Oomycetes, such as Phytophthora sojae, are plant pathogens that employ protein effectors that enter host cells to facilitate infection. Plants may overcome infection by recognizing pathogen effectors via intracellular receptors (R proteins) that form part of their defense system. Entry of some effector proteins into plant cells is mediated by conserved RxLR motifs in the effectors and phosphoinositides (PIPs) resident in the host plasma membrane such as phosphatidylinositol 3-phosphate (PtdIns(3)P). Recent reports differ regarding the regions on RxLR effector proteins involved in PIP recognition. To clarify these differences, I have structurally and functionally characterized the P. sojae effector, avirulence homolog-5 (Avh5). Using NMR spectroscopy, I demonstrate that Avh5 is helical in nature with a long N-terminal disordered region. Heteronuclear single quantum coherence titrations of Avh5 with the PtdIns(3)P head group, inositol 1,3-bisphosphate (Ins(1,3)P2), allowed us to identify a C-terminal lysine-rich helical region (helix 2) as the principal lipid-binding site in the protein, with the N-terminal RxLR (RFLR) motif playing a more minor role. Furthermore, mutations in the RFLR motif slightly affected PtdIns(3)P binding, while mutations in the basic helix almost abolished it. Avh5 exhibited moderate affinity for PtdIns(3)P, which increased the thermal stability of the protein. Mutations in the RFLR motif or in the basic region of Avh5 both significantly reduced protein entry into plant and human cells. Both regions independently mediated cell entry via a PtdIns(3)P-dependent mechanism. My findings support a model in which Avh5 transiently interacts with PtdIns(3)P by electrostatic interactions mainly through its positively charged helix 2 region, providing stability to the protein during RFLR-mediated host entry. / Ph. D.

Pentameriska ligandstyrda jonkanaler (pLGICs) är avgörande för omvandlingen av kemisk till elektrisk signalöverföring i djurs nervsystem. Dysfunktion i dessa kanaler har visat sig vara kopplad till flera sjukdomar inklusive epilepsi, schizofreni, Alzheimers och autism, vilket gör dem till en måltavla för en mängd olika läkemedel. Att studera eukaryota kanaler är dock mycket utmanande, så upptäckten av prokaryota homologer, som är mycket lättare att studera, har därmed bidragit mycket till förståelsen för struktur och funktion hos proteiner i denna familj. I detta projekt producerades och renades en prokaryotisk pLGIC kallad DeCLIC från Escherichia coli. Strukturell bestämning av kanalen genomfördes med användning av kryo-elektronmikroskopi vid lågt pH och i närvaro av kalcium. En elektrontäthet med 3.4 Å upplösning uppnåddes och jämfördes med tidigare bestämda strukturer vid olika förhållanden i ett försök att bestämma hur proteinets struktur moduleras av kalcium och pH. Resultaten visar flera skillnader i kanalens konformation i närvaro och frånvaro av kalcium såväl som vid olika pH-värden. Dessutom antyder analys av den bestämda elektrontätheten ett möjligt intermediärt tillstånd vid lågt pH i närvaro av kalcium. / Pentameric ligand-gated ion channels (pLGICs) are crucial for the conversion of chemical to electrical signaling in the nervous system of mammals. Dysfunction in these channels has been found to be connected to several diseases including epilepsy, schizophrenia, Alzheimer’s, and autism, making them the target of a wide variety of therapeutic agents. However, studying eukaryotic channels is challenging so the discovery of prokaryotic homologs that are much easier to study has thus greatly helped in the understanding of the structure and function in this family of proteins. In this project, a prokaryotic pLGIC called DeCLIC was produced and purified from Escherichia coli. Structural determination of the channel was pursued using cryo-electron microscopy at a low pH and in the presence of calcium. An electron density at 3.4 Å resolution was achieved and compared to previously determined structures at different conditions in an attempt to determine the structural modulation of calcium and pH. Results show multiple differences in channel conformation in the presence and absence of calcium as well as in different pH conditions. Furthermore, analysis of the determined electron density suggests a possible intermediate state at low pH in the presence of calcium.

