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A comprehensive quantum chemical analysis of the influence of backbone torsion angles on 31P chemical shifts in DNAs has been carried out. An extensive DFT study employed snapshots obtained from the molecular dynamics simulation of [d(CGCGAATTCGCG)]2 to construct geometries of a hydrated dimethyl phosphate, which was used as a model for the phosphodiester linkage. Our calculations provided differences of 2.1 ± 0.3 and 1.6 ± 0.3 ppm between the BI and BII chemical shifts in two B-DNA residues of interest, which is in a very good agreement with the difference of 1.6 ppm inferred from experimental data. A more negative 31P chemical shift for a residue in pure BI conformation compared to residues in mixed BI/BII conformation states is provided by DFT, in agreement with the NMR experiment. Statistical analysis of the MD/DFT data revealed a large dispersion of chemical shifts in both BI and BII regions of DNA structures. δP ranges within 3.5 ± 0.8 ppm in the BI region and within 4.5 ± 1.5 ppm in the BII region. While the 31P chemical shift becomes more negative with increasing α in BI-DNA, it has the opposite trend in BII-DNA when both α and ζ increase simultaneously. The 31P chemical shift is dominated by the torsion angles α and ζ, while an implicit treatment of β and ε is sufficient. The presence of an explicit solvent leads to a damping and a 2−3 ppm upfield shift of the torsion angle dependences.
As a part of the innate immunity, NK (Natural Killer) cells provide an early immune response to different stimuli, e.g. viral infections and tumor growths. However, their functions are more complex; they play an important role in reproduction, alloimmunity, autoimmunity and allergic diseases. NK cell activities require an intricate system of regulation that is ensured by many different receptors on a cell surface which integrate signals from interacting cells and soluble factors. One way to understand NK cell biology is through the structure of NK receptors, which can reveal ligand binding conditions. We present a modified protocol for recombinant expression in Escherichia coli and in vitro refolding of the ligand-binding domain of the inhibitory Nkrp1b (SJL/J) protein. Nkrp1b identity and folding was confirmed using mass spectrometry (accurate mass of the intact protein and evaluation of disulfide bonds) and one-dimensional nuclear magnetic resonance spectroscopy. The intention is to provide the basis for conducting structural studies of the inhibitory Nkrp1b protein, since only the activating Nkrp1a receptor structure is known.
Purpose: Mild hyperthermia can improve tumour oxygenation and enhance radiosensitivity. Imaging the hypoxic fraction of a tumour can guide hyperthermia treatment planning and facilitate treatment optimization. 64Cu-ATSM (Copper-diacetyl-bis(N4-methylthiosemicarbazone)) is a positron emitting compound that has been demonstrated to have rapid uptake and selective retention in hypoxic cells and has been used for imaging human and animal tumours. The purpose of the present report is to establish methodology that will allow one to use Cu-ATSM PET scanning to detect the impact of hyperthermia on tumour physiology in as little time as possible.Materials and methods: EMT6 tumours (mouse mammary carcinoma) were implanted into the subcutaneous tissue of both thighs of 10 BALB/c mice (one heated, one control tumour per animal). The target thermal dose was 41.5°C × 45 min. Without interrupting heating, 64Cu-ATSM (mean activity 1.8 mCi) was then injected and serial PET scans were obtained. In a sub-group of four animals, a low administered activity (∼0.3 mCi) 64Cu-ATSM scan was also conducted before heating to permit a direct comparison of the effects of hyperthermia on the same tumours. In another sub-group of five animals, a low activity (∼0.3 mCi) 64Cu-PTSM (pyruvaldehyde-bis(N*-methylthiosemicarbazone)) scan was conducted before heating, to confirm a posited correlation between perfusion and early 64Cu-ATSM uptake.Results: This study corrected for perfusion differences by dividing tumour uptake by the average early (first minute) uptake (‘self-normalized uptake’). The 10 heated tumours showed a significantly (p = 0.007) lower self-normalized uptake than control tumours by 2 min. For the four mice with low activity Cu-ATSM scans performed before hyperthermia, the tumours to be heated demonstrated self-normalized uptake consistent with the unheated control tumours and which departed significantly (p ≤ 0.02) from their post-hyperthermia scans by 5 min. Comparisons between scans and needle electrode surveys were performed in an additional four animals with eight tumours. For technical reasons electrode surveys were done after the end of hyperthermia—and, therefore, these animals also had comparison scans taken after hyperthermia. Reduced self-normalized uptake on scans was associated with increased pO2 on electrode surveys. These data also suggested a substantial degradation of the effect on tumour hypoxia by ∼15–45 min after the end of mild hyperthermia.Conclusion: Short imaging times of ∼5 min with modest (∼4–10) numbers of mice can discriminate the effects of mild hyperthermia on tumour physiology. The long-term objective is to use this tool to identify as short and mild a hyperthermia session as possible.
