Η Νουκλεολίνη είναι μια πρωτεΐνη που λειτουργεί ως υποδοχέας για διάφορα μόρια, όπως αυξητικοί παράγοντες, μόρια κυτταρικής προσκόλλησης, ακόμα και ιοί (HIV, coxsackie B, κ.ά.), που εμπλέκονται σε παθοφυσιολογικές διεργασίες. Συμμετέχει στις διαδικασίες της ογκογένεσης και της αγγειογένεσης και ανιχνεύεται στον πυρήνα, το κυτταρόπλασμα των κυττάρων, καθώς επίσης και στην επιφάνεια καρκινικών και μιτωτικά ενεργών ενδοθηλιακών κυττάρων. Το συνθετικό πεπτίδιο ΗΒ‐19 συνδέεται στη Νουκλεολίνη της επιφάνειας και καταστέλλει την ογκογένεση, αλλά και την αγγειογένεση Η ερευνητική ομάδα του εργαστηρίου έδειξε, σε προηγούμενες εργασίες, ότι το ΗΒ‐19 καταστέλλει την in vivo αγγειογένεση στο μοντέλο μελέτης της CAM σε έμβρυο όρνιθας. Βρέθηκε, ακόμη, ότι καταστέλλει την in vitro προσκόλληση, μετανάστευση και τον πολλαπλασιασμό των HUVEC, χωρίς να προκαλεί την απόπτωσή τους. Οι βιολογικές δράσεις αυτές φαίνεται να διαμεσολαβούνται και από τις κινάσες Src, ERK1/2, Akt και FAK, αφού παρατηρήθηκε ότι το ΗΒ‐19 καταστέλλει την ενεργοποίησή τους στα κύτταρα αυτά. Προκειμένου να διερευνηθεί ο μηχανισμός με τον οποίο το ΗΒ‐19 ασκεί τις βιολογικές του δράσεις, καθώς επίσης και η συμμετοχή της Νουκλεολίνης στη διαδικασία αυτή, HUVEC υπέστησαν παροδικό μετασχηματισμό με χρήση siRNA που στοχεύει το mRNA της Νουκλεολίνης. Η αποσιώπηση της Νουκλεολίνης εμπόδισε την κατασταλτική δράση του ΗΒ‐19 στον πολλαπλασιασμό και την προσκόλληση των HUVEC και, από το ίδιο πείραμα, φάνηκε ότι η Νουκλεολίνη είναι ένα κομβικό μόριο, καθώς η αποσιώπησή της ήταν από μόνη της ικανή να καταστείλει τις παραπάνω λειτουργίες. Για να επιβεβαιωθεί ότι η μεταγωγή σήματος του ΗΒ‐19 ενεργοποιείται από την αλληλεπίδρασή του με τη Νουκλεολίνη, μελετήθηκαν οι επιπτώσεις του στην ενεργοποίηση των κινασών Src και ERK1/2, σε παροδικά μετασχηματισμένα (με siRNA για Νουκλεολίνη) HUVEC.Παρατηρήθηκαν μειωμένα επίπεδα φωσφορυλίωσης των κινασών αυτών στα μετασχηματισμένα κύτταρα, συγκριτικά με τα φυσιολογικά. Ακόμη, η αποσιώπηση της Νουκλεολίνης εμπόδισε το ΗΒ‐19 να ασκήσει την κατασταλτική του δράση στη φωσφορυλίωσή τους. Συνοψίζοντας τα παραπάνω αποτελέσματα, φαίνεται ότι η αλληλεπίδραση του ΗΒ‐19 με τη Νουκλεολίνη πυροδοτεί οδούς μεταγωγής σήματος που μειώνουν τα επίπεδα φωσφορυλίωσης των Src και ERK1/2 και προκαλούν την καταστολή της προσκόλλησης και του πολλαπλασιασμού των HUVEC. Λαμβάνοντας υπ' όψιν και την υπάρχουσα βιβλιογραφία, φαίνεται πως το ΗΒ‐19 θα μπορούσε να χρησιμοποιηθεί στην αντικαρκινική θεραπεία ως μέρος μιας στρατηγικής με στόχο τη Νουκλεολίνη. / Nucleolin is a protein serving as a receptor for various ligands such as growth factors, cell adhesion molecules and viruses (e.g. HIV, coxsackie B), implicated in pathophysiological processes. It is overexpressed on the surface of tumour and mitotic endothelial cells and recent studies have demonstrated its involvement in tumour growth and angiogenesis. The synthetic peptide HB‐19 binds to the cell surface expressed Nucleolin and inhibits tumour growth and angiogenesis