Indirect Readout in Protein-Peptide Recognition: A Different Story from Classical Biomolecular Recognition

Protein-peptide interactions are prevalent and play essential roles in many living activities. Peptides recognize their protein partners by direct nonbonded interactions and indirect adjustment of conformations. Although processes of protein-peptide recognition have been comprehensively studied in both sequences and structures recently, flexibility of peptides and the configuration entropy penalty in recognition did not get enough attention. In this study, 20 protein-peptide complexes and their corresponding unbound peptides were investigated by molecular dynamics simulations. Energy analysis revealed that configurational entropy penalty introduced by restriction of the degrees of freedom of peptides in indirect readout process of protein-peptide recognition is significant. Configurational entropy penalty has become the main content of the indirect readout energy in protein-peptide recognition instead of deformation energy which is the main source of the indirect readout energy in classical biomolecular recognition phenomena, such as protein–DNA binding. These results provide us a better understanding of protein-peptide recognition and give us some implications in peptide ligand design

Aromatic Heterocyclic Organic Spacer Cation-Assisted Growth of Large-Grain-Size 2DRP Perovskite Film for Enhanced Solar Cell Performance

Organic spacer cations play an important role in the aggregation of two-dimensional Ruddlesden–Popper (2DRP) L′2Ln–1BnX3n+1 perovskite precursors. Therefore, it is necessary to study how mixed A′ spacer cations affect the aggregation behavior of precursors and the carrier transport properties of 2DRP films. Herein, a novel spacer cation 4-pyridinylmethylammonium (PyA) is introduced to prepare a new mixed spacer cation 2DRP (BA1–xPyAx)2MA3Pb4I13 perovskite. The incorporation of PyA suppresses the precursor aggregation and reduces the transformation energy of the sol–gel to the directional three-dimensional phase, leading to the formation of large-grain-size 2DRP perovskite films. The PyA-based 2DRP perovskite exhibits efficient carrier transport owing to fewer defects and suppressed nonradiative recombination. Thus, the champion efficiency of 13.01% is achieved for BA- and PyA-based devices. The unencapsulated PyA-based devices maintain 98% of their initial efficiency after storage under nitrogen atmosphere for 1200 h. This work paves the way for preparing a large-grain-size 2DRP perovskite by suppressing precursor aggregation

Direct Fluorescence Detection of RNA on Microarrays by Surface-Initiated Enzymatic Polymerization

We report the first demonstration of surface-initiated enzymatic polymerization (SIEP) for the direct detection of RNA in a fluorescence microarray format. This new method incorporates multiple fluorophores into an RNA strand using the two-step sequential and complementary reactions catalyzed by yeast poly­(A) polymerase (PaP) to incorporate deoxyadenosine triphosphate (dATP) at the 3′–OH of an RNA molecule, followed by terminal deoxynucleotidyl transferase (TdT) to catalyze the sequential addition of a mixture of natural and fluorescent deoxynucleotides (dNTPs) at the 3′–OH of an RNA–DNA hybrid. We found that the 3′-end of RNA can be efficiently converted into DNA (∼50% conversion) by polymerization of dATP using yeast PaP, and the short DNA strand appended to the end of the RNA by PaP acts as the initiator for the TdT-catalyzed polymerization of longer DNA strands from a mixture of natural and fluorescent dNTPs that contain up to ∼45 Cy3 fluorophores per 1 kb DNA. We obtained an ∼2 pM limit of detection (LOD) and a 3 log-linear dynamic range for hybridization of a short 21 base-long RNA target to an immobilized peptide nucleic acid probe, while fragmented mRNA targets from three different full length mRNA transcripts yielded a ∼10 pM LOD with a similar dynamic range in a microarray format

HTT regulates apical vesicular trafficking of PAR3-aPKC during cystogenesis.

