Biochemical analysis of an in vivo immunoprecipitation complex of the prion protein

Das Prion-Protein (PrPC) ist ein Glykosyl-Phosphatidylinositol-verankertes Membranprotein, welches durch Konformationsänderung zur beta-Faltblatt-reichen und proteaseresistenten Skrapie-Isoform (PrPSc) zum Auslöser der transmissiblen spongiformen Enzephalopathien (TSE) werden kann. Die physiologische Funktion von PrP bleibt hingegen weitgehend unbekannt. PrP-Expression und Verankerung an der Zellmembran sind für die Entwicklung der Pathologie unerlässlich. In zahlreichen Arbeiten wurde die Rolle von PrP in Signaltransduktionswegen anhand von direkten Protein-Protein Interaktionen untersucht. Durch Immunpräzipitation mithilfe der monoklonalen Antikörper POM2 wurde für diesen Zweck ein in vivo Immunpräzipitationskomplex (IP-Komplex) isoliert, welcher anschliessend mittels synthetischer Peptide spezifisch für die POM2-Bindungsstelle eluiert wurde. Durch native Gele und Gel-Permeations-Chromatographie (GPC) wurde die Grösse des IP-Komplexes (800kD) bestätigt. Anschliessende massenspektroskopische Analysen deuteten eine Beteiligung mehrerer Proteine im Komplex an. In dieser Studie habe ich versucht die Interaktion der Kandidatenproteine mit PrP im IP-Komplex mithilfe des Western Blotting nachzuprüfen. Eine spezifische Interaktion konnte bis jetzt nicht bewiesen werden. Ausserdem konnte nach der Silberfärbung unter denaturierenden Bedingungen im SDS-PAGE Gel nur eine einzige Bande nachgewiesen werden, die der Grösse des PrP selbst entspricht und praktisch die Präsenz anderer Proteine ausschliesst. Kontrollexperimente mit monomerem detergenzfrei aufgereinigtem bPrP zeigten, dass die 800kD Bande nicht infolge der beeinträchtigten elektrophoretischen Migration entstanden ist. Zusätzlich haben in vitro Experimente unter Einsatz von chemischen Crosslinkern nahegelegt, dass PrP in multimeren Clustern an der Zelloberfläche vorkommen könnte. Zusammenfassend weisen diese Ergebnisse auf eine homotypische Interaktion des zellulären PrP hin, die möglicherweise für die Ausübung der physiologischen Funktion von Bedeutung ist. Anderseits könnte eine Unterbrechung der physiologischen Signaltransduktion zu Neurotoxizität führen, worauf genetische Ablationen bereits hindeuten. Die Erkenntnis der multimeren Organisation von PrP könnte helfen die Physiologie und viel wichtiger die Aktivierung der pathologischen Vorgänge besser zu verstehen.The prion protein is a cell surface GPI-anchored protein, best known for causing transmissible spongiform encephalopathies (TSE) when conformationally converted into its protease resistant and beta-sheet rich scrapie form (PrPSc). Its physiological function remains largely unknown, however its expression and membrane anchoring is indispensable for the development of TSEs. Numerous biochemical efforts have been made recently to address PrP’s involvement in signaling pathways, by testing direct protein-protein interactions of PrP. To this end, an in vivo immunoprecipitation (IP) complex was isolated using mouse monoclonal antibodies (POM2) and eluted with synthetic peptides specific for the POM2 binding region. Native gels (and size-exclusion chromatography) revealed a high-molecular weight complex of approximately 800kD. Subsequent mass spectrometry analyses suggested the presence of multiple proteins in this complex. Here, I tried to verify the interaction of the candidate proteins with PrP in the complex via Western blotting. So far, no specific interaction could be verified. In addition, silver staining under denaturing conditions revealed a single band on a SDS-PAGE corresponding to PrP, suggesting an absence of other proteins/peptides in the complex. Control experiments with monomeric detergent-free purified and phospholipase-cleaved bovine PrP further confirmed that the HMW band is not a mere electrophoretic migration artifact. Furthermore, chemical cross linking with cell impermeable agents in cell culture yielded multiple band shifts, potentially indicating a multimeric organization of PrP at the cell surface. Taken together, these results suggest that there is a homotypic interaction of cellular PrP, which might be necessary for exerting its physiological function in signal transduction. Conversely, a disruption of this type of interaction might subvert PrP-mediated signal transduction thus causing severe neurotoxicity reminiscent of the phenotype resulting from genetic ablation of the central domain. In summary, the multimeric organization of PrP might provide new insights in understanding PrP physiology and more importantly help to better understand the initiation of toxic events in prion-mediated pathologies

Challenging Tumor Heterogeneity with HER2, p16 and Somatostatin Receptor 2 Expression in a Case of EBV-Associated Lymphoepithelial Carcinoma of the Salivary Gland

