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Identification and characterization of CEP131 as a novel BBSome interacting proteinChamling, Xitiz 01 May 2014 (has links)
Bardet-Biedl syndrome (BBS) is a pleiotropic and genetically heterogeneous disorder, and a well-known ciliopathy. Nineteen different genes have been reported for BBS, mutations in which cause characteristic phenotypes including retinal degeneration, obesity, polydactyly, renal abnormalities, hypogenitalism and cognitive impairment. Protein products of eleven BBS genes are part of two major complexes: the BBSome complex and a CCT/CTRiC/BBS complex. The CCT/CTRiC/BBS complex assists in the formation of the BBSome complex, which in turn traffics numerous receptor proteins to the cilia. However, the precise mechanism by which BBSome ciliary trafficking activity is regulated is not fully understood. In fact, a complete picture of the cellular functions of BBS proteins is still missing, and gaps remain in our understanding of the pleiotropy and heterogeneity of the disease. With the aim of bridging those gaps, this thesis project was designed to identify tissue specific cargoes of the BBSome and to characterize their BBS-related functions. To this end, we generated a transgenic LAP-BBS4 mouse, which expresses the transgene in various tissues including brain, eye, testis, heart, kidney, and adipose tissue. We found that despite tissue specific variable expression, LAP-BBS4 was able to complement the deficiency of Bbs4 and rescue all the BBS phenotypes in the Bbs4 null mice. The finding provides an encouraging prospective for gene therapy for BBS related phenotypes and potentially for other ciliopathies. We also utilized the transgenic mice to search for tissue specific BBSome cargo proteins and identified CEP131 as a novel BBSome interacting protein. Using in vitro cell culture models we show that CEP131 interacts with the BBSome through BBS4. CEP131 is not involved in BBSome assembly, but accumulation of the BBSome in cilia is enhanced upon CEP131 depletion. Our in vitro data implicate CEP131 as a negative regulator of ciliary BBSome trafficking. Finally, we show that cep131 knockdown in zebrafish embryos results in typical BBS phenotypes including Kupffer's vesicle abnormalities and melanosome transport delay. This finding confirms the association of CEP131 with the BBS pathway. Overall, the work performed for this thesis provides further insight into the regulation of BBSome ciliary trafficking and suggests CEP131 as a BBS candidate gene.
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Architecture of the BBSome and its role in ciliary protein trafficking / BBSomeの構築様式と繊毛内タンパク質輸送における役割Nozaki, Shohei 25 March 2019 (has links)
京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第21709号 / 薬科博第100号 / 新制||薬科||11(附属図書館) / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 中山 和久, 教授 竹島 浩, 教授 土居 雅夫 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM
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Structural Analysis of Cell Signaling ComplexesAoba, Takuma 01 December 2016 (has links)
Bardet-Biedl syndrome (BBS) is a rare genetic disease that causes retinal degradation, obesity, kidney dysfunction, polydactyly, and other cilium-related disorders. To date, more than 20 BBS genes, whose mutants cause BBS phenotypes, have been identified, and eight of those (BBS1-2, 4-5, 7-9, and 18) are known to form the BBSome complex. Recent studies have revealed that the BBSome is closely involved in the trafficking of signaling proteins in the primary cilium. Mutations in BBS genes are highly pathogenic because trafficking in the primary cilium is not fully functional when BBS mutations impair assembly of the BBSome. However, the functional links between onset of BBS and BBSome assembly are not well understood. To address this gap in knowledge, we examined the structure of a BBSome assembly intermediate, the BBSome core complex (BBS2, 7, and 9). We employed a combination of chemical crosslinking coupled with mass spectrometry (XL-MS) and electron microscopy (EM) to determine the structure. We applied this structural information to BBS mutations in the core complex to understand how these mutations might cause the disease. These results provide the first structural model of the BBSome core complex and give insight into the molecular basis of Bardet-Biedl syndrome. We have also investigated the mechanism of assembly of the two mTOR kinase complexes (mTORC1 and 2). mTOR is a master regulator of cell metabolism, growth and proliferation. As such, mTOR is a high-value drug target. We investigated the mechanism of assembly of these mTOR complexes and found that the cytosolic chaperonin CCT contributes to mTOR signaling by assisting in the folding of mLST8 and Raptor, components of mTORC1 and mTORC2. To understand the function of CCT in mTOR complex assembly at the molecular level, we have isolated the mLST8-CCT complex and performed a structural analysis using chemical cross-linking couple with mass spectrometry (XL-MS) and cryogenic EM. We found that mLST8 binds CCT deep in its folding cavity, making specific contacts with the CCTα and γ subunits and forming a near-native β-propeller conformation. This information can be used to develop new therapeutics that regulate mTOR activity by controlling mTOR complex assembly.
