Water dynamics and stability of major blood proteins at pre-denaturation stage

We investigate the temperature effect on the size and stability of two major blood plasma proteins, human serum albumin and fibrinogen in aqueous NaCl solution. Dynamic Light Scattering measurements were carried out in the physiological temperature range up to 45 degrees C. The analysis of the results provided the temperature dependences of the macromolecular hydrodynamic radius and the potential. For albumin the hydrodynamic radius remained unchanged, while the zeta-potential increased sharply at approximately 40 degrees C. For fibrinogen the radius increased significantly above 45 degrees C and the zeta-potential increased similar to albumin at slightly below 40 degrees C. The dynamics of albumin macromolecule was simulated using classical Molecular Dynamics, which showed no change in the gyration radius, root mean square deviation, and the composition of disulfide and salt bridges, but substantial change in the secondary structure of the protein. We conclude that these changes in the structure and dynamics of the proteins are correlated with the qualitative change of water dynamics at 42 degrees C in the hydration shell of the proteins

The Soluble Heparin-Binding EGF-Like Growth Factor Stimulates EGF Receptor Trafficking to the Nucleus.

Most ligands of epidermal growth factor receptor (EGFR) have the ability to induce EGFR translocation into the nucleus, where EGFR acts as an important transcriptional regulator. Soluble form of heparin-binding EGF-like growth factor (sHB-EGF) is one of the ligands for EGFR in many cell types; however, there is no evidence for the ability of sHB-EGF to induce EGFR nuclear importation. Here, we demonstrated that treatment of A431 cells with sHB-EGF resulted in nuclear localization of EGFR and such translocation occurs via retrograde pathway. It was shown by confocal microscopy and co-immunoprecipitation assay that the translocation complex consisted of both ligand and receptor. The chromatin immunoprecipitation assay showed the association of sHB-EGF-EGFR complex with promoter region of cyclin D1 in the cell nucleus and this association was prevented by application of EGFR kinase inhibitor AG-1478. The obtained data suggest that sHB-EGF acts similarly to other EGFR ligands and is capable to induce EGFR nuclear translocation as a part of ligand-receptor complex in a tyrosine phosphorylation-dependent manner

A time-dependent co-localization of mCherry-sHB-EGF with CGN-ER membranes in A431 cells.

<p>Cells were transfected with p23-EYFP and treated with sHB-EGF during different time periods. AG1478 was added during 60 min of sHB-EGF treatment. (mCherry-sHB-EGF <i>(Red)</i>, CGN-ER membranes <i>(green)</i>; co-localization of mCherry-sHB-EGF with CGN-ER membranes <i>(yellow</i> and <i>white)</i>). Co-localization data was calculated with FIJI software. White scale bar corresponding to 5 um.</p

The EGFR localization in nuclear and cytoplasmic fractions after treatment of A431 cells with sHB-EGF.

<p>Non-nuclear and nuclear cellular extracts were subjected to western-blot analysis after sHB-EGF treatment in the absence or presence of EGFR tyrosine kinase inhibitor AG1478. Down-regulation of EGFR activity by kinase inhibitor AG1478 suppressed the EGFR nuclear localization in A431 cells under sHB-EGF treatment.</p

Chromatin immunoprecipitation with anti-GST antibodies to identify the nuclear localization of GST-sHB-EGF.

<p>Down-regulation of EGFR activity by kinase inhibitor AG1478 and endocytosis inhibitor (PAO) suppressed the association of GST—sHB-EGF with the cyclin D1 promoter in A431 cells. <i>Inp</i>—input nuclear DNA.</p

Chromatin immunoprecipitation with anti-EGFR monoclonal antibody.

<p><b>A</b>, <b>B.</b> Nuclear localization of EGFR following sHB-EGF (A) and H₂O₂ (B) treatment. Increased association of nuclear EGFR and the cyclin D1 promoter region following sHB-EGF treatment is shown. <b>C</b>, Down-regulation of EGFR activity by kinase inhibitor AG1478 suppressed the association of EGFR with the cyclin D1 promoter in A431 cells under sHB-EGF treatment.</p