DNAとカチオン性ポリペプチドの液-液相分離機構に関する研究

筑波大学University of Tsukuba博士（工学）Doctor of Philosophy in Engineering2021doctoral thesi

Molecular Modelling of a novel G-quadruplex structure and its interaction with ligands

DNA can exist under many different forms. Lately, G-quadruplexes, which are one example of the non-canonical DNA forms, have been getting a lot of attention due to the role they play in certain biological processes and as potential targets for therapeutic interventions. For example, these structures can exist in certain parts of the telomeres, structures responsible for cell replication. In cancer cells, if the enzyme telomerase could be inhibited, by inducing the formation of a G-quadruplex structure in guanine-rich telomere sequences, the spread of cancer cells would cease. For this and other reasons, it becomes important to be able to induce the formation of G-quadruplex structures and/or stabilize them, and one of the ways of doing so consists of targeting these sequences with ligands that have good affinity to G-quadruplex structures. However, few G-quadruplex ligands demonstrated the needed properties to fulfill the clinical needs, and further efforts to determine which would be better suited to target any particular sequence are needed. This work aimed at comparing the affinity to the pre-miR-149 G-quadruplex structure of seven promising ligands found in the literature, through the latest techniques fit for that purpose. The seven ligands tested were: [16]phenN2, [32]phen2N4, phen-DC3, pyridostatin, acridine orange derivatives C8 and C8-NH2 and L-arginine. Firstly, they underwent computational tests, with the molecular structure of the quadruplex and the ligand being simulated, and their optimal binding site and conformation found. Their binding energies were compared, and they underwent molecular dynamics runs to simulate their behavior in an environment with solvent, followed by another binding energy comparison. The trend obtained in order of decreasing binding affinity was: pyridostatin > [32]phen2N4 > [16]phenN2 > Phen-DC3 > L-arginine > C8 > C8-NH2. Biophysical techniques were then performed, to determine the binding affinities experimentally. First, circular dichroism spectroscopy and melting studies (performed on four ligands) established the following trend: C8 > pyridostatin > C8-NH2 > [16]phenN2. Fluorescence spectroscopy titration (performed on three) revealed a similar trend: C8 > C8-NH2 > [16]phenN2. Lastly, affinity chromatography experiments were held to test how other DNA sequences would bind to C8-NH2. The results revealed that the ligand has better binding affinity with parallel quadruplexes over antiparallel ones, and poor binding with a duplex sequence. Overall, the best ligands identified for binding to the G-quadruplex structure were the acridine orange derivatives C8 and C8-NH2, and pyridostatin. These three ligands should be considered prime candidates for further research in this area.ADN pode existir sob a forma de diversas estruturas, contrariamente ao que a vasta maioria da população pensa, ao imaginar a dupla hélice de Watson e Crick. Uma das formas que tem sido mais investigada ultimamente consiste no G-quadruplex. Esta estrutura não canónica do DNA ocorre quando guaninas se emparelham e organizam em estruturas cíclicas através de pontes de hidrogénio Hoogsteen, chamadas G-quartetos. Estas estruturas formam-se por empilhamento p-p entre elas próprias, originando o G-quadruplex, desde que haja um catião (preferivelmente K+) para assumir uma localização central entre todos os quartetos. Estas estruturas desempenham funções importantes a nível de regulação da transcrição e replicação do DNA. Alguns estudos indicam também que podem ser relevantes a nível de manutenção do DNA, e que várias secções do DNA humano se encontram num estado de equilíbrio entre a forma de G-quadruplex e duplex. São também considerados alvos para certas abordagens terapêuticas a nível do cancro. Por exemplo, vários oncogenes como c-kit e c-myc têm a capacidade de formar G-quadruplexes nos seus promotores. Controlando a forma que estes genes assumem, seria possível controlar a sua transcrição, e possivelmente impedir a formação de cancro. Outra possibilidade cinge-se à inibição da telomerase, uma enzima responsável pela replicação celular, que está sobreexpressa em células cancerígenas. Se uma parte do telómero assumir uma