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Molecular basis of insulin resistance in Bardet Biedl syndromeStarks, Rachel Diaz 01 May 2015 (has links)
Bardet Biedl Syndrome (BBS) displays heterogeneity in the genes involved and clinical features. Mutations in 19 genes have been associated with BBS. Eight BBS proteins (BBS1, 2, 4, 5, 7, 8, 9 and 18) form the BBSome. Assembly of the BBSome is mediated by three BBS proteins (BBS6, 10, 12) in a complex with the CCT/Tric chaperonins. The BBSome is involved in formation and maintenance of primary cilia and vesicle trafficking. The clinical features of BBS include obesity, degenerative retinopathy, polydactyly, renal dysfunction, hypogonadism, and learning disability. Diabetes mellitus is commonly associated with BBS, but the mechanisms remain unknown.
Our objective was to understand the molecular mechanism of BBS-associated diabetes. The role of BBS in insulin receptor (IR) signaling in Bbs4-/-mice was tested by preventing obesity using calorie restriction. These studies demonstrate the genetic defect in BBS directly contributes to the diabetes phenotype independently from the obesity phenotype.
Emerging evidence implicating neuronal mechanisms in various BBS phenotypes led us to test the possibility that loss of Bbs1 in the central nervous system (CNS) disrupts glucose homeostasis. We found that deletion of the Bbs1 gene throughout the CNS or in specific hypothalamic neurons leads to hyperglycemia, glucose intolerance and insulin resistance. Our data demonstrate the critical role of neuronal Bbs1 in the regulation of glucose in an insulin-independent manner.
Finally, the IR was found to interact with BBS proteins. The loss of BBSome proteins leads to a specific reduction in the amount of IR at the cell surface. The results demonstrate that BBSome proteins are required to maintain adequate levels of IR at the cell surface. The role of BBS proteins in transporting IR has not been previously described. Loss of the BBSome appears to be a novel mechanism of insulin resistance.
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The Role of the Cytosolic Chaperonin CCT in Folding β-Propeller ProteinsLudlam, William Grant 14 June 2021 (has links)
Many Proteins require the aid of molecular chaperones to achieve a stable folding state and avoid misfolding pathologies. A major eukaryotic chaperone is the cytosolic chaperonin CCT. While CCT is known to fold a significant portion of all cytosolic proteins, there is no general model for the mechanism CCT uses to fold substrate proteins. One class of proteins that CCT is known to fold are β-propeller containing proteins. Here, we present structural and biochemical data on the processes that CCT uses to fold three distinct β-propeller proteins: the G-protein Beta 5 (Gβ5) subunit of the Gβ5-RGS complex, mLST8 of the mTOR complexes, and BBS2, 7, and 9 of the BBSome. We also explore the mechanisms by which these proteins are assembled into their respective signaling complexes after being folded by CCT. We found that each CCT substrate follows a unique folding trajectory and posit that the major determinants underlying each trajectory are governed by interactions between the substrate and CCT and interactions with downstream binding partners.
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Études fonctionnelles de deux nouvelles protéines centrosomales, NPHP5 et Cep76, et leurs implications dans les maladies humainesBarbelanne, Marine 08 1900 (has links)
Les centrosomes sont de petits organites qui régulent divers processus cellulaires
comme la polarité ou la mitose dans les cellules de mammifères. Ils sont composés de deux
centrioles entourés par une matrice péricentriolaire. Ces centrosomes sont les principaux
centres organisateurs de microtubules. De plus, ils favorisent la formation de cils, des
protubérances sur la surface des cellules quiescentes qui sont critiques pour la transduction du
signal. Une grande variété de maladies humaines telles que les cancers ou les ciliopathies sont
liées à un mauvais fonctionnement des centrosomes et des cils. C’est pourquoi le but de mes
projets de recherche est de comprendre les mécanismes nécessaires à la biogénèse et au
fonctionnement des centrosomes et des cils.
