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Universiẗat, Diss., 2006--Frankfurt (Main). / Zsfassung in dt. und engl. Sprache.

The E3 ubiquitin ligase RNF43 (RING finger protein 43) is an important negative modulator of the WNT signalling pathway that acts at the plasma membrane by targeting Frizzled and its co-receptor LRP for degradation. In the small intestine, this prevents uncontrolled expansion of the stem cell compartment and so it is essential to the maintenance of normal tissue homeostasis. However, despite its crucial role in fine-tuning the WNT pathway and its role as a tumour suppressor, it is unclear whether RNF43 has further binding partners and what their functional relevance is to the modulation of WNT signalling. Here, I describe the development of a new screening strategy which combines CRISPR/Cas9 technology with 3D-intestinal organoid culture for the identification of novel molecular interactors of RNF43. Overall, this study and the technology developed provide a tool to enable the detailed description of the mechanism of action of RNF43, which is important not only in order to increase our understanding of WNT pathway regulation but also to gain potential new insights into RNF43 paralogs, by analogy. The investigation of paralogs is crucial as RNF43 belongs to a newly identified family of E3 ubiquitin ligases, named the PA-RING family, whose members are still poorly characterised. The majority of PA-RING family members have not been linked to any signalling pathway, most of their targets are still unknown and in many cases their in vivo function has not been addressed. In this context, my work has specifically focused on the investigation of the potential involvement of additional PA-RING family members in WNT pathway modulation and also on target identification for selected members. The results summarised in this dissertation show that no other PA-RING family member plays a prominent role in WNT pathway modulation aside from Rnf43 and its homologue Znrf3, however, different classes of adhesion molecules are likely to be regulated by certain of these E3 ligases. In conclusion, my work has contributed to unravelling previously unexplored aspects of this protein family, with particular regard to RNF43 and its mechanism of action. Thanks to this original approach, it was possible to identify potential new players involved either in membrane clearance of Frizzled or in RNF43 maturation. In particular, my thesis focuses on the characterisation of the role of DAAM in RNF43-mediated Frizzled internalisation.

Pin1 is an enzyme central to cell signaling pathways because it catalyzes the cis–trans isomerization of the peptide ω-bond in phosphorylated serine/threonine-proline motifs in many proteins. This regulatory function makes Pin1 a drug target in the treatment of various diseases. The effects of phosphorylation on Pin1 substrates and the basis for Pin1 recognition are not well understood. The conformational consequences of phosphorylation on Pin1 substrate analogues and the mechanism of recognition by the catalytic domain of Pin1 were determined using molecular dynamics simulations. Phosphorylation perturbs the backbone conformational space of Pin1 substrate analogues. It is also shown that Pin1 recognizes specific conformations of its substrate by conformational selection. Dynamical correlated motions in the free Pin1 enzyme are present in the enzyme of the enzyme–substrate complex when the substrate is in the transition state configuration. This suggests that these motions play a significant role during catalysis. These results provide a detailed mechanistic understanding of Pin1 substrate recognition that can be exploited for drug design purposes and further our understanding of the subtleties of post-translational phosphorylation and cis–trans isomerization. Results from accelerated molecular dynamics simulations indicate that catalysis occurs along a restricted path of the backbone configuration of the substrate, selecting specific subpopulations of the conformational space of the substrate in the active site of Pin1. The simulations show that the enzyme–substrate interactions are coupled to the state of the prolyl peptide bond during catalysis. The transition-state configuration of the substrate binds better than the cis and trans states to the catalytic domain of Pin1. This suggests that Pin1 catalyzes its substrate by noncovalently stabilizing the transition state. These results suggest an atomistic detail understanding of the catalytic mechanism of Pin1 that is necessary for the design of novel inhibitors and the treatment of several diseases. Additionally, a set of constant force biased molecular dynamics simulations are presented to explore the kinetic properties of a Pin1 substrate and its unphosphorylated analogue. The simulations indicate that the phosphorylated Pin1 substrate isomerizes slower than the unphosphorylated analogue. This is due to the lower diffusion constant for the phosphorylated Pin1 substrate.

