http://www.dspace.cam.ac.uk:80/handle/1810/221629 Sat, 18 May 2013 10:41:13 GMT 2013-05-18T10:41:13Z http://www.dspace.cam.ac.uk:80/handle/1810/243433 Title: Characterisation of the AP-3 adaptor-like complex Authors: Peden, Andrew Alexander Abstract: Clathrin coated vesicles were the first type of coated vesicle to be characterised. The coat consists of two components, clathrin and adaptor (or AP) complexes, the AP-1 complex is associated with the clathrin coated vesicles that bud from the TGN and the AP-2 complex is associated with the clathrin coated vesicles that bud from the plasma membrane. A new type of adaptor-like complex was discovered in our laboratory and was published in 1996. The complex has been shown to consist of two known proteins, beta3B and mu3B, and two unknown proteins of 160kD and 22kD. Unlike the conventional adaptor complexes this complex is not associated with clathrin. The aim of this thesis was to complete the characterisation of the adaptor-like complex and to establish its function. My studies have shown that, the adaptor-like complex consist of an alpha/gamma like subunit, delta, a beta subunit (beta3A/B), a mu subunit (mu3A/B) and a sigma subunit (sigma3A/B). We named the adaptor-like complex AP-3, by analogy with the AP-1 and AP-2 complexes. The AP-3 complex is localised to perinuclear and more peripheral membranes in non-neuronal cells, with little overlap with endocytic markers. The beta subunit of the AP-3 complex is the major target for phosphorylation. Analysis of mice with mutations in the beta3A subunit, and in the delta subunit of the AP-3 complex, have revealed that the beta subunit is required for the stability of the mu subunit and that the delta subunit is essential for the stability of the whole complex. Further analysis of the mutant mice indicated that the mice lack significant levels of functional AP-3 complex. Studies on fibroblasts generated from these mice revealed that the AP-3 complex plays a role in the trafficking of LAMPI to lysosomes. Tue, 15 Feb 2000 00:00:00 GMT http://www.dspace.cam.ac.uk:80/handle/1810/243433 2000-02-15T00:00:00Z http://www.dspace.cam.ac.uk:80/handle/1810/239299 Title: Carbon-nanoparticle-triggered acute lung inflammation and its resolution are not altered in PPARgamma-defective (P465L) mice Authors: Gotz, Alexander A; Vidal-Puig, Antonio; Rodel, Heiko G; Hrabe de Angelis, Martin; Stoeger, Tobias Abstract: Abstract Background The alveolar macrophage (AM) - first line of innate immune defence against pathogens and environmental irritants - constitutively expresses peroxisome-proliferator activated receptor γ (PPARγ). PPARγ ligand-induced activation keeps the AM quiescent, and thereby contributes to combat invaders and resolve inflammation by augmenting the phagocytosis of apoptotic neutrophils and inhibiting an excessive expression of inflammatory genes. Because of these presumed anti-inflammatory functions of PPARγ we tested the hypothesis, whether reduced functional receptor availability in mutant mice resulted in increased cellular and molecular inflammatory response during acute inflammation and/or in an impairment of its resolution. Methods To address this hypothesis we examined the effects of a carbon-nanoparticle (CNP) lung challenge, as surrogate for non-infectious environmental irritants, in a murine model carrying a dominant-negative point mutation in the ligand-binding domain of PPARγ (P465L/wt). Animals were instilled intratracheally with Printex 90 CNPs and bronchoalveolar lavage (BAL) was gained 24 h or 72 h after instillation to investigate its cellular and protein composition. Results Higher BAL cell numbers - due to higher macrophage counts - were found in mutants irrespective of treatment. Neutrophil numbers in contrast were slightly lower in mutants. Intratracheal CNP instillation resulted in a profound recruitment of inflammatory neutrophils into the alveolus, but genotype related differences at acute inflammation (24 h) and resolution (72 h) were not observed. There were no signs for increased alveolar-capillary membrane damage or necrotic cell death in mutants as determined by BAL protein and lactate-dehydrogenase content. Pro-inflammatory macrophage-derived cytokine osteopontin was higher, but galectin-3 lower in female mutants. CXCL5 and lipocalin-2 markers, attributed to epithelial cell stimulation did not differ. Conclusions Despite general genotype-related differences, we had to reject our hypothesis of an increased CNP induced lung inflammation and an impairment of its resolution in PPARγ defective mice. Although earlier studies showed ligand-induced activation of nuclear receptor PPARγ to promote resolution of lung inflammation, its reduced activity did not provide signs of resolution impairment in the settings investigated here. Description: RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are. Mon, 19 Sep 2011 23:00:00 GMT http://www.dspace.cam.ac.uk:80/handle/1810/239299 2011-09-19T23:00:00Z http://www.dspace.cam.ac.uk:80/handle/1810/238244 Title: Human autoantibodies against the 54 kDa protein of the signal recognition particle block function at multiple