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961.
Mycobacterium tuberculosis prcBA genes encode a gated proteasome with broad oligopeptide specificity
Lin G Hu G Tsu C Kunes YZ Li H Dick L Parsons T Li P Chen Z Zwickl P Weich N Nathan C 《Molecular microbiology》2006,59(5):1405-1416
Genes predicted to be associated with the putative proteasome of Mycobacterium tuberculosis (Mtb) play a critical role in defence of the bacillus against nitrosative stress. However, proteasomes are uncommon in eubacteria and it remains to be established whether Mtb's prcBA genes in fact encode a proteasome. We found that coexpression of recombinant PrcB and PrcA in Escherichia coli over a prolonged period at 37 degrees C allowed formation of an alpha(7)beta(7)beta(7)alpha(7), 750 kDa cylindrical stack of four rings in which all 14 beta-subunits were proteolytically processed to expose the active site threonine. In contrast to another Actinomycete, Rhodococcus erythropolis, Mtb's beta-chain propeptide was not required for particle assembly. Peptidolytic activity of the 750 kDa particle towards a hydrophobic oligopeptide was nearly two orders of magnitude less than that of the Rhodococcus 20S proteasome, and unlike eukaryotic and archaeal proteasomes, activity of the Mtb 750 kDa particle could not be stimulated by SDS, Mg(2+) or Ca(2+). Electron microscopy revealed what appeared to be obstructed alpha-rings in the Mtb 750 kDa particle. Deletion of the N-terminal octapeptide from Mtb's alpha-chain led to disappearance of the apparent obstruction and a marked increase of peptidolytic activity. Unlike proteasomes isolated from other Actinomycetes, the open-gate Mtb mutant 750 kDa particle cleaved oligopeptides not only after hydrophobic residues but also after basic, acidic and small, neutral amino acids. Thus, Mtb encodes a broadly active, gated proteasome that may work in concert with an endogenous activator. 相似文献
962.
Becknell NC Zulli AL Angeles TS Yang S Albom MS Aimone LD Robinson C Chang H Hudkins RL 《Bioorganic & medicinal chemistry letters》2006,16(20):5368-5372
A novel series of C-3 urea, amide, and carbamate fused dihydroindazolocarbazole (DHI) analogs are reported as highly potent dual inhibitors of TIE-2 and VEGF-R2 receptor tyrosine kinases with excellent cellular potency. Structure-activity relationship (SAR) studies indicate the optimal N-13 alkyl substitutions are n-propyl and i-butyl. The isopropyl carbamate 39 displayed good dual enzyme, cell potency, and rat pharmacokinetic properties for advancement to in vivo evaluation. 相似文献
963.
Dardonville C Fernandez-Fernandez C Gibbons SL Ryan GJ Jagerovic N Gabilondo AM Meana JJ Callado LF 《Bioorganic & medicinal chemistry》2006,14(19):6570-6580
Two series of fentanyl-derived hybrid molecules bearing potent I2-imidazoline binding site (IBS) ligands (i.e., guanidine and BU224 moieties) linked with an aliphatic (m=2, 3, 4, 6, 7, 8, 9 and 12 methylene units) or aromatic spacer were prepared. Their affinities for the mu-opioid receptors and for the I2-IBS were determined through competition binding studies on human postmortem brain membranes. Whereas the BU224 hybrid molecules bound to the mu-opioid receptor and the I2-IBS in the micromolar to low micromolar range, the alkaneguanidine series exhibited remarkable affinities in the nanomolar range for both receptors. [35S]GTPgammaS functional assays were performed on human postmortem brain membranes with selected ligands from each series (4f and 8g) showing the highest dual affinity for the mu-opioid receptor and I2-IBS affinities. Both compounds displayed agonist properties: at the mu-opioid receptor for the alkaneguanidine derivative 4f (spacer: six methylene units) and at a G-protein coupled receptor (GPCR) which remains to be determined for 8g. The lack of analgesic properties of 4f in vivo (i.e., hot plate and writhing tests in mice), discordant with the good in vitro binding data (Ki mu=1.04+/-0.28 nM, Ki I2=409+/-238 nM), may possibly be due to the low intrinsic efficacy of the compound. Alternatively, a low access to the central nervous system for this kind of hybrid molecules cannot be ruled out. Two new compounds reported here (9f and 13), which were not dual acting, are worth mentioning for their outstanding binding affinities; 9f bound to the mu-opioid receptor with a picomolar affinity (Ki=0.0098+/-0.0033 nM), whereas 13 presented an I2-IBS affinity (Ki=18+/-11 nM) similar to the reference compound BU224. 相似文献
964.