Neuraminidase (NA) is one of two major antigens on the surface of influenza A viruses. It is comprised of a single N-terminal transmembrane domain (TMD), a stalk domain, and a C-terminal enzymatic head domain that cleaves sialic acid, most notably to release new particles from the host cell surface. NA is only enzymatically active as a homo-tetramer. However, it is not known which properties facilitate the oligomerization of NA during assembly. Our results show that, apart from anchoring the protein to the membrane, the NA TMD also contributes to the assembly process by keeping the stalk in a tetrameric conformation. The ability of the TMD to oligomerize is shown to be dependent on its amphiphilic characteristics that was largely conserved across the nine NA subtypes (N1-N9). Over time the NA TMDs in human H1N1 viruses were found to have become more amphiphilic, which correlated with stronger oligomerization. An old H1N1 virus with a more recent N1 TMD had impaired growth, but readily acquired compensatory mutations in the TMD to restore growth, by reverting the TMD oligomerization strength back to that of the old TMD, demonstrating a biological role of the TMD in folding and assembly. NA and the other viral proteins are spatially and temporally coordinated to achieve optimal viral production. By using a co-transfection analysis, the high AU-content in the NA and HA ER-targeting sequence coding regions (for NA TMD as well as the HA signal sequence) were found to inhibit their expression. The inhibition was alleviated by the early expressed influenza RNA-binding protein NS1, which promoted translation and showed enriched foci at the endoplasmic reticulum (ER). NS1, which expresses early during infection, is therefore likely the regulator of NA and HA to prevent premature expression. These results show that the NA TMD is under substantial selection pressure at both the nucleotide and amino acid level to accommodate its roles in ER-targeting, protein folding, and post-transcriptional regulation. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Accepted.</p>

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Allostery is a regulatory process whereby a perturbation by an effector at one discrete locus creates a conformational change that stimulates a functional change at another. The two sites communicate through networks of interacting residues that respond in a concerted manner to the allosteric perturbation. These allosteric networks are traditionally mapped with high resolution structure determination techniques to understand the conformational changes that regulate protein function as well as its modulation by allosteric ligands and its dysfunction caused by disease-related mutations (DRMs). However, high resolution structural determination techniques, such as X-ray crystallography, cryo-electron microscopy and nuclear Overhauser effect NMR spectroscopy are not always amenable for systems plagued by poor solubility and line broadening caused by μs-ms dynamics or systems where allostery relies primarily on dynamical rather than structural changes. This dissertation discusses methodologies to map the allosteric sites and pathways for such challenging systems. The foundation of this approach is to model allosteric pathways in the context of their respective thermodynamic cycles. In chapter 2, the thermodynamic cycle of a DRM in the hyperpolarization-activated cyclic nucleotide-gated ion channel 4 (HCN4) is analyzed with respect to structure, dynamics and kinetics, revealing how the DRM remodels the free energy landscape of HCN4 and results in a loss-of-function disease phenotype. In chapter 3, the mechanism of action of an uncompetitive inhibitor for the exchange protein activated by cAMP is elucidated by characterizing its selectivity for distinct conformations within the thermodynamic cycle that are trapped using a combination of mutations and ligand analogs. In chapter 4, we discuss two new protocols for the chemical shift covariance analysis (CHESCA). The CHESCA is an approach that identifies allosteric signaling pathways by measuring concerted residue responses to a library of chemical perturbations that stabilize conformational equilibria at different positions. Overall, the approaches discussed in this dissertation are widely applicable for mapping the mechanisms of allosteric perturbations that arise from ligand binding, post-translational modifications and mutations, even in systems where traditional structure determination techniques remain challenging to implement. / Thesis / Doctor of Philosophy (PhD) / Allostery is a regulatory mechanism for proteins, which controls functional properties of one distinct site through the perturbation of another distinct, and often distant, site. The two sites are connected via a series of residues that undergo conformational changes once perturbed by the allosteric effector. Mapping these communication pathways reveals mechanisms of protein regulation, which are invaluable for developing pharmacological modulators to target these pathways or for understanding the mechanisms of disease mutations that disrupt these pathways. Allosteric pathways have been traditionally determined using structure determination approaches that provide a static snapshot of the protein’s structure. However, these approaches are typically not effective when allostery relies extensive changes in dynamics. The goal of this thesis was to develop methods to characterize systems that are dynamic or otherwise unsuitable for traditional structure determination. Herein, we utilize NMR spectroscopy to analyze the allosteric mechanisms of three cAMP-binding proteins involved in cardiovascular health.

