Mechanistic Scrutiny Identifies a Kinetic Role for Cytochrome b5 Regulation of Human Cytochrome P450c17 (CYP17A1, P450 17A1)

Cytochrome P450c17 (P450 17A1, CYP17A1) is a critical enzyme in the synthesis of androgens and is now a target enzyme for the treatment of prostate cancer. Cytochrome P450c17 can exhibit either one or two physiological enzymatic activities differentially regulated by cytochrome b5. How this is achieved remains unknown. Here, comprehensive in silico, in vivo and in vitro analyses were undertaken. Fluorescence Resonance Energy Transfer analysis showed close interactions within living cells between cytochrome P450c17 and cytochrome b5. In silico modeling identified the sites of interaction and confirmed that E48 and E49 residues in cytochrome b5 are essential for activity. Quartz crystal microbalance studies identified specific protein-protein interactions in a lipid membrane. Voltammetric analysis revealed that the wild type cytochrome b5, but not a mutated, E48G/E49G cyt b5, altered the kinetics of electron transfer between the electrode and the P450c17. We conclude that cytochrome b5 can influence the electronic conductivity of cytochrome P450c17 via allosteric, protein-protein interactions.     

Effects of rottlerin on silica-exacerbated systemic autoimmune disease in New Zealand mixed mice

Environmental crystalline silica exposure has been associated with formation of autoantibodies and development of systemic autoimmune disease, but the mechanisms leading to these events are unknown. Silica exposure in autoimmune-prone New Zealand mixed (NZM) mice results in a significant exacerbation of systemic autoimmunity as measured by increases in autoantibodies and glomerulonephritis. Previous studies have suggested that silica-induced apoptosis of alveolar macrophages (AM) contributes to the generation of the autoantibodies and disease. Rottlerin has been reported to inhibit apoptosis in many cell types, possibly through direct or indirect effects on PKCdelta. In this study, rottlerin reduced silica-induced apoptosis in bone marrow-derived macrophages as measured by DNA fragmentation. In NZM mice, RNA and protein levels of PKCdelta were significantly elevated in AM 14 wk after silica exposure. Therefore, rottlerin was used to reduce apoptosis of AM and evaluate the progress of silica-exacerbated systemic autoimmune disease. Fourteen weeks after silica exposure, NZM mice had increased levels of anti-histone autoantibodies, high proteinuria, and glomerulonephritis. However, silica-instilled mice that also received weekly instillations of rottlerin had significantly lower levels of proteinuria, anti-histone autoantibodies, complement C3, and IgG deposition within the kidney. Weekly instillations of rottlerin in silica-instilled NZM mice also inhibited the upregulation of PKCdelta in AM. Together, these data demonstrate that in vivo treatment with rottlerin significantly decreased the exacerbation of autoimmunity by silica exposure.     

One-step amino acid selective isotope labeling of proteins in prototrophic Escherichia coli strains

Amino acid selective isotope labeling is a useful approach to simplification of nuclear magnetic resonance (NMR) spectra of large proteins. Cell-free protein synthesis offers essentially unlimited flexibility of labeling patterns but is labor-intensive and expensive. In vivo labeling is simple in principle but generally requires auxotrophic strains, inhibitors of amino acid synthesis, or complex media formulations. We describe a simple procedure for amino acid selective labeling of proteins expressed in prototrophic Escherichia coli strains. Excellent labeling selectivity was achieved for histidine, lysine, methionine, and alanine. Simplicity and robustness of this protocol make it a useful tool for protein NMR.     

Membrane docking geometry of GRP1 PH domain bound to a target lipid bilayer: an EPR site-directed spin-labeling and relaxation study

The second messenger lipid PIP(3) (phosphatidylinositol-3,4,5-trisphosphate) is generated by the lipid kinase PI3K (phosphoinositide-3-kinase) in the inner leaflet of the plasma membrane, where it regulates a broad array of cell processes by recruiting multiple signaling proteins containing PIP(3)-specific pleckstrin homology (PH) domains to the membrane surface. Despite the broad importance of PIP(3)-specific PH domains, the membrane docking geometry of a PH domain bound to its target PIP(3) lipid on a bilayer surface has not yet been experimentally determined. The present study employs EPR site-directed spin labeling and relaxation methods to elucidate the membrane docking geometry of GRP1 PH domain bound to bilayer-embedded PIP(3). The model target bilayer contains the neutral background lipid PC and both essential targeting lipids: (i) PIP(3) target lipid that provides specificity and affinity, and (ii) PS facilitator lipid that enhances the PIP(3) on-rate via an electrostatic search mechanism. The EPR approach measures membrane depth parameters for 18 function-retaining spin labels coupled to the PH domain, and for calibration spin labels coupled to phospholipids. The resulting depth parameters, together with the known high resolution structure of the co-complex between GRP1 PH domain and the PIP(3) headgroup, provide sufficient constraints to define an optimized, self-consistent membrane docking geometry. In this optimized geometry the PH domain engulfs the PIP(3) headgroup with minimal bilayer penetration, yielding the shallowest membrane position yet described for a lipid binding domain. This binding interaction displaces the PIP(3) headgroup from its lowest energy position and orientation in the bilayer, but the headgroup remains within its energetically accessible depth and angular ranges. Finally, the optimized docking geometry explains previous biophysical findings including mutations observed to disrupt membrane binding, and the rapid lateral diffusion observed for PIP(3)-bound GRP1 PH domain on supported lipid bilayers.     

