STRUCTURAL INSIGHTS INTO FOLDED, UNFOLDED AND NASCENT PROTEIN STATES USING ENSEMBLE SAMPLING AND CLUSTER EXPANSION

Ph.DDOCTOR OF PHILOSOPH

Role of tertiary interactions in determining RNA architecture

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal

Structural and Biophysical Characterisation of Denatured States and Reversible Unfolding of Sensory Rhodopsin II

Our understanding of the folding of membrane proteins lags behind that of soluble proteins due to the challenges posed by the exposure of hydrophobic regions during in vitro chemical denaturation and refolding experiments. While different folding models are accepted for soluble proteins, only the two-stage model and the long-range interactions model have been proposed so far for helical membrane proteins. To address our knowledge gap on how different membrane proteins traverse their folding landscapes, Chapter 2 investigates the structural features of SDS-denatured states and the kinetics for reversible unfolding of sensory rhodopsin II (pSRII), a retinal-binding photophobic receptor from Natronomonas pharaonis. pSRII is difficult to denature, and only SDS can dislodge the retinal chromophore without rapid aggregation. Even in 30% SDS (0.998 $\mathit{\Chi}_{SDS}$), pSRII retains the equivalent of six out of seven transmembrane helices, while the retinal binding pocket is disrupted, with transmembrane residues becoming more solvent-exposed. Folding of pSRII from an SDS-denatured state harbouring a covalently-bound retinal chromophore shows deviations from an apparent two-state behaviour. SDS denaturation to form the sensory opsin apo-protein is reversible. This chapter establishes pSRII as a new model protein which is suitable for membrane protein folding studies and has a unique folding mechanism that differs from those of bacteriorhodopsin and bovine rhodopsin. In Chapter 3, SDS-denatured pSRII, acid-denatured pSRII and sensory opsin obtained by hydroxylamine-mediated bleaching of pSRII were characterised by solution state NMR. 1D $^1$H and $^{19}$F NMR were first used to characterise global changes in backbone amide protons and tryptophan side-chains. Residue-specific changes in backbone amide chemical shifts and peak intensities in 2D [$^1$H,$^{15}$N]-correlation spectra were analysed. While only small changes in the chemical environment of backbone amides were detected, changes in backbone amide dynamics were identified as an important feature of SDS- and acid-denatured pSRII and sensory opsin. $^{15}$N relaxation experiments were performed to study the backbone amide dynamics of SDS-denatured pSRII, reflecting motions on different timescales, including fast fluctuations of NH bond vectors on the ps–ns timescale and the lack of exchange contributions on the µs timescale. These studies shed insight on differences in the unfolding pathways under different denaturing conditions and the crucial role of the retinal chromophore in governing the structural integrity and dynamics of the pSRII helical bundle. Hydrogen bonds play fundamental roles in stabilising protein secondary and tertiary structure, and regulating protein function. Successful detection of hydrogen bonds in denatured states and during protein folding would contribute towards our understanding on the unfolding and folding pathways of the protein. Previous studies have demonstrated residue-specific detection of stable and transient hydrogen bonds in small globular proteins by measuring $^1{\it J}_{NH}$ scalar coupling constants using NMR. In Chapter 4, different methods for measuring $^1{\it J}_{NH}$ scalar coupling were explored using RalA, a small GTPase with a mixed alpha/beta fold, as proof-of-concept. Detection of hydrogen bonds was then attempted with OmpX, a beta-barrel membrane protein, both in its folded state in DPC micelles and in the urea-denatured state. While $^1{\it J}_{NH}$ measurement holds promise for studying hydrogen bond formation, further optimisation of NMR experiments and utilisation of perdeuterated samples are required to improve the precision of such measurements in large detergent-membrane protein complexes. Naturally occurring split inteins can mediate spontaneous trans-splicing both in vivo and in vitro. Previous studies have demonstrated successful assembly of proteorhodopsin from two separate fragments consisting of helices A–B and helices C–G via a splicing site in the BC loop. To complement the in vitro unfolding/folding studies, pSRII assembly in vivo was attempted by introducing a splicing site in the loop region of the beta-hairpin constituting the BC loop of pSRII. The expression conditions for the N- and C-terminal pSRII-intein segments were optimised, and the two segments co-expressed. However, the native chromophore was not observed. Further optimisation is required for successful in vivo trans-splicing of pSRII and application of this approach towards understanding the roles of helices and loops in the folding of pSRII.Cambridge Commonwealth, European & International Trus

