Search results for "recombinant"
showing 10 items of 1150 documents
Identification and characterization of amphiphysin II as a novel cellular interaction partner of the hepatitis C virus NS5A protein.
2003
The hepatitis C virus (HCV) NS5A protein is highly phosphorylated by cellular protein kinases. To study how NS5A might be integrated in cellular kinase signalling, we isolated phosphoproteins from HuH-7 hepatoma cells that specifically interacted with recombinant NS5A protein. Subsequent mass spectrometry identified the adaptor protein amphiphysin II as a novel interaction partner of NS5A. Mutational analysis revealed that complex formation is primarily mediated by a proline-rich region in the C-terminal part of NS5A, which interacts with the amphiphysin II Src homology 3 domain. Importantly, we could further demonstrate specific co-precipitation and cellular co-localization of endogenous a…
Cytoglobin is a respiratory protein in connective tissue and neurons, which is up-regulated by hypoxia.
2004
Cytoglobin is a recently discovered vertebrate globin distantly related to myoglobin, and its function is unknown. Here we present the first detailed analysis of the distribution and expression of cytoglobin. Northern and Western blotting experiments show the presence of cytoglobin mRNA and protein in a broad range of tissues. Quantitative PCR demonstrates an up-regulation of cytoglobin mRNA levels in rat heart and liver under hypoxic conditions (22 and 44 h of 9% oxygen). Immunofluorescence studies with three antibodies directed against different epitopes of the protein consistently show cytoglobin in connective tissue fibroblasts as well as in hepatic stellate cells. Cytoglobin is also pr…
Rhodopsin's carboxy-terminal cytoplasmic tail acts as a membrane receptor for cytoplasmic dynein by binding to the dynein light chain Tctex-1.
1999
AbstractThe interaction of cytoplasmic dynein with its cargoes is thought to be indirectly mediated by dynactin, a complex that binds to the dynein intermediate chain. However, the roles of other dynein subunits in cargo binding have been unknown. Here we demonstrate that dynein translocates rhodopsin-bearing vesicles along microtubules. This interaction occurs directly between the C-terminal cytoplasmic tail of rhodopsin and Tctex-1, a dynein light chain. C-terminal rhodopsin mutations responsible for retinitis pigmentosa inhibit this interaction. Our results point to an alternative docking mechanism for cytoplasmic dynein, provide novel insights into the role of motor proteins in the pola…
Selective targeting of avidin/mannose 6-phosphate receptor chimeras to early or late endosomes
2000
Summary In this study we have used the Semliki forest virus expression system to transiently express chimeric proteins that contain transmembrane and cytoplasmic domains of the cation-independent mannose 6-phosphate receptor (CI-MPR) fused to chicken avidin. Immunofluorescence and electron microscopy studies showed that the chimeric protein with the entire cytoplasmic domain of CI-MPR was transported to late endosomes, where it accumulated. We made use of the biotin-binding capacity of lumenal avidin, and found that, in agreement with this distribution, the chimeric protein could be labelled with biotinylated HRP endocytosed for a long, but not a brief, period of time. However, truncation o…
Amyloid Precursor-like Protein 1 Influences Endocytosis and Proteolytic Processing of the Amyloid Precursor Protein
2005
Ectodomain shedding of the amyloid precursor protein (APP) is a key regulatory step in the generation of the Alzheimer disease amyloid beta peptide (Abeta). The molecular mechanisms underlying the control of APP shedding remain little understood but are in part dependent on the low density lipoprotein receptor-related protein (LRP), which is involved in APP endocytosis. Here, we show that the APP homolog APLP1 (amyloid precursor-like protein 1) influences APP shedding. In human embryonic kidney 293 cells expression of APLP1 strongly activated APP shedding by alpha-secretase and slightly reduced beta-secretase cleavage. As revealed by domain deletion analysis, the increase in APP shedding re…
ENO1 gene product binds to the c-myc promoter and acts as a transcriptional repressor: relationship with Myc promoter-binding protein 1 (MBP-1).
