Successful data recovery from oscillation photographs containing strong polycrystalline diffraction rings from an unknown small-molecule contaminant: preliminary structure solution of Salmonella typhimurium pyridoxal kinase (PdxK)

Pyridoxal kinase (PdxK; EC 2.7.1.35) belongs to the phosphotransferase family of enzymes and catalyzes the conversion of the three active forms of vitamin B-6, pyridoxine, pyridoxal and pyridoxamine, to their phosphorylated forms and thereby plays a key role in pyridoxal 5 `-phosphate salvage. In the present study, pyridoxal kinase from Salmonella typhimurium was cloned and overexpressed in Escherichia coli, purified using Ni-NTA affinity chromatography and crystallized. X-ray diffraction data were collected to 2.6 angstrom resolution at 100 K. The crystal belonged to the primitive orthorhombic space group P2(1)2(1)2(1), with unitcell parameters a = 65.11, b = 72.89, c = 107.52 angstrom. The data quality obtained by routine processing was poor owing to the presence of strong diffraction rings caused by a polycrystalline material of an unknown small molecule in all oscillation images. Excluding the reflections close to powder/polycrystalline rings provided data of sufficient quality for structure determination. A preliminary structure solution has been obtained by molecular replacement with the Phaser program in the CCP4 suite using E. coli pyridoxal kinase (PDB entry 2ddm) as the phasing model. Further refinement and analysis of the structure are likely to provide valuable insights into catalysis by pyridoxal kinases.

Alkyl chain substituted 1,9-pyrazoloanthrones exhibit prominent inhibitory effect on c-Jun N-terminal kinase (JNK)

N-Alkyl substituted pyrazoloanthrone derivatives were synthesized, characterized and tested for their in vitro inhibitory activity over c-Jun N-terminal kinase (JNK). Among the tested molecules, a few derivatives showed significant inhibitory activity against JNK with minimal off-target effect on other mitogen-activated protein kinase (MAP kinase) family members such as MEK1/2 and MKK3,6. These results suggested that N-alkyl (propyl and butyl) bearing pyrazoloanthrone scaffolds provide promising therapeutic inhibitors for JNK in regulating inflammation associated disorders.

Interferon-gamma induced cell death: Regulation and contributions of nitric oxide, cJun N-terminal kinase, reactive oxygen species and peroxynitrite

Interferon-gamma (Ifn gamma), a known immunomodulatory cytokine, regulates cell proliferation and survival. In this study, the mechanisms leading to the selective susceptibility of some tumor cells to Ifn gamma were deciphered. Seven different mouse tumor cell lines tested demonstrated upregulation of MHC class I to variable extents with Ifn gamma; however, only the cell lines, H6 hepatoma and L929 fibrosarcoma, that produce higher amounts of nitric oxide (NO) and reactive oxygen species (ROS) are sensitive to Ifn gamma-induced cell death. NO inhibitors greatly reduce Ifn gamma-induced ROS; however, ROS inhibitors did not affect the levels of Ifn gamma-induced NO, demonstrating that NO regulates ROS. Consequently, NO inhibitors are more effective, compared to ROS inhibitors, in reducing Ifn gamma-induced cell death. Further analysis revealed that Ifn gamma induces peroxynitrite and 3-nitrotyrosine amounts and a peroxynitrite scavenger, FeTPPS, reduces cell death. Ifn gamma treatment induces the phosphorylation of c-jun N-terminal kinase (Jnk) in H6 and L929 but not CT26, a colon carcinoma cell line, which is resistant to Ifn gamma-mediated death. Jnk activation downstream to NO leads to induction of ROS, peroxynitrite and cell death in response to Ifn gamma. Importantly, three cell lines tested, i.e. CT26, EL4 and Neuro2a, that are resistant to cell death with Ifn gamma alone become sensitive to the combination of Ifn gamma and NO donor or ROS inducer in a peroxynitrite-dependent manner. Overall, this study delineates the key roles of NO as the initiator and Jnk, ROS, and peroxynitrite as the effectors during Ifn gamma-mediated cell death. The implications of these findings in the Ifn gamma-mediated treatment of malignancies are discussed. (C) 2014 Elsevier B.V. All rights reserved.

