10 research outputs found

    Sterol metabolism regulates neuroserpin polymer degradation in the absence of the unfolded protein response in the dementia FENIB.

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    Mutants of neuroserpin are retained as polymers within the endoplasmic reticulum (ER) of neurones to cause the autosomal dominant dementia familial encephalopathy with neuroserpin inclusion bodies or FENIB. The cellular consequences are unusual in that the ordered polymers activate the ER overload response (EOR) in the absence of the canonical unfolded protein response. We use both cell lines and Drosophila models to show that the G392E mutant of neuroserpin that forms polymers is degraded by UBE2j1 E2 ligase and Hrd1 E3 ligase while truncated neuroserpin, a protein that lacks 132 amino acids, is degraded by UBE2g2 (E2) and gp78 (E3) ligases. The degradation of G392E neuroserpin results from SREBP-dependent activation of the cholesterol biosynthetic pathway in cells that express polymers of neuroserpin (G392E). Inhibition of HMGCoA reductase, the limiting enzyme of the cholesterol biosynthetic pathway, reduced the ubiquitination of G392E neuroserpin in our cell lines and increased the retention of neuroserpin polymers in both HeLa cells and primary neurones. Our data reveal a reciprocal relationship between cholesterol biosynthesis and the clearance of mutant neuroserpin. This represents the first description of a link between sterol metabolism and modulation of the proteotoxicity mediated by the EOR

    Proteomic plasma membrane profiling reveals an essential role for gp96 in the cell surface expression of LDLR family members, including the LDL receptor and LRP6.

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    The endoplasmic reticulum chaperone gp96 is required for the cell surface expression of a narrow range of proteins, including toll-like receptors (TLRs) and integrins. To identify a more comprehensive repertoire of proteins whose cell surface expression is dependent on gp96, we developed plasma membrane profiling (PMP), a technique that combines SILAC labeling with selective cell surface aminooxy-biotinylation. This approach allowed us to compare the relative abundance of plasma membrane (PM) proteins on gp96-deficient versus gp96-reconstituted murine pre-B cells. Analysis of unfractionated tryptic peptides initially identified 113 PM proteins, which extended to 706 PM proteins using peptide prefractionation. We confirmed a requirement for gp96 in the cell surface expression of certain TLRs and integrins and found a marked decrease in cell surface expression of four members of the extended LDL receptor family (LDLR, LRP6, Sorl1 and LRP8) in the absence of gp96. Other novel gp96 client proteins included CD180/Ly86, important in the B-cell response to lipopolysaccharide. We highlight common structural motifs in these client proteins that may be recognized by gp96, including the beta-propeller and leucine-rich repeat. This study therefore identifies the extended LDL receptor family as an important new family of proteins whose cell surface expression is regulated by gp96

    Development of a database with web-based user interface for taqman assay design

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    TaqMan RT-PCR (reverse transcription-polymerase chain reaction) is a technique used to measure the relative gene expression in a biological sample and is one of the core technologies used by the Molecular Pathology and Toxicology (MPT) Group at GlaxoSmithKline. Conducting TaqMan experiments is a complex process which involves the design of a TaqMan assay specific to a gene of interest. A wealth of data has been generated during assay design, but systems are not currently available to readily share this data within the MPT group. There is a need for a central data storage repository so that data associated with assay design can be organised efficiently and rapidly accessed. Experiments are conducted within limited timeframes and resource is often limited so this would be of great benefit to the MPT group. This thesis describes the development of a database to house data associated with TaqMan assay design, software to populate the database with minimal user interaction and a web based CGI application for members of the MPT group to query and submit data to the database. Finally, the output from testing the software is provided and discussed

