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1.
Tishchenko VM 《Molekuliarnaia biologiia》2011,45(6):1055-1064
Isolated constant domains from two Bence-Jones proteins VAD and BIR able to form amyloid fibrils, whereas only the first of them to keep specific ability of the intact protein. Studies of conformation and stability of these proteins by scanning microcalorimetry, circular dichroism, fluorescence and analytical centrifugation at physiological conditions (10 mM phosphate buffer, pH 7.0, 100 mM NaCl) showed that the stability of isolated pair of constant domains (C(L)-C(L)) VAD and BIR is reduced by compared with standard (nonamyloidogenic) Bence-Jones protein. However, in the intact protein BIR stability of his constant domains increases sharply, which correlated with the loss of the protein ability to form amyloid fibrils. 相似文献
2.
V. M. Tishchenko 《Biochemistry. Biokhimii?a》2013,78(4):368-376
Intact Bence-Jones proteins TIM and LUS under simulated physiological conditions (10 mM phosphate buffer, pH 7.0, 100 mM NaCl, 37°C) did not display amyloidogenic properties. However, their isolated variable domains exhibit these qualities in full measure. Therefore, both intact proteins and their variable domains were studied using a complex of physical methods (scanning microcalorimetry, analytical centrifugation, optics) that allowed us to assess the stability of their tertiary and quaternary structures. The experimentally obtained thermodynamic functions indicated that the stability of iso-lated variable domains of TIM and LUS was comparable to the stability of similar domains in amyloidogenic proteins described earlier. However, inside the whole protein their stability was comparable to the stability of VL domains of ordinary Bence-Jones proteins. The decreased stability of the isolated variable domains of TIM and LUS was shown to be due both to weak interactions between a pair of variable domains (trans -interaction) and to a natural lack of interaction with the con-stant domains (cis-interaction). 相似文献
3.
Multiple myeloma nephropathy occurs due to the aggregate formation by monoclonal immunoglobulin light chains (Bence-Jones proteins) in kidneys of patients with multiple myeloma. The mechanism of amyloid deposit formation is still unclear. Earlier, the key role in the fibril formation has been assigned to the variable domains that acquired amyloidogenic properties as a result of somatic mutations. However, fibril formation by the Bence-Jones protein BIF was found to be the function of its constant domain. The substitution of Ser177 by Asn in the constant domain of the BIF protein is most likely an inherited than a somatic mutation. To study the role of this mutation in amyloidogenesis, the recombinant Bence-Jones protein BIF and its mutant with the N177S substitution typical for the known immunoglobulin Cκ allotypes Km1, Km1,2, and Km3 were isolated. The morphology of aggregates formed by the recombinant proteins under conditions similar to those occurring during the protein transport in bloodstream and its filtration into the renal glomerulus, in the distal tubules, and in the proximal renal tubules was analyzed by atomic force microscopy. The nature of the aggregates formed by BIF and its N177S mutant during incubation for 14 days at 37°C strongly differed and depended on both pH and the presence of a reducing agent. BIF formed fibrils at pH 7.2, 6.5, and 10.1, while the N177S mutant formed fibrils only at alkaline pH 10.1. The refolding of both proteins in the presence of 5 mM dithiothreitol resulted in the formation of branched structures. 相似文献
4.
V. M. Tischenko 《Molecular Biology》2011,45(6):967-975
Isolated constant domains of two Bence Jones proteins, VAD and BIR, are able to form amyloid fibrils, but only the first one
retains this feature within the intact protein. The conformation and stability of these proteins were studied using scanning
microcalorimetry, circular dichroism, fluorescence spectroscopy, and analytical centrifugation at physiological conditions
(10 mM phosphate buffer, pH 7.0, 100 mM NaCl), and it was shown that isolated pairs of constant domains (CL-CL) of VAD and BIR had reduced stability in comparison to ordinary (nonamyloidogenic) Bence Jones proteins. However, in the
intact BIR protein, the stability of the constant domain block increased dramatically, in agreement with the loss of ability
to form amyloid fibrils. 相似文献
5.
