The Inhibitor of Apoptosis (IAP) proteins are a highly conserved group of anti-apoptotic proteins. Cellular IAP 1 and 2 (cIAP1 and 2) are two members of the IAP protein family that regulate the activity of the Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) transcription factor family. The role and mechanism of the IAPs in ubiquitination are not yet completely understood due to the complexity of this posttranslational modification process. Additionally, The IAPs are involved in a myriad of cellular processes, and many of the process-specific mechanisms by which the IAPs are involved is unknown. I aim to delve deeper into the signalling pathways that are controlled by cIAP1 and cIAP2 by discovering currently unknown protein-protein interactions. In doing so, I will determine which proteins interact with the cIAPs and what signalling pathways these proteins are involved in. Using a BioID approach, I sought to characterize the cIAP1 interactors involved in the canonical and non-canonical NF-κB pathways. I generated a stable cell line expressing TurboID-cIAP1 fusion protein in HEK 293T cells that expressTurboID-cIAP1 at levels comparable to endogenous cIAP1. I identified multiple potential cIAP1 interactors that have ties to the NF-κB pathway. These proteins regulate NF-κB signalling in multiple ways including influencing acetylation and nuclear retention of the NF-κB transcription factors, phosphorylation of NF-κB transcription factors, and RNA splicing of genes involved in the TNFR1 complex I. Further work needs to be done to confirm these interactions and to discover the mechanisms by which these interactions occur. NF-κB signalling is known to have widespread function within the cell and within diseases such as cancer; it will be beneficial to study these interactions to better understand how cancer develops and how to treat it best, especially in patients with a poor prognosis.

La littérature indique que les miARN sont régulés à plusieurs niveaux de leur biogenèse et de leur activité. Cependant, il existe très peu d’information concernant la régulation de la stabilité des miARN. Le projet de thèse a consisté à étudier la dégradation spécifique d’un miRNA cellulaire (miR-27) induite par un transcrit viral (m169) au cours de l’infection par le cytomégalovirus murin (MCMV). Ce miARN est déstabilisé par un mécanisme moléculaire appelé ‘target-RNA directed miRNA degradation’ (TDMD). En suivant deux grands axes de recherche j’ai entrepris : premièrement l’étude et la caractérisation des déterminants moléculaires et des facteurs cellulaires impliqués dans le mécanisme de TDMD ; puis dans un second temps, la mise en place d’une approche protéomique permettant l’identification des partenaires de la protéine AGO2 potentiellement impliqué dans le TDMD dans des cellules infectées ou non par le MCMV. / Several regulatory mechanisms have been uncovered at every level of the biogenesis and the activity of miRNAs. However, there is less information about the regulation of the stability of miRNAs. The PhD project entailed the study of a process, which specifically enables the degradation of a cellular miRNA (miR-27) induced by a viral transcript (m169) during an infection by the mouse cytomegalovirus (MCMV). This miRNA is destabilized by a process called ‘target-RNA directed miRNA degradation’ (TDMD). I first undertook the study and the characterization of the molecular determinants and the cellular factors implicated in TDMD. Moreover, I started to set up a protocol in order to identify AGO2 partners of viral or host origin during MCMV infection, which would potentially be implicated in TDMD.

The actin and microtubule cytoskeleton play critical roles in Golgi and cilia assembly. Inverted-Formin 1 (INF1) is a novel, microtubule-associated protein that regulates both actin and microtubule dynamics and affects Golgi and cilia assembly. A non-biased discovery based approach was used to investigate the interactome of INF1 using BioID in combination with stable isotopic labeling in cell culture (SILAC). A number of INF1-interacting proteins were identified and validated in co-IP experiments. The INF1 interaction domains were mapped using an extensive set of INF1 deletion and point mutation derivatives. Functional characterization of these interactions suggests a mechanism for the effects of INF1 on ciliogenesis. The establishment and maintenance of cellular architecture requires the coordinated, dynamic regulation of actin and microtubule networks. Our data suggests that INF1 plays a crucial role in connecting these two cytoskeletal networks for the regulated assembly of the Golgi ribbon and the primary cilium.

