In contrast to the wide spectrum of cytochrome P450 monooxygenases, there are only 2 heme-based dioxygenases in humans: tryptophan dioxygenase (hTDO) and indoleamine 2,3-dioxygenase (hIDO). hTDO and hIDO catalyze the same oxidative ring cleavage reaction of L-tryptophan to N-formyl kynurenine, the initial and rate-limiting step of the kynurenine pathway. Despite immense interest, the mechanism by which the 2 enzymes execute the dioxygenase reaction remains elusive. Here, we report experimental evidence for a key ferryl intermediate of hIDO that supports a mechanism in which the 2 atoms of dioxygen are inserted into the substrate via a consecutive 2-step reaction. This finding introduces a paradigm shift in our understanding of the heme-based dioxygenase chemistry, which was previously believed to proceed via simultaneous incorporation of both atoms of dioxygen into the substrate. The ferryl intermediate is not observable during the hTDO reaction, highlighting the structural differences between the 2 dioxygenases, as well as the importance of stereoelectronic factors in modulating the reactions. indoleamine 2,3-dioxygenase ͉ reasonance Raman spectroscopy ͉ tryptophan dioxygenase

Indoleamine 2,3-dioxygenase (IDO) is a nonhepatic intracellular heme-containing enzyme. 1 It catalyzes the conversion of Ltryptophan (L-Trp) to N-formylkynurenine (NFK), the initial and rate-determining step of the kynurenine pathway, by inserting both atoms of dioxygen across the C 2 dC 3 bond of the indole moiety of L-Trp.2 IDO was first discovered by Hayaishi et al. more than four decades ago. 3 Since then, its structural and functional properties were extensively studied until the early 1990s. This field of research was invigorated recently due to the discoveries that IDO is linked to a variety of immune-related pathophysiological conditions and that IDO is a potential target for pharmacological intervention against cancer. 4 Here we report evidence supporting the presence of an inhibitory substrate binding site (S i site) in human IDO (hIDO), which is capable of binding substrates (L-Trp and 1-methy-L-tryptophan), an effector (3-indole ethanol), and an uncompetitive inhibitor (Mitomycin C). The structures of these molecules are illustrated in Scheme 1.

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Arabidopsis thaliana HYPONASTIC LEAVES1 (HYL1) is a microRNA (miRNA) biogenesis protein that contains two N-terminal double-stranded RNA binding domains (dsRBDs), a putative nuclear localization site (NLS), and a putative protein-protein interaction domain. The interaction of HYL1 with DICER-LIKE1 is important for the efficient and precise processing of miRNA primary transcripts in plant miRNA biogenesis. To define the roles of the various domains of HYL1 in miRNA processing and the miRNA-directed phenotype, we transferred a series of HYL1 deletion constructs into hyl1 null mutants. The N-terminal region containing dsRBD1 and dsRBD2 completely rescued the mutant phenotype of hyl1, triggering the accumulation of miR166 and miR160 and resulting in reduced mRNA levels of the targeted genes. In vivo biochemical analysis of the HYL1-containing complexes from the transgenic plants revealed that the N-terminal dsRBDs of HYL1 were sufficient for processing miRNA precursors and the generation of mature miRNA. Transient and stable expression analysis demonstrated that the putative NLS domain was indeed the nuclear localization signal, whereas the N-terminal region containing the dsRBDs was not restricted to the nucleus. We suggest that the N-terminal dsRBDs fulfill the function of the whole HYL1 and thus play an essential role in miRNA processing and miRNA-directed silencing of targeted genes.