Yu Feng Tianchen Hu Jianping Ding Hualiang Jiang Dongxiang Liu 《Archives of biochemistry and biophysics》2009,484(1):46-35317
Bcl-2 family proteins regulate apoptosis through their homo- and heterodimerization. By protein sequence analysis and structural comparison, we have identified a conserved hydrophobic core at the BH1 and BH2 domains of Bcl-2 family proteins. The hydrophobic core is stabilized by hydrophobic interactions among the residues of Trp137, Ile140, Trp181, Ile182, Trp188 and Phe191 in Bcl-xL. Destabilization of the hydrophobic core can promote the protein unfolding and pore formation in synthetic lipid vesicles. Interestingly, though the hydrophobic core does not participate in binding with BH3 domain of pro-apoptotic proteins, disruption of the hydrophobic core can reduce the affinity of Bcl-xL with BH3-domain peptide by changing the conformation of Bcl-xL C-terminal residues that are involved in the peptide interaction. The BH3-domain peptide binding affinity and pore forming propensity of Bcl-xL were correlated to its death-repressor activity, which provides new information to help study the regulatory mechanism of anti-apoptotic proteins. Meanwhile, as the tryptophans are conserved in the hydrophobic core, in vitro binding assay based on FRET of “Trp → AEDANS” can be devised to screen for new modulators targeting anti-apoptotic proteins as well as “multi-BH domains” pro-apoptotic proteins. 相似文献
6.
Zhang L Xing D Chen M 《Apoptosis : an international journal on programmed cell death》2008,13(7):950-958
Bcl-2 family proteins are implicated as essential regulators in tumor necrosis factor-α (TNFα)-induced apoptosis. BimL, a BH3-only member of Bcl-2 family, can directly or indirectly activate the proapoptotic Bax and the subsequent mitochondrial
apoptotic pathway. However, the molecular mechanism of BimL activating Bax activation during TNFα-induced apoptosis is not fully understood. In this study, the role of BimL in Bax activation during TNFα-induced apoptosis was investigated in differentiated PC12 and MCF7 cells, with real-time single-cell
analysis. The experimental results show that Bax translocated to mitochondria and cytochrome c (Cyt c) released from mitochondria
after TNFα treatment. Furthermore, SP600125 (specific inhibitor of JNK) could inhibit the Cyt c release from mitochondria.
Co-immunoprecipitation results show that, the interaction between Bcl-xL and Bax decreased after TNFα treatment, while that between Bcl-xL and BimL increased. Bax did not co-immunoprecipitate with BimL before or after the TNFα treatment. In addition, the increased interaction between BimL and Bcl-xL was dynamically monitored by using fluorescence resonance energy transfer (FRET) technique. Most importantly, there was no
evidence of BimL redistribution to mitochondria until cell apoptosis. By comprehensively analyzing these data, it is concluded that BimL displaces Bcl-xL in the mitochondria and promotes Bax translocation during TNFα-induced apoptosis. 相似文献
7.
Alejandra Hernández-Santoyo Luis del Pozo Yauner Deyanira Fuentes-Silva Enrique Rudiño-Piñera Eduardo Horjales Adela Rodríguez-Romero 《Journal of molecular biology》2010,396(2):280-292
Systemic amyloid light-chain (LC) amyloidosis is a disease process characterized by the pathological deposition of monoclonal LCs in tissue. All LC subtypes are capable of fibril formation although λ chains, particularly those belonging to the λ6 type, are overrepresented. Here, we report the thermodynamic and in vitro fibrillogenic properties of several mutants of the λ6 protein 6aJL2 in which Pro7 and/or His8 was substituted by Ser or Pro. The H8P and H8S mutants were almost as stable as the wild-type protein and were poorly fibrillogenic. In contrast, the P7S mutation decreased the thermodynamic stability of 6aJL2 and greatly enhanced its capacity to form amyloid-like fibrils in vitro. The crystal structure of the P7S mutant showed that the substitution induced both local and long-distance effects, such as the rearrangement of the VL (variable region of the light chain)-VL interface. This mutant crystallized in two orthorhombic polymorphs, P212121 and C2221. In the latter, a monomer that was not arranged in the typical Bence-Jones dimer was observed for the first time. Crystal-packing analysis of the C2221 lattice showed the establishment of intermolecular β-β interactions that involved the N-terminus and β-strand B and that these could be relevant in the mechanism of LC fibril formation. Our results strongly suggest that Pro7 is a key residue in the conformation of the N-terminal sheet switch motif and, through long-distance interactions, is also critically involved in the contacts that stabilized the VL interface in λ6 LCs. 相似文献
8.
