Les cellules de Schwann (CS) sont les cellules gliales myélinisantes du Système Nerveux Périphérique (SNP). Il existe une communication étroite entre ces cellules et les axones auxquels elles s’associent et ce dès les stades les plus précoces de leur développement. Elles migrent tout en se divisant le long des axones; cette division migratoire est suivie d’une deuxième division post-migratoire dans le but d’établir un ratio 1:1 avec les axones pour ensuite les myéliniser. Ce travail vise à analyser, in vivo, le comportement des CS chez le poisson zèbre au cours de leurs divisions.Nous avons remarqué que les CS se divisent parallèlement aux axones le long du nerf de la Ligne Latérale Postérieure (PLL). En analysant les deux mutants has et nok, nous avons montré que les gènes de polarité apicale aPKC et pals1 ne sont pas requis pour la migration et la division des CS, ni pour leur capacité à myéliniser. Nous avons mis en évidence, en analysant le mutant cassiopeia qui présente des défauts d’organisation du fuseau mitotique et en utilisant l’agent pharmacologique le nocodazole, que l’assemblage du fuseau mitotique au cours de la division des CS est essentiel pour la myélinisation.En parallèle, nous avons analysé le rôle du gène rgs4 (regulator of G-protein Signaling 4) dans le développement du SNP chez le poisson zèbre. Nous avons généré un mutant stable rgs4 par la technique CRISPR/Cas9 et montré un rôle de ce gène dans le développement du ganglion de la PLL et des motoneurones, et ce en agissant en amont de la voie PI3K/Akt/mTOR.Contrairement à l’inhibition pharmacologique qui suggère un rôle de rgs4 dans la myélinisation périphérique, le mutant ne présente pas de défauts de myéline. / Schwann cells (SCs) are the myelinating glial cells of the Peripheral Nervous System (PNS). They derive from neural crest cells during development, then migrate and divide along the axons of the peripheral nerves. This migratory division is followed by a post-migratory division in order to radially sort the axons in a 1:1 ratio and wrap them with a myelin sheath. This work provides an analysis of the polarity of SC divisions, in vivo, in intact zebrafish embryos.We showed that SCs divide parallel to the axons along the Posterior Lateral Line nerve (PLL). By analyzing the two mutants has and nok, we revealed that the apical polarity genes aPKC and pals1, are neither required for the migration and division of SCs, nor for their capacity to myelinate. By studying the cassiopeia mutant that shows defects in mitotic spindle, we revealed that the assembly of the mitotic spindle is essential for SC myelination.We have also analysed the role of rgs4 (regulator of G-protein Signaling 4) in PNS development. We generated a stable rgs4 mutant using the CRISPR/Cas9 technology. We showed that rgs4 plays an essentiel role in PLLg and motoneurons development by acting upstream of PI3K/Akt/mTOR pathway. Pharmacological analysis suggested a role for rgs4 in peripheral myelination, however, the rgs4 mutant do not show any myelin defects.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第19048号 / 農博第2126号 / 新制||農||1032(附属図書館) / 学位論文||H27||N4930(農学部図書室) / 31999 / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 植田 充美, 教授 植田 和光, 教授 小川 順 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Phosducin-like protein (PhLP) binds G-protein beta gamma subunits and is thought to assist in assembly of the G-protein beta gamma dimer. Phosphorylation of PhLP at serine residues 18-20 by the casein kinase 2 (CK2) appears to play an essential role in this process. PhLP has also been shown to interact with the chaperonin containing TCP-1 (CCT) atop its apical domain, not entering the substrate folding cavity. However, the physiological role of the PhLP-CCT interaction in G-protein beta gamma dimer formation remains unclear. This study addresses the mechanism of G-protein beta gamma assembly by exploring the specific roles of CCT and CK2 phosphorylation of PhLP in the assembly process. Both overexpressed and endogenous Gbeta were shown to co-immunoprecipitate with CCT to a similar extent as PhLP, indicating that CCT may be involved in the folding of Gbeta. In addition, Ggamma overexpression enhanced the binding of PhLP to CCT, suggesting the formation of a ternary PhLP-Gbeta-CCT complex. In contrast, overexpression of PhLP caused the release of G-beta from CCT. This release was blocked by a PhLP S18-20A variant that lacks the S18-20 CK2 phosphorylation site. PhLP S18-20A has been previously shown to negatively affect the G-protein beta gamma dimer formation, suggesting a correlation between PhLP-mediated release of Gbeta from CCT and G protein beta gamma assembly. Experiments investigating the role of Ggamma in this process show that Ggamma does not interact with CCT nor is it the essential factor in the release of Gbeta from CCT. A new model is therefore proposed for the G-protein beta gamma subunits' assembly involving the formation of a PhLP-Gbeta-CCT ternary complex followed by the release of a phosphorylated PhLP-Gbeta complex from CCT. In the PhLP-Gbeta complex, the Ggamma binding face of Gbeta is exposed, allowing for the formation of the G-protein beta gamma dimer.

