Obtaining protein crystals suitable for X-ray diffraction studies comprises the greatest challenge in the determination of protein crystal structures, especially for membrane proteins and protein ...complexes. Although high purity has been broadly accepted as one of the most significant requirements for protein crystallization, a recent study of the Escherichia coli proteome showed that many proteins have an inherent propensity to crystallize and do not require a highly homogeneous sample (Totir et al., 2012). As exemplified by RPE65 (Kiser, Golczak, Lodowski, Chance, & Palczewski, 2009), there also are cases of mammalian proteins crystallized from less purified samples. To test whether this phenomenon can be applied more broadly to the study of proteins from higher organisms, we investigated the protein crystallization profile of bovine rod outer segment (ROS) crude extracts. Interestingly, multiple protein crystals readily formed from such extracts, some of them diffracting to high resolution that allowed structural determination. A total of seven proteins were crystallized, one of which was a membrane protein. Successful crystallization of proteins from heterogeneous ROS extracts demonstrates that many mammalian proteins also have an intrinsic propensity to crystallize from complex biological mixtures. By providing an alternative approach to heterologous expression to achieve crystallization, this strategy could be useful for proteins and complexes that are difficult to purify or obtain by recombinant techniques.
G protein-coupled receptor (GPCR) kinases (GRKs) phosphorylate activated heptahelical receptors, leading to their uncoupling from G proteins. Here we report six crystal structures of rhodopsin kinase ...(GRK1), revealing not only three distinct nucleotide-binding states of a GRK but also two key structural elements believed to be involved in the recognition of activated GPCRs. The first is the C-terminal extension of the kinase domain, which was observed in all nucleotide-bound GRK1 structures. The second is residues 5-30 of the N terminus, observed in one of the GRK1·(Mg2+)2·ATP structures. The N terminus was also clearly phosphorylated, leading to the identification of two novel phosphorylation sites by mass spectral analysis. Co-localization of the N terminus and the C-terminal extension near the hinge of the kinase domain suggests that activated GPCRs stimulate kinase activity by binding to this region to facilitate full closure of the kinase domain.
An association of acetaminophen use and asthma was observed in the International Study of Asthma and Allergies in Childhood study. However there are no clear mechanisms to explain an association ...between acetaminophen use and immunologic pathology. In acidic conditions like those in the stomach and inflamed airway, tyrosine residues are nitrated by nitrous and peroxynitrous acids. The resulting nitrotyrosine is structurally similar to 2,4-dinitrophenol and 2,4-dinitrochlorobenzene, known haptens that enhance immune responses by covalently binding proteins. Nitrated acetaminophen shares similar molecular structure.
We hypothesized the acetaminophen phenol ring undergoes nitration under acidic conditions, producing 3-nitro-acetaminophen which augments allergic responses by acting as a hapten for environmental allergens.
3-nitro-acetaminophen was formed from acetaminophen in the presence of acidified nitrite, purified by high performance liquid chromatography, and assayed by gas-chromatography mass spectrometry. Purified 3-nitro-acetaminophen was reacted with Dermatophagoides pteronyssinus (Der p1) and analyzed by mass spectrometry to identify the modification site. Human peripheral blood mononuclear cells proliferation response was measured in response to 3-nitro-acetaminophen and to 3-nitro-acetaminophen-modified Der p1.
Acetaminophen was modified by nitrous acid forming 3-nitro-acetaminophen over a range of different acidic conditions consistent with airway inflammation and stomach acidity. The Der p1 protein-hapten adduct creation was confirmed by liquid chromatography-mass spectrometry proteomics modifying cysteine 132. Peripheral blood mononuclear cells exposed to 3-nitro-acetaminophen-modified Der p1 had increased proliferation and cytokine production compared to acetaminophen and Der p1 alone (n = 7; p < 0.05).
These data suggests 3-nitro-acetaminophen formation and reaction with Der p1 provides a mechanism by which stomach acid or infection-induced low airway pH in patients could enhance the allergic response to proteins such as Der p1.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Single crystal lanthanum hexaboride (LaB6) field emission arrays with uniform tip structures have been achieved using the electrochemical etching method and the field emission characteristics are ...evaluated. A direct current voltage source is introduced in the etching process and phosphoric acid is used as the electrolyte solution. The SEM observation of the emitters reflects the feasibility of the electrochemical etching method for the fabrication of single crystal LaB6 FEA. The turn-on field of LaB6 FEA is low at 3.2 Vmum-1 and the emission current exhibits high stability during the 20-minute operation. Furthermore, even at low vacuum ( > 1.5 X 10-5 Torr) it still exhibits good emission properties and a strong ability to withstand the ion bombardment, which is useful for low operation vacuum microelectronic devices.
