Neurogenin 2 (Ngn2) is a basic Helix-Loop-Helix (bHLH) transcription factor regulating differentiation and cell cycle exit in the developing brain. By transcriptional upregulation of a cascade of other bHLH factors, neural progenitor cells exit the cell cycle and differentiate towards a neuronal fate. Xenopus laevis Ngn2 (xNgn2) is a short-lived protein, targeted for degradation by the 26S proteasome. I have investigated the stability of Ngn2 mediated by post-translational modifications and structural disorder. Firstly I will describe work focused on ubiquitylation of xNgn2, targeting it for proteasomal degradation. xNgn2 is ubiquitylated on lysines, the recognized site of modification. I will discuss the role of lysines in ubiquitylation and stability of xNgn2. In addition to canonical ubiquitylation on lysines, I describe ubiquitylation of xNgn2 on non-canonical sites, namely its amino-terminal amino group, and cysteine, serine and threonine residues. I show that the ubiquitylation of cysteines in particular exhibits cell cycle dependence and is also observed in mammalian cell lines, resulting in cell cycle-dependent regulation of stability. I will then discuss whether phosphorylation, a regulator of xNgn2 activity, also affects xNgn2 stability. I will provide evidence of cell cycle-dependent phosphorylation of cyclin dependent kinase (cdk) consensus sites affecting the stability of xNgn2. Finally I describe studies on the folding properties of Ngn2 to assess their role in protein stability. xNgn2 associates with DNA and its heterodimeric binding partner xE12 and may interact directly with the cyclin-dependent kinase inhibitor Xic1. I will discuss the role of these interaction partners in xNgn2 stability. xNeuroD, a downstream target of xNgn2, is a related bHLH transcription factor which is stable. Here I describe domain swapping experiments between these two proteins highlighting regions conferring instability on the chimeric protein. Finally I will provide nuclear magnetic resonance (NMR) data looking at the effect of phosphorylation on protein structure in mouse Ngn2 (mNgn2).

Normal DNA replication is blocked by DNA damage in the template strand. Translesion synthesis is a major pathway for overcoming these replication blocks. In this process, multiple non-classical DNA polymerases form a complex at the stalled replication fork called the mutasome. This complex is structurally organized by the replication accessory factor PCNA and the non-classical DNA polymerase Rev1. One of the non-classical DNA polymerases within the mutasome then catalyzes replication through the damage. Each non-classical DNA polymerase has one or more cognate lesions, which the enzyme bypasses with high accuracy and efficiency. Thus, the accuracy and efficiency of translesion synthesis depends on which non-classical DNA polymerase within the mutasome is chosen to bypass the damage. In this thesis, I discuss how the most appropriate polymerase is chosen. In so doing, I examine the components of the mutasome; the structural motifs that mediate the protein interactions in the mutasome; the methods used to study translesion synthesis; the definition of a cognate lesion; the intrinsically disordered regions that tether the polymerases to PCNA and to one another; the multiple architectures that the mutasome can adopt, such as PCNA tool belts and Rev1 bridges; and the kinetic selection model in which the most appropriate polymerase is chosen via a competition among the multiple polymerases within the mutasome. Taken together, this thesis provides and inclusive review of the current state of what is known about translesion synthesis with conclusions at its end suggesting what major questions remain and ideas of how to answer them.

