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91.
Seunghyi Kook Xuanzhi Zhan Tamer S. Kaoud Kevin N. Dalby Vsevolod V. Gurevich Eugenia V. Gurevich 《The Journal of biological chemistry》2013,288(52):37332-37342
Non-visual arrestins scaffold mitogen-activated protein kinase (MAPK) cascades. The c-Jun N-terminal kinases (JNKs) are members of MAPK family. Arrestin-3 has been shown to enhance the activation of JNK3, which is expressed mainly in neurons, heart, and testes, in contrast to ubiquitous JNK1 and JNK2. Although all JNKs are activated by MKK4 and MKK7, both of which bind arrestin-3, the ability of arrestin-3 to facilitate the activation of JNK1 and JNK2 has never been reported. Using purified proteins we found that arrestin-3 directly binds JNK1α1 and JNK2α2, interacting with the latter comparably to JNK3α2. Phosphorylation of purified JNK1α1 and JNK2α2 by MKK4 or MKK7 is increased by arrestin-3. Endogenous arrestin-3 interacted with endogenous JNK1/2 in different cell types. Arrestin-3 also enhanced phosphorylation of endogenous JNK1/2 in intact cells upon expression of upstream kinases ASK1, MKK4, or MKK7. We observed a biphasic effect of arrestin-3 concentrations on phosphorylation of JNK1α1 and JNK2α2 both in vitro and in vivo. Thus, arrestin-3 acts as a scaffold, facilitating JNK1α1 and JNK2α2 phosphorylation by MKK4 and MKK7 via bringing JNKs and their activators together. The data suggest that arrestin-3 modulates the activity of ubiquitous JNK1 and JNK2 in non-neuronal cells, impacting the signaling pathway that regulates their proliferation and survival. 相似文献
92.
Cytokines are pivotal to a balanced innate or cell-mediated immune response, and can be indicative of disease progression and/or resolution. Methods to measure key cytokines rapidly with accuracy, precision, and sensitivity are consequently important. The current assay technologies, which are based on RT-PCR, immunoassays, or bioassays, are limited in their use in the clinic, in particular because of the long time (1-3 h) required to carry out the assays. An alternative, semi-quantitative approach described here, uses an immunological capture step and a mass spectral readout. The goal of the assay is speed rather than sensitivity or precision. 相似文献
93.
Yoichi Tamai Hisako Kojima Shintaro Saito† Kikue Uchida Rie Kitajima Hiroshi Komatsu Tsuneo Moriya‡ 《Journal of neurochemistry》1993,60(5):1854-1863
Abstract: The metamorphic changes in levels of glycolipids and myelin proteins and 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) in the brains of bullfrog tadpoles, adult frogs, and axolotls were investigated, with particular emphasis on myelin maturation. The concentrations of cerebroside. sulfatide, and galactosyldiacylglycerol gradually increased from the onset of prometamorphosis throughout the active metamorphic period and then greatly increased after metamorphosis was completed. The ratio of glucocerebroside to galactocerebroside increased greatly in the prometamorphic period and then rapidly decreased to the frog level during the climax period. The fatty acid compositions of cerebroside and sulfatide showed a developmental change, with 24:1 being more predominant in the later metamorphic stage. The proportion of hydroxy fatty acids increased up to the onset of the prometamorphic stage and thereafter remained constant at ∼ 50% of the total. The CNP activity remained unchanged throughout metamorphosis at 60% that in frog myelin and increased in the adult frog. The composition of tadpole myelin proteins remained constant during metamorphosis, with large basic protein being the most abundant, and in the frog, proteolipid protein and large basic protein were present in comparable amounts. The two adult forms of axolotl, i.e., the neotenous and metamorphosed forms, exhibited almost identical myelin constituents, and CNP activity in the neotenous form amounted to one-fifth that in the bullfrog. These results indicate that active biosynthesis of myelin marker components occurs as metamorphosis proceeds, but more pronounced changes of myelin components occur after metamorphosis is completed. 相似文献
94.
Kaji H Isobe T 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2003,787(1):91-99
Whole genome sequencing of the free-living nematode Caenorhabditis elegans is a prominent achievement in genomics and uncovers the existence of enormous known and unknown gene products. Characterization and linking of all gene products are the next challenging theme of biology. Genome-wide researches are already progressing on C. elegans and the fruits of these efforts are accessible through the internet. To link the sequence-function relationship, proteomic research has been applied to provide comprehensive information of the worm proteins. In addition to 2-dimensional gel electrophoresis for visualization of the proteome, recent advances in liquid chromatography (LC)-based technologies have allowed the large-scale analysis of proteins and are at cutting-edge of high-throughput analysis of focused proteome. 相似文献
95.
