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Spores are single-celled dispersal units surrounded by a wall of the highly resistant biopolymer sporopollenin. All land plants produce spores. Spore development is described in Physcomitrella patens, a moss with single-celled spores, and Dendroceros, a hornwort with multicellular spores. Correlated light, fluorescence and immuno-electron microscopy localizes callose in the aperture of developing spores in the model moss Physcomitrella. Twelve copies of callose synthase genes were annotated bioinformatically and compared with Arabidopsis callose synthase genes. This study identifies a suspect gene involved in moss spore exine development. Unicellular spores of Dendroceros following meiosis remain in tetrads, fill the intercapsular space, and are surrounded by a convoluted, homogeneous electron-opaque outer wall and narrow fibrillar inner wall. No precise pattern of cell division leads to multicellular spores of variable shape and cell number. Evolution of precocious endospory in epiphytic hornworts is a means to protect nascent spores while it develops biochemical and structural machinary to withstand drying. To advance knowledge of genetic control of spore wall development, the sequenced genome of Physcomitrella is probed using a bioinformatic approach to decipher the evolution of five selected genes putatively involved in spore wall formation. Those encoding for callose synthase provide the most complete results. Callose involvement in spore development is a plesiomorphic feature of land plants. Phylogenomic analysis of callose synthases in land plants with sequenced genomes revealed a single moss callose synthase basal in a clade containing the only Arabidopsis callose synthase implicated in exine development of pollen walls as well as two clades of moss specific callose synthase proteins. A predicted protein-protein interactome was constructed to investigate the protein landscape in Physcomitrella for proteins involved in sporogenesis. Orthologous genes were identified between Physcomitrellaand several other species to map orthologous interactions and predict the first bryophyte interactome. The Physcomitrella predicted protein-protein interactome contains 41,936 unique interactions for 4062 different proteins, none of which are associated with sporogenesis. Rather the most conserved interactions among proteins were those associated with metabolic processes. The utility of predicted protein interactions to infer biological roles, providing provisional molecular roadmaps is demonstrated to generate hypotheses for experimental approaches.

Bioinformatics is a valuable tool to understand gene regulatory networks. Cis-regulatory elements (CREs) previously found in promoter regions are known to recruit transcription in signaling pathways. In this work it has been hypothesized to consider CREs as a family of related words that interact/bind to a family of related transcription factors, and thus have similar but distinct regulation patterns. A 1460 microarray gene expression collection was obtained via online databases to create a transcriptomic meta-dataset. A novel bioinformatic algorithm was applied to annotate all 65536 (64k) potential 8-letter CREs in the 500 bp upstream promoter region of all A. thaliana genes across the transcriptomic meta-dataset. Of the possible words, only 2,498 were significantly associated with a pattern of regulation in any of the 1,460 microarrays tested whereas the remaining motifs appeared not to be regulatory. Unique CREs were categorized into 4 regulatory types: inducer, suppressor, biregulator and insulator. A predicted protein protein interactome was created for an economically important plant Coffea canephora. Here, it has been hypothesized that evolutionary conservation of many core biological processes enable generation of predicted protein interactome for species with few resources other than sequenced genome. Of over 12,000 genes identified, 939 were predicted to have 4,587 interactions. Gene Ontology analysis revealed enrichment of processes conserved in all eukaryotes but depletion in unique plant processes. A third study was conducted to determine if homology modeling, evolutionary analysis, and structural evolution could determine key factors involved in function, and interaction specificity in Pus10 (EC 5.4.99.25) found in Archaea and Eukaryotes. Redundancy of Pus10 and the bacterial TrmA and TruB orthologs appear to have resulted in significant molecular evolution of Pus10 function. Neofunctionalization was identified in animal kingdom where thiouridine synthase, methylases and PSUSs (THUMP)-domain modification in early animal evolution coincides with appearance of TNF-related apoptosis-inducing ligand (TRAIL) apoptosis components. Subfunctionalization was identified for Thermococcales lineage of Archaea where a shorter forefinger-loop coincides with the loss of Ψ54 specificity as experimentally verified in P. furiosus. Absence of Pus10 was observed in Sulfolobus and higher fungi whereas in plant kingdom Pus10 function remains unknown with possible pseudogene in some lineages

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.

