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1.
Kinetics of Cdc42 membrane extraction by Rho-GDI monitored by real-time fluorescence resonance energy transfer 总被引:6,自引:0,他引:6
The mechanisms underlying the ability of the Rho-GDP dissociation inhibitor (RhoGDI) to elicit the release of Rho-related GTP-binding proteins from membranes is currently unknown. In this report, we have set out to address this issue by using fluorescence resonance energy transfer approaches to examine the functional interactions of the RhoGDI with membrane-associated Cdc42. Two fluorescence assays were developed to monitor the interactions between these proteins in real time. The first involved measurements of resonance energy transfer between N-methylanthraniloyl GDP (MantGDP) bound to Cdc42 and fluorescein maleimide covalently attached to cysteine 79 of RhoGDI (RhoGDI-FM). This assay allowed us to directly monitor the binding of RhoGDI to membrane-associated Cdc42. The second fluorescence assay involved measurements of resonance energy transfer between membrane-associated Cdc42-MantGDP and hexadecyl(amino) fluorescein that was randomly inserted into the membrane bilayer. This assay enabled us to directly monitor the (GDI-induced) release of Cdc42 from membranes. Analyses of the rates of change in the fluorescence of Cdc42-MantGDP, which serves as a resonance energy transfer donor in both of these assays, as a function of RhoGDI concentration suggests a two-step mechanism to explain the ability of RhoGDI to stimulate the release of Cdc42 from membranes. Specifically, we propose that the GDI first binds rapidly to membrane-associated Cdc42 and then a slower isomerization occurs which represents the rate-limiting step for the dissociation of the Cdc42-RhoGDI complex from membranes. We propose that this slow step in the observed kinetics reflects the time-course of translocation of the geranyl-geranyl lipid tail of Cdc42 from the outer leaflet of the membrane to the isoprenyl binding site observed in the previously reported NMR structure of the Cdc42-RhoGDI complex [Gosser et al. (1997) Nature 387, 814]. 相似文献
2.
Nomanbhoy T Morales AJ Abraham AT Vörtler CS Giegé R Schimmel P 《Nature structural biology》2001,8(4):344-348
Transfer RNA (tRNA) is a small nucleic acid (typically 76 nucleotides) that forms binary complexes with proteins, such as aminoacyl tRNA synthetases (RS) and Trbp111. The latter is a widely distributed structure-specific tRNA-binding protein that is incorporated into cell signaling molecules. The structure of Trbp111 was modeled onto to the outer, convex side of the L-shaped tRNA. Here we present RNA footprints that are consistent with this model. This binding mode is in contrast to that of tRNA synthetases, which bind to the inside, or concave side, of tRNA. These opposite locations of binding for these two proteins suggest the possibility of a ternary complex. The formation of a tRNA synthetase--tRNA--Trbp111 ternary complex was detected by two independent methods. The results indicate that the tRNA is sandwiched between the two protein molecules. A thermodynamic and functional analysis is consistent with the tRNA retaining its native structure in the ternary complex. These results may have implications for how the translation apparatus is linked to other cellular machinery. 相似文献
3.
Some aminoacyl-tRNA synthetases have two catalytic centers that together achieve fine-structure discrimination of closely similar amino acids. The role of tRNA is to stimulate translocation of a misactivated amino acid from the active site to the editing site where the misactivated substrate is eliminated by hydrolysis. Using isoleucyl-tRNA synthetase as an example, we placed mutations in the catalytic center for editing at residues strongly conserved from bacteria to humans. A particular single substitution and one double substitution resulted in production of mischarged tRNA, by interfering specifically with the chemical step of hydrolytic editing. The substitutions affected neither amino acid activation nor aminoacylation, with the cognate amino acid. Thus, because of the demonstrated functional independence of the two catalytic sites, errors of aminoacylation can be generated by selective mutations in the center for editing. 相似文献
4.
Deng X Dzamko N Prescott A Davies P Liu Q Yang Q Lee JD Patricelli MP Nomanbhoy TK Alessi DR Gray NS 《Nature chemical biology》2011,7(4):203-205
Mutations in leucine-rich repeat kinase 2 (LRRK2) are strongly associated with late-onset autosomal dominant Parkinson's disease. We employed a new, parallel, compound-centric approach to identify a potent and selective LRRK2 inhibitor, LRRK2-IN-1, and demonstrated that inhibition of LRRK2 induces dephosphorylation of Ser910 and Ser935 and accumulation of LRRK2 within aggregate structures. LRRK2-IN-1 will serve as a versatile tool to pharmacologically interrogate LRRK2 biology and study its role in Parkinson's disease. 相似文献
5.
Shreder KR Wong MS Corral S Yu Z Winn DT Wu M Hu Y Nomanbhoy T Alemayehu S Fuller SR Rosenblum JS Kozarich JW 《Bioorganic & medicinal chemistry letters》2005,15(19):4256-4260
Dipeptide-based inhibitors with C-substituted (alkyl or aminoalkyl) alpha-amino acids in the P2 position and boro-norleucine (boro-Nle) in the P1 position were synthesized. Relative to boro-proline, boro-Nle as a P1 residue was shown able to significantly dial out DPP4, FAP, DPP8, and DPP9 activity. Dab-boro-Nle (4g) proved to be the most selective and potent DPP7 inhibitor with a DPP7 IC50 value of 480 pM. 相似文献
6.
