Proteine Funktion Prädiktion im Licht der Evolution

1\. Introduction ............................................................................................................................................... 8 1.1. The place of evolutionary theory in modern biology ............................................................................. 8 1.1.1. First observation: nothing in experimental biology makes sense, except in the light of evolution .... 8 1.1.2. Second observation: new solutions to biological problems using concepts from evolutionary biology are emerging .................................................................................................................................... 9 1.2. Brief reminder of major concepts of evolutionary biology .................................................................. 10 1.3. Evolvability and robustness of living systems ..................................................................................... 12 1.3.1. At the level of genomes .................................................................................................................... 12 1.3.2. At higher degrees of cellular complexity .......................................................................................... 13 1.4. Strategies that promote evolvability .................................................................................................... 14 1.4.1. First strategy: Innovation by gene duplication .................................................................................. 14 1.4.1.1. Concept of gene duplication .......................................................................................................... 14 1.4.1.2. Emergence of protein repeats ......................................................................................................... 14 1.4.2. Second strategy: Evolving new physiological compartments ........................................................... 16 1.4.2.1. Compartmentalization in metazoans .............................................................................................. 16 1.4.2.2. Evolution of physiological systems ............................................................................................... 17 1.5. Methods to study protein mutations using structural and evolutionary information ........................... 18 1.5.1. Mutations in the context of genetic diseases ..................................................................................... 18 1.5.2. Tools to explore the effect of mutations on protein structure and function ...................................... 19 1.5.2.1. Sequence alignment ....................................................................................................................... 19 1.5.2.2. Prediction tools .............................................................................................................................. 19 1.5.2.3. Protein visualization ....................................................................................................................... 19 1.6. Thesis outline ....................................................................................................................................... 20 2\. Emergence of proteins with alpha-solenoids .......................................................................................... 21 2.1. Introduction .......................................................................................................................................... 21 2.1.1. Functional and genomic analyses of alpha-solenoid proteins ........................................................... 21 2.1.2. Function of huntingtin alpha-solenoid region and prediction of consequence of mutations for its structure ...................................................................................................................................................... 23 2.2. Detection of alpha-solenoids ................................................................................................................ 23 2.2.1. Introduction to artificial neural networks .......................................................................................... 24 2.2.2. Presentation of the neural network of ARD ...................................................................................... 28 2.2.3. Improvements of ARD ...................................................................................................................... 29 2.2.4. Evaluation of ARD2 performance ..................................................................................................... 31 2.3. Structure of alpha- solenoids................................................................................................................. 34 2.3.1. Some types of alpha-helical repeats are newly classified as alpha- solenoids ................................... 34 2.3.2. Alpha-solenoids can interact with nucleic acids and lipids ............................................................... 35 2.3.3. Alpha-solenoids can be located outside as well as inside of proteins ............................................... 37 2.4. Functions of alpha- solenoids ............................................................................................................... 37 2.4.1. Alpha-solenoid proteins are promiscuous ......................................................................................... 38 2.4.2. Alpha-solenoid proteins are primarily involved in intracellular trafficking...................................... 39 2.4.3. Some proteins are newly detected as containing alpha- solenoids..................................................... 41 2.5. Distribution of alpha-solenoid proteins across the tree of life ............................................................. 43 2.6. Modeling of an alpha-solenoid region of protein huntingtin ............................................................... 47 2.6.1. Introduction ....................................................................................................................................... 47 2.6.2. Methods............................................................................................................................................. 49 2.6.3. Results and discussion ...................................................................................................................... 51 2.6.4. Conclusion ........................................................................................................................................ 59 2.7. General conclusion of chapter 2 ........................................................................................................... 