No description available.

Introduction. The inflammatory bowel diseases (IBD), Crohn’s disease (CD) and ulcerative colitis (UC), are common in Western Europe (200-400 cases /100,000) and associated with substantial morbidity, although mortality is now low. There is presently a great unmet need for novel therapeutics in IBD as present agents are limited by lack of efficacy, toxicity and poor patient acceptance. Recent findings from genome-wide association studies (GWAS) have characterised the genetic architecture of CD and UC. Defects in innate and adaptive immunity have been clearly established, and substantial novel insights into disease pathogenesis have been gained. Over 30 genes / loci are now associated with CD; a number of these, along with a few specific loci, are also associated with UC. The hedgehog (HH) signalling pathway is critical to gastrointestinal development and plays key roles in intestinal and immune homeostasis. Furthermore, in addition to well described roles in tumorigenesis, it is evident that recapitulation of embryonic HH signals play critical roles in response to acute and chronic inflammatory challenge in diverse tissues. Aims. The main aims of the work presented in this thesis were to characterise the expression of key HH signalling components in the healthy and inflamed human intestine, establish whether germline variation in HH genes is associated with IBD and describe the in vitro responses of intestinal epithelial cells to pathogen associated molecular patterns. The WNT pathway, antagonised by HH in the intestine, and two HH target genes (NKX2.3 and CCL20) were also analysed for evidence of association with IBD. Methods. Expression of HH and WNT signalling components was described by immunohistochemistry and microarray analysis in healthy controls (HC), CD, UC, and non- IBD inflamed terminal ileal and colonic samples. Gene-wide haplotype-tagging studies were performed for GLI1 in Scottish, English and Swedish CD and UC, and Scottish early-onset colo-rectal cancer, IHH in Scottish IBD, NKX2.3 in Scottish and UK IBD, and CCL20 in Scottish, Swedish and Japanese IBD. Evidence for association of all HH (n=13) and WNT (n=27) signalling genes in CD was established by analysis of UK GWAS data and metaanalysis from UK, French/Belgium and N American studies. The effect of lipopolysaccharide (LPS) and muramyl dipeptide (MDP) on HH signalling was assessed in colonic epithelial cells (SW480). The effect of HH pathway agonists and antagonists on NFκB activity and cytokine expression was analysed in SW480 cells and peripheral blood mononuclear cells (HC and IBD patients) in vitro. Results. The expression of HH pathway ligand is present in the intestinal epithelium and the pathway response network in the lamina propria demonstrating the paracrine nature of HH signalling in the intestine. Immunohistochemical studies and microarray analysis demonstrates that HH pathway activity is decreased in all forms of colonic inflammation studied in man. Variation in Glioma-associated oncogene homolog 1 (GLI1), a key HH transcription factor located at 12q13 (IBD2), was associated with IBD (p<0.0001), UC (p<0.0001) and to a lesser extent CD (p=0.03) in Scotland, a finding replicated in English IBD and UC. This association was attributed to a non-synonymous SNP (rs2228226C→G) with pools odds ratio of 1.194 in meta-analysis of over 5000 individuals from Scotland, England and Sweden (p=0.0002). There was association of this SNP with early-onset colorectal cancer, but of borderline significance (p=0.05). The variant protein (Q1100E) is 50% less active than wild-type protein in vitro. IHH was not associated with CD or UC. Preliminary evidence was produced for association at SUFU (10q24; p=0.005), a GLI1- binding protein, and at the WNT3 / WNT9B locus (17q21; p=0.0005). MDP stimulation of colonic epithelial cells decreased HH pathway activity. Exogenous HH increased expression of CCL20. CCL20 promoter polymorphisms were associated with UC in Japanese patients (p=0.018) but not in Scotland or Sweden. NKX2.3 was associated with IBD in Scotland (UC>CD), but there was insufficient power for fine-mapping of causative variants. Conclusions. Multiple lines of evidence presented here demonstrate that the HH signalling pathway is involved in IBD pathogenesis. In key complementary in vivo studies (conceived by CWL; conducted in collaboration with the Gumucio lab in Ann Arbor) we have demonstrated that Gli1+/- mice develop early, severe colitis with high mortality in response to acute inflammatory challenge. Furthermore, lamina propria antigen presenting cells are identified as the key HH target cells. With HH agonists and antagonists in extensive preclinical and early clinical testing, these studies have real potential to translate into novel therapeutics for patients with IBD.

