endopeptidase for coeliac disease therapy

The digestion of gluten generates toxic peptides, among which a highly immunogenic proline-rich 33-mer from wheat α-gliadin, that trigger coeliac disease. Neprosin from the pitcher plant is a reported prolyl endopeptidase. Here, we produce recombinant neprosin and its mutants, and find that full-length neprosin is a zymogen, which is self-activated at gastric pH by the release of an all-β pro-domain via a pH-switch mechanism featuring a lysine plug. The catalytic domain is an atypical 7+8-stranded β-sandwich with an extended active-site cleft containing an unprecedented pair of catalytic glutamates. Neprosin efficiently degrades both gliadin and the 33-mer in vitro under gastric conditions and is reversibly inactivated at pH > 5. Moreover, co-administration of gliadin and the neprosin zymogen at the ratio 500:1 reduces the abundance of the 33-mer in the small intestine of mice by up to 90%. Neprosin therefore founds a family of eukaryotic glutamate endopeptidases that fulfils requisites for a therapeutic glutenase.This study was supported in part by grants from Spanish and Catalan public and private bodies (grant/fellowship references PID2019-107725RG-I00 to F.X.G.-R., A.R.B., U.E. and T.G.; BES-2016-076877 to S.R.M., BES-2015-074583 to L.A.M., Beatriu de Pinós 2018BP00163 to U.E., 2017SGR3 and Fundació La Marató de TV3 201815 to F.X.G.-R., U.E., A.R.B. and T.G.)

Structure and mechanism of a bacterial host-protein citrullinating virulence factor, Porphyromonas gingivalis peptidylarginine deiminase.

Citrullination is a post-translational modification of higher organisms that deiminates arginines in proteins and peptides. It occurs in physiological processes but also pathologies such as multiple sclerosis, fibrosis, Alzheimer's disease and rheumatoid arthritis (RA). The reaction is catalyzed by peptidylarginine deiminases (PADs), which are found in vertebrates but not in lower organisms. RA has been epidemiologically associated with periodontal disease, whose main infective agent is Porphyromonas gingivalis. Uniquely among microbes, P. gingivalis secretes a PAD, termed PPAD (Porphyromonas peptidylarginine deiminase), which is genetically unrelated to eukaryotic PADs. Here, we studied function of PPAD and its substrate-free, substrate-complex, and substrate-mimic-complex structures. It comprises a flat cylindrical catalytic domain with five-fold α/β-propeller architecture and a C-terminal immunoglobulin-like domain. The PPAD active site is a funnel located on one of the cylinder bases. It accommodates arginines from peptide substrates after major rearrangement of a "Michaelis loop" that closes the cleft. The guanidinium and carboxylate groups of substrates are tightly bound, which explains activity of PPAD against arginines at C-termini but not within peptides. Catalysis is based on a cysteine-histidine-asparagine triad, which is shared with human PAD1-PAD4 and other guanidino-group modifying enzymes. We provide a working mechanism hypothesis based on 18 structure-derived point mutants

De novo design of immunoglobulin-like domains

Antibodies, and antibody derivatives such as nanobodies, contain immunoglobulin-like (Ig) β-sandwich scaffolds which anchor the hypervariable antigen-binding loops and constitute the largest growing class of drugs. Current engineering strategies for this class of compounds rely on naturally existing Ig frameworks, which can be hard to modify and have limitations in manufacturability, designability and range of action. Here, we develop design rules for the central feature of the Ig fold architecture—the non-local cross-β structure connecting the two β-sheets—and use these to design highly stable Ig domains de novo, confirm their structures through X-ray crystallography, and show they can correctly scaffold functional loops. Our approach opens the door to the design of antibody-like scaffolds with tailored structures and superior biophysical properties.This research was supported by grants from the Spanish Ministry of Science and Innovation (RYC2018-025295-I, EUR2020-112164, and PID2020-120098GA-I00). This study was also supported in part by grants from Spanish and Catalan public and private bodies (grant/fellowship references MCIN/AEI/10.13039/501100011033/PID2019-107725RG-I00, 2017SGR3 and Fundació “La Marató de TV3” 201815). S.R.M. acknowledges grant BES2016-076877 from the Spanish State Agency for Research (MCIN/AEI/10.13039/501100011033) and the European Social Fund “ESF invests in your future”. U.E. was funded by a Beatriu de Pinós post-doctoral fellowship (AGAUR-MSCA COFUND 2018BP00163. J.R.T. was supported by an EMBO postdoctoral fellowship (under grant agreement ALTF 145-2021). J.C.K. was supported by a National Science Foundation Graduate Research Fellowship (grant DGE-1256082). D.B. and T.M.C. acknowledge the Howard Hughes Medical Institute. We thank the Princess Margaret Cancer Centre for funding of the NMR facility. The Structural Genomics Consortium is a registered charity (no: 1097737) that receives funds from Bayer AG, Boehringer Ingelheim, Bristol Myers Squibb, Genentech, Genome Canada through Ontario Genomics Institute [OGI-196], EU/EFPIA/OICR/McGill/KTH/Diamond Innovative Medicines Initiative 2 Joint Undertaking [EUbOPEN grant 875510], Janssen, Merck KGaA (aka EMD in Canada and US), Pfizer and Takeda