as well. The lab group has shown, in previous projects, that HB‐19 inhibits in vivo angiogenesis in the chicken embryo CAM assay. Evidence has been provided, in addition, that HB‐19 inhibits the in vitro adhesion, proliferation and migration of HUVEC, while not inducing their apoptosis. The aforementioned biological actions seem to be regulated by Src, ERK1/2, Akt and FAK kinases, as HB‐19 was found to inhibit their activation in HUVEC. In order to investigate the mechanism through which HB‐19 exerts the above described actions, as well as the involvement of cell‐surface Nucleolin in them, HUVEC were transiently transfected using siRNA targeting the mRNA of Nucleolin. The inhibitory effect of HB‐19 in cell proliferation and adhesion was blocked by the knockdown of Nucleolin and, as was shown by the same experiment, Nucleolin is a crucial factor to HUVEC proliferation and adhesion, since both processes were inhibited by the down‐regulation of Nucleolin expression. In order to confirm that HB‐19 signaling is activated by its reaction with Nucleolin, its effect on the activation of Src and ERK1/2 was tested on transiently transfected HUVEC (using siRNA for Nucleolin). A decrease in phosphorylation levels of Src and ERK1/2 was induced on the transfected cells, compared to wild type cells. Furthermore, Nucleolin knockdown blocked HB‐19‐induced Src and ERK1/2 inactivation. In conclusion, these results indicate that HB‐19's interaction with Nucleolin triggers a signal transduction pathway that reduces the phosphorylation levels of Src and ERK1/2 and causes inhibition of HUVEC proliferation and adhesion. Thereof, HB‐19 could constitute a potentially interesting tool for tumour therapy, as part of a Nucleolin targeting strategy.

Class of 2016 Abstract / Objectives: The purpose of this experiment is to compare NUC1234 expression levels from E. coli cultures by manipulating cell cultures according to the optical density (OD595) at which protein expression is induced, and the harvest time after induction. Methods: E. coli BL21(DE3) cells transformed with DNA to produce NUC1234 were plated and then grown in minimal media for protein expression. They were induced at a variety of optical densities and harvested at different times post-induction. Protein quantities from each sample were then compared using a protein determination assay and an SDS-PAGE gel. Results: The sample induced at an OD595 of 0.5 yielded a lower concentration of protein (0.874409mg/ml) compared to other samples; however, it visualized as a stronger band in the SDS-PAGE gel. The sample harvested at 6 hours yielded the largest protein concentration (1.347215mg/ml) among all active samples and appeared as bold as, or bolder than, those harvested at other times. Conclusions: The results of the protein assay study and gel visualization suggest that the optimal conditions for the production of NUC1234 are growth to an OD595 of 0.5 before induction of protein expression and a harvest time of 6 hours after induction.

