Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0344329 (collapse)
 28,634 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Apoptosis is orchestrated by a family of cysteine proteases known as the caspases. Fourteen mammalian caspases have been identified, three of which (caspase-3, -6, and -7) are thought to coordinate the execution phase of apoptosis by cleaving multiple structural and repair proteins. However, the relative contributions that the "executioner" caspases make to the demolition of the cell remains speculative. Here we have used cell-free extracts immuno-depleted of either caspase-3, -6, or -7 to examine the caspase requirements for apoptosis-associated proteolysis of 14 caspase substrates as well as nuclear condensation, chromatin margination, and DNA fragmentation. We show that caspase-3 is the primary executioner caspase in this system, necessary for cytochrome c/dATP-inducible cleavage of fodrin, gelsolin, U1 small nuclear ribonucleoprotein, DNA fragmentation factor 45 (DFF45)/inhibitor of caspase-activated DNase (ICAD), receptor-interacting protein (RIP), X-linked inhibitor of apoptosis protein (X-IAP), signal transducer and activator of transcription-1 (STAT1), topoisomerase I, vimentin, Rb, and lamin B but not for cleavage of poly(ADP-ribose) polymerase (PARP) or lamin A. In addition, caspase-3 was also essential for apoptosis-associated chromatin margination, DNA fragmentation, and nuclear collapse in this system. Surprisingly, although caspase-6 and -7 are considered to be important downstream effector caspases, depletion of either caspase had minimal impact on any of the parameters investigated, calling into question their precise role during the execution phase of apoptosis.
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PMID:Executioner caspase-3, -6, and -7 perform distinct, non-redundant roles during the demolition phase of apoptosis. 1105 99

To investigate the functional and structural roles of the proximal thiolate ligand in cytochrome P450cam, we prepared the C357H mutant of the enzyme in which the axial cysteine residue (Cys357) was replaced with a histidine residue. We obtained the unstable C357H mutant by developing a new preparation procedure involving in vitro folding of P450cam from the inclusion bodies. The C357H mutant in the ferrous-CO form exhibited the Soret peak at 420 nm and the Fe-CO stretching line at 498 cm-1, indicating a neutral histidine residue as the axial ligand. However, another internal ligand is coordinated to the heme iron as the sixth ligand in the ferric and ferrous forms of the C357H mutant, suggesting the collapse of the substrate-binding site. The C357H mutant showed no catalytic activity for camphor hydroxylation and the reduced heterolytic/homolytic ratio of the O-O bond scission in the reaction with cumene hydroperoxide. The present observations indicate that the thiolate coordination in P450cam is important for the construction of the heme pocket and the heterolysis of the O-O bond.
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PMID:Proximal cysteine residue is essential for the enzymatic activities of cytochrome P450cam. 1116 58

Drug-induced acute liver failure (ALF) is a devastating and often fatal disease mainly caused by poisoning by acetaminophen (APAP). The toxic metabolite, N-acetyl-p-benzoquinone-imine (NAPQI), that leads to gluthatione depletion has been suspected to be the main effector of hepatocyte apoptosis during APAP-induced ALF. We have investigated whether reactive oxygen species (ROS) also play a role in APAP-induced ALF, and whether manganese III tetrakis (5,10,15,20 benzoic acid) (MnTBAP), a mimic of superoxide dismutase (SOD) with catalase-like activity, can treat the disease in mice. The effects of MnTBAP were tested on APAP-intoxicated mice and on isolated hepatocytes incubated with APAP. MnTBAP preventively and curatively administered significantly improved survival times, and dramatically reduced serum transaminase activity levels and parenchymal lesions in APAP-intoxicated mice. Whereas pretreatment with N-acetyl-L-cysteine (NAC) prevented ALF in a dose-dependent manner, the molecule was ineffective when curatively administered. The significant increase in glutathione peroxidase (Gpx) activity following APAP administration, and the beneficial effects of MnTBAP suggested that ROS were produced during APAP-induced ALF. A direct evidence of ROS generation was provided by flow cytometry of isolated hepatocytes incubated with APAP. In vitro, ROS production was associated with mitochondrial damage characterized by the collapse of transmembrane potential and the loss of cardiolipin content. In livers of intoxicated mice, ALF was associated with cytochrome c release that led to the activation of caspases-9 and -3. The capacity of MnTBAP to abrogate all those alterations suggests that ROS play a role in APAP-induced apoptosis of hepatocytes, and explains the beneficial effects of MnTBAP, which could be of interest in APAP-induced ALF in humans.
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PMID:Detoxification of reactive oxygen species by a nonpeptidyl mimic of superoxide dismutase cures acetaminophen-induced acute liver failure in the mouse. 1134 46