The C-terminal --COOH of prenylated proteins is methylated to --COOCH3. The --COOCH3 ester forms are hydrolyzed by prenylated methylated protein methyl esterase (PMPMEase) to the original acid forms. This is the only reversible step of the prenylation pathway. PMPMEase has not been purified and identified and is therefore understudied. Using a prenylated-L-cysteine methyl ester as substrate, PMPMEase was purified to apparent homogeneity from porcine liver supernatant. SDS-PAGE analysis revealed an apparent mass of 57 kDa. Proteomics analyses identified 17 peptides (242 amino acids). A Mascot database search revealed these as portions of the Sus scrofa carboxylesterase, a 62-kDa serine hydrolase with the C-terminal HAEL endoplasmic reticulum-retention signal. It is at least 71% identical to such mammalian carboxylesterases as human carboxylesterase 1 with affinities toward hydrophobic substrates and known to activate prodrugs, metabolize active drugs, as well as detoxify various substances such as cocaine and food-derived esters. The purified enzyme hydrolyzed benzoyl-Gly-farnesyl-L-cysteine methyl ester and hydrocinamoyl farnesyl-L-cysteine methyl ester with Michaelis-Menten constant (K(m)) values of 33 +/- 4 and 25 +/- 4 microM and V(max) values of 4.51 +/- 0.28 and 6.80 +/- 0.51 nmol/min/mg of protein, respectively. It was inhibited by organophosphates, chloromethyl ketones, ebelactone A and B, and phenylmethylsulfonyl fluoride. 相似文献
96.
Alexander W. Lohman Janelle L. Weaver Marie Billaud Joanna K. Sandilos Rachael Griffiths Adam C. Straub Silvia Penuela Norbert Leitinger Dale W. Laird Douglas A. Bayliss Brant E. Isakson 《The Journal of biological chemistry》2012,287(47):39602-39612
S-Nitrosylation is a post-translational modification on cysteine(s) that can regulate protein function, and pannexin 1 (Panx1) channels are present in the vasculature, a tissue rich in nitric oxide (NO) species. Therefore, we investigated whether Panx1 can be S-nitrosylated and whether this modification can affect channel activity. Using the biotin switch assay, we found that application of the NO donor S-nitrosoglutathione (GSNO) or diethylammonium (Z)-1–1(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA NONOate) to human embryonic kidney (HEK) 293T cells expressing wild type (WT) Panx1 and mouse aortic endothelial cells induced Panx1 S-nitrosylation. Functionally, GSNO and DEA NONOate attenuated Panx1 currents; consistent with a role for S-nitrosylation, current inhibition was reversed by the reducing agent dithiothreitol and unaffected by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, a blocker of guanylate cyclase activity. In addition, ATP release was significantly inhibited by treatment with both NO donors. To identify which cysteine residue(s) was S-nitrosylated, we made single cysteine-to-alanine substitutions in Panx1 (Panx1C40A, Panx1C346A, and Panx1C426A). Mutation of these single cysteines did not prevent Panx1 S-nitrosylation; however, mutation of either Cys-40 or Cys-346 prevented Panx1 current inhibition and ATP release by GSNO. This observation suggested that multiple cysteines may be S-nitrosylated to regulate Panx1 channel function. Indeed, we found that mutation of both Cys-40 and Cys-346 (Panx1C40A/C346A) prevented Panx1 S-nitrosylation by GSNO as well as the GSNO-mediated inhibition of Panx1 current and ATP release. Taken together, these results indicate that S-nitrosylation of Panx1 at Cys-40 and Cys-346 inhibits Panx1 channel currents and ATP release. 相似文献
97.
Song Q Pallikkuth S Bossuyt J Bers DM Robia SL 《The Journal of biological chemistry》2011,286(11):9120-9126
Na/K-ATPase (NKA) activity is dynamically regulated by an inhibitory interaction with a small transmembrane protein, phospholemman (PLM). Inhibition is relieved upon PLM phosphorylation. Phosphorylation may alter how PLM interacts with NKA and/or itself, but details of these interactions are unknown. To address this, we quantified FRET between PLM and its regulatory target NKA in live cells. Phosphorylation of PLM was mimicked by mutation S63E (PKC site), S68E (PKA/PKC site), or S63E/S68E. The dependence of FRET on protein expression in live cells yielded information about the structure and binding affinity of the PLM-NKA regulatory complex. PLM phosphomimetic mutations altered the quaternary structure of the regulatory complex and reduced the apparent affinity of the PLM-NKA interaction. The latter effect was likely due to increased oligomerization of PLM phosphomimetic mutants, as suggested by PLM-PLM FRET measurements. Distance constraints obtained by FRET suggest that phosphomimetic mutations slightly alter the oligomer quaternary conformation. Photon-counting histogram measurements revealed that the major PLM oligomeric species is a tetramer. We conclude that phosphorylation of PLM increases its oligomerization into tetramers, decreases its binding to NKA, and alters the structures of both the tetramer and NKA regulatory complex. 相似文献
98.