The activated product of the c-MYC proto-oncogene is one of the strongest known activators of carcinogenesis. It has been estimated that as many as one-seventh of all cancer deaths are associated with alterations in the c-MYC gene or its expression [1]. Therefore, understanding the regulation of c-MYC expression is a key factor in understanding carcinogenesis in many histologic classes of malignancy. The nuclease hypersensitive element (NHE) III₁ region of the c-MYC promoter has been shown to be particularly important in regulating c-MYC expression. Specifically, the formation of a G-quadruplex structure appears to promote repression of c-MYC transcription. In this dissertation, we investigate the role that nucleolin, a critical player in ribosome biogenesis and cell stress sensing, plays on the transcriptional regulation of the c-MYC promoter through its interaction with the c-MYC G-quadruplex structure. Our studies initiated with the design of a c-MYC G-quadruplex affinity column intended to trap potential c-MYC G-quadruplex-binding proteins that were then identified by LC-MS/MS. After careful examination of the literature of the list of potential c-MYC G-quadruplexbinding proteins, we realized that several of the proteins identified had been previously reported to interact directly with nucleolin. Consequently, we chose to focus our studies on nucleolin, as it could be a central regulator of the (NHE) III region. By performing chromatin immunoprecipitation in HeLa cells, we found that nucleolin indeed interacts with the c-MYC promoter region containing the NHE III₁ element. This binding activity was confirmed by both electromobility shift assay and polymerase stop assay. We provide evidence that nucleolin can induce the formation of the c-MYC G-quadruplex structure from single-stranded DNA, both in linear and circular DNA forms. We show that upon binding, nucleolin increases the stability of the c-MYC G-quadruplex structure leading to repression of c-MYC promoter activity. We also show that nucleolin binds with much higher affinity to G-quadruplex structures with topology similar to that of the parallel c-MYC G-quadruplex, such as those found in the VEGF and PDGF-A promoters; in comparison to G-quadruplexes found in telomeres or the c-MYB promoter, whose have significantly different topology. Interestingly, we also demonstrate that nucleolin binds with higher affinity to the c-MYC G-quadruplex than to its consensus RNA substrate, the nucleolin recognition element (NRE). Furthermore, we show that the C-terminal domain of nucleolin is critical for its interaction and stabilization of the c-MYC G-quadruplex structure. Lastly, we show that the binding of nucleolin to the (NHE) III region causes repression of c-MYC transcription. On the basis of these results, we propose that nucleolin may play an important role in the transcriptional regulation of c-MYC in vivo by inducing the formation of the c-MYC G-quadruplex structure.

<p>Current standard cancer treatments such as chemotherapeutics, and radiation therapy are nearly as likely to kill the patient as cure the cancer. Therapies that have such a narrow window of efficacy are necessary for the treatment of aggressive diseases, but safer alternatives must be created. By discovering novel therapeutics that target specific disease processes within specific diseased cells, while leaving healthy cells unaltered, we can improve the lives of millions of cancer sufferers and their families. A therapeutic's window of efficacy can be measured by the therapeutic index. For many anti-cancer therapeutics, the therapeutic index is very small, the dose of treatment that kills cancer cells and shrinks tumors is nearly the dose that causes toxicity. In cancer patients, this toxicity causes many serious conditions such as gastrointestinal distress, organ damage, and death. </p><p>Recently, the model of cancer treatment has evolved from non-specific cytotoxic agents to more selective therapeutics that target cellular processes necessary for cancer cell survival. If a therapy can be targeted to selectively bind and internalize targeted cells, its toxicity would only impact the targeted cells and healthy cells in the immediate vicinity, which would greatly reduce the toxic effects on the rest of the body. Targeting cancer cells can be done through cancer biomarkers, which are cell surface proteins, expressed exclusively, or are much more abundant on the surface of cancer cells than on somatic cells. </p><p>Advances in antibody and aptamer technology have enabled researchers to design those molecules to bind specifically to cancer cells, and deliver drugs that alter specific cellular processes. An aptamer designed to bind PSMA, a prostate cancer biomarker, only bound to a specific subset of cancer cells, and delivered a therapeutic siRNA that prevented a specific survival process from occurring. While this technology is promising, it is currently limited to targeting small subsets of cancer types. To generate an aptamer therapeutic that would have greater utility and efficacy, I have examined the properties of a nucleolin aptamer-mediated delivery system that targets multiple types of cancer cells, and delivers various oligonucleotide therapeutics. </p><p>The nucleolin aptamer targeted cancer cells by binding to membrane&ndash;associated nucleolin. Nucleolin, a conserved protein found in all eukaryotes, shuttles from the nucleus, through the cytoplasm to the cell membrane. Cancer cells express a far greater amount of membrane&ndash;associated nucleolin than somatic cells, making nucleolin an ideal cancer biomarker. The shuttling, and oligonucleotide binding attributes of the protein enable it to deliver aptamer chimeras from the cell surface to the nucleus. Therefore the nucleolin aptamer has unique access to the nuclei of cancer cells, and can deliver therapeutic oligonucleotide cargoes through nucleolin binding.