Littérature scientifique sur le sujet « 4S pathway »

Stage 4S neuroblastoma is a childhood cancer occurring in infants (<12 months at diagnosis) with metastases limited to liver, skin, and bone marrow (<10%). It is associated with an excellent outcome, due to its notable ability to undergo spontaneous regression without any therapeutic intervention. However, a subgroup of patients is doomed to relapse and eventually to die in spite of aggressive therapies. Stage 4S neuroblastoma shows characteristic hypermethylation of genes involved in the telomere maintenance, indicating that the dysregulation of these genes might serve as prognostic marker. The retinoblastoma tumor suppressor protein (RB)-E2F transcription factors pathway is one of the critical tumor-suppressor/oncogene pathways involved in regulating telomerase expression. We have interrogated in silicopublic neuroblastoma databases for regulators involved in the RB-E2F pathway especially for E2F factors themselves, and we identified the E2F transcription factor 3 (E2F3) expression as a potential prognostic marker in stage 4S neuroblastoma. In order to confirm this finding, we screened 38 paraffin-embedded tissue samples stage 4S neuroblastoma for E2F3 protein expression using immunofluorescence, and we observed that augmented expression was strongly associated with impaired event-free survival. These results indicate that E2F3 expression might serve as prognostic marker in patients with stage 4S disease.

IscA/SufA paralogues are the members of the iron-sulfur cluster assembly machinery in Escherichia coli. Whereas deletion of either IscA or SufA has only a mild effect on cell growth, deletion of both IscA and SufA results in a null-growth phenotype in minimal medium under aerobic growth conditions. Here we report that cell growth of the iscA/sufA double mutant (E. coli strain in which both iscA and sufA had been in-frame-deleted) can be partially restored by supplementing with BCAAs (branched-chain amino acids) and thiamin. We further demonstrate that deletion of IscA/SufA paralogues blocks the [4Fe-4S] cluster assembly in IlvD (dihydroxyacid dehydratase) of the BCAA biosynthetic pathway in E. coli cells under aerobic conditions and that addition of the iron-bound IscA/SufA efficiently promotes the [4Fe-4S] cluster assembly in IlvD and restores the enzyme activity in vitro, suggesting that IscA/SufA may act as an iron donor for the [4Fe-4S] cluster assembly under aerobic conditions. Additional studies reveal that IscA/SufA are also required for the [4Fe-4S] cluster assembly in enzyme ThiC of the thiamin-biosynthetic pathway, aconitase B of the citrate acid cycle and endonuclease III of the DNA-base-excision-repair pathway in E. coli under aerobic conditions. Nevertheless, deletion of IscA/SufA does not significantly affect the [2Fe-2S] cluster assembly in the redox transcription factor SoxR, ferredoxin and the siderophore-iron reductase FhuF. The results suggest that the biogenesis of the [4Fe-4S] clusters and the [2Fe-2S] clusters may have distinct pathways and that IscA/SufA paralogues are essential for the [4Fe-4S] cluster assembly, but are dispensable for the [2Fe-2S] cluster assembly in E. coli under aerobic conditions.

ABSTRACT Strain DCL14, which is able to grow on limonene as a sole source of carbon and energy, was isolated from a freshwater sediment sample. This organism was identified as a strain of Rhodococcus erythropolis by chemotaxonomic and genetic studies. R. erythropolis DCL14 also assimilated the terpenes limonene-1,2-epoxide, limonene-1,2-diol, carveol, carvone, and (−)-menthol, while perillyl alcohol was not utilized as a carbon and energy source. Induction tests with cells grown on limonene revealed that the oxygen consumption rates with limonene-1,2-epoxide, limonene-1,2-diol, 1-hydroxy-2-oxolimonene, and carveol were high. Limonene-induced cells of R. erythropolis DCL14 contained the following four novel enzymatic activities involved in the limonene degradation pathway of this microorganism: a flavin adenine dinucleotide- and NADH-dependent limonene 1,2-monooxygenase activity, a cofactor-independent limonene-1,2-epoxide hydrolase activity, a dichlorophenolindophenol-dependent limonene-1,2-diol dehydrogenase activity, and an NADPH-dependent 1-hydroxy-2-oxolimonene 1,2-monooxygenase activity. Product accumulation studies showed that (1S,2S,4R)-limonene-1,2-diol, (1S,4R)-1-hydroxy-2-oxolimonene, and (3R)-3-isopropenyl-6-oxoheptanoate were intermediates in the (4R)-limonene degradation pathway. The opposite enantiomers [(1R,2R,4S)-limonene-1,2-diol, (1R,4S)-1-hydroxy-2-oxolimonene, and (3S)-3-isopropenyl-6-oxoheptanoate] were found in the (4S)-limonene degradation pathway, while accumulation of (1R,2S,4S)-limonene-1,2-diol from (4S)-limonene was also observed. These results show thatR. erythropolis DCL14 metabolizes both enantiomers of limonene via a novel degradation pathway that starts with epoxidation at the 1,2 double bond forming limonene-1,2-epoxide. This epoxide is subsequently converted to limonene-1,2-diol, 1-hydroxy-2-oxolimonene, and 7-hydroxy-4-isopropenyl-7-methyl-2-oxo-oxepanone. This lactone spontaneously rearranges to form 3-isopropenyl-6-oxoheptanoate. In the presence of coenzyme A and ATP this acid is converted further, and this finding, together with the high levels of isocitrate lyase activity in extracts of limonene-grown cells, suggests that further degradation takes place via the β-oxidation pathway.