estrutura em G-quadruplex, a ação desta enzima fica inibida, efetivamente parando a progressão do cancro. Portanto, torna-se necessário induzir e estabilizar a formação de estruturas do G-quadruplex. A estratégia é utilizar ligandos que interajam por interações intermoleculares de forma a estabilizar a estrutura do G-quadruplex, e outra topologia que esteja em equilíbrio. No entanto, analisando a literatura, conclui-se que apenas alguns grupos de ligandos são efetivamente ligandos de G-quadruplex. Este trabalho de investigação teve como objetivo comparar 7 ligandos promissores da estrutura de G-quadruplex designada por pre-miR-149 literatura. Os ligandos selecionados foram macrociclos derivados de fenantrolina ([16]phenN2, [32]phen2N4, Phen-DC3, e derivados de laranja de acridina C8 e C8-NH2. Determinou-se a afinidade e a estabilização destes ligandos com a estrutura do RNA G-quadruplex, a pre-miR-149. Isso será feito em duas etapas principais. Primeiro, foram realizadas simulações computacionais para determinar quais os ligandos mais promissores e quais os seus métodos de interação com a estrutura G-quadruplex. Estas dividiram-se em três passos: primeiro, foram geradas as estruturas da sequência e de cada ligando em software adequado. Segundo, foram feitas simulações de docking de modo a averiguar os locais de ligação de cada ligando ao G-quadruplex, e a conformação e interações entre o ligando e o quadruplex, sendo também calculadas energias de ligação entre o ligando e o G-quadruplex. Finalmente, foram feitas simulações de dinâmica molecular sobre como essa conformação evoluiria num ambiente fisiológico simulado e calculadas novas energias de ligação, que comparadas entre si, revelam diferenças de afinidades entre os ligandos. Após estas técnicas computacionais, foram executadas técnicas biofísicas, como espetroscopia de dicroísmo circular e estudos de desnaturação térmica, e espectroscopia de fluorescência para determinar experimentalmente as afinidades de cada ligando para com a estrutura escolhida. Foram também executadas experiências de cromatografia de afinidade para determinar o comportamento de um ligando para com sequência do RNA G-quadruplex, a pre-miR-149. O programa usado para avaliar as conformações iniciais gerou estruturas demasiado rígidas e pouco flexíveis com os ligandos macrocíclicos [16]phenN2 e [32]phen2N4. As energias de ligação obtidas revelaram a nível de afinidade a seguinte ordem decrescente: piridostatina > [32]phen2N4 > [16]phenN2 > PhenDC3 > L-arginina > C8 > C8-NH2. Esta tendência não foi a mesma verificada experimentalmente, e logo, foi descartada. A nível destas experiências, retiram-se maioritariamente apenas as conformações dos ligandos que não são macrociclos. A nível das experiências de dicroísmo circular mencionadas, as variações de temperatura de desnaturação térmica ligando-quadruplex foram diferentes,verificando-se a seguinte ordem: C8 > piridostatina > C8-NH2 > [16]phenN2. Seguidamente, foram realizadas titulações por espectroscopia de fluorescência as quais revelaram a seguinte tendência: C8 > C8-NH2 > [16]phenN2. De notar que apenas quatro dos sete ligandos ([16]phenN2, [32]phen2N4, C8 and C8-NH2) possuíam fluorescência intrínseca, e que desses, apenas estes três puderam ser selecionados. Estes resultados mostraram que a piridostatina, e derivados de laranja de acridina C8 e C8-NH2 apresentaram maior afinidade para esta estrutura de G-quadruplex. Por último, os resultados de cromatografia de afinidade revelaram que o ligando C8-NH2 tem maior afinidade com o RNA G-quadruplex pre-miR-149 . Das seis sequências testadas, três delas (c-myc, c-kit e pre-miR-149) formam G-quadruplexes com topologia paralela, e tiveram tempos de retenção mais altos. Outras sequências (TBA e AG23) formam G-quadruplexes com topologia antiparalela, e mostram tempos de retenção mais baixos. A sequência ds26 (duplex) teve o tempo de retenção mais baixo. Conclui-se que este ligando tem maior especificidade para com G-quadruplexes com topologia paralela em detrimento do duplex. As simulações de docking corroboram esta conclusão. Deste modo, conclui-se que os melhores ligandos a nível de afinidade para com a sequência pre-miR-149 são os derivados de laranja de acridina C8 e C8-NH2 e a piridostatina, de modo que futura investigação nesta área deve considerar estes três como fortes candidatos a ligandos de RNA G-quadruplex