Interferon-gamma induced cell death: Regulation and contributions of nitric oxide, cJun N-terminal kinase, reactive oxygen species and peroxynitrite

Interferon-gamma (Ifn gamma), a known immunomodulatory cytokine, regulates cell proliferation and survival. In this study, the mechanisms leading to the selective susceptibility of some tumor cells to Ifn gamma were deciphered. Seven different mouse tumor cell lines tested demonstrated upregulation of MHC class I to variable extents with Ifn gamma; however, only the cell lines, H6 hepatoma and L929 fibrosarcoma, that produce higher amounts of nitric oxide (NO) and reactive oxygen species (ROS) are sensitive to Ifn gamma-induced cell death. NO inhibitors greatly reduce Ifn gamma-induced ROS; however, ROS inhibitors did not affect the levels of Ifn gamma-induced NO, demonstrating that NO regulates ROS. Consequently, NO inhibitors are more effective, compared to ROS inhibitors, in reducing Ifn gamma-induced cell death. Further analysis revealed that Ifn gamma induces peroxynitrite and 3-nitrotyrosine amounts and a peroxynitrite scavenger, FeTPPS, reduces cell death. Ifn gamma treatment induces the phosphorylation of c-jun N-terminal kinase (Jnk) in H6 and L929 but not CT26, a colon carcinoma cell line, which is resistant to Ifn gamma-mediated death. Jnk activation downstream to NO leads to induction of ROS, peroxynitrite and cell death in response to Ifn gamma. Importantly, three cell lines tested, i.e. CT26, EL4 and Neuro2a, that are resistant to cell death with Ifn gamma alone become sensitive to the combination of Ifn gamma and NO donor or ROS inducer in a peroxynitrite-dependent manner. Overall, this study delineates the key roles of NO as the initiator and Jnk, ROS, and peroxynitrite as the effectors during Ifn gamma-mediated cell death. The implications of these findings in the Ifn gamma-mediated treatment of malignancies are discussed. (C) 2014 Elsevier B.V. All rights reserved.

Calcium binding properties of calcium dependent protein kinase 1 (CaCDPK1) from Cicer arietinum

Calcium plays a crucial role as a secondary messenger in all aspects of plant growth, development and survival. Calcium dependent protein kinases (CDPKs) are the major calcium decoders, which couple the changes in calcium level to an appropriate physiological response. The mechanism by which calcium regulates CDPK protein is not well understood. In this study, we investigated the interactions of Ca2+ ions with the CDPK1 isoform of Cicer arietinum (CaCDPK1) using a combination of biophysical tools. CaCDPK1 has four different EF hands as predicted by protein sequence analysis. The fluorescence emission spectrum of CaCDPK1 showed quenching with a 5 nm red shift upon addition of calcium, indicating conformational changes in the tertiary structure. The plot of changes in intensity against calcium concentrations showed a biphasic curve with binding constants of 1.29 mu M and 120 mu M indicating two kinds of binding sites. Isothermal calorimetric (ITC) titration with CaCl2 also showed a biphasic curve with two binding constants of 0.027 mu M and 1.7 mu M. Circular dichroism (CD) spectra showed two prominent peaks at 208 and 222 nm indicating that CaCDPK1 is a alpha-helical rich protein. Calcium binding further increased the alpha-helical content of CaCDPK1 from 75 to 81%. Addition of calcium to CaCDPK1 also increased fluorescence of 8-anilinonaphthalene-1-sulfonic acid (ANS) indicating exposure of hydrophobic surfaces. Thus, on the whole this study provides evidence for calcium induced conformational changes, exposure of hydrophobic surfaces and heterogeneity of EF hands in CaCDPK1. (C) 2015 Elsevier GmbH. All rights reserved.

Imaging cellular signalling: many `moving tales' in MAP kinase odyssey

Cellular signalling events are at the core of every adaptive response. Signalling events link environmental changes to physiological responses, consequently allowing cellular and organismal sustenance and survival. Classical approaches to study cellular signalling have relied on a variety of cell disruptive techniques which yield limited kinetic information, while the underlying events are much more complex. In this article, we discuss how modern live cell imaging microscopy has found increasing utilization in revealing spatio temporal dynamics of various signalling pathways. Utilizing the well studied mitogen-activated protein kinase (MAPK) signalling cascade as a template, the design, construction and utilization of `mobile' (translocation proficient) biosensors, suitable for studying MAPK signalling in living cells are described in detail. Experimental setup and results obtained from these biosensors, based on different proteins involved in the MAPK signalling cascade, have been described along with the setup of a microscope optimal for live cell imaging applications. Utilizing the ability to activate or deactivate signalling pathways using defined activators and specific pharmacological inhibitors, we also show how these sensors can yield unique spatial and temporal kinetic information of signalling in living cells.

