scholarly journals W196 and the β-Hairpin Motif Modulate the Redox Switch of Conformation and the Biomolecular Interaction Network of the Apoptosis-Inducing Factor

2021 ◽  
Vol 2021 ◽  
pp. 1-19
Author(s):  
Silvia Romero-Tamayo ◽  
Ruben Laplaza ◽  
Adrian Velazquez-Campoy ◽  
Raquel Villanueva ◽  
Milagros Medina ◽  
...  
Keyword(s):  
Interaction Network ◽  
Kinetic Studies ◽  
Protein Dimerization ◽  
Redox Sensor ◽  
Mitochondrial Respiratory Complex ◽  
Redox Switch ◽  
Molecular Therapies ◽  
Low Efficiency ◽  
Apoptosis Inducing Factor ◽  
Hairpin Conformation

The human apoptosis-inducing factor (hAIF) is a moonlight flavoprotein involved in mitochondrial respiratory complex assembly and caspase-independent programmed cell death. These functions might be modulated by its redox-linked structural transition that enables hAIF to act as a NAD(H/+) redox sensor. Upon reduction with NADH, hAIF undergoes a conformational reorganization in two specific insertions—the flexible regulatory C-loop and the 190-202 β-harpin—promoting protein dimerization and the stabilization of a long-life charge transfer complex (CTC) that modulates its monomer-dimer equilibrium and its protein interaction network in healthy mitochondria. In this regard, here, we investigated the precise function of the β-hairpin in the AIF conformation landscape related to its redox mechanism, by analyzing the role played by W196, a key residue in the interaction of this motif with the regulatory C-loop. Mutations at W196 decrease the compactness and stability of the oxidized hAIF, indicating that the β-hairpin and C-loop coupling contribute to protein stability. Kinetic studies complemented with computational simulations reveal that W196 and the β-hairpin conformation modulate the low efficiency of hAIF as NADH oxidoreductase, contributing to configure its active site in a noncompetent geometry for hydride transfer and to stabilize the CTC state by enhancing the affinity for NAD+. Finally, the β-hairpin motif contributes to define the conformation of AIF’s interaction surfaces with its physiological partners. These findings improve our understanding on the molecular basis of hAIF’s cellular activities, a crucial aspect for clarifying its associated pathological mechanisms and developing new molecular therapies.

Effects of Scopoletin and Aflatoxin on B1 on Bovine Hepatic Mitochondrial Respitatory Complexes, 2: a-Ketoglutarate Cytochrome c and Succinate Cytochrome c Reductases

2001 ◽  
Vol 56 (3-4) ◽  
pp. 278-282 ◽  
Author(s):  
Sebastian C. Obasi
Keyword(s):  
Cytochrome C ◽  
Mitochondrial Respiration ◽  
Bos Indicus ◽  
Kinetic Studies ◽  
Complex Iii ◽  
Mitochondrial Respiratory Complex ◽  
In Vitro Effects ◽  
Coumarin Compounds ◽  
Aflatoxin B

Abstract The in vitro effects of the toxin coumarin compounds scopoletin and aflatoxin B1 (AFB1) on bovine (Bos indicus) hepatic mitochondrial respiratory complex III enzymes, succinate cytochrome c and a-ketoglutarate cytochrome c reductases, were examined. Kinetic studies on the interaction of the toxins with the enzymes were also carried out. The results showed that although the observed inhibitory and stimulatory effects of the two toxins were consistent with the changes in the kinetic parameters (Km and Vmax values), these parameters were not consistent with the observed effects of the toxins at certain concentrations. These observations are discussed in terms of the relative locations of the enzymes in the mito­chondria, and the previously reported inhibitory and uncoupling effects of the toxins on cow liver mitochondrial respiration.

scholarly journals The metabolism of nitrosothiols in the mycobacteria: identification and characterization of S-nitrosomycothiol reductase

2003 ◽  
Vol 374 (3) ◽  
pp. 657-666 ◽  
Author(s):  
Ryan N. VOGT ◽  
Daniel J. STEENKAMP ◽  
Renjian ZHENG ◽  
John S. BLANCHARD
Keyword(s):  
Affinity Chromatography ◽  
Metal Ion ◽  
Kinetic Studies ◽  
Formaldehyde Dehydrogenase ◽  
Class Iii ◽  
Steady State Kinetic ◽  
Sequential Mechanism ◽  
A Subunit ◽  
Low Efficiency