Recent work using chemical cross-linking to define interresidue distance constraints in proteins has shown that these constraints are useful for testing tertiary structural models. We applied this approach to the G-protein-coupled receptor bovine rhodopsin in its native membrane using lysine- and cysteine-targeted bifunctional cross-linking reagents. Cross-linked proteolytic peptides of rhodopsin were identified by combined liquid chromatography and FT-ICR mass spectrometry with automated data-reduction and assignment software. Tandem mass spectrometry was used to verify cross-link assignments and locate the exact sites of cross-link attachment. Cross-links were observed to form between 10 pairs of residues in dark-state rhodopsin. For each pair, cross-linkers with a range of linker lengths were tested to determine an experimental distance-of-closest-approach (DCA) between reactive side-chain atoms. In all, 28 cross-links were identified using seven different cross-linking reagents. Molecular mechanics procedures were applied to published crystal structure data to calculate energetically achievable theoretical DCAs between reactive atoms without altering the position of the protein backbone. Experimentally measured DCAs are generally in good agreement with the theoretical DCAs. However, a cross-link between C316 and K325 in the C-terminal region cannot be rationalized by DCA simulations and suggests that backbone reorientation relative to the crystal coordinates occurs on the timescale of cross-linking reactions. Biochemical and spectroscopic data from other studies have found that the C-terminal region is highly mobile in solution and not fully represented by X-ray crystallography data. Our results show that chemical cross-linking can provide reliable three-dimensional structural information and insight into local conformational dynamics in a membrane protein.
The discrete activation of individual caspases is essential during T-cell development, activation, and apoptosis. Humans carrying nonfunctional caspase-8 and caspase-8 conditional knockout mice exhibit several defects in the progression of naive CD4+ T cells to the effector stage. MST1, a key kinase of the Hippo signaling pathway, is often presented as a substrate of caspases, and its cleavage by caspases potentiates its activity. Several studies have focused on the involvement of MST1 in caspase activation and also reported several defects in the immune system function caused by MST1 deficiency. Here, we show the rapid activation of the MEK-ERK-MST1 axis together with the cleavage and activation of caspase-3, -6, -7, -8, and -9 after PI3K signaling blockade by the selective inhibitor GDC-0941 in Jurkat T cells. We determined the phosphorylation pattern of MST1 using a phosphoproteomic approach and identified two amino acid residues phosphorylated in an ERK-dependent manner after GDC-0941 treatment together with a novel phosphorylation site at S21 residue, which was extensively phosphorylated in an ERK-independent manner during PI3K signaling blockade. Using caspase inhibitors and the inhibition of MST1 expression using siRNA, we identified an exclusive role of the MEK-ERK-MST1 axis in the activation of initiator caspase-8, which in turn activates executive caspase-3/-7 that finally potentiate MST1 proteolytic cleavage. This mechanism forms a positive feed-back loop that amplifies the activation of MST1 together with apoptotic response in Jurkat T cells during PI3K inhibition. Altogether, we propose a novel MEK-ERK-MST1-CASP8-CASP3/7 apoptotic pathway in Jurkat T cells and believe that the regulation of this pathway can open novel possibilities in systemic and cancer therapies.
