Méthodes d’apprentissage et approches expérimentales appliqués aux réseaux d’interfaces protéiques

The aim of this study is to understand protein assembly mechanisms. The assembly of a protein in an oligomer is particularly important because it is involved in many pathologies going from bacterial infection, Alzheimer like diseases or even some cancers. Protein assembly is the combination of two or more protein chains to induce a biological activity. The B subunit of the cholera toxin pentamer (CtxB5), which belongs to the family of AB5 toxins, is studied as the main model of assembly. Experimental results have provided information on the assembly of the toxin highlighting the involvement of certain amino acids. The first problem addressed in my thesis is to understand their role and see if network approaches are relevant to such investigation. I was able to show using amino acid mutations, that amino acids influence each other by cascade or "peer to peer" mechanisms in order to coordinate the various steps of the assembly (Chapters 4, 5 and 6). The structure and function of the proteins are defined by amino acid sequences which naturally vary due to genetic mutation. So I decided to expand this field of investigation to see if the cascade mechanism was generalized as a mean of disrupting a protein structure. Here it is to understand how a protein loses its function by way of a significant change of structure upon mutation. First, I studied dataset to know the characteristics of healthy protein networks (Chapter 7, 8 and 9), and after I looked at the effects of the systematic mutation of each amino acid of CtxB5 on its overall structure (Chapter 10 and 11). Mutations led from moderate to very large structural changes around the mutated amino acid or at long distances. These results are consistent with known effects of mutation: robustness (maintenance function), evolution or adaptation (emergence of a new feature) and fragility (pathologies). The results also show a weak correlation between the number of amino acid contacts of the mutated amino acid and the amount of structural change induced by its mutation. It is therefore not easy to anticipate the effect of a mutation: The last chapter of my thesis addresses this problem (Chapter 12).Cette étude s’inscrit dans le cadre d’un problème biologique et son objectif est de comprendre les mécanismes d’assemblage des protéines. L’assemblage d’une protéine en oligomère est particulièrement important car il est impliqué dans de nombreuses pathologies allant de l’infection bactérienne aux maladies de type Alzheimer ou même des cancers. L’assemblage protéique est un mécanisme de combinaison de deux ou plusieurs chaînes protéiques, il est aussi par ailleurs souvent utilisé par les organismes vivants pour déclencher une activité biologique. La sous unité B de la toxine du choléra(CtxB5), qui appartient à la famille des toxines AB5, est étudiée comme modèle principal de l’assemblage. Des résultats expérimentaux ont fourni des informations sur l’assemblage de la toxine mettant en avant l’implication de certains acides aminés. La première question que j’ai abordée pendant ma thèse était de comprendre leur rôle et de voir si les approches réseaux étaient pertinentes pour y répondre. J’ai pu montrer en utilisant des mutations d’acides aminés que ces derniers s’influençaient entre eux suivant des mécanismes en cascade ou de « Peer to Peer » afin de coordonner les étapes de l’assemblage (les chapitres 4, 5 et 6). La structure et la fonction des protéines sont définies par des séquences d’acides aminés qui varient naturellement en raison de mutation génétique. J’ai donc décidé d’élargir ce champ d’investigation pour voir si le mécanisme en cascade était généralisable comme moyen de perturber une structure de protéine par le biais d’une mutation. Ici il s’agit de comprendre les changements de structure liés à des mutations et pouvant menés à des maladies. J’ai tout d’abord étudié des jeux de données pour connaître les caractéristiques réseaux de protéines saines (chapitre 7, 8 et 9), avant de regarder l’effet de la mutation systématique de chacun des acides aminés de CtxB5 sur sa structure globale (chapitre 10 et 11). Les mutations peuvent engendrer des changements de structure modérés ou très grand autour de l’acide aminé muté ou à des distances très éloignées. Ces résultats sont consistants avec tous les effets connus de mutation : robustesse (maintien de la fonction), évolution ou adaptation (émergence d’une nouvelle fonction) et fragilité (pathologies). Les résultats montrent aussi une faible corrélation entre le nombre de contacts d’un acide aminé et la quantité de changement structuraux induit par sa mutation. Il n’est donc pas simple d’anticiper l’effet d’une mutation : Le dernier chapitre de ma thèse aborde ce problème (chapitre 12)