This thesis comprises three published papers and one manuscript, all focused on the role of juvenile hormone (JH), the JH receptor Methoprene-tolerant (Met) and its target gene Krüppel-homolog 1 (Kr-h1) in insect development and reproduction. The JH-Met-Kr-h1 pathway is critical for metamorphic transition in hemimetabolan Pyrrhocoris apterus (Hemiptera) and holometabolan Bombyx mori (Lepidoptera) but seems to be dispensable during early larval postembryonic development. The results also show that Met and its heterodimeric partner Taiman (Tai) but not Kr-h1 are critical for ovarian development and vitellogenesis in Pyrrhocoris females. In vitro, in vivo and cell-based techniques in Drosophila melanogaster have demonstrated that Met and its paralog Gce are a bona fide receptor for JH. Only Gce capable of binding JH rescued Drosophila deficient for Met and Gce proteins, and the capacity of Gce to bind JH was necessary for JH-dependent transcriptional activation by Gce and Tai.

Phosphorylation mediated inactivation of PTEN leads to multiple malignancies with increased severity. However, the consequence of such inactivation on downstream functions of PTEN are poorly understood. Therefore, the objective of my thesis is to ascertain the molecular mechanisms by which PTEN phosphorylation drives lung cancer. PTEN phosphorylation at the C-terminal serine/threonine cluster abrogates its tumor suppressor function. Despite the critical role of the PTEN C-tail in regulating its function, the crystal structure of the C-tail remains unknown. Using bioinformatics and structural analysis, I determined that the PTEN C-tail is an intrinsically disordered region and is a hot spot for post-translational modifications (particularly phosphorylation) and protein-protein interactions. Evolutionary analysis of PTEN and its interacting proteins revealed that the PTEN C-tail has only recently evolved to acquire the ability to engage in a myriad of protein-protein interactions, resulting in its versatile functions. Replacement of the PTEN C-tail serine/threonine residues with alanines generated an artificial mutant, PTEN-4A, which remained “phospho-deficient” and therefore constitutively active. Interestingly, PTEN-4A suppressed cell proliferation and migration to a greater extent than PTEN-WT. PTEN-4A preferentially localized to the nucleus where it suppressed E2F-mediated transcription of cell cycle genes. PTEN physically interacted with the E2F1 protein and at E2F1-binding sites on chromatin, a likely mechanism for its transcriptional function. Further, deletion analysis on various PTEN domains revealed that the C2 domain of PTEN is indispensable for suppression of E2F-related genes. Systematic transcriptional promoter-reporter assays identified disease-associated C2 domain mutations that lose their ability to suppress E2F-mediated transcription, supporting the concept that these mutations are oncogenic in patients. Consistent with my findings, I observed increased level of PTEN phosphorylation and reduced nuclear PTEN levels in lung cancer patient samples. Further, to determine whether the enhanced growth-suppressive properties of PTEN-4A may be due to differential protein-protein interactions, I performed a comparative proteomic profiling of PTEN-WT and PTEN-4A interactomes using the SILAC methodology. Galectin-1 was identified as a candidate protein that binds preferentially to PTEN-WT and inhibits its tumor suppressive function. Taken together, the various tumor suppressive mechanisms of PTEN-4A may be harnessed therapeutically as adjunctive cancer therapy. Use of small molecule inhibitors that hinder PTEN C-tail phosphorylation is a plausible approach to activate PTEN function to reduce tumor burden.

