Time-Resolved Assembly of Chiral Uranyl Peroxo Cage Clusters Containing Belts of Polyhedra

Two chiral cage clusters built from uranyl polyhedra and (HPO₃)^2– groups have been synthesized in pure yield and characterized structurally and spectroscopically in the solid state and aqueous solution. Synthesis reactions under ambient conditions in mildly acidic aqueous solutions gave clusters <i>U</i>₂₂<i>PO</i>₃ and <i>U</i>₂₈<i>PO</i>₃ that contain belts of four uranyl peroxide pentagonal and hexagonal bipyramids, in contrast to earlier reported uranyl peroxide cage clusters that are built from four-, five-, and six-membered rings of uranyl hexagonal bipyramids. <i>U</i>₂₂<i>PO</i>₃ and <i>U</i>₂₈<i>PO</i>₃ are also the first chiral uranyl-based cage clusters, the first that contain uranyl pentagonal bipyramids that contain no peroxide ligands, and the first that incorporate (HPO₃)^2– bridges between uranyl ions. They are built from 22 uranyl polyhedra and 20 (HPO₃)^2– groups, or 28 uranyl polyhedra and 24 (HPO₃)^2– groups, with the outer and inner surfaces of the cages passivated by the O atoms of uranyl ions. Small-angle X-ray scattering (SAXS) profiles demonstrated that <i>U</i>₂₂<i>PO</i>₃ clusters formed in solution within 1 h after mixing of reactants, and remained in solution for 2 weeks prior to crystallization. Time-resolved electrospray ionization mass spectrometry and SAXS demonstrated that <i>U</i>₂₈<i>PO</i>₃ clusters formed in solution within 1 h of mixing the reactants, and remained in solution 1 month before crystallization. Crystallization of <i>U</i>₂₂<i>PO</i>₃ and <i>U</i>₂₈<i>PO</i>₃ is accelerated by addition of KNO₃. Clusters of <i>U</i>₂₂<i>PO</i>₃ with and without encapsulated cations exhibit markedly different aqueous solubility, reflecting the importance of cluster surface charge in fostering linkages through counterions to form a stable solid

Time-Resolved Assembly of Chiral Uranyl Peroxo Cage Clusters Containing Belts of Polyhedra

Two chiral cage clusters built from uranyl polyhedra and (HPO₃)^2– groups have been synthesized in pure yield and characterized structurally and spectroscopically in the solid state and aqueous solution. Synthesis reactions under ambient conditions in mildly acidic aqueous solutions gave clusters <i>U</i>₂₂<i>PO</i>₃ and <i>U</i>₂₈<i>PO</i>₃ that contain belts of four uranyl peroxide pentagonal and hexagonal bipyramids, in contrast to earlier reported uranyl peroxide cage clusters that are built from four-, five-, and six-membered rings of uranyl hexagonal bipyramids. <i>U</i>₂₂<i>PO</i>₃ and <i>U</i>₂₈<i>PO</i>₃ are also the first chiral uranyl-based cage clusters, the first that contain uranyl pentagonal bipyramids that contain no peroxide ligands, and the first that incorporate (HPO₃)^2– bridges between uranyl ions. They are built from 22 uranyl polyhedra and 20 (HPO₃)^2– groups, or 28 uranyl polyhedra and 24 (HPO₃)^2– groups, with the outer and inner surfaces of the cages passivated by the O atoms of uranyl ions. Small-angle X-ray scattering (SAXS) profiles demonstrated that <i>U</i>₂₂<i>PO</i>₃ clusters formed in solution within 1 h after mixing of reactants, and remained in solution for 2 weeks prior to crystallization. Time-resolved electrospray ionization mass spectrometry and SAXS demonstrated that <i>U</i>₂₈<i>PO</i>₃ clusters formed in solution within 1 h of mixing the reactants, and remained in solution 1 month before crystallization. Crystallization of <i>U</i>₂₂<i>PO</i>₃ and <i>U</i>₂₈<i>PO</i>₃ is accelerated by addition of KNO₃. Clusters of <i>U</i>₂₂<i>PO</i>₃ with and without encapsulated cations exhibit markedly different aqueous solubility, reflecting the importance of cluster surface charge in fostering linkages through counterions to form a stable solid

AROS and DBC1 overexpression impacts the transcription of <i>hsp70</i> and initiation of the HSR.