Macroautophagy (hereafter referred to as autophagy) is the bulk degradation and recycling of cytoplasmic materials by forming a double membrane vesicle called the autophagosome. Autophagosome formation is regulated by the coordinated action of a set of proteins. ATG9A is the only multispanning transmembrane protein that plays an essential role in autophagosome formation, yet its function is largely elusive. Previous studies have shown that the C-terminus of ATG9A plays an important role in regulating its trafficking and proper function in autophagy. In line with that idea, we previously identified an AMPK- and ULK1- mediated phosphorylation on the C terminus of ATG9A at S761, which is required for proper ATG9A trafficking and autophagic flux. In our current study, we employed a BioID-based proteomics approach and identified a network of ATG9A C terminal interactors that include members of the ULK1 complex, ATG13, and ATG101, as well as protein complexes within the ER, Golgi, and endosomal trafficking pathways, many of which provide new insight into ATG9A trafficking mechanisms. We discovered that ATG9A exists with ATG13 and ATG101 in a separate subcomplex outside the canonical ULK1 complex. We show that the ATG13-ATG101 subcomplex regulates ATG9A trafficking and basal P62 degradation.

Autism spectrum disorder (ASD) encompasses a broad range of neurodevelopmental disorders, with two core symptoms: deficits in social communication, and restrictive interests and repetitive behaviors. Genetics is thought to play a large role in ASD and currently there are hundreds of associated genes. We first studied the thousand and one amino acid kinase gene (TAOK2), which plays an important role in neurodevelopment. We found that loss of TAOK2 causes deficits in neuron development and activity, leading to morphological changes in various mouse brain regions and ASD-related behaviors. We studied the impact of de novo mutations identified in TAOK2, which caused aberrant neuron dendritic arborization and formation of synapses. To elucidate how TAOK2 regulates neuron development we used a proximity-labeling proteomics technique (BioID) to identify its protein-protein interaction (PPI) network. We applied this same methodology to a total of 41 ASD-risk genes and observed multiple convergent biological processes, including the less-studied mitochondrial and metabolic pathways. ASD-risk genes, including TAOK2, associated with mitochondrial proteins were found to have altered cellular respiration. The shared ASD-risk gene PPI network enriched for other ASD-risk genes and was used to group genes based on their shared PPI networks. These gene groups showed correlation between the clinical behavior scores of individuals that had mutations within the distinct gene groups. Lastly, we identified changes in the PPI networks of multiple ASD-risk genes through BioID, which we validated with various functional assays. In summary, we developed a proximity-labeling proteomics method that identified multiple convergent biological pathways associated with ASD. Studying the function of TAOK2 revealed multiple disease-relevant pathologies associated with the disorder, however proximity labeling has the potential to categorize multiple ASD-risk genes and elucidate their shared signaling pathways, which together, can advance the development of robust treatments for ASD. / Thesis / Doctor of Philosophy (PhD) / Autism spectrum disorder (ASD) is a group of brain disorders that affect more than 1% of children. Genetic variants are thought to cause ASD pathology, however there are currently hundreds of genes that have not been studied. We studied how disruption of one of those genes, TAOK2, alters brain development in mice and identified TAOK2 variants in multiple children with ASD. We then used BioID to find the shared disease-related mechanisms between multiple ASD-risk genes, and found that mitochondrial function and activity were connected to many of these genes. We showed that BioID can be used to study the effect of mutations in multiple ASD-risk genes simultaneously. Last, we could group children with ASD with similar behavior test scores based on the shared mechanisms of ASD-risk genes. Together our findings could be used to advance the development of robust treatments or new diagnostic tools for ASD.

Protein kinase A (PKA) regulates a diverse array of cellular activities including metabolism, differentiation, actomyosin contractility, and migration. The multifunctionality of this ubiquitous enzyme is achieved, in part, through subcellular targeting mediated by the A Kinase Anchoring Proteins (AKAP) family of proteins. AKAPs serve as scaffolding proteins that localize PKA to various cellular compartments and bring together specific targets and modulators of PKA activity. The importance of spatially restricted PKA signaling is particularly apparent in the context of cell motility. It has been observed that both anchoring through AKAPs and the subsequent localized activation of PKA at the leading edge of migrating cells are required for directed migration in multiple cell types. Despite the significant body of evidence linking PKA to the regulation of cellular adhesion, contractility, and migration, the mechanisms governing the spatiotemporal control of PKA signaling during these activities is not fully understood. Focal adhesion complexes, which connect the actin cytoskeleton to the extracellular matrix and are thus intimately involved in the adhesive and contractile state of the cell, are attractive potential sites of PKA signaling. We have evidence indicating that PKA is active within these complexes, and that this activity impacts focal adhesion dynamics. To address the question of how PKA may be recruited to adhesive complexes, we have developed a targeted screen to identify PKA interacting proteins within adhesive and cytoskeletal structures. This method utilizes proximity-dependent biotin labeling in combination with a focal adhesion purification preparation and downstream proteomic analysis. The results of this screen will be used to identify candidate AKAPs and will serve as the foundation for future inquiry into the complex role of PKA in the regulation of cell migration.

