排序方式: 共有18条查询结果,搜索用时 15 毫秒
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Dinh M Grunberger D Ho H Tsing SY Shaw D Lee S Barnett J Hill RJ Swinney DC Bradshaw JM 《The Journal of biological chemistry》2007,282(12):8768-8776
Bruton's tyrosine kinase (BTK) is a member of the Tec non-receptor tyrosine kinase family that is involved in regulating B cell proliferation. To better understand the enzymatic mechanism of the Tec family of kinases, the kinetics of BTK substrate phosphorylation were characterized using a radioactive enzyme assay. We first examined whether autophosphorylation regulates BTK activity. Western blotting with a phosphospecific antibody revealed that BTK rapidly autophosphorylates at Tyr(551) within its activation loop in vitro. Examination of a Y551F BTK mutant indicated that phosphorylation of Tyr(551) causes a 10-fold increase in BTK activity. We then proceeded to characterize the steady state kinetic mechanism of BTK. Varying the concentrations of ATP and S1 peptide (biotin-Aca-AAAEEIY-GEI-NH2) revealed that BTK employs a ternary complex mechanism with KmATP = 84 +/- 20 microM and KmS1 = 37 +/- 8 microM. Inhibition studies were also performed to examine the order of substrate binding. The inhibitors ADP and staurosporine were both found to be competitive with ATP and non-competitive with S1, indicating binding of ATP and S1 to BTK is either random or ordered with ATP binding first. Negative cooperativity was also found between the S1 and ATP binding sites. Unlike ATP site inhibitors, substrate analog inhibitors did not inhibit BTK at concentrations less than 1 mm, suggesting that BTK may employ a "substrate clamping" type of kinetic mechanism whereby the substrate Kd is weaker than Km. This investigation of BTK provides the first detailed kinetic characterization of a Tec family kinase. 相似文献
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Anna Tsing Shiho Satsuka for the Matsutake Worlds Research Group 《Economic botany》2008,62(3):244-253
Diverging Understandings of Forest Management in Matsutake Science. As high-value gourmet mushrooms, the matsutake complex of the genus Tricholoma has been the subject of extensive research. This article reviews two trajectories of matsutake research, showing how distinctive
regional nodes may develop within a cosmopolitan modern science. The global center of matsutake research is in Japan, where
problems of artificial cultivation and the “orchard-style” enhancement of production under forest conditions stimulate basic
research. U.S. Pacific Northwest research forms a contrasting regional node, with a focus on sustainable yields in the context
of timber production. Regional differences in research design and results point to the importance of distinctive scientific
legacies, in this case formed in relation to divergent histories of forest management. Attention to regional distinctions
in the framing of scientific problems is particularly important as scientific frameworks are exported to new places; for example,
both Japanese and American forms of matsutake science have been extended to China.
高価なグルメきのこであるマツタケとその近縁種群のTricholoma属は広範囲に渡る科学的研究の対象となってきた。本論では二つの地域特徴的なマツタケ研究の軌跡を概観し、文化的差異を超えて世界的に通用する近代科学においても地域固有の関心に応じて特徴のある知識が結節し発展することを示す。マツタケ研究の世界的な中心地である日本では人工増殖やマツタケを殖やすための「果樹園的」な山林作りへの関心が基礎研究の方向性に刺激を与えてきた。一方日本とは対照的に、米国北西岸州では木材の持続的産出に主眼をおいた山林管理の流れの中で研究が進んできた。こうした研究計画や結果的に得られる知識の違いは、地域ごとに特徴のある科学的遺産
- 本件の場合は森林管理の歴史が多様に枝分かれしていること - に注目することが重要であることを知らせてくれる。近年日本や米国で発展したマツタケ研究の方法や成果が中国での研究にも影響を与えているが、特に新しい研究の場を広げる場合には科学的な関心、問題がどのような枠組で組み立てられるか地域によって多様であることを考慮することが重要である。 相似文献
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Cheng T Hitomi K van Vlijmen-Willems IM de Jongh GJ Yamamoto K Nishi K Watts C Reinheckel T Schalkwijk J Zeeuwen PL 《The Journal of biological chemistry》2006,281(23):15893-15899
Cystatin M/E is a high affinity inhibitor of the asparaginyl endopeptidase legumain, and we have previously reported that both proteins are likely to be involved in the regulation of stratum corneum formation in skin. Although cystatin M/E contains a predicted binding site for papain-like cysteine proteases, no high affinity binding for any member of this family has been demonstrated so far. We report that human cathepsin V (CTSV) and human cathepsin L (CTSL) are strongly inhibited by human cystatin M/E. Kinetic studies show that Ki values of cystatin M/E for the interaction with CTSV and CTSL are 0.47 and 1.78 nM, respectively. On the basis of the analogous sites in cystatin C, we used site-directed mutagenesis to identify the binding sites of these proteases in cystatin M/E. We found that the W135A mutant was rendered inactive against CTSV and CTSL but retained legumain-inhibiting activity. Conversely, the