京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21993号 / 医博第4507号 / 新制||医||1037(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 長船 健二, 教授 竹内 理, 教授 横出 正之 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・論文博士 / 博士(生命科学) / 乙第13281号 / 論生博第19号 / 新制||生||55(附属図書館) / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 井垣 達吏, 教授 垣塚 彰, 教授 藤田 尚志 / 学位規則第4条第2項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

Innate leukocytes manifest dynamic and distinct inflammatory responses upon challenges with rising dosages of pathogen associated molecular pattern molecules (PAMPs) such as lipopolysaccharide (LPS). To differentiate signal strengths, innate leukocytes may utilize distinct intra-cellular signaling circuitries modulated by adaptor molecules. Toll-interacting protein (Tollip) is one of the critical adaptor molecules in Toll-like receptor 4 (TLR4) signaling and potentially playing key roles in modulating the dynamic adaptation of innate leukocytes to varying dosages of external stimulants. While Tollip may serve as a negative regulator of NFkB signaling pathway in cells challenged with higher dosages of LPS, it acts as a positive regulator for low-grade chronic inflammation in leukocytes programmed by subclinical low-dosages of LPS. We aim to show recent progress in our understanding of complex innate leukocyte dynamics and its relevance in the pathogenesis of resolving versus non-resolving chronic inflammatory diseases. / Ph. D. / White blood cells, or leukocytes, have a dynamic inflammatory response to rising doses of bacterial cell wall components. Lipopolysaccharide (LPS) is a ubiquitous component of gram negative bacteria that is recognized by Toll-like receptor 4 (TLR4) and can shed into the blood stream, causing low-grade non-resolving inflammation. In order to differentiate between varying signal strengths of LPS, leukocytes utilize signaling within the cell, which is often regulated by adaptor molecules. Toll-interacting protein (Tollip) is one of the critical adaptor molecules in TLR4 signaling and potentially plays key roles in modulating the dynamic adaptation of innate leukocytes to varying dosages of external stimulants. While Tollip serves to inhibit the pro-inflammatory NFκB signaling in cells challenged with higher dosages of LPS, it acts to increase low-grade chronic inflammation in leukocytes programmed by low-dosages of LPS. In these studies we show recent progress in elucidating the mechanism for Tollip involvement in low-grade non-resolving inflammation in mouse fibroblast cells.

This thesis describes the cloning and characterisation of a novel human gene and its protein products, which have been designated SAPK- and Ras-interacting protein (SinRIP). SinRIP shares identity with JC310, a partial human cDNA that was previously identified a candidate Ras-inhibitor (Colicelli et al., 1991, Proc Natl Acad Sci USA 88, p. 2913). In this study, it was shown that SinRIP is a member of an orthologous family of proteins that is conserved from yeast to mammals and contains proteins involved in Ras- and SAPK-mediated signalling pathways. Comparison of this family of proteins showed that human SinRIP contains a potential Ras-binding domain (RBD; residues 279-354), a PH-like domain (PHL; 376-487), and a highly conserved novel region designated the CRIM (134-265). Several other potential targeting sites, such as nuclear localisation signals and target sites for kinases, were identified within the SinRIP sequence. The human SinRIP gene is unusually large (>280 kbp) and is located on chromosome 9 at 9q34. SinRIP mRNA was detected in a wide variety of tissue-types and cell lines by RT-PCR, and the SinRIP sequences in the EST database were derived from an diverse array of tissues, suggesting a widespread or ubiquitous expression. Northern blot analysis revealed the highest levels in skeletal muscle and heart tissue. However, the steady-state levels of SinRIP mRNA vary greatly from cell to cell, and SinRIP expression is likely to be regulated at multiple post-transcriptional levels. It was shown that SinRIP mRNA is likely to be translated inefficiently by the normal cap-scanning mechanism, due to the presence of a GC-rich and structured 5’-UTR, which also contains upstream ORFs. Alternative polyadenylation signals in the SinRIP 3’-UTR can be used, resulting in the expression of short and long SinRIP mRNA isoforms. Several potential A/T-rich regulatory