This article discusses how nitrate assimilation is integrated with nitrate uptake, with ammonium assimilation and amino acid synthesis, with pH regulation, and with the sugar supply in tobacco leaves. During the first part of the light period, nitrate assimilation exceeds nitrate uptake by 2-fold and ammonium assimilation by 50%, leading to rapid depletion of nitrate and accumulation of ammonium, glutamine, glycine and serine. NIA, NII and PPC expression show a shared maximum early in the diurnal cycle to direct carbon towards malate synthesis for pH regulation. Later in the diurnal cycle an orchestrated increase of GLN2, PKc, CS, and ICDH-1 expression re-establishes a balance between nitrate assimilation and ammonium metabolism. Nitrate uptake continues throughout the night, replenishing the leaf nitrate pool. These diurnal changes are attenuated or abolished in mutants with low NIA activity, and modified in wild-type plants growing on different nitrogen sources or elevated [CO(2)]. Comparison across genotypes and conditions reveals that NIA transcript levels are always closely related to the balance between nitrate influx and assimilation, but are unrelated to changes of glutamine or 2-oxoglutarate. In a systematic search for other downstream regulators, a wide range of downstream metabolites was fed to detached leaves and glutamate, cysteine, asparagine, and malate identified as candidates. Low sugars totally inhibit nitrate assimilation, overriding signals from nitrogen metabolism. Moderate changes act post-transcriptionally, and larger changes lead to a collapse of the NIA transcript. Low sugars also lead to a collapse of minor amino acids and a dramatic decrease of phenylpropanoids and nicotine. Consequently, wild-type plants growing in unfavourable light regimes and antisense RBCS transformants are simultaneously carbon- and nitrogen-limited.
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PMID:Steps towards an integrated view of nitrogen metabolism. 1191 38

Methionine sulfoxide reductases (Msr) protect against oxidative damage that can contribute to cell death. The tandem Msr domains (MsrA and MsrB) of the pilB protein from Neisseria gonorrhoeae each reduce different epimeric forms of methionine sulfoxide. The overall fold of the MsrB domain revealed by the 1.85 A crystal structure shows no resemblance to the previously determined MsrA structures from other organisms. Despite the lack of homology, the active sites show approximate mirror symmetry. In each case, conserved amino acid motifs mediate the stereo-specific recognition and reduction of the substrate. Unlike the MsrA domain, the MsrB domain activates the cysteine or selenocysteine nucleophile through a unique Cys-Arg-Asp/Glu catalytic triad. The collapse of the reaction intermediate most likely results in the formation of a sulfenic or selenenic acid moiety. Regeneration of the active site occurs through a series of thiol-disulfide exchange steps involving another active site Cys residue and thioredoxin. These observations have broad implications for modular catalysis, antibiotic drug design and continuing longevity studies in mammals.
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PMID:The mirrored methionine sulfoxide reductases of Neisseria gonorrhoeae pilB. 1193 52