Evaluation of anti-metastatic therapeutics targeting SEC62 in head and neck cancer using newly established murine in vivo metastasis models and functional characterization of stable SEC62 knock-out head and neck cancer cell lines generated by CRISPR/Cas9

For several years, research has been concerned with the clinical and molecular role of SEC62, a protein located in the ER membrane, which has been considered as a potential oncogene. Over time, significant overexpression of the SEC62 protein has been shown in various human tumour entities, including squamous cell carcinoma of the head and neck (HNSCC) which display a SEC62 overexpression in approximately 86% of cases. In general, head and neck tumours are among the seventh most common tumour entities worldwide with a mostly consistent and poor 5-year survival rate for affected patients of about 50 to 60 %. Head and neck tumours are often diagnosed at an advanced stage of disease and about 50 % of all patients already display lymph node metastases of the neck at the time of diagnosis. About 10% of patients even have distant metastases. Studies have shown that the ER membrane protein SEC62 is not only overexpressed in the majority of HNSCC patients but is also associated with a significantly worse prognosis and an advanced TNM status. In functional in vitro studies of the SEC62 protein, significant changes in migration were found, depending on an artificially altered SEC62 expression. Thus, plasmid-mediated overexpression of SEC62 led to a significantly increased migration. A transient knock-down of SEC62 via siRNA significantly decreased the ability of migration in HNSCC cells. Based on these properties, SEC62 was considered as a potential new target for directed therapy of SEC62-overexpressing HNSCC tumours. Since a direct transfer from those in vitro experiments to human is not possible, this work aimed at a functional knock-down of SEC62 by the administration of TFP and TG. TFP leads to an increased cytosolic Ca2+ content by blocking CaM, which would close the protein translocation channel SEC61 in the ER membrane. A strong concentration gradient causes a flow of Ca2+ from the ER Ca2+-store into the cytosol. Moreover, the simultaneous administration of TG irreversibly inhibits SERCA so that no Ca2+ can be actively transported back into the ER. These two compounds in combination are thought to significantly upset Ca2+ homeostasis in SEC62-overexpressing cancer cells, causing the cells to undergo apoptosis. Since these effects could be proven in vitro, two murine xenograft metastasis models were established within this thesis in order to be able to test the efficacy of both substances in vivo on the metastasis of HNSCC cells. Therefore, on the one hand tumour cells were injected orthotopically into the tongue of mice to test the anti-metastatic ability of TFP and TG. Here, TFP treatment resulted in a reduction of the number of cervical lymph node metastases, whereas TG treatment was able to reduce the size of detectable metastases. On the other hand, tumour cells were injected i.v. into the tail vein of the animals to investigate the impact of both substances on haematogenous metastasis. Here, the evaluation of the combinatory treatment on haematogenous metastasis achieved a reduction in metastatic burden in the lung. In addition to the main aspect of the effects of TFP and TG on metastasis, various imaging techniques and their evaluations including MRI, micro-CT, and histology were also established for monitoring the emerging metastases. In a second part of this work stable SEC62-knockout clones of an immortalized HNSCC cell line were generated using CRISPR-Cas9 technology, which should provide deeper insights into the role of SEC62 and the SEC62-associated processes inside the cell. These SEC62-ko clones were validated using various methods such as immunofluorescence, Western blot and next generation sequencing (NGS), and were also characterised based on whole-RNA-sequencing. Furthermore, first functional studies with SEC62-ko clones and their tolerance to TFP and TG were applied. In summary, two murine xenograft models for lymphatic and haematogenous metastasis of head and neck cancer were successfully established, on which first investigations on the efficacy of two potential new compounds for a targeted therapy of metastatic SEC62-overexpressing head and neck tumours could be performed. Herein, TG and TFP showed promising effects on lymphatic and haematogenous metastasis in vivo motivating for further investigations. Furthermore, two stable SEC62-ko HNSCC cell lines could be generated, validated and characterised using CRISPR-Cas9. Further analyses to understand the influence and function of SEC62 in more detail are the subject of future research.Die Forschung beschäftigt sich bereits seit mehreren Jahren