《Biocatalysis and Biotransformation》2013,31(4):377-384
AbstractThe direct electron transfer of immobilized haemoglobin (Hb) on nano-TiO2 and dodecyltrimethylammonium bromide (DTAB) film modified carbon paste electrode (CPE) and its application as a hydrogen peroxide (H2O2) biosensor were investigated. On nano-TiO2/DTAB/Hb/CPE, Hb displayed a rapid electron transfer process with participation of one proton and with an electron transfer rate constant which estimated as 0.29 s??1. Thus, the proposed biosensor exhibited a high sensitivity and excellent electrocatalytic activity for the reduction of H2O2. The catalytic reduction current of H2O2 was proportional to H2O2 concentration in the range of 0.2–4.0 mM with a detection limit of 0.07 mM. The apparent Michaelis–Menten constant (Kmapp) of the biosensor was calculated to be 0.127 mM, exhibiting a high enzymatic activity and affinity. This sensor for H2O2 can potentially be applied in determination of other reactive oxygen species as well. 相似文献
9.
Spinal muscular atrophy (SMA) is a genetic disorder characterized by degeneration of spinal cord motoneurons (MNs), resulting in muscular atrophy and weakness. SMA is caused by mutations in the Survival Motor Neuron 1 (SMN1) gene and decreased SMN protein. SMN is ubiquitously expressed and has a general role in the assembly of small nuclear ribonucleoproteins and pre-mRNA splicing requirements. SMN reduction causes neurite degeneration and cell death without classical apoptotic features, but the direct events leading to SMN degeneration in SMA are still unknown. Autophagy is a conserved lysosomal protein degradation pathway whose precise roles in neurodegenerative diseases remain largely unknown. In particular, it is unclear whether autophagosome accumulation is protective or destructive, but the accumulation of autophagosomes in the neuritic beadings observed in several neurite degeneration models suggests a close relationship between the autophagic process and neurite collapse. In the present work, we describe an increase in the levels of the autophagy markers including autophagosomes, Beclin1 and light chain (LC)3-II proteins in cultured mouse spinal cord MNs from two SMA cellular models, suggesting an upregulation of the autophagy process in Smn (murine survival motor neuron protein)-reduced MNs. Overexpression of Bcl-xL counteracts LC3-II increase, contributing to the hypothesis that the protective role of Bcl-xL observed in some SMA models may be mediated by its role in autophagy inhibition. Our in vitro experimental data indicate an upregulation in the autophagy process and autophagosome accumulation in the pathogenesis of SMA, thus providing a valuable clue in understanding the mechanisms of axonal degeneration and a possible therapeutic target in the treatment of SMA. 相似文献
10.
11.
Hydrogen peroxide (H2O2)-induced aggregation of calf platelets and its modification by agents with specific properties were characterized employing
a spectrophotometric assay. An Arrhenius activation energy of 20 ± 1 kcal/mol was found in the temperature range of 25‡-36‡C.
Rate inhibition occurred on either side of this temperature range, and under anaerobic conditions. Exogenous Ca2+ ions were not required but Ca2+ ions, at 1 mM-concentration, optimally increased rates and extent of aggregation at suboptimal H2O2 concentrations but only extent of aggregation at optimal H2O2 concentrations. Ba2+, Sr2+, Cd2+, Mn2+ and Ni2+ ions (1 mM) and Zn2+, Pb2+ and Hg2+ ions (10 mM) were inhibitory. The cyclo-oxygenase inhibitor, indomethacin (10-30 mM) exerted only mild inhibition by a competitive
mechanism. Another cyclo-oxygenase inhibitor, aspirin, functioned to increase aggregation. Ligands acting directly at the
prostaglandin H2/thromboxane A, receptor (5Z. 9, 11, 13E, 15(S) 15-hydroxy 9(11) epoxy methano prosta 5, 13-dien-1-oic acid, pinane thromboxane
A2, arachidonic acid, eicosapentaenoic acid, and N-ethylmaleimide) functioned as competitive inhibitors. Another platelet-activating
sulphydryl reagent, thimerosal, also inhibited competitively while the protein kinase C inhibitor, sphingosine, and the protein
kinase C modulator, Zn2+ ions, inhibited by different mechanisms. The results indicate direct action of H2O2 at the prostaglandin H2/thromboxane A2 receptor, possibly its sulphydryls, to activate the protein kinase C pathway, independently of cyclo-oxygenase products.
