Gene/Protein Disease Symptom Drug Enzyme Compound
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The formation mechanism of an alanine-based peptide has been studied by all-atom molecular dynamics simulations with a recently developed all-atom point-charge force field and the Generalize Born continuum solvent model at an effective salt concentration of 0.2M. Thirty-two simulations were conducted. Each simulation was performed for 100 ns. A surprisingly complex folding process was observed. The development of the helical content can be divided into three phases with time constants of 0.06-0.08, 1.4-2.3, and 12-13 ns, respectively. Helices initiate extreme rapidly in the first phase similar to that estimated from explicit solvent simulations. Hydrophobic collapse also takes place in this phase. A folding intermediate state develops in the second phase and is unfolded to allow the peptide to reach the transition state in the third phase. The folding intermediate states are characterized by the two-turn short helices and the transition states are helix-turn-helix motifs-both of which are stabilized by hydrophobic clusters. The equilibrium helical content, calculated by both the main-chain Phi-Psi torsion angles and the main-chain hydrogen bonds, is 64-66%, which is in remarkable agreement with experiments. After corrected for the solvent viscosity effect, an extrapolated folding time of 16-20 ns is obtained that is in qualitative agreement with experiments. Contrary to the prevailing opinion, neither initiation nor growth of the helix is the rate-limiting step. Instead, the rate-limiting step for this peptide is breaking the non-native hydrophobic clusters in order to reach the transition state. The implication to the folding mechanisms of proteins is also discussed.
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PMID:Breaking non-native hydrophobic clusters is the rate-limiting step in the folding of an alanine-based peptide. 1257 80

We have studied the action of some membranotropic agents (MTAs) on the parameters of mono- and multilayers of dipalmitoylphosphatidylcholine (DPPC). The MTAs used included an antimicrobial drug, decamethoxinum, the model amphiphilic agent stearoyl-L-alpha-alanine, and cholesterol as a reference substance. Using differential scanning calorimetry and the Langmuir monolayer technique, we measured the temperature and enthalpy of the main phase transition of DPPC, the mean molecular area, the collapse pressure and the free energy of the mixed monolayers of DPPC and MTA. A good correlation has been obtained between the structure of the MTA used and changes in the parameters of both mono- and multilayers. Thus, for cholesterol, its well-known condensing effect in the L alpha phase correlates with its behavior in the mixed monolayers. The disturbing action of decamethoxinum (depression of the phase transition in DPPC multilayers and relatively high free energy of mixing in monolayers) is presumably connected with interaction of its charged ammonium moieties with polar phospholipid heads. At the same time, stearoyl-L-alpha- alpha-alanine condensed the lipid layers and increased the melting point of DPPC, owing to its interaction with both polar and non-polar lipid moieties. One can conclude that the three MTAs used can really be considered as representative examples of three different types of behavior in mono- and multilayers.
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PMID:Effects of membranotropic agents on mono- and multilayer structures of dipalmitoylphosphatidylcholine. 1260 41

Deuterium isotope effects at C2 of aspartate and heavy atom isotope effects at C2, C3, and the amino group of aspartate were determined for the reaction of the lysine-258 to alanine mutant of Escherichia coli rescued with exogenous ammonia. We were able to calculate an (15)N intrinsic isotope effect of 1.034. The intrinsic (13)C isotope effect at C3 is 1.0060, and the (13)C isotope effect at C2 is 1.0016. These isotope effects reveal that collapse of the carbinolamine (or gem-diamine) to give the final product is the rate-determining step in this system. Furthermore, these results indicate that lysine-258 is critical to the catalysis of the final breakdown to give product, and in fact this step is more strongly affected by mutation of lysine-258 than the deprotonation of the external aldimine.
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PMID:2H, 13C, and 15N kinetic isotope effects on the reaction of the ammonia-rescued K258A mutant of aspartate aminotransferase. 1284 86

