Academic Senate - Meeting Minutes, 4/18/2017

<p>All values are presented with SD. Differences between <i>LDLR−/−</i> and the other two genotypes are significant where indicated, ANOVA: *p<0.05, **p<0.01.</p

ENC1-mediated down-regulation of Nrf2 was independent of proteasomal or lysosomal degradation.

<p>A. MDA-MB-231 cells were transfected with an empty vector or a vector for ENC1-Myc. Transfected cells were treated with proteasome inhibitors MG132 (M) (10 µM), clasto-lactacystin β-lactone (Lac) (10 µM) or epoxomicin (Ep) (1 µM), or lysosome inhibitors chloroquine (Ch) (50 µM) or ammonium chloride (Ac) (50 mM) for 4 hr before cells were lysed at 48 hr post-transfection. Cell lysates were subjected to immunoblot analysis with anti-HA, anti-Myc and anti-β-actin antibodies. B. MDA-MB-231 cells were transfected with HA-Nrf2 and with an empty vector or ENC1-Myc. Transfected cells were treated with proteasome and lysosome inhibitors as described above. Immunoblot analysis was performed. C. <i>In vivo</i> ubiquitination assay was performed in MDA-MB-231 cells transfected with plasmids for HA-Ub and Gal4-Neh2, along with either ENC1-Myc or Keap1-CBD. Transfected cells were treated with 10 µM MG132 for 4 hr prior to cell lysis. Cell lysates were denatured by heating and subjected to immunoprecipitation with anti-Gal4 antibodies. The precipitated protein complexes were subjected to immunoblot analysis with anti-HA antibodies for detecting ubiquitin-conjugated Gal4-Neh2 (top panel). Small aliquots of total cell lysates were immunoblotted with the indicated antibodies (bottom three panels).</p

ENC1 down-regulated Nrf2 at the translational level.

<p>A. Pulse-chase assay was conducted with MDA-MB-231 cells transfected with either an empty vector or an expression vector for ENC1-Myc. Cells were incubated for 30 minutes with medium containing [³⁵S]-methionine and [³⁵S]-cysteine to label proteins. Cells were then washed and incubated in normal complete medium for the indicated time periods prior to cell lysis. Cell lysates were subjected to immunoprecipitation with anti-Nrf2 antibodies and immunoprecipitates were resolved in SDS-PAGE gel and detected by autoradiography. B. Nrf2 band intensities were quantified using Quantity One (BIO-RAD) and the half-life was plotted and calculated. C. Pulse-chase assay was conducted in the same way, except that cells were treated with 100 µM tBHQ for 4 hr. D. Quantified data of Fig. 5C.</p

Keap1 was not essential in ENC1-mediated down-regulation of Nrf2.

<p>A. MDA-MB-231 cells were transfected with an expression vector for ENC1-Myc, along with an empty vector or an expression vector for Keap1-CBD. Cell lysates were subjected to chitin pull-down assay. Precipitated proteins were subjected to immunoblot analysis with anti-Myc and anti-CBD antibodies for detection of Keap1-CBD and ENC1-Myc (top panel). Small aliquots of total lysates were analyzed by immunoblot with the indicated antibodies (bottom panel). B. Cell lysates from MDA-MB-231 cells were immunoprecipitated with anti-Keap1 antibody. The precipitated protein complexes were subjected to immunoblot analysis with anti-Nrf2 and anti-ENC1 antibodies. IgG was included in the immunoprecipitation analysis as a negative control. C. Plasmids for full-length ENC1 (E-FL) and two ENC1 deletion mutants E320 and E570, Nrf2 and Luciferase (Luc) were used for <i>in vitro</i> transcription/translation to synthesize [³⁵S]-labeled proteins. The proteins were incubated with Keap1-His purified from <i>E. Coli</i>. Protein complexes containing Keap1 were pulled-down with nickel beads and resolved in SDS-PAGE and detected by autoradiography. Nrf2 and Luciferase were used as a positive and a negative control. 20% of [³⁵S]-labeled proteins were resolved by SDS-PAGE gel to show equivalent input of each protein (right panel). D. Keap1-/- and wild-type MEF cells were transfected with plasmids for HA-Nrf2 and ENC1-Myc. Cells were lysed at 48 hr post-transfection. Cell lysates were analyzed by immunoblot with anti-HA, anti-Myc and anti-β-actin. E. Cell lysates from MDA-MB-231 cells transfected with plasmids for Keap1-CBD, HA-Nrf2, and ENC1-Myc were used for pull-down assay with chitin beads Precipitated proteins were subjected to immunoblot analysis with anti-HA and anti-CBD antibodies (left figure, top two panels). Small aliquots of total lysates were analyzed by immunoblot with the indicated antibodies (left figure, bottom four panels). Nrf2 and β-Actin band intensities were quantified using Quantity One (BIO-RAD). The intensity of Nrf2 was normalized to that of β-actin (right graph).</p