The genetic disease cystic fibrosis is the most common lethal genetic disease in Western countries. People born with cystic fibrosis suffer from many health issues including severe respiratory problems, inflammation and recurrent lung infections that can become fatal. The disease is caused by the loss of function of a protein called the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is an chloride ion channel and, in healthy people, its activity assures correct water and salt transport across the cell membrane. Most cases of cystic fibrosis are caused by a genetic defect that leads to the deletion of phenylalanine 508 (F508del) in the amino acid sequence of the protein. The molecular mechanism by which F508del leads to loss of function of the CFTR channel is still poorly understood. The mutation is found in the first nucleotide binding domain (NBD1) and studies have shown that it causes misfolding of CFTR and subsequent degradation of the protein by the cellular quality control system. It is established that the mutation affects stability and dynamics of NBD1 but does not alter its structure significantly. This destabilizing effect of F508del can be compensated by specific mutations distributed over different regions of NBD1, leading to recovery of membrane expression of a functional channel. A surprising example involves the regulatory insertion (RI), a 32-residue long segment found in all CFTR orthologs but not in related channels or transporters. The RI is not resolved in crystal structures of NBD1 nor cryo-EM structures of CFTR and has been described as intrinsically disordered. Its functional role in CFTR is unknown. Removal of the RI increases the stability of the NBD1 domain and, in the context of F508del-CFTR, this deletion restores maturation, cell surface expression and activity of the mutant channel. We probed the effect of the RI on NBD1 structure, dynamics and allostery using X-ray crystallography, single molecule FRET and hydrogen-deuterium exchange. We discovered that the RI enables an alternative NBD1 fold which departs markedly from the canonical fold previously observed for this domain and the NBDs of other ABC transporters. The conformational equilibrium between these states is regulated by ATP binding and affected by disease-associated conditions. Aside from clear alterations to structure and dynamics of NBD1, the RI also affects allostery, i.e. how NBD1 structure and dynamics respond to perturbations such as ligand binding. Finally, we show that the RI-enabled conformation is adopted in full-length CFTR and associated with increased channel activity in electrophysiological assays. We then identify an allosteric network that links the structural hotspots of the conformational changes to F508 and its surroundings. Lastly, we argue that these conformational changes lead to unfolding of NBD1 in the context of F508del, providing a new model for the molecular mechanism leading to pathogenesis. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Hsp70 molecular chaperones protect proteins from aggregation, assist in their native structure formation, and regulate stress responses in the cell. A mechanistic understanding of Hsp70 function will be necessary to explain its physiological roles and guide the therapeutic modulation of various disease states. To this end, several fundamental features of the Hsp70 structure-function relationship are investigated. The central component of Hsp70 chaperone function is its capacity for allosteric signaling between structural domains and tunable binding of misfolded protein substrates. In order to identify a cooperative network of sites that mediates interdomain allostery within Hsp70, a mutational correlation analysis is performed using genetic data. Evolutionarily correlations that describe an allosteric network are validated by examining roles for implicated sites in cellular fitness and molecular function. In a second component of the Hsp70 molecular mechanism, a novel function is discovered for the disordered C-terminal tail. This region of the protein enhances the refolding efficiency of substrate proteins independently of interdomain allostery and is required in the cell upon depletion of compensatory chaperones, suggesting a previously undescribed mode of chaperone action. Finally, experiments are initiated to assess the dynamic assembly of Hsp70 domains in various allosteric states and how domain orientations may be guided through interaction with partner co-chaperone proteins.

• Argonaute proteins are important effectors in RNA silencing pathways, but they must interact with other machinery to trigger silencing. Ago hooks have emerged as a conserved motif responsible for interaction with Argonaute proteins, but little is know about the sequence surrounding Ago hooks that must restrict or enable interaction with specific Argonautes. • Here we investigated the evolutionary dynamics of an Argonaute-binding platform in NRPE1, the largest subunit of RNA Polymerase V. We compared NRPE1 sequences from more than 50 species, including dense sampling of two plant lineages. • This study demonstrates that the Argonaute-binding platform of NRPE1 retains Ago-hooks, intrinsic disorder, and repetitive character while being highly labile at the sequence level. We reveal that loss of sequence conservation is due to relaxed selection and frequent expansions and contractions of tandem repeat arrays. These factors allow a complete restructuring of the Ago-binding platform over 50-60 million years. This evolutionary pattern is also detected in a second Ago-binding platform, suggesting it is a general mechanism. • The presence of labile repeat arrays in all analyzed NRPE1 Ago-binding platforms indicates that selection maintains repetitive character, potentially to retain the ability to rapidly restructure the Ago-binding platform.

