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1.
FASEB J ; 31(3): 1226-1237, 2017 03.
Article in English | MEDLINE | ID: mdl-28003343

ABSTRACT

The adhesion molecule CD99 is essential for the transendothelial migration of leukocytes. In this study, we used biochemical and cellular assays to show that CD99 undergoes ectodomain shedding by the metalloprotease meprin ß and subsequent intramembrane proteolysis by γ-secretase. The cleavage site in CD99 was identified by mass spectrometry within an acidic region highly conserved through different vertebrate species. This finding fits perfectly to the unique cleavage specificity of meprin ß with a strong preference for aspartate residues and suggests coevolution of protease and substrate. We hypothesized that limited CD99 cleavage by meprin ß would alter cellular transendothelial migration (TEM) behavior in tissue remodeling processes, such as inflammation and cancer. Indeed, meprin ß induced cell migration of Lewis lung carcinoma cells in an in vitro TEM assay. Accordingly, deficiency of meprin ß in Mep1b-/- mice resulted in significantly increased CD99 protein levels in the lung. Therefore, meprin ß could serve as a therapeutic target, given that in a proof-of-concept approach we showed accumulation of CD99 protein in lungs of meprin ß inhibitor-treated mice.-Bedau, T., Peters, F., Prox, J., Arnold, P., Schmidt, F., Finkernagel, M., Köllmann, S., Wichert, R., Otte, A., Ohler, A., Stirnberg, M., Lucius, R., Koudelka, T., Tholey, A., Biasin, V., Pietrzik, C. U., Kwapiszewska, G., Becker-Pauly, C. Ectodomain shedding of CD99 within highly conserved regions is mediated by the metalloprotease meprin ß and promotes transendothelial cell migration.


Subject(s)
12E7 Antigen/metabolism , Conserved Sequence , Metalloendopeptidases/metabolism , Proteolysis , Transendothelial and Transepithelial Migration , 12E7 Antigen/chemistry , Animals , Carcinoma, Lewis Lung/metabolism , HEK293 Cells , HeLa Cells , Humans , Mice , Mice, Inbred C57BL
2.
Virology ; 502: 1-12, 2017 02.
Article in English | MEDLINE | ID: mdl-27951436

ABSTRACT

For human hepatitis B virus eight distinct and two candidate genotypes are described. These genotypes differ with respect to geographic distribution, molecular virology and virus-associated pathogenesis. Comparative analysis of HBV genotypes revealed, with exception of HBV/G that shows impaired HBsAg release, that no fundamental disparities between genotypes exist regarding glycosylation, subcellular distribution, release of HBsAg and formation of subviral particles. However, there are distinctions regarding the proportion of L to M to S HBs proteins detected intra- and extracellularly for different genotypes. 2D electrophoresis revealed different posttranslational modification patterns for LHBs. In light of the relevance of HBsAg as diagnostic marker, detectability of purified recombinant HBsAg of various genotypes by HBsAg-specific detection systems licensed in Europe was investigated, showing similar sensitivities for genotypes included in this analysis. These data indicate that recombinant HBsAg reproducibly purified following a defined protocol might be used as an alternative to reference materials currently established.


Subject(s)
Hepatitis B Surface Antigens/genetics , Hepatitis B virus/genetics , Hepatitis B/virology , Genotype , Glycosylation , Hepatitis B Surface Antigens/metabolism , Hepatitis B virus/classification , Hepatitis B virus/isolation & purification , Hepatitis B virus/metabolism , Humans
3.
Sci Rep ; 6: 26616, 2016 05 27.
Article in English | MEDLINE | ID: mdl-27229711

ABSTRACT

Deoxynucleotide triphosphates (dNTPs) are essential for efficient hepatitis B virus (HBV) replication. Here, we investigated the influence of the restriction factor SAMHD1, a dNTP hydrolase (dNTPase) and RNase, on HBV replication. We demonstrated that silencing of SAMHD1 in hepatic cells increased HBV replication, while overexpression had the opposite effect. SAMHD1 significantly affected the levels of extracellular viral DNA as well as intracellular reverse transcription products, without affecting HBV RNAs or cccDNA. SAMHD1 mutations that interfere with the dNTPase activity (D137N) or in the catalytic center of the histidine-aspartate (HD) domain (D311A), and a phospho-mimetic mutation (T592E), abrogated the inhibitory activity. In contrast, a mutation diminishing the potential RNase but not dNTPase activity (Q548A) and a mutation disabling phosphorylation (T592A) did not affect antiviral activity. Moreover, HBV restriction by SAMHD1 was rescued by addition of deoxynucleosides. Although HBV infection did not directly affect protein level or phosphorylation of SAMHD1, the virus upregulated intracellular dATPs. Interestingly, SAMHD1 was dephosphorylated, thus in a potentially antiviral-active state, in primary human hepatocytes. Furthermore, SAMHD1 was upregulated by type I and II interferons in hepatic cells. These results suggest that SAMHD1 is a relevant restriction factor for HBV and restricts reverse transcription through its dNTPase activity.


