<p>Ankyrin repeats (ARs) are polypeptide motifs identified in thousands of proteins. Many AR proteins play a function as scaffolds in protein-protein interactions which may require specific mechanical properties. Also, a number of AR proteins have been proposed to mediate mechanotransduction in a variety of different functional settings. The folding and stability of a number of AR proteins have been studied in detail by chemical and temperature denaturation experiments, yet the mechanic of AR proteins remain largely unknown. In this dissertation, we have researched the mechanical properties of AR proteins by using protein engineering and a combination of atomic force microscopy (AFM)-based single-molecule force spectroscopy and steered molecular dynamics (SMD) simulations. Three kinds of AR proteins were investigated: NI6C (synthetic AR protein), D34 (of ankyrin-R) and gankyrin (oncoprotein). While the main focus of this research was to characterize the response of AR proteins to mechanical forces, our results extended beyond the protein nanomechanics to the understanding of protein folding mechanisms.</p> / Dissertation

The transient receptor potential ankyrin 1 (TRPA1) ion channel is expressed in a subset of primary afferent neurones where it is activated by a variety of pungent and chemically reactive compounds such as allyl isothiocyanate or cinnamaldehyde. This voltage- dependent channel is activated through covalent modification of cytoplasmic cysteines and, from the cytoplasmic side, is also critically regulated by calcium ions. Both, amino (N-) and carboxyl (C-) termini have been shown to be involved in these processes. Using electrophysiological and molecular-biology techniques, we explored the role of specific cytoplasmic domains in the activation of TRPA1. By measuring chemically-, voltage-, and calcium-activated membrane TRPA1-mediated currents, we identified highly conserved serine and threonine residues along the N-terminal ankyrin repeat domain, mutation of which strongly affected responses of the channel. In addition, using C-terminally truncated construct previously reported to be involved in calcium regulation, we present a new finding that the distal C-terminal tail contributes to voltage-dependent activation of TRPA1.

No description available.

Hypoxia-inducible factor (HIF) is the master regulator of genes involved in adaptation to hypoxia. The stability and transcriptional activity of HIF are regulated by post-translational hydroxylations: prolyl hydroxylation by the prolyl hydroxylase domain-containing enzymes PHD1 – 3 earmarks HIF for proteasomal degradation, whilst asparaginyl hydroxylation by factor inhibiting HIF (FIH) blocks the interaction of HIF with the transcriptional coactivators p300/CBP. The PHDs and FIH hydroxylate HIF directly from molecular oxygen and are therefore oxygen sensors. Recent literature shows that FIH also hydroxylates a number of proteins containing an ankyrin-repeat domain (ARD). Together with reports suggesting that the PHDs are involved in HIF-independent pathways, this suggests that the HIF hydroxylases may have a wide range of non-HIF targets. This thesis describes my investigations into novel substrates of the HIF hydroxylases. This work has characterized the FIH-dependent hydroxylation of the ARD-containing protein Notch1, and defined a consensus sequence for hydroxylation that corresponds to the ankyrin-repeat consensus. Using this consensus potential sites of hydroxylation in a novel ARD FIH substrate, myosin phosphatase targeting subunit 1 (MYPT1), were identified then subsequently confirmed and characterized. Notch1 competes with HIF for FIH hydroxylation. My experiments show that this occurs because Notch1 is a more efficient substrate than HIF, whilst studies on MYPT1 and other proteins indicate that competitive inhibition of FIH may be a general property of ARDs. There are more than 300 ARD proteins in the human genome, and this thesis demonstrates that FIH may hydroxylate a significant percentage of these. In addition to the analysis of ARD hydroxylation a proteomic investigation into novel PHD3 substrates has identified two candidate proteins, suggesting that the PHDs may also have multiple targets. These results have important implications for oxygen sensing, and indicate that post-translational hydroxylation is likely to be a widespread modification in cell biology.