The Smoothened (SMO) receptor is the most druggable target in the Hedgehog (HH) pathway for anticancer compounds. However, SMO antagonists such as vismodegib rapidly develop drug resistance. In this study, new SMO antagonists having the versatile purine ring as a scaffold were designed, synthesised, and biologically tested to provide an insight to their mechanism of action. Compound 4s was the most active and the best inhibitor of cell growth and selectively cytotoxic to cancer cells. 4s induced cell cycle arrest, apoptosis, a reduction in colony formation and downregulation of PTCH and GLI1 expression. BODIPY-cyclopamine displacement assays confirmed 4s is a SMO antagonist. In vivo, 4s strongly inhibited tumour relapse and metastasis of melanoma cells in mice. In vitro, 4s was more efficient than vismodegib to induce apoptosis in human cancer cells and that might be attributed to its dual ability to function as a SMO antagonist and apoptosis inducer.

The reaction of N (4S) radical with NCO (X2Π) radical has been studied theoretically using density functional theory and ab initio quantum chemistry method. The triplet electronic state [ N 2 CO ] potential energy surface (PES) is calculated at the G3B3 and CCSD(T)/aug-cc-pVDZ//B3LYP/6-311++G(d,p) levels of theory. All the energies of the transition states and isomers in the pathway RP1 are lower than that of the reactants; the rate of this pathway should be very fast. Thus, the novel reaction N + NCO can proceed effectively even at low temperatures and it is expected to play a role in both combustion and interstellar processes. On the basis of the analysis of the kinetics of all pathways through which the reactions proceed, we expect that the competitive power of reaction pathways may vary with experimental conditions for the title reaction.

ABSTRACT For the ornithine fermentation pathway, described more than 70 years ago, genetic and biochemical information are still incomplete. We present here the experimental identification of the last four missing genes of this metabolic pathway. They encode l-ornithine racemase, (2R,4S)-2,4-diaminopentanoate dehydrogenase, and the two subunits of 2-amino-4-ketopentanoate thiolase. While described only for the Clostridiaceae to date, this pathway is shown to be more widespread.

We discuss structural and mechanistic aspects of the Dsz enzymes in the 4S pathway, with a focus on rational molecular strategies for enzyme engineering, aiming at enzyme catalytic rate and efficiency improvement to meet industrial demands.

The non-mevalonate or also called MEP pathway is an essential route for the biosynthesis of isoprenoid precursors in most bacteria and in microorganisms belonging to the Apicomplexa phylum, such as the parasite responsible for malaria. The absence of this pathway in mammalians makes it an interesting target for the discovery of novel anti-infectives. As last enzyme of this pathway, IspH is an oxygen sensitive [4Fe-4S] metalloenzyme that catalyzes 2H+/2e- reductions and a water elimination by involving non-conventional bioinorganic and bioorganometallic intermediates. After a detailed description of the discovery of the [4Fe-4S] cluster of IspH, this review focuses on the IspH mechanism discussing the results that have been obtained in the last decades using an approach combining chemistry, enzymology, crystallography, spectroscopies, and docking calculations. Considering the interesting druggability of this enzyme, a section about the inhibitors of IspH discovered up to now is reported as well. The presented results constitute a useful and rational help to inaugurate the design and development of new potential chemotherapeutics against pathogenic organisms.