G-protein coupled receptors (GPCRs) play a key role in a broad range of biological processes by binding to a wide variety of signaling molecules, which have resulted in 34% of all FDA-approved drugs which target GPCRs. The human genome encodes for approximately 800 GPCR members of which about 140 non-olfactory receptors remain orphans with an unknown function and endogenous ligand. Despite prolonged efforts to deorphanize the unresolved receptors, they remain orphans until this day. By studying scientific publications, this thesis has clarified the challenges with the deorphanization of GPCRs to explain why there are still so many orphan GPCRs when they have confirmed involvement in so many human disorders.

Specifically addressing cell surface molecules on cancer cells facilitates targeted cancer therapies that offer the potential to selectively destroy malignant cells, while sparing healthy tissue. Thus, undesired side-effects in tumor patients are highly reduced. Peptide-binding receptors are frequently overexpressed on cancer cells and therefore promising targets for selective tumor therapy. In this review, peptide-binding receptors for anti-cancer drug delivery are summarized with a focus on peptide ligands as delivery agents. In the first part, some of the most studied peptide-binding receptors are presented, and the ghrelin receptor and the Y1 receptor are introduced as more recent targets for cancer therapy. Furthermore, nonpeptidic small molecules for receptor targeting on cancer cells are outlined. In the second part, peptide conjugates for the delivery of therapeutic cargos in cancer therapy are described. The essential properties of receptor-targeting peptides are specified, and recent developments in the fields of classical peptide-drug conjugates with toxic agents, radiolabeled peptides for radionuclide therapy, and boronated peptides for boron neutron capture therapy are presented.

No description available.

付記する学位プログラム名: 充実した健康長寿社会を築く総合医療開発リーダー育成プログラム / 京都大学 / 新制・課程博士 / 博士(医学) / 甲第23789号 / 医博第4835号 / 新制||医||1057(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 渡邊 直樹, 教授 松田 道行, 教授 寺田 智祐 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Breast cancer is the second leading cause of death in United States. Using 2D-HPLE, a novel separation technology, G-protein coupled receptor-associated sorting protein 1(GASP-1) was identified in sera of patients with early stage cancer, while it could not be detected in sera from healthy individuals. This was the first indication that GASP-1 was positively correlated with breast cancer. However, the function of GASP-1 in breast cancer was unknown. In this study, I verified the 2D-HPLE results by quantifying the expression level of GASP-1 in sera and tissue specimens of cancer patients using specific antibodies against GASP-1. A GASP-1 specific ELISA was developed and used to quantify GASP-1 levels in cancer patient sera. Immunohistochemistry was performed to verify and localize GASP-1 expression in tumor. I also characterized the tumorigenic potential of GASP-1 andidentified the signaling pathways mediated by GASP-1 in breast cancer cells in vitro.GASP-1 expression levelsin MDA-MB-231 cells were modified by transfecting cells with anti-GASP-1 shRNA and over-expression plasmids. Stable cell lines were prepared and their tumorigenic potential was evaluated using cell proliferation, migration, and colony formation assays. These cells were analyzed for markers used to identify epithelial to mesenchymal transition (EMT) using RT-PCR and western blot. They were also analyzed for NFkappaB activity, src phosphorylation, and GPR30 expression. The results showed that GASP-1 was over-expressed in sera and tissue specimens of breast cancer patients and other cancer types including brain, lung, liver and pancreatic cancer and that it correlated with early stage disease. GASP-1 positively regulated migration, and is required for cell proliferation and colony formation. GASP-1 is also necessary for the expression of EMT marker slug, increases NFkappaB activity and GPR30 expression level, while decreases the inhibitory phospho-src Tyr 530. I conclude that GASP-1 is a nearly marker for multiple cancer types. GASP-1 promotes tumorigenesis in breast cancer, possibly through multiple cancer related signaling pathways. These findings may contribute to our understanding of the mechanism of breast cancer tumorigenesis and identify new biomarkers that can be used for diagnosis and therapy of cancer. / Biology

