Amino acid sequence comparison of TRIM16 and MID1 and modeling of TRIM16 B-boxes.

<p>The conserved residues in the zinc binding regions are in bold underlined type and are; cysteine; C, histidine; H, alanine; A aspartic acid; D. (<b>A</b>) TRIM16 and MID1 share the zinc binding consensus sequence for B-box1. (B) TRIM16 and MID1 share the Zinc binding consensus sequence for B-box2. (C/D) Modeling of B-boxes reveals zinc binding capability. Superimposition of the alpha-carbon backbone of the B-boxes from the MDM1 NMR structure (purple) and the homology model of TRIM16 (blue-grey). These two structures overlay with an average root-mean-square deviation of 0.4 Å.</p

TRIM16 can heterodimerize with MID1, TRIM24 and PML.

<p>(A) Schematic structures of TRIM proteins used in heterodimerization studies. (B) TRIM16 binds MID1. Co-transfection of MID1-GFP and TRIM16-myc-His in HEK293 cells and subsequent immunoprecipitation by anti-myc antibody and Western blot with anti-GFP antibody. (C) MID1 was pulled down via its GFP antibody and a Western blot was performed to detect TRIM16-myc-His in the immunoprecipitated protein complex. (D) Whole cell lysates (WCL) of HEK293 cells transfected with empty vector (EV) or TRIM16-GFP were immunopreciptated with anti-GFP antibody. An anti-PML antibody was used to detect PML as a binding partner of TRIM16. (E) Lysates containing both TRIM16-GFP and TRIM24-His proteins were immunopreciptated with anti-GFP antibody. Anti-His antibody was used to detect the presence of TRIM24 in the TRIM16-associated complex.</p

B-boxes are required for TRIM16’s E3 ligase activity.

<p>(A) TRIM16 <i>in vivo</i> polyubiquitination assay. In HEK293 cells, HA-Ub was co-transfected with various TRIM16-GFP domain deletion expression plasmids. The protein lysate was subjected to immunoprecipitation by GFP antibody, and subjected to Western blot and probed with anti-HA antibody for Ub (right panel) and anti-GFP antibody for TRIM16 (left and middle panel). Two exposure times are shown. GFP antibodies detect both un-ubiquitinated and polyubiquitinated forms of TRIM16. Polyubiquitinated smear is present in the sample transfected with wild-type TRIM16 and shown by anti-GFP and anti-HA antibodies. (B) <i>In vitro</i> ubiquitination assay with myc-His tagged TRIM16 together with a panel of E2 enzymes, showing activity with the UbcH5 family. (C) <i>In vitro</i> ubiquitination assay with full-length TRIM16, TRIM16 domain deletion mutants or empty vector showing extensive polyubiquitination with full-length TRIM16 as detected by Western blot with anti-myc antibodies. Numbers indicate protein size in kDa. (D) Recombinant TRIM16 (Abnova) was evaluated for E3 activity in the presence of recombinant E1, UbcH5b, and HA-Ub as indicated. The capacity to catalyse auto-ubiquitination <i>in vitro</i> was observed only in the presence of ZnCl₂ and ATP. Western Blot (lower panel) with TRIM16 antibody showed amount of TRIM16 protein in each lane.</p

TRIM16 homodimerizes through its coiled-coil domain

<p>. (A) TRIM16-GFP domain deletion plasmids. (B) Co-transfection of TRIM16-GFP and TRIM16-myc-His in HEK293 cells and subsequent immunoprecipitation by anti-myc antibody (Ab) and Western blot with anti-GFP antibody. Whole cell extract (WCE) used as total input. (C) TRIM16 homodimerizes through its coiled-coil domain. GFP deletion mutants were co-transfected with the TRIM16-myc-His vector. Anti-GFP antibody was used to pull down proteins binding the GFP tagged proteins and the TRIM16-myc-His was used to detect self-association via its different tag (right panel). Transfection efficiency was confirmed (left panel). TRIM16-GFP mutants were efficiently pulled down (middle panel). * non-specific bands, # refer to text.</p

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

<div><p>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 <i>in silico</i>, <i>in vivo</i> and <i>in vitro</i> analyses were undertaken. Fluorescence Resonance Energy Transfer analysis showed close interactions within living cells between cytochrome P450c17 and cytochrome b5. <i>In silico</i> 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.</p></div

Reversible potentials (Eac0) of the surface confined Fe^3+/2+ redox couple derived from the a.c. voltammograms (<i>f</i> = 219 Hz)<i>^{^a}</i> obtained from CNT electrodes modified with hemin, wt cyt b5, E48G/E49G cyt b5, P450c17, and their combinations.