Important receptor for sensing painful stimuli is ion channel TRPA1, which is expressed in peripheral endings of nociceptive neurons, where it serves as transducer of physical and chemical environmental signals to the language of the nervous system. The effort to understand the mechanisms of its activity on a molecular level is driven by the vision of progress in treatment of chronic pain in humans. Our work focused on C-terminal cytoplasmic domain of TRPA1 receptor, where we described i.a. the probable binding site for calcium, which is the most important TRPA1 modulator. Using the combination of homology modeling and molecular dynamic simulations with electrophysiological measurements we were able to explain molecular basis of familial pain syndrome caused by TRPA1 point mutation. We contributed to the understanding of the TRPA1 voltage-dependent activation mechanism by describing the amino acids in proximal C-terminus and in S4-S5 linker of transmembrane domain that are directly involved in voltage-dependent gating. Powered by TCPDF (www.tcpdf.org)

Designade ankyrinupprepningsproteiner (DARPiner) är små, mycket stabila antikroppsmimetiska proteiner. I det här projektet användes anti-EpCAM-DARPiner tillsammans med mikrofluidik för att avgära om de kunde fånga upp HCT116-celler mer effektivt än anti-EpCAM-antikroppar. Ytorna på insidan av mikroffluidikkanaler förändrades genom bindning av N-γ-maleimidobutyryl-oxysuccinimidester (GMBS) och merkaptopropyltrietoxysilan (MPTES) för anti-EpCAM-antikroppar och GMBS och (3-aminopropyl)trietoxysilan (APTES) för DARPiner. Båda kanaltyperna testades genom inflöde av cancerceller och helblod blandat med cancerceller. Ingen effektiv och konsekvent celluppfångst åstadkoms trots att det visades att antikropparna och DARPinerna kunde binda till cellerna direkt och att test med fluorescenta DARPiner och antikroppar visade att ytförändringskemin var fungerande. Slutsatsen blev att de mest troliga orsakerna till misslyckandena var att ytförändringskemin påverkade proteinernas bindningsförmåga negativt eller att proteinerna bands till kanalernas yta i fel riktning. DARPiner är fortfarande intressanta för tillämpningar inom mikrofluidik, men vidare förbättring av det experimentella protokollet behövs. / Designed ankyrin repeat proteins (DARPins) are small and highly stable antibody mimetics. In this project, anti-EpCAM DARPins were used in conjunction with microfluidics to determine if they could capture HCT116 cells more effectively than anti-EpCAM antibodies. The inside surfaces of microfluidic chips were modified using N-γ-maleimidobutyryl-oxysuccinimide ester (GMBS) and mercaptopropyltriethoxysilane (MPTES) for anti-EpCAM antibodies, and surface modifications for anti-EpCAM DARPins were made using GMBS and (3-aminopropyl)triethoxysilane (APTES). Both chip types were tested using cancer cells and whole blood mixed with cancer cells. No effective and consistent cell capture was achieved, despite the antibodies and DARPins being shown to be able to bind to the cells directly and tests with fluorescently labelled DARPins and antibodies showing that the surface modification chemistry used was functional. It was concluded that the most likely causes of the failures were surface modifications interfering with the binding ability of the proteins, or improper orientation of the bound proteins. The DARPin remains a protein of interest for microfluidic applications, but further changes and optimisation of the experimental protocol is necessary.