BACKGROUND Lymphoepithelial carcinoma of the salivary glands (LECSG) is a rare disease in the Western hemisphere that is typically associated with an EBV infection. The molecular mechanisms of LECSG tumorigenesis are poorly understood. RESULTS Here we report a case of EBV-associated LECSG with an unusual immunophenotype. The tumor exhibited bi-morphic histological features with a mutually exclusive expression of HER2 and p16. The p16-positive domain of the tumor immunohistochemically co-expressed late membrane protein 1 (LMP-1), while the HER2 positive domain did not. Both tumor regions expressed SSTR2. METHODS In situ hybridization confirmed the EBV origin of the tumor while extensive immunohistochemical characterization and the recently established RNA-based next generation sequencing panel ("SalvGlandDx" panel) did not reveal evidence for another salivary gland neoplasm. No HPV co-infection was detected by in situ hybridization or PCR-based screenings and no ERBB2 gene amplification was detected by fluorescence in situ hybridization. CONCLUSION These findings suggest tumor heterogeneity and lack of genomic aberrations in EBV-associated LECSGs. The heterogenous and unusual immunohistochemical features explain the diagnostic difficulties and simultaneously extend the immunophenotype spectrum of this tumor entity

Metabolic Activation of Intrahepatic CD8+ T Cells and NKT Cells Causes Nonalcoholic Steatohepatitis and Liver Cancer via Cross-Talk with Hepatocytes

SummaryHepatocellular carcinoma (HCC), the fastest rising cancer in the United States and increasing in Europe, often occurs with nonalcoholic steatohepatitis (NASH). Mechanisms underlying NASH and NASH-induced HCC are largely unknown. We developed a mouse model recapitulating key features of human metabolic syndrome, NASH, and HCC by long-term feeding of a choline-deficient high-fat diet. This induced activated intrahepatic CD8+ T cells, NKT cells, and inflammatory cytokines, similar to NASH patients. CD8+ T cells and NKT cells but not myeloid cells promote NASH and HCC through interactions with hepatocytes. NKT cells primarily cause steatosis via secreted LIGHT, while CD8+ and NKT cells cooperatively induce liver damage. Hepatocellular LTβR and canonical NF-κB signaling facilitate NASH-to-HCC transition, demonstrating that distinct molecular mechanisms determine NASH and HCC development

Sublethal necroptosis signaling promotes inflammation and liver cancer

It is currently not well known how necroptosis and necroptosis responses manifest in vivo. Here, we uncovered a molecular switch facilitating reprogramming between two alternative modes of necroptosis signaling in hepatocytes, fundamentally affecting immune responses and hepatocarcinogenesis. Concomitant necrosome and NF-κB activation in hepatocytes, which physiologically express low concentrations of receptor-interacting kinase 3 (RIPK3), did not lead to immediate cell death but forced them into a prolonged "sublethal" state with leaky membranes, functioning as secretory cells that released specific chemokines including CCL20 and MCP-1. This triggered hepatic cell proliferation as well as activation of procarcinogenic monocyte-derived macrophage cell clusters, contributing to hepatocarcinogenesis. In contrast, necrosome activation in hepatocytes with inactive NF-κB-signaling caused an accelerated execution of necroptosis, limiting alarmin release, and thereby preventing inflammation and hepatocarcinogenesis. Consistently, intratumoral NF-κB-necroptosis signatures were associated with poor prognosis in human hepatocarcinogenesis. Therefore, pharmacological reprogramming between these distinct forms of necroptosis may represent a promising strategy against hepatocellular carcinoma

Metabolic activation of intrahepatic CD8(+) T cells and NKT cells causes nonalcoholic steatohepatitis and liver cancer via cross-talk with hepatocytes

Hepatocellular carcinoma (HCC), the fastest rising cancer in the United States and increasing in Europe, often occurs with nonalcoholic steatohepatitis (NASH). Mechanisms underlying NASH and NASH-induced HCC are largely unknown. We developed a mouse model recapitulating key features of human metabolic syndrome, NASH, and HCC by long-term feeding of a choline-deficient high-fat diet. This induced activated intrahepatic CD8(+) T cells, NKT cells, and inflammatory cytokines, similar to NASH patients. CD8(+) T cells and NKT cells but not myeloid cells promote NASH and HCC through interactions with hepatocytes. NKT cells primarily cause steatosis via secreted LIGHT, while CD8(+) and NKT cells cooperatively induce liver damage. Hepatocellular LTβR and canonical NF-κB signaling facilitate NASH-to-HCC transition, demonstrating that distinct molecular mechanisms determine NASH and HCC development

Characterization of HCC Mouse Models: Towards an Etiology-Oriented Subtyping Approach