Aminoacyl-tRNA synthetases establish the rules of the genetic code by catalyzing attachment of amino acids to specific transfer RNAs (tRNAs) that bear the anticodon triplets of the code. Each of the 20 amino acids has its own distinct aminoacyl-tRNA synthetase. Here we use energy-transfer-dependent fluorescence from the nucleotide probe N-methylanthraniloyl dATP (mdATP) to investigate the active site of a specific aminoacyl-tRNA synthetase. Interaction of the enzyme with the cognate amino acid and formation of the aminoacyl adenylate intermediate were detected. In addition to providing a convenient tool to characterize enzymatic parameters, the probe allowed investigation of the role of conserved residues within the active site. Specifically, a residue that is critical for binding could be distinguished from one that is important for the transition state of adenylate formation. Amino acid binding and adenylate synthesis by two other aminoacyl-tRNA synthetases was also investigated with mdATP. Thus, a key step in the synthesis of aminoacyl-tRNA can in general be dissected with this probe. 相似文献
7.
Novel mechanistic class of fatty acid amide hydrolase inhibitors with remarkable selectivity 总被引:5,自引:0,他引:5
Ahn K Johnson DS Fitzgerald LR Liimatta M Arendse A Stevenson T Lund ET Nugent RA Nomanbhoy TK Alexander JP Cravatt BF 《Biochemistry》2007,46(45):13019-13030
Fatty acid amide hydrolase (FAAH) is an integral membrane enzyme that degrades the fatty acid amide family of signaling lipids, including the endocannabinoid anandamide. Genetic or pharmacological inactivation of FAAH leads to analgesic, anti-inflammatory, anxiolytic, and antidepressant phenotypes in rodents without showing the undesirable side effects observed with direct cannabinoid receptor agonists, indicating that FAAH may represent an attractive therapeutic target for treatment of pain, inflammation, and other central nervous system disorders. However, the FAAH inhibitors reported to date lack drug-like pharmacokinetic properties and/or selectivity. Herein we describe piperidine/piperazine ureas represented by N-phenyl-4-(quinolin-3-ylmethyl)piperidine-1-carboxamide (PF-750) and N-phenyl-4-(quinolin-2-ylmethyl)piperazine-1-carboxamide (PF-622) as a novel mechanistic class of FAAH inhibitors. PF-750 and PF-622 show higher in vitro potencies than previously established classes of FAAH inhibitors. Rather unexpectedly based on the high chemical stability of the urea functional group, PF-750 and PF-622 were found to inhibit FAAH in a time-dependent manner by covalently modifying the enzyme's active site serine nucleophile. Activity-based proteomic profiling revealed that PF-750 and PF-622 were completely selective for FAAH relative to other mammalian serine hydrolases. We hypothesize that this remarkable specificity derives, at least in part, from FAAH's special ability to function as a C(O)-N bond hydrolase, which distinguishes it from the vast majority of metabolic serine hydrolases in mammals that are restricted to hydrolyzing esters and/or thioesters. The piperidine/piperazine urea may thus represent a privileged chemical scaffold for the synthesis of FAAH inhibitors that display an unprecedented combination of potency and selectivity for use as potential analgesic and anxiolytic/antidepressant agents. 相似文献
8.
Li B Cociorva OM Nomanbhoy T Li Q Nakamura K Nomura M Okada K Yumoto K Liyanage M Zhang MC Aban A Szardenings AK Kozarich JW Kohno Y Shreder KR 《Bioorganic & medicinal chemistry letters》2012,22(2):1005-1008
We previously disclosed tricylic, 6-carboxylic acid-bearing 4-quinolones as GSK-3β inhibitors. Herein we discuss the optimization of this series to yield a series of more potent 6-nitrile analogs with insignificant anti-microbial activity. Finally, kinase profiling indicated that members of this class were highly specific GSK-3 inhibitors. 相似文献
9.
Shreder KR Liu Y Nomanhboy T Fuller SR Wong MS Gai WZ Wu J Leventhal PS Lill JR Corral S 《Bioconjugate chemistry》2004,15(4):790-798
The design and synthesis of AX7574, a microcystin-derived probe for serine/threonine phosphatases, is described. A key step in the synthesis was the conjugation under basic conditions of a tetramethylrhodamine 1,3-diketone derivative to the arginine side chain present in microcystin-LR. The resulting conjugate specifically labeled the active site of protein phosphatases 1 (PP-1) with a 1:1 stoichiometry and IC50 of 4.0 nM. AX7574 was used to isolate and identify PP-1, PP-2A, PP-4, and PP-6 in Jurkat cells. Finally, AX7574 was able to record changes in the phosphatase activity levels of calyculin A treated Jurkat cells versus untreated control cells. 相似文献
10.
Douglas S. Johnson Kay Ahn Suzanne Kesten Scott E. Lazerwith Yuntao Song Mark Morris Lorraine Fay Tracy Gregory Cory Stiff James B. Dunbar Marya Liimatta David Beidler Sarah Smith Tyzoon K. Nomanbhoy Benjamin F. Cravatt 《Bioorganic & medicinal chemistry letters》2009,19(10):2865-2869
The synthesis and structure–activity relationships (SAR) of a series of benzothiophene piperazine and piperidine urea FAAH inhibitors is described. These compounds inhibit FAAH by covalently modifying the enzyme’s active site serine nucleophile. Activity-based protein profiling (ABPP) revealed that these urea inhibitors were completely selective for FAAH relative to other mammalian serine hydrolases. Several compounds showed in vivo activity in a rat complete Freund’s adjuvant (CFA) model of inflammatory pain. 相似文献