</p><p>The nucleolin aptamer delivered splice&ndash;switching oligonucleotides, a form of antisense technology, improving their efficacy, and potentially increasing their therapeutic viability. The ability to deliver antisense oligonucleotides to the nuclei of cancer cells has the potential for other therapeutic possibilities including the inhibition of transcription with antisense triplexes.</p><p>The nucleolin aptamer can also deliver therapeutic aptamers. The nucleolin aptamer&ndash;&beta;&ndash;arrestin aptamer chimera prevented the stem cell renewal phenotype necessary for leukemia progression in human patient tissue samples. The ability to effectively deliver therapeutic aptamers may lead to clinical applications for many of the aptamers that have been selected against intracellular targets including transcriptional activators.</p><p>Oligonucleotide research continues to advance our understanding of potentially therapeutic oligonucleotides. Long non&ndash;coding RNAs for example, may impact epigenetics, and transcription. Additionally, locked nucleic acids have been developed to improve binding affinity, thus increasing the efficacy of antisense oligonucleotides. In order to bring these discoveries into the clinic, they must be safely and specifically delivered to their target cells. </p><p>This work demonstrated that the nucleolin aptamer could deliver oligonucleotide therapeutics to specific cancer cells. Nucleolin aptamer chimeras have the potential to develop into safe and effective cancer therapies, thus improving the treatment options for cancer sufferers.</p> / Dissertation

Chez les eucaryotes, les gènes d’ARNr 45S sont présents en un grand nombre de copies organisées dans des régions chromosomiques appelées NOR pour « Nucleolus Organizer Region ». Cependant, seule une fraction de ces gènes est activement exprimée et leur activation/répression est majoritairement contrôlée par des mécanismes épigénétiques. Parmi les facteurs requis pour l’expression de ces gènes, se trouve la nucléoline, une protéine majeure du nucléole. Chez A. thaliana, la protéine NUC1 est nécessaire pour le maintien de la méthylation et le control de l’expression de variants spécifiques des gènes d’ARNr. De manière intéressante, contrairement aux animaux et aux levures, le génome des plantes possède un deuxième gène codant la nucléoline : NUC2. Au cours de cette étude, nous avons montré que les deux gènes NUC1 et NUC2 sont nécessaires pour la survie de la plante. L’étude de plantes mutées pour le gène NUC2 a révélé que cette protéine est impliquée dans l’organisation et l’expression des ADNr mais par des mécanismes antagonistes à ceux de son homologue NUC1. En effet, l’absence de la NUC2 induit une hyperméthylation des ADNr ainsi qu’une réorganisation spatiale et une variation du nombre de copie des différents variants des gènes d’ARNr. Par ailleurs, la protéine NUC1 se lie aux gènes actifs alors que la protéine NUC2 est associée à la chromatine condensée en périphérie du nucléole. En parallèle, nous avons montré que l’expression des ADNr est affectée en réponse à la chaleur et que le gène NUC2 est fortement induit. L’ensemble de ces données suggèrent un potentiel rôle de la NUC2 dans la répression des gènes d’ARNr au cours du développement et en réponse au stress. / In eukaryotes, 45S rRNA genes are highly repeated and localize in chromosomal regions known as NOR for “Nucleolus Organizer Regions”. However, only a small proportion of these genes is transcriptionally active and their activation and/or repression depends on epigenetic mechanisms. One of the factors involved in rDNA expression is nucleolin, a major nucleolar protein. In A. thaliana, nucleolin protein NUC1 is required to maintain rDNA methylation and control expression of specific rDNA variants. Interestingly, in contrast to animals and yeast, plants encode a second nucleolin gene: NUC2. Here, we show that NUC1 and NUC2 genes are both required for plant survival. Analysis of nuc2 mutant plants reveals that NUC2 protein is required for rDNA organization and expression but with mechanisms antagonistic to those described for its homologue NUC1. In fact, loss of NUC2 induces rDNA hypermethylation and a spatial reorganization of rRNA genes with changes in copy numbers of rDNA variants. Moreover, NUC1 protein binds transcriptionally active rRNA genes while NUC2 protein associates with condensed chromatin in the periphery of the nucleolus. Furthermore, we show that rRNA gene expression is affected in response to heat shock and that the NUC2 gene is strongly induced. Altogether, our results suggest a potential role of NUC2 protein in rDNA repression during development and/or in response to stress.

