A structural model of a P450-ferredoxin complex from orientation-selective double electron-electron resonance spectroscopy

This research was supported by the Engineering & Physical Sciences Research Council (EPSRC) and the Biotechnology & Biological Sciences Research Council (BBSRC), UK (EP/D048559). AMB and EOJD were supported by graduate studentships from the BBSRC (BB/F01709X/1) and NJH and JEL were supported by graduate studentships from the EPSRC, and JEL after her DPhil by EP/D048559. AMB gratefully acknowledges her current fellowship support from the Royal Society and EPSRC for a Dorothy Hodgkin Fellowship (DH160004). JRH acknowledges support from the ARC (FT120100421) and the Centre for Advanced Imaging, The University of Queensland.Cytochrome P450 (CYP) monooxygenases catalyze the oxidation of chemically inert carbon-hydrogen bonds in diverse endogenous and exogenous organic compounds by atmospheric oxygen. This C–H bond oxy-functionalization activity has huge potential in biotechnological applications. Class I CYPs receive the two electrons required for oxygen activation from NAD(P)H via a ferredoxin reductase and ferredoxin. The interaction of Class I CYPs with their cognate ferredoxin is specific. In order to reconstitute the activity of diverse CYPs, structural characterization of CYP-ferredoxin complexes is necessary, but little structural information is available. Here we report a structural model of such a complex (CYP199A2-HaPux) in frozen solution derived from distance and orientation restraints gathered by the EPR technique of orientation-selective double electron-electron resonance (os-DEER). The long-lived oscillations in the os-DEER spectra were well modeled by a single orientation of the CYP199A2-HaPux complex. The structure is different from the two known Class I CYP-Fdx structures: CYP11A1-Adx and CYP101A1-Pdx. At the protein interface, HaPux residues in the [Fe2S2] cluster-binding loop and the α3 helix, and the C-terminus residue interact with CYP199A2 residues in the proximal loop and the C helix. These residue contacts are consistent with biochemical data on CYP199A2-ferredoxin binding and electron transfer. Electron-tunneling calculations indicate an efficient electron-transfer pathway from the [Fe2S2] cluster to the heme. This new structural model of a CYP-Fdx complex provides the basis for tailoring CYP enzymes for which the cognate ferredoxin is not known, to accept electrons from HaPux and display monooxygenase activity.PostprintPeer reviewe

Noninvasive prenatal diagnosis for X-linked disease by maternal plasma sequencing in a family of Hemophilia B

Objective: To apply a Hidden Markov Model to test Hemophilia B in a fetus by maternal plasma sequencing only employing proband and maternal haplotypes. Case Report: A family at risk for Hemophilia B was recruited in this study. We performed genetic diagnosis on the proband using our targeted capture system (containing F9 gene coding region, highly heterozygous SNPs and a 13-kb chromosome Y specific region), and revealed a causative F9 gene mutation (c.190T>C, p.Cys64Arg). Maternal plasma cell-free DNA obtained at 8 weeks of gestation was targeted-captured and sequenced using the customized system. The fetus inherited the F9 (c.190T>C, p.Cys64Arg) mutation according to the Hidden Markov Model. The mother continued the pregnancy. Conclusions: This study is the first report of a haplotype-based approach in NIPD of Hemophilia B. With further evaluation, this method might be useful for NIPD of Hemophilia B and for other X-linked single-gene disorders

A structural model of a P450-ferredoxin complex from orientation-selective double electron-electron resonance spectroscopy

Cytochrome P450 (CYP) monooxygenases catalyze the oxidation of chemically inert carbon-hydrogen bonds in diverse endogenous and exogenous organic compounds by atmospheric oxygen. This C–H bond oxy-functionalization activity has huge potential in biotechnological applications. Class I CYPs receive the two electrons required for oxygen activation from NAD(P)H via a ferredoxin reductase and ferredoxin. The interaction of Class I CYPs with their cognate ferredoxin is specific. In order to reconstitute the activity of diverse CYPs, structural characterization of CYP-ferredoxin complexes is necessary, but little structural information is available. Here we report a structural model of such a complex (CYP199A2-HaPux) in frozen solution derived from distance and orientation restraints gathered by the EPR technique of orientation-selective double electron-electron resonance (os-DEER). The long-lived oscillations in the os-DEER spectra were well modeled by a single orientation of the CYP199A2-HaPux complex. The structure is different from the two known Class I CYP-Fdx structures: CYP11A1-Adx and CYP101A1-Pdx. At the protein interface, HaPux residues in the [Fe2S2] cluster-binding loop and the α3 helix, and the C-terminus residue interact with CYP199A2 residues in the proximal loop and the C helix. These residue contacts are consistent with biochemical data on CYP199A2-ferredoxin binding and electron transfer. Electron-tunneling calculations indicate an efficient electron-transfer pathway from the [Fe2S2] cluster to the heme. This new structural model of a CYP-Fdx complex provides the basis for tailoring CYP enzymes for which the cognate ferredoxin is not known, to accept electrons from HaPux and display monooxygenase activity

Integrating longitudinal clinical laboratory tests with targeted proteomic and transcriptomic analyses reveal the landscape of host responses in COVID-19

Abstract The pathophysiology of coronavirus disease 19 (COVID-19) involves a multitude of host responses, yet how they unfold during the course of disease progression remains unclear. Here, through integrative analysis of clinical laboratory tests, targeted proteomes, and transcriptomes of 963 patients in Shanghai, we delineate the dynamics of multiple circulatory factors within the first 30 days post-illness onset and during convalescence. We show that hypercortisolemia represents one of the probable causes of acute lymphocytopenia at the onset of severe/critical conditions. Comparison of the transcriptomes of the bronchoalveolar microenvironment and peripheral blood indicates alveolar macrophages, alveolar epithelial cells, and monocytes in lungs as the potential main sources of elevated cytokines mediating systemic immune responses and organ damages. In addition, the transcriptomes of patient blood cells are characterized by distinct gene regulatory networks and alternative splicing events. Our study provides a panorama of the host responses in COVID-19, which may serve as the basis for developing further diagnostics and therapy

Defective anchoring of JNK1 in the cytoplasm by MKK7 in Jurkat cells is associated with resistance to Fas-mediated apoptosis

MKK7 works as a cytoplasmic anchoring protein for JNK1 in various cell lines but exhibits aberrant nuclear entry in Jurkat cells, which leads to resistance to Fas-mediated apoptosis