The novel enzyme 4-methyl-2-enelactone methyl-isomerase was detected in, and purified to electrophoretic homogeneity from, p-toluate-grown cells of Rhodococcus rhodocrous N75, a nocardioform actinomycete. The enzyme was very thermostable and had a native Mr of 75,500; as the monomer had an Mr of 17,000, the enzyme is probably tetrameric. The new isomerase is highly specific with respect to its lactone substrate, only accepting (+)-(4S)-4-methylmuconolactone (4-carboxymethyl-4-methylbut-2-en-1,4-olide), and the putative isomerization reaction intermediate 1-methylbislactone ((-)-1-methyl-3,7-dioxo-2,6-dioxabicyclo-[3.3.0]octane) as substrates, and yielding (-)-(4S)-3-methylmuconolactone (4-carboxymethyl-3-methylbut-2-en-1,4-olide) as product. Some other lactone analogues acted as competitive inhibitors. Our data suggest that the isomerization does not involve actual methyl migration, but proceeds via the 1-methybislactone.

L’enorme quantità di pneumatici fuori uso accumulati costituisce un grave problema ambientale. Per porvi rimedio si cercano soluzioni che mirano al riutilizzo della gomma naturale (NR), principale materia prima utilizzata nella produzione di pneumatici. Per poter essere riutilizzata, la NR viene generalmente macinata finemente e sottoposta ad un trattamento di devulcanizzazione, cioè alla rottura dei ponti trasversali zolfo-zolfo. Diversi metodi chimici o meccanici sono utilizzabili per devulcanizzare il polverino. Tuttavia, ciascuno di essi presenta degli svantaggi. Sarebbe quindi auspicabile la messa a punto di processi in cui reazioni specifiche siano realizzate in condizioni moderate di temperatura e pressione. In questo scenario l’utilizzo di biocatalizzatori potrebbe costituire un’alternativa valida e di minor impatto ambientale. Questo studio si occupa di verificare la possibilità di trattamenti enzimatici di “biodevulcanizzazione”. Non essendo noti enzimi in grado di devulcanizzare la gomma, questo lavoro di tesi è partito dall’analisi di microrganismi isolati da campioni ambientali e con potenziali proprietà desolforanti che sono state saggiate su un substrato modello, il dibenzotiofene (DBT). Il primo screening sui microrganismi ha portato alla selezione di un nuovo ceppo di Rhodococcus sp. AF21875. L’attività desolforante di questo microrganismo è stata studiata con due approcci paralleli, da un lato verificando la presenza di geni codificanti per enzimi che desolforano il DBT e dall’altro individuando nuovi enzimi con abilità desolforanti. In batteri attivi in processi di desolforazione, i geni dsz codificano per 4 enzimi: DszA, DszB, DszC e DszD. Nel DNA genomico di Rhodococcus sp. AF21875 è stata verificata la presenza dei quattro geni dsz, che sono stati clonati per consentire la produzione ricombinante delle proteine corrispondenti in un ceppo del batterio Escherichia coli. Tre delle proteine, DszA, DszC e DszD, sono abbondantemente espresse in forma solubile e sono state purificate con successo. In previsione di un impiego biotecnologico delle proteine DszA, DszC e DszD è stata intrapresa l’analisi di alcune caratteristiche strutturali e di stabilità. In particolare, è stata analizzata la composizione della struttura secondaria e la stabilità al calore mediante dicroismo circolare; la stabilità in presenza di diversi solventi organici, attraverso spettrofluorimetria. L’attività degli enzimi è stata monitorata mediante cromatografia liquida (HPLC) che consente di rilevare la formazione di 2-idrossibifenile (HBP) come prodotto finale di reazione. L’attività di desolforazione dei quattro enzimi è stata infine saggiata su gomma naturale vulcanizzata. È emerso che il trattamento enzimatico provoca modificazioni chimiche della gomma. Sebbene le analisi condotte evidenzino cambiamenti di modesta entità e non associabili univocamente ad un processo di desolforazione, gli enzimi individuati costituiscono un buon punto di partenza per approcci di ingegneria proteica volti a migliorare l’attività e la stabilità degli enzimi Dsz da Rhodococcus sp. AF21875. Inoltre, per individuare nuove attività enzimatiche è stata eseguita un’analisi di proteomica differenziale delle cellule di Rhodococcus sp. AF21875. Da cellule cresciute in terreno privo o addizionato di DBT sono state estratte le proteine totali che sono state analizzate mediante elettroforesi bidimensionale. Quando Rhodococcus sp. AF21875 cresce in presenza di DBT, produce un pool di proteine che non si ritrovano tra le proteine espresse in assenza di DBT. Tre delle proteine sono state analizzate tramite digestione triptica in gel e analisi di spettrometria di massa. Questa analisi ha permesso di identificare due enzimi che non sono coinvolti nel metabolismo dello zolfo ma appartengono ad una stessa via catabolica distinta da quella cui appartengono gli enzimi Dsz.