Ephrin-As are repulsive axonal guidance cues that regulate retinotectal projection. EphA tyrosine kinases, which are the receptors of ephrin-As, activate signaling cascades leading to cytosckeleton reorganization. Here, we address the role of cyclin-dependent kinase (Cdk) 5 in Eph receptor signaling induced by ephrin-A5. Ephrin-A5 induced a cell morphological response in PC-3M cells that endogenously express Cdk5 and EphA2, a receptor for ephrin-A5. This response was augmented by the transfection of p35, which is a neuronal regulator of Cdk5. While the morphological response of native PC-3M cells was not affected by olomoucine, an inhibitor of Cdk, the response was inhibited in the p35-transfected cells. In retinal ganglion cells, either olomoucine at 20 microM or Y-27632 at 10 microM, an inhibitor of Rho-kinase/ROKalpha/ROCKII, showed maximum inhibitory effect against ephrin-A5 (10 microg/ml)-induced growth cone collapse. Combined application of olomoucine and Y-27632 further suppressed the ephrin-A5-induced response. Ephrin-A5 evoked phosphorylation of Cdk5 at Tyr15 and tau, a substrate of Cdk5 in retinal growth cones. Recombinant herpes simplex virus expressing Cdk5 mutant (kinase-negative or Tyr15 to Ala) showed a dominant-negative effect on the ephrin-A5-induced growth cone collapse. These findings demonstrate that both Cdk5 and the Rho kinase pathway independently contribute to the downstream of ephrin-A-induced signaling in retinal ganglion cells.
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PMID:Cdk5/p35 and Rho-kinase mediate ephrin-A5-induced signaling in retinal ganglion cells. 1466 14

Cadmium (Cd) is an environmental pollutant of global concern with a 10-30-year biological half-life in humans. Accumulating evidence suggests that the lung is one of the major target organs of inhaled Cd compounds. Our previous report demonstrated that 100 microM Cd induces MRC-5 cells, normal human lung fibroblasts, to undergo caspase-independent apoptosis mediated by mitochondrial membrane depolarization and translocation of apoptosis-inducing factor (AIF) from mitochondria into the nucleus. Here, using benzyloxycarbonyl-Val-Ala-Asp-(ome) fluoromethyl ketone (Z-VAD.fmk) as a tool, we further demonstrated that Cd could induce caspase-independent apoptosis at concentrations varied from 25 to 150 microM, which was modulated by reactive oxygen species (ROS) scavengers, such as N-acetylcysteine (NAC), mannitol, and tiron, indicating that ROS play a crucial role in the apoptogenic activity of Cd. Consistent with this notion, the intracellular hydrogen peroxide (H2O2) was 2.9-fold elevated after 3 h of Cd treatment and diminished rapidly within 1 h as detected by flow cytometry with 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) staining. Using inhibitors of the mitochondrial electron transport chain (ETC) (oligomycin A and rotenone for complex I and V, respectively) and mitochondrial permeability transition pore (MPTP) (cyclosporin A and aristolochic acid), we coincidently found the ROS production, mitochondrial membrane depolarization, and apoptotic content were almost completely or partially abolished. As revealed by confocal microscopy staining with chloromethyl-X-rosamine (CMXRos) and an anti-AIF antibody, the collapse of mitochondrial membrane potential induced by Cd (3 h-treatment) was a prelude to the translocation of caspase-independent pro-apoptotic factor, AIF, into the nucleus (after 4 h of Cd treatment). In summary, this study demonstrated that, in MRC-5 fibroblasts, Cd induced caspase-independent apoptosis through a mitochondria-ROS pathway. More importantly, we provide several lines of evidence supporting a role of mitochondrial ETC and MPTP in the regulation of caspase-independent cell death triggered by Cd.
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PMID:Mediating of caspase-independent apoptosis by cadmium through the mitochondria-ROS pathway in MRC-5 fibroblasts. 1474 97