Sialoadhesin expressed on IFN-induced monocytes binds HIV-1 and enhances infectivity

Background

HIV-1 infection dysregulates the immune system and alters gene expression in circulating monocytes. Differential gene expression analysis of CD14⁺ monocytes from subjects infected with HIV-1 revealed increased expression of sialoadhesin (Sn, CD169, Siglec 1), a cell adhesion molecule first described in a subset of macrophages activated in chronic inflammatory diseases.

Methodology/Principal Findings

We analyzed sialoadhesin expression on CD14⁺ monocytes by flow cytometry and found significantly higher expression in subjects with elevated viral loads compared to subjects with undetectable viral loads. In cultured CD14⁺ monocytes isolated from healthy individuals, sialoadhesin expression was induced by interferon-α and interferon-γ but not tumor necrosis factor-α. Using a stringent binding assay, sialoadhesin-expressing monocytes adsorbed HIV-1 through interaction with the sialic acid residues on the viral envelope glycoprotein gp120. Furthermore, monocytes expressing sialoadhesin facilitated HIV-1 trans infection of permissive cells, which occurred in the absence of monocyte self-infection.

Conclusions/Significance

Increased sialoadhesin expression on CD14⁺ monocytes occurred in response to HIV-1 infection with maximum expression associated with high viral load. We show that interferons induce sialoadhesin in primary CD14⁺ monocytes, which is consistent with an antiviral response during viremia. Our findings suggest that circulating sialoadhesin-expressing monocytes are capable of binding HIV-1 and effectively delivering virus to target cells thereby enhancing the distribution of HIV-1. Sialoadhesin could disseminate HIV-1 to viral reservoirs during monocyte immunosurveillance or migration to sites of inflammation and then facilitate HIV-1 infection of permissive cells.     

Temperature-sensitive vaccinia virus mutants identify a gene with an essential role in viral replication.

Vaccinia virus mutants ts2 and ts25, members of the same complementation group, exhibit a temperature-dependent arrest at the stage of viral DNA replication. The lesions responsible for the mutant phenotypes have been localized to the far left region of the HindIII B genomic fragment by marker rescue studies. Hybrid selection analyses established that the DNA fragments positive for rescue represented the first open reading frame of the HindIII B fragment and encoded a 30-kilodalton protein. The gene is expressed early after infection as a rightwardly transcribed 1-kilobase-pair mRNA whose coordinates were determined by S1 nuclease mapping. To further the phenotypic analysis of the mutants, the accumulation of viral DNA sequences during permissive and nonpermissive infections was quantitated. The extent of the DNA- phenotype was shown to vary in different cell types. In mouse L cells at either high or low multiplicity of infection, nonpermissive DNA synthesis was less than 5% of that seen in permissive infections. This severe defect was mirrored by correspondingly low viral yields. In infections of BSC40 monkey cells, however, the deficiencies in both DNA synthesis and virus production were far less severe. For one mutant (ts2), the temperature sensitivity in BSC40 cells varied inversely with the multiplicity of infection.     

Unusual sugar nucleotide recognition elements of mesophilic vs. thermophilic glycogen synthases

The glycogen synthase found in Pyrococcus furiosus is a hyperthermophilic biocatalyst that transfers the glucose portion of nucleotide-diphosphoglucose onto a growing carbohydrate biopolymer chain at 80 degrees C. In contrast to the mesophilic rabbit muscle glycogen synthase, the biocatalyst from P. furiosus possesses unusually broad nucleotide tolerance. The enzyme accepts all four common glucose-containing nucleotide-diphosphosugars: ADP-glucose, GDP-glucose, dTDP-glucose, and UDP-glucose. Using an electrospray ionization-mass spectroscopy (ESI-MS) assay, we determined the K(M) and Vmax for GDP-glucose to be 3.9 +/- 0.6 mM and 0.243 +/- 0.009 mM/min, and for dTDP-glucose to be 4.0 +/- 0.5 mM and 0.216 +/- 0.008 mM/min. A related nucleotide sugar, UDP-galactose, was not a reactive substrate, but was instead a competitive inhibitor with a Ki of 17 +/- 2 mM. The glycogen synthase from P. furiosus was shown not to have phosphorylase activity. The DeltaDeltaG of substrate binding was compared between the mesophilic rabbit muscle and the hyperthermophilic P. furiosus glycogen synthase to dissect any differences in sugar nucleotide recognition strategies at elevated temperatures. Both biocatalysts were shown to gain most of their substrate affinity through electrostatic interactions between the enzyme and the alpha-phosphate.     

Crystal structure of human PACRG in complex with MEIG1 reveals roles in axoneme formation and tubulin binding

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