60 5 3\. Emergence and evolution of the renin-angiotensin-aldosterone system ................................................. 63 3.1. Introduction .......................................................................................................................................... 63 3.1.1. Introduction to regulation of blood pressure ..................................................................................... 64 3.1.1.1. Definition of blood pressure .......................................................................................................... 64 3.1.1.2. Sensors of blood pressure variation ............................................................................................... 65 3.1.1.3. Effectors of blood pressure ............................................................................................................ 65 3.1.2. Presentation of the renin-angiotensin-aldosterone system ................................................................ 67 3.1.2.1. Anatomical and physiological features .......................................................................................... 67 3.1.2.2. Molecular features ......................................................................................................................... 68 3.1.3. Putative mechanisms leading to hypertension .................................................................................. 70 3.2. Evolution of anatomical and physiological features of the renin- angiotensin-aldosterone system (RAAS) ....................................................................................................................................................... 70 3.3. Analysis of DNA sequences of proteins of the renin-angiotensin- aldosterone system ....................... 70 3.3.1. Angiotensinogen ............................................................................................................................... 72 3.3.2. Angiotensin-converting enzymes ...................................................................................................... 76 3.3.3. Renin ................................................................................................................................................. 80 3.3.4. Evolution of RAAS targets ............................................................................................................... 82 3.3.4.1. AT1 and AT2 ................................................................................................................................... 82 3.3.4.2. (P)RR ............................................................................................................................................. 83 3.3.4.3. MAS ............................................................................................................................................... 83 3.3.4.4. Mineralocorticoid receptor ............................................................................................................. 84 3.4. Conclusion ........................................................................................................................................... 86 4\. Methods to study the impact of mutations on proteins related to disease using structural and evolutionary information ............................................................................................................................ 88 4.1. Introduction .......................................................................................................................................... 88 4.2. PDBpaint, a visualization tool to display proteins using functional annotations ................................. 89 4.2.1. Introduction ....................................................................................................................................... 89 4.2.2. Functionalities of PDBpaint .............................................................................................................. 90 4.2.3. Technical specifications of PDBpaint ............................................................................................... 93 4.2.4. Comparison with other tools ............................................................................................................. 93 4.2.5. Conclusion of section 4.2. ................................................................................................................. 94 4.3. Study of deleterious mutations in huntingtin interacting protein CRMP-1 ......................................... 95 4.3.1. Introduction ....................................................................................................................................... 95 4.3.2. Methods............................................................................................................................................. 96 4.3.3. Results and discussion ...................................................................................................................... 96 4.3.3.1. Design of CRMP-1 mutants ........................................................................................................... 96 4.3.3.2. Impact of mutation D408V on the function of CRMP-1 ............................................................... 99 4.3.4. Conclusion ...................................................................................................................................... 101 4.4. Study of myosin mutations involved in cardiac septal defects. ......................................................... 102 4.4.1. Introduction ..................................................................................................................................... 102 4.4.2. Methods........................................................................................................................................... 102 4.4.3. Results and discussion .................................................................................................................... 103 4.4.4. Conclusion ...................................................................................................................................... 105 4.5. Conclusion to chapter 4 ..................................................................................................................... 105 5\. General conclusion ................................................................................................................................ 106 Summary ................................................................................................................................................... 