Human indoleamine 2,3-dioxygenase (hIDO) is an intracellular heme-containing enzyme, which catalyzes the initial and rate-determining step of L-tryptophan (L-Trp) metabolism via the kynurenine pathway in nonhepatic tissues. Steady-state kinetic data showed that hIDO exhibits substrate inhibition behavior, implying the existence of a second substrate binding site in the enzyme, although so far there is no direct evidence supporting it. The kinetic data also revealed that the Km of L-Trp (15 μM) is ~27-fold lower than the Kd of L-Trp (0.4 mM) for the ligand-free ferrous enzyme, suggesting that O2 binding proceeds L-Trp binding during the catalytic cycle. With cyanide as a structural probe, we have investigated the thermodynamic and kinetic parameters associated with ligand and substrate binding to hIDO. Equilibrium titration studies show that the cyanide adduct is capable of binding two L-Trp molecules, with Kd values of 18 μM and 26 mM. The data offer the first direct evidence of the second substrate binding site in hIDO. Kinetic studies demonstrate that prebinding of L-Trp to the enzyme retards cyanide binding by ~13-fold, while prebinding of cyanide to the enzyme facilitates L-Trp binding by ~22-fold. The data support the view that during the active turnover of the enzyme it is kinetically more favored to bind O2 prior to L-Trp.

Background Schistosoma japonicum is a parasitic flatworm that causes human schistosomiasis. Secreted extracellular vesicles (EVs) play a key role in pathogen-host interfaces. Previous studies have shown that S. japonicum adult worms can release microRNA (miRNA)-containing EVs, which can transfer their cargo to mammalian cells and regulate gene expression in recipient cells. Tissue-trapped eggs are generally considered the major contributor to the severe pathology of schistosomiasis; however, the interactions between the host and parasite in this critical stage remain largely unknown.MethodsThe culture medium for S. japonicum eggs in vitro was used to isolate EVs. Transmission electron microscopy (TEM) analysis was used to confirm that vesicles produced by the eggs were EVs based on size and morphology. Total RNA extracted from EVs was analyzed by Solexa technology to determine the miRNA profile. The in vitro internalization of the EVs by mammalian cells was analyzed by confocal microscopy. The presence of EVs associated miRNAs in the primary hepatocytes of infected mice was determined by quantitative real-time PCR (qRT-PCR).ResultsEVs were isolated from the culture medium of in vitro cultivated S. japonicum eggs. TEM analysis confirmed that nanosized vesicles were present in the culture medium. RNA-seq analysis showed that the egg-derived EVs contained small non-coding RNA (sncRNA) populations including miRNAs, suggesting a potential role in host manipulation. This study further showed that Hepa1-6, a murine liver cell line, internalized the purified EVs and their cargo miRNAs that were detectable in the primary hepatocytes of mice infected with S. japonicum.Conclusions Schistosoma japonicum eggs can release miRNA-containing EVs, and the EVs can transfer their cargo to recipient cells in vitro. These results demonstrate the regulatory potential of S. japonicum egg EVs at the parasite-host interface.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1845-2) contains supplementary material, which is available to authorized users.

Background: The aim of this study was to characterize the disorder of lipid metabolism in hepatocellular carcinoma (HCC). HCC is a worldwide disease. The research into the disorder of lipid metabolism in HCC is very limited. Study of lipid metabolism in liver cancer tissue may have the potential to provide new insight into HCC mechanisms. Methods: A lipidomics study of HCC based on Ultra high performance liquid chromatography-electronic spray ionization-QTOF mass spectrometer (UPLC-ESI-QTOF MS) and Matrix assisted laser desorption ionization-fourier transform ion cyclotron resonance mass spectrometer (MALDI-FTICR MS) was performed. Results: Triacylglycerols (TAGs) with the number of double bond (DB) > 2 (except 56:5 and 56:4 TAG) were significantly down-regulated; conversely, others (except 52:2 TAG) were greatly up-regulated in HCC tissues. Moreover, the more serious the disease was, the higher the saturated TAG concentration and the lower the polyunsaturated TAG concentration were in HCC tissues. The phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol (PI) were altered in a certain way. Sphingomyelin (SM) was up-regulated and ceramide (Cer) were down-regulated in HCC tissues. Conclusions: To our knowledge, this is the first such report showing a unique trend of TAG, PC, PE and PI. The use of polyunsaturated fatty acids, like eicosapentanoic and docosahexanoic acid, as supplementation, proposed for the treatment of Non-alcoholic steatohepatitis (NASH), may also be effective for the treatment of HCC.

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