京都大学 / 新制・課程博士 / 博士(理学) / 甲第23044号 / 理博第4721号 / 新制||理||1677(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 沼田 英治, 教授 曽田 貞滋, 准教授 森 哲 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

<p>The PRLs are a subfamily of dual specificity phosphatases that appear to play important roles in oncogenesis. Much of the current understanding of PRL function has been either correlative, and deduced from observed PRL overexpression in pathological conditions, or from in vitro analysis of signaling pathways following PRL deletion or overexpression. Such studies, necessitated by the general lack of synthetic inhibitors or compounds to probe the substrate specificity and biological interactions of the PRLs, are nonetheless now providing critical insight into potential biological substrates and roles of the PRL phosphatases. The recent identification of PTEN as a substrate for PRL2 provided the foundation for studies to further define the role of PRL2 in oncogenesis and, by analogy, the normal physiological function of PRL2. In the studies described herein, a novel PRL2 conditional knock-out animal was generated and used to validate the PRL2/PTEN interaction in a leukemic phenotype, and further demonstrated that PRL2 inhibition can restore dysregulated PTEN/AKT pathways to significantly attenuate disease progression. Inhibition of PRL2 therefore represents a novel potential therapeutic strategy in the management and treatment of AML. This thesis project also sought to further examine the role of the PRLs in oncogenesis through their regulation and interaction of targets within the TME. Functional analyses revealed that PRL3 was the only PRL to have a prominent role in host response to TME development, and that previously proposed roles for PRL3 in angiogenesis and immune cell recruitment is dependent upon PRL3 expression and activity in cells external to the TME. The study also revealed a previously unrecognized synergism between VEGF and PRL3 in the host in promoting TME angiogenesis. The studies of PRL3 in the TME suggest the potential physiological role of PRL3 in wound healing.</p>

To survive, an organism must constantly adjust its internal state to changes in the environment from which it receives signals. The signals set off a chain of events referred to signal transduction. Signal transduction systems are especially important in multicellular organisms, such as plants and animals, because of the need to coordinate the activities of hundreds to trillions of cells. Plants, in particular, have a special need for perceiving signals from their environment because of their static nature. As in the animal cell, the first steps in perception of a signal include signal interaction with a receptor, signal amplification through second messenger production, and signal termination through second messenger hydrolysis. Myo-inositol polyphosphate 5-phosphatases (5PTases) (EC 3.1.3.56) have unique signal terminating abilities toward the second messenger inositol trisphosphate (Ins (1,4,5)P3, InsP3). In Arabidopsis thaliana there are 15 members of the 5PTase family, the majority of which contain a single 5PTase catalytic domain. Four members of the Arabidopsis 5PTase family, however, have a unique protein domain structure, with additional N-terminal WD40 repeats that are implicated in protein-protein interactions. The research presented here focused on the identification of 5PTase interacting proteins and the characterization of their functional role in Arabidopsis. To accomplish this goal, I examined a 5PTase13-interacting protein, the sucrose (Suc) nonfermenting-1-related kinase, SnRK1.1, an important energy sensor that is highly conserved among eukaryotes. My identification of a 5PTase13:SnRK1.1 complex points to the novel interaction of this metabolic modulator and inositol signaling/metabolism. 5PTase13 , however, plays a regulatory role in other plant specific processes as well, since I also identified the Arabidopsis homolog (Atp80) of the human WDR48 (HsWDR48, Hsp80) as a novel protein interactor of 5PTase13. My results indicate that Atp80 is important for leaf emergence, development and senescence likely via a regulatory interaction with 5PTase13 and PINOID â binding protein (PBP1). / Ph. D.

