Modulation of p53 N-terminal transactivation domain 2 conformation ensemble and kinetics by phosphorylation

Phosphorylation of protein is critical for various cell processes, which preferentially happens in intrinsically disordered proteins (IDPs). How phosphorylation modulates structural ensemble of disordered peptide remains largely unexplored. Here, using replica exchange molecular dynamics (REMD) and Markov state model (MSM), the conformational distribution and kinetics of p53 N-terminal transactivation domain (TAD) 2 as well as its dual-site phosphorylated form (pSer46, pThr55) were simulated. It reveals that the dual phosphorylation does not change overall size and secondary structure element fraction, while a change in the distribution of hydrogen bonds induces slightly more pre-existing bound helical conformations. MSM analysis indicates that the dual phosphorylation accelerates conformation exchange between disordered and order-like states in target-binding region. It suggests that p53 TAD2 after phosphorylation would be more apt to bind to both the human p62 pleckstrin homology (PH) domain and the yeast tfb1 PH domain through different binding mechanism, where experimentally it exhibits an extended and α-helix conformation, respectively, with increased binding strength in both complexes. Our study implies except binding interface, both conformation ensemble and kinetics should be considered for the effects of phosphorylation on IDPs. AbbreviationsIDPsintrinsically disordered proteinsREMDreplica exchange molecular dynamicsMSMMarkov state modelTADtransactivation domainPHpleckstrin homologyPRRproline-rich regionDBDDNA-binding domainTETTetramerization domainREGregulatory domainMDmolecular dynamicsPMEparticle-mesh EwaldTICAtime-lagged independent component analysisCKChapman–KolmogorovGMRQgeneralized matrix Rayleigh quotientSARWself-avoiding random walkKIDkinase-inducible domainMFPTmean first passage timeDSSPdefinition of secondary structure of proteinsRMSDroot mean square deviationRgradius of gyrationReeend to end distance intrinsically disordered proteins replica exchange molecular dynamics Markov state model transactivation domain pleckstrin homology proline-rich region DNA-binding domain Tetramerization domain regulatory domain molecular dynamics particle-mesh Ewald time-lagged independent component analysis Chapman–Kolmogorov generalized matrix Rayleigh quotient self-avoiding random walk kinase-inducible domain mean first passage time definition of secondary structure of proteins root mean square deviation radius of gyration end to end distance Communicated by Ramaswamy H. Sarma</p

Conjugated Polymers/DNA Hybrid Materials for Protein Inactivation

Chromophore-assisted light inactivation (CALI) is a powerful tool for analyzing protein functions due to the high degree of spatial and temporal resolution. In this work, we demonstrate a CALI approach based on conjugated polymers (CPs)/DNA hybrid material for protein inactivation. The target protein is conjugated with single-stranded DNA in advance. Single-stranded DNA can form CPs/DNA hybrid material with cationic CPs via electrostatic and hydrophobic interactions. Through the formation of CPs/DNA hybrid material, the target protein that is conjugated with DNA is brought into close proximity to CPs. Under irradiation, CPs harvest light and generate reactive oxygen species (ROS), resulting in the inactivation of the adjacent target protein. This approach can efficiently inactivate any target protein which is conjugated with DNA and has good specificity and universality, providing a new strategy for studies of protein function and adjustment of protein activity

Visual Detection of Multiplex MicroRNAs Using Cationic Conjugated Polymer Materials

A simple, visual, and specific method for simultaneous detection of multiplex microRNAs (miRNAs) has been developed by integrating duplex-specific nuclease (DSN)-induced amplification with cationic conjugated polymer (CCP) materials. The probe DNA with a complementary sequence to target miRNA is labeled with fluorescein dye (FAM). Without target miRNA, the single-strand DNA probe cannot be digested by DSN. Upon adding CCPs, efficient fluorescence resonance energy transfer (FRET) from CCP to FAM occurs owing to strong electrostatic interactions between CCP and the DNA probe. In the presence of target miRNA, the DNA probe hybridizes with target miRNA followed by digestion to small nucleotide fragments by DSN; meanwhile, the miRNA is released and subsequently interacts again with the probe, resulting in the cycled digestion of the DNA probe. In this case, weak electrostatic interactions between oligonucleotide fragments and CCP lead to inefficient FRET from CCP to FAM. Thus, by triggering the FRET signal from CCP to FAM, miRNA can be specially detected, and the fluorescence color change based on FRET can be visualized directly with the naked eye under an UV lamp. Furthermore, an energy transfer cascade can be designed using CCP and DNA probes labeled at the 5′-terminus with FAM and Cy3 dyes, and the multistep FRET processes offer the ability of simultaneous detection of multiplex miRNAs