G-quadruplexes in the HSV-1 and HHV-6 genomes as antiviral targets

Guanine-rich nucleic acids can fold into G-quadruplexes, four-stranded secondary structures which are implicated in important regulatory functions at the genomic level in humans, prokaryotes and viruses. Because the herpes simplex virus-1 (HSV-1) genome is remarkably rich in guanines, we aimed at investigating both the presence of G-quadruplex forming sequences at the viral genome level and the possibility to target them with G-quadruplex ligands to obtain anti-HSV-1 effects with novel mechanisms of action. Here we show that HSV-1 displays six clusters of repeated sequences that form very stable G-quadruplexes. These sequences are located in the inverted repeats and in two gene-coding regions (ICP0 and UL36) of the HSV-1 genome. One G-quadruplex repeat is located in the promoter region of the multifunctional protein γ134.5. Treatment of HSV-1 infected cells with the G-quadruplex ligands BRACO-19 and TMPyP4 induced significant inhibition of virus production and reduction of viral transcripts. BRACO-19 was able to inhibit Taq polymerase processing at G-quadruplex forming sequences in the HSV-1 genome, and caused a decreased intracellular viral DNA in infected cells. The last step targeted by BRACO-19 was viral DNA replication, while no effect on virus entry in the cells was observed. A different TMPyP4-mediated mechanism of action was on the contrary observed. Despite its capability to affect Taq polymerase processing, TMPyP4 did not inhibit intracellular viral DNA and it appeared to prevent HSV-1 maturation/egress by stimulating the autophagy process. As a second part of the study, we extended this innovative antiviral approach to human herpes virus-6 (HHV-6). One of the main HHV-6 features is the presence of tandem repeats of the telomeric sequence (TTAGGG)n at the genome termini (DR). This peculiarity is thought to be responsible for the viral integration in specific human chromosomes, occurring in the 1-2% of the world population. To date, the telomeric G-quadruplex structure had been extensively characterized. We showed that BRACO-19 and TMPyP4 displayed a great antiviral activity against both HHV-6A and HHV-6B. In the third part of this study, by using specific DNA G-quadruplex-interacting antibodies, for the first time we visualized viral DNA G-quadruplexes in infected cells at crucial time points for the viral replication cycle, in which viral DNA is likely in a single-stranded state. This work, besides presenting the first evidence of extended G-quadruplex sites in key regions of the HSV-1 and HHV-6 genomes, points out G-quadruplexes as innovative potential antiviral targets in novel therapeutic interventions, based on the use of G-quadruplex ligands

In What Ways Do Synthetic Nucleotides and Natural Base Lesions Alter the Structural Stability of G-Quadruplex Nucleic Acids?

Synthetic analogs of natural nucleotides have long been utilized for structural studies of canonical and noncanonical nucleic acids, including the extensively investigated polymorphic G-quadruplexes (GQs). Dependence on the sequence and nucleotide modifications of the folding landscape of GQs has been reviewed by several recent studies. Here, an overview is compiled on the thermodynamic stability of the modified GQ folds and on how the stereochemical preferences of more than 70 synthetic and natural derivatives of nucleotides substituting for natural ones determine the stability as well as the conformation. Groups of nucleotide analogs only stabilize or only destabilize the GQ, while the majority of analogs alter the GQ stability in both ways. This depends on the preferred syn or anti N-glycosidic linkage of the modified building blocks, the position of substitution, and the folding architecture of the native GQ. Natural base lesions and epigenetic modifications of GQs explored so far also stabilize or destabilize the GQ assemblies. Learning the effect of synthetic nucleotide analogs on the stability of GQs can assist in engineering a required stable GQ topology, and exploring the in vitro action of the single and clustered natural base damage on GQ architectures may provide indications for the cellular events

Targeting receptors and DNA secondary structures with small molecules and calix[4]arene conjugates