The interaction of palytoxin with the Na,K-ATPase was studied by the electrochromic styryl dye RH421, which monitors the amount of ions in the membrane domain of the pump. The toxin affected the pump function in the state P-E2, independently of the type of phosphorylation (ATP or inorganic phosphate). The palytoxin-induced modification of the protein consisted of two steps: toxin binding and a subsequent conformational change into a transmembrane ion channel. At 20 degrees C, the rate-limiting reaction had a forward rate constant of 10(5) M(-1)s(-1) and a backward rate constant of about 10(-3) s(-1). In the palytoxin-modified state, the binding affinity for Na+ and H+ was increased and reached values between those obtained in the E1 and P-E2 conformation under physiological conditions. Even under saturating palytoxin concentrations, the ATPase activity was not completely inhibited. In the Na/K mode, approximately 50% of the enzyme remained active in the average, and in the Na-only mode 25%. The experimental findings indicate that an additional exit from the inhibited state exists. An obvious reaction pathway is a slow dephosphorylation of the palytoxin-inhibited state with a time constant of approximately 100 s. Analysis of the effect of blockers of the extracellular and cytoplasmic access channels, TPA+ and Br2-Titu3+, respectively, showed that both access channels are part of the ion pathway in the palytoxin-modified protein. All experiments can be explained by an extension of the Post-Albers cycle, in which three additional states were added that branch off in the P-E2 state and lead to states in which the open-channel conformation is introduced and returns into the pump cycle in the occluded E2 state. The previously suggested molecular model for the channel state of the Na,K-ATPase as a conformation in which both gates between binding sites and aqueous phases are simultaneously in their open state is supported by this study. 相似文献
965.
966.
Powell MS Barnes NC Bradford TM Musgrave IF Wines BD Cambier JC Hogarth PM 《Journal of immunology (Baltimore, Md. : 1950)》2006,176(12):7489-7494
The aggregation of cell surface FcRs by immune complexes induces a number of important Ab-dependent effector functions. However, despite numerous studies that examine receptor function, very little is known about the molecular organization of these receptors within the cell. In this study, protein complementation, mutagenesis, and ligand binding analyses demonstrate that human FcgammaRIIa is present as a noncovalent dimer form. Protein complementation studies found that FcgammaRIIa molecules are closely associated. Mutagenesis of the dimer interface, as identified by crystallographic analyses, did not affect ligand binding yet caused significant alteration to the magnitude and kinetics of receptor phosphorylation. The data suggest that the ligand binding and the dimer interface are distinct regions within the receptor, and noncovalent dimerization of FcgammaRIIa may be an essential feature of the FcgammaRIIa signaling cascade. 相似文献
967.
Invertebrate herbivory along a gradient of plant species diversity in extensively managed grasslands
Increasing plant diversity has long been hypothesized to negatively affect levels of invertebrate herbivory due to a lower number of specialist insect herbivores in more diverse sites, but studies of natural systems have been rare. We used a planned comparison to study herbivory in a set of 19 semi-natural montane grasslands managed as hay meadows. Herbivory was measured in transects through the plant communities, and in individuals of Plantago lanceolata and Trifolium pratense that were transplanted into each meadow. In addition, plant community biomass and arthropod abundances were determined in the grasslands. Before the first mowing in June, mean herbivory levels correlated negatively with plant species richness, as predicted by theory, but they were also significantly affected by plant community biomass and plant community composition. After mowing, herbivory levels were only significantly related to plant community composition. Damage levels in the transplants were lower than herbivory levels in the established plant communities. Most insect herbivores were generalists and not specialists. The number of insect herbivores and spiders were positively correlated and tended to increase with increasing plant species richness. Herbivory levels were correlated negatively with spider abundances. We conclude that while the predicted negative relationship between plant species richness and insect herbivory can be found in grasslands, the underlying mechanism involves generalist rather than specialist herbivores. Our data also suggest a role of natural enemies in generalist herbivore activities.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at . 相似文献
968.
Russell JA Conforti ML Connor NP Hartig GK 《Plastic and reconstructive surgery》2006,117(7):2259-66; discussion 2267-8
969.