107 Zusammenfassung ..................................................................................................................................... 108 Appendix ................................................................................................................................................... 109 Bibliography ............................................................................................................................................. 128 List of publications ................................................................................................................................... 141 Curriculum vitae ....................................................................................................................................... 142Evolution is a major actor of function of living beings. Studying biological processes with the perspective of an evolutionary biologist is important in order to have the most complete picture possible of the processes acting in nature. Following this idea, we have studied protein sequences to study two different biological systems. Such information was used to build evolutionary scenarios for our two questions. We first studied alpha-solenoid repeat proteins. We have improved a method to detect such motives inside of protein sequences and applied this updated method to all sequences available in protein databases. The study of the distribution of such sequences in the tree of life shows that eukaryota are the taxons displaying the most this type of structure, as well as two groups of bacteria, cyanobacteria and planctomycetes. Importantly, the three groups of alpha-solenoid show limit similarity. We speculate that they appeared independently. Equally important, the three groups, eukaryota and the two bacteria taxons, are associated with increased cellular complexity versus classical bacteria groups. We hypothesized that the increased demand of protein important to protein transport and synthesis in living beings with compartmentalized cells induces a higher recruitment of alpha-solenoid proteins, as they require more complex protein machinery in order to be built. This high pressure could have occurred in the three groups independently, increased the recruitment of alpha-solenoid proteins. The second evolutionary scenario we tried to put together is about the renin-angiotensin system which regulates hypertension in higher vertebrates. To perform this, we conducted a phylogenetic analysis of a dozen of proteins involved in the system, from higher vertebrates to invertebrates. We found that contrary to naïve thinking, some of the components of the system appeared before the set of the system, and had a complete different function, showing orthologue sequences in invertebrates. Some proteins, present in taxons with no regulation of hypertension such as Drosophila, were previously used for development a long time before being co-opted for homeostasis regulation in vertebrates. We could confirm the onset of the system around the appearance of cartilage fishes, around 400 million years ago. Both analysis, of alpha-solenoid repeat proteins and protein sequences from the renin- angiotensin system, showed the importance of using evolutionary cues in order to better comprehend how living being work. Aside from these evolutionary scenarios, we also used evolutionary along with structural information in order to study the impact of mutations for the structure of various proteins, and the relation of such mutations to disease and function. For these analyses, we have developed a tool called PDBpaint to visualize various annotations on the structure of proteins.Evolution prägt die Funktionsweise aller Lebewesen. Es ist notwendig biologische Prozesse aus der Perspektive eines Evolutionsbiologen zu betrachten, wenn man ein möglichst vollständiges Bild von den natürlichen Vorgängen erhalten möchte. In diesem Sinne haben wir Proteinsequenzen herangezogen um zwei verschiedene biologische Systeme zu untersuchen und um Evolutionsszenarios für unsere beiden Fragestellungen zu entwickeln. Zunächst analysierten wir Proteine mit sich wiederholenden Alpha-Solenoidsequenzen. Wir verbesserten eine Methode zur Detektion solcher Motive innerhalb von Proteinsequenzen und wandten die verbesserte Methode auf alle Sequenzen an, die in Proteindatenbanken erhältlich waren. Die Untersuchung der Verteilung solcher Motive im phylogenetischen Stammbaum der Arten zeigte, dass besonders Eukaryonten und zwei Gruppen von Bakterien, die Cyanobakterien und Planctomyceten, diese Struktur tragen. Wichtig hierbei ist, dass die drei Gruppen von Alpha-Solenoidsequenzen nur begrenzte Ähnlichkeit aufweisen. Wir nehmen an, dass sie unabhängig voneinander entstanden sind. Genauso wichtig ist, dass die drei Gruppen, Eukaryonten und die zwei bakteriellen Taxa, ein erhöhtes Maß an zellulärer Komplexität aufweisen im Vergleich mit anderen bakteriellen Gruppen. Unsere Hypothese ist, dass Alpha-Solenoid Proteine durch den zunehmendem Bedarf an Proteinen für Transport und Synthese in Lebewesen mit kompartmentalisieren Zellenvermehrt entstehen, da sie eine komplexe Proteinmaschinerie benötigen, um gebildet zu werden. Dieser Druck könnte in allen drei Gruppen unabhängig bewirkt haben, dass Alpha-Solenoid Proteine in immer größerer Zahl hervorgebracht wurden. Das zweite evolutionäre Szenario, das wir untersuchten, das Renin-Angiotensin System, reguliert Bluthochdruck in höheren Vertebraten. Wir unternahmen eine phylogenetische Analyse von zwölf Proteinen, die in das System involviert sind, von höheren Vertebraten bis hin zu Invertebraten. Wir fanden heraus, dass entgegen unseren Erwartungen einige Komponenten lange vor dem eigentlichen Regulationssystem auftraten, mit völlig anderen Funktionen, gezeigt anhand von orthologen Sequenzen in Invertebraten. Einige Proteine, die in Taxa ohne Regulation von Bluthuchdruck zu finden sind, so wie Drosophila, wurden schon lange vorher für die Regulation von Entwicklung verwendet. Wir konnten bestätigen, dass das System etwa gleichzeitig mit den Knorpelfischen entstand, ca. vor 400 Millionen Jahren. Beide Analysen, sowohl die der Alpha-Solenoid Proteine als auch der Proteinsequenzen des Renin-Angiotensin Systems, haben gezeigt, wie wichtig es ist, Hinweise aus der Evolution in die Betrachtungen mit einzubeziehen, bei dem Versuch Lebewesen und ihre Funktionsweise, zu verstehen. Neben diesen Evolutionsszenarios verwendeten wir evolutionäre gleichzeitig mit strukturellen Informationen um den Einfluss von Mutationen auf die Struktur verschiedener Proteine zu erforschen, sowie die Beziehung solcher Mutationen zu Krankheit und Funktion. Für solche Analysen haben wir PDBpaint entwickelt, ein Werkzeug zur Visualisierung von Annotationen in Proteinstrukturen