Sub-lethal excitotoxic injury to dendrites can elicit loss or shrinkage of dendritic spines. Here, we used a cell culture model of sub-lethal NMDA-induced injury to investigate a role for proteolysis in spine collapse. Transient incubation with NMDA-induced spine collapse and spine F-actin loss within 10 min, an effect not mimicked by the actin assembly inhibitor latrunculin A. NMDA-induced spine collapse was significantly attenuated by preincubation with broad-spectrum cysteine protease inhibitors. Results obtained using several class-specific protease inhibitors suggested that this protective effect was due to specific blockade of cathepsin B/L type protease activity, since selective inhibitors of only these proteases significantly attenuated spine loss. Cathepsin B-like immunoreactivity was observed at synaptic sites, but lysosomes were not. Immunoblot analysis showed that MARCKS (myristoylated-alanine-rich C-kinase substrate), a known substrate of cathepsin B, was specifically degraded in response to intense NMDA receptor stimulation. This effect was blocked by preincubation with a cathepsin B-selective inhibitor. Together these data suggest a model in which NMDA-induced spine collapse involves cathepsin B-like proteolysis of MARCKS, and possibly other proteins that regulate the actin-based cytoskeleton.
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PMID:Cathepsin B-like proteolysis and MARCKS degradation in sub-lethal NMDA-induced collapse of dendritic spines. 1545 42

Rasburicase (Fasturtec/Elitek) is a new generation of recombinant urate oxidase administred therapeutically by intravenous infusion for the prevention or treatment of hyperuricemia during chemotherapy. To ensure a long storage period, a freeze-dried formulation was developed to guarantee the molecular integrity and enzyme activity. Screening of potential excipients was the first stage of the preformulation study. The selection was based on stability results (rasburicase solution with excipient) obtained with the isoelectric focusing profiles and residual enzyme activity. The different excipients were classified as stabilising, neutral or destabilising. A stability study was then carried out on different freeze-dried formulations containing the usual bulking agents for freeze-drying, excipients with a high glass transition temperature or competitive enzyme inhibitors having a stabilising effect. A mannitol/alanine mixture in phosphate buffer was selected from these preliminary results. Finally, the optimal content of mannitol and alanine in the freeze-dried powder was determined by an experimental design study. The water content and the appearance of the "cake", the osmolality, pH, clarity, and enzyme activity of the reconstituted solution were assessed. The formula with a mannitol/alanine ratio of 0.7 was found to be the best composition. Differential scanning calorimetry and ThermoStimulated Current technique experiments were carried out to study the amorphous phase. A glass transition temperature of about 45-500 degrees C was found. Glassy state is known to preserve stability, which was verified by the real stability data. X-ray diffraction studies have shown that alanine is in a crystallised state and that mannitol remains amorphous. Crystallised excipients participate in forming the structure of the powder and therefore help to prevent any collapse. Amorphous mannitol creates a surrounding medium favourable to the stability of the protein.
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PMID:Stabilization of rasburicase and physico-chemical characterization of the resulting injectable formulation. 1552 33

Collapsin response mediating protein-2 (CRMP2) has been identified as an intracellular protein mediating Semaphorin3A (Sema3A), a repulsive guidance molecule. In this study, we demonstrate that cyclin-dependent kinase 5 (Cdk5) and glycogen synthase kinase 3beta (GSK3beta) plays a critical role in Sema3A signalling. In In vitro kinase assay, Cdk5 phosphorylated CRMP2 at Ser522, while GSK3beta did not induce any phosphorylation of CRMP2. Phosphorylation by GSK3beta was exclusively observed in Cdk5-phosphorylated CRMP2, but barely in CRMP2T509A. These results indicate that Cdk5 primarily phosphorylates CRMP2 at Ser522 and GSK3beta secondarily phosphorylates at Thr509. The dual-phosphorylated CRMP2, but not non-phosphorylated or single-phosphorylated CRMP2, is recognized with the antibody 3F4, which is highly reactive with the neurofibrillary tangles of Alzheimer's disease. 3F4 recognized the CRMP2 in the wild-type but not cdk5-/- mouse embryonic brain lysates. The phosphorylation of CRMP2 at Ser522 caused reduction of its affinity to tubulin. In dorsal root ganglion neurones, Sema3A stimulation enhanced the levels of the phosphorylated form of CRMP2 detected by 3F4. Over-expression of CRMP2 mutant substituting either Ser522 or Thr509 to Ala attenuates Sema3A-induced growth cone collapse response. These results suggest that the sequential phosphorylation of CRMP is an important process of Sema3A signalling and the same mechanism may have some relevance to the pathological aggregation of the microtubule-associated proteins.
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PMID:Semaphorin3A signalling is mediated via sequential Cdk5 and GSK3beta phosphorylation of CRMP2: implication of common phosphorylating mechanism underlying axon guidance and Alzheimer's disease. 1567 27