Les poly(ADP-ribose) polymérases (PARP) sont des enzymes qui modifient les protéines ou l’ADN par ADP-ribosylation à l’aide du nicotinamide adénine dinucléotide (NAD+). Tankyrase est une PARP qui régule divers processus cellulaires, tels que la maintenance des télomères, la signalisation Wnt et le traitement de l’ARN. Elle est constituée de groupes de répétitions d’ankyrine (ARC) N-terminaux qui se lient aux protéines substrats, d’un domaine de motif α stérile intermédiaire qui médie la polymérisation avec d’autres molécules de tankyrase et d’un domaine catalytique C-terminal. Étant donné que l’activité de la tankyrase favorise la signalisation pro-oncogène Wnt/β-caténine, des inhibiteurs sous forme de petites molécules ont été développés pour cibler son domaine catalytique et se sont révélés prometteurs pour le traitement du cancer colorectal. Certaines protéines se lient à la tankyrase, mais plutôt que d’être ciblées pour l’ADP-ribosylation, elles inhibent son activité catalytique. Ces partenaires de liaison comprennent la GDP-mannose 4,6-déshydratase (GMD) et le gène 4 associé à la prostate (PAGE4). Il est important de noter que la structure de l’enzyme tankyrase complète n’a pas encore été déterminée, et on ignore actuellement pourquoi certaines protéines qui se lient à la tankyrase sont modifiées par l’ADP-ribose, tandis que d’autres restent inchangées et inhibent plutôt son activité catalytique. Comprendre ce mécanisme d’inhibition par analyse structurale pourrait fournir de nouvelles voies thérapeutiques bloquant la signalisation Wnt/β-caténine en ciblant le domaine N-terminal de la tankyrase. Notre objectif était d’utiliser la microscopie à coloration négative et la cryomicroscopie électronique pour déterminer les structures de la tankyrase seule et en complexe avec GMD et PAGE4. Nous avons observé que la tankyrase 1 s’assemble en double hélice lors de la polymérisation, créant des contacts interdomaines susceptibles de faciliter ses rôles catalytiques et d’échafaudage dans la signalisation cellulaire. De plus, GMD compactée et ARC1–5 de la tankyrase 1 se lient dans un rapport molaire 1:1 pour former un complexe bilobé, ce qui aide à expliquer pourquoi la GMD reste inchangée. Ces connaissances permettront le développement d’interventions cliniques plus efficaces ciblant les ARC N-terminaux ainsi que les contacts interdomaines de la tankyrase humaine. / Poly(ADP-ribose) polymerases (PARPs) are enzymes that modify proteins or DNA by ADP-ribosylation using nicotinamide adenine dinucleotide (NAD+). Tankyrase is a PARP that regulates diverse cellular processes, such as telomere maintenance, Wnt signaling, and RNA processing. It is made up of N-terminal ankyrin repeat clusters (ARCs) that bind substrate proteins, a middle sterile α motif domain that mediates polymerization with other tankyrase molecules, and a C-terminal catalytic domain. Due to the fact that tankyrase activity promotes pro-oncogenic Wnt/β-catenin signaling, small molecule inhibitors have been developed that target its catalytic domain and have shown promise for the treatment of colorectal cancer. Certain proteins bind tankyrase, but rather than being targeted for ADP-ribosylation, they inhibit its catalytic activity. These binding partners include GDP-mannose 4,6-dehydratase (GMD) and prostate-associated gene 4 (PAGE4). Importantly, the structure of the full-length tankyrase enzyme has yet to be determined, and it is currently unknown why some proteins that bind to tankyrase are modified with ADP-ribose, while others remain unmodified and instead inhibit its catalytic activity. Understanding this mechanism of inhibition by structural analysis could provide new therapeutic avenues that oppose Wnt/β-catenin signaling by targeting the N-terminal domain of tankyrase. We aimed to use negative stain and cryo-electron microscopy to determine the structures of tankyrase alone and in complex with GMD and PAGE4. We observed that tankyrase 1 assembles as a double helix upon polymerization, creating interdomain contacts that may facilitate its catalytic and scaffolding roles in cellular signaling. Moreover, compacted GMD and ARC1–5 of tankyrase 1 bind in a 1:1 molar ratio to form a bilobal complex, which aids in explaining why GMD remains unmodified. These insights will allow for the development of more effective clinical interventions targeting the N-terminal ARCs as well as interdomain contacts of human tankyrase.