Les protéines sont des acteurs majeurs dans les processus moléculaires et cellulaires d’un organisme. La remise en question des modalités associées aux fonctions de ces macromolécules a récemment été apportée par le concept de désordre intrinsèque. Celui-ci définit l’absence (transitoire ou permanente) de structure tridimensionnelle de certaines protéines ou régions protéiques comme étant directement liée à leurs fonctions. Chez les virus à ARN, les propriétés des protéines ou régions désordonnées semblent associées aux capacités de ces micro-organismes à détourner la machinerie cellulaire de l’hôte en interagissant avec de multiples partenaires, et à s’adapter aux nombreuses contraintes auxquelles ils doivent faire face en tant que parasites obligatoires. Ce travail porte sur les potyvirus, figurant parmi les pathogènes de plantes les plus dommageables étudiés à ce jour. L'objectif de cette thèse a été d’explorer les fonctions associées au désordre intrinsèque dans le cycle infectieux des potyvirus ainsi que dans le processus d’adaptation. Notre approche a ainsi démontré que : i) le désordre est ubiquitaire chez le genre Potyvirus ; ii) les régions de désordre conservées chez plusieurs protéines de potyvirus semblent être associées à leur(s) fonction(s) pendant l'infection ; iii) les régions désordonnées sont généralement associées à moins de contraintes évolutives, suggérant ainsi leur implication dans les processus adaptatifs des potyvirus ; iv) les régions prédites comme désordonnées semblent privilégier l’apparition de mutations et donc la capacité d’un virus à accumuler de la diversité génétique au cours de l'évolution sur son hôte naturel ; v) ce travail a permis de corréler le taux en désordre de la protéine viral genome-linked (VPg) du Potato virus Y à sa capacité à s’adapter à la résistance récessive pvr23 du piment. / Proteins are essential actors involved in a majority of molecular and cellular processes. The features associated with the functions of these macromolecules have been recently questioned with the emergence of the intrinsic disorder concept. It defines the transitory or permanent lack of 3D structure in some proteins or regions as directly related to their functions. Among RNA viruses, the properties of disordered proteins may be linked to the ability of these microorganisms to hijack the host machinery by interacting with multiple partners, as well as to adapt to the multiple constraints they must face as obligatory parasites. This work focuses on the Potyvirus genus, which includes some of the most damaging plant pathogens studied to date. The goal of this thesis was to explore the functions associated with intrinsic disorder in the infectious cycle of this viral genus as well as in its process of adaptation. Our studies have shown that i) intrinsic disorder is ubiquitous in potyviruses; ii) intrinsically disordered regions (IDR) of some of potyviral proteins are likely to be associated with important functions for the viral cycle ; iii) IDR are generally less evolutionary constrained, suggesting an adaptive potential of these regions ; iv) predicted IDR seem to favor the appearance of mutations and therefore virus ability to accumulate genetic diversity during its evolution in natural host ; v) an experimental disorder modulation within the Viral genome-linked (VPg) protein has been demonstrated as positively correlated with the adaptive ability of the Potato virus Y to overcome the pvr23 recessive resistance in pepper.

Phosphorylation mediated inactivation of PTEN leads to multiple malignancies with increased severity. However, the consequence of such inactivation on downstream functions of PTEN are poorly understood. Therefore, the objective of my thesis is to ascertain the molecular mechanisms by which PTEN phosphorylation drives lung cancer. PTEN phosphorylation at the C-terminal serine/threonine cluster abrogates its tumor suppressor function. Despite the critical role of the PTEN C-tail in regulating its function, the crystal structure of the C-tail remains unknown. Using bioinformatics and structural analysis, I determined that the PTEN C-tail is an intrinsically disordered region and is a hot spot for post-translational modifications (particularly phosphorylation) and protein-protein interactions. Evolutionary analysis of PTEN and its interacting proteins revealed that the PTEN C-tail has only recently evolved to acquire the ability to engage in a myriad of protein-protein interactions, resulting in its versatile functions. Replacement of the PTEN C-tail serine/threonine residues with alanines generated an artificial mutant, PTEN-4A, which remained “phospho-deficient” and therefore constitutively active. Interestingly, PTEN-4A suppressed cell proliferation and migration to a greater extent than PTEN-WT. PTEN-4A preferentially localized to the nucleus where it suppressed E2F-mediated transcription of cell cycle genes. PTEN physically interacted with the E2F1 protein and at E2F1-binding sites on chromatin, a likely mechanism for its transcriptional function. Further, deletion analysis on various PTEN domains revealed that the C2 domain of PTEN is indispensable for suppression of E2F-related genes. Systematic transcriptional promoter-reporter assays identified disease-associated C2 domain mutations that lose their ability to suppress E2F-mediated transcription, supporting the concept that these mutations are oncogenic in patients. Consistent with my findings, I observed increased level of PTEN phosphorylation and reduced nuclear PTEN levels in lung cancer patient samples. Further, to determine whether the enhanced growth-suppressive properties of PTEN-4A may be due to differential protein-protein interactions, I performed a comparative proteomic profiling of PTEN-WT and PTEN-4A interactomes using the SILAC methodology. Galectin-1 was identified as a candidate protein that binds preferentially to PTEN-WT and inhibits its tumor suppressive function. Taken together, the various tumor suppressive mechanisms of PTEN-4A may be harnessed therapeutically as adjunctive cancer therapy. Use of small molecule inhibitors that hinder PTEN C-tail phosphorylation is a plausible approach to activate PTEN function to reduce tumor burden.