Kürzlich wurde mithilfe von Ribo-seq Experimenten die Translation hunderter Mikroproteine in menschlichen Herzen entdeckt. Diese blieben zuvor aufgrund ihrer geringen Größe (< 100 Aminosäuren) unentdeckt, und ihre physiologische Rolle ist noch weitgehend unbekannt. Ziel dieser Promotionsarbeit ist es, potentielle Funktionen dieser neuartigen Mikroproteine zu entschlüsseln. Dabei sollen insbesondere die Aufklärung ihrer evolutionären Konservierungssignatur, subzellulären Lokalisierung und ihres Proteininteraktoms helfen. Die Konservierungsanalyse ergab, dass fast 90% der Mikroproteine nur in Primaten konserviert ist. Weiterhin konnte ich die Produktion von Mikroproteine in vitro und in vivo nachweisen, die subzelluläre Lokalisierung von 92 Mikroproteinen definieren, und Interaktionspartner für 60 Mikroproteine identifizieren. Dutzende dieser Mikroproteine lokalisieren in Mitochondrien. Dazu gehörte ein im Herzen angereichertes Mikroprotein, das aufgrund der Interaktions- und Lokalisationsdaten einen neuartigen Modulator der mitochondrialen Proteintranslation darstellen könnte. Der Interaktom-Screen zeigte außerdem, dass evolutionär junge Mikroproteine ähnliche Interaktionsfähigkeiten wie konservierte Kandidaten haben. Schließlich wurden kurze Sequenzmotive identifiziert, die Mikroprotein-Protein-Wechselwirkungen vermitteln, wodurch junge Mikroproteine mit zellulären Prozessen – wie z.B. Endozytose und Spleißen – in Verbindung gebracht werden konnten. Zusammenfassend wurde die Produktion vieler kleiner Proteine im menschlichen Herzen bestätigt, von denen die meisten lediglich in Primaten konserviert sind. Zusätzlich verknüpften umfangreiche Lokalisierungs- und Interaktionsdaten mehrere Mikroproteine mit Prozessen wie Spleißen, Endozytose und mitochondrialer Translation. Weitere Untersuchungen dieses zuvor verborgenen Teils des Herzproteoms werden zu einem besseren Verständnis von evolutionär jungen Proteinen und kardiologischen Prozessen beitragen. / Recently, the active translation of hundreds of previously unknown microproteins was detected using ribosome profiling on tissues of human hearts. They had remained undetected due to their small size (< 100 amino acids), and their physiological roles are still largely unknown. This dissertation aims to investigate these novel microproteins and validate their translation by independent methods. Particularly, elucidating their conservation signature, subcellular localization, and protein interactome shall aid in deciphering their potential biological role. Conservation analysis revealed that sequence conservation of almost 90% of microproteins was restricted to primates. I next confirmed microprotein production in vitro and in vivo by in vitro translation assays and mass spectrometry-based approaches, defined the subcellular localization of 92 microproteins, and identified significant interaction partners for 60 candidates. Dozens of these microproteins localized to the mitochondrion. These included a novel cardiac-enriched microprotein that may present a novel modulator of mitochondrial protein translation based on its interaction profile and subcellular localization. The interactome screen further revealed that evolutionarily young microproteins have similar interaction capacities to conserved candidates. Finally, it allowed identifying short linear motifs that may mediate microprotein-protein interactions and implicated several young microproteins in distinct cellular processes such as endocytosis and splicing. I conclude that many novel small proteins are produced in the human heart, most of which exhibit poor sequence conservation. I provide a substantial resource of microprotein localization and interaction data that links several to cellular processes such as splicing, endocytosis, and mitochondrial translation. Further investigation into this hidden part of the cardiac proteome will contribute to our understanding of recently evolved proteins and heart biology.

Proteins and their intricate network of interactions are fundamental to many molecular processes that govern life. Mass spectrometry-based structural proteomics represents a powerful set of techniques for characterizing protein structures and interactions. The last decade has witnessed a large-scale adoption in the application of these techniques toward solving a variety of biological questions. Addressing these questions has often been coincident with the further development of these techniques. Insight into the structures of individual proteins and their interactions with other proteins in a proteome-wide context has been made possible by recent developments in the relatively new field of chemical crosslinking combined with mass spectrometry. In these experiments crosslinking reagents are used to capture protein-protein interactions by forming covalent linkages between proximal amino acid residues. The crosslinked proteins are then enzymatically digested into peptides, and the covalently-coupled crosslinked peptides are identified by mass spectrometry. These identified crosslinked peptides thus provide evidence of interacting regions within or between proteins. In this dissertation the development of tools and methods that facilitate this powerful technique are described. The primary arc of this work follows the development and application of mass spectrometry-based approaches for the identification of protein crosslinks ranging from those which exist endogenously to those which are introduced synthetically. Firstly, the development of a novel strategy for comprehensive determination of naturally occurring protein crosslinks in the form of disulfide bonds is described. Secondly, the application of crosslinking reagents to create synthetic crosslinks in proteins coupled with molecular dynamics simulations is explored in order to structurally characterize the intrinsically disordered tau protein. Thirdly, improvements to a crosslinking-mass spectrometry method for defining a protein-protein interactome in a complex sample is developed. Altogether, these described approaches represent a toolset to allow researchers to access information about protein structure and interactions. / Graduate