Tumor suppressor p53, the guardian of the genome, has been intensely studied molecule owing to its central role in maintaining cellular integrity. While the level of p53 protein is maintained low in unstressed conditions, there is a rapid increase in the functional p53 protein levels during stress conditions. It is now well documented in literature that p53 protein accumulates in the cells following DNA damage by posttranslational modifications leading to increased stability and half life of protein. Additionally, recent studies have also highlighted the significance of increased p53 translation during stress conditions. Interestingly, an alternative initiation codon has been shown to be present within the coding region of p53 mRNA. Translation initiation from this internal AUG results in an N-terminally truncated p53 isoform, described as ΔN-p53. However, the mechanisms underlying co-translational regulation of p53 and ΔN-p53 are still poorly understood. Studies have suggested that synthesis of both p53 and its ΔN-p53 isoform is regulated during cell cycle and also stress and cell-type specific manner. Interestingly, reports also demonstrate continued synthesis of both p53 isoforms during stress conditions. In contrast, global rates of cap-dependent translation initiation are shown to be reduced during stress conditions. This translation attenuation is observed mainly due to restricted availability of critical initiation factors. Interestingly, preferential synthesis of a vital pool of survival factors persists even during these circumstances. Studies have suggested that this selective translation is mediated via alternative mechanisms of translation initiation. One of the important mechanisms used for protein synthesis during these conditions is internal initiation. In this mechanism, the ribosomes are recruited to a complex RNA structural element known as ‘Internal Ribosome Entry Site (IRES)’, generally present in the 5’ untranslated region (UTR) of mRNA. Therefore, it is possible that the translation of p53 and ΔN-p53 could also be regulated by IRES mediated translation, especially during stress conditions. In this thesis the role of internal initiation in translational control of p53 and ΔN-p53 has been investigated. Additionally, the putative secondary structure of p53 IRES RNA has been determined. Further, it has been shown that polypyrimidine tract binding (PTB) protein acts as an important regulator of p53 IRES activities. The probable mechanism of action of PTB protein has also been investigated. The results suggest that interaction with PTB alters the p53 IRES conformation which could facilitate translation initiation. Finally, the possible physiological significance of existence of p53 IRES elements has been addressed. In the first part of the thesis, the presence of internal ribosome entry site within p53 mRNA has been investigated. As a first step, the 5’UTRs mediating the translation of both p53 and ΔN-p53 were cloned in the intercistronic regions of bicistronic constructs. Results of in vivo transfection of these bicistronic constructs suggested the presence of two IRES elements within p53 mRNA, with activities comparable to known viral and cellular IRESs. The IRES directing the translation of p53 is in the 5'-untranslated region of the mRNA, whereas the IRES mediating the translation of ΔN-p53 extends further into the protein-coding region. To further validate, stringent assays were performed to rule out the possibility of any cryptic promoter activity, re-initiation/scanning or alternative splicing in the p53 mRNA. Transfection of in vitro synthesized bicistronic RNAs confirmed the presence of IRES elements within p53 mRNA. Incidentally, this constitutes the first report on translational control of p53 by internal initiation. In the second part of the thesis, the secondary structure of p53 IRES RNA has been investigated. Structural analysis of p53 RNA was performed using structure-specific nucleases and modifying chemicals. The results obtained from chemical modification and nuclease probing experiments were used to constrain Mfold predicted structures. Based on this, a putative secondary structure model for p53 IRES RNA has been derived. Sequence alignment suggested that the p53 IRES RNA showed significant sequence conservation across mammalian species. To study the effect of mutations on the IRES structure, mutant p53 IRESs were used that harbor silent mutations at critical locations within the p53 IRES element. Incidentally, one of the mutant constructs used in the study was observed to be a naturally occurring mutation in a chronic lymphocyte leukemia patient. RNA structure analyses of these two mutant p53 IRES RNAs were performed. The nuclease mapping data suggested conformational alteration in these mutant RNAs with respect to wild type. Consistently, a comparative Circular-Dichroism spectroscopy of the Wt and mutant RNAs also validated the