N64A mutant lost legumain-inhibiting activity but remained active against the papain-like cysteine proteases. We conclude that legumain and papain-like cysteine proteases are inhibited by two distinct non-overlapping sites. Using immunohistochemistry on normal human skin, we found that cystatin M/E co-localizes with CTSV and CTSL. In addition, we show that CTSL is the elusive enzyme that processes and activates epidermal transglutaminase 3. The identification of CTSV and CTSL as novel targets for cystatin M/E, their (co)-expression in the stratum granulosum of human skin, and the activity of CTSL toward transglutaminase 3 strongly imply an important role for these enzymes in the differentiation process of human epidermis. 相似文献
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Kuglstatter A Wong A Tsing S Lee SW Lou Y Villaseñor AG Bradshaw JM Shaw D Barnett JW Browner MF 《Protein science : a publication of the Protein Society》2011,20(2):428-436
Bruton's tyrosine kinase (BTK) plays a key role in B cell receptor signaling and is considered a promising drug target for lymphoma and inflammatory diseases. We have determined the X-ray crystal structures of BTK kinase domain in complex with six inhibitors from distinct chemical classes. Five different BTK protein conformations are stabilized by the bound inhibitors, providing insights into the structural flexibility of the Gly-rich loop, helix C, the DFG sequence, and activation loop. The conformational changes occur independent of activation loop phosphorylation and do not correlate with the structurally unchanged WEI motif in the Src homology 2-kinase domain linker. Two novel activation loop conformations and an atypical DFG conformation are observed representing unique inactive states of BTK. Two regions within the activation loop are shown to structurally transform between 3(10)- and α-helices, one of which collapses into the adenosine-5'-triphosphate binding pocket. The first crystal structure of a Tec kinase family member in the pharmacologically important DFG-out conformation and bound to a type II kinase inhibitor is described. The different protein conformations observed provide insights into the structural flexibility of BTK, the molecular basis of its regulation, and the structure-based design of specific inhibitors. 相似文献
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Shaw D Wang SM Villaseñor AG Tsing S Walter D Browner MF Barnett J Kuglstatter A 《Journal of molecular biology》2008,383(4):885-893
c-Jun N-terminal kinase (JNK) 2 is a member of the mitogen-activated protein (MAP) kinase group of signaling proteins. MAP kinases share a common sequence insertion called “MAP kinase insert”, which, for ERK2, has been shown to interact with regulatory proteins and, for p38α, has been proposed to be involved in the regulation of catalytic activity. We have determined the crystal structure of human JNK2 complexed with an indazole inhibitor by applying a high-throughput protein engineering and surface-site mutagenesis approach. A novel conformation of the activation loop is observed, which is not compatible with its phosphorylation by upstream kinases. This activation inhibitory conformation of JNK2 is stabilized by the MAP kinase insert that interacts with the activation loop in an induced-fit manner. We therefore suggest that the MAP kinase insert of JNK2 plays a role in the regulation of JNK2 activation, possibly by interacting with intracellular binding partners. 相似文献
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Andreas Kuglstatter Manjiri Ghate Stan Tsing Armando G. Villaseñor David Shaw Jim W. Barnett Michelle F. Browner 《Bioorganic & medicinal chemistry letters》2010,20(17):5217-5220
JNK2 and p38α are closely related mitogen-activated protein kinases that regulate various cellular activities and are considered drug targets for inflammatory diseases. We have determined the X-ray crystal structure of the clinical phase II p38α inhibitor BIRB796 bound to its off-target JNK2. This shows for the first time a JNK subfamily member in the DFG-out conformation. The fully resolved activation loop reveals that BIRB796 inhibits JNK2 activation by stabilizing the loop in a position that does not allow its phosphorylation by upstream kinases. The structure suggests that substituents at the BIRB796 morpholino group and modifications of the t-butyl moiety should further increase the p38α to JNK2 potency ratio. For the design of selective DFG-out binding JNK2 inhibitors, the binding pocket of the BIRB796 tolyl group may have the best potential. 相似文献
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Liu SS Chan KY Leung RC Chan KK Tam KF Luk MH Lo SS Fong DY Cheung AN Lin ZQ Ngan HY 《PloS one》2011,6(5):e19244