ELUCIDATION OF THE STRUCTURE OF THE RECOMBINANT PRPSC PRIONS (Q3165564)
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Project Q3165564 in Spain
| Language | Label | Description | Also known as |
|---|---|---|---|
| English | ELUCIDATION OF THE STRUCTURE OF THE RECOMBINANT PRPSC PRIONS |
Project Q3165564 in Spain |
Statements
96,800.0 Euro
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121,000.0 Euro
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80.0 percent
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1 January 2018
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31 December 2020
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UNIVERSIDAD DE SANTIAGO DE COMPOSTELA
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42°52'49.51"N, 8°32'45.10"W
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15078
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LOS PRIONES SON PROTEINAS INFECCIOSAS. SON VERSIONES MAL-PLEGADA DE PRTEINAS ISOSECUENCIALES CON PLEGAMIENTO NORMAL QUE SE PROPAGAN FORZANDO SU CONFORMACION EN SU PRECURSOR. LA PRPSC (PROTEINA PRIONICA, ISOFORMA SCRAPIE), EL PRIMER PRION QUE SE DESCUBRIO, ES EL AGENTE CAUSAL DE ENFERMEDADES NEURODEGENERATIVAS MORTALES QUE AFECTAN A LOS SERES HUMANOS, COMO LA ENFERMEDAD DE CREUTZFELDT-JAKOB, Y A LOS ANIMALES, COMO LA ENCEFALOPATIA ESPONGIFORME BOVINA (EEB), O ¿ENFERMEDAD DE LAS VACAS LOCAS¿. INVESTIGACIONES LLEVADAS A CABO POR NUESTRO GRUPO Y OTROS HAN DEMOSTRADO QUE LA ARQUITECTURA DE LA PRPSC CONSISTE EN UN SOLENOIDE- DE 4 PISOS, QUE SE APILA PARA FORMAR PROTOFILAMENTOS AMILOIDES. EL CONOCIMIENTO DE ESTAS CARACTERISTICAS ESTRUCTURALES, HA PERMITIDO COMPRENDER COMO SE PROPAGA LA PRPSC: LAS LAMINAS- DESAPAREADAS DE LOS PISOS SUPERIOR E INFERIOR DEL PROTOFILAMENTO ACTUAN DE MOLDE PARA CUALQUIER MOLECULA DE PRP DESPLEGADA QUE SE ACERQUE, FORZANDOLA A FORMAR LAMINAS- SIMILARES, Y POSTERIORMENTE ENROLLARSE PARA FORMAR PISOS ADICIONALES A MEDIDA QUE APARECEN NUEVAS SUPERFICIES DE LAMINAS- DESAPAREADAS. SIN EMBARGO, HAY MUCHOS ASPECTOS DE LA BIOLOGIA DE LA PRPSC QUE NO PUEDEN EXPLICARSE CON EL GRADO DE CONOCIMIENTO ESTRUCTURAL ACTUAL. EN PARTICULAR, LA EXISTENCIA DE BARRERAS DE TRANSMISION ENTRE ESPECIES, O LA EXISTENCIA DE ¿CEPAS¿ DE PRPSC QUE TENIENDO LA MISMA SECUENCIA, EXHIBEN PROPIEDADES BIOQUIMICAS Y BIOLOGICAS DIFERENTES. EN ESTE CONTEXTO, NUESTRA PROPUESTA BUSCA LA OBTENCION DE MAS INFORMACION SOBRE LA ESTRUCTURA DE LA PRPSC , PARA PODER ASI REFINAR NUESTRO MODELO ESTRUCTURAL ACTUAL. ES LA CONTINUACION DE NUESTRO PROYECTO DEL PLAN NACIONAL BFU2013-48436-C2-1-P. A TRAVES DE ESE PROYECTO, ABRIMOS EL CAMINO PARA USAR PRPSC RECOMBINANTE (RECPRPSC) COMO