Mishra NK Peleg Y Cirri E Belogus T Lifshitz Y Voelker DR Apell HJ Garty H Karlish SJ 《The Journal of biological chemistry》2011,286(11):9699-9712
FXYD proteins are a family of seven small regulatory proteins, expressed in a tissue-specific manner, that associate with Na,K-ATPase as subsidiary subunits and modulate kinetic properties. This study describes an additional property of FXYD proteins as stabilizers of Na,K-ATPase. FXYD1 (phospholemman), FXYD2 (γ subunit), and FXYD4 (CHIF) have been expressed in Escherichia coli and purified. These FXYD proteins associate spontaneously in vitro with detergent-soluble purified recombinant human Na,K-ATPase (α1β1) to form α1β1FXYD complexes. Compared with the control (α1β1), all three FXYD proteins strongly protect Na,K-ATPase activity against inactivation by heating or excess detergent (C12E8), with effectiveness FXYD1 > FXYD2 ≥ FXYD4. Heating also inactivates E1 ↔ E2 conformational changes and cation occlusion, and FXYD1 protects strongly. Incubation of α1β1 or α1β1FXYD complexes with guanidinium chloride (up to 6 m) causes protein unfolding, detected by changes in protein fluorescence, but FXYD proteins do not protect. Thus, general protein denaturation is not the cause of thermally mediated or detergent-mediated inactivation. By contrast, the experiments show that displacement of specifically bound phosphatidylserine is the primary cause of thermally mediated or detergent-mediated inactivation, and FXYD proteins stabilize phosphatidylserine-Na,K-ATPase interactions. Phosphatidylserine probably binds near trans-membrane segments M9 of the α subunit and the FXYD protein, which are in proximity. FXYD1, FXYD2, and FXYD4 co-expressed in HeLa cells with rat α1 protect strongly against thermal inactivation. Stabilization of Na,K-ATPase by three FXYD proteins in a mammalian cell membrane, as well the purified recombinant Na,K-ATPase, suggests that stabilization is a general property of FXYD proteins, consistent with a significant biological function. 相似文献
99.
100.
Characterization of the interactions between the nucleoprotein and the phosphoprotein of Henipavirus
Habchi J Blangy S Mamelli L Jensen MR Blackledge M Darbon H Oglesbee M Shu Y Longhi S 《The Journal of biological chemistry》2011,286(15):13583-13602
The Henipavirus genome is encapsidated by the nucleoprotein (N) within a helical nucleocapsid that recruits the polymerase complex via the phosphoprotein (P). In a previous study, we reported that in henipaviruses, the N-terminal domain of the phosphoprotein and the C-terminal domain of the nucleoprotein (N(TAIL)) are both intrinsically disordered. Here we show that Henipavirus N(TAIL) domains are also disordered in the context of full-length nucleoproteins. We also report the cloning, purification, and characterization of the C-terminal X domains (P(XD)) of Henipavirus phosphoproteins. Using isothermal titration calorimetry, we show that N(TAIL) and P(XD) form a 1:1 stoichiometric complex that is stable under NaCl concentrations as high as 1 M and has a K(D) in the μM range. Using far-UV circular dichroism and nuclear magnetic resonance, we show that P(XD) triggers an increase in the α-helical content of N(TAIL). Using fluorescence spectroscopy, we show that P(XD) has no impact on the chemical environment of a Trp residue introduced at position 527 of the Henipavirus N(TAIL) domain, thus arguing for the lack of stable contacts between the C termini of N(TAIL) and P(XD). Finally, we present a tentative structural model of the N(TAIL)-P(XD) interaction in which a short, order-prone region of N(TAIL) (α-MoRE; amino acids 473-493) adopts an α-helical conformation and is embedded between helices α2 and α3 of P(XD), leading to a relatively small interface dominated by hydrophobic contacts. The present results provide the first detailed experimental characterization of the N-P interaction in henipaviruses and designate the N(TAIL)-P(XD) interaction as a valuable target for rational antiviral approaches. 相似文献