This body of research is focused on developing calixarene conjugates targeting i-motif structures and integrin receptors. In Chapter 1, a general background of DNA secondary structures and i-motifs was described, mainly focused on the biological relevance, the experimental techniques and known interacting ligands in i-motif studies. In Chapter 2, a high-throughput i-motif ligand screen method was established, based on fluorescent intercalator displacement. Thiazole orange was used as the fluorescent intercalator in the screen against human telomeric i-motif. Its binding was studied using several spectroscopic techniques. A compound library was used to evaluate the newly developed high-throughput screen method using a plate reader and tobramycin was found as the most valuable hit compound in this screen. In Chapter 3, a family of water soluble, DNA-targeting calixarene conjugates were synthesis and characterised. They were functionalised with and DNA-binding moiety on the lower rim. It was found that two of the calixarene conjugates, 28 and 54, were able to condense G-quadruplex and i-motif forming sequences from human telomere and c-MYC promoter. The calixarene induced condensation was stable under acidic pH, but behave reversible by heating at neutral pH. Chapter 4 discussed the possibility to develop a calixarene based tumour recognising ligand. In order to achieve tumour targeting, a novel cyclic RGD peptide bearing alkyne was made and tested in the ‘click’ reaction. Later on, a route to conjugate the novel cyclic RGD peptide with a calixarene tethered with a fluorescent tag was established. It was found that a linker between calixarene and peptide moiety or the copper (I) catalyst was crucial in making this calixarene-peptide conjugate. Chapter 5 described the experimental procedures used in Chapter 2, 3 and 4. Chapter 6 summarised the key findings in Chapter 2, 3 and 4, as well as proposing the future work for all three chapters

Inhibition of Ribosome Biogenesis Through Genetic and Chemical Approaches

In order to maintain the ability to generate proteins, proliferating cells must continuously generate ribosomes, designating up to 80% of their energy to ribosome biogenesis (RBG). RBG involves transcription of rDNA by RNA polymerases I (Pol I) and III (Pol III), expression of approximately 80 ribosomal proteins, and assembly of these components in a process referred to as ribosome maturation. During maturation, the Pol I transcribed 47S pre-rRNA undergoes a number of processing events, while simultaneously interacting with processing factors and ribosomal proteins that drive pre-ribosome assembly. Inhibition of RBG has become one of the pursued targets for cancer therapy in the past 15 years (Drygin et al., 2010; Hannan et al., 2013), prompted by the observation that cancer cells upregulate RBG (Brighenti et al 2015). This project was aimed to determine whether RBG inhibition can induce reversible cell cycle arrest, a key feature in a cancer fighting strategy known as cyclotherapy (van Leeuwen, 2012). Cyclotherapy aims to halt proliferation of healthy host cells prior to treatment with chemotherapeutic agents that target dividing cells. This approach ensures selective targeting of deregulated cells that continue to proliferate, while the arrested healthy host cells are protected. To test this approach, I inhibited RBG by knocking down expression of ribosomal proteins and by 9-aminoacridine (9-AA) treatment. I demonstrated that in addition to inhibiting Pol I transcription, 9-AA also impedes pre-rRNA processing in a dose-dependent manner. Further, I found that the association between pre-ribosomes and snoRNAs required for rRNA maturation is disrupted due to 9-AA treatment. This finding suggested a mechanism by which 9-AA may inhibit pre-rRNA processing. Significantly, this work demonstrated that RBG inhibition can cause a stress response that selectively protects p53 positive cells during treatment with cytotoxic agents targeting proliferating cells. Finally, I initiated characterization of two previously unknown potential factors of RBG, PSMA3 and Nol7. These proteins are required for a pre-rRNA processing step, however the molecular mechanisms of their function in RBG are not known. PSMA3 and Nol7 may present themselves as exploitable targets for RBG inhibition