Les thérapies ciblées utilisent des agents thérapeutiques qui interfèrent specifiquement avec les molécules nécessaires pour la croissance et la progression tumorale. Les chimiothérapies classiques sont toxiques pour les cellules qui se divisent rapidement du à leur interaction avec les cellules en division. Le premier but des thérapies ciblées est de combattre plus précisement les cellules cancereuses et ainsi éviter les effets indesirables.La thérapie anti-cancereuse utilisant les anticorps a été développé depuis environ 15 ans et est actuellement une des plus efficaces des thérapies ciblées. Dans certains cas des anticorps monoclonaux sont conjugués avec des isotopes radioactifs ou des toxines pour permettre une délivrance ciblée de ces derniers dans les cellules cancereuses. De plus en plus, pour remplacer les anticorps, les thérapies ciblées utilisent des peptides ou des acides nucleiques comme agent ciblant.Dans ce travail, nous avons utilisé des stratégies diverses de ciblage pour permettre l’internalisation de substance toxique (une toxine ou son gène) specifiquement dans les cellules cancereuses. Nous avons travaillé sur le modèle du glioblastome.Notre groupe a publié un article décrivant l’utilisation du domaine PDZ d’une proteine hCASK permettant la liaison a un biomarqueur surexprimé dans les cellules cancereuses. En effet, ce domaine PDZ est capable de se lier à la partie C-terminale de la proteine CD98. Le produit de fusion hCASK-PDZ a été génétiquement lié a une toxine (la saporine). Nous avons démontré une activité in vitro évidente de ce conjugué dans les cellules de glioblastome.Dans cette étude, nous avons utilisé d’autres agents ciblant une autre proteine surexprimée à la surface des cellules cancereuses: la nucléoline. Dans ce contexte, un aptamer et un pseudopetide se liant specifiquement à cette dernière ont été developpé et étudié.L’aptamer AS1411 (Antisoma, UK) est un agent ciblant la nucleolin approuvé par la FDA (food and drug administration). Il se lie à la nucleoline et est internalisé, perturbant ainsi l’interaction de cette dernière avec ses partenaires inhibant ainsi proliferation cellulaire.En parallèle, notre groupe a developpé un pseudopeptide, antagoniste de la nucleoline (Nucant, N6L). Il a été montré qu’il inhibe drastiquement la croissance tumorale dans le cancer du sein en induisant l’apoptose et il est actuellement en préparation pour une phase II d’essai clinique (IPP-204106). Nous avons démontré l’effet anti-proliferatif du N6L in vitro sur des cellules primaires de glioblastome.La surexpression de la nucleoline à la surface des cellules de glioblastomes couplée à la specificité de l’aptamere et du N6L pour cette deniere nous ont amené à vouloir augmenter leur efficacité d’action en les liant à une toxine. Le gène codant pour la saporine (proteine inhibitrice des ribosomes ) ou la saporine elle-meme a donc été lié à l’aptamere et au N6L.Nous avons donc dans ce travail etudié l’activité cytotoxique de l’aptamere et du N6L liés à la saporine. Les résultats obtenus sont evalués pour des futurs publications.Toutes les approches ciblées décrites, en dépit de certains problèmes, semblent prometteuses et nécessitent d'autres recherches, mais confirment que l'exploitation de cibles pour fournir des substances toxiques est l'avenir de la thérapie pour les formes cancéreuses difficiles à battre avec les thérapies conventionnelles. / Targeted cancer therapies are drugs designed to interfere with specific molecules necessary for tumor growth and progression. Traditional cytotoxic chemotherapies usually kill rapidly dividing cells in the body by interfering with cell division. A primary goal of targeted therapies is to fight cancer cells with more precision and potentially fewer side effects.Antibody-based therapy for cancer has become established over the past 15 years and is now one of the most successful and important targeted strategies. In some cases, monoclonal antibodies are conjugated to radio-isotopes or toxins (immunotoxin) to allow specific delivery of these cytotoxic agents to the intended cancer cell target. Furthermore thargeted therapies may be based also on the use of targeting molecues other than antibodies, such as peptides, growth factors, and also nucleic acids.Indeed, in this work we studyed a multi targeting strategy to deliver toxic substances (protein toxin or its gene) to cancer cells (glioblastoma).Our group published a paper describing the use of PDZ protein domain of hCASK (serine kinase calcium/calmodulin-dependent of MAGUK family) and to exploit the ability of this protein to bind to the C-terminus of hCD98 in the extracellular space. CD98 is an interesting target because it is overexpressed in different types of tumors (Giansanti F., 2015). hCASK-PDZ was genetically fused to the toxin saporin and this chimeric toxin proved to be active on glioblastoma cells in vitro.Other cell killing agents were designed to recognize and bind specifically nucleolin (NCL). This multifunctional protein is overexpressed on the surface of activated endothelial and tumor cells. In this context, compounds targeting NCL, such an aptamer, and a multivalent pseudopeptide, have been developed and investigated for cancer therapy.The aptamer against NCL, NCL-APT also known as AS1411 (Antisoma, UK), is a US Food and Drug Administration (FDA)-approved NCL targeting agent. It binds to NCL on the cell surface, preferentially gets internalized, and inhibits cancer cell growth sparing normal cells (Bates PJ, 2009).In parallel, our group, recently developed a multivalent synthetic pseudopeptide N6L that selectively binds to nucleolin (Destouches D., 2011). N6L strongly inhibits breast cancer growth by inducing apoptosis of tumor cells and is currently in preparation for phase II clinical trials (IPP-204106). We demonstrated the anti-proliferative effect of N6L on human glioblastoma cells in primary culture prepared form post-surgical specimens (Benedetti E, 2015).The overexpression of NCL on glioblastoma cell surface and the recognized selectivity of AS1411 and N6L prompted us to study a way to increase the efficiency of these ligands binding them Saporin coding gene or the protein toxin Saporin-S6, a type 1 RIP (Ribosome-Inactivating Protein) widely studied because of its potential therapeutic application in a variety of human diseases as toxic moiety of a conjugate.The characterization of the toxic activity of AS1411 linked to saporin gene (APT-SAP) and of NCL linked to saporin protein (SAP-N6L) is therefore described. Both these researches are under evaluation for publication.All the described thargeted approaches, nothwithstanding some problems, look promising and need further research, but confirm the fact that exploiting targets to deliver toxic substances is the future of therapy for cancer forms that are difficult to beat with conventional therapies.

京都大学 / 0048 / 新制・課程博士 / 博士(人間・環境学) / 甲第21874号 / 人博第903号 / 新制||人||215(附属図書館) / 2018||人博||903(吉田南総合図書館) / 京都大学大学院人間・環境学研究科相関環境学専攻 / (主査)准教授 小林 純也, 教授 宮下 英明, 准教授 三浦 智行 / 学位規則第4条第1項該当 / Doctor of Human and Environmental Studies / Kyoto University / DGAM

<p>c-Myc is one of the most important oncogenes. G-quadruplex DNA secondary structure formed in the proximal promoter region of c-Myc functions as a transcription silencer and is targetable by small molecules. Therefore, the c-Myc promoter G-quadruplex (MycG4) is an attractive anticancer drug target. Protein recognition of MycG4 is essential for its transcriptional regulating. Nucleolin was discovered as a major MycG4 binding protein in 2009. It shows a remarkably higher binding affinity for MycG4 over its known substrate NRE_RNA and overexpression of nucleolin represses the activity of the c-Myc promoter. However, little is known about its molecular recognition of MycG4. Here, we use X-ray crystallography combined with other biochemical and biophysical methods to understand how nucleolin recognizes MycG4. Nucleolin is a 77 kD protein with a modular organization. The four RNA-binding domains (RBD) of nucleolin are the minimal domains for high affinity binding with MycG4. We show that nucleolin prefers the c-Myc parallel G-quadruplex with a 6-nt central loop (Myc161) that is the thermodynamically favored conformation. Using a custom G4 DNA microarray, we optimized the MycG4 sequence with over 10-fold increased binding affinity to nucleolin. Fabs are widely used tools to facilitate crystallization and we have discovered Fabs that specifically bind the nucleolin-MycG4 complex using a phage display screening. This approach enabled us to obtain crystals of the nucleolin-MycG4-Fab ternary complex diffracted at 2.6 Å and we determined the crystal structure. In the