Media 2: Field-matter integral overlap to estimate the sensitivity of surface plasmon resonance biosensors

Originally published in JOSA A on 01 July 2012 (josaa-29-7-1367

Media 1: Field-matter integral overlap to estimate the sensitivity of surface plasmon resonance biosensors

Originally published in JOSA A on 01 July 2012 (josaa-29-7-1367

Inverse correlation between RANTES mRNA and miR-UL148D expression at a later stage during Toledo infection.

<p>(A) HFF cells were infected with WT-AD169 (triangles) or WT-Toledo (circles). RANTES mRNA at indicated time of post-infection (hpi) was quantified by quantitative RT-PCR and data were normalized to GAPDH mRNA (RANTES/GAPDH). Values represent the average ± S.D. of triplicate experiments. (B) The predicted duplex of the 3′UTR of RANTES and miR-UL148D-WT. The indicated free energy value was calculated using RNA22 (<a href="http://cbcsrv.watson.ibm.com/rna22.html" target="_blank">http://cbcsrv.watson.ibm.com/rna22.html</a>). (C) Alignment of miR-UL148D stem-loop sequence of various HCMV clinical strains. We analyzed the genomic sequences of several HCMV clinical strains from GenBank (Toledo GU937742.1, TB40/E AY446866.1, Merlin AY446894.2, JHC HQ380895.1, VR1814 GU179289.1). The conserved sequence (gray) and non-conserved sequence (blue) is shown. Mature miR-UL148D sequence is highlighted in red. (D) The detection of miR-UL148D in Toledo-infected HFF cells was assessed using the RNase protection assay as described under “<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002577#s4" target="_blank">Materials and Methods</a>.” 5S rRNA bands stained with ethidium bromide are presented as a loading control. Using the same RNA samples, the RANTES mRNA and GAPDH mRNA were measured by RT-PCR (lower panel). NI indicates non-infected control.</p

miR-UL148D inhibits RANTES expression during viral infection.

<p>(A) Genomic location of UL150 and miR-UL148D (upper panel). The predicted mature sequence of miR-UL148D (blue) and its mutated residues at wobble position are shown (red) (bottom panel). (B) HFF cells were infected with Toledo-WT, ToledoΔmiR-UL148D or Toledo-Revertant. After extracting miRNAs from the infected cells, the detection of miR-UL148D was assessed using the RNase protection assay as described under “<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002577#s4" target="_blank">Materials and Methods</a>.” 5S rRNA was presented as a loading control stained by ethidium bromide. IE1 and gB gene expression was analyzed by RT-PCR. (C) Growth curves of Toledo-WT, Toledo-ΔmiR-UL148D and Toledo-Revertant. HFF cells were infected with wild-type, mutant and revertant viruses at an MOI of 2. The total number of cell-free viruses in the supernatants of infected cultures was determined by limiting dilution analyses. (D, E) After HFF cells were infected with Toledo-WT (white bars), ToledoΔmiR-UL148D (light gray bars) and Toledo-Revertant (dark gray bars), culture supernatants were harvested at the indicated post-infection time. The accumulated RANTES in supernatants was quantified by ELISA (D). RANTES mRNA level was detected by qRT-PCR (E). NI indicates non-infected control. Similar data were obtained in three independent experiments and the bars indicate mean ±S.D.</p

PNA-based antisense oligonucleotides specific to miR-UL148D revert Toledo-induced inhibition of RANTES production.

<p>2 days before HCMV infection, PNA-control or PNA-anti-miR-UL148D was transfected to HFF. After 48 h of infection, culture media and total RNA were analyzed by ELISA (A) and qRT-PCR (B). Down-regulation of miR-UL148D in the presence of PNA was detected by RNase protection assay (C). NI indicates non-infected control. Similar data were obtained in three independent experiments and the bars indicate mean ±S.D.</p

RANTES-3′UTR is a target of miR-UL148D.

<p>(A) 293T cells were co-transfected with the Renilla luciferase expression vector and firefly luciferase vector expressing normal RANTES-3′UTR (RANTES-3′UTR-WT) and either miR-UL148D, miR-UL148D-mut, or siGFP as a negative control (mock, white bar). The relative luciferase activity was calculated as a ratio of firefly to Renilla luciferase activity. (B) Mutations in the 3′-UTR of RANTES and miR-UL148D. (C) After transfection with RANTES-3′UTR-WT (white bars) or RANTES-3′UTR-mut (gray bars), cells were re-transfected with miR-UL148D-WT or miR-UL148D-mut. The cells were lysed, and firefly luciferase activity was measured and normalized to Renilla luciferase. Data represent the mean ± S.E. of four independent experiments (*statistically significant difference between cells expressing miR-control and those expressing miR-UL148D (P<0.05 by Student's t-test, NS; non-specific).</p

