Annexins-a family of proteins with distinctive tastes for cell signaling and membrane dynamics

Annexins are cytosolic proteins with conserved three-dimensional structures that bind acidic phospholipids in cellular membranes at elevated Ca2+ levels. Through this they act as Ca2+-regulated membrane binding modules that organize membrane lipids, facilitating cellular membrane transport but also displaying extracellular activities. Recent discoveries highlight annexins as sensors and regulators of cellular and organismal stress, controlling inflammatory reactions in mammals, environmental stress in plants, and cellular responses to plasma membrane rupture. Here, we describe the role of annexins as Ca2+-regulated membrane binding modules that sense and respond to cellular stress and share our view on future research directions in the field.Work in our laboratories was supported by grants from the German Research Foundation to V.G. (CRC1348/A04, GE514/6-3) and U.R. (CRC1009/A06, CRC1348/A11); the Interdisciplinary Center for Clinical Research of the Münster Medical School to U.R. (Re2/022/20); the Danish Council for Independent Research (6108-00378A, 9040-00252B), Scientific Committee Danish Cancer Society (R90-A5847-14-S2, R269-A15812), and the Novo Nordisk Foundation (NNF18OC0034936) to J.N.; the Royal Society and Wolfson Foundation (RSWF\R3\183001) to F.N.E.G.; the National Institute of Health (NIH R01AR055686) to J.K.J.; the Ara Parseghian Medical Research Fund (G217137) to T.G

Canine respiratory coronavirus employs caveolin-1-mediated pathway for internalization to HRT-18G cells

Canine respiratory coronavirus (CRCoV), identified in 2003, is a member of the Coronaviridae family. The virus is a betacoronavirus and a close relative of human coronavirus OC43 and bovine coronavirus. Here, we examined entry of CRCoV into human rectal tumor cells (HRT-18G cell line) by analyzing co-localization of single virus particles with cellular markers in the presence or absence of chemical inhibitors of pathways potentially involved in virus entry. We also targeted these pathways using siRNA. The results show that the virus hijacks caveolin-dependent endocytosis to enter cells via endocytic internalization

Comparative Proteomics Analyses Reveal the virB of B. melitensis Affects Expression of Intracellular Survival Related Proteins

BACKGROUND: Brucella melitensis is a facultative, intracellular, pathogenic bacterium that replicates within macrophages. The type IV secretion system encoded by the virB operon (virB) is involved in Brucella intracellular survival. However, the underlying molecular mechanisms, especially the target proteins affected by the virB, remain largely unclear. METHODOLOGY/PRINCIPAL FINDINGS: In order to define the proteins affected by virB, the proteomes of wild-type and the virB mutant were compared under in vitro conditions where virB was highly activated. The differentially expressed proteins were identified by MALDI-TOF-MS. Forty-four down-regulated and eighteen up-regulated proteins which exhibited a 2-fold or greater change were identified. These proteins included those involved in amino acid transport and metabolism, lipid metabolism, energy production, cell membrane biogenesis, translation, post-translational modifications and protein turnover, as well as unknown proteins. Interestingly, several important virulence related proteins involved in intracellular survival, including VjbR, DnaK, HtrA, Omp25, and GntR, were down-regulated in the virB mutant. Transcription analysis of virB and vjbR at different growth phase showed that virB positively affect transcription of vjbR in a growth phase dependent manner. Quantitative RT-PCR showed that transcription of these genes was also affected by virB during macrophage cell infection, consistent with the observed decreased survival of the virB mutant in macrophage. CONCLUSIONS/SIGNIFICANCE: These data indicated that the virB operon may control the intracellular survival of Brucella by affecting the expression of relevant proteins

Interaction of Virstatin with Human Serum Albumin: Spectroscopic Analysis and Molecular Modeling

Virstatin is a small molecule that inhibits Vibrio cholerae virulence regulation, the causative agent for cholera. Here we report the interaction of virstatin with human serum albumin (HSA) using various biophysical methods. The drug binding was monitored using different isomeric forms of HSA (N form ∼pH 7.2, B form ∼pH 9.0 and F form ∼pH 3.5) by absorption and fluorescence spectroscopy. There is a considerable quenching of the intrinsic fluorescence of HSA on binding the drug. The distance (r) between donor (Trp214 in HSA) and acceptor (virstatin), obtained from Forster-type fluorescence resonance energy transfer (FRET), was found to be 3.05 nm. The ITC data revealed that the binding was an enthalpy-driven process and the binding constants Ka for N and B isomers were found to be 6.09×105 M−1 and 4.47×105 M−1, respectively. The conformational changes of HSA due to the interaction with the drug were investigated from circular dichroism (CD) and Fourier Transform Infrared (FTIR) spectroscopy. For 1∶1 molar ratio of the protein and the drug the far-UV CD spectra showed an increase in α- helicity for all the conformers of HSA, and the protein is stabilized against urea and thermal unfolding. Molecular docking studies revealed possible residues involved in the protein-drug interaction and indicated that virstatin binds to Site I (subdomain IIA), also known as the warfarin binding site

Structural analysis of vitamin A transport system

The method of isoraorphous replacement has been used to determine the crystal structure of human plasma prealbumin (Mwt. 56 000) Three derivatives were measured to a resolution of 2.5 Å and a mercury derivative was re-measured with a long x-ray count time to improve the quality of the estimates of anomalous scatter. A protein Fourier synthesis has been calculated using phases determined from the anomalous and isomorphous differences. The conformation of the two polypeptide chains in the asymmetric unit of P21212 (the crystal space group) has been determined. The chains in each monomer have an apparently identical conformation They are nearly fully extended and form an extensive network of (beta)-structure, Eight strands of (beta)-sheet in each monomer form a flattened cup shape the interior of which appears to be entirely hydrophobic. The ends and bottom of the cup are closed by loops in a random coil conformation. One loop contains a right-handed (alpha)-helix of about two turns. The top of the cup is closed by the second monomer,so that the front of this unit is a continuous 8-stranded (beta)-pleated sheet. The back of this is also formed by an 8-stranded p-sheet,but this is less regular. The dimers formed in this way interact so that their front sheets are opposed at a distance of 10 Å. The thyroxine-binding site has been identified within the channel formed by the (beta)-sheets of opposed dimers. There are two possible binding sites, only one of which may be occupied in one molecule (on chemical evidence).</p