STAT3 differential scanning fluorimetry and differential scanning light scattering assays: Addressing a missing link in the characterization of STAT3 inhibitor interactions

STAT3 protein is an established target for the development of new cancer therapeutic agents. Despite lacking a traditional binding site for small molecule inhibitors, many STAT3 inhibitors have been identified and explored for their anti-cancer activity. Because STAT3 signaling is mediated by protein-protein interactions, indirect methods are often employed to determine if proposed STAT3 inhibitors bind to STAT3 protein. While established STAT3 inhibition assays (such as the fluorescence polarization assay, electrophoretic mobility shift assay and ELISAs) have been used to identify novel inhibitors of STAT3 signaling, methods that directly assess STAT3 protein-inhibitor interactions could facilitate the development of novel inhibitors. In this context, we herein report new STAT3 binding assays based on differential scanning fluorimetry (DSF) and differential scanning light scattering (DSLS) to characterize interactions between STAT3 protein and inhibitors. Several peptide and small molecule STAT3 inhibitors have been evaluated, and new insight into how these compounds may interact with STAT3 is provided

Nonquasiparticle states in half-metallic ferromagnets

Anomalous magnetic and electronic properties of the half-metallic ferromagnets (HMF) have been discussed. The general conception of the HMF electronic structure which take into account the most important correlation effects from electron-magnon interactions, in particular, the spin-polaron effects, is presented. Special attention is paid to the so called non-quasiparticle (NQP) or incoherent states which are present in the gap near the Fermi level and can give considerable contributions to thermodynamic and transport properties. Prospects of experimental observation of the NQP states in core-level spectroscopy is discussed. Special features of transport properties of the HMF which are connected with the absence of one-magnon spin-flip scattering processes are investigated. The temperature and magnetic field dependences of resistivity in various regimes are calculated. It is shown that the NQP states can give a dominate contribution to the temperature dependence of the impurity-induced resistivity and in the tunnel junction conductivity. First principle calculations of the NQP-states for the prototype half-metallic material NiMnSb within the local-density approximation plus dynamical mean field theory (LDA+DMFT) are presented.Comment: 27 pages, 9 figures, Proceedings of Berlin/Wandlitz workshop 2004; Local-Moment Ferromagnets. Unique Properties for Moder Applications, ed. M. Donath, W.Nolting, Springer, Berlin, 200

Optimizing heterologous protein production in the periplasm of E. coli by regulating gene expression levels

Background: In Escherichia coli many heterologous proteins are produced in the periplasm. To direct these proteins to the periplasm, they are equipped with an N-terminal signal sequence so that they can traverse the cytoplasmic membrane via the protein-conducting Sec-translocon. For poorly understood reasons, the production of heterologous secretory proteins is often toxic to the cell thereby limiting yields. To gain insight into the mechanism(s) that underlie this toxicity we produced two secretory heterologous proteins, super folder green fluorescent protein and a single-chain variable antibody fragment, in the Lemo21(DE3) strain. In this strain, the expression intensity of the gene encoding the target protein can be precisely controlled.Results: Both SFGFP and the single-chain variable antibody fragment were equipped with a DsbA-derived signal sequence. Producing these proteins following different gene expression levels in Lemo21(DE3) allowed us to identify the optimal expression level for each target gene. Too high gene expression levels resulted in saturation of the Sec-translocon capacity as shown by hampered translocation of endogenous secretory proteins and a protein misfolding/aggregation problem in the cytoplasm. At the optimal gene expression levels, the negative effects of the production of the heterologous secretory proteins were minimized and yields in the periplasm were optimized.Conclusions: Saturating the Sec-translocon capacity can be a major bottleneck hampering heterologous protein production in the periplasm. This bottleneck can be alleviated by harmonizing expression levels of the genes encoding the heterologous secretory proteins with the Sec-translocon capacity. Mechanistic insight into the production of proteins in the periplasm is key to optimizing yields in this compartment. © 2013 Schlegel et al.; licensee BioMed Central Ltd

Binding of Pro Gly Pro at the active site of leukotriene A 4 hydrolase aminopeptidase and development of an epoxide hydrolase selective inhibitor

Leukotriene (LT) A(4) hydrolase/aminopeptidase (LTA4H) is a bifunctional zinc metalloenzyme that catalyzes the committed step in the formation of the proinflammatory mediator LTB(4). Recently, the chemotactic tripeptide Pro-Gly-Pro was identified as an endogenous aminopeptidase substrate for LTA(4) hydrolase. Here, we determined the crystal structure of LTA(4) hydrolase in complex with a Pro-Gly-Pro analog at 1.72 Å. From the structure, which includes the catalytic water, and mass spectrometric analysis of enzymatic hydrolysis products of Pro-Gly-Pro, it could be inferred that LTA(4) hydrolase cleaves at the N terminus of the palindromic tripeptide. Furthermore, we designed a small molecule, 4-(4-benzylphenyl)thiazol-2-amine, denoted ARM1, that inhibits LTB(4) synthesis in human neutrophils (IC(50) of ∼0.5 μM) and conversion of LTA(4) into LTB(4) by purified LTA4H with a K(i) of 2.3 μM. In contrast, 50- to 100-fold higher concentrations of ARM1 did not significantly affect hydrolysis of Pro-Gly-Pro. A 1.62-Å crystal structure of LTA(4) hydrolase in a dual complex with ARM1 and the Pro-Gly-Pro analog revealed that ARM1 binds in the hydrophobic pocket that accommodates the ω-end of LTA(4), distant from the aminopeptidase active site, thus providing a molecular basis for its inhibitory profile. Hence, ARM1 selectively blocks conversion of LTA(4) into LTB(4), although sparing the enzyme’s anti-inflammatory aminopeptidase activity (i.e., degradation and inactivation of Pro-Gly-Pro). ARM1 represents a new class of LTA(4) hydrolase inhibitor that holds promise for improved anti-inflammatory properties