Neurodevelopmental disorders (NDDs) are a heterogeneous set of disorders that are characterized by early disruptions to brain development and include autism spectrum disorder (ASD), attention deficit/hyperactivity disorder (ADHD), developmental delay (DD), intellectual disability (ID), epilepsy and schizophrenia (SZ). Although thousands of genetic risk variants have been identified, there is a lack of understanding of how they impact cellular and molecular mechanisms that underlie the clinical presentation and heterogeneity of NDDs. To investigate this, we used a combination of cellular, molecular, bioinformatic and omics methods to study NDD-associated molecular pathways in distinct neuronal populations. First, we studied the interaction between the high-confidence SZ risk genes DISC1 and NRG1-ErbB4 in cortical inhibitory neurons and found that NRG1-ErbB4 functions through DISC1 to regulate dendrite growth and excitatory synapses onto inhibitory neurons. Next, we studied the 15q13.3 microdeletion, a recurrent copy number variation (CNV) that is associated with multiple NDDs. Using a heterozygous mouse model [Df(h15q13)/+] and human sequencing data we identified OTUD7A (encoding a deubiquitinase) as an important gene driving neurodevelopmental phenotypes in the 15q13.3 microdeletion syndrome. Due to the paucity of literature on the function of OTUD7A in the brain, we used a proximity-labeling approach (BioID2) to elucidate the OTUD7A protein interaction network (PIN) in cortical neurons, and to examine how patient mutations affect the OTUD7A PIN. We found that the OTUD7A PIN was enriched for postsynaptic and axon initial segment proteins, and that distinct patient mutations have shared and distinct effects on the OTUD7A PIN. Further, we identified the interaction of OTUD7A with a high-confidence bipolar risk gene ANK3, which encodes AnkyrinG. We identified decreased levels of AnkyrinG in Df(h15q13)/+ neurons, and synaptic phenotypes were rescued by increasing AnkyrinG levels or targeting the Wnt pathway. Future investigation should include examination of the role of OTUD7A deubiquitinase activity in neural development. / Dissertation / Doctor of Philosophy (PhD) / Neurodevelopmental disorders result from disruptions to early brain development and include autism spectrum disorder (ASD), developmental delay (DD), epilepsy, and schizophrenia (SZ). These disorders affect more than 3% of children worldwide and can have a significant impact on an individual’s quality of life, including an increased risk of death in some cases. There is currently a lack of understanding of how these disorders develop and how to effectively treat them. Neurodevelopmental disorders are thought to arise from alterations in the connections between brain cells (neurons) and one of the major risk factors for these disorders is having certain variations in regions of the genome (DNA sequences), with more than 1000 of these risk variants having been identified so far. In this thesis, we analyzed how genetic risk factors interact in neurons to regulate neural connectivity. We discovered that risk variants found in individuals with different disorders actually work together to regulate similar processes important for neural connectivity, which suggests that distinct disorders may share a common underlying cause. Additionally, we established the importance of a new ASD risk gene and discovered that it interacts with other known risk genes to regulate neural connectivity. This thesis provides new insights into the processes in the brain that lead to neurodevelopmental disorders and has implications for future development of effective therapies for individuals affected by these disorders.

Studies demonstrated ADAMTS13 possesses unique properties with a mystifying regulatory mechanism. ADAMTS13’s role is in its proteolytic function to its VWF. The disparity in the hemostatic balance between ADAMTS13 activity and the distribution of VWF multimers could result in the bleeding disorder Von Willebrand Disease (VWD) or the thrombotic disorder thrombotic thrombocytopenic purpura (TTP). ADAMTS13 is constitutively secreted as an active protease, yet VWF retains its capacity to recruit platelets. This ability makes ADAMTS13 an enigmatic protease with an unknown regulatory mechanism. Currently, the postulated regulatory mechanism of ADAMTS13 is in its open/closed conformation, yet ADAMTS13 activity is retained in both forms. Literature showed that few proteases are capable of degrading ADAMTS13 in-vitro. We hypothesize that the partial degradation of ADAMTS13 regulates its activity, thereby stabilizing VWF and promoting thrombosis. The goals of this project were to develop and optimize in-vitro plasma BioID to identify novel interactions to ADAMTS13, validate novel interactions, identify proteases capable of degrading ADAMTS13 and their proteolytic sites, and develop protease-resistant ADAMTS13 mutants as novel therapeutics to thrombotic disorders. We optimized the BioID technique to be used in-vitro in plasma, to study novel interactions with ADAMTS13. Our results identified novel potential interactions with vitronectin or plasminogen. Validation studies disregarded vitronectin’s interaction and confirmed plasminogen’s interaction through the CUB and Kringle domains in a lysine-dependent manner. Further, the list of proteases capable of degrading ADAMTS13 was expanded to include FXIa and neutrophil-derived proteases including Cathepsin G, elastase, and hPR3. Activated neutrophils played a stronger role than coagulation proteases in degrading ADAMTS13 in vivo, while also demonstrating that elastase is a more potent regulator. Proteolytically degraded sites on ADAMTS13 were identified and proteolytic-resistant ADAMTS13 mutants were produced accordingly, which we aim to be utilized as a novel therapeutic to thrombotic disorders. / Thesis / Doctor of Philosophy (Medical Science)