A Protoberberine Derivative HWY336 Selectively Inhibits MKK4 and MKK7 in Mammalian Cells: The Importance of Activation Loop on Selectivity

<div><p>A protoberberine derivative library was used to search for selective inhibitors against kinases of the mitogen-activated protein kinase (MAPK) cascades in mammalian cells. Among kinases in mammalian MAPK pathways, we identified a compound (HWY336) that selectively inhibits kinase activity of mitogen-activated protein kinase kinase 4 and 7 (MKK4 and MKK7). The IC₅₀ of HWY336 was 6 µM for MKK4 and 10 µM for MKK7 <i>in vitro</i>. HWY336 bound to both kinases reversibly via noncovalent interactions, and inhibited their activity by interfering with access of a protein substrate to its binding site. The binding affinity of HWY336 to MKK4 was measured by surface plasmon resonance to determine a dissociation constant (<i>K_d</i>) of 3.2 µM. When mammalian cells were treated with HWY336, MKK4 and MKK7 were selectively inhibited, resulting in inhibition of c-Jun NH₂-terminal protein kinases <i>in vivo</i>. The structural model of HWY336 bound to either MKK4 or MKK7 predicted that HWY336 was docked to the activation loop, which is adjacent to the substrate binding site. This model suggested the importance of the activation loop of MKKs in HWY336 selectivity. We verified this model by mutating three critical residues within this loop of MKK4 to the corresponding residues in MKK3. The mutant MKK4 displayed similar kinase activity as wild-type kinase, but its activity was not inhibited by HWY336 compared to wild-type MKK4. We propose that the specific association of HWY336 to the activation loop of MKK4/MKK7 is responsible for its selective inhibition.</p></div

MKK4 and MKK7 homology in mammalian MAPK pathways.

<p>A) Phylogenetic tree for human MKK4, MKK7, MKK3, MKK6, MEK1, p38, JNK, and ERK. B) Amino acid sequence alignment starting from the hinge region to the activation loop are shown for MKK3 (UniProtKB accession code: P46734), MKK6 (UniProtKB accession code: P52564), MKK4 (UniProtKB accession code: P45985), and MKK7 (UniProtKB accession code: O14733). Green denotes a highly conserved region among these MKKs. Residues highlighted in yellow designate sequence variations in the activation loop. C, D) Three-dimensional structure of MKK4 suggests that HWY336 interacts with the activation loop through hydrogen bonding. C) (top) Amino acid sequence variations within the activation loop of MKKs. (bottom) Proposed docked pose of ATP in MKK4. The arrow designates different amino acids that may determine MKK selectivity. The MKK4 structure is shown in the background with the activation loop (white) containing the varying amino acids (Arg²⁶²) at the respective positions (generated with the Pymol program; <a href="http://www.pymol.org" target="_blank">www.pymol.org</a>). D) Hydrophobic interactions between HWY336 and the MKK4 active site are shown. Hydrophobic residues within the active site are designated by the cap-stick model and HWY336 is displayed using transparent hydrophobic surfaces. The hydrophobicity index is displayed on the left, where brown and blue denote highly hydrophobic and hydrophilic areas, respectively. Pro²⁶⁸, Phe³⁰⁵, Pro³⁰⁸, and Val³¹³ interact with HWY336 side chains. HWY336 interacts with the activation loop of MKK4 through hydrogen bonding via the hydroxyl group of Thr²⁶¹.</p

HWY336 does not inhibit the activity of MKK4 and MKK7 mutants of the proposed docking residues.

<p>Flag-tagged MKK4, MKK4-Q253Y I258V R262M, HA-tagged MKK7, and MKK7-R283Y K288V R292M were immunoprecipitated from activated HEK293 cells as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091037#s2" target="_blank">Materials and Methods</a>. Usage of the same amount of MKK4, MKK4 mutant, MKK7, and MKK7 mutant was confirmed by western blots with anti-flag antibody and anti-HA antibody, respectively. A, B) Kinase activity of immunoprecipitated (A) MKK4 and the MKK4 mutant, (B) MKK7 and the MKK7 mutant was assayed by measuring γ-P³² phosphorylation of JNK as a substrate. C–F) Effect of HWY336 on the kinase activity of (C, D) wild-type (top) and mutant (bottom) MKK4, (E, F) wild-type (top) and mutant (bottom) MKK7 was assayed after treatment with increasing concentrations of HWY336 (10, 30, 50 µM) or DMSO. D, F) Kinase activity of wild-type and mutant MKK4 and MKK7 with increasing concentrations of HWY336 shown in (C, E), was quantified by NIH ImageJ software.</p

SPR detects the interaction of HWY336 with MKK4.

<p>The concentration of HWY336 was varied from 100-MKK4 complex with time was measured over the course of the interaction between HWY336 and MKK4.</p