Phototransduction is carried out by a signaling pathway that links photoactivation of visual pigments in retinal photoreceptor cells to a change in their membrane potential. Upon photoactivation, the ...second messenger of phototransduction, cyclic GMP, is rapidly degraded and must be replenished during the recovery phase of phototransduction by photoreceptor guanylate cyclases (GCs) GC1 (or GC-E) and GC2 (or GC-F) to maintain vision. Here, we present data that address the role of the GC kinase homology (KH) domain in cyclic GMP production by GC1, the major cyclase in photoreceptors. First, experiments were done to test which GC1 residues undergo phosphorylation and whether such phosphorylation affects cyclase activity. Using mass spectrometry, we showed that GC1 residues Ser-530, Ser-532, Ser-533, and Ser-538, located within the KH domain, undergo light- and signal transduction-independent phosphorylation in vivo. Mutations in the putative Mg2+ binding site of the KH domain abolished phosphorylation, indicating that GC1 undergoes autophosphorylation. The dramatically reduced GC activity of these mutants suggests that a functional KH domain is essential for cyclic GMP production. However, evidence is presented that autophosphorylation does not regulate GC1 activity, in contrast to phosphorylation of other members of this cyclase family.
Indolemine 2,3-dioxygenase (IDO)-mediated oxidation of tryptophan produces kynurenines (KYNs), which may play a role in cataract formation. The molecular mechanisms by which KYNs cause cellular ...changes are poorly understood. The effects of KYNs on mouse lens epithelial cells by overexpression of human IDO were investigated.
Lens epithelial cells (mLECs) derived from human IDO-overexpressing hemizygous transgenic (hemTg) and wild-type (Wt) mice were used. IDO activity was measured by quantifying kynurenine (KYN) by HPLC. KYN-mediated protein modifications were detected by immunocytochemistry and measured by ELISA. Cell proliferation and apoptosis were measured with commercially available kits. Cell distribution between cell cycle phases was examined with flow cytometric analysis. Immunoprecipitation followed by LC/MS was used to identify kynurenine-modified proteins.
mLECs derived from hemTg animals exhibited considerable IDO immunoreactivity and enzyme activity, which were barely detectable in Wt mLECs. KYN and KYN-mediated protein modification were detected in hemTg but not in Wt mLECs; the modified proteins were myosin II and alpha/gamma-actin. HemTg mLECs displayed reduced viability and proliferation. Cell cycle analysis of hemTg mLEC cultures showed approximately a twofold increase in cells at G(2)/M or in both phases, relative to Wt mLECs. Blocking IDO activity with 1-methyl-d,l-tryptophan in hemTg mLECs prevented KYN formation, KYN-mediated protein modification, and G(2)/M arrest.
Excess IDO activity in mLECs results in KYN production, KYN-mediated modification of myosin II and alpha/gamma-actin, and cell cycle perturbation. Modification of myosin II and gamma-actin by KYN may interfere with cytokinesis, leading to defective epithelial cell division and thus a decreased number of fiber cells.
The HIV trans-activator Tat recruits the host transcription elongation factor P-TEFb to stimulate proviral transcription. Phosphorylation of Thr-186 on the activation loop (T-loop) of ...cyclin-dependent kinase 9 (CDK9) is essential for its kinase activity and assembly of CDK9 and cyclin T1 (CycT1) to form functional P-TEFb. Phosphorylation of a second highly conserved T-loop site, Ser-175, alters the competitive binding of Tat and the host recruitment factor bromodomain containing 4 (BRD4) to P-TEFb. Here, we investigated the intracellular mechanisms that regulate these key phosphorylation events required for HIV transcription. Molecular dynamics simulations revealed that the CDK9/CycT1 interface is stabilized by intramolecular hydrogen bonding of pThr-186 by an arginine triad and Glu-96 of CycT1. Arginine triad substitutions that disrupted CDK9/CycT1 assembly accumulated Thr-186–dephosphorylated CDK9 associated with the cytoplasmic Hsp90/Cdc37 chaperone. The Hsp90/Cdc37/CDK9 complex was also present in resting T cells, which lack CycT1. Hsp90 inhibition in primary T cells blocked P-TEFb assembly, disrupted Thr-186 phosphorylation, and suppressed proviral reactivation. The selective CDK7 inhibitor THZ1 blocked CDK9 phosphorylation at Ser-175, and in vitro kinase assays confirmed that CDK7 activity is principally responsible for Ser-175 phosphorylation. Mutation of Ser-175 to Lys had no effect on CDK9 kinase activity or P-TEFb assembly but strongly suppressed both HIV expression and BRD4 binding. We conclude that the transfer of CDK9 from the Hsp90/Cdc37 complex induced by Thr-186 phosphorylation is a key step in P-TEFb biogenesis. Furthermore, we demonstrate that CDK7-mediated Ser-175 phosphorylation is a downstream nuclear event essential for facilitating CDK9 T-loop interactions with Tat.