Tout d'abord, j’ai caractérisé une nouvelle protéine centrosomale nommée
nephrocystine - 5 (NPHP5). Cette protéine est localisée dans les cellules en interphase au
niveau de la région distale des centrioles. Sa déplétion inhibe la migration des centrosomes à
la surface cellulaire lors de l’étape précoce de la formation des cils. NPHP5 interagit avec la
protéine CEP290 via sa région C-terminale qui est essentielle pour la ciliogenèse. Elle
interagit également avec la calmoduline ce qui empêche son auto-agrégation. J’ai démontré
que les domaines de liaison de NHPH5 à CEP290 et à la calmoduline, ainsi que son domaine
de localisation centrosomale sont séparables. De plus, j’ai démontré que les protéines NPHP5
présentant des mutations pathogènes ne peuvent plus interagir avec CEP290 et ne sont plus
localisées aux centrosomes, rendant ainsi ces protéines non fonctionnelles. Enfin, en utilisant
une approche pharmacologique pour moduler les événements en aval dans la voie
ciliogénique, j’ai montré que la formation des cils peut être restaurée même en absence de
NPHP5.
D’autre part, j’ai étudié le rôle de NPHP5 dans l'assemblage et le trafic du complexe
BBSome dans le cil. Le BBSome est composé de huit sous-unités différentes qui s’assemblent
en un complexe fonctionnel dont on sait peu de chose sur la régulation spatiotemporelle de son
processus d'assemblage. J’ai précédemment montré que NPHP5 favorisait la formation des cils
et que son dysfonctionnement contribuait au développement de néphronophtise (NPHP).
Bien que la NPHP et le syndrome de Bardet-Biedl (BBS) soient des ciliopathies qui partagent
des caractéristiques cliniques communes, la base moléculaire de ces ressemblances
phénotypiques n’est pas comprise. J’ai constaté que NPHP5, localisé à la base du cil, contient
deux sites de liaison distincts pour le BBSome. De plus, j’ai démontré que NPHP5 et son
partenaire CEP290 interagissent de façon dynamique avec le BBSome pendant la transition de
la prolifération à la quiescence. La déplétion de NPHP5 ou CEP290 conduit à la dissociation
d’au moins deux sous-unités du BBSome formant alors un sous-complexe dont la capacité de
migration dans le cil n’est pas compromise. J’ai montré que le transport des cargos vers le
compartiment ciliaire par ce sous-complexe n’est que partiellement altéré.
Enfin, j’ai également concentré mes recherches sur une autre protéine centrosomale
peu caractérisée. La protéine centrosomale de 76 kDa (Cep76) a été précédemment impliquée
dans le maintien d’une duplication unique des centrioles par cycle cellulaire, et dans une
interaction avec la kinase cycline-dépendante 2 (CDK2). Cep76 est préférentiellement
phosphorylée par le complexe cycline A/CDK2 sur le site unique S83. Cet événement est
essentiel pour supprimer l'amplification des centrioles en phase S. J’ai démontré que Cep76
inhibe cette amplification en bloquant la phosphorylation de Plk1 au niveau des centrosomes.
D’autre part, Cep76 peut être acétylée au site K279 en phase G2, ce qui régule négativement
son activité et sa phosphorylation sur le site S83.
Ces études permettent d'améliorer notre compréhension de la biologie des
centrosomes et des cils et pourraient conduire au développement de nouvelles applications
diagnostiques et thérapeutiques. / Centrosomes are small organelles that regulate diverse cellular processes such as
polarity or mitosis in mammalian cells. They are composed of two centrioles surrounded by a
pericentriolar matrix. These centrosomes are the major microtubule organizing centers.