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.

Das CCAAT/Enhancer-Binding-Protein α (C/EBPα) ist ein Transkriptionsfaktor, der das Zellschicksal des hämatopoetischen Systems bestimmt. C/EBPα reguliert die Selbsterneuerung hämatopoetischer Stammzellen und bestimmt die myelomonozytäre Zelldifferenzierung. C/EBPα besitzt zudem die Eigenschaft lymphoide Zellen in myeloischen Zellen zu transdifferenzieren. Die von C/EBPα induzierte lymphoid-myeloische Transdifferenzierung kann als Modellsystem dienen, um die Vorgänge der Linienfestlegung, der zellulären Plastizität sowie der Funktionen von C/EBPα in der Zelldifferenzierung und Leukämogenese zu untersuchen. C/EBPα ist ein unstrukturiertes Protein, das seine Funktionen durch wechselnde Proteininteraktionen ausübt. Intrinsisch unstrukturierte Proteine, wie C/EBPα, sind prädestiniert an schwachen, multivalenten und hochdynamischen Proteininteraktionen teilzunehmen. Solche Inter-aktionen werden hauptsächlich durch kurze lineare Motive der Protein-Primärstruktur vermittelt. Motiv-basierte Proteininteraktionen sind mit den herkömmlichen Methoden der Interaktom-Analyse schwer zu analysieren. Die Anwendung der neuartigen Biotin-Ligase basierten TurboID Methode mit schneller Enzym-Kinetik ermöglichte nun die Analyse eines dynamischen C/EBPα Interaktoms während der lymphoid-myeloiden Transdifferenzierung. Es wurde festgestellt, dass sich die Proteinexpression der meisten C/EBPα interagierenden Proteine während der Transdifferenzierung kaum änderte, trotz erheblicher Änderungen des C/EBPα Interaktoms, was eine Regulation durch alternative Mechanismen nahelegt. Es wurden mehrere epigenetische Komplexe gefunden, einschließlich Mediator, SWI/SNF und CAF-1, die als mögliche Verbindung zwischen Interaktom, Transkriptom, Chromatinstruktur und Phänotyp in Betracht gezogen werden können. / The CCAAT/enhancer binding protein α (C/EBPα) is a key lineage-instructive transcription factor in the haematopoietic system. C/EBPα regulates the self-renewal of haematopoietic stem cells and is one of the main determinants of myeloid commitment. C/EBPα induces transdifferentiation of B cells into myeloid cells. C/EBPα-induced lymphoid-myeloid transdifferentiation may serve as a model system to study lineage commitment and cellular plasticity as well as address open questions related to functions of C/EBPα in differentiation and leukaemogenesis. C/EBPα is an intrinsically disordered protein that exerts its functions through protein interactions. Intrinsically disordered proteins (IDPs) engage in highly dynamic, weak, and multivalent protein interactions, which are mediated by short linear motifs (SLiMs). SLiMs-based protein interactions are difficult to analyse by conventional methods of interactome analysis. However, to understand the connection between the C/EBPα interactome and its functions, analysis in the cellular context is required. The application of the novel biotin ligase TurboID with faster kinetics enabled the analysis of the dynamic interactome of C/EBPα in the process of lymphoid-myeloid transdifferentiation. For most of the interacting proteins, the protein level did not change, yet variable interactions were found, indicating regulated interactions. TurboID identified changes in the composition of the SWI/SNF complex during transdifferentiation, including the exchange of subunits specific for BAF and PBAF subcomplexes, Brg1 and Brm ATPases, and cell type-specific subunits. The analysis also identified the interaction pattern of the histone demethylase Kdm6b and functional assays confirmed its role in transdifferentiation. TurboID enabled the comparative analysis of the interactomes of C/EBPα isoforms and mutants, that alter the balance between differentiation and proliferation and its oncogenic functions.