Identification of Novel Interaction between ADAM17 (a Disintegrin and Metalloprotease 17) and Thioredoxin-1

ADAM17, which is also known as TNFα-converting enzyme, is the major sheddase for the EGF receptor ligands and is considered to be one of the main proteases responsible for the ectodomain shedding of surface proteins. How a membrane-anchored proteinase with an extracellular catalytic domain can be activated by inside-out regulation is not completely understood. We characterized thioredoxin-1 (Trx-1) as a partner of the ADAM17 cytoplasmic domain that could be involved in the regulation of ADAM17 activity. We induced the overexpression of the ADAM17 cytoplasmic domain in HEK293 cells, and ligands able to bind this domain were identified by MS after protein immunoprecipitation. Trx-1 was also validated as a ligand of the ADAM17 cytoplasmic domain and full-length ADAM17 recombinant proteins by immunoblotting, immunolocalization, and solid phase binding assay. In addition, using nuclear magnetic resonance, it was shown in vitro that the titration of the ADAM17 cytoplasmic domain promotes changes in the conformation of Trx-1. The MS analysis of the cross-linked complexes showed cross-linking between the two proteins by lysine residues. To further evaluate the functional role of Trx-1, we used a heparin-binding EGF shedding cell model and observed that the overexpression of Trx-1 in HEK293 cells could decrease the activity of ADAM17, activated by either phorbol 12-myristate 13-acetate or EGF. This study identifies Trx-1 as a novel interaction partner of the ADAM17 cytoplasmic domain and suggests that Trx-1 is a potential candidate that could be involved in ADAM17 activity regulation.     

Limits of detection of a total reflection x-ray fluorescence system with double reflection module

An X-ray tube with a Mo target and Zr filter, operated at 45 kV/20 mA, was used to excite samples (5 ΜL deposited on a quartz support) and the total reflection angle condition was obtained with a double reflector module built with two 10-cm-long 7-mm-thick quartz crystals placed 50 Μm apart. A high-resolution spectrometer based on a Si(Li) detector coupled to a multichannel analyzer was used for X-ray detection and the spectra were interpreted with the AXIL software. The system was calibrated with standard chemical solutions containing Cr, Fe, Cu, Zn, and Pb, and Y was used as an internal standard to correct eventual geometric errors and high-voltage instabilities of the X-ray generator. The limits of detection were 19, 9, 5, and 4 ng/mL for Cr, Fe, Cu, and Zn, respectively, analyzed through characteristicKk _α X-rays, and 7 ng/mL for Pb, throughLk _α X-rays, considering 50 ΜL samples deposited and dried on a quartz support, to be excited/ detected for 1000 s.     

The human nucleolar protein FTSJ3 associates with NIP7 and functions in pre-rRNA processing

NIP7 is one of the many trans-acting factors required for eukaryotic ribosome biogenesis, which interacts with nascent pre-ribosomal particles and dissociates as they complete maturation and are exported to the cytoplasm. By using conditional knockdown, we have shown previously that yeast Nip7p is required primarily for 60S subunit synthesis while human NIP7 is involved in the biogenesis of 40S subunit. This raised the possibility that human NIP7 interacts with a different set of proteins as compared to the yeast protein. By using the yeast two-hybrid system we identified FTSJ3, a putative ortholog of yeast Spb1p, as a human NIP7-interacting protein. A functional association between NIP7 and FTSJ3 is further supported by colocalization and coimmunoprecipitation analyses. Conditional knockdown revealed that depletion of FTSJ3 affects cell proliferation and causes pre-rRNA processing defects. The major pre-rRNA processing defect involves accumulation of the 34S pre-rRNA encompassing from site A' to site 2b. Accumulation of this pre-rRNA indicates that processing of sites A0, 1 and 2 are slower in cells depleted of FTSJ3 and implicates FTSJ3 in the pathway leading to 18S rRNA maturation as observed previously for NIP7. The results presented in this work indicate a close functional interaction between NIP7 and FTSJ3 during pre-rRNA processing and show that FTSJ3 participates in ribosome synthesis in human cells.     

Production of polyclonal antibodies against the human respiratory syncytial virus nucleoprotein and phosphoprotein expressed in Escherichia coli

The nucleoprotein (N) and the phosphoprotein (P) of the human respiratory syncytial virus (HRSV), A2 strain, were cloned into pMAL-c2e vector. The proteins were expressed fused with the maltose-binding protein (MBP) and were preferentially found in the soluble fraction of the bacterial lysate. After their purification using amylose resin, almost no other protein was detected in SDS-PAGE. The fused proteins were cleaved by digestion with enterokinase and then used as antigens for BALB/c mice immunization. The obtained polyclonal antibodies were tested against HRSV infected cells in immunofluorescence assays. The results indicate that the antibodies generated against the recombinant proteins were able to recognize the virus proteins. We now intend to purify the cleaved N and P proteins and use them in structural studies. The recombinant proteins will also be tested as potential inducers of a protective immunity against the HRSV.