    The Hedgehog signalling pathway regulates autophagy

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    Autophagy is a highly conserved degradative process that removes damaged or unnecessary proteins and organelles, and recycles cytoplasmic contents during starvation. Autophagy is essential in physiological processes such as embryonic development but how autophagy is regulated by canonical developmental pathways is unclear. Here we show that the Hedgehog signalling pathway inhibits autophagosome synthesis, both in basal and in autophagy-induced conditions. This mechanism is conserved in mammalian cells and in Drosophila, and requires the orthologous transcription factors Gli2 and Ci, respectively. Furthermore, we identify that activation of the Hedgehog pathway reduces PERK levels, concomitant with a decrease in phosphorylation of the translation initiation factor eukaryotic initiation factor 2α, suggesting a novel target of this pathway and providing a possible link between Hedgehog signalling and autophagy

    Proteomic Plasma Membrane Profiling Reveals an Essential Role for gp96 in the Cell Surface Expression of LDLR Family Members, Including the LDL Receptor and LRP6

    No full text
    The endoplasmic reticulum chaperone gp96 is required for the cell surface expression of a narrow range of proteins, including toll-like receptors (TLRs) and integrins. To identify a more comprehensive repertoire of proteins whose cell surface expression is dependent on gp96, we developed plasma membrane profiling (PMP), a technique that combines SILAC labeling with selective cell surface aminooxy-biotinylation. This approach allowed us to compare the relative abundance of plasma membrane (PM) proteins on gp96-deficient versus gp96-reconstituted murine pre-B cells. Analysis of unfractionated tryptic peptides initially identified 113 PM proteins, which extended to 706 PM proteins using peptide prefractionation. We confirmed a requirement for gp96 in the cell surface expression of certain TLRs and integrins and found a marked decrease in cell surface expression of four members of the extended LDL receptor family (LDLR, LRP6, Sorl1 and LRP8) in the absence of gp96. Other novel gp96 client proteins included CD180/Ly86, important in the B-cell response to lipopolysaccharide. We highlight common structural motifs in these client proteins that may be recognized by gp96, including the beta-propeller and leucine-rich repeat. This study therefore identifies the extended LDL receptor family as an important new family of proteins whose cell surface expression is regulated by gp96

    A genomewide scan for loci predisposing to type 2 diabetes in a U.K. population (the Diabetes UK Warren 2 Repository): analysis of 573 pedigrees provides independent replication of a susceptibility locus on chromosome 1q

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    Improved molecular understanding of the pathogenesis of type 2 diabetes is essential if current therapeutic and preventative options are to be extended. To identify diabetes-susceptibility genes, we have completed a primary (418-marker, 9-cM) autosomal-genome scan of 743 sib pairs (573 pedigrees) with type 2 diabetes who are from the Diabetes UK Warren 2 repository. Nonparametric linkage analysis of the entire data set identified seven regions showing evidence for linkage, with allele-sharing LOD scores > or =1.18 (P< or =.01). The strongest evidence was seen on chromosomes 8p21-22 (near D8S258 [LOD score 2.55]) and 10q23.3 (near D10S1765 [LOD score 1.99]), both coinciding with regions identified in previous scans in European subjects. This was also true of two lesser regions identified, on chromosomes 5q13 (D5S647 [LOD score 1.22] and 5q32 (D5S436 [LOD score 1.22]). Loci on 7p15.3 (LOD score 1.31) and 8q24.2 (LOD score 1.41) are novel. The final region showing evidence for linkage, on chromosome 1q24-25 (near D1S218 [LOD score 1.50]), colocalizes with evidence for linkage to diabetes found in Utah, French, and Pima families and in the GK rat. After dense-map genotyping (mean marker spacing 4.4 cM), evidence for linkage to this region increased to a LOD score of 1.98. Conditional analyses revealed nominally significant interactions between this locus and the regions on chromosomes 10q23.3 (P=.01) and 5q32 (P=.02). These data, derived from one of the largest genome scans undertaken in this condition, confirm that individual susceptibility-gene effects for type 2 diabetes are likely to be modest in size. Taken with genome scans in other populations, they provide both replication of previous evidence indicating the presence of a diabetes-susceptibility locus on chromosome 1q24-25 and support for the existence of additional loci on chromosomes 5, 8, and 10. These data should accelerate positional cloning efforts in these regions of interest
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