Slow inactivation of voltage-gated Na channels is kinetically and structurally distinct from fast inactivation. Whereas structures that participate in fast inactivation are well described and include the cytoplasmic III-IV linker, the nature and location of the slow inactivation gating mechanism remains poorly understood. Several lines of evidence suggest that the pore regions (P-regions) are important contributors to slow inactivation gating. This has led to the proposal that a collapse of the pore impedes Na current during slow inactivation. We sought to determine whether such a slow inactivation-coupled conformational change could be detected in the outer pore. To accomplish this, we used a rapid perfusion technique to measure reaction rates between cysteine-substituted side chains lining the aqueous pore and the charged sulfhydryl-modifying reagent MTS-ET. A pattern of incrementally slower reaction rates was observed at substituted sites at increasing depth in the pore. We found no state-dependent change in modification rates of P-region residues located in all four domains, and thus no change in aqueous accessibility, between slow- and nonslow-inactivated states. In domains I and IV, it was possible to measure modification rates at residues adjacent to the narrow DEKA selectivity filter (Y401C and G1530C), and yet no change was observed in accessibility in either slow- or nonslow-inactivated states. We interpret these results as evidence that the outer mouth of the Na pore remains open while the channel is slow inactivated.
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PMID:Slow inactivation does not block the aqueous accessibility to the outer pore of voltage-gated Na channels. 1235 53

A detailed kinetic analysis of metal encapsulation by a de novo-designed protein is described. The kinetic mechanism of Hg(II) encapsulation in the three-stranded coiled coil formed by the peptide CH(3)CO-G LKALEEK CKALEEK LKALEEK G-NH(2) (Baby L9C) is derived by global analysis. The mechanism involves rapid initial collapse of two peptides by Hg(II) forming Hg(Baby L9C(-H))(2) with a linear thiolato Hg(II) bound to the cysteine sulfur atoms. Here, Baby L9C(-H) denotes Baby L9C with the cysteine thiol deprotonated. Addition of the third peptide, forming the three-stranded coiled coil, is the rate-determining step and results in an intermediate state involving two separate species. One of the species, termed the properly folded intermediate, undergoes rapid deprotonation of the third cysteine thiol, yielding the desired three-stranded coiled coil with an encapsulated trigonal thiolato Hg(II). The other species, termed the misfolded intermediate, rearranges in an experimentally distinguishable step to the properly folded intermediate. The order of the reaction involving the addition of the third peptide with respect to the concentration of Baby L9C indicates that addition of the third helix only occurs through reaction of Hg(Baby L9C(-H))(2) and Baby L9C that is unassociated with a coiled coil. Temperature dependence of the reaction afforded activation parameters for both the addition of the third helix (deltaH = 20(2) kcalmol; deltaS= 40(5) calmol K) and the rearrangement of the misfolded intermediate steps (deltaH = 23(2) kcalmol; deltaS= 27(5) calmol K). The mechanism is discussed with regard to metalloprotein folding and metalloprotein design.
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PMID:Hg(II) binding to a weakly associated coiled coil nucleates an encoded metalloprotein fold: a kinetic analysis. 1255 28

A method for thermally induced switching of enzyme activity has been developed, based on the site-directed conjugation of end-reactive temperature-responsive polymers to a unique cysteine (Cys) residue positioned near the enzyme active site. The reversible temperature-induced collapse of N,N-dimethylacrylamide (DMA)/N-4-phenylazo-phenylacrylamide (AZAAm) copolymers (DMAAm) has been used as a molecular switch to control the catalytic activity of endoglucanase 12A (EG 12A). The polymer was conjugated to the EG 12A site-directed mutant N55C, directly adjacent to the cellulose binding cleft, and to the S25C mutant, where the conjugation site is more distant. The N55C conjugate displayed a larger activity shutoff efficiency in the collapsed polymer state than the S25C conjugate. Increasing the polymer molecular weight was also shown to increase the shutoff efficiency of the switch. Related to these effects of conjugation site and polymer size, the switching efficiency was found to be strongly dependent on substrate size. With a small substrate, o-nitrophenyl-beta-d-cellobioside (ONPC), there was minimal blocking of enzyme activity when the polymer was in the expanded state. With a large substrate, hydroxyethyl cellulose (HEC), there was a large reduction of enzyme activity in the polymer expanded state, even with relatively small polymer chains, and a further reduction when the polymer was collapsed. Similar general trends for the interactive effects of conjugation site, polymer size, and substrate size were observed for immobilized conjugates. Kinetic studies demonstrated that the switching activity was due to the blocking of substrate association by the collapsed polymers. These investigations provide mechanistic insight that can be utilized to design molecular switches for a variety of stimuli-responsive polymer-protein conjugates.
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PMID:Temperature-induced switching of enzyme activity with smart polymer-enzyme conjugates. 1275 74