structure, the parallel MycG4 is very well-defined with two K⁺ between the three G-treads. The central 6-nt loop residue protrude from the G4-core and extensively recognized by the nucleolin. Only RBD1 and RBD2 of nucleolin are seen in the crystal structures and interact extensively with the 6-nt central loop and 5′-flanking of MycG4. The binding surface and area of the globular MycG4 by nucleolin is much more extensive than NRE_RNA and involves an extra binding site. Fab binds to both RBD1 and 3′-end of MycG4 to stabilize the complex. The well-defined partial RBD2-3 linker and a cavity close to the 1-nt T19 loop suggest that the missing RBD3 likely binds the 3^rd loop of MycG4. This structure is the first MycG4-protein complex structure. It will help understand MycG4 and nucleolin interactions and the development of MycG4 targeted cancer therapeutics. This structure also provides novel insights into how proteins recognize the globular G-quadruplexes, highlighting the potential of G-quadruplexes as a platform for multivalent interactions such as with multiple tandem RBDs.</p>

Positive strand RNA viruses are intracellular parasites, and their genome replication and infection involves complex virus-host interactions. Therefore, identification of host factors and dissection of their functions during virus replication could facilitate our understanding of the mechanism of virus infection. Those host factors may also provide new targets for viral disease control. Tomato bushy stunt virus (TBSV) has recently become one of the model viruses to study positive strand RNA virus replication and hostvirus interactions. To identify host factors involved in TBSV replication we used yeast as a model host. Co-expression of the replication proteins and a replicon RNA (DI RNA) via plasmids in yeast resulted in robust replication of the viral RNA. Previous work using a yeast single gene deletion library (YKO) revealed 96 yeast genes affecting virus replication. The essential yeast genes could not be deleted so we used the Yeast Tet Promoters Hughes Collection (yTHc) where the original promoter was replaced by Tetracyclin-titratable promoter. I tested the 800 essential host genes available in yTHc. In total, we found 30 new host genes whose down-regulated expression either increased or decreased the accumulation of a TBSV repRNA. The identified essential yeast genes fall into different categories on the basis of the cellular processes they are involved in, such as RNA transcription/metabolism, protein metabolism/transport etc. Detailed analysis of the effects of some of the identified yeast genes revealed that they might affect RNA replication by altering (i) the amounts of p33 and p92(pol) viral replication proteins, (ii) the activity of the tombusvirus replicase complex, and (iii) the ratio of plus- versus minus-stranded RNA replication products. Altogether, this and previous YKO screening of yeast led to the identification of 126 host genes (out of ~5,600 genes that represent ~95% of all the known and predicted yeast genes) that affected the accumulation of tombusvirus RNA. In the YKO screening, we found NSR1 (homologous to plant nucleolin) gene, whose deletion led to increased TBSV repRNA accumulation. Nucleolin is an abundant RNA binding protein, which shuttles between the nucleolus, the nucleoplasm and the cytoplasm. This protein is involved in rRNA maturation, ribosome assembly and regulation of cellular RNA metabolism.We found that over-expression of Nsr1p in yeast or nucleolin in Nicotiana benthamiana inhibited the accumulation of tombusvirus RNA by ~10-fold. Temporal regulation of Nsr1p over-expression revealed that the inhibitory effect of Nsr1p was more profound when it was expressed at early stages of viral replication. In vitro binding experiments showed that Nsr1p binds preferably to the RIII in the repRNA (which is derived from 3’ UTR of viral genome). Consistent with its RIII specific binding, over-expression of Nsr1p only reduced 40% of the accumulation of TBSVΔRIII repRNA in yeast. The purified recombinant Nsr1p inhibited the in vitro replication of the viral RNA in a yeast cell-free assay when pre-incubated with the viral RNA before the in vitro replication assay. Our data suggest that Nsr1p/nucleolin inhibits tombusvirus replication by interfering with the recruitment of the viral RNA for replication.