<p>(A) <i>aros</i> and <i>dbc1</i> mRNA expression levels do not undergo a significant change upon 0–3 hours of HS, while small changes in levels are observed between 3–6 hours of HS. HEK293 cells were exposed to a 42°C HS from 0 to 6 hours and mRNA levels were determined by qRT-PCR. (B) AROS overexpression enhances HS induction of <i>hsp70</i> mRNA, while (C) DBC1 overexpression inhibits HS induction of <i>hsp70</i> mRNA. For B and C, HEK293 cells were transfected with empty vector (EV), AROS, or DBC1 and then exposed to a 42°C HS from 0 to 6 hours. The mRNA levels were determined for <i>hsp70</i> by qRT-PCR. Results in A-C are in technical triplicates and are representative of biological duplicates. Statistical significance was measured by Student’s t test as compared to 0 hr HS (*<i>P<0.05;</i> **<i>P<0.01;</i> ***<i>P<0.001).</i></p

AROS and DBC1 impact HSF1 acetylation, DNA recruitment, and activity at the <i>hsp70</i> promoter.

<p>(A) AROS and DBC1 overexpression impact HSF1 acetylation. HEK293 cells were transfected with p300 and FLAG-HSF1 as well as empty vector, SIRT1-MYC, DBC1-HA, or AROS as indicated and exposed to 0 or 2 hours of HS at 42°C. A FLAG immunoprecipitation was run on an SDS-PAGE gel and the acetylation status of HSF1 was determined using an acetyl-lysine antibody. Results are representative of biological triplicates. (B) The expression of transfected SIRT1-MYC, DBC1-HA, and AROS was verified by Western analysis. (C) Overexpression of HSF1, DBC1, and AROS do not significantly impact cell viability compared to the empty vector (EV) control as measured by PrestoBlue. The cell viability assay was performed in biological and technical triplicates and statistical significance was measured by Student’s t test compared to EV. (D) AROS and DBC1 overexpression impact HSF1 recruitment to the <i>hsp70</i> promoter. AROS, DBC1, or empty vector (EV) was overexpressed in HEK293 cells prior to HS for 0, 2, or 6 hours and chromatin immunoprecipitation (ChIP) was performed with an HSF1 antibody. The purified DNA was then analyzed by qPCR and DNA levels were determined for the <i>hsp70</i> promoter. The qPCR results are in technical triplicates and statistical significance was measured by Student’s t test as compared to EV at 0 hr HS (*<i>P<0.0;</i> ** <i>P<0.01).</i> ChIP was performed in biological duplicates. (E) AROS and DBC1 knockdown impact <i>hsp70.1</i> promoter-luciferase reporter activity. HeLa <i>hsp70.1</i> promoter-luciferase reporter cells were transfected with 50 nM of Dharmacon SmartPool DBC1, AROS, HSF1, or non-targeting (NT) control siRNA and HS was induced with celastrol (5 µM). Luciferase activity was measured and compared to that of the NT control. Luciferase assays were performed in biological triplicate. Statistical significance was measured by Student’s t test as compared to the NT siRNA control with celastrol (5 µM) treatment (*<i>P<0.05).</i></p

List of PCR primers used in this study.

*<p>F: forward, R: reverse.</p>1<p>Primers from Folz <i>et al. Am. J. Respir. Cell Mol. Biol.</i> 2008 <b>39</b>:2, 243.</p>2<p>Primers from Yao <i>et al. Journal of Biomedical Science</i> 2010 <b>17</b>:30.</p>3<p>Primers from Westerheide <i>et al. Science</i> 2009 <b>323,</b> 1063.</p>4<p>Primers from McLean <i>et al. Biochemical and Biophysical Research Communications</i> 2006 <b>351</b>:3.</p

SIRT1 is not regulated by a change in expression upon HS.