mit der klinischen als auch molekularen Rolle von SEC62, einem Protein, dass sich in der ER Membran befindet und als potentielles Onkogen gehandelt wird. In den letzten ca. 15 Jahren konnte in verschiedenen Tumorentitäten eine signifikante Überexpression des SEC62-Proteins nachgewiesen werden, so auch in ca. 86% aller Plattenepithelkarzinome des Kopf-Hals-Bereiches (head and neck squamous cell carinoma, HNSCC). Im Allgemeinen zählen Kopf-Hals Tumore zu den sieben häufigsten Tumorerkrankungen weltweit mit einer weitestgehend gleichbleibenden, schlechten 5-Jahres Überlebensrate für betroffene Patienten von etwa 50-60%. In der Mehrzahl der Fälle werden Kopf-Hals-Tumore erst in einem späten Stadium der Erkrankung diagnostiziert. Daher weisen zum Diagnosezeitpunkt bereits ca. 50% aller HNSCC-Patienten Hals-Lymphknotenmetastasen auf, ca. 10% der Patienten zeigen sogar bereits Fernmetastasen. In Studien konnte nachgewiesen werden, dass das ER-membranständige Protein SEC62 nicht nur von einem Großteil aller HNSCC Patienten überexprimiert wird, sondern auch mit einer deutlich schlechteren Prognose, sowie mit einem fortgeschrittenen TNM-Stadium assoziiert ist. In funktionellen in vitro Untersuchungen des SEC62 Proteins konnten signifikante Veränderungen in der Migrationsfähigkeit festgestellt werden, je nach artifiziell veränderter SEC62-Expression. So führte eine Plasmid-vermittelte Überexpression von SEC62 zu einer deutlich gesteigerten Migration, bzw. ein transienter knock-down zu einer signifikanten Abnahme der Migrationsfähigkeit in HNSCC Zellen. Aufgrund dieser Eigenschaften bietet sich SEC62 als mögliches neues Target für eine zielgerichtete Therapie von SEC62-überexprimierenden HNSCCs an. Da ein direkter Transfer der genannten in vitro Experimente auf den Menschen nicht möglich ist, wurde in dieser Arbeit ein funktioneller knock-down von SEC62 durch die Gabe von TFP und TG angestrebt. TFP führt zu einem gesteigerten zytosolischen Ca2+-Gehalt durch die Blockade von CaM, das normalerweise den Proteintranslokationskanal SEC61 in der ER-Membran verschließen würde. Durch ein starkes Konzentrationsgefälle von Ca2+ strömen die Ionen aus dem ER Ca2+-Speicher ins Zytosol. Durch die gleichzeitige Gabe von TG wird zudem auch die SERCA irreversible inhibiert, sodass kein Ca2+ energieabhängig ins ER zurückgepumpt werden kann. Diese beiden Substanzen in Kombination bringen die Ca2+-Homöostase in SEC62-überexprimierenden Krebszellen signifikant aus dem Gleichgewicht, sodass eine Apoptose der Tumorzellen erreicht werden kann. Da in vitro diese Effekte gezeigt werden konnten, wurden in dieser Arbeit zwei murine Xenograft Metastasierungs-Modell etabliert, um die Wirksamkeit der beiden Substanzen auch in vivo auf die Metastasierung von HNSCC Zellen testen zu können. Eine TFP-Behandlung orthothop injizierter Tiere zur Analyse der Auswirkungen beider Substanzen auf die lymphogene Metastasierung, führte zu einer Reduktion der Metastasenanzahl. Eine Behandlung mit TG konnte eine Verkleinerung der detektierten Metastasen erzeugen. Die Auswertung der Kombinationsbehandlung auf die hämatogene Metastasierung im zweiten etablierten Tiermodell erzielte eine Reduktion der Metastasenlast in der Lunge. Neben dem Hauptaspekt der Auswirkungen von TFP und TG auf die Metastasierung wurden auch verschiedene Bildgebungsverfahren und deren Auswertungen (Kleintier-MRT, micro-CT, Histologie) zum Monitoring der entstehenden Metastasen etabliert. In einem zweiten Teil dieser Arbeit wurden stabile SEC62-knockout Klone einer immortalisierten HNSCC-Zelllinie mittels CRISPR-Cas9 generiert, die tiefere Erkenntnisse über Rolle von SEC62 und die mit SEC62-assoziierten Prozesse in der Zelle liefern sollten. Diese SEC62-ko Klone wurden mittels verschiedenster Methoden wie Immunfluoreszenz, Western Blot und next generation sequencing (NGS) validiert, sowie basierend auf einer whole-RNA-Sequenzierung charakterisiert. Des Weiteren wurden erste funktionelle Untersuchungen mit den SEC62-ko Klonen und ihre Toleranz gegenüber TFP und TG durchgeführt. Zusammenfassend konnten zwei murine Xenograft Modelle zur lymphogenen und hämatogenen Metastasierung von Kopf-Hals-Tumoren erfolgreich etabliert werden, an denen erste Untersuchungen zur Wirksamkeit zweier potentieller neuer Substanzen zur Therapie von metastasierenden, SEC62-überexpreimierenden Kopf-Hals-Tumoren durchgeführt werden konnten. Hierbei zeigten sich die beiden Substanzen TG und TFP als vielversprechende metastasierungshemmende Wirkstoffe, was zu weiteren Untersuchungen motiviert. Zudem konnten zwei stabile SEC62-ko Zelllinien mittels CRISPR-Cas9 generiert, validiert und charakterisiert werden. Weitere Analysen, um den Einfluss und die Funktion von SEC62 genauer verstehen zu können sind Gegenstand zukünftiger Forschung.Else-Kröner-Fresenius Stiftung, HOMFO