Guanosine-borate hydrogels- Form and function

Due to their biocompatibility and stimuli-responsive nature, supramolecular hydrogels derived from natural products are attractive for a number of biomedical applications, including diagnostics, targeted drug delivery and tissue engineering. Nucleosides, the building blocks of nucleic acids, are desirable candidates for forming supramolecular gels as they readily engage in reversible, noncovalent interactions. Guanosine (G 1), in particular, is unique in that it has multiple faces for noncovalent interactions and can self-associate into stable higher-order assemblies, such as G4-quartets and G-quadruplexes. This self-assembly of G 1 and its derivatives into G4-quartets has long been known to induce hydrogelation. However, the requirement of excess salt and the propensity of G 1 to crystallize persist as limitations for G4-hydrogels. Thus, recent interest has focused on developing G4-hydrogels with improved lifetime stabilities and lower salt concentrations. The work described here focuses on a long-lived G4-hydrogel made from G 1 and 0.5 equiv. of KB(OH)4. Gelation occurs through the formation of guanosine-borate (GB) diesters and subsequent assembly into cation-templated G4•K+-quartets. The physical properties and stability of the GB hydrogel can be readily manipulated by varying the gelation components. For example, merely altering the identity of the cation drastically alters the gel’s physical properties. Namely, while GB hydrogels formed with K+ are self-supporting and robust, mixing G 1 with LiB(OH)4 results in a weak gel that readily dissociates upon physical agitation. Small molecules, such as cationic dyes and nucleosides, could be selectively incorporated into the GB hydrogel through reversible noncovalent and covalent interactions. One such dye and known G4-quartet binding ligand, thioflavin T (ThT) fluoresces in the presence of the GB hydrogel. The ThT fluorescence increases as a function of gelator concentration with a sharp increase correlating to the gel point. Thus, this ThT fluorescence assay is a new method for probing the formation of G4-hydrogels. Additionally, ThT acts as a molecular chaperone for Li+ GB hydrogelation. Substoichiometric amounts of ThT results in faster hydrogelation, increased gel strength and improved recovery of a hydrogel destroyed by external stress. Insights gained from this research have implications towards development of biomaterials, biomolecule sensing, and drug delivery

The Effects Of Stress On The Mammalian Nucleolus And Ribosome Synthesis

Ribosomes are responsible for translating every protein in the cell and are essential in all domains of life. Ribosome biosynthesis (RB) takes place in the nucleolus and is an intricate hierarchical process involving over 200 factors, including ribosomal proteins, ribosomal RNA (rRNA), and trans-acting ribosome biogenesis factors (RBFs). Inhibiting RB can disrupt nucleolar integrity, causing ribosomal- and nucleolar-factors to delocalize. This can stabilize the transcription factor p53, which is normally degraded rapidly, ultimately causing cell cycle arrest or apoptosis, through a mechanism termed the nucleolar stress response (NSR). This thesis explores the effects of inhibiting RB post rRNA transcription and discusses its role in human diseases. Using a dominate-negative RBF mutant, this work demonstrates that targeting RB after rRNA transcription is a viable approach to increase the efficacy of the chemotherapeutic agent camptothecin (CPT) against p53-negative cancers. Importantly, this model of targeting RB is independent of DNA damage, unlike currently developed molecular inhibitors of RB. In addition to exploring the combinational effects of CPT and post-transcriptional inhibition of RB, this thesis also investigated the impact of oxidative stress on nucleolar function. The onset of several diseases, including cancer and neurodegeneration, has been linked to both oxidative stress and nucleolar stress. However, very little has been reported on the effects of oxidative stress on the nucleolus. Using a newly generated nucleolar-specific redox sensor, this work demonstrates an increase in the oxidative state of the nucleolus and oxidative damage in nucleolar RNA when cells are challenged with a variety of chemical pro-oxidants. Additionally, rRNA processing and ribosomal subunit synthesis is inhibited by pro-oxidants. This phenotype was exacerbated when the antioxidant glutathione was depleted. The significance of this work demonstrates the susceptibility of the nucleolus to oxidative stress