Murine liver tumors often fail to recapitulate the complexity of human hepatocellular carcinoma (HCC), which might explain the difficulty to translate preclinical mouse studies into clinical science. The aim of this study was to evaluate a subtyping approach for murine liver cancer models with regard to etiology-defined categories of human HCC, comparing genomic changes, histomorphology, and IHC profiles. Sequencing and analysis of gene copy-number changes [by comparative genomic hybridization (CGH)] in comparison with etiology-dependent subsets of HCC patients of The Cancer Genome Atlas (TCGA) database were conducted using specimens (75 tumors) of five different HCC mouse models: diethylnitrosamine (DEN) treated wild-type C57BL/6 mice, c-Myc and AlbLTαβ transgenic mice as well as TAK1 and Mcl-1 mice. Digital microscopy was used for the assessment of morphology and IHC of liver cell markers (A6-CK7/19, glutamine synthetase) in mouse and = 61 human liver tumors. Tumor CGH profiles of DEN-treated mice and c-Myc transgenic mice matched alcohol-induced HCC, including morphologic findings (abundant inclusion bodies, fatty change) in the DEN model. Tumors from AlbLTαβ transgenic mice and TAK1 models revealed the highest overlap with NASH-HCC CGH profiles. Concordant morphology (steatosis, lymphocyte infiltration, intratumor heterogeneity) was found in AlbLTαβ murine livers. CGH profiles from the Mcl-1 model displayed similarities with hepatitis-induced HCC and characteristic human-like phenotypes (fatty change, intertumor and intratumor heterogeneity). IMPLICATIONS: Our findings demonstrate that stratifying preclinical mouse models along etiology-oriented genotypes and human-like phenotypes is feasible. This closer resemblance of preclinical models is expected to better recapitulate HCC subgroups and thus increase their informative value

Cerebellar pathology in FTgpi mice.

<p>All histological investigations show FTgpi177 <i>Prnp</i>^o/o mice; age-matched <i>Prnp</i>^o/o mice were used as controls. (<b>A</b>) H&E staining showing severe atrophy of the cerebellum and conspicuous loss of CGNs in FTgpi177 mice (right panels, arrows) compared to controls (left panels). (<b>B</b>) GFAP-stained cerebellum showing widespread astrogliosis in FTgpi177 mice. (<b>C</b>) Both FTgpi155 and FTgpi177 showed increased FTgpi expression in the cerebellum. Western blot was decorated with POM2 antibody. (<b>D</b>) Cerebellar frozen sections were stained with DAPI (blue) and TUNEL (red) to reveal nuclei and fragmented DNA, respectively. A gradual loss of CGNs is evident in the granular layer. Numerous TUNEL⁺ cells were detected at 10 weeks in FTgpi mice. Frozen sections of age-matched <i>Prnp</i>^o/o controls were stained at all respective time points (shown at 28 weeks) and did not reveal any TUNEL⁺ cells. (<b>E</b>) Cerebellar frozen sections were stained with DAPI (blue), Iba1 (yellow) and TUNEL (purple) to reveal nuclei, fragmented DNA, and microglia respectively. TUNEL signal localized to the cytoplasm of microglia. (<b>F</b>) Cerebellar frozen sections were stained with DAPI (blue), Iba1 (red) and POM11 (yellow) to reveal nuclei, microglia, and FTgpi. A strong FTgpi signal was detected in the cytoplasm of microglia. (<b>G</b>) Western blot using POM11 showed FTgpi protein expression decreasing over time in the cerebellum, possibly as a consequence of the progressively reduced number of neurons.</p

FTgpi induces ER stress in HPL cells.

<p>Cell lysates were analyzed by Western blotting. Numbers denote independent experiments. p-PERK (<b>A</b>) and p-eIF2α (<b>B</b>) were significantly higher in HPL-FTgpi than in HPL-GFP cells. p-PERK blot: non-consecutive lanes of the same blot. p-PERK/PERK and p-eIF2α/eIF2α ratios were calculated after values were normalized with actin. (<b>C</b>) CHOP was also found to be increased in HPL-FTgpi cells. (<b>D</b>) CHOP mRNA was quantified by RT-PCR. Thapsigargin (Tg)-treated cells were used as positive control. CHOP was significantly upregulated in HPL cells expressing FTgpi. (<b>A-B-C</b>) Each bar indicates the average ± SEM of 3 or 6 biological replicates. ** P<0.01 and *P<0.05 by unpaired two-tails t-test (each bar is compared to HPL-GFP). (<b>D</b>) Each sample is representative of 4 biological replicates. Error bars indicate averages ± SEM. **P<0.01 by one-way ANOVA with Bonferroni multiple comparisons post-test (each sample is compared to HPL-GFP).</p

Neurodegeneration and Unfolded-Protein Response in Mice Expressing a Membrane-Tethered Flexible Tail of PrP

<div><p>The cellular prion protein (PrP^C) consists of a flexible N-terminal tail (FT, aa 23–128) hinged to a membrane-anchored globular domain (GD, aa 129–231). Ligation of the GD with antibodies induces rapid neurodegeneration, which is prevented by deletion or functional inactivation of the FT. Therefore, the FT is an allosteric effector of neurotoxicity. To explore its mechanism of action, we generated transgenic mice expressing the FT fused to a GPI anchor, but lacking the GD (PrP_Δ141–225, or “FTgpi”). Here we report that FTgpi mice develop a progressive, inexorably lethal neurodegeneration morphologically and biochemically similar to that triggered by anti-GD antibodies. FTgpi was mostly retained in the endoplasmic reticulum, where it triggered a conspicuous unfolded protein response specifically activating the PERK pathway leading to phosphorylation of eIF2α and upregulation of CHOP ultimately leading to neurodegeration similar to what was observed in prion infection.</p></div