The ubiquitous inducible transcription factor NF-KB plays central roles in immune and inflammatory responses and in tumorigenesis. Complex posttranslational modifications of the p65 subunit (RelA) ...are a major aspect of the extremely flexible regulation of NF-KB activity. Although phosphorylation, acetylation, ubiquitination, and lysine methylation of NF-KB have been well described, arginine methylation has not yet been found. We now report that, in response to IL-1β, the p65 subunit of NF-KB is dimethylated on arginine 30 (R30) by protein-arginine methyltransferase 5 (PRMT5). Expression of the R30A and R30K mutants of p65 substantially decreased the ability of NF-KB to bind to KB elements and to drive gene expression. A model in which dimethyl R30 is placed into the crystal structure of p65 predicts new van der Waals contacts that stabilize intraprotein interactions and indirectly increase the affinity of p65 for DNA. PRMT5 was the only arginine methyltransferase that coprecipitated with p65, and its overexpression increased NF-KB activity, whereas PRMT5 knockdown had the opposite effect. Microarray analysis revealed that ~85% of the NF-KB-inducible genes that are down-regulated by the R30A mutation are similarly down-regulated by knocking PRMT5 down. Many cytokine and chemokine genes are among these, and conditioned media from cells expressing the R30A mutant of p65 had much less NF-KB-inducing activity than media from cells expressing the wild-type protein. PRMT5 is overexpressed in many types of cancer, often to a striking degree, indicating that high levels of this enzyme may promote tumorigenesis, at least in part by facilitating NF-KB-induced gene expression. PUBLICATION ABSTRACT
Hydroxyl radical protein footprinting (HRPF) using synchrotron radiation is a well-validated method to assess protein structure in the native solution state. In this method, X-ray radiolysis of water ...generates hydroxyl radicals that can react with solvent accessible side chains of proteins, with mass spectrometry used to detect the resulting labeled products. An ideal footprinting dose provides sufficient labeling to measure the structure but not so much as to influence the results. The optimization of hydroxyl radical dose is typically performed using an indirect Alexa488 fluorescence assay sensitive to hydroxyl radical concentration, but full evaluation of the experiment's outcome relies upon bottom-up liquid chromatography mass spectrometry (LC-MS) measurements to directly determine sites and extent of oxidative labeling at the peptide and protein level. A direct evaluation of the extent of labeling to provide direct and absolute measurements of dose and “safe” dose ranges in terms of, for example, average numbers of labels per protein, would provide immediate feedback on experimental outcomes prior to embarking on detailed LC-MS analyses. To this end, we describe an approach to integrate intact MS screening of labeled samples immediately following exposure, along with metrics to quantify the extent of observed labeling from the intact mass spectra. Intact MS results on the model protein lysozyme were evaluated in the context of Alexa488 assay results and a bottom-up LC-MS analysis of the same samples. This approach provides a basis for placing delivered hydroxyl radical dose metrics on firmer technical grounds for synchrotron X-ray footprinting of proteins, with explicit parameters to increase the likelihood of a productive experimental outcome. Further, the method directs approaches to provide absolute and direct dosimetry for all types of labeling for protein footprinting.
•Intact MS was used to directly assess hydroxyl radical labeling outcome.•An oxidation events per protein (OEP) metric was defined to analyze MS spectra.•Classical bottom-up MS analysis validates the intact MS results.•Intact MS and OEP analysis can be used to optimize X-ray footprinting experiments.
Aqueous redox polymerization using a surfactant was studied to prepare submicrometer-sized polyelectrolyte gel particles from a monomer solution containing N-isopropylacrylamide (NIPA), N,N ...‘-methylenebisacrylamide (Bis), and acrylic acid (AAc) or 1-vinylimidazole (VI). Sodium dodecylbenzenesulfonate and ammonium persulfate were used as the surfactant and the initiator, respectively. The gel particles purified via dialysis were characterized by photon correlation spectroscopy. Since the AAc and VI contents of the monomer solutions were controllable within 0−30 mol %, the diameter of the gel particles with 30 mol % of AAc or VI increased from 125 to 600 nm at 25 °C when converting the carboxyl or imidazolyl groups into the corresponding salt form. However, a complete elimination of charges from the anionic or cationic gel particles gave rise to a neutral gel, the size of which varied from 125 nm at 25 °C (swollen state) to 50 nm at temperatures >35 °C (fully collapsed state). These swelling behaviors were the same as those of NIPA-based ionic bulk gels. Detailed examinations of the gel particles with 30 mol % AAc by potentiometric titration and electrophoretic light scattering showed that almost all of the COOH groups in the pregel solution were incorporated into the particle interiors without concentration on the particle surface.
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