Subject(s)
Hepatitis B virus/physiology , Hepatocytes , Mutation, Missense , SAM Domain and HD Domain-Containing Protein 1/metabolism , Virus Replication/physiology , Amino Acid Substitution , Hep G2 Cells , Hepatocytes/enzymology , Hepatocytes/pathology , Hepatocytes/virology , Humans , SAM Domain and HD Domain-Containing Protein 1/genetics
4.
Biochem J ; 473(2): 145-55, 2016 Jan 15.
Article in English | MEDLINE | ID: mdl-26527738

ABSTRACT

Although it is well established that the release of HCV (hepatitis C virus) occurs through the secretory pathway, many aspects concerning the control of this process are not yet fully understood. α-Taxilin was identified as a novel binding partner of syntaxin-4 and of other members of the syntaxin family, which are part of SNARE (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor) complexes and so are involved in intracellular vesicle traffic. Since α-taxilin prevents t-SNARE (target SNARE) formation by binding exclusively to free syntaxin-4, it exerts an inhibitory effect on the vesicular transport. HCV-replicating Huh7.5 cells and HCV-infected primary human hepatocytes and liver samples of patients suffering from chronic HCV contain significantly less α-taxilin compared with the controls. HCV impairs the expression of α-taxilin via NS5A-dependent interruption of the Raf/MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase] signal transduction cascade. Moreover, the half-life of α-taxilin is significantly reduced in HCV-replicating cells. Whereas modulation of α-taxilin expression does not significantly affect genome replication, the overexpression of α-taxilin prevents the release of HCV. In contrast with this, silencing of α-taxilin expression leads to increased release of infectious viral particles. This is due to the negative effect of α-taxilin on t-SNARE formation that leads to impaired vesicular trafficking. Accordingly, overexpression of the t-SNARE component syntaxin-4 increases release of HCV, whereas silencing leads to an impaired release. These data identify α-taxilin as a novel factor that controls the release of HCV and reveal the mechanism by which HCV controls the activity of α-taxilin.


Subject(s)
Hepacivirus/metabolism , Vesicular Transport Proteins/biosynthesis , Hep G2 Cells , Humans , Synaptic Vesicles/metabolism
5.
J Hepatol ; 62(4): 791-8, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25445396

ABSTRACT

BACKGROUND & AIMS: Hepatitis B virus genotype G (HBV/G) is characterized by a lack of HBeAg secretion and very low HBsAg secretion. This study aimed at (1) comparing HBV genotype G and A2 with respect to morphogenesis and release of HBV-derived particles, (2) characterizing factors contributing to HBV/G-associated pathogenesis. METHODS: HBV/G- and HBV/A-expressing hepatoma cells and infected HepaRG cells were analyzed by confocal laser scanning microscopy, Western blot, real-time PCR, density gradient centrifugation, and electron microscopy. Modulation of the transcription factors Nrf2 and AP-1 was analyzed. RESULTS: While the release of viral particles is not affected in HBV/G replicating cells, the secretion of subviral particles is impaired, although they are produced in high amounts. These subviral particles, which display an increased density and a predominantly filamentous morphology, accumulate at the endoplasmic reticulum. The PreS1PreS2 domain of genotype G, which forms aggregates, causes the block of HBsAg-secretion at the ER and leads to decreased transcriptional activator function of LHBs. Intracellular accumulation of HBsAg and impaired induction of the cytoprotective transcription factor Nrf2 lead to an elevated level of ROIs. This results in activation of JNK and as a consequence in Ser-phosphorylation of IRS-1, which is known to impair insulin signaling, a key factor for liver regeneration. CONCLUSIONS: Although competent for release of viral particles, secretion of subviral particles is impaired in HBV/G expressing cells leading to ER-stress. In parallel, HBV-induced Nrf2 activation diminishes, which causes a decrease of the capacity to inactivate ROIs. This might be related to genotype-specific pathogenesis.


Subject(s)
Hepatitis B Surface Antigens/immunology , Hepatitis B e Antigens/immunology , Hepatitis B virus/genetics , Hepatitis B/immunology , NF-E2-Related Factor 2/metabolism , Transcription Factor AP-1/metabolism , Cell Line, Tumor , Genotype , Hepatitis B virus/immunology , Hepatocytes/immunology , Humans , Virion/metabolism
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