PhD THESIS USING RECOMBINANT HUMAN CARBAMOYL PHOSPHATE SYNTHETASE 1 (CPS1) FOR STUDYING THIS ENZYME’S FUNCTION, REGULATION, PATHOLOGY AND STRUCTURE Author: CARMEN DÍEZ FERNÁNDEZ Directors: Drs. Vicente Rubio Zamora y Javier Cervera Miralles May 2015 INSTITUTO DE BIOMEDICINA DE VALENCIA (IBV) Vicente Rubio Zamora, Doctor en Medicina, Profesor de Investigación del Consejo Superior de Investigaciones Científicas (CSIC) y Profesor Titular de Bioquímica (en Excedencia) de la Universidad de Valencia, y Javier Cervera Miralles, Doctor en Biología, Investigador jubilado del Centro de Investigación Príncipe Felipe de Valencia, de la Fundación Valenciana de Investigaciones Biomédicas de Valencia, CERTIFICAN: Que Dª, Carmen Díez Fernández, Ingeniera Agrónoma por la Universidad Politécnica de Valencia, con DNI 29208872E, ha realizado bajo su dirección el trabajo de Tesis Doctoral que lleva por título (en lengua inglesa): “Using recombinant human carbamoyl phosphate synthetase 1 (CPS1) for studying this enzyme’s function, regulation, pathology and structure”. Que están de acuerdo con los contenidos del trabajo, realizado en la modalidad de acopio de publicaciones. Que entienden que el trabajo reúne los requisitos para ser susceptible de defensa ante el Tribunal apropiado, por incluir dos publicaciones ya aparecidas y una sometida a evaluación, en revistas internacionales de amplia difusión y en las que la doctoranda es primera autora; así como porque el contenido de las otras secciones de dicho trabajo de tesis reflejan adecuadamente el estado del conocimiento y discuten apropiadamente el contenido y las implicaciones del trabajo realizado; porque las conclusiones recogen un avance suficiente en el campo objeto de estudio como para hacer a la candidata merecedora del doctorado; y, finalmente, porque les consta que ninguno de los coautores de dichas publicaciones, todos ellos ya doctores, han utilizado ni utilizarán las mismas para confeccionar otro trabajo de tesis doctoral. Valencia, a 3 de Marzo de 2015. Fdo. Vicente Rubio Zamora Fdo. Javier Cervera Miralles C/ JAUME ROIG, 11 46010 VALENCIA ESPAÑA TEL.: 96 339 17 60 FAX: 96 369 08 00 ACEPTACIÓN DE LOS COAUTORES PARA QUE LA DOCTORANDA PRESENTE LOS SIGUIENTES ARTÍCULOS COMO TESIS; Y RENUNCIA EXPRESA A PRESENTARLOS COMO PARTE DE OTRA TESIS DOCTORAL ACCEPTANCE OF THE COAUTHORS FOR THE SUBMISSION BY THE DOCTORAL CANDIDATE OF THE FOLLOWING RESEARCH PUBLICATIONS OR MANUSCRIPTS AS PART OF HER DOCTORAL THESIS, DECLINING THE USE OF THESE PUBLICATIONS AS A PART OF ANOTHER THESIS WORK Los/las coautores/coautoras de los artículos científicos citados al pie, Drs. Ana Isabel Martinez, Satu Pekkala, Belén Barcelona, Isabel Pérez- Arellano, Ana María Guadalajara, Marshall Summar, Liyan Hu, José Gallego, Johannes Häberle, Javier Cervera Miralles y Vicente Rubio Zamora, aceptan que la doctoranda Dª. Carmen Díez Fernández, con DNI 29208872E, presente los artículos listados al pie como parte de la Tesis Doctoral con título “USING RECOMBINANT HUMAN CARBAMOYL PHOSPHATE SYNTHETASE 1 (CPS1) FOR STUDYING THIS ENZYME’S FUNCTION, REGULATION, PATHOLOGY AND STRUCTURE”, y renuncian a presentarlos como parte de otra tesis doctoral. The coauthors of the research papers listed below, Drs. Ana Isabel Martinez, Satu Pekkala, Belén Barcelona, Isabel Pérez-Arellano, Ana María Guadalajara, Marshall Summar, Liyan Hu, José Gallego, Johannes Häberle, Javier Cervera Miralles and Vicente Rubio Zamora, agree with the inclusion of the below listed research papers as a part of the Doctoral Thesis of Ms. Carmen Díez Fernández (Spanish DNI 29208872E), entitled “USING RECOMBINANT HUMAN CARBAMOYL PHOSPHATE SYNTHETASE 1 (CPS1) FOR STUDYING THIS ENZYME’S FUNCTION, REGULATION, PATHOLOGY AND STRUCTURE”. They decline to use them as a part of another doctoral thesis. Artículos científicos: Scientific articles: [1] Diez-Fernandez C, Martínez AI, Pekkala S, Barcelona B, Pérez-Arellano I, Guadalajara AM, Summar M, Cervera J, Rubio V. 2013. Molecular characterization of carbamoyl-phosphate synthetase (CPS1) deficiency using human recombinant CPS1 as a key tool. Hum Mutat. 34:1149-1159. [2] Díez-Fernández C, Hu L, Cervera J, Häberle J, Rubio V. (2014) Understanding carbamoyl phosphate synthetase (CPS1) deficiency by using the recombinantly purified human enzyme: effects of CPS1 mutations that concentrate in a central domain of unknown function. Mol Genet Metab. 