UN ANALOGO ADECUADO DE LA PRPSC DE CEREBRO EN ESTUDIOS ESTRUCTURALES. PARA ELLO COLABORAMOS CON JOAQUIN CASTILLA (CIC-BIOGUNE, BILBAO) EN EL DESARROLLO DE METODOS PARA PRODUCIR CANTIDADES SIGNIFICATIVAS DE RECPRPSC INFECCIOSA. POSTERIORMENTE, DETERMINAMOS QUE LA RECPRPSC Y LA PRPSC DE CEREBRO COMPARTEN UNA MISMA ARQUITECTURA BASICA Y DESARROLLAMOS METODOS DE RMN EN SOLUCION PARA ESTUDIAR LA RECPRPSC. _x000D_ PROVISTOS DE TODO ESTO, NOS PROPONEMOS: 1) DILUCIDAR SI EL APILAMIENTO DE LAS UNIDADES DE RECPRPSC A LO LARGO DEL PROTOFILAMENTO DE RECPRPSC ES ¿CABEZA-CABEZA¿ O ¿CABEZA-COLA¿; 2: EXPANDIR LA INFORMACION OBTENIDA HASTA LA FECHA MEDIANTE PROTEOLISIS LIMITADA, ACOPLANDO LA TECNICA A UN DESPLEGAMIENTO PARCIAL DE LA RECPRPSC. ESPECIFICAMENTE, BUSCAMOS CONFIRMAR LA IDENTIDAD DE LAS LAMINAS- Y LAZOS QUE HEMOS IDENTIFICADO TENTATIVAMENTE, E IDENTIFICAR MAS, PARTICULARMENTE EN LOS 2 PISOS C-TERMINALES; 3: EXPANDIR LA METODOLOGIA DE RMN EN SOLUCION DESARROLLADA EN BFU2013-48436-C2-1-P UTILIZANDO APROXIMACIONES EXPERIMENTALES DIFERENTES PARA DESPLEGAR PARCIALMENTE LA RECPRPSC, Y ASI PODER CONFIRMAR Y AMPLIAR LOS DATOS OBTENIDOS; 4: INICIAR ESTUDIOS DE RMM DE ESTADO SOLIDO DE LA RECPRPSC; NOS PROPONEMOS EMPEZAR CON EXPERIMENTOS SIMPLES 1D PARA CALCULAR CUANTOS AMINOACIDOS DE CADA TIPO ESTAN LOCALIZADOS EN LAMINAS- Y CUANTOS EN LAZOS; 5: INTEGRAR TODOS LOS DATOS OBTENIDOS EN UN MODELO ESTRUCTURAL CADA VEZ MAS REFINADO DE LA RECPRPSC, QUE INCLUIRA UN ALINEAMIENTO CON ESQUELETOS -SOLENOIDEOS CONOCIDOS. TODO ESTO DEBERIA CONDUCIR A UN MODELO ATOMISTICO DE LA PRPSC QUE ESPERAMOS SE CONVIERTA EN UNA REFERENCIA PARA EL CAMPO. (Spanish)
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PRIONS ARE INFECTIOUS PROTEINS. THEY ARE MISFOLDED VERSIONS OF NORMALLY FOLDED ISOSEQUENTIAL PROTEINS. PRIONS PROPAGATE BY FORCING THEIR CONFORMATION INTO THEIR NORMALLY FOLDED PRECURSORS. PRPSC (PRION PROTEIN, SCRAPIE ISOFORM), THE FIRST PRION TO BE DISCOVERED, IS THE CAUSAL AGENT OF DEADLY NEURODEGENERATIVE DISEASES AFFECTING HUMANS, LIKE CREUTZFELDT-JAKOB DISEASE, AND ANIMALS, LIKE BOVINE SPONGIFORM ENCEPHALOPATHY (BSE), ALSO KNOWN AS ¿MAD COW DISEASE¿. RESEARCH FROM OUR AND OTHER GROUPS HAS SHOWN THAT THE ARCHITECTURE OF PRPSC CONFORMS TO A 4-RUNG -SOLENOID, THAT STACKS TO FORM AMYLOID PROTOFILAMENTS. KNOWLEDGE OF THESE STRUCTURAL CHARACTERISTICS, STILL OF A LOW RESOLUTION, ALLOW, NEVERTHELESS, AN UNDERSTANDING OF HOW PRPSC PROPAGATES: THE UNPAIRED -STRANDS IN THE UPPER- AND LOWERMOST RUNGS OF THE PROTOFILAMENT CAN TEMPLATE AN INCOMING UNFOLDED PRP MOLECULE TO FORM SIMILAR -STRANDS, AND THEN COIL TO FORM ADDITIONAL RUNGS AS FRESH UNPAIRED -STRANDS ARE