stages Authors: Romisch, Karin; Miller, Frederick W; Dobberstein, Bernhard; High, Stephen Abstract: Abstract The 54 kDa subunit of the signal recognition particle (SRP54) binds to the signal sequences of nascent secretory and membrane proteins and it contributes to the targeting of these precursors to the membrane of the endoplasmic reticulum (ER). At the ER membrane, the binding of the signal recognition particle (SRP) to its receptor triggers the release of SRP54 from its bound signal sequence and the nascent polypeptide is transferred to the Sec61 translocon for insertion into, or translocation across, the ER membrane. In the current article, we have characterized the specificity of anti-SRP54 autoantibodies, which are highly characteristic of polymyositis patients, and investigated the effect of these autoantibodies on the SRP function in vitro. We found that the anti-SRP54 autoantibodies had a pronounced and specific inhibitory effect upon the translocation of the secretory protein preprolactin when analysed using a cell-free system. Our mapping studies showed that the anti-SRP54 autoantibodies bind to the amino-terminal SRP54 N-domain and to the central SRP54 G-domain, but do not bind to the carboxy-terminal M-domain that is known to bind ER signal sequences. Nevertheless, anti-SRP54 autoantibodies interfere with signal-sequence binding to SRP54, most probably by steric hindrance. When the effect of anti-SRP autoantibodies on protein targeting the ER membrane was further investigated, we found that the autoantibodies prevent the SRP receptor-mediated release of ER signal sequences from the SRP54 subunit. This observation supports a model where the binding of the homologous GTPase domains of SRP54 and the α-subunit of the SRP receptor to each other regulates the release of ER signal sequences from the SRP54 M-domain. Description: RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are. Thu, 26 Jan 2006 00:00:00 GMT http://www.dspace.cam.ac.uk:80/handle/1810/238244 2006-01-26T00:00:00Z http://www.dspace.cam.ac.uk:80/handle/1810/238124 Title: Identification of a novel gene family that includes the interferon-inducible human genes 6–16and ISG12 Authors: Parker, Nadeene; Porter, Andrew C G Abstract: Abstract Background The human 6–16 and ISG12 genes are transcriptionally upregulated in a variety of cell types in response to type I interferon (IFN). The predicted products of these genes are small (12.9 and 11.5 kDa respectively), hydrophobic proteins that share 36% overall amino acid identity. Gene disruption and over-expression studies have so far failed to reveal any biochemical or cellular roles for these proteins. Results We have used in silico analyses to identify a novel family of genes (the ISG12 gene family) related to both the human 6–16 and ISG12 genes. Each ISG12 family member codes for a small hydrophobic protein containing a conserved ~80 amino-acid motif (the ISG12 motif). So far we have detected 46 family members in 25 organisms, ranging from unicellular eukaryotes to humans. Humans have four ISG12 genes: the 6–16 gene at chromosome 1p35 and three genes (ISG12(a), ISG12(b) and ISG12(c)) clustered at chromosome 14q32. Mice have three family members (ISG12(a), ISG12(b1) and ISG12(b2)) clustered at chromosome 12F1 (syntenic with human chromosome 14q32). There does not appear to be a murine 6–16 gene. On the basis of phylogenetic analyses, genomic organisation and intron-alignments we suggest that this family has arisen through divergent inter- and intra-chromosomal gene duplication events. The transcripts from human and mouse genes are detectable, all but two (human ISG12(b) and ISG12(c)) being upregulated in response to type I IFN in the cell lines tested. Conclusions Members of the eukaryotic ISG12 gene family encode a small hydrophobic protein with at least one copy of a newly defined motif of ~80 amino-acids (the ISG12 motif). In higher eukaryotes, many of the genes have acquired a responsiveness to type I IFN during evolution suggesting that a role in resisting cellular or environmental stress may be a unifying property of all family members. Analysis of gene-function in higher eukaryotes is complicated by the possibility of functional redundancy between family-members. Genetic studies in organisms (e.g. Dictyostelium discoideum) with just one family member so far identified may be particularly helpful in this respect. Description: RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are. Mon, 19 Jan 2004 00:00:00 GMT http://www.dspace.cam.ac.uk:80/handle/1810/238124 2004-01-19T00:00:00Z http://www.dspace.cam.ac.uk:80/handle/1810/237673 Title: Bioinformatics strategies for lipidomics analysis: characterization of obesity related hepatic steatosis Authors: Yetukuri, Laxman; Katajamaa, Mikko; Medina-Gomez, Gema; Seppanen-Laakso, Tuulikki; Vidal-Puig, Antonio; Oresic, Matej Abstract: Abstract Background Lipids are an important and highly diverse class of molecules having structural, energy storage and signaling roles. Modern analytical technologies afford screening of many lipid molecular species in parallel. One of the biggest challenges of lipidomics