Chemokines are chemotactic cytokines which play an important role in the migration of immune cells to distant tissues or compartments within tissues. These proteins have also been demonstrated to play a major role in cancer metastasis. The C-C chemokine receptor type 7 (CCR7) is a member of the chemokine receptor family. CCR7 along with its ligands CCL19 and CCL21 plays an important role in innate immune response by trafficking of lymphocytes. In cancer, tumour cells expressing CCR7 migrate to lymphoid organs and thus disseminate to other organs. Neutralizing the interactions between CCL21/CCR7 would therefore be expected to inhibit the progression and metastasis of many different types of cancer to regional lymph nodes or distant organs. Our objective was to identify a potent small molecule antagonist of CCR7 as a prelude to the investigation of the role of this axis in cancer metastasis. In this study, we provided a brief description of chemokines and their role in health and disease with an emphasis on the CCR7/CCL19/CCL21 axis, as well as identification of a CCR7 antagonist “hit”. The potency of the CCR7 antagonist “hit” was optimised by synthesizing different CCR7 antagonist analogues. The “hit” optimization process has led to discover the most active compound amongst a series of different analogues which have the ability to bind and block CCR7 receptor. The efficacy of the most active compound and other analogues were evaluated in vitro using a calcium flux assay which is based on detecting fluorescent light emitted upon release of calcium ions. To identify a suitable cell line, which expresses CCR7 and capably respond to it, amongst a panel of cell lines for in vitro assessment of potency of synthesised compounds, we used Western blot assay and later by flow cytometry assay. The activity and selectivity of the most effective compound against CCR7 receptor was evaluated in vitro by other functional assays such as “configured agarose spot assay” and scratch assay. We first configured the existing under agarose assay to fulfil our requirements and then used it to assess activity and selectivity of compounds. The configured agarose spot assay also describes the application of the agarose spot for evaluation of cells chemotactic response to multiple chemokines under identical experiment conditions.

Introduction Adhesion GPCRs (aGPCRs) constitute the second largest family of the GPCR superfamily, and yet their properties are also the least understood. Growing research on the biological functions of aGPCRs suggest their implications in various (patho)physiological processes, such as cell migration, organ development and cancers. Moreover, due to the unique architecture of a large extracellular region (ECR) containing a plethora of adhesion motifs, aGPCRs are vital as a mechanosensor which transduces extracellular mechanical stimuli into intracellular signal transduction. One distinct feature of aGPCRs among the GPCR superfamily is the possession of a conserved extracellular fold termed GPCR autoproteolysis-inducing (GAIN) domain in perhaps all members within the class. The cleavage at the last loop of the GAIN domain leads to the formation of two non-covalently associated N- and C-terminal fragments (NTF/CTF). A peptide stretch in the start of the CTF acts as a tethered agonist (TA) which is responsible for at least part of the signaling volumes of an activated receptor. Despite the strict conservation of the GAIN domain and its importance in the activation mechanism of aGPCRs, some other fundamental properties of the receptors, with reference to GAIN domain cleavage, have not been rigorously analysed in a biological context. Thus, this study aims to: 1. Explore the structural and molecular determinants that affects GAIN domain cleavage; 2. Investigate the consequences of GAIN domain cleavage towards (i) surface trafficking, and (ii) phosphorylation of receptors. Results Abolishment of GAIN domain cleavage in Polycystin-1, the only other protein family possessing the GAIN domain, was found to eliminate its surface expression, which is a cause of polycystic kidney/liver disease. However, whether such relationship is also true for aGPCRs has not been systematically analysed. Therefore, the study started with profiling the kinetics of surface delivery of several members of aGPCRs. Mutations on the -2 or +1 residues of the GPCR proteolytic site (GPS) (thereby abolishing GAIN domain cleavage) affected the steady-state surface and total expressions of the receptors differently, and had variable effect towards different receptor members. However, the observations from steady-state kinetics are also a resultant output from numerous processes involved in proteostasis. To further dissect whether GPS mutations affect the surface trafficking of the receptors, a pulse-chase assay called the ‘Retention Upon Selective Hook’ (RUSH) assay was employed, wherein the synthesised receptor molecules conjugated to a streptavidin-binding peptide are withheld in the ER by the co-expressed, ER-resident streptavidin, and are only released upon the addition of biotin that outcompete the receptor-streptavidin binding, creating a synchronised transport. By adapting the RUSH assay on some aGPCR members, the attenuation of surface trafficking by GPS mutations has become more apparent. The tested receptors were found to have a deficit in the quantity of surface population, rather than a change in rate of trafficking, upon the introduction of GPS mutations. This implies that the cells may utilise GAIN domain cleavage as a quality checkpoint for ER exit of aGPCRs. As the GAIN domains of at least some aGPCRs were found to be cleaved before ER exit, and as the rate of surface delivery was generally not affected by GAIN domain cleavage, the influence of GAIN domain cleavage may arise earlier during the receptor maturation