The results underscored the power of the kinetic approach for investigating mechanisms of platelet activation. 相似文献
12.
M-C. Diemert V. Tricottet L. Benel G. Descamps E. Escolano J. Galli M. Reynès F. Rousselet 《In vitro cellular & developmental biology. Animal》1995,31(9):716-723
Summary The cytotoxicity of a Bence-Jones protein was assessed using a porcine renal tubule cell line (LLC-PK1), with the aim of developing
a model for studying the potential nephrotoxicity of these proteins.
The effects of a kappa Bence-Jones protein on cell viability were studied by means of biochemical methods (supravital dye
uptake and measurement of cellular enzyme activities) and morphological electron microscopy.
After a 24-h-treatment with Bence-Jones protein, a moderate cytotoxicity (about 15%) was noted but only a minor difference
compared to treatment with bovine albumin in the same conditions.
The morphological study showed a few cells in the process of lysis, but their numbers were insufficient for the demonstration
of a clear cytotoxic effect. Immunocytochemical studies showed Bence-Jones protein fixation on some cells, especially on the
outer membrane. Labeling of the hyaloplasm and basal pole of a few cells pointed to internalization of protein by LLC-PK1
cells.
Although the cytotoxicity of the Bence-Jones protein tested here was only moderate, the use of this model enabled its cytotoxic
effect to be distinguished from that ofβ-lactoglobulin. This isolate could serve as a “moderate control” for a later study with a BJP having caused acute renal failure. 相似文献
13.
The total amino acid sequence of a lambda Bence-Jones protein has been established. The protein contains 211 residues, which include two methionine residues. Splitting with cyanogen bromide gave three fragments, the largest of which included the C-terminal half, which is common to other Bence-Jones proteins of the same type. The peptides obtained by tryptic, chymotryptic and peptic digestion were isolated and purified by paper-electrophoretic and chromatographic techniques. Reduction followed by carboxymethylation of the cysteine residues with radioactive iodoacetate was found to be a powerful tool in the isolation of some insoluble peptides. Unusual features of the molecule are the fact that it contains six cysteine residues and not five as observed in both kappa and lambda Bence-Jones proteins studied previously, and its size, which seems two residues smaller than the smallest Bence-Jones protein studied hitherto. The similarities and differences between this and other Bence-Jones proteins are discussed. 相似文献
14.
Chen SP Wu JL Su YC Hong JR 《Apoptosis : an international journal on programmed cell death》2007,12(6):1043-1060
Nervous necrosis virus (NNV)-induced, host cell apoptosis mediates secondary necrosis by an ill-understood process. In this
study, redspotted grouper nervous necrosis virus (RGNNV) is shown to induce mitochondria-mediated necrotic cell death in GL-av
cells (fish cells) via cytochrome c release, and anti-apoptotic proteins are shown to protect these cells from death. Western blots revealed that cytochrome
c release coincided with disruption of mitochondrial ultrastructure and preceded necrosis, but did not correlate with caspases
activation. To identify the mediator(s) of this necrotic process, a protein synthesis inhibitor (cycloheximide; CHX; 0.33 μg/ml)
was used to block cytochrome c release as well as PS exposure and mitochondrial membrane permeability transition pore (MMP) loss. CHX (0.33 μg/ml) completely
blocked viral protein B2 expression, and partly blocked protein A, protein α, and a pro-apoptotic death protein (Bad) expression.
Overexpression of B2 gene increased necrotic-like cell death up to 30% at 48 h post-transfection, suggesting that newly synthesized protein (B2)
may be involved in this necrotic process. Finally, necrotic death was prevented by overexpression of Bcl-2 family proteins,
zfBcl-xL and xfMcl-1a. Thus, new protein synthesis and release of cytochrome c are required for RGNNV-induced necrotic cell death, which can be blocked by anti-apoptotic Bcl-2 members.