2000
The Myc promoter-binding protein-1 (MBP-1) is a 37-38 kDa protein that binds to the c-myc P2 promoter and negatively regulates transcription of the protooncogene. MBP-1 cDNA shares 97% similarity with the cDNA encoding the glycolytic enzyme alpha-enolase and both genes have been mapped to the same region of human chromosome 1, suggesting the hypothesis that the two proteins might be encoded by the same gene. We show here data indicating that a 37 kDa protein is alternatively translated from the full-length alpha-enolase mRNA. This shorter form of alpha-enolase is able to bind the MBP-1 consensus sequence and to downregulate expression of a luciferase reporter gene under the control of the c…
Interaction of Mitogen-activated Protein Kinases with the Kinase Interaction Motif of the Tyrosine Phosphatase PTP-SL Provides Substrate Specificity …
1999
ERK1 and ERK2 associate with the tyrosine phosphatase PTP-SL through a kinase interaction motif (KIM) located in the juxtamembrane region of PTP-SL. A glutathione S-transferase (GST)-PTP-SL fusion protein containing the KIM associated with ERK1 and ERK2 as well as with p38/HOG, but not with the related JNK1 kinase or with protein kinase A or C. Accordingly, ERK2 showed in vitro substrate specificity to phosphorylate GST-PTP-SL in comparison with GST-c-Jun. Furthermore, tyrosine dephosphorylation of ERK2 by the PTP-SLDeltaKIM mutant was impaired. The in vitro association of ERK1/2 with GST-PTP-SL was highly stable; however, low concentrations of nucleotides partially dissociated the ERK1/2.P…
A novel regulatory mechanism of MAP kinases activation and nuclear translocation mediated by PKA and the PTP-SL tyrosine phosphatase
1999
Protein tyrosine phosphatase PTP-SL retains mitogen-activated protein (MAP) kinases in the cytoplasm in an inactive form by association through a kinase interaction motif (KIM) and tyrosine dephosphorylation. The related tyrosine phosphatases PTP-SL and STEP were phosphorylated by the cAMP-dependent protein kinase A (PKA). The PKA phosphorylation site on PTP-SL was identified as the Ser231 residue, located within the KIM. Upon phosphorylation of Ser231, PTP-SL binding and tyrosine dephosphorylation of the MAP kinases extracellular signal–regulated kinase (ERK)1/2 and p38α were impaired. Furthermore, treatment of COS-7 cells with PKA activators, or overexpression of the Cα catalytic subunit …
Generation of tumor-reactive CTL against the tumor-associated antigen HER2 using retrovirally transduced dendritic cells derived from CD34+ hemopoiet…
2000
Abstract Ag-specific CD8+ CTL are crucial for effective tumor rejection. Attempts to treat human malignancies by adoptive transfer of tumor-reactive CTL have been limited due to the difficulty of generating and expanding autologous CTL with defined Ag specificity. The current study examined whether human CTL can be generated against the tumor-associated Ag HER2 using autologous dendritic cells (DC) that had been genetically engineered to express HER2. DC progenitors were expanded by culturing CD34+ hemopoietic progenitor cells in the presence of the designer cytokine HyperIL-6. Proliferating precursor cells were infected by a retroviral vector encoding the HER2 Ag and further differentiated…
Cytotoxic T lymphocytes define multiple peptide isoforms derived from the melanoma-associated antigen MART-1/Melan-A
1999
Peptides derived from the melanoma-associated MART-1/Melan-A antigen are currently implemented in immunotherapy for inducing or augmenting T-cell responses directed against peptides expressed by autologous tumor cells in HLA-A2+ patients with melanoma. Here, we describe the specificity of the T-cell clone SK29-FFM1.1, which secretes GM-CSF in response to a panel of synthetic MART-1/Melan-A-derived peptides, including the naturally presented ILTVILGVL32–40, but exhibits cytotoxicity and IFN-γ secretion exclusively to the MART-1/Melan-A derived peptide AAGIGILTV27–35. In addition, cytotoxic T-lymphocyte (CTL) clone SK29-FFM1.1 recognizes 3 different naturally processed and presented peptides …