Descripción del proyecto
ESTE PROYECTO CONTINUA NUESTRAS CONTRIBUCIONES PREVIAS E INTERESES, EL IP ES UN EXPERTO EN EL CICLO DE L UREA Y SUS ENFERMEDADES, CON PARTICULAR INTERES EN CARBAMIL FOSFATO SINTETASA 1 (CPS1), UN HITO MUY RECIENTE DEL GRUPO HA SIDO LA DETERMINACION DE LA ESTRUCTURA CRISTALINA DE LA CPS1 HUMANA EN FORMAS INACTIVA Y ACTIVADA POR N-ACETYL-L-GLUTAMATE (NAG), VARIOS DE LOS OBJETIVOS TRATAN DE AVANZAR EL CONOCIMIENTO EN CPS1, EXPLOTANDO NUESTRA INFORMACION ESTRUCTURAL, ACLARAREMOS POR QUE LA CPS1, A DIFERENCIA DE LAS DEMAS CPSS, USA AMONIO CONA ALTA AFINIDAD Y NO UTILIZA GLUTAMINA, LA ESTRUCTURA DE CPS1 DA PISTAS QUE VAMOS A CONFIORMAR, LA FALTA DE ACTIVIDAD GLUTAMINASA DEJA SIN FUINCION APARENTE A LA PORCION N-TERMINAL DE 40 KDA, SI BIEN ALGUNOS PACIENTES CON DEFICIT DE CPS1 TIENEN MUTACIONES EN ESTA REGION, USAREMOS MUTAGENESIS DIRIGIDA, INGENIERIA DE PROTEINAS Y LA PRODUCCION DE UNA CPS PROXIMA QUE SI USA GLUTAMINA, LA CPSIII PARA INVESTIGAR EL PAPEL DE ESTA PORCION N-TERMINAL, DETERMINANDO A LA VEZ LA PATOGENICIDAD DE LAS MUTACIONES ENCONTRADAS EN ELLA EN PACIENTES CONB DEFICIENCIA, ADEMAS TRATAREMOS DE DETERMINAR LA ESTRUCTURA CRISTALINA DE CPSIII, PARA ACLARAR LA EVOLUCION DESDE LA BIOSINTESIS DE ARGININA (CPS BACTERIANA) A LA UREOSMOSIS (CPSIII) Y AL UREOTELISMO (CPS1), APROVECHANDO NO SOLO LA NUEVA INFORMACION ESTRUCTURAL SINO TAMBIEN NUESTRA CAPACIDAD DE PRODUCIR CPS1 RECOMBINANTE, PURA Y ACTIVA, TRATAREMOS DE ACLARAR LA DISPUTA SOBRE SI LA ACILACION DE LA CPS1 EN SUS LISINAS DE SUPERFICIE Y SU DEACILACION POR SIRTUINA5, SUPUESTAMENTE CONEXION IMPORTANTE CON LA BIOLOGIA DEL "AGING", REALMENTE CONTROLA LA ACTIVIDAD CPS1 Y LA DEL CICLO DE LA UREA, LA MISMA APPROXIMACION, COMBINADA WITH THE FACT THAT WE ALSO PRODUCE THE HUMAN ENZYME FOR MAKING NAG, NAG SYNTHASE, WILL GIVE US A UNIQUE OPPORTUNITY TO EXAMINE EXPERIMENTALLY THE MECHANISMS OF SECONDARY HYPERAMMONEMIAS SUCH AS THOSE FOUND IN THE ORGANIC ACIDEMIAS AND IN VAPROIC ACID HYPERAMMONEMIA, WE WILL EXPLOIT ALSO OUR ABILITY TO MAKE PURE NAGS TO TRY TO DETERMINE ITS STRUCTURE BY A COMBIANTION OF LOW-RESOLUTION TECHNIQUES (EM) PLUS FITTING OF DOMAIN STRUCTURES, A SIMILAR APPROACH WILL BE USED FOR TRYING TO DETERMINE THE ARCHITECTURE OF THE BIFUNCTIONAL ORNITHINE AND PROLINE-MAKING ENZYME PYRROLINE 5-CARBOXYLATE SYNTHETASE (P5CS), WHICH IS OF PARTICULAR INTEREST GIVEN THE FACT THAT SOME OF THE MUTATIONS AFFECTING THIS ENZYME CAUSE DOMINAT PATHOLOGY, WHEREAS THE MAJORITY OF THEM ARE TRANSMITTED RECESSIVELY, NEVERTHELESS, WE WILL CONTINUE OUR EFFORTS TO DETERMINE THE CRYSTAL STRUCTURE OF P5CS, USING FRESH PROTEIN FROM APLANT SOURCE, GIVEN THE EXISTING DIFFICULTIES WITH THE HUMAN ENZYME, ANOTHER CENTER OF INTEREST OF OUR GROUP RELATED TO NITROGEN METBOLIC CONTROL IS THE SIOGNALING PROTEIN PII, A FIELD IN WHICH WE HAVE MADE VERY IMPRTANT CONTRIBUTIONS, THIS INTEREST IS REPRESENTED IN THIS PROJECTS IN TWO WAYS: BY TRYING TO UNDERTAND THE SIGNIFICANCE OF PII ADENYLYLATION, WHICH WE WILL STUDY BY TRYING TO DETERMINE THE STRUCTURE OF THE COMPLEX OF THE ADENYLYLATED PROTEIN WITH THE GENE REGULATOR AMTR, AND ALSO CONCERNING A NON-CANONIC FORM OF PII THAT IS VERY WIDESPREAD AND PRESENT IN ANIMALS, CALLED CUTA, AND FOR WHICH DESPITE ITS BIOLOGCIAL CONSTANCY, NO FUNCTION IS KNOWN, WE WILL USE A BACTERIAL MODEL AND A SIMPLE ANIMAL MODEL (C, ELEGANS) TO INVESTIGATE THE FUNCTION OF CUTA, CARBAMIL FOSFATO SINTETASA\PIRROLIN 5-CARBOXILATO SINTETASA\ACETILGLUTAMATO SINTASA\PROTEÍNA PII\AMTR\PROTEÍNA CUTA\UREOGÉNESIS\SIRTUINA 5