J.-L. Wu and J.-R. Hong contributed equally to the research. 相似文献
15.
Shivakumar A Nagaraja P Chamaraja NA Krishna H Avinash K 《Journal of biotechnology》2011,155(4):406-411
A biocatalatic pathway involving chromogenic probe has been proposed for the determination of catalase activity by means of iso-nicotinicacidhydrazide (INH) and pyrocatechol (PC). The assay is based on the enzymatic consumption of hydrogen peroxide using INH-PC system. The response of the catalase activity was ascertained by the rate of the reaction involving 14.10 mM H2O2. On addition of H2O2, INH-PC indicator system formed a chromogenic product with absorbance maxima at 490 nm. Hence the activity of catalase was directly measured by the chromogenic response in the formation of the coupled product. The catalase assay was elaborated by the kinetic response of the INH-PC system. The linearity of the catalase activity and H2O2 was in the range 0.2-7.0 units and 1.76-7.0 mM, respectively in 3 ml solution. The catalytic efficiency and catalytic power were calculated. The Michaelis-Menten constant of INH, PC and H2O2 were found to be 0.344, 0.176 and 8.82 mM, respectively. The indicator reaction was applied in the determination of catalase activity in mycelia mats and culture media. 相似文献
16.
Prema B. Rapuri J.C. Gallagher Zafar Nawaz 《The Journal of steroid biochemistry and molecular biology》2007,103(3-5):368
Of the various risk factors contributing to osteoporosis, dietary/lifestyle factors are important. In a clinical study we reported that women with caffeine intakes >300 mg/day had higher bone loss and women with vitamin D receptor (VDR) variant, tt were at a greater risk for this deleterious effect of caffeine. However, the mechanism of how caffeine effects bone metabolism is not clear. 1,25-Dihydroxy vitamin D3 (1,25(OH)2D3) plays a critical role in regulating bone metabolism. The receptor for 1,25(OH)2D3, VDR has been demonstrated in osteoblast cells and it belongs to the superfamily of nuclear hormone receptors. To understand the molecular mechanism of the role of caffeine in relation to bone, we tested the effect of caffeine on VDR expression and 1,25(OH)2D3 mediated actions in bone. We therefore examined the effect of different doses of caffeine (0.2, 0.5, 1.0 and 10 mM) on 1,25(OH)2D3 induced VDR protein expression in human osteoblast cells. We also tested the effect of different doses of caffeine on 1,25(OH)2D3 induced alkaline phosphatase (ALP) activity, a widely used marker of osteoblastic activity. Caffeine dose dependently decreased the 1,25(OH)2D3 induced VDR expression and at concentrations of 1 and 10 mM, VDR expression was decreased by about 50–70%, respectively. In addition, the 1,25(OH)2D3 induced alkaline phosphatase activity was also reduced at similar doses thus affecting the osteoblastic function. The basal ALP activity was not affected with increasing doses of caffeine. Overall, our results suggest that caffeine affects 1,25(OH)2D3 stimulated VDR protein expression and 1,25(OH)2D3 mediated actions in human osteoblast cells. 相似文献
17.
Yakov M. Milgrom 《BBA》2010,1797(10):1768-1774
The effect of inorganic phosphate (Pi) on uni-site ATP binding and hydrolysis by the nucleotide-depleted F1-ATPase from beef heart mitochondria (ndMF1) has been investigated. It is shown for the first time that Pi decreases the apparent rate constant of uni-site ATP binding by ndMF1 3-fold with the Kd of 0.38 ± 0.14 mM. During uni-site ATP hydrolysis, Pi also shifts equilibrium between bound ATP and ADP + Pi in the direction of ATP synthesis with the Kd of 0.17 ± 0.03 mM. However, 10 mM Pi does not significantly affect ATP binding during multi-site catalysis. 相似文献
18.
