Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.4.4 (kinesin)
5,033 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Treatment of proteins in vitro with sulfhydryl (SH)-reactive compounds has been used successfully to determine protein regions critical for normal function. To probe structure-function relationships in the microtubule (MT) motor kinesin, the motor was treated with two SH reactive compounds, n-ethylmaleimide and ethacrynic acid, and its function was assayed by motility and co-sedimentation techniques. In the motility assay, treatment of kinesin either before or after adsorption to the glass surfaces of a flow cell was found to inhibit the ability of coverslip-bound kinesin to bind to MTs. Inactivation of MT binding was slow, required high molar excess of the SH-reactive drug, and was very sensitive to temperature. Inhibition of MT binding occurred well after complete modification of kinesin light chain, but paralleled modification of the kinesin heavy chain. The results point to a model in which one critical cysteine per kinesin heavy chain is relatively inaccessible to solvent. Surprisingly, when the interaction between modified kinesin and MTs was examined by a co-sedimentation assay, kinesin retained the ability to bind MTs. These contrasting results may be due to conformational differences in the kinesin molecule that exist in the two assays.
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PMID:n-ethylmaleimide and ethacrynic acid inhibit kinesin binding to microtubules in a motility assay. 925 2

To probe the structural changes within kinesin molecules, we made the mutants of motor domains of two-headed kinesin (4-411 aa) in which either all the five cysteines or all except Cys45 were mutated. A residual cysteine (Cys45) of the kinesin mutant was labeled with an environment-sensitive fluorescent probe, acrylodan. ATPase activity, mechanical properties, and fluorescence intensity of the mutants were measured. Upon acrylodan-labeled kinesin binding to microtubules in the presence of 1 mM AMPPNP, the peak intensity was enhanced by 3.4-fold, indicating the structural change of the kinesin head by the binding. Substitution of cysteines decreased both the maximum microtubule-activated ATPase and the sliding velocity to the same extent. However, the maximum force and the step size were not affected; the force produced by a single molecule was 6-6.5 pN, and a step size due to the hydrolysis of one ATP molecule by kinesin molecules was about 10 nm for all kinesins. This step size was close to a unitary step size of 8 nm. Thus, the mechanical events of kinesin are tightly coupled with the chemical events.
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PMID:Mechanical and chemical properties of cysteine-modified kinesin molecules. 1044 Nov 25

Kinesin motor proteins use ATP hydrolysis for transport along microtubules in the cell. We sought to identify small organic ligands to inhibit kinesin's activity. Candidate molecules were identified by computational docking of commercially available compounds using the computer program DOCK. Compounds were docked at either of two sites, and a selection was tested for inhibition of microtubule-stimulated ATPase activity. Twenty-two submillimolar inhibitors were identified. Several inhibitors appeared to be competitive for microtubule binding and not for ATP binding, and three compounds showed 50% inhibition down to single-digit micromolar levels. Most inhibitors grouped into four distinct classes (fluoresceins, phenolphthaleins, anthraquinones, and naphthylene sulfonates). We measured the binding of one inhibitor, rose bengal lactone (RBL), to kinesin (dissociation constant 2.5 microM) by its increase in steady-state fluorescence anisotropy. The RBL binding site on kinesin was localized by fluorescent resonance energy transfer (FRET) using a donor fluorophore (coumarin) covalently attached at unique, surface-exposed cysteine residues engineered at positions 28, 149, 103, 220, or 330. RBL was found to bind in its original docked site: the pocket cradled by loop 8 and beta-strand 5 in kinesin's three-dimensional structure. These results confirm this region's role in microtubule binding and identify this pocket as a novel binding site for kinesin inhibition.
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PMID:Inhibitors of kinesin activity from structure-based computer screening. 1070 33