is elucidation of important pathobiological phenomena from the integration of the large amounts of new data becoming available. Results We present computational and informatics approaches to study lipid molecular profiles in the context of known metabolic pathways and established pathophysiological responses, utilizing information obtained from modern analytical technologies. In order to facilitate identification of lipids, we compute the scaffold of theoretically possible lipids based on known lipid building blocks such as polar head groups and fatty acids. Each compound entry is linked to the available information on lipid pathways and contains the information that can be utilized for its automated identification from high-throughput UPLC/MS-based lipidomics experiments. The utility of our approach is demonstrated by its application to the lipidomic characterization of the fatty liver of the genetically obese insulin resistant ob/ob mouse model. We investigate the changes of correlation structure of the lipidome using multivariate analysis, as well as reconstruct the pathways for specific molecular species of interest using available lipidomic and gene expression data. Conclusion The methodology presented herein facilitates identification and interpretation of high-throughput lipidomics data. In the context of the ob/ob mouse liver profiling, we have identified the parallel associations between the elevated triacylglycerol levels and the ceramides, as well as the putative activated ceramide-synthesis pathways. Description: RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are. Thu, 15 Feb 2007 00:00:00 GMT http://www.dspace.cam.ac.uk:80/handle/1810/237673 2007-02-15T00:00:00Z http://www.dspace.cam.ac.uk:80/handle/1810/224775 Title: The mechanism of HIV-1 Nef-mediated downregulation of CD4 Authors: Chaudhuri, Rittik Abstract: Nef, an accessory protein of HIV-1, is a critical determinant of viral pathogenicity. The pathogenic effects of Nef are in large part dependent on its ability to decrease the amount of CD4 on the surface of infected cells. Early studies suggested that Nef induces downregulation by linking the cytosolic tail of CD4 to components of the host-cell protein-trafficking machinery. However, the specific sorting pathway that Nef uses to modulate CD4 expression remained uncertain. According to one model, Nef was thought to interfere with the transport of newly synthesized CD4 from the TGN to the cell-surface. Another model claimed that Nef facilitated the removal of CD4 from the plasma membrane. The primary goal of this thesis was to determine which of these models was correct. To accomplish this objective, a novel Nef-CD4 system was developed in Drosophila S2 cells. Nef was not only able to downregulate human CD4 in S2 cells, but it did so in a manner that was phenotypically indistinguishable from its activity in human cells. An RNAi screen targeting protein-trafficking genes in S2 cells revealed a requirement for clathrin and the clathrin-associated, plasma membrane-localized AP-2 complex in the Nef-mediated downregulation of CD4. In contrast, depletion of the related AP-1 and AP-3 complexes, which direct transport from the TGN and endosomes, had no effect. The requirement for AP-2 was subsequently confirmed in a human cell line. Yeast three-hybrid and GST pull-down assays were then used to demonstrate a robust, direct interaction between Nef and AP-2. This interaction was found to depend on a [D/E]xxxL[L/I]-type dileucine motif, located in the C-terminal loop of Nef, that is essential for CD4 downregulation. While mapping the binding site of AP-2 on Nef, a second determinant of interaction in the C-terminal loop was identified. Mutation of this motif, which conforms to a consensus [D/E]D diacidic sequence, prevented Nef from binding to AP-2 and down-regulating CD4. However, the same mutations did not affect the ability of Nef to interact with either AP-1 or AP-3, providing further evidence that these complexes are not required for the modulation of CD4 expression. Additional experiments indicated that the Nef diacidic motif most likely binds to a basic patch on AP-2 α-adaptin that is not present in the homologous AP-1 γ and AP-3 δ subunits. As with the Nef diluecine and diacidic motifs, the α-adaptin basic patch was shown to be necessary for CD4 downregulation. Moreover, all three of these motifs were needed for the cooperative assembly of a CD4-Nef-AP-2 tripartite complex, which was observed here for the first time using a yeast four-hybrid system. The data in this thesis uniformly support an endocytic model of Nef-mediated CD4 downregulation. Indeed, there is now strong evidence that Nef simultaneously binds CD4 and AP-2, thereby connecting the receptor to the cellular endocytic machinery and promoting its rapid internalization from the plasma membrane. In addition, the identification of novel motifs required for this process has provided new insights on endocytosis, and may facilitate the development of pharmacological inhibitors of Nef function. Tue, 09 Feb 2010 00:00:00 GMT http://www.dspace.cam.ac.uk:80/handle/1810/224775 2010-02-09T00:00:00Z