The environmental hazard posed by the accumulation of huge amounts of used tires might be partly relieved by the implementation of methods for recycling natural rubber (NR) from waste tires. This approach requires rubber grinding and a process of devulcanization that breaks the sulfur-sulfur crosslinks among polymer chains. Several chemic al or mechanical methods are already used to devulcanize ground-rubber. However, each of them has drawbacks related either to the lack of specificity or to the use of hazardous chemicals. It would therefore be desirable to develop processes in which selective and specific reactions are carried out in mild conditions of temperature and pressure, without the use of hazardous compounds. In this view, the use of biocatalysts could be a valuable and ecological alternative. This study explores the possibility of applying enzymes to devulcanize rubber in a process of “biodevulcanization”. Since enzymes active in rubber devulcanization were not available at the beginning of this thesis, this research started with the analysis of microorganisms isolated from environmental samples contaminated with waste tires. The desulfuring properties of several bacteria were tested on the model substrate dibenzothiophene (DBT). A first in-vivo screening of microorganisms allowed the selection of a new strain of Rhodococcus sp. referred as AF21875. This microorganism was studied with two aims: assessing the presence of a metabolic pathway for DBT desulfurization already described in other bacteria and identifying new metabolic abilities and enzymes. In bacteria active in desulfurization, four enzymes co-operate in the reaction of desulfurization: DszA, DszB, DszC and DszD. The presence of the four corresponding dsz genes in the genomic DNA of Rhodococcus sp. AF21875 has been assessed. The four genes have been cloned in a strain of the bacterium Escherichia coli to allow for the production of recombinant Dsz enzymes. The three recombinant proteins DszA, DszC and DszD are soluble and were successfully purified. More difficult was the production of DszB that is poorly expressed in any experimental condition. In view of a biotechnological application, structural and stability studies were carried out on DszA, DszC and DszD enzymes. In particular, we investigated secondary structure and heat stability by circular dichroism, while protein stability in the presence of different organic solvents was studied by spectrofluorimetry. Enzymes activity on DBT was assessed by high performance liquid chromatography (HPLC) by detecting the formation of 2 -hydroxybiphenyl (HBP), the reaction product of DBT desulfurization. The desulfurization activity of the four enzymes was then tested on vulcanized natural rubber using Rubber Process Analyzer and Fourier Transform Infrared Spectroscopy to detect chemical modifications induced by the enzymatic treatment. These analyses revealed minor changes. Other studies should be performed to attribute such modifications to desulfurization. Overall, Dsz enzymes from Rhodococcus sp. AF21875 were found to be an interesting starting point for the application of protein engineering approaches aimed to improve not only their activity but also their stability. A differential proteomic analysis of Rhodococcus sp. AF21875 was performed to identify enzymatic activities related to sulfur metabolism and different from Dsz proteins. Total proteins, extracted from cells grown either in the presence or in the absence of DBT, were separated by two-dimensional electrophoresis, showing that DBT induces a few changes in the proteome of Rhodococcus sp. AF21875. Three proteins, belonging to a metabolic pathway different from the Dsz one were identified by in-gel tryptic digestion and mass spectrometry.

S-Adenosylmethionine (SAM), with the unique thermodynamically activated but kinetically stable trivalent sulfonium cation in its side chain, is the second most widely used coenzyme after ATP. SAM can engage in a multitude of two-electron paths, where transfer of the methyl group as a nascent cation equivalent to O, N, S, and even carbon nucleophiles dominates natural product tailoring flux. The aminobutyryl group of SAM is also activated at C4 as an electrophilic carbon. Equally important, if not more so, in natural product biosynthetic pathways are one-electron redox routes where SAM, coordinated to an [4Fe–4S] cube, undergoes fragmentation to leave methionine coordinated to the iron–sulfur cluster, while generating the 5′-deoxyadenosyl radical (5′-dA&lt;o&gt;˙) in situ. This homolytic fragmentation leads to 5′-dA˙ as initiator of a variety of radical-based scaffold transformations in cosubstrates. More than 500 000 so-called radical SAM enzymes have been catalogued in protein databases, although only a few dozen have yet been characterized for the scope of their radical chemistry practised on specific cosubstrates.