<p>(A–C) <i>hsp70</i>, <i>hsp27,</i> and <i>hsp90</i> mRNA expression levels are induced during a HS timecourse. HeLa cells were exposed to a 42°C HS from 0 to 6 hours and mRNA levels were determined by qRT-PCR. (D) <i>sirt1</i> mRNA expression levels are not altered in the early stages of HS. HeLa cells were exposed to a 42°C HS from 0 to 6 hours and mRNA levels were determined qRT-PCR. Results for A–D are in technical triplicates and are representative of biological duplicates. Statistical significance was measured by Student’s t test as compared to 0 hr HS (*<i>P<0.05;</i> ** <i>P<0.01).</i> (E) SIRT1 protein levels are not altered during a HS timecourse. HeLa cells were treated with a HS timecourse and SIRT1, HSP70, HSP27, and β-Tubulin protein levels were determined by Western analysis. (F) Protein levels from E were quantified and plotted using ImageJ.</p

HS results in an increase in SIRT1 recruitment that is specific to the HSE within the <i>hsp70</i> promoter.

<p>(A) Schematic representation of primers designed to amplify the HSE site and a nonspecific (NS) site at the <i>hsp70</i> promoter. (B) SIRT1 is recruited to the <i>hsp70</i> promoter HSE site during HS. HEK293 cells were exposed to a 42°C HS from 0 to 6 hours prior to chromatin immunoprecipitation (ChIP) with a SIRT1 antibody. SIRT1 is not recruited upon HS to a non-specific (NS) upstream <i>hsp70</i> promoter site (C) or to the <i>gapdh</i> promoter (D). Results in B-D are in technical triplicates and are representative of biological duplicates. Statistical significance was measured by Student’s t test as compared to - HS (*<i>P<0.05;</i> ** <i>P<0.01;</i> ***<i>P<0.001).</i></p

Cation Templating and Electronic Structure Effects in Uranyl Cage Clusters Probed by the Isolation of Peroxide-Bridged Uranyl Dimers

The self-assembly of uranyl peroxide polyhedra into a rich family of nanoscale cage clusters is thought to be favored by cation templating effects and the pliability of the intrinsically bent U–O₂–U dihedral angle. Herein, the importance of ligand and cationic effects on the U–O₂–U dihedral angle were explored by studying a family of peroxide-bridged dimers of uranyl polyhedra. Four chemically distinct peroxide-bridged uranyl dimers were isolated that contain combinations of pyridine-2,6-dicarboxylate, picolinate, acetate, and oxalate as coordinating ligands. These dimers were synthesized with a variety of counterions, resulting in the crystallographic characterization of 15 different uranyl dimer compounds containing 17 symmetrically distinct dimers. Eleven of the dimers have U–O₂–U dihedral angles in the expected range from 134.0 to 156.3°; however, six have 180° U–O₂–U dihedral angles, the first time this has been observed for peroxide-bridged uranyl dimers. The influence of crystal packing, countercation linkages, and π–π stacking impact the dihedral angle. Density functional theory calculations indicate that the ligand does not alter the electronic structure of these systems and that the U–O₂–U bridge is highly pliable. Less than 3 kcal·mol^–1 is required to bend the U–O₂–U bridge from its minimum energy configuration to a dihedral angle of 180°. These results suggest that the energetic advantage of bending the U–O₂–U dihedral angle of a peroxide-bridged uranyl dimer is at most a modest factor in favor of cage cluster formation. The role of counterions in stabilizing the formation of rings of uranyl ions, and ultimately their assembly into clusters, is at least as important as the energetic advantage of a bent U–O₂–U interaction

Summary of hit compounds that were characterized in this study.

<p>Structures were generated by CambridgeSoft ChemDraw Ultra 7.0 (Cambridge, MA). NI = No Inhibition. NA = Not Applicable.</p

Dose-dependent inhibition of fluorescent reporters.

<p><i>Pgst-4::GFP</i> and <i>Pdop-3::RFP</i> fluorescence was measured after 21 h exposure to 38 µM juglone (A) or 2.8 mM acrylamide (B) in 384 well plates (<i>n = </i>32 wells). (C) <i>Phsp-16.2::GFP</i> and <i>Pdop-3::RFP-</i>expressing worms were heat-shocked at 35°C for 1 h followed by 5 h recovery at 20°C (<i>n = </i>32 wells). Values are means ± SEM.</p