Protein-coding genes evolve together with their genome and acquire changes, some of which affect the length of their protein products. This explains why equivalent proteins from different species can exhibit length differences. Variation in length of proteins during evolution arguably presents a large number of possibilities for improvement and innovation of protein structure and function. In order to contribute to an increased understanding of this process, we have studied variation caused by tandem domain duplications and insertions or deletions of intrinsically disordered residues. The study of two proteins, Nebulin and Filamin, together with a broader study of long repeat proteins (&gt;10 domain repeats), began by confirming that tandem domains evolve by internal duplications. Next, we show that vertebrate Nebulins evolved by duplications of a seven-domain unit, yet the most recent duplications utilized different gene parts as duplication units. However, Filamin exhibits a checkered duplication pattern, indicating that duplications were followed by similarity erosions that were hindered at particular domains due to the presence of equivalent binding motifs. For long repeat proteins, we found that human segmental duplications are over-represented in long repeat genes. Additionally, domains that have formed long repeats achieved this primarily by duplications of two or more domains at a time. The study of homologous protein pairs from the well-characterized eukaryotes nematode, fruit fly and several fungi, demonstrated a link between variation in length and variation in the number of intrinsically disordered residues. Next, insertions and deletions (indels) estimated from HMM-HMM pairwise alignments showed that disordered residues are clearly more frequent among indel than non-indel residues. Additionally, a study of raw length differences showed that more than half of the variation in fungi proteins is composed of disordered residues. Finally, a model of indels and their immediate surroundings suggested that disordered indels occur in already disordered regions rather than in ordered regions. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: In press. Paper 4: Manuscript.</p>

The heat shock response (HSR) is a robust cellular reaction to mitigate protein damage from heat and other challenges to the proteome. This protective molecular program in humans is controlled by heat shock transcription factor 1 (HSF1). Activation of HSF1 leads to the induction of an array of cytoprotective genes, many of which code for chaperones. These chaperones, known as heat shock proteins (HSPs), are responsible for maintaining the functional integrity of the proteome. HSPs achieve this by promoting proper folding and assembly of nascent proteins, refolding denatured proteins, and processing for degradation proteins and aggregates which cannot be returned to a functional conformation. The powerful ability of the heat shock response to promote cell survival makes its master regulator, HSF1, an important point of research. To garner a better understanding of HSF1, we reviewed the role of the highly dynamic HSF1 protein structure and investigated how HSF1 affects cancer cell behavior and drug response. Cancers can be characterized in part by abhorrent replication, self-sufficient growth signaling, invasion, and evasion of apoptosis. HSF1 has been found to promote proliferation, invasion, and drug resistance in several types of cancer; including lung and ovarian cancer. Ovarian cancer has elevated levels of HSF1, but the role of HSF1 in ovarian cancer behavior had not been previously examined. Researching the role of HSF1 in ovarian cancer is merited, because treatment outcomes are poor due to the high frequency of late stage detection and drug resistance. We hypothesized that HSF1 is important in the malignant growth and drug resistance of ovarian cancer. We have created ovarian cancer cell lines with inducible knockdown of HSF1 to investigate how HSF1 contributes to the behavior of ovarian cancer. This allowed us to examine the behavior of cells in the absence HSF1. Both 2D and 3D spheroid tissue culture models were used to study how HSF1 contributes to the growth and invasion of ovarian cancer cells after treatment with the transforming growth factor β (TGFβ) cytokine. Additionally, we studied how HSF1 reduction modulates the response to multiple therapeutic drugs. Our research shows that HSF1 induces epithelial-mesenchymal transition (EMT) in a 3D growth model. Our work also demonstrates that reduction of HSF1 sensitizes ovarian cancer cells to multiple drugs.