FORMED. HOWEVER, THERE ARE MANY KEY ASPECTS OF PRPSC BIOLOGY THAT MUST BE OBVIOUSLY ENCIPHERED IN ITS STRUCTURE AND THEREFORE CANNOT BE EXPLAINED WITH THE CURRENT LEVEL OF STRUCTURAL RESOLUTION. IN PARTICULAR, THE EXISTENCE OF TRANSMISSION BARRIERS BETWEEN SPECIES OR PRPSC STRAINS, WHICH HAVING THE SAME SEQUENCE, EXHIBIT DIFFERENT BIOCHEMICAL AND BIOLOGICAL PROPERTIES. FURTHERMORE, A MORE DETAILED ELUCIDATION OF THE STRUCTURE OF PRPSC WILL HELP RATIONALLY DESIGN DRUGS FOR THESE CURRENTLY FATAL DISEASES._x000D_ IN THIS CONTEXT, OUR PROPOSAL AIMS AT OBTAINING FURTHER STRUCTURAL INFORMATION ON THE STRUCTURE OF PRPSC PRIONS, SO AS TO REFINE THE CURRENT STRUCTURAL MODEL. IT IS A CONTINUATION OF OUR CURRENT NATIONAL GRANT, BFU2013-48436-C2-1-P. THROUGH THIS PROJECT, WE PAVED THE WAY TO USE RECOMBINANT PRPSC (RECPRPSC) AS AN APPROPRIATE SURROGATE OF BRAIN-DERIVED PRPSC FOR STRUCTURAL STUDIES. FOR THIS, WE COLLABORATED WITH JOAQUIN CASTILLA (CIC-BIOGUNE, BILBAO) TO DEVELOP METHODS TO PRODUCE SIGNIFICANT AMOUNTS OF FULLY INFECTIOUS RECPRPSC. THEN, USING LIMITED PROTEOLYSIS AND ELECTRON MICROSCOPY STUDIES, WE OBTAINED STRONG EVIDENCE INDICATING THAT BRAIN AND RECPRPSC SHARE A COMMON BASIC ARCHITECTURE. FINALLY, WE DEVELOPED SOLUTION NMR METHODS TO STUDY RECPRPSC. _x000D_ ARMED WITH ALL THIS, WE PROPOSE NOW: 1) TO ELUCIDATE WHETHER THE STACKING OF RECPRPSC SUBUNIT ALONG THE RECPRPSC PROTOFILAMENT IS HEAD-TO-HEAD OR HEAD-TO-TAIL; 2: TO EXPAND THE LIMITED PROTEOLYSIS DATA OBTAINED THUS FAR, COUPLING IT TO PARTIAL UNFOLDING. WE SPECIFICALLY SEEK TO CONFIRM THE IDENTITY OF -STRANDS AND LOOPS THAT WE HAVE ALREADY TENTATIVELY IDENTIFIED, AND TO IDENTIFY ADDITIONAL -STRANDS AND LOOPS IN THE 2 C-TERMINAL RUNGS; 3: TO EXPAND THE SOLUTION NMR METHODOLOGY DEVELOPED THROUGH BFU2013-48436-C2-1-P USING ALTERNATIVE APPROACHES TO RECPRPSC PARTIAL UNFOLDING, SO AS TO CONFIRM AND EXTEND THE DATA THAT WE HAVE OBTAINED THUS FAR; 4: TO INITIATE SOLID STATE NMR APPROACHES TO STUDY RECPRPSC DIRECTLY. IN PARTICULAR, WE PROPOSE TO BEGIN WITH A SIMPLE 1D SOLID NMR APPROACH TO ASSESS HOW MANY AMINO ACIDS OF EACH TYPE ARE LOCATED IN -STRANDS VS. COILS; 5: TO INTEGRATE ALL STRUCTURAL RESTRAINTS OBTAINED THROUGH THE PRECEDING AIMS INTO AN EVER MORE REFINED STRUCTURAL MODEL OF RECPRPSC, INCLUDING THREADING AND MODELING USING KNOWN -SOLENOID SCAFFOLDS.ALL THIS SHOULD LEAD TO AN IMPROVED, ATOMISTIC MODEL OF PRPSC WHICH SHOULD BECOME A REFERENCE IN THE FIELD. (English)
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Santiago de Compostela
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Identifiers
BFU2017-86692-P
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