Genetically-encoded affinity tags constitute an important strategy for purifying proteins. Here, we have designed a novel affinity matrix based on the his-arsenical fluorescein dye FlAsH, which specifically recognizes short alpha-helical peptides containing the sequence CCXXCC (Griffin BA, Adams SR, Tsien RY, 1998, Science 281:269-272). We find that kinesin tagged with this cysteine-containing helix binds specifically to FlAsH resin and can be eluted in a fully active form. This affinity tag has several advantages over polyhistidine, the only small affinity tag in common use. The protein obtained with this single chromatographic step from crude Escherichia coli lysates is purer than that obtained with nickel affinity chromatography of 6xHis tagged kinesin. Moreover, unlike nickel affinity chromatography, which requires high concentrations of imidazole or pH changes for elution, protein bound to the FlAsH column can be completely eluted by dithiothreitol. Because of these mild elution conditions, FlAsH affinity chromatography is ideal for recovering fully active protein and for the purification of intact protein complexes.
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PMID:A novel method of affinity-purifying proteins using a bis-arsenical fluorescein. 1071 73

Human Eg5, a member of the kinesin superfamily, plays a key role in mitosis, as it is required for the formation of a bipolar spindle. We describe here the first in vitro microtubule-activated ATPase-based assay for the identification of small-molecule inhibitors of Eg5. We screened preselected libraries obtained from the National Cancer Institute and identified S-trityl-L-cysteine as the most effective Eg5 inhibitor with an IC50 of 1.0 micromol/L for the inhibition of basal ATPase activity and 140 nmol/L for the microtubule-activated ATPase activity. Subsequent cell-based assays revealed that S-trityl-L-cysteine induced mitotic arrest in HeLa cells (IC50, 700 nmol/L) with characteristic monoastral spindles. S-trityl-L-cysteine is 36 times more potent for inducing mitotic arrest than the well-studied inhibitor, monastrol. Gossypol, flexeril, and two phenothiazine analogues were also identified as Eg5 inhibitors, and we found that they all result in monoastral spindles in HeLa cells. It is notable that all the Eg5 inhibitors identified here have been shown previously to inhibit tumor cell line growth in the NCI 60 tumor cell line screen, and we conclude that their antitumor activity may at least in part be explained by their ability to inhibit Eg5 activity.
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PMID:In vitro screening for inhibitors of the human mitotic kinesin Eg5 with antimitotic and antitumor activities. 1536 2

Human Eg5, a mitotic motor of the kinesin superfamily, is involved in the formation and maintenance of the mitotic spindle. The recent discovery of small molecules that inhibit HsEg5 by binding to its catalytic motor domain leading to mitotic arrest has attracted more interest in Eg5 as a potential anticancer drug target. We have used hydrogen-deuterium exchange mass spectrometry and directed mutagenesis to identify the secondary structure elements that form the binding sites of new Eg5 inhibitors, in particular for S-trityl-l-cysteine, a potent inhibitor of Eg5 activity in vitro and in cell-based assays. The binding of this inhibitor modifies the deuterium incorporation rate of eight peptides that define two areas within the motor domain: Tyr125-Glu145 and Ile202-Leu227. Replacement of the Tyr125-Glu145 region with the equivalent region in the Neurospora crassa conventional kinesin heavy chain prevents the inhibition of the Eg5 ATPase activity by S-trityl-l-cysteine. We show here that S-trityl-l-cysteine and monastrol both bind to the same region on Eg5 by induced fit in a pocket formed by helix alpha3-strand beta5 and loop L5-helix alpha2, and both inhibitors trigger similar local conformational changes within the interaction site. It is likely that S-trityl-l-cysteine and monastrol inhibit HsEg5 by a similar mechanism. The common inhibitor binding region appears to represent a "hot spot" for HsEg5 that could be exploited for further inhibitor screening.
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PMID:Identification of the protein binding region of S-trityl-L-cysteine, a new potent inhibitor of the mitotic kinesin Eg5. 1547 1

Autosomal dominant hereditary spastic paraplegia (AD HSP) linked to chromosome 12q (SPG10) is caused by mutations in the neuronal kinesin heavy-chain KIF5A gene. This is a rare cause of AD HSP, and only two disease-causing mutations have been reported thus far. In both instances, affected individuals harboring mutations in the KIF5A gene displayed symptom onset at a very early age. Here we present the results of clinical and genetic analyses of a large kindred with uncomplicated AD HSP. We were able to establish a definitive linkage to the SPG10 locus, and sequencing of the KIF5A gene revealed a heterozygous missense mutation 1,035 A>G in exon 10, resulting in tyrosine-to-cysteine substitution. This mutation is located in a highly conserved kinesin motor domain of the neuronal kinesin heavy-chain protein, but in contrast to two previously reported missense mutations, the age of symptom onset in our family was much later, with an average age of 36.1+/-4 years. Our results demonstrate that mutations in the KIF5A gene can also be associated with an adult age of onset of AD HSP.
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PMID:Mutation in KIF5A can also cause adult-onset hereditary spastic paraplegia. 1648 70

