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Analysis of Different Evolutionary Strategies to Prevent Protein Aggregation

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Analysis of Different Evolutionary Strategies to Prevent Protein Aggregation Universitat Autònoma de Barcelona Departament de Bioquímica i Biologia Molecular and Institut de Biotecnologia i Biomedicina Analysis of Different Evolutionary Strategies to Prevent Protein Aggregation Ricardo Graña Montes Bellaterra, December 2014 Universitat Autònoma de Barcelona Departament de Bioquímica i Biologia Molecular and Institut de Biotecnologia i Biomedicina Analysis of Different Evolutionary Strategies to Prevent Protein Aggregation Doctoral thesis submitted by Ricardo Graña Montes in candidacy for the degree of Ph.D. in Biochemistry, Molecular Biology and Biomedicine from the Universitat Autònoma de Barcelona. The work described herein has been performed at the Department de Bioquímica i Biologia Molecular and at the Institut de Biotecnologia i Biomedicina, under the supervision of Prof. Salvador Ventura Zamora. Ricardo Graña Montes Prof. Salvador Ventura Zamora Bellaterra, December 2014 SUMMARY IN ENGLISH In the last 15 years, the study of protein aggregation has evolved from a mostly neglected topic of protein chemistry to a highly dynamic research area which has expanded its implications through different fields including biochemistry, biotechnology, nanotechnology and biomedicine. The analysis of protein aggregation has attracted a particular interest in the biomedical and biotechnological areas. Because, on one side, the formation of insoluble protein deposits is associated to an increasing number of human disorders, many of which present fatal pathological consequences. And on the other hand, aggregation is a frequent shortcoming in the recombinant expression of proteins at the industrial level, such as in the production of proteinaceous therapeutic agents like antibodies. Consequently, the survey of mechanisms to prevent protein aggregation is currently the focus of deep investigation with the aim to develop preventive or therapeutic methods for the intervention of these depositional disorders and to enhance the yield in the biotechnological production of proteins. The power of the computational tools developed to predict protein aggregation has fostered the identification of the determinants influencing the aggregation of polypeptides and has allowed to investigate how the selective pressure to avoid aggregation has shaped the cellular proteomes along evolution. From these analyses, different mechanisms to prevent protein aggregation have emerged ranging from negative design strategies found in polypeptide sequences and structures, to the characterization of the factors governing the cellular machinery in charge of the protein quality control. The present thesis provides a multiperspective analysis for the detailed characterization of several of these mechanisms evolved to confront the risk of protein aggregation. In this sense, the use of a variety of specific proteic models of aggregation or different sets of proteins with related properties has allowed to analyze particular strategies to avoid aggregation in depth. At the same time, the approach based on the study of closely related ensembles of proteins has allowed to identify functional constraints that limit the evolutionary selection against aggregation. More specifically, the work presented here addresses the effect of restricting the configurational freedom of the polypeptide chain by disulfide cross-linking on the aggregation process, as well as the impact over this phenomenon exerted by the presence of intrinsically disordered protein regions. Additionally, the regulation of cellular protein abundance as a function of protein aggregation propensity has also been surveyed. On the other hand the analysis centered on the study of closely related proteins has revealed how the requirements to fold efficiently and to maintain the catalytic activity constrain the minimization of the aggregation propensity of proteins. These analyses highlight particularly the interplay between folding and aggregation, in such a way that the analysis of the aggregational properties of polypeptides allows to forecast the mechanism of folding of certain kind of proteins. RESUM EN CATALÀ En els darrers 15 anys, l'estudi de l'agregació de proteïnes ha evolucionat de ser una part de la química de proteïnes que tradicionalment generava poc interès, a convertir-se en una àrea d'investigació dinàmica que ha ampliat el seu abast a diferents camps de recerca incloent- hi la bioquímica, la biotecnologia, la nanotecnologia y la biomedicina. Dins d'aquests camps, l'anàlisi de l'agregació de proteïnes un interès particularment rellevant en les àrees biomèdica i biotecnològica. Això es degut, per una banda, a que la aparició de depòsits proteics insolubles está relacionada