When grown in culture Mycobacterium smegmatis metabolized S-nitrosoglutathione to oxidized glutathione and nitrate, which suggested a possible involvement of an S-nitrosothiol reductase and mycobacterial haemoglobin. The mycothiol-dependent formaldehyde dehydrogenase from M. smegmatis was purified by a combination of Ni2+-IMAC (immobilized metal ion affinity chromatography), hydrophobic interaction, anion-exchange and affinity chromatography. The enzyme had a subunit molecular mass of 38263 kDa. Steady-state kinetic studies indicated that the enzyme catalyses the NAD+-dependent conversion of S-hydroxymethylmycothiol into formic acid and mycothiol by a rapid-equilibrium ordered mechanism. The enzyme also catalysed an NADH-dependent decomposition of S-nitrosomycothiol (MSNO) by a sequential mechanism and with an equimolar stoichiometry of NADH:MSNO, which indicated that the enzyme reduces the nitroso group to the oxidation level of nitroxyl. Vmax for the MSNO reductase reaction indicated a turnover per subunit of approx. 116700 min−1, which was 76-fold faster than the formaldehyde dehydrogenase activity. A gene, Rv2259, annotated as a class III alcohol dehydrogenase in the Mycobacterium tuberculosis genome was cloned and expressed in M. smegmatis as the C-terminally His6-tagged product. The purified recombinant enzyme from M. tuberculosis also catalysed both activities. M. smegmatis S-nitrosomycothiol reductase converted MSNO into the N-hydroxysulphenamide, which readily rearranged to mycothiolsulphinamide. In the presence of MSNO reductase, M. tuberculosis HbN (haemoglobin N) was converted with low efficiency into metHbN [HbN(Fe3+)] and this conversion was dependent on turnover of MSNO reductase. These observations suggest a possible route in vivo for the dissimilation of S-nitrosoglutathione.

scholarly journals NS3 Peptide, a Novel Potent Hepatitis C Virus NS3 Helicase Inhibitor: Its Mechanism of Action and Antiviral Activity in the Replicon System

2007 ◽  
Vol 52 (2) ◽  
pp. 393-401 ◽  
Author(s):  
Agnieszka Gozdek ◽  
Igor Zhukov ◽  
Agnieszka Polkowska ◽  
Jaroslaw Poznanski ◽  
Anna Stankiewicz-Drogon ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Antiviral Activity ◽  
Protein Interactions ◽  
Kinetic Studies ◽  
Current Therapy ◽  
Hcv Rna ◽  
Chronic Infections ◽  
Nmr Studies ◽  
Low Efficiency

ABSTRACT Hepatitis C virus (HCV) chronic infections represent one of the major and still unresolved health problems because of low efficiency and high cost of current therapy. Therefore, our studies centered on a viral protein, the NS3 helicase, whose activity is indispensable for replication of the viral RNA, and on its peptide inhibitor that corresponds to a highly conserved arginine-rich sequence of domain 2 of the helicase. The NS3 peptide (p14) was expressed in bacteria. Its 50% inhibitory activity in a fluorometric helicase assay corresponded to 725 nM, while the ATPase activity of NS3 was not affected. Nuclear magnetic resonance (NMR) studies of peptide-protein interactions using the relaxation filtering technique revealed that p14 binds directly to the full-length helicase and its separately expressed domain 1 but not to domain 2. Changes in the NMR chemical shift of backbone amide nuclei (1H and 15N) of domain 1 or p14, measured during complex formation, were used to identify the principal amino acids of both domain 1 and the peptide engaged in their interaction. In the proposed interplay model, p14 contacts the clefts between domains 1 and 2, as well as between domains 1 and 3, preventing substrate binding. This interaction is strongly supported by cross-linking experiments, as well as by kinetic studies performed using a fluorometric assay. The antiviral activity of p14 was tested in a subgenomic HCV replicon assay that showed that the peptide at micromolar concentrations can reduce HCV RNA replication.