Protein subunit association: NOT a social network

International audienceMost proteins cannot function as single unit but associate subunits via the formation of protein interfaces, to be biologically active. How the amino acids involved in subunit association, so-called hot spots, regulate the formation of a protein interface is still an open question. Here, we show how network and graph theories can help addressing the role of hot spots. We built a MatLab code called SpectralPro which identifies hot spots and reconstructs the protein interface as a subnetwork of hot spots in interaction, with the hot spots as nodes and the bonds between hot spots as links. Using as a case study, the cholera toxin B pentamer (five subunits), we investigate if the degree of a node, namely the number of contacts of a hot spot, is important in the formation of an interface. The degree of a node is known to be important in many real networks. For example in social networks, hubs control the communication between most nodes and as such are vulnerable to changes. But our result shows that in the toxin interface sub-graph hub-like nodes are less vulnerable to change than single link node

Beta-Strand Interfaces of Non-Dimeric Protein Oligomers Are Characterized by Scattered Charged Residue Patterns

Protein oligomers are formed either permanently, transiently or even by default. The protein chains are associated through intermolecular interactions constituting the protein interface. The protein interfaces of 40 soluble protein oligomers of stœchiometries above two are investigated using a quantitative and qualitative methodology, which analyzes the x-ray structures of the protein oligomers and considers their interfaces as interaction networks. The protein oligomers of the dataset share the same geometry of interface, made by the association of two individual β-strands (β-interfaces), but are otherwise unrelated. The results show that the β-interfaces are made of two interdigitated interaction networks. One of them involves interactions between main chain atoms (backbone network) while the other involves interactions between side chain and backbone atoms or between only side chain atoms (side chain network). Each one has its own characteristics which can be associated to a distinct role. The secondary structure of the β-interfaces is implemented through the backbone networks which are enriched with the hydrophobic amino acids favored in intramolecular β-sheets (MCWIV). The intermolecular specificity is provided by the side chain networks via positioning different types of charged residues at the extremities (arginine) and in the middle (glutamic acid and histidine) of the interface. Such charge distribution helps discriminating between sequences of intermolecular β-strands, of intramolecular β-strands and of β-strands forming β-amyloid fibers. This might open new venues for drug designs and predictive tool developments. Moreover, the β-strands of the cholera toxin B subunit interface, when produced individually as synthetic peptides, are capable of inhibiting the assembly of the toxin into pentamers. Thus, their sequences contain the features necessary for a β-interface formation. Such β-strands could be considered as ‘assemblons’, independent associating units, by homology to the foldons (independent folding unit). Such property would be extremely valuable in term of assembly inhibitory drug development

Learning methods and experimental approaches applied on protein interface networks