elements were also identified in SinRIP mRNA, which may target specific SinRIP mRNA isoforms for rapid degradation. Importantly, it was shown that SinRIP mRNA is alternatively spliced, resulting in the production of distinct SinRIP protein isoforms. Three isoforms, SinRIP2-4, were definitively identified by RT-PCR and full-length cloning. The SinRIP isoforms contain deletions in conserved regions, and are likely to have biochemical characteristics that are different to full-length SinRIP1. SinRIP2 is C-terminally truncated and lacks the PHL domain and part of the RBD, and relatively high levels of SinRIP2 expression arelikely to occur in kidneys. The RBD is disrupted in SinRIP3, but all other domains are intact, and RT-PCR analyses suggest that SinRIP3 is present in some cells at levels comparable to SinRIP1. A rabbit polyclonal antiserum against SinRIP was generated and detected endogenous SinRIP proteins. Using the anti-SinRIP antibody in immunoblots, multiple SinRIP isoforms were observed in most cell types. SinRIP1 and another endogenous SinRIP protein, likely to be SinRIP3, were detected in most cell lines, and appear to be are the major SinRIP proteins expressed in most cells. The subcellular localisation of both recombinant and endogenous SinRIP proteins was investigated by immunofluorescence assays and biochemical fractionation. Recombinant SinRIP1 protein was found in the cytoplasm and associated with the plasma membrane. In contrast, the SinRIP2 protein was predominantly nuclear, with only low-level cytoplasmic staining observed. The endogenous SinRIP proteins, likely to comprise these and other SinRIP isoforms, were found in both the nucleus and cytoplasm. SinRIP1 interacted with GTP-bound (active) Ras, but not GDP-bound (inactive) Ras, in an in vitro assay, and also co-localised with activated H- and K-Ras in cells. The binding profile observed is typical of Ras-effectors, and SinRIP did not inhibit signalling by the Ras proteins, suggesting that it is not likely to be a Ras-inhibitor. It was also shown that SinRIP1 and SinRIP2 both interact and colocalise with c-Jun NH2- terminal kinase (JNK). Both SinRIP proteins were able to recruit JNK to their respective sub-cellular compartments. These interactions suggest an adaptor role for SinRIP in the Ras and/or JNK pathways. In addition, Sam68 was isolated as a SinRIP-binding protein in a yeast two-hybrid screen. Sam68 was shown to colocalise with SinRIP2 and endogenous SinRIP proteins, but not SinRIP1. Further colocalisation studies showed that endogenous SinRIP proteins localise in nuclear structures that may be associated with pre-mRNA splicing. Likely functions for SinRIP, as indicated by experimental results and studies of the orthologues of SinRIP in other species, are discussed.

Le protéasome est responsable de la dégradation régulée d'une majeure partie des protéines intracellulaires. Cette machinerie multimoléculaire est composée d'un cœur catalytique, le protéasome 20S, qui peut être activé par plusieurs types de protéines régulatrices, en particulier la particule régulatrice 19S ou PA700, les complexes heptamériques formés par les membres de la famille 11S (ou PA28) et PA200. Au cours de ce travail, nous nous sommes focalisés sur PA28gamma, un régulateur nucléaire du protéasome, qui active la dégradation de plusieurs substrats par le protéasome 20S de façon indépendante de l'ubiquitine et de l'ATP. Malgré de multiples études montrant l'implication de PA28gamma dans de nombreux processus cellulaires essentiels tels que le cycle cellulaire, la prolifération, l'apoptose, l'architecture nucléaire, la dynamique de la chromatine, les infections virales et la réponse au stress, ses fonctions exactes ne sont pas encore comprises. De plus, les mécanismes impliqués dans la régulation de l'activité de PA28gamma et de son association avec le protéasome 20S restent mystérieux. Une analyse SILAC des partenaires d'interaction de PA28gamma endogène a révélé l'existence d'un nouveau facteur, non caractérisé, que nous avons appelé PIP30 (PA28gamma Interacting Protein 30 kDa). Le gène PIP30 contient un domaine très conservé chez les Eucaryotes. Nous avons produit et purifié la protéine PIP30 recombinante et montré qu'elle est faiblement structurée, malgré le fait qu'elle puisse se dimériser. Nous avons confirmé, aussi bien in vitro qu'in cellulo, que PIP30 interagit directement et spécifiquement avec PA28gamma. En analysant