conformational alteration of the mutant RNAs. This also suggested that the presence of mutations in p53 IRES might result in decreased induction of p53 protein following DNA damage due to altered RNA structure. This might constitute as one of the mechanisms leading to tumor development in some types of cancers. In the third part of the thesis, the role of important cellular proteins that might modulate p53 IRES mediated translation has been studied. These cellular proteins act as IRES interacting trans-acting factors (ITAFs). Polypyrimidine tract binding (PTB) protein is an important ITAF implicated in regulating IRES mediated gene expression during apoptosis. It was observed that PTB protein specifically interacts with both the IRES elements within p53 mRNA. Interestingly, the affinity of interaction of PTB protein with both p53 IRES RNAs was observed to be significantly different. In order to determine the contact points of PTB on p53 IRES, a foot-printing assay using structure specific nuclease and recombinant-PTB protein was performed on p53 RNA. The data from foot-printing as well as primer extension inhibition assay (toe-printing analysis) suggested the presence of multiple PTB binding sites on p53 IRES RNA. Based on these results, a deletion mutant was generated that showed reduced PTB binding and also reduced IRES activity as compared to wild type. Further, to study the role of PTB in mediating p53 translation, the expression of PTB gene was partially silenced by using PTB specific siRNA. Partial depletion of endogenous PTB protein showed a significant decrease in the p53 IRES activities. These results suggest that PTB protein is essential for the p53 IRES activities. To understand the probable mechanism by which PTB regulates p53 IRES mediated translation, CD spectroscopy analysis of p53 IRES RNA was performed in the absence and presence of PTB protein. Interestingly, CD spectra analysis of the p53 RNA in the presence of PTB suggested a specific conformational change in p53 IRES, which might probably facilitate ribosome loading during internal initiation. This also suggests that abnormal expression of p53 ITAFs might lead to reduced p53 induction following DNA damage conditions. It could also be another event leading to malignant transformation of cells bearing wild type p53. It is highly tempting to speculate that the levels of p53 ITAFs could also be used as tumor biomarkers. In the fourth part of the thesis, the physiological relevance of existence of IRES elements within p53 mRNA has been investigated. The levels of p53 and ΔN-p53 proteins are known to be regulated in a cell cycle phase-dependent manner. The IRES activities of both p53 IRES elements were investigated at different phases of cell cycle. The activity of the IRES responsible for translation of p53 protein was found to be highest at G2-M transition and the maximum IRES activity corresponding to ΔN-p53 synthesis was observed at G1-S transition. These results suggested that the p53 IRES activities are regulated in a cell-cycle phase-dependent manner. Next, the regulation of p53 IRES mediated translation during stress conditions was studied. Human lung carcinoma cell line, A549 cells (that endogenously express both the p53 isoforms), were exposed to DNA damaging drug, doxorubicin. The level of p53 protein was observed to increase in a time-dependent manner. Interestingly, PTB protein, which is predominantly nuclear, was found to translocate to the cytoplasm during stress condition in a time-dependent manner. Under similar conditions, p53 protein was observed to reverse translocate from the cytoplasm to nucleus, probably to function as a transcription factor. Next, the influence of partial PTB silencing on p53 isoforms in the presence of cell stress (mediated by doxorubicin) was investigated. The data indicated reduced levels of both p53 and ΔN-p53 when PTB gene expression was partially silenced. These observations constitute “the proof of concept” that relative abundance of an ITAF, such as PTB protein, might contribute to regulating the coordinated expression of the p53 isoforms. The thesis reveals the presence as well as the physiological relevance of existence of IRES elements within p53 mRNA. The novel discovery of p53 IRES elements may provide new insights into the underlying mechanism of translational regulation. The modulation of the p53 IRES activities by PTB protein suggests that the regulated expression of p53 isoforms depends on the integrity of IRES elements and availability of cellular proteins that can serve as p53 ITAFs. Thus, studies pertaining to the identification of mutations within p53 IRES region as well as abnormal expression of p53 ITAFs such as PTB in cancer cells may have far reaching implications. These studies might lead to further advances in the field of cancer detection, prognosis and design of novel therapeutic strategies.

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