in the ER. However, while the mechanisms of GAIN domain cleavage have been elucidated previously, they rely heavily on purified domains. The fundamental questions of when exactly the GAIN domain is cleaved and what additional determinants apart from the GPS sequence contribute to GAIN domain cleavage during receptor biogenesis have still not been answered. In combination with molecular dynamics (MD) simulation studies on the GAIN domain of rat isoform of ADGRL1, F803 was found to be crucial in the proteolysis by forming an edge-π interaction with H836 (-2 position of the cleavage site), such that H836 is in close proximity to the hydroxyl group of T838 for the initiation of the nucleophilic attack. Reconstruction of the edge-π interaction into ADGRB3, a naturally uncleavable receptor, partially reinstates its GAIN domain cleavage; but similar reintroduction on ADGRB2 has no effect on restoring the proteolysis. Nonetheless, this observation highlights the vitality of a proper folding of the GAIN domain, specifically the microenvironment of the cleavage site, in assisting in cleavage. The study continued with a systematic series of experiments that ultimately discover the roles of the CTF towards GAIN domain cleavage of aGPCRs. Firstly, to mimic the biogenesis of the receptor, the seven transmembrane (7TM) region of ADGRE2 (E2) was stepwisely truncated and then analysed for GAIN domain cleavage. It was observed that the extent of GAIN domain cleavage increases when the ECR of E2 precedes with more number of TMs. The proteolysis occurs, although less efficiently, as early as the first TM is synthesised. Interestingly, GAIN domain cleavage is unaffected when the TM region of the E2-1TM mutant was replaced by other single-pass TM, and whether it is trafficked to the surface or held in the ER, while the proteolysis of TM-less ECR mutants is largely impeded. Based on this observation, the ECR and the TM region was spaced either by a fluorophore moiety or a variable number of helical turns. Remarkably, the extent of GAIN domain cleavage of all tested receptors declined upon the increase in displacement with the lumenal side of the ER membrane, defining the importance of membrane proximity in the completion of proteolysis during the maturation of GAIN domain. In that, a new model of GAIN domain cleavage during biogenesis has been proposed, with appreciation of the GAIN domain as part of a higher-order stuctural organisation rather than an independent domain. A physiological extent of GAIN domain cleavage does not only require the folding of the GAIN domain, but also the membrane tethering property of the CTF, allowing a partial cleavage as little as one TM is generated, and a dynamic stability provided by the full CTF. In some aGPCRs, the contributions from CTF are more significant than the autonomous GAIN domain folding. The findings implicate more complex requirements for GAIN domain cleavage in a biological context, and hence supporting a possibility that GAIN domain cleavage is the rate-determining step for ER exit of the receptor, leading to the observations obtained in the kinetic study. Phosphorylation of L3 by PKC activated by distant signaling cascade(s) The last part of the study focused on characterising the mechanism of phosphorylation of ADGRL3 (L3) at Thr1140 (pT1140), which is a poorly explored field of aGPCRs. It was made possible by exploiting a phosphospecific antibody developed in collaboration. Coincidently, pT1140 was not dependent on the examined GPCR properties of the receptor, such as G protein coupling, dependence of the TA, and GRK-mediated phosphorylation. Instead, by series of pharmacological inhibitions, it was discovered that pT1140 originates from the action of novel PKCs (nPKCs). Co-expression of L3 and dominant-negative mutants or the catalytic domains of individual members of nPKCs reveals that PKC acts as a master regulator of the phosphorylation event, by directly phosphorylating the receptor and priming other members of the nPKCs for pT1140. Finally, possible origins of the PKC activation were explored. It was found that the stimulation of PKC occurs via actin disassembly, which can act downstreams of VEGF-A/VEGFR2 signaling, although the physiological relevance is still yet to be deciphered. Nonetheless, the observations opened up new directions of research in the aspect of crosstalks between different signaling cascades and the possible modulations of the signaling fidelity of aGPCRs. Additionally, the complexity of aGPCR signaling has been clearly demonstrated. Conclusion This study has further defined the importance of GAIN domain cleavage for surface trafficking of aGPCRs, a process crucial for extracellular interactions. Moreover, a novel mechanistic model of GAIN domain cleavage in relevance to biogenesis and maturation of the receptors has been postulated. Characterisation of a site-specific phosphorylation mechanism of L3 has illustrated the potential of complex interactions of aGPCRs with other signaling pathways in cells. The results collectively shed light on the structure-function relationship of aGPCRs, and pave ways for numerous potential areas for explorations in the future.