Investigation of the sequences and structural elements required for HIV-2 infectivity and genome dimerisation

A unique feature of retroviruses is that two copies of the genomic RNA are packaged in each particle. The selective encapsidation of viral genomes is ensured by the binding of the Nucleocapsid to a specific motif on the RNA genome, the packaging signal (Psi). The Psi regions of many retroviruses overlap with sequences that promote the dimerisation of the genome, the dimerisation initiation site (DIS), and it has been suggested that the two mechanisms are closely linked. The aim of the research presented herein was to identify the sequences and structural elements required for the dimerisation of HIV-2 genomic RNA and to investigate the relationship between HIV-2 genome dimerisation and encapsidation, infectivity and particle morphogenesis. Mutations of two palindromic sequences, introduced in an infectious molecular clone of the HIV-2rod isolate, revealed that a palindrome within HIV-2 Psi was important for genome dimerisation. In contrast with previous studies, the palindrome termed DIS is not required for genome dimerisation and viral replication. Viruses bearing mutations within the Psi region failed to dimerise and to replicate in T-cells, a defect that could not be rescued by targeting more genomes to the cells. Psi-deleted viruses also displayed a defect in particle morphogenesis. A reduced packaging efficiency, combined with the presence of RNA monomers or unstable dimers in these virions, resulted in the production of fewer mature particles. However an increase in the number of particles containing two cores was observed. Further characterisation of the sequences and structural elements required for RNA dimerisation, packaging and viral replication showed that the formation of stem B is not critical for viral replication. However, a GGAG purine-rich motif at position 392-395 of the HIV-2rod genome is absolutely essential for genome dimerisation and viral infectivity, and a correlation was observed between dimer formation and viral replication

Global energy minimisation and cotranslational protein folding of HP models

The globally minimum energy configurations of simpleHPlatticemodels (which use only two amino acid types, positioned on the vertices of a square lattice) of proteins have been established for short sequences. Here we investigate the folding of such proteins to this globally minimum energy configuration, both cotranslationally (as they are manufactured, sequentially, in the ribosome) and starting from a fully extended state. In order to do this we model the folding process and develop a heuristic method for finding local energy minima. Two main results emerge. First, some sequences do fold better cotranslationally than from a fully extended state and second, this can be due to cotranslational folding leading to an initial local energy minimum from which movement to the global minimum is efficient. Sequences for which this is true tend to have a higher density of hydrophobic residues at the start than at the finish. Structural properties of sequences that fold better cotranslationally than from a fully extended state are also identified.16 page(s