Loop L5 of kinesin is located near the ATPase site, in common with kinesins of various animal species. The rice plant-specific kinesin K16 also has a corresponding loop that is slightly shorter than that of mouse brain kinesin. The present study was designed to monitor conformational changes in loop L5 during ATP hydrolysis. For this purpose, we introduced one reactive cysteine into the L5 of rice kinesin and modified it with fluorescent probes. The cysteine in L5 was labeled with a fluorescent probe 2-(4'(iodoacetamide) anilino-naphthalene-6-sulfonic acid sodium salt) [IAANS]. IAANS was incorporated into L5 at an almost equimolar ratio in the absence of nucleotides. In contrast, the incorporated amount was reduced to 0.62 and 0.32 mol IAANS/mol motor domain in the presence of ATP and ADP, respectively. Upon nucleotide addition, the fluorescent intensity of IAANS incorporated into L5 was significantly reduced to 63% and 51% for ATP and ADP, respectively. These results suggest that L5 of rice kinesin significantly changes its conformation during ATP hydrolysis.
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PMID:Conformational change of the loop L5 in rice kinesin motor domain induced by nucleotide binding. 1675 93

A variety of bifunctional crosslinking agents have been explored for stabilizing microtubule shuttles used for the active transport of nanomaterials in artificial environments. Crosslinking agents that target amine residues form intertubulin crosslinks that produce crosslinked microtubules (CLMTs) with structural and functional lifetimes that can be up to four times as long as those achieved with taxol stabilization. Such CLMTs are stable at temperatures down to -10 degrees C, are resistant to depolymerization induced by metal ions such as Ca2+, and yet continue to be adsorbed and transported by self-assembled monolayers containing the motor protein kinesin. However, crosslinkers that target cysteine residues depolymerize the MTs, probably by interfering with the guanosine triphosphate binding site. The impact of crosslink attributes, including terminal group chemistry, chain length, crosslink density, and specific location on the tubulin surface, on microtubule stability and functionality are discussed.
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PMID:The stability and functionality of chemically crosslinked microtubules. 1719 24

HsEg5 (Eg5) is a kinesin required for proper execution of mitosis. Several compounds that specifically block Eg5 are in clinical development and have the potential to be used in the treatment of breast cancer. In this study, we investigated the interaction between Eg5 and estrogen receptor signaling. We observed decreased Eg5 expression after treatment of estrogen receptor-positive human breast cancer MCF-7 cells with the estrogen receptor downregulator fulvestrant. Downregulation of Eg5 expression in response to fulvestrant was also observed in another estrogen receptor-positive cell line ZR-75, but not in the estrogen receptor-negative breast cancer cell line MDA-231. Moreover, in MCF-7 cells previously arrested in the G0/G1 phase of the cell cycle by fulvestrant, addition of estrogen increased Eg5 expression. This upregulation correlated with progression through S-phase. Nevertheless, the effect of fulvestrant in Eg5 expression could not be explained solely by cell cycle arrest, because treatments that blocked cell cycle progression did not consistently decrease Eg5 expression. Pharmacological inhibition of Eg5 function, with either S-trityl-L-cysteine or monastrol, prevented growth of estrogen-treated MCF-7 cells with an IC50 of 0.46 and 29.71 micromol/l, respectively. Simultaneous inhibition of estrogen receptor function with fulvestrant increased the IC50 for S-trityl-L-cysteine to 2.30 micromol/l and for monastrol to 112.69 micromol/l. Our results suggest that pharmacological inhibition of Eg5 may be an effective treatment for estrogen receptor-positive breast cancer, even without concomitant hormonal therapy.
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PMID:Estrogen-dependent regulation of Eg5 in breast cancer cells. 1758 Dec 99


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