Flavin radicals, conformational sampling and robust design principles in interprotein electron transfer: the trimethylamine dehydrogenase-electron-transferring flavoprotein complex

2004 ◽  
Vol 71 ◽  
pp. 1-14
Author(s):  
David Leys ◽  
Jaswir Basran ◽  
François Talfournier ◽  
Kamaldeep K. Chohan ◽  
Andrew W. Munro ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Dimensional Space ◽  
Three Dimensional ◽  
Kinetic Studies ◽  
Molecular Assembly ◽  
Conformational Sampling ◽  
Trimethylamine Dehydrogenase ◽  
Key Residues ◽  
Electron Transferring Flavoprotein ◽  
Electron Transfer Complex

TMADH (trimethylamine dehydrogenase) is a complex iron-sulphur flavoprotein that forms a soluble electron-transfer complex with ETF (electron-transferring flavoprotein). The mechanism of electron transfer between TMADH and ETF has been studied using stopped-flow kinetic and mutagenesis methods, and more recently by X-ray crystallography. Potentiometric methods have also been used to identify key residues involved in the stabilization of the flavin radical semiquinone species in ETF. These studies have demonstrated a key role for 'conformational sampling' in the electron-transfer complex, facilitated by two-site contact of ETF with TMADH. Exploration of three-dimensional space in the complex allows the FAD of ETF to find conformations compatible with enhanced electronic coupling with the 4Fe-4S centre of TMADH. This mechanism of electron transfer provides for a more robust and accessible design principle for interprotein electron transfer compared with simpler models that invoke the collision of redox partners followed by electron transfer. The structure of the TMADH-ETF complex confirms the role of key residues in electron transfer and molecular assembly, originally suggested from detailed kinetic studies in wild-type and mutant complexes, and from molecular modelling.

Tc-99m Labeled Complexes of Aldehydes and Glutamate as Cholescintigraphic Agents

1975 ◽  
Vol 14 (04) ◽  
pp. 330-338
Author(s):  
L. G. Colombetti ◽  
J. S. Arnold ◽  
W. E. Barnes
Keyword(s):  
Nuclear Medicine ◽  
Gall Bladder ◽  
Rose Bengal ◽  
Valence State ◽  
Monosodium Glutamate ◽  
Kinetic Studies ◽  
Scintillation Camera ◽  
Blood Clearance ◽  
Lower Valence ◽  
Wire Basket

SummaryTc-99m pyridoxylidene glutamate has proven to be an excellent biliary scanning agent, far superior in many respect to the commonly used 1-131 rose bengal. The preparation of the compound as previously reported by Baker et al is too time consuming and requires the use of an autoclave which is not available in most nuclear medicine departments. In our facility, we have been preparing similar compounds using several aldehydes and monosodium glutamate to make labeled complexes having the same pharmacological characteristics. The mixture of monosodium glutamate, aldehyde, and Tc-99m pertechnetate is made slightly alkaline, purged with helium, and placed in a sealed vial. The vial, which is protected by a wire basket, is then heated in a laboratory oven at 130° C for a period of 15 to 20 minutes. During this time, the technetium is reduced to a lower valence state and bound to the complex formed. Chromatographic data show that these compounds are chemically similar to that previously reported. The compounds prepared concentrate in the gall bladder of the rabbit in less than 10 minutes. Kinetic studies have been performed on dogs with a scintillation camera and small digital computer to measure rates of blood clearance, liver and gall bladder uptake, and excretion into the intestine. The aldehyde — glutamate complex promises to be a useful scanning agent for the diagnosis of biliary and hepatocellular diseases.

Factor V from Human Plasma

1961 ◽  
Vol 05 (01) ◽  
pp. 001-020
Author(s):  
Douglas M. Surgenor ◽  
Nancy A. Wilson ◽  
Anne S. Henry
Keyword(s):  
Human Serum ◽  
Human Plasma ◽  
Kinetic Data ◽  
Human Factor ◽  
Kinetic Studies ◽  
Partial Purification ◽  
Factor V ◽  
Two Stage ◽  
Plasma Factor ◽  
Tissue Thromboplastin

SummaryA method is described for the partial purification of a human plasma factor which accelerates the conversion of prothrombin to thrombin in the presence of tissue thromboplastin. This factor may be dried from the frozen state, and may be kept in stable dry form for long periods of time. The quantitative assay of this activity is done in a classical two-stage prothrombin system using tissue thromboplastin and calcium. From its properties, it is concluded that this activity corresponds to factor V, labile factor and plasma Ac-globulin.Chemical and kinetic studies reveal that human factor V is active in plasma and is destroyed by thrombin. Human serum has little or no factor V activity.These results thus fail to support the postulated activation of factor V during clotting. All of the kinetic data are consistent with an enzymatic role for factor V in the formation of tissue prothrombin activator (thromboplastin).

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