H νουκλεολίνη είναι μια πρωτεΐνη μοριακού βάρους 110 kDa και απαντάται στον πυρήνα, το κυτταρόπλασμα αλλά και στην επιφάνεια των κυττάρων, ενώ υπερεκφράζεται σε καρκινικά κύτταρα και σε ενεργά ενδοθηλιακά κύτταρα. Πρόσφατες έρευνες υποδεικνύουν την συμμετοχή της στις διαδικασίες ανάπτυξης καρκινικών όγκων και στην αγγειογένεση. Στην επιφάνεια του κυττάρου δρα ως υποδοχέας χαμηλής συγγένειας αυξητικών παραγόντων, μορίων κυτταρικής προσκόλλησης (ιντεγκρίνες και σελεκτίνες) λιποπρωτεϊνών και ορισμένων ιών (HIV-1 και coxsackie B). Παρουσιάζει πλήθος λειτουργιών, αλληλεπιδρώντας τόσο με πρωτεΐνες, όσο και με νουκλεϊνικά οξέα. Το HB-19 και το Ν6L είναι συνθετικά πεπτίδια που προσδένονται στην νουκλεολίνη της κυτταρικής επιφάνειας και καταστέλλουν τόσο την ανάπτυξη καρκίνου όσο και την αγγειογένεση. Στη συγκεκριμένη εργασία μελετήσαμε την βιολογική δράση των πεπτιδίων αυτών και βρέθηκε ότι αναστέλλουν τον in vitro πολλαπλασιασμό και την in vitro προσκόλληση των κυττάρων HUVEC. Τα πεπτίδια αυτά παρουσιάζουν in vivo ογκοκατασταλτική δράση και βρέθηκε να αναστέλλουν την αγγειογένεση στο σύστημα της χοριοαλλαντοϊδικής μεμβράνης εμβρύου όρνιθας. Επίσης αναστέλλουν τη μετανάστευση των κυττάρων HUVEC στο in vitro σύστημα μελέτης της μετανάστευσης Boyden chamber. Το πεπτίδιο HB-19 αναστέλλει την έκφραση διαφόρων αγγειογενετικών αυξητικών παραγόντων όπως ο FGF, VEGF και των υποδοχέων τους σε επίπεδο mRNA. Το HB-19, όπως και το Ν6L επηρεάζουν την έκφραση της μεταλλοπρωτεϊνάσης MMP-2 τόσο σε επίπεδο πρωτεΐνης όσο και σε επίπεδο έκφρασης γονιδίου. Επιπλέον, κανένα από τα δύο πεπτίδια δεν προκαλεί απόπτωση in vitro στα κύτταρα HUVEC. Τα πεπτίδια HB-19 και Ν6L δεσμεύονται στη νουκλεολίνη της επιφάνειας του κυττάρου και φαίνεται ότι για την άσκηση των βιολογικών τους δράσεων διαμεσολαβούν οι κινάσες SRC, ERK1/2, AKT και FAK καθώς αναστέλλουν την ενεργοποίησή τους σε κύτταρα HUVEC. Τέλος, μείωση της έκφρασης της νουκλεολίνης με χρήση siRNA επιβεβαίωσε τον ρόλο της νουκλεολίνης στην βιολογική δράση των πεπτιδίων HB-19 και N6L. Συνοψίζοντας τα παραπάνω αποτελέσματα, φαίνεται ότι τόσο το HB-19 όσο και το Ν6L εμφανίζουν βιολογικές δράσεις που τα καθιστούν υποψήφια μόρια για αντινεοπλασματική χρήση. / Nucleolin is a 110 kDa protein, located in the nucleus and the cytoplasm while it is over expressed on the surface of tumor and endothelial cells. Recent studies have demonstrated the involvement of nucleolin in tumor growth and angiogenesis. Thus, this cell surface molecule serves as a receptor for various ligands implicated in pathophysiological processes such as growth factors, cell adhesion molecules (integrins and selectins), laminin-1, lipoproteins and viruses (HIV and coxsackie B). HB-19 and N6L are synthetic peptides that bind cell surface expressed nucleolin and inhibit both tumor growth and angiogenesis. In the present work, we investigated the biological actions of peptides HB-19 and N6L. Our results show that both peptides inhibit the in vivo angiogenesis on the chicken embryo CAM assay and inhibit the in vitro adhesion, proliferation, migration and motility of HUVEC cells. HB-19 peptide inhibits the expression of growth factors FGF and VEGF mRNA as well as the expression of their receptors FGFR1 and FLT-1 respectively. We also found that both enzyme activity and expression of MMP-2 was inhibited by HB-19 and N6L. Furthermore, we found that HB-19 and N6L treatment shows no toxicity in HUVEC cells in vitro. The above biological actions seem to be regulated by SRC, ERK1/2, AKT and FAK kinases as we found that HB-19, as well as N6L, inhibit their activation in HUVEC cells. Finally, down regulation of nucleolin using siRNA confirmed the implication of nucleolin in the biological actions of these peptides. Taken all the above results into account, it is indicated that HB-19 and N6L could constitute an interesting tool for tumor therapy strategy targeting nucleolin.