Der Transkriptionsfaktor CEBPA reguliert Differenzierung und Proliferation in verschiedenen Zelltypen und spielt eine herausragende Rolle in der Hämatopoese. Die CEBPA RNA kann in die lange P42-Isoform oder die N-terminal verkürzte P30-Isoform translatiert werden. Während P42-CEBPA differenzierungsinduzierend wirkt, ist P30 als Inhibitor von P42 und als Onkogen in akuter myeloider Leukämie beschrieben. Die Modularität und Multifunktionalität von CEBPA, die ihn zahlreichen Studien beobachtet wurde, lässt sich möglicherweise durch differentielle Protein–Protein-Interaktionen erklären. Zahlreiche post-translationale Modifikationen (PTMs) und die intrinsisch ungeordnete, flexible Struktur von CEBPA stellen jedoch eine Herausforderung für traditionelle Ansätze in Proteininteraktionsstudien dar. In der vorliegenden Arbeit wird ein neuer, alternativer Ansatz präsentiert, der auf einem in vitro Proteininteraktions-screen auf einer Peptidmatrix (PRISMA) und Biotinligase proximity labelling (BioID) in lebenden Zellen basiert. In einem PRISMA-screen wurden 120 CEBPA Peptide auf Proteininteraktionen mit Proteinextrakt aus myeloiden Zellen untersucht. Im Screen wurden 40 verschiedene CEBPA PTMs inkludiert, unter anderem auch die hier erstmals neu beschriebenen Methylierungen der CEBPA Argininreste R12 und R142. Daten aus dem PRISMA-screen wurden mit BioID Experimenten in myeloiden Zellen validiert, um eine Proteininteraktionslandkarte von CEBPA zu generieren, die 52 bekannte und 68 neue CEBPA Proteininteraktoren umfasst. Hotspots für Proteininteraktionen fallen in evolutionär konservierte CEBPA Regionen und der Vergleich des Bindungsprofils mit publizierten Daten zeigt Ähnlichkeiten zu verwandten Transkriptionsfaktoren der CEBP Familie. Die Ergebnisse legen nahe, dass die Multifunktionalität von CEBPA von multivalenten Proteininteraktionen in Abhängigkeit von PTMs koordiniert wird, um CEBPA mit dem epigenetischen und transkriptionellen Apparat der Zelle verknüpfen. / The pioneering transcription factor CEBPA plays a lineage-instructing role during haematopoiesis and also regulates proliferation and differentiation in many other cell types. The CEBPA RNA can be translated into a full length (P42-CEBPA) or N-terminally truncated isoform (P30-CEBPA). While P42 induces differentiation in various cell types, the P30 isoform is mostly regarded as a dominant inhibitor of P42-CEBPA and acts as an oncogene in acute myeloid leukaemia. Protein interactions may be the key to explaining the functional plasticity and modularity of CEBPA that has been demonstrated in diverse experimental settings. However, the disordered structure and the numerous post-translational modification sites (PTMs) of CEBPA pose a challenge to traditional protein interaction studies. In the present work, a novel alternative approach is presented that combines an in vitro protein interaction screen on a peptide matrix (PRISMA) with biotin ligase proximity labelling (BioID) in living cells. To this end, 120 CEBPA peptides were probed for protein interactions with PRISMA. The screen comprised 40 different PTMs, including newly identified CEBPA arginine methylation sites. PRISMA data was validated with BioID experiments and generated a detailed CEBPA protein interaction map in myeloid cells. The interactome presented here contains 52 known and 68 novel CEBPA interactors that can now be mapped across the CEBPA sequence in a PTM dependent fashion. Hotspots of protein interaction correlated with conserved regions and comparison with previously published data revealed related binding profiles of homologous CEBP regions. Taken together, the data indicates that the functional plasticity of CEBPs is orchestrated by multivalent protein interactions and PTMs to configure a dynamic CEBP hub that interacts with many partners of the transcriptional and epigenetic machinery.