<p>(A) Four-day MDCK 3-D cultures stained for HTT 4C8 and PAR3 or aPKC. Arrowheads indicate localization of HTT and PAR3 or aPKC to vesicular-like structures. Colocalization of HTT and PAR3 or aPKC is displayed in yellow (merge). (B) Illustration showing the HTT/PAR3/PAR6/aPKC complex localization on apical membrane and vesicles. (C) Western blotting of MDCK cell extracts. The histogram corresponds to the quantification of HTT levels. (D) 24 h MDCK 3-D structures stained for ß-catenin and PAR3 or aPKC. HTTFL is tagged with mCherry and staining is displayed in magenta. Arrows indicate the basolateral compartment and dashed ellipses indicate the apical surface. (E) Representative line-scan analysis (relative fluorescence intensity; at least 20 cells were analyzed per condition). (F) Illustration showing the role of HTT in PAR3-aPKC apical vesicular trafficking. (G) Four-day MDCK 3-D structures stained for E-cadherin and PAR3 or aPKC. (H) Four-day MDCK 3-D structures stained for aPKC. PAR3-GFP staining is displayed in magenta and the colocalization of aPKC and PAR3-GFP appears in white. (I) Percentage of acini with normal lumen. (J) Quantification of acini size. (I and J) Control: <i>n</i> = 125 acini, Control + PAR3: <i>n</i> = 102 acini, shHTT1: <i>n</i> = 149 acini, shHTT2: <i>n</i> = 114 acini, shHTT2 + HTT: <i>n</i> = 163 acini, shHTT2 + PAR3: <i>n</i> = 89 acini. All scale bars, 10 μm. Error bars, SEM. *** <i>p</i><0.001. Complete statistical analyses with number of measures are detailed in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002142#pbio.1002142.s001" target="_blank">S1 Data</a>.</p

HTT is required for apical localization of PAR3 and aPKC in vivo.

<p>(A) Mammary gland sections stained for E-cadherin and PAR3 or aPKC. Arrows indicate cytoplasmic accumulation and impaired localization of PAR3 and aPKC to the apical surface; asterisks indicate small lumens. (B) Mammary gland sections stained for keratin 5 (K5) and GM130. (C) Percentage of LCs showing ribbon-like and fragmented localization of GM130 (control: <i>n</i> = 4 mice; mutant: <i>n</i> = 4 mice). (D) Mammary gland sections stained for HTT 4C8 and PAR3 or aPKC. (E) HTT/PAR3/PAR6/aPKC/RAB11A complexes were immunoprecipitated from MCF-10A cells. Mouse IgG (mIgG) was used as a negative control. The immunoprecipitates (IP) were analyzed by western blotting. All scale bars, 10 μm. Error bars, SEM. *** <i>p</i><0.001. Complete statistical analyses with number of measures are detailed in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002142#pbio.1002142.s001" target="_blank">S1 Data</a>.</p

Model for HTT-mediated regulation of apical polarity.

<p>During epithelial morphogenesis, HTT modulates the activation of RAB11A (1). HTT-RAB11A forms a complex with PAR3-aPKC, which may be recruited to HTT-kinesin 1 apical vesicles (2). HTT coordinates apical recycling of PAR3-aPKC vesicles (3). PAR3-aPKC accumulation at the pre-apical patches (PAP) (4) triggers the expansion of the apical membrane, leading to the formation of a central lumen (5).</p

HTT regulates apical vesicular trafficking in a microtubule-dependent manner.

<p>(A–C) FM64-4 4-day MDCK 3-D structures were video-recorded. Maximum intensity and z projections are shown. Magnifications are shown in (D) (left; 120 min). (D) Representative line-scan analysis (relative fluorescence intensity; at least 20 cells were analyzed per condition). (E) Four-day MDCK 3-D structures stained for E-cadherin and PAR3, aPKC, or F-actin. (F) Percentage of acini with normal lumen (control: <i>n</i> = 94 acini, Noco 10 μM 90 min: <i>n</i> = 67 acini, Noco 5 μM 16h: <i>n</i> = 72 acini). (G) Twenty-four–hour and four-day MDCK 3-D structures stained for HTT and kinesin 1. Arrows indicate the basolateral compartment, and dashed ellipses indicate the apical surface. Colocalization of HTT and kinesin 1 is displayed in yellow (merge). (H) Percentage of 3-D structures with vesicular kinesin 1 staining (control: <i>n</i> = 22 24h-acini and <i>n</i> = 26 day 4-acini, shHTT1: <i>n</i> = 25 24h-acini and <i>n</i> = 25 day 4-acini). (I) Representative line-scan analysis (relative fluorescence intensity; at least 20 cells were analyzed per condition). (J) FM64-4 4-day MDCK 3-D structures were video-recorded. Maximum intensity and z projections are shown. (K) Left: percentage of acini with normal lumen (si-Control: <i>n</i> = 30 acini, si-kinesin 1: <i>n</i> = 28 acini), right: western blotting of MDCK cell extracts. (L) Illustration showing HTT and kinesin 1 during microtubule-dependent apical vesicular trafficking. All scale bars, 10 μm. Error bars, SEM. *** <i>p</i><0.001. Complete statistical analyses with number of measures are detailed in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002142#pbio.1002142.s001" target="_blank">S1 Data</a>.</p

MMTV-driven loss of HTT affects ductal morphogenesis.