The intravenous injection of crystalline papain into young rabbits results in depletion of cartilage matrix throughout the body, with loss of rigidity and collapse of the ears, provided the enzyme is inactivated by oxidation or sulfhydryl blocking agents prior to administration. Cysteine-activated crystalline papain, when injected intravenously, produces little or no change in cartilage. The changes which occur in cartilage following an injection of inactivated crystalline papain are indistinguishable from those produced by crude papain. Activation of crude papain by cysteine prior to injection results in loss of its capacity to produce in vivo changes in cartilage. The progressive changes which take place in cartilage in vivo also occur in vitro in isolated rabbit ears removed shortly after an injection of crude papain or inactivated crystalline papain. In vitro ear collapse occurs rapidly at 37 degrees C. and does not occur at 4 degrees C. Collapse is enhanced by exposing the cartilage to cysteine and prevented by exposure to iodoacetamide or p-chloromercuribenzoate. The direct action of crystalline papain on plates of normal cartilage, in vitro, results in the same gross and histological changes which were observed in vivo. The direct action is accelerated by cysteine and inhibited by iodoacetamide or p-chloromercuribenzoate. The intravenous injection of iodoacetamide-treated bromelin produces the same in vivo changes in cartilage as papain. Untreated bromelin has no demonstrable effect on cartilage. It is suggested that the reason for the failure of activated papain to enter cartilage, after being injected intravenously, is that it probably reacts with a substrate or substrates in the blood. Oxidized or otherwise inactivated papain, in contrast, is readily taken up by cartilage and there converted to its active form.
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PMID:The removal of cartilage matrix, in vivo, by papain; identification of crystalline papain protease as the cause of the phenomenon. 1357 73

The positively charged S4 transmembrane segment of voltage-gated channels is thought to function as the voltage sensor by moving charge through the membrane electric field in response to depolarization. Here we studied S4 movements in the mammalian HCN pacemaker channels. Unlike most voltage-gated channel family members that are activated by depolarization, HCN channels are activated by hyperpolarization. We determined the reactivity of the charged sulfhydryl-modifying reagent, MTSET, with substituted cysteine (Cys) residues along the HCN1 S4 segment. Using an HCN1 channel engineered to be MTS resistant except for the chosen S4 Cys substitution, we determined the reactivity of 12 S4 residues to external or internal MTSET application in either the closed or open state of the channel. Cys substitutions in the NH2-terminal half of S4 only reacted with external MTSET; the rates of reactivity were rapid, regardless of whether the channel was open or closed. In contrast, Cys substitutions in the COOH-terminal half of S4 selectively reacted with internal MTSET when the channel was open. In the open state, the boundary between externally and internally accessible residues was remarkably narrow (approximately 3 residues). This suggests that S4 lies in a water-filled gating canal with a very narrow barrier between the external and internal solutions, similar to depolarization-gated channels. However, the pattern of reactivity is incompatible with either classical gating models, which postulate a large translational or rotational movement of S4 within a gating canal, or with a recent model in which S4 forms a peripheral voltage-sensing paddle (with S3b) that moves within the lipid bilayer (the KvAP model). Rather, we suggest that voltage sensing is due to a rearrangement in transmembrane segments surrounding S4, leading to a collapse of an internal gating canal upon channel closure that alters the shape of the membrane field around a relatively static S4 segment.
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PMID:Changes in local S4 environment provide a voltage-sensing mechanism for mammalian hyperpolarization-activated HCN channels. 1467 86


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