Nadine Hagen Paul P. Van Veldhoven Richard L. Proia Hyejung Park Alfred H. Merrill Jr. Gerhild van Echten-Deckert 《The Journal of biological chemistry》2009,284(17):11346-11353
Cerebellar granule cells from sphingosine 1-phosphate (S1P) lyase-deficient
mice were used to study the toxicity of this potent sphingolipid metabolite in
terminally differentiated postmitotic neurons. Based on earlier findings with
the lyase-stable, semi-synthetic, cis-4-methylsphingosine phosphate,
we hypothesized that accumulation of S1P above a certain threshold induces
neuronal apoptosis. The present studies confirmed this conclusion and further
revealed that for S1P to induce apoptosis in lyase-deficient neurons it must
also be produced by sphingosine-kinase2 (SK2). These conclusions are based on
the finding that incubation of lyase-deficient neurons with either sphingosine
or S1P results in a similar elevation in cellular S1P; however, only S1P
addition to the culture medium induces apoptosis. This was not due to S1P
acting on the S1P receptor but to hydrolysis of S1P to sphingosine that was
phosphorylated by the cells, as described before for
cis-4-methylsphingosine. Although the cells produced S1P from both
exogenously added sphingosine as well as sphingosine derived from exogenous
S1P, the S1P from these two sources were not equivalent, because the former
was primarily produced by SK1, whereas the latter was mainly formed by SK2 (as
also was cis-4-methylsphingosine phosphate), based on studies in
neurons lacking SK1 or SK2 activity. Thus, these investigations show that, due
to the existence of at least two functionally distinct intracellular origins
for S1P, exogenous S1P can be neurotoxic. In this model, S1P accumulated due
to a defective lyase, however, this cause of toxicity might also be important
in other cases, as illustrated by the neurotoxicity of
cis-4-methylsphingosine phosphate.Sphingosine 1-phosphate
(S1P)2 is a potent
lipid mediator that has been shown to regulate a wide range of physiological
processes, including proliferation, differentiation, motility, cytoskeleton
rearrangements, and calcium homeostasis
(1,
2). There is convincing
experimental evidence that this bioactive sphingolipid can act both
extracellularly, as a ligand for a family of five specific G protein-coupled
receptors, and inside the cells, as a second messenger
(3,
4). In most cell types
described so far, S1P and its metabolic precursor ceramide exert antagonistic
effects on cell survival with S1P being generally regarded as a survival
signal, whereas ceramide and sphingosine are generally toxic
(5,
6). Interestingly, generation
of sphingosine and S1P is generally thought to be dependent on the
availability of ceramide (7),
however, relatively high amounts of S1P are also present in blood, lymph, and
cerebrospinal fluid (8,
9) and may serve as additional
sources for some cells.More than a decade ago, we introduced the synthetic sphingosine analog
cis-4-methylsphingosine as a tool for studies of sphingoid base
metabolism and function (10).
When added to the culture medium, this analog is taken up and mainly
phosphorylated to the respective cis-4-methylsphingosine phosphate,
which accumulates intracellularly, because it is poorly cleaved (if at all) by
S1P lyase (10). Intriguingly,
this compound promoted proliferation of quiescent Swiss 3T3 fibroblasts
(11), as does S1P
(12), but induced apoptosis in
postmitotic terminally differentiated primary cultured neurons
(13).Despite the fact that neither S1P nor sphingosine were able to induce
apoptosis in neurons, we proposed that cis-4-methylsphingosine is
phosphorylated by cells yielding a metabolically stable analog of S1P. This
prediction was based on experimental results indicating that the different
physiological effects, apoptosis in the case of the accumulating metabolically
stable synthetic compound versus no apoptosis in the case of the
short living S1P, rely only on nuances of impact
(13). Both sphingoid
phosphates affected similar pathways. However, the effect of the synthetic
accumulated compound was more pronounced and persistent when compared with the
more transient and less pronounced effect of the short living physiological
counterpart (13). We therefore
assumed that conditions that allow sufficient accumulation of S1P in primary
cultured neurons should end up in neuronal apoptosis.To explore this hypothesis, which might be relevant to neurodegenerative
processes, we attempted to elevate intracellular S1P using siRNAs directed to
S1P lyase (encoded by the Sgpl1 gene). However, suppression of lyase
by ∼70% did not result in an accumulation of endogenous S1P in primary
cultured neurons (14).The central aim of the present study was to evaluate the hypothesis that
endogenous S1P induces neuronal apoptosis when it exceeds a certain threshold
by a more effective method for lyase activity suppression. We thus used
primary cultured neurons prepared from cerebella of 6-day-old lyase-deficient
mice (15). The present studies
not only confirmed that elevation of S1P induced cell death but also revealed
that the origin of the S1P was important. Intriguingly, neuronal apoptosis was
induced only by S1P derived from exogenous S1P that was dephosphorylated and
then resynthesized to S1P by sphingosine kinase 2 (SK2). Interestingly, we
then found that this is also the kinase responsible for synthesis of
cis-4-methylsphingosine phosphate. In addition, our data document
that the pro-apoptotic effect of S1P is independent of cellular ceramide
content. 相似文献
970.
Regina Rabl Vincent Soubannier Roland Scholz Frank Vogel Nadine Mendl Andreja Vasiljev-Neumeyer Christian K?rner Ravi Jagasia Thomas Keil Wolfgang Baumeister Marek Cyrklaff Walter Neupert Andreas S. Reichert 《The Journal of cell biology》2009,185(6):1047-1063
Crista junctions (CJs) are important for mitochondrial organization and function, but the molecular basis of their formation and architecture is obscure. We have identified and characterized a mitochondrial membrane protein in yeast, Fcj1 (formation of CJ protein 1), which is specifically enriched in CJs. Cells lacking Fcj1 lack CJs, exhibit concentric stacks of inner membrane in the mitochondrial matrix, and show increased levels of F1FO–ATP synthase (F1FO) supercomplexes. Overexpression of Fcj1 leads to increased CJ formation, branching of cristae, enlargement of CJ diameter, and reduced levels of F1FO supercomplexes. Impairment of F1FO oligomer formation by deletion of its subunits e/g (Su e/g) causes CJ diameter enlargement and reduction of cristae tip numbers and promotes cristae branching. Fcj1 and Su e/g genetically interact. We propose a model in which the antagonism between Fcj1 and Su e/g locally modulates the F1FO oligomeric state, thereby controlling membrane curvature of cristae to generate CJs and cristae tips. 相似文献