112:123-132. [3] Díez-Fernández C, Gallego J, Häberle J, Cervera J, Rubio V. (2015) Experimental studies on the inherited metabolic error carbamoyl phosphate synthetase 1 deficiency shed light on the mechanism for switching on/off the urea cycle. Submitted to Journal of Genetics and Genomics. Valencia, 3 de marzo 2015. Valencia, March 3, 2015. Dr. Javier Cervera Miralles Dr. Vicente Rubio Zamora Dr. Ana Isabel Martinez Dr. Satu Pekkala Dr. Belén Barcelona Dr. Isabel Pérez-Arellano Dr. Ana María Guadalajara Dr. Marshall Summar Dr. Liyan Hu Dr. Johannes Häberle Dr. José Gallego To carry out this PhD work entitled "USING RECOMBINANT HUMAN CARBAMOYL PHOSPHATE SYNTHETASE 1 (CPS1) FOR STUDYING THIS ENZYME’S FUNCTION, REGULATION, PATHOLOGY AND STRUCTURE", the doctoral candidate, Carmen Díez-Fernández was supported first by a Predoctoral fellowship of the Centro de Investigación Príncipe Felipe and then by an FPU fellowship (AP2009-2261) of the Ministry of Education of the Spanish Govenment, with additional funds from the same last source for a short-stay work visit in the Children's Hospital of Zürich. The work was carried out within group 739 of CIBERER-Instituto de Salud Carlos III (IP, V. Rubio) under the auspices of the following research projects: - BFU 2011-30407 of the Ministerio de Ciencia e Innovación of the Spanish Government, entitled "Luz estructural sobre señalización y regulación por nitrógeno y sobre biosíntesis de arginina/urea, sus errores congénitos y su conexión con biología del envejecimiento" (IP, V.Rubio). - Prometeo/2009/051 of the Consellería d'Educació de la Generalitat Valenciana, entitled: "BioMeder. Genes, proteínas y rutas de señalización en enfermedades raras" (investigators, F. Palau, P. Sanz y V. Rubio). - Fundación Alicia Koplowitz 2012: "Encefalopatía hiperamoniémica por déficit de carbamil fosfato sintetasa 1 (CPS1). Hacia el diagnóstico, consejo genético y tratamiento a través de la estructura atómica y estudios funcionales de CPS1" (IPs, J. Cervera y V. Rubio). - In Zurich, the work was carried out with the Swiss National Science Foundation grant 310030_127184 to Dr. Johannes Häberle. Summary Carbamoyl phosphate synthetase 1 (CPS1), a 1462-residue mitochondrial enzyme, catalyzes the entry of ammonia into the urea cycle, which converts ammonia, the neurotoxic waste product of protein catabolism, into barely toxic urea. The urea cycle inborn error and rare disease CPS1 deficiency (CPS1D) is inherited with mendelian autosomal recessive inheritance, being due to CPS1 gene mutations (>200 mutations reported), and causing life-threatening hyperammonemia. We have produced recombinantly human CPS1 (hCPS1) in a baculovirus/insect cell expression system, isolating the enzyme in active and highly purified form, in massive amounts. This has allowed enzyme crystallization for structural studies by X-ray diffraction (an off-shoot of the present studies). This hCPS1 production system allows site-directed mutagenesis and enzyme characterization as catalyst (activity, kinetics) and as protein (stability, aggregation state, domain composition). We have revealed previously unexplored traits of hCPS1 such as its domain composition, the ability of glycerol to replace the natural and essential CPS1 activator N-acetyl-L- glutamate (NAG), and the hCPS1 protection (chemical chaperoning) by NAG and by its pharmacological analog N-carbamyl-L-glutamate (NCG). We have exploited this system to explore the effects on the activity, kinetic parameters and stability/folding of the enzyme, and to test the disease-causing nature, of mutations identified in patients with CPS1 deficiency (CPS1D). These results, supplemented with those obtained with other non-clinical mutations, have provided novel information on the functions of three non- catalytic domains of CPS1. We have introduced three CPS1D-associated mutations and one trivial polymorphism in the glutaminase-like domain of CPS1, supporting a stabilizing and an activity-enhancing function of this non-catalytic domain. Two mutations introduced into the bicarbonate phosphorylation domain have shed light on bicarbonate binding and have directly confirmed the importance of this domain for NAG binding to the distant (in the sequence) C-terminal CPS1 domain. The introduction of 18 CPS1D-associated missense mutations mapping in a clinically highly eloquent central non-catalytic domain have proven the disease- causing nature of most of these mutations while showing that in most of the cases they trigger enzyme misfolding and/or destabilization. These results, by proving an important role of this domain in the structural integration of the multidomain CPS1 protein, have led us to call this domain the Integrating Domain. Finally, we have examined the effects of eight CPS1D-associated mutations, of one trivial polymorphism and of five non-clinical mutations, all of them mapping in the C-terminal domain of the enzyme where NAG binds, whereas we have re-analyzed prior results with another four clinical and five non- clinical mutations affecting this domain. We have largely confirmed the pathogenic nature of the clinical mutations, predominantly because