Light-Controllable Cucurbit[7]uril-Based Molecular Shuttle

The design and construction of novel artificial molecular machines can be categorized as a currently important field of modern chemistry. In the present work, a novel photoresponsive [3]­rotaxane containing two cucurbit[7]­uril (CB[7]) rings and a dumbbell component consisting of one <i>trans</i>-azobenzene unit along with two viologen units was developed. Each viologen group was encircled by a CB[7] ring with a rapid shuttling equilibration distribution extended to the <i>trans</i>-azobenzene unit located in the middle of the dumbbell component. Upon the <i>trans</i>-to-<i>cis</i> photoisomerization of the azobenzene unit under UV light irradiation, a shuttling restriction of the CB[7] rings along the dumbbell component was observed. The equilibration distribution of the macrocycles on the dumbbell component can be recovered by the <i>cis</i>-to-<i>trans</i> photoisomerization of the azobenzene unit under visible light irradiation. Such a controllable shuttling process was fully characterized by ¹H NMR spectroscopy and was easily indicated by fluorescent changes of the [3]­rotaxane

Electron density is shown around Cys136 in <i>sm</i>PncA (A,C,E,G) and Cys264 in Casp6 (B,D,F,H).

<p>A, B: SAD phases from <i>phaser</i> prior to any refinement steps. C, D: SAD phases form <i>resolve</i> after DM and NCS averaging. E, F: 2Fo-Fc map with the final phase from <i>phenix.refine</i> and G, H: Fo-Fc omit density map. This figure was generated by <i>PyMol</i>. Atoms are shown as stick and colored as follows: carbon, green; oxygen, red; nitrogen, blue; zinc, grey; arsenic, purple. The electron density is contoured at 1.5 σ, except that in the omit map (G, H), the positive density (Green) was contoured at 4 σ.</p

A putative three-stage cysteine modification mechanism for <i>Sm</i>PncA.

<p>In stage 1, proton abstraction by Asp9 would increase the nucleophilicities of Cys136 and facilitate nucleophilic attack at cacodylic acid (CAD); Proton donation from Asp9 to the hydroxyl group of the CAD would promote As-O bond cleavage and release of H₂O as the intermediate in stage 2 collapses to give a dimethylarsinoyl fixed Cys136 in stage 3.</p

Anomalous difference Fourier map calculated with final phases at 5 σ.

<p>A. Atoms are shown as sticks model, and colored as follows: carbon, green; oxygen, red; nitrogen, blue; zinc, grey; arsenic, purple. B. The Casp6 structure is shown as a backbone representation. The cysteines modified by cacodylic acid are presented in the model.</p

Substructure solution of anomalous scatterers from <i>Phaser</i>, with each peak annotated with the corresponding atom.

<p>Substructure solution of anomalous scatterers from <i>Phaser</i>, with each peak annotated with the corresponding atom.</p

Anomalous differences between arsenic and zinc.

<p>A. The anomalous signal reduction as a function of resolution. The peak heights of the heavy atoms were derived from the anomalous Fourier map, which was calculated using final phases. B. SAD phase errors (after density modification) against the final model as a function of resolution cutoff. Different colors represent the phases calculated from different anomalous scatterers: phases calculated from zinc atoms are shown in silver, those from arsenic atoms are shown in purple and from both are shown in orange.</p