Search

Flavin-binding photoreceptor proteins sense blue-light (BL) in diverse organisms and have become core elements in recent optogenetic applications. The light-oxygen-voltage (LOV) protein Vivid (VVD) from the filamentous fungus Neurospora crassa is a classic BL photoreceptor, characterized by effecting a photocycle based on light-driven formation and subsequent spontaneous decay of a flavin-cysteinyl adduct. Here we report that VVD presents alternative outcomes to light exposure that result in protein self-oxidation and, unexpectedly, rise of stability through kinetic control. Using optical absorbance and mass spectrometry we show that purified VVD develops amorphous aggregates with the presence of oxidized residues located at the cofactor binding pocket. Light exposure increases oxidative levels in VVD and specific probe analysis identifies singlet oxygen production by the flavin. These results indicate that VVD acts alternatively as a photosensitizer, inducing self-oxidative damage and subsequent aggregation. Surprisingly, BL illumination has an additional, opposite effect in VVD. We show that light-induced adduct formation establishes a stable state, delaying protein aggregation until photoadduct decay occurs. In accordance, repeated BL illumination suppresses VVD aggregation altogether. Furthermore, photoadduct formation confers VVD stability against chemical denaturation. Analysis of the aggregation kinetics and testing of stabilizers against aggregation reveal that aggregation in VVD proceeds through light-dependent kinetic control and dimer formation. These results uncover the aggregation pathway of a photosensor, where light induces a remarkable interplay between protein damage and stability. [ABSTRACT FROM AUTHOR]

Busulphan (Bu) is an alkylating agent used in the conditioning regimen prior to hematopoietic stem cell transplantation (HSCT). Bu is extensively metabolized in the liver via conjugations with glutathione to form the intermediate metabolite (sulfonium ion) which subsequently is degraded to tetrahydrothiophene (THT). THT was reported to be oxidized forming THT-1-oxide that is further oxidized to sulfolane and finally 3-hydroxysulfolane. However, the underlying mechanisms for the formation of these metabolites remain poorly understood. In the present study, we performed in vitro and in vivo investigations to elucidate the involvement of flavin-containing monooxygenase-3 (FMO3) and cytochrome P450 enzymes (CYPs) in Bu metabolic pathway. Rapid clearance of THT was observed when incubated with human liver microsomes. Furthermore, among different recombinant microsomal enzymes, the highest intrinsic clearance for THT was obtained via FMO3 followed by several CYPs including 2B6, 2C8, 2C9, 2C19, 2E1 and 3A4. In Bu- or THT-treated mice, inhibition of FMO3 by phenylthiourea significantly suppressed the clearance of both Bu and THT. Moreover, the simultaneous administration of a high dose of THT (200μmol/kg) to Bu-treated mice reduced the clearance of Bu. Consistently, in patients undergoing HSCT, repeated administration of Bu resulted in a significant up-regulation of FMO3 and glutathione-S-transfrase -1 (GSTA1) genes. Finally, in a Bu-treated patient, additional treatment with voriconazole (an antimycotic drug known as an FMO3-substrate) significantly altered the Bu clearance. In conclusion, we demonstrate for the first time that FMO3 along with CYPs contribute a major part in busulphan metabolic pathway and certainly can affect its kinetics. The present results have high clinical impact. Furthermore, these findings might be important for reducing the treatment-related toxicity of Bu, through avoiding interaction with other concomitant used drugs during conditioning and hence improving the clinical outcomes of HSCT. [ABSTRACT FROM AUTHOR]

The guidance cue UNC-6/Netrin regulates both attractive and repulsive axon guidance. Our previous work showed that in C. elegans, the attractive UNC-6/Netrin receptor UNC-40/DCC stimulates growth cone protrusion, and that the repulsive receptor, an UNC-5:UNC-40 heterodimer, inhibits growth cone protrusion. We have also shown that inhibition of growth cone protrusion downstream of the UNC-5:UNC-40 repulsive receptor involves Rac GTPases, the Rac GTP exchange factor UNC-73/Trio, and the cytoskeletal regulator UNC-33/CRMP, which mediates Semaphorin-induced growth cone collapse in other systems. The multidomain flavoprotein monooxygenase (FMO) MICAL (Molecule Interacting with CasL) also mediates growth cone collapse in response to Semaphorin by directly oxidizing F-actin, resulting in depolymerization. The C. elegans genome does not encode a multidomain MICAL-like molecule, but does encode five flavin monooxygenases (FMO-1, -2, -3, -4, and 5) and another molecule, EHBP-1, similar to the non-FMO portion of MICAL. Here we show that FMO-1, FMO-4, FMO-5, and EHBP-1 may play a role in UNC-6/Netrin directed repulsive guidance mediated through UNC-40 and UNC-5 receptors. Mutations in fmo-1, fmo-4, fmo-5, and ehbp-1 showed VD/DD axon guidance and branching defects, and variably enhanced unc-40 and unc-5 VD/DD axon guidance defects. Developing growth cones in vivo of fmo-1, fmo-4, fmo-5, and ehbp-1 mutants displayed excessive filopodial protrusion, and transgenic expression of FMO-5 inhibited growth cone protrusion. Mutations suppressed growth cone inhibition caused by activated UNC-40 and UNC-5 signaling, and activated Rac GTPase CED-10 and MIG-2, suggesting that these molecules are required downstream of UNC-6/Netrin receptors and Rac GTPases. From these studies we conclude that FMO-1, FMO-4, FMO-5, and EHBP-1 represent new players downstream of UNC-6/Netrin receptors and Rac GTPases that inhibit growth cone filopodial protrusion in repulsive axon guidance. [ABSTRACT FROM AUTHOR]

As an important enzyme in Phase I drug metabolism, the flavin-containing monooxygenase (FMO) also metabolizes some xenobiotics with soft nucleophiles. The site of metabolism (SOM) on a molecule is the site where the metabolic reaction is exerted by an enzyme. Accurate prediction of SOMs on drug molecules will assist the search for drug leads during the optimization process. Here, some quantum mechanics features such as the condensed Fukui function and attributes from circular fingerprints (called Molprint2D) are computed and classified using the support vector machine (SVM) for predicting some potential SOMs on a series of drugs that can be metabolized by FMO enzymes. The condensed Fukui function fA− representing the nucleophilicity of central atom A and the attributes from circular fingerprints accounting the influence of neighbors on the central atom. The total number of FMO substrates and non-substrates collected in the study is 85 and they are equally divided into the training and test sets with each carrying roughly the same number of potential SOMs. However, only N-oxidation and S-oxidation features were considered in the prediction since the available C-oxidation data was scarce. In the training process, the LibSVM package of WEKA package and the option of 10-fold cross validation are employed. The prediction performance on the test set evaluated by accuracy, Matthews correlation coefficient and area under ROC curve computed are 0.829, 0.659, and 0.877 respectively. This work reveals that the SVM model built can accurately predict the potential SOMs for drug molecules that are metabolizable by the FMO enzymes. [ABSTRACT FROM AUTHOR]