Competing views of the products of sub-millisecond folding reactions observed in many globular proteins have been ascribed either to the formation of discrete, partially folded states or to the random collapse of the unfolded chain under native-favoring conditions. To test the validity of these alternative interpretations for the stopped-flow burst-phase reaction in the (betaalpha)8, TIM barrel motif, a series of alanine replacements were made at five different leucine or isoleucine residues in the alpha subunit of tryptophan synthase (alphaTS) from Escherichia coli. This protein has been proposed to fold, in the sub-millisecond time range, to an off-pathway intermediate with significant stability and approximately 50% of the far-UV circular dichroism (CD) signal of the native conformation. Individual alanine replacements at any of three isoleucine or leucine residues in either alpha1, beta2 or beta3 completely eliminate the off-pathway species. These variants, within 5 ms, access an intermediate whose properties closely resemble those of an on-pathway equilibrium intermediate that is highly populated at moderate urea concentrations in wild-type alphaTS. By contrast, alanine replacements for leucine residues in either beta4 or beta6 destabilize but preserve the off-pathway, burst-phase species. When considered with complementary thermodynamic and kinetic data, this mutational analysis demonstrates that the sub-millisecond appearance of CD signal for alphaTS reflects the acquisition of secondary structure in a distinct thermodynamic state, not the random collapse of an unfolded chain. The contrasting results for replacements in the contiguous alpha1/beta2/beta3 domain and the C-terminal beta4 and beta6 strands imply a heterogeneous structure for the burst-phase species. The alpha1/beta2/beta3 domain appears to be tightly packed, and the C terminus appears to behave as a molten-globule-like structure whose folding is tightly coupled to that of the alpha1/beta2/beta3 domain.
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PMID:Specific structure appears at the N terminus in the sub-millisecond folding intermediate of the alpha subunit of tryptophan synthase, a TIM barrel protein. 1602 36

The folding kinetics of a 16-residue beta-hairpin (trpzip4) and five mutants were studied by a laser-induced temperature-jump infrared method. Our results indicate that mutations which affect the strength of the hydrophobic cluster lead to a decrease in the thermal stability of the beta-hairpin, as a result of increased unfolding rates. For example, the W45Y mutant has a phi-value of approximately zero, implying a folding transition state in which the native contacts involving Trp45 are not yet formed. On the other hand, mutations in the turn or loop region mostly affect the folding rate. In particular, replacing Asp46 with Ala leads to a decrease in the folding rate by roughly 9 times. Accordingly, the phi-value for D46A is determined to be approximately 0.77, suggesting that this residue plays a key role in stabilizing the folding transition state. This is most likely due to the fact that the main chain and side chain of Asp46 form a characteristic hydrogen bond network with other residues in the turn region. Taken together, these results support the folding mechanism we proposed before, which suggests that the turn formation is the rate-limiting step in beta-hairpin folding and, consequently, a stronger turn-promoting sequence increases the stability of a beta-hairpin primarily by increasing its folding rate, whereas a stronger hydrophobic cluster increases the stability of a beta-hairpin primarily by decreasing its unfolding rate. In addition, we have examined the compactness of the thermally denatured and urea-denatured states of another 16-residue beta-hairpin, using the method of fluorescence resonance energy transfer. Our results show that the thermally denatured state of this beta-hairpin is significantly more compact than the urea-denatured state, suggesting that the very first step in beta-hairpin folding, when initiated from an extended conformation, probably corresponds to a process of hydrophobic collapse.
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PMID:Understanding the mechanism of beta-hairpin folding via phi-value analysis. 1648 60


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