la co-immunoprécipitation de PA28gamma avec différents mutants tronqués de GFP-PIP30, nous avons pu identifier la séquence de PIP30 responsable de l'interaction avec PA28gamma dans sa partie C-terminale. Nous essayons maintenant d'identifier la séquence de PA28gamma impliquée dans la liaison de PIP30 et de cristalliser le complexe PA28gamma/PIP30. L'élaboration d'un anticorps anti-PIP30 « maison » nous a permis de montrer que PIP30 est une protéine nucléaire stable. Son niveau d'expression diminue en réponse à la déplétion de PA28gamma, ce qui suggère que PIP30 est stabilisée par son interaction avec PA28gamma in cellulo. Nous avons démontré in vitro que PIP30 inhibe partiellement l'activation médiée par PA28gamma des activités de type chymotrypsine et caspase, mais pas trypsine, du protéasome. Cependant, nous avons montré, par une approche ELISA, que PIP30 n'affecte pas la liaison de PA28gamma au protéasome 20S. Par ailleurs, nous avons testé l'effet de PIP30 sur la dégradation de p21 par le complexe PA28gamma/protéasome 20S et observé que PIP30 augmente la vitesse de dégradation de p21 dans ce test. Nos tentatives pour élucider la fonction exacte de PIP30 in cellulo n'ont jusqu'ici pas abouti à une conclusion convaincante. L'ensemble de ces résultats suggère que PIP30 pourrait être impliqué dans le recrutement sélectif des substrats de PA28gamma et/ou dans la modulation de l'activation du protéasome par PA28gamma. / The proteasome is responsible for the regulated degradation of most intracellular proteins. This multi-subunit machinery is composed of a common catalytic core, the 20S proteasome, which can be activated by various types of regulators, notably the 19S regulatory particle or PA700, the heptameric complexes formed by the members of the 11S (or PA28) family and PA200. This work has been focused on PA28gamma, a nuclear regulator of the proteasome, which has been shown to activate degradation of several proteasomal substrates in an ATP- and ubiquitin- independent manner. Despite many evidences revealing the involvement of PA28gamma in many essential cellular processes, such as cell cycle progression, proliferation, apoptosis, nuclear architecture, chromatin dynamics, viral infection and stress response, its exact function(s) remain to be understood. In addition, how PA28gamma activity and association to the 20S proteasome are regulated is completely unclear. A SILAC-based analysis of endogenous PA28gamma interaction partners revealed the existence of a novel, completely uncharacterized protein, which we called PIP30 (PA28gamma Interacting Protein 30 kDa). Evolutionary analysis indicates that PIP30 gene contains a domain highly conserved in Eukaryotes, without any alternative splicing or gene duplication evidences. We produced and purified the recombinant PIP30 protein and showed that it is poorly structured, although it is able to make dimers. We confirmed both in vitro and in cellulo that PIP30 directly and specifically interacts with PA28gamma. By analyzing the co-immunoprecipitation of PA28gamma with various GFP-PIP30 truncation mutants, we identified the sequence of PIP30 responsible for PA28gamma binding in its C-terminal part. Ongoing analyses now focus on the identification of PIP30 binding motif on PA28gamma sequence and the crystallization of the PA28gamma-PIP30 complex. Using homemade anti-PIP30 antibodies, we showed that PIP30 is a stable nuclear protein. Its expression level is decreased in response to PA28gamma depletion, suggesting that it is stabilized by its interaction with PA28gamma in cellulo. We demonstrated in vitro that PIP30 partially inhibits PA28gamma-mediated activation of the chymotrypsin- and caspase-, but not the trypsin-like, activities of the proteasome. However, we showed by an ELISA-based approach that PIP30 does not affect PA28gamma binding to 20S. Considering the limitations of probing proteasome activity with small fluorogenic substrates, we tested the effect of PIP30 on the PA28gamma-dependent proteasomal degradation of in vitro translated p21, a known protein substrate of PA28gamma. We unexpectedly found that PIP30 enhanced the rate of p21 degradation. Our attempts to elucidate the exact functions of PIP30 in cellulo were unsuccessful so far. Altogether, our results suggest that PIP30 could be involved in the selective recruitment of PA28gamma protein substrates and/or modulate PA28gamma-mediated proteasome activation.