Cette étude s’inscrit dans le cadre d’un problème biologique et son objectif est de comprendre les mécanismes d’assemblage des protéines. L’assemblage d’une protéine en oligomère est particulièrement important car il est impliqué dans de nombreuses pathologies allant de l’infection bactérienne aux maladies de type Alzheimer ou même des cancers. L’assemblage protéique est un mécanisme de combinaison de deux ou plusieurs chaînes protéiques, il est aussi par ailleurs souvent utilisé par les organismes vivants pour déclencher une activité biologique. La sous unité B de la toxine du choléra(CtxB5), qui appartient à la famille des toxines AB5, est étudiée comme modèle principal de l’assemblage. Des résultats expérimentaux ont fourni des informations sur l’assemblage de la toxine mettant en avant l’implication de certains acides aminés. La première question que j’ai abordée pendant ma thèse était de comprendre leur rôle et de voir si les approches réseaux étaient pertinentes pour y répondre. J’ai pu montrer en utilisant des mutations d’acides aminés que ces derniers s’influençaient entre eux suivant des mécanismes en cascade ou de « Peer to Peer » afin de coordonner les étapes de l’assemblage (les chapitres 4, 5 et 6). La structure et la fonction des protéines sont définies par des séquences d’acides aminés qui varient naturellement en raison de mutation génétique. J’ai donc décidé d’élargir ce champ d’investigation pour voir si le mécanisme en cascade était généralisable comme moyen de perturber une structure de protéine par le biais d’une mutation. Ici il s’agit de comprendre les changements de structure liés à des mutations et pouvant menés à des maladies. J’ai tout d’abord étudié des jeux de données pour connaître les caractéristiques réseaux de protéines saines (chapitre 7, 8 et 9), avant de regarder l’effet de la mutation systématique de chacun des acides aminés de CtxB5 sur sa structure globale (chapitre 10 et 11). Les mutations peuvent engendrer des changements de structure modérés ou très grand autour de l’acide aminé muté ou à des distances très éloignées. Ces résultats sont consistants avec tous les effets connus de mutation : robustesse (maintien de la fonction), évolution ou adaptation (émergence d’une nouvelle fonction) et fragilité (pathologies). Les résultats montrent aussi une faible corrélation entre le nombre de contacts d’un acide aminé et la quantité de changement structuraux induit par sa mutation. Il n’est donc pas simple d’anticiper l’effet d’une mutation : Le dernier chapitre de ma thèse aborde ce problème (chapitre 12).The aim of this study is to understand protein assembly mechanisms. The assembly of a protein in an oligomer is particularly important because it is involved in many pathologies going from bacterial infection, Alzheimer like diseases or even some cancers. Protein assembly is the combination of two or more protein chains to induce a biological activity. The B subunit of the cholera toxin pentamer (CtxB5), which belongs to the family of AB5 toxins, is studied as the main model of assembly. Experimental results have provided information on the assembly of the toxin highlighting the involvement of certain amino acids. The first problem addressed in my thesis is to understand their role and see if network approaches are relevant to such investigation. I was able to show using amino acid mutations, that amino acids influence each other by cascade or "peer to peer" mechanisms in order to coordinate the various steps of the assembly (Chapters 4, 5 and 6). The structure and function of the proteins are defined by amino acid sequences which naturally vary due to genetic mutation. So I decided to expand this field of investigation to see if the cascade mechanism was generalized as a mean of disrupting a protein structure. Here it is to understand how a protein loses its function by way of a significant change of structure upon mutation. First, I studied dataset to know the characteristics of healthy protein networks (Chapter 7, 8 and 9), and after I looked at the effects of the systematic mutation of each amino acid of CtxB5 on its overall structure (Chapter 10 and 11). Mutations led from moderate to very large structural changes around the mutated amino acid or at long distances. These results are consistent with known effects of mutation: robustness (maintenance function), evolution or adaptation (emergence of a new feature) and fragility (pathologies). The results also show a weak correlation between the number of amino acid contacts of the mutated amino acid and the amount of structural change induced by its mutation. It is therefore not easy to anticipate the effect of a mutation: The last chapter of my thesis addresses this problem (Chapter 12)

Intermolecular β-strand networks avoid hub residues and favor low interconnectedness: a potential protection mechanism against chain dissociation upon mutation.

Altogether few protein oligomers undergo a conformational transition to a state that impairs their function and leads to diseases. But when it happens, the consequences are not harmless and the so-called conformational diseases pose serious public health problems. Notorious examples are the Alzheimer's disease and some cancers associated with a conformational change of the amyloid precursor protein (APP) and of the p53 tumor suppressor, respectively. The transition is linked with the propensity of β-strands to aggregate into amyloid fibers. Nevertheless, a huge number of protein oligomers associate chains via β-strand interactions (intermolecular β-strand interface) without ever evolving into fibers. We analyzed the layout of 1048 intermolecular β-strand interfaces looking for features that could provide the β-strands resistance to conformational transitions. The interfaces were reconstructed as networks with the residues as the nodes and the interactions between residues as the links. The networks followed an exponential decay degree distribution, implying an absence of hubs and nodes with few links. Such layout provides robustness to changes. Few links per nodes do not restrict the choices of amino acids capable of making an interface and maintain high sequence plasticity. Few links reduce the "bonding" cost of making an interface. Finally, few links moderate the vulnerability to amino acid mutation because it entails limited communication between the nodes. This confines the effects of a mutation to few residues instead of propagating them to many residues via hubs. We propose that intermolecular β-strand interfaces are organized in networks that tolerate amino acid mutation to avoid chain dissociation, the first step towards fiber formation. This is tested by looking at the intermolecular β-strand network of the p53 tetramer

beta-Strand Interfaces of Non-Dimeric Protein Oligomers are Characterized by Scattered Charge Residues Pattern