Genome instability induced by structured DNA and replication fork restart

DNA replication is a central mechanism to all forms of life. Errors occurring during DNA replication can result in mutagenesis and genome rearrangements, which can cause various diseases. In this work I have investigated the stability of direct tandem repeats (TRs) in the context of replication and replication-associated repair mechanisms. During DNA replication the replication fork encounters many obstacles, such as DNA-protein barriers, secondary DNA structures and DNA lesions. How and if replication resumes or restarts in these circumstances in order to complete genome replication is not well understood and the fidelity of replication in response to such obstacles remains unclear. I have developed TR assays to assess replication errors in the context of replication fork restart and secondary structures. The results suggest that structured DNA (G4) can cause instability of TRs in the context of normal replication and that restarted replication can be intrinsically error-prone. Surprisingly, the mutagenic effect of G4-DNA on TR stability was not elevated in the context of replication fork restart. Therefore, deletions of TRs containing G4-DNA are not more susceptible to the compromised fidelity of a restarted replication fork. Structures such as stalled replication forks can induce checkpoint responses to maintain genome stability. The stabilisation of replication forks is central in the response to replication stress. These protective mechanisms include the regulation of enzymatic activities. Mus81-Eme1 is a structurespecific endonuclease which is regulated by the DNA replication checkpoint, but has also been shown to be required for replication fork restart in certain circumstances. In collaboration with Professor Neil McDonald I analysed a novel domain identified in Mus81-Eme1. Mutagenesis of key residues deduced from the protein structure and comparison of their genetic analysis to known phenotypes of Mus81-Eme1 suggests distinct requirements for this domain

Mitochondrial DNA gene expression and consequences of its loss in mammals

Mitochondria are intracellular organelles that fulfil multiple essential functions, including the generation of the vast majority of the cellular energy currency adenosine triphosphate (ATP) via the oxidative phosphorylation (OXPHOS) system. Mitochondrial dysfunction is found in severe inherited metabolic disorders with a broad clinical spectrum, in several common age-related diseases, and has been associated with the ageing process itself. Mitochondrial biogenesis depends on the expression of two cellular genomes. While the mitochondrial genome (mtDNA) encodes a few essential subunits of the OXPHOS system and the transfer and ribosomal RNAs required to translate these subunits in mitochondria, most mitochondrial proteins are encoded in the nuclear genome (nDNA). The mechanisms regulating the expression of mtDNA are still largely unknown and how disrupting this process leads to pathogenic phenotypes is poorly understood. The first aim of this thesis was to elucidate the in vivo function of the mitochondrial RNA polymerase (POLRMT) in the regulation mtDNA gene expression. We generated and characterized transgenic mouse models with varying gene dosage of Polrmt to investigate the role of POLRMT and the splice variants of the Polrmt gene in mammals. These Polrmt models included a whole-body knockout, a heart and skeletal muscle knockout, a heterozygous knockout, and an overexpressing mouse strain. Our findings reveal that Polrmt only codes for a mitochondrial isoform, POLRMT, and that no other RNA polymerase can replace its function. We show that POLRMT has two essential roles in mammalian mitochondria, i) it is the only RNA polymerase transcribing mtDNA, and ii) it synthesizes the RNA primers required for mtDNA replication. Moreover, our data suggests that POLRMT is part of a mechanism involving promoter-specific transcription initiation that coordinates the switch between mtDNA replication and transcription. Finally, we show that POLRMT is the limiting factor for transcription initiation and that it is frequently loading at the promoters to initiate transcription, whereas productive near-genome length mitochondrial RNA (mt-RNA) synthesis is regulated at the elongation level. The second aim of this thesis was to investigate the cellular consequences of disrupting mtDNA gene expression and the progression of OXPHOS deficiency. We performed a systematic comparison of the mitoproteome and total cellular transcriptome from heart of five knockout mouse models, each deficient in an essential factor acting at a specific level of gene expression regulation. We also studied the mitoproteome changes during normal post-natal development in mouse heart and followed proteome changes in a model with progressive OXPHOS deficiency in the heart. Surprisingly, we found a decline in the intra-mitochondrial Q biosynthesis pathway that correlates with the progressive OXPHOS deficiency. Furthermore, we found that cellular stress responses, likely mediated by the myc proto-oncogene protein (MYC) and the cyclic AMP-dependent transcription factor ATF4 (ATF4) transcription factors, are activated very early in the progression of OXPHOS deficiency. These cellular responses include the upregulation of enzymes of the mitochondrial one-carbon (1C) pathway, proline synthesis, and mitochondrial proteases and chaperones. In conclusion, the work presented in this thesis has identified fundamental mechanisms of regulation of mtDNA gene expression in vivo and pinpointed primary and secondary consequences of impaired expression of mtDNA. This work has not only important implications for our understanding of the function of this essential organelle but it has also identified novel mechanisms involved in mitochondrial pathology that can be relevant for patient diagnosis and future treatment strategies