Kinesin-1 transports various cargos along the axon, while the light chain subunits play a role in selecting the types of cargos to transport. However, the mechanisms of cargo recognition and interaction have yet to be characterized. Both c-Jun kinase-interacting protein-1 (JIP1) and alcadein-1 (ALC1) are kinesin-1 cargos and compete with each other for the axonal transport machinery. I identified two polar patches of KLC1 that play a role in the interactions with JIP1 and ALC1, respectively. The main components of these two polar patches are asparagine “clamps” surrounded by positively charged lysines. Consistent with this finding, negatively charged residues of JIP1 and ALC1 are required to interact with KLC1. By structural modeling, I narrowed down the possible key residues of KLC1 that are required for interaction with c-Jun kinase interacting protein-3 (JIP3). Together, these findings reveal the versatility of KLC in the mode of interaction with many different cargos.

Kinesin-1 transports various cargos along the axon, while the light chain subunits play a role in selecting the types of cargos to transport. However, the mechanisms of cargo recognition and interaction have yet to be characterized. Both c-Jun kinase-interacting protein-1 (JIP1) and alcadein-1 (ALC1) are kinesin-1 cargos and compete with each other for the axonal transport machinery. I identified two polar patches of KLC1 that play a role in the interactions with JIP1 and ALC1, respectively. The main components of these two polar patches are asparagine “clamps” surrounded by positively charged lysines. Consistent with this finding, negatively charged residues of JIP1 and ALC1 are required to interact with KLC1. By structural modeling, I narrowed down the possible key residues of KLC1 that are required for interaction with c-Jun kinase interacting protein-3 (JIP3). Together, these findings reveal the versatility of KLC in the mode of interaction with many different cargos.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20616号 / 医博第4265号 / 新制||医||1023(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 長船 健二, 教授 渡邊 直樹, 教授 岩田 想 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Receptor-Interacting Serine/Threonine Protein Kinase 2 (RIP2) is a kinase which modulates signaling downstream of the bacterial peptidoglycan sensors NOD1 and NOD2. It is known that activation of RIP2 by engaging NOD receptors increases the production of pro-inflammatory cytokine and lipid mediators. We have some data indicating that RIP2 may also be involved in specialized pro-resolution lipid mediator (SPM) production. However, the molecular mechanisms by which RIP2 is involved in lipid mediator biosynthesis, are currently unknown. Understanding this process may have significant implications for RIP2-targeted therapies, which may not only inhibit pro-inflammatory cytokine and lipid mediator production but may also disrupt SPM production and resolution programs. This thesis aims to demonstrate that RIP2 is involved in promoting the activation of ALOX5 in a transient overexpression setting but also in an endogenous setting using relevant bacterial stimuli. These aims were accomplished through the optimization of a fluorescent assay to assess ALOX5 enzymatic activity, by optimization of ALOX5 enzyme purification and through molecular cloning of ALOX5 into a retroviral vector followed by viral transduction of the THP-1 human monocytic cell line. We find that co-expression of RIP2 with ALOX5 significantly enhances the enzymatic activity of ALOX5. We have successfully cloned NTAP-tagged ALOX5 into the pBABE retroviral vector and are currently selecting transduced cells so that we might test if this effect also occurs endogenously. Understanding the mechanisms underlying the production and regulation of SPMs would provide greater insight into potential new therapeutic approaches to promote resolution in chronic inflammatory diseases.

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