International audienceProtein oligomers are formed either permanently, transiently or even by default. The protein chains are associated through intermolecular interactions constituting the protein interface. The protein interfaces of 40 soluble protein oligomers of stœchiometries above two are investigated using a quantitative and qualitative methodology, which analyzes the x-ray structures of the protein oligomers and considers their interfaces as interaction networks. The protein oligomers of the dataset share the same geometry of interface, made by the association of two individual β-strands (β-interfaces), but are otherwise unrelated. The results show that the β-interfaces are made of two interdigitated interaction networks. One of them involves interactions between main chain atoms (backbone network) while the other involves interactions between side chain and backbone atoms or between only side chain atoms (side chain network). Each interaction network has its own characteristics which can be associated to a distinct role. The secondary structure of the β-interfaces is implemented through the backbone networks which are enriched with the hydrophobic amino acids favored in intramolecular β-sheets (MCWIV). The intermolecular specificity is provided by the side chain networks via positioning different types of charge residues at the extremities (arginine) and in the middle (glutamic acid and histidine) of the interface. Such charge distribution helps discriminating between sequences of intermolecular β-strands, of intramolecular β- strands and of β-strands forming β-amyloid fibers. This might open new venues for drug designs and predictive tool developments. Moreover, the β-strands of the cholera toxin B subunit interface, when produced individually as synthetic peptides, are capable of inhibiting the assembly of the toxin into pentamers. Thus, their sequences contain the features necessary for a β-interface formation. Such β-strands could be considered as 'assemblons', independent associating units, by homology to the foldons (independent folding unit). Such property would be extremely valuable in term of assembly inhibitory drug development

Cross-Talk Between Intramolecular and Intermolecular Amino Acid Networks Orchestrates the Assembly of the Cholera Toxin B Pentamer via the Residue His94

Protein assembly is the mechanism of combining two or more protein chains. Living organisms to trigger biological activity often uses this mechanism. The cholera toxin B subunit pentamer (CtxB 5), the binding moiety of CtxAB 5, a member of the AB 5 toxin family, is presented here as a model for studying protein assembly. Experimental results showed the importance of histidine residues in CtxB 5 assembly. Nevertheless, the histidines are located outside the pentamer interfaces suggesting an indirect role. Gemini and Spectral-Pro, two networkbased models developped in our team, were used in combination with in silico mutations produced with Fold X to investigate this question. All the residues of the pentamer interfaces, so-called hotspots, were identified and some appeared to be chemical neighbors of the His 94, making possible a propagation path of conformational changes from the His 94 to the interface to regulate the pentamer assembly. Mutations of the residues along intra-to-inter amino acid paths produce non additive mutational perturbations of the interface energy, indicating influences of His 94 on its hotspot neighbors to regulate the interface. The role of intrato-inter amino acid paths in regulating the pentamer interface was confirmed by applying similar approach to the heat labile toxin B pentamer (LTB 5), which share 84% sequence identity, structural and functional similarity with CtxB 5. Different intra-to-inter amino acid paths were identified for LTB 5 consistently with the different assembly mechanisms followed by both toxin pentamers. These results open up avenues for understanding why these two toxins follow different assembly mechanisms

Observed SC Pair occurrences.

<p>The values in a given row are the occurrences of the residue <i>a</i> in contact with the residues <i>b_i</i>, cited on columns. Thus, there are twenty rows <i>a_i</i> and twenty column <i>b_i,</i> - <i>i</i>- covering the 20 different amino acids. Due to the counting procedure the table is read row-wise (material and methods).</p

Local preferences of the charged amino acids in the SC hot spots.

<p>Local preferences of the charged amino acids in the SC hot spots.</p

Number of contacts of the hot spots.

<p>A. The degree distributions of the BB and SC hot spots are plotted on a semi-log scale. The degree distribution <i>P(k)</i> of the SC hot spots decreases exponentially (R² = 0.99). B. Linear correlation between the number of atoms of a SC hot spot and its tendency to have more than one contact. The ratio of the frequency of an amino acid in multiple contacts to its frequency in single contact is plotted against the number of its side chain atoms. C. Probability of a SC hot spot to have <i>k</i> contacts. The probabilities for a SC hot spot to have <i>k</i>>3 (♦) or <i>k</i> = 1 (○) are plotted against the number of atoms of its respective amino acid. The horizontal line indicates the probability at which every amino acid has the same probability to have <i>k</i> contacts (0.05 = 1/20). The vertical line indicates a number of atoms equals to 14.</p