Xiaohua Chen Jianqiang Hu Zhiwu Chen Xiumei Feng Aiqing Li 《Biosensors & bioelectronics》2009,24(12):3448-3454
A layered inorganic perovskite sub-micrometer-scale material, nanoplated bismuth titanate (Bi4Ti3O12) sub-microspheres (NBTSMs) constructed with tens of Bi4Ti3O12 nanoplates, was for the first time synthesized by a facile hydrothermal synthesis strategy. The NBTSMs were employed as a supporting matrix to explore a novel immobilization and biosensing platform of redox proteins through a combined hydrogen bond and electrostatic assembly process. Biocompatibility, stability, reproducibility, and electrochemical and electrocatalytic properties of the resulting NBTSMs-based composite were studied by UV–vis absorption, FTIR, and electrochemical methods. The research results revealed that the NBTSMs-based composite was a satisfying matrix for proteins to effectively retain their native structure and bioactivity. With advantages of the Bi4Ti3O12 layered material, facilitated direct electron transfer of the metalloenzymes with an apparent heterogeneous electron transfer rate constant (ks) of 20.0 ± 3.8 s−1 was acquired on the NBTSMs-based enzyme electrode. The NBTSMs-based biosensor demonstrated significant electrocatalytic activity for the reduction of hydrogen peroxide with an apparent Michaelis–Menten constant (204 μM), wide linear range (2–430 μM), and low detection limit (0.46 μM, S/N = 3). These indicated that the nanoplate-constructed Bi4Ti3O12 sub-microspheres were one of ideal candidate materials for direct electrochemistry of redox proteins and the construction of the related enzyme biosensors, and may find potential applications in biomedical, food, and environmental analysis and detection. 相似文献
19.
In Acanthamoeba castellanii mitochondria, the apparent affinity values of alternative oxidase for oxygen were much lower than those for cytochrome c oxidase. For unstimulated alternative oxidase, the KMox values were around 4-5 μM both in mitochondria oxidizing 1 mM external NADH or 10 mM succinate. For alternative oxidase fully stimulated by 1 mM GMP, the KKMox values were markedly different when compared to those in the absence of GMP and they varied when different respiratory substrates were oxidized (KMox was around 1.2 μM for succinate and around 11 μM for NADH). Thus, with succinate as a reducing substrate, the activation of alternative oxidase (with GMP) resulted in the oxidation of the ubiquinone pool, and a corresponding decrease in KMox. However, when external NADH was oxidized, the ubiquinone pool was further reduced (albeit slightly) with alternative oxidase activation, and the KMox increased dramatically. Thus, the apparent affinity of alternative oxidase for oxygen decreased when the ubiquinone reduction level increased either by changing the activator or the respiratory substrate availability. 相似文献
20.
Jafari Anarkooli I Sankian M Vahedi F Bonakdaran S Varasteh AR Haghir H 《Cellular and molecular neurobiology》2009,29(1):133-140
Aims Effects of insulin and ascorbic acid on expression of Bcl-2 family proteins and caspase-3 activity in hippocampus of diabetic
rats were evaluated in this study. Methods Diabetes was induced in Wistar male rats by streptozotocin (STZ). Six weeks after verification of diabetes, the animals were
treated for 2 weeks with insulin or/and ascorbic acid in separate groups. Hippocampi of rats were removed and evaluation of
Bcl-2, Bcl-xL, and Bax proteins expression in frozen hippocampi tissues were done by SDS-PAGE electrophoresis and blotting. The Bcl-2,
Bcl-xL, and Bax proteins bands were visualized after incubation with specific antibodies using enhanced chemiluminescences method.
Caspase-3 activity was determined using the caspase-3/CPP32 Fluorometric Assay Kit. Results Diabetic rats showed increase in Bax protein expression and decrease in Bcl-2 and Bcl-xL proteins expression. The Bax/Bcl-2 and Bax/Bcl-xL ratios were found higher compared with non-diabetic control group. Treatments with insulin and/or ascorbic acid were resulted
in decrease in Bax protein expression and increase in Bcl-2 and Bcl-xL proteins expression. The Bcl-2/Bax and Bcl-xL/Bax ratios were found higher in treated groups than untreated diabetic group. Caspase-3 activity level was found higher in
diabetic group compared with non-diabetic group. Treatment with insulin and ascorbic acid did downregulated caspase-3 activity.
Conclusions Our data provide supportive evidence to demonstrate the antiapoptotic effects of insulin and ascorbic acid on hippocampus
of STZ-induced diabetic rats. 相似文献