amb un nombre creixent de malalties humanes, moltes d'elles amb conseqüències fatals per als malalts. Per l'altre costat, l'agregació proteica es una complicació habitual en la expresió recombinant de proteïnes a nivell industrial, com ara a la producció d'agents terapèutics de naturalessa proteica, com els anticossos. Conseqüentment, l'exploració de mecanismes que permetin prevenir l'agregació proteica es subjecte actualment d'una intensa tasca d'investigació adreçada a desenvolupar métodes per a la prevenció i l'intervenció terapèutiques d'aquestes malalties greus; i també amb l'objectiu d'incrementar els rendiments en la producció biotechnològica de proteïnes. La gran capacitat de les eines computacionals desenvolupades per tal de predir l'agregació proteica ha donat un gran impuls als esforços destinats a identificar els determinants de l'agregació de polipèptids, i també ha permès investigar com la pressió selectiva contra l'agregació ha moldeat els proteomes cel·lulars al llarg de l'evolució. A partir d’aquests anàlisis, s’han pogut identificar diferents mecanismes evolucionats per prevenir l’agregació proteica que van des de estratègies de disseny negatiu que s’han detectat en seqüències i estructures de proteïnes, fins a la caracterització dels factors que governen la maquinària cel•lular encarregada del control de qualitat proteic. En aquesta tesi es proporciona un anàlisis des de diferents perspectives per assolir una caracterització detallada de diversos d’aquests mecanismes que han sorgit al llarg de l’evolució per fer front al risc d’agregació. Amb aquest objectiu, s’han fet servir tant models proteics d’agregació específics com diferents conjunts de proteïnes amb propietats relacionades, que han permès analitzar en profunditat estratègies contra l’agregació. Al mateix temps, l’aproximació basada en l’estudi de conjunts de proteïnes íntimament relacionats també ha permès identificar limitacions funcionals que limiten la selecció a nivell evolutiu contra l’agregació. De manera més específica, el treball que es presenta aquí aborda l’efecte de la restricció de la llibertat conformacional de la cadena polipeptídica, causada per l’establiment d’un pont disulfur, sobre el procés d’agregació; també s’ha analitzat l’impacte de regions intrínsicament desestructurades en aquest mateix fenomen d’agregació. A més també s’ha investigat la regulació de l’abundància proteica dins la cèl·lula en funció de la tendència a agregar específica de les proteïnes. Per altra banda, l’anàlisi centrat en l’estudi de proteïnes relacionades ha revelat com els requeriments per assolir un plegament eficient i per mantenir l’activitat catalítica limiten la disminució de la càrrega d’agregació de les proteïnes. Aquests anàlisis posen especialment en relleu el balanç entre plegament funcional i agregació, de manera que la caracterització de les propietats aggregatives de determinats polipèptids permet predir el seus mecanismes de plegament. LIST OF PUBLICATIONS This thesis is composed of the following published works: I. Ricardo Graña-Montes, Virginia Castillo, &. Salvador Ventura: The Aggregation Properties of Escherichia coli Proteins is Associated with their Cellular Abundance. Biotechnology Journal 6(6) pp. 752–760 (2011) II. Ricardo Graña-Montes, Natalia Sánchez de Groot, Virginia Castillo, Javier Sancho, Adrian Velázquez-Campoy & Salvador Ventura: Contribution of Disulfide Bonds to Stability, Folding, and Amyloid Fibril Formation: the PI3-SH3 Domain Case. Antioxidants & Redox Signaling 16(1) pp. 1- 15 (2012) III. Ricardo Graña-Montes, Ricardo Sant'Anna de Oliveira & Salvador Ventura: Protein Aggregation Profile of the Human Kinome. Frontiers in Physiology 3 – 438, (2012) IV. Ricardo Graña-Montes, Patrizia Marinelli, David Reverter & Salvador Ventura: N-terminal Protein Tails Act as Aggregation Protective Entropic Bristles: the SUMO Case. Biomacromolecules 15 (4) pp 1194-1203, (2014) V. Ricardo Graña-Montes, Hugo Fraga, Ricard Illa, Giovanni Covaleda & Salvador Ventura:Association between Foldability and Aggregation Propensity in Small Disulfide-Rich Proteins. Antioxidants & Redox Signaling, 21(3) pp 368-383, (2014) Other articles co-authored which are not part of this thesis: VI. Virginia Castillo, Ricardo Graña-Montes, Raimon Sabaté &. Salvador Ventura: Prediction of the Aggregation Propensity of Proteins from the Primary Sequence: Aggregation Properties of Proteomes. Biotechnology Journal 6(6) pp. 674-685 (2011) review article VII. Raimon Sabaté, Alba Espargaró, Ricardo Graña-Montes, David Reverter & Salvador Ventura: Native Structure Protects SUMO Proteins from Aggregation into Amyloid Fibrils. Biomacromolecules 13(6) pp. 1916-1926 (2012) VIII. Ricardo Graña-Montes, & Salvador Ventura: About Targets and Causes in Protein Folding. Journal of Biomolecular Structure and Dynamics 31(9) pp. 970-972 (2013) IX. Ricardo Sant'Anna,
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