<p>^<i>a</i> Average of the potentials of the central minimum in the 6^th a.c. harmonic (envelope presentation, in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141252#pone.0141252.g003" target="_blank">Fig 3</a>) from the forward and backward d.c. potential sweep directions.</p><p>^<i>b</i> Values are reproducible to ±0.001 V.</p><p>^<i>c</i> Hemin adsorbed on the electrode pre-modified with wt cyt b5.</p><p>^<i>d</i> either wt or E48G/E49G cyt b5 adsorbed on the electrode pre-modified with P450c17.</p><p>Reversible potentials (<math><msubsup><mrow><mi>E</mi></mrow><mrow><mi>a</mi><mi>c</mi></mrow><mrow><mn>0</mn></mrow></msubsup></math>) of the surface confined Fe^3+/2+ redox couple derived from the a.c. voltammograms (<i>f</i> = 219 Hz)<i>^{^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141252#t001fn001" target="_blank">a</a>}}</i> obtained from CNT electrodes modified with hemin, wt cyt b5, E48G/E49G cyt b5, P450c17, and their combinations.</p

Quartz Crystal Microbalance data for proteins binding to a membrane layer.

<p><b>a,</b> QCM data showing changes in frequency (lines, left axes) and dissipation (symbols, right axes) (7^th harmonic; Δ<i>f</i>_QCM data are normalized to the overtone number) derived from deposition of proteins onto a DMPC-cholesterol membrane pre-deposited on a mpa-layer adhered to a Au-coated quartz crystal. QCM profiles obtained with protein mixtures 20 nM P450c17+20 nM CPR+20 nM wt cyt b5 (blue) and 20 nM P450c17 + 20 nM CPR + 100 nM wt cyt b5 (black) were similar to each other and also to that for 20 nM P450c17+20 nM CPR (Figure H in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141252#pone.0141252.s001" target="_blank">S1 File</a>). In contrast, the E48G/E49G cyt b5 was deposited non-specifically and competitively with P450c17+CPR, as determined with 20 nM P450c17+20 nM CPR+20 nM E48G/E49G cyt b5 (green) and especially 20 nM P450c17+20 nM CPR+100 nM E48G/E49G cyt b5 (red) mixtures. <b>b,</b> Analysis of the QCM data using Δ<i>f</i>_QCM<i>vs</i>. Δ<i>D</i> ‘fingerprint’ plots of the temporally resolved data from panel (a). The traces were divided into characteristic deposition stages labeled with roman numerals (I–III). DMPC = 1,2-dimyristoyl-<i>sn</i>-glycero-3-phosphocholine; mpa = mercaptopropionic acid.</p

Cyclic voltammetric analysis of P450c17, wt cyt b5, E48G/E49G cyt b5 and sequential additions.

<p><b>a, c,</b> d.c. voltammograms and <b>b, d,</b> the 6^th harmonic of the a.c. voltammograms for the bare (<i>grey</i>; left axis) and modified CNT electrodes. <b>a</b>, <b>b,</b> Adsorption of hemin (black, right axes) produced a much larger faradaic current and an <math><msubsup><mrow><mi>E</mi></mrow><mrow><mi>a</mi><mi>c</mi></mrow><mrow><mn>0</mn></mrow></msubsup></math> value (dashed lines) that were at least 0.02 V more negative than those derived from adsorption of proteins (left axes): wt cyt b5 (green), E48G/E49G cyt b5 (red) or P450c17 (blue). <b>c,</b> Adsorption of E48G/E49G cyt b5 on P450c17/CNT (blue) produced a substantial increase in the surface concentration of hemin (orange), but negligible changes in <i>Γ</i> were derived from adsorption of wt cyt b5 (magenta) on P450c17/CNT. <b>d,</b> The faradaic current in the a.c. components for P450c17 (blue) was suppressed upon interaction with wt cyt b5 (magenta); the a.c. data for wt cyt b5 (green) is shown as a control. (<b>e</b>) Schematic representation of the modes of interaction of the wt cyt b5 and the E48G/E49G cyt b5 with the P450c17/CNT electrode. All currents were normalized to the geometric electrode surface area (a-c) or to the amount of electroactive heme (d); The electrolyte solution in each case was a deoxygenated aqueous 0.20 M NaCl+0.02 M (K₂HPO₄+KH₂PO₄), pH = 7.0.</p