Moreover, they promote the formation of cilia, protrusions on the surface of quiescent cells
that are critical for signal transduction. A wide variety of human diseases such as cancers or
ciliopathies are linked to a malfunction of centrosomes and cilia. Therefore the aim of my
research is to understand the mechanisms necessary for the biogenesis and function of
centrosomes and cilia.
First, I have characterized a novel centrosomal protein called nephrocystin - 5
(NPHP5). This protein is localized, in interphase cells, in the distal region of centrioles. Its
depletion inhibits the migration of centrosomes to the cell surface during the early stage of
cilia formation. NPHP5 interacts with CEP290 via its C-terminal region that is essential for
ciliogenesis. It also interacts with calmodulin, which prevents its self-aggregation. I have
demonstrated that the Cep290- and CaM-binding domains as well as the centrosomal
localization domain of NPHP5 are separable. Moreover, I have shown that NPHP5 proteins
with pathogenic mutations can no longer interact with CEP290 and are not localized to
centrosomes, rendering these proteins non-functional. Finally, using a pharmacological
approach to modulate the downstream events in the ciliogenic pathway, I showed that cilia
formation can be restored even without NPHP5.
On the other hand, I studied the role of NPHP5 in the assembly and trafficking of the
BBSome into the cilium. The BBSome consists of eight different subunits that assemble into a
functional complex of which little is known about the spatiotemporal regulation of its
assembly process. I have previously shown that NPHP5 favored the formation of cilia and its
dysfunction contributes to the development of nephronophthisis (NPHP). Although the NPHP
and BBS syndrome (BBS) are ciliopathies that share common clinical features, molecular
basis of these phenotypic similarities is not understood. I found that NPHP5, located at the
base of the cilium, contains two separate binding sites for BBSome.
Furthermore, I demonstrated that NPHP5 and his partner CEP290 interact dynamically with
the BBSome during the transition from quiescence to proliferation. Depletion NPHP5 or
CEP290 leads to the dissociation of at least two subunits of BBSome forming a sub-complex
that can still traffic into the cilium. I have shown that the transport of cargo to the ciliary
compartment through this sub-complex is only partially altered.
Finally, I have also focused my research on another centrosomal protein poorly
characterized. The centrosomal protein of 76 kDa (Cep76) was previously involved in the
maintenance of a single duplication of centrioles per cell cycle, and interacts with the cyclindependent
kinase 2 (CDK2). Cep76 is preferentially phosphorylated by cyclin A/CDK2 on the
single site S83. This event is essential to suppress centrioles amplification in S phase. I have
demonstrated that Cep76 inhibits amplification by blocking the phosphorylation of Plk1 at the
centrosome. Moreover, Cep76 can be acetylated at the K279 site in G2 phase, which
negatively regulates its activity and phosphorylation on the site S83.
These studies will improve our understanding of the biology of centrosomes and cilia
and could lead to development of new diagnostic and therapeutic applications.
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Role proteinového komplexu BBS v T lymfocytech / Role of Bardet-Biedl syndrome (BBS) protein complex in T cellsNiederlová, Veronika January 2018 (has links)
BBSome is a protein complex crucial for trafficking of specific cargoes to the primary cilium. Although primary cilia are typically not present in cells of haematopoietic origin, such as T cells, recent research has revealed striking parallels between the primary cilium and the immunological synapse. Amongst other similarities, both structures are supposed to use the same transport machinery involving Rab8 and IFT20, the close interaction partners of BBSome. The first goal of this thesis was to investigate the role of BBSome in the biology of T cells. Using RT-qPCR, we have shown that BBSome subunits are expressed in lymphoid tissues and T cells. Studies of localization of BBSome subunits in Jurkat cell line and primary OT-I T cells revealed that the subunits have distinct localization patterns with BBS4 localizing to the centrosome and BBS1, BBS5, and BBip10 having dispersed localization. After the contact with an antigen presenting cell, BBS4 re-localizes to the immunological synapse. Mutations in BBSome encoding genes cause Bardet-Biedl syndrome (BBS), a rare ciliopathy presenting with multiorganic symptoms. The second goal of this thesis was to examine the associations between BBS and the immune system. Examination of medical records of more than 450 BBS patients revealed that autoimmune...
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