<p>(A) Quantitative real-time RT-PCR analysis of <i>Cre</i> and <i>Htt</i> gene in basal and luminal mammary epithelial cells from 16-wk-old virgin mice. Data are presented as means obtained in three independent experiments (control: five mice per experiment, mutant: five mice per experiment). (B) Carmine-stained whole mounts of mammary glands and hematoxylin and eosin (H&E) staining. (C) Degree of ductal invasion of the fat pad in virgin mammary glands. (D) Number of branches in virgin mammary glands. (E) Number of terminal end buds (TEBs) in virgin mammary glands. (F) H&E staining of mammary gland sections. (G) Mammary gland sections stained for E-cadherin and cleaved caspase 3. (H) Percentage of cleaved caspase 3-positive cells and number of intraluminal cells per duct. (I) Mammary gland sections stained for KI67. (J) Percentage of KI67-positive cells. Number of mice analyzed are the same in C-E, H, J: control: <i>n</i> = 5 mice; mutant: <i>n</i> = 7 mice. All scale bars, 10 μm. Error bars, standard error of the mean (SEM). *<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001. Complete statistical analyses with number of measures are detailed in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002142#pbio.1002142.s001" target="_blank">S1 Data</a>.</p

HTT colocalizes with RAB11A and regulates RAB11A activity during apical vesicular trafficking.

<p>(A) Mammary gland section stained for HTT 4C8 and RAB11A. (B) Mammary gland section stained for E-cadherin and RAB11A. (C) Twenty-four–hour and four-day MDCK 3-D cultures stained for HTT 4C8 and RAB11A. Colocalization of HTT and RAB11A is displayed in yellow (merge). (D) Twenty-four–hour and four-day MDCK 3-D cultures stained for RAB11A. HTTFL is tagged with mCherry and fluorescence is displayed in magenta and the colocalization of RAB11A and HTTFL appears in white. (E) Twenty-four–hour and four-day MDCK 3-D cultures transfected with RAB11A^WT, RAB11A^Q70L or RAB11A^S22N, stained for PAR3. RAB11 is tagged with GFP and fluorescence is displayed in magenta. The colocalization of aPKC and RAB11A appears in white. (F) Representative line-scan analysis (relative fluorescence intensity; at least 20 cells were analyzed per condition). (G) Percentage of acini with normal lumen. (H) Quantification of acini size. (G and H) Control+RAB11A^WT: <i>n</i> = 59 acini, Control+RAB11A^Q70L: <i>n</i> = 54 acini, Control+RAB11A^S22N: <i>n</i> = 66 acini, shHTT2+RAB11A^WT: <i>n</i> = 60 acini, shHTT2+RAB11A^Q70L: <i>n</i> = 72 acini, shHTT2+RAB11A^S22N: <i>n</i> = 93 acini. (I) FM64-4 4-day MDCK 3-D structures were video recorded. Maximum intensity and z projections are shown. All scale bars, 10 μm. Error bars, SEM. ** <i>p</i><0.01; *** <i>p</i><0.001. Complete statistical analyses with number of measures are detailed in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002142#pbio.1002142.s001" target="_blank">S1 Data</a>.</p

Multi-level Reactive Oxygen Species Amplifier to Enhance Photo-/Chemo-Dynamic/Ca²⁺ Overload Synergistic Therapy

Reactive oxygen species (ROS)-involved photodynamic therapy (PDT) and chemodynamic therapy (CDT) hold great promise for tumor treatment. However, hypoxia, insufficient H2O2, and overexpressed glutathione (GSH) in the tumor microenvironment (TME) hinder ROS generation significantly. Herein, we reported CaO2@Cu-TCPP/CUR with O2/H2O2/Ca2+ self-supply and GSH depletion for enhanced PDT/CDT and Ca2+ overload synergistic therapy. CaO2 nanospheres were first prepared and used as templates for guiding the coordination between the carboxyl of tetra-(4-carboxyphenyl)porphine (TCPP) and Cu2+ ions as hollow CaO2@Cu-TCPP, which facilitated GSH-activated TCPP-based PDT and Cu+-mediated CDT. The hollow structure was then loaded with curcumin (CUR) to form CaO2@Cu-TCPP/CUR composites. Cu-TCPP prevented degradation of CaO2, while Cu2+ ions reacted with GSH to deplete GSH, produce Cu+ ions, and release TCPP, CaO2, and CUR. CaO2 reacted with H2O to generate O2, H2O2, and Ca2+ to achieve O2/H2O2/Ca2+ self-supply for TCPP-based PDT, Cu+-mediated CDT, and CUR-enhanced Ca2+ overload therapy. Thus, this multilevel ROS amplifier enhances synergistic therapy with fewer side effects and drug resistance