Impact of intracellular ionic strength on dimer binding in the NF-kB Inducing kinase.

Improper signaling of the nuclear factor-κB (NF-κB) pathway plays a critical role in many inflammatory disease states including cancer, stroke, and viral infections. Although the signaling pathways are known, how these molecular mechanisms respond to changes in the intracellular microenvironment such as pH, ionic strength, and temperature, remains elusive. Molecular dynamics simulations were employed to differentiate the structural dynamics of the NF-κB Inducing Kinase (NIK), a protein kinase responsible for invoking the non-canonical NF-κB pathway, in its native and mutant form, and in the absence and presence of salt concentration in efforts to probe whether changes in the ionic environment stabilize or destabilize the NIK dimer. Analyses of structure-activity and conformational-activity relationships indicate that the protein-protein interactions are sensitive to changes in the ionic strength. Ligand binding pockets as well as regions between the oligomer interface either compress or expand, affecting both local and distal intermolecular interactions that result in stabilization or destabilization in the protein assembly.

Adaptive redox response of mesenchymal stromal cells to stimulation with lipopolysaccharide inflammagen: mechanisms of remodeling of tissue barriers in sepsis.

Acute bacterial inflammation is accompanied by excessive release of bacterial toxins and production of reactive oxygen and nitrogen species (ROS and RNS), which ultimately results in redox stress. These factors can induce damage to components of tissue barriers, including damage to ubiquitous mesenchymal stromal cells (MSCs), and thus can exacerbate the septic multiple organ dysfunctions. The mechanisms employed by MSCs in order to survive these stress conditions are still poorly understood and require clarification. In this report, we demonstrated that in vitro treatment of MSCs with lipopolysaccharide (LPS) induced inflammatory responses, which included, but not limited to, upregulation of iNOS and release of RNS and ROS. These events triggered in MSCs a cascade of responses driving adaptive remodeling and resistance to a "self-inflicted" oxidative stress. Thus, while MSCs displayed high levels of constitutively present adaptogens, for example, HSP70 and mitochondrial Sirt3, treatment with LPS induced a number of adaptive responses that included induction and nuclear translocation of redox response elements such as NFkB, TRX1, Ref1, Nrf2, FoxO3a, HO1, and activation of autophagy and mitochondrial remodeling. We propose that the above prosurvival pathways activated in MSCs in vitro could be a part of adaptive responses employed by stromal cells under septic conditions.

Macroscopic 2D networks self-assembled from nanometer-sized protein/DNA complexes.

We demonstrate the self-assembly of DNA and DNA binding proteins into two-dimensional networks that are then addressable by sending a second protein to a specific recognition site on the DNA network. These networks cover centimeters in area but can be addressed with nanometer precision. This hierarchical self-assembly of specific DNA protein complexes will be the basis for complex positioning of single molecules in two and three dimensions.

A novel DNA recognition mode by the NF-kappa B p65 homodimer.

The crystal structure of the NF-kappa B p65 (RelA) homodimer in complex with a DNA target has been determined to 2.4 A resolution. The two p65 subunits are not symmetrically disposed on the DNA target. The homodimer should optimally bind to a pseudo-palindromic nine base pair target with each subunit recognizing a 5'GGAA-3' half site separated by a central A-T base pair. However, one of the subunits (subunit B) encounters a half site of 5'-GAAA-3'. The single base-pair change from G-C to A-T results in highly unfavorable interactions between this half site and the base contacting protein residues in subunit B, which leads to an 18 degrees rotation of the N-terminal terminal domain from its normal conformation. Remarkably, subunit B retains all the interactions with the sugar phosphate backbone of the DNA target. This mode of interaction allows the NF-kappa B p65 homodimer to recognize DNA targets containing only one cognate half site. Differences in the sequence of the other half site provide variations in conformation and affinity of the complex.

Engineering of diffraction-quality crystals of the NF-kappaB P52 homodimer:DNA complex.

The eukaryotic transcription factors NF-kappaB P50 and NF-kappaB P52 are closely related members of the Rel family. Growth of diffraction-quality NF-kappaB P52:DNA co-crystals crucially depended on (a) extensive screens for the DNA fragment of optimal length and (b) engineering of the protein based on the two known NF-kappaB P50:DNA co-crystal structures [Müller et al. (1995) Nature 373, 311-317; Ghosh et al. (1995) Nature 373, 303-310]; namely, deletion of 12 C-terminal amino acid residues. These residues are part of the Rel homology region and comprise the nuclear localization signal. The approach might be of general use for the crystallization of other Rel protein: DNA complexes and in our case yielded co-crystals which diffract beyond 2.0 A resolution.

DNA binding alters the protease susceptibility of the p50 subunit of NF-kappa B.

The subdomain structure of the p50 subunit of NF-kappa B (amino acids 35-381) was investigated by partial proteolysis of the native protein. Trypsin cleaves p50 at a limited number of sites with an initial cleavage at low trypsin concentration occurring after R362 and a second cleavage taking place at higher trypsin concentration after K77. The cleavage after R362 does not alter the DNA binding characteristics of p50 but removes the nuclear localisation signal indicating that this region occupies a highly exposed position on the surface of the protein. The second cleavage after K77 generates a protein that although dimeric is incapable of binding DNA, thus emphasising the importance of residues 35-77 in DNA recognition. However p50 dimers containing one molecule cleaved after K77 and one molecule with this region intact are capable of binding DNA. When very high concentrations of trypsin are employed p50 is completely degraded. However if p50 is bound tightly to DNA containing its specific recognition site prior to trypsin addition the cleavage after K77 is almost completely blocked and the protein becomes highly resistant to proteolysis. These data suggest that bound DNA may mask critical trypsin cleavage sites or that DNA binding is accompanied by a conformational change in protein structure that renders the protein resistant to proteolysis.