Structures of substrate- and nucleotide-bound propionate kinase from Salmonella typhimurium: substrate specificity and phosphate-transfer mechanism

Kinases are ubiquitous enzymes that are pivotal to many biochemical processes. There are contrasting views on the phosphoryl-transfer mechanism in propionate kinase, an enzyme that reversibly transfers a phosphoryl group from propionyl phosphate to ADP in the final step of non-oxidative catabolism of L-threonine to propionate. Here, X-ray crystal structures of propionate- and nucleotide-bound Salmonella typhimurium propionate kinase are reported at 1.8-2.0 angstrom resolution. Although the mode of nucleotide binding is comparable to those of other members of the ASKHA superfamily, propionate is bound at a distinct site deeper in the hydrophobic pocket defining the active site. The propionate carboxyl is at a distance of approximate to 5 angstrom from the -phosphate of the nucleotide, supporting a direct in-line transfer mechanism. The phosphoryl-transfer reaction is likely to occur via an associative S(N)2-like transition state that involves a pentagonal bipyramidal structure with the axial positions occupied by the nucleophile of the substrate and the O atom between the - and the -phosphates, respectively. The proximity of the strictly conserved His175 and Arg236 to the carboxyl group of the propionate and the -phosphate of ATP suggests their involvement in catalysis. Moreover, ligand binding does not induce global domain movement as reported in some other members of the ASKHA superfamily. Instead, residues Arg86, Asp143 and Pro116-Leu117-His118 that define the active-site pocket move towards the substrate and expel water molecules from the active site. The role of Ala88, previously proposed to be the residue determining substrate specificity, was examined by determining the crystal structures of the propionate-bound Ala88 mutants A88V and A88G. Kinetic analysis and structural data are consistent with a significant role of Ala88 in substrate-specificity determination. The active-site pocket-defining residues Arg86, Asp143 and the Pro116-Leu117-His118 segment are also likely to contribute to substrate specificity.

Ac2PIM-responsive miR-150 and miR-143 Target Receptor-interacting Protein Kinase 2 and Transforming Growth Factor Beta-activated Kinase 1 to Suppress NOD2-induced Immunomodulators

Specific and coordinated regulation of innate immune receptor-driven signaling networks often determines the net outcome of the immune responses. Here, we investigated the cross-regulation of toll-like receptor (TLR)2 and nucleotide-binding oligomerization domain (NOD)2 pathways mediated by Ac2PIM, a tetra-acylated form of mycobacterial cell wall component and muramyl dipeptide (MDP), a peptidoglycan derivative respectively. While Ac2PIM treatment of macrophages compromised their ability to induce NOD2-dependent immunomodulators like cyclooxygenase (COX)-2, suppressor of cytokine signaling (SOCS)-3, and matrix metalloproteinase (MMP)-9, no change in the NOD2-responsive NO, TNF-alpha, VEGF-A, and IL-12 levels was observed. Further, genome-wide microRNA expression profiling identified Ac2PIM-responsive miR-150 and miR-143 to target NOD2 signaling adaptors, RIP2 and TAK1, respectively. Interestingly, Ac2PIM was found to activate the SRC-FAK-PYK2-CREB cascade via TLR2 to recruit CBP/P300 at the promoters of miR-150 and miR-143 and epigenetically induce their expression. Loss-of-function studies utilizing specific miRNA inhibitors establish that Ac2PIM, via the miRNAs, abrogate NOD2-induced PI3K-PKC delta-MAPK pathway to suppress beta-catenin-mediated expression of COX-2, SOCS-3, and MMP-9. Our investigation has thus underscored the negative regulatory role of Ac2PIM-TLR2 signaling on NOD2 pathway which could broaden our understanding on vaccine potential or adjuvant utilities of Ac2PIM and/or MDP.