Binding of the Rev protein with Rev Response Element (RRE) RNA present in singly- and unspliced mRNA transcripts is necessary for the replication of HIV-1. This interaction transports the mRNA transcripts from the nucleus to the cytoplasm for translation of the necessary structural and enzymatic proteins for the newly budding virus as well as for providing its genetic material. Given the high rate of mutation in HIV-1, the highly conserved and pertinent RRE RNA is of high interest for pharmaceutical intervention. Consequently, a branched peptide library containing unnatural amino acids was developed to target RRE RNA with the goal of increasing stability, potency, selectivity, and in vivo activity for RRE RNA. An unnatural amino acid branched peptide library (46,656 sequences) was synthesized and screened against RRE IIB and several hits in the sub-micromolar regime were found. A number of hits demonstrated selectivity in the presence of other RNAs in addition to two hits, 4A5 and 4B3, significantly inhibiting HIV-1 growth in vitro. These peptides inhibited HIV-1 replication in a concentration dependent manner and were demonstrated to be non-toxic. Further analysis of 4A5 and 4B3 via footprinting and SHAPE-MaP experiments determined that these peptides blocked binding of Rev through binding at the primary and secondary binding sites of RRE RNA. Sphingosine 1-phosphate (S1P) is a signaling molecule that plays a role in various biological processes including immunity, neurogenesis, and angiogenesis. The role S1P plays is largely determined by its location, in which Spinster homolog 2 (spns2) and mfsd2b are the two known transporters. The two transporters exist in different cell types and cellular localizations, with spns2-produced S1P being responsible for trafficking of lymphocytes. As such, spns2 has become of interest for therapeutic targeting in autoimmune and inflammatory diseases. To validate spns2 as a target in pharmaceutical intervention, a series of spns2 inhibitors were developed. A screening of a library of inhibitors found that compound SLP7120922 demonstrated inhibition of spns2 transport activity. The design, synthesis, and biological evaluation of inhibitors based on SLP7120922 is described. Modifications to the lipophilic tail region were performed with one compound 4.40f discovered to be potent, minimally toxic, and active in vivo. A series of modifications to the head region were then conducted that evaluated linear head derivatives with alkyl-, amide-, and amino acid-based groups. A number of compounds are reported that demonstrate good in vitro activity and minimal toxicity with two compounds, 4.48b and 4.52c, showing favorable in vivo activity in mice. / Doctor of Philosophy / Human immunodeficiency virus (HIV-1) has a high rate of mutation, which commonly leads to the need for many types of medications throughout the lifetime of a patient. In order to design a therapeutic that the virus has a low chance of growing resistance to, a target needs to be chosen with a low mutation rate. One such target is the Rev Response Element (RRE) RNA and it is necessary for the virus to replicate. A protein named Rev binds to RRE RNA in order for RRE to carry out its pertinent function. To block this function we have chosen branched peptides to target the RNA. Peptides are made of the same building blocks of proteins, but are much shorter than proteins. The peptides described here are made up of modified building blocks, called unnatural amino acids. This work describes the generation of an unnatural amino acid branched peptide library and how it was screened in order to find branched peptides that bind RRE RNA. Many peptides were found to bind RRE RNA but two in particular, 4A5 and 4B3, were the best binders that inhibited HIV-1 growth. The remainder of the work describes how these peptides bind to RRE RNA, while demonstrating that they are non-toxic and bind HIV-1 in a concentration dependent manner. A transporter protein termed Spinster homolog 2 (spns2) transports a signaling molecule known as sphingosine 1-phosphate (S1P). For our immune system to function properly, spns2 has to transport S1P to the appropriate places to signal to immune cells. Unfortunately, this is a problem in autoimmune and inflammatory diseases, such as multiple sclerosis, due to these diseases having an overactive immune system. A potential way to treat these diseases would be by inhibiting spns2. This work describes the design, synthesis, and biological evaluation of spns2 inhibitors. Many compounds were found to inhibit spns2 to a degree, but three compounds, in particular, show potent and effective inhibition in mice.