Antitumor effects of anti-CD137/PD-1 mAbs againstne ID8 ovarian cancer.

<p>Mice (5/group) transplanted i.p. with 3 × 10⁶ ID8 cells 10 days previously were injected i.p. twice at 4 days interval with the indicated mAb combinations (0.5 mg of each mAb/mouse); survival was recorded (A, C) and mean survival time was calculated (B, D). The experiment was repeated once with similar results. E, Mice (8-9/group) transplanted i.p. with 3 × 10⁶ ID8 cells 3 days previously were injected i.p. twice at 4 days interval with 0.5 mg of control, anti-PD-1, anti-CD137 and anti-PD-1/CD137 mAb and their survival was recorded. *P < 0.05, **P < 0.01, compared with control mAb treated mice. </p

Combinatorial PD-1 Blockade and CD137 Activation Has Therapeutic Efficacy in Murine Cancer Models and Synergizes with Cisplatin

<div><p>There is an urgent need for improved therapy for advanced ovarian carcinoma, which may be met by administering immune-modulatory monoclonal antibodies (mAbs) to generate a tumor-destructive immune response. Using the ID8 mouse ovarian cancer model, we investigated the therapeutic efficacy of various mAb combinations in mice with intraperitoneal (i.p.) tumor established by transplanting 3 × 10⁶ ID8 cells 10 days previously. While most of the tested mAbs were ineffective when given individually or together, the data confirm our previous finding that 2 i.p. injections of a combination of anti-CD137 with anti-PD-1 mAbs doubles overall survival. Mice treated with this mAb combination have a significantly increased frequency and total number of CD8⁺ T cells both in the peritoneal lavage and spleens, and these cells are functional as demonstrated by antigen-specific cytolytic activity and IFN-γ production. While administration of anti-CD137 mAb as a single agent similarly increases CD8⁺ T cells, these have no functional activity, which may be attributed to up-regulation of co-inhibitory PD-1 and TIM-3 molecules induced by CD137. Addition of the anti-cancer drug cisplatin to the 2 mAb combination increased overall survival >90 days (and was probably curative) by a mechanism which included a systemic CD8⁺ T cell response with tumor specificity and immunological memory. Strikingly, combined treatment of cisplatin and CD137/PD-1 mAb also gave rise to the long-term survival of mice with established TC1 lung tumors. A similar combination of the 2 mAbs and cisplatin should be considered for clinical ‘translation’. </p> </div

Combining anti-PD-1/CD137 mAb with cisplatin induced complete remission of established ID8 cancer with long-lasting systemic tumor-specific immunity.

<p>Mice (10/group) transplanted i.p. with 3 x 10⁶ ID8 cells 10 days earlier were injected i.p. with two doses of control, anti-PD-1, anti-CD137 or anti-PD-1/CD137 mAb (0.5 mg per dose per mouse) at 4 days interval with or without coadministration of cisplatin (10mg/kg) at the first treatment and their survival was evaluated (A). Mice (10/group) treated with combined anti-PD-1/CD137/cisplatin were depleted of lymphocyte subsets by injection of anti-CD4 (0.2 mg/mouse), anti-CD8 (0.2 mg/mouse), anti-NK1.1 (0.1 mg/mouse) or control mAb (0.2 mg/mouse) 48 and 72 hours prior to the first treatment and every 3-4 days thereafter for the duration of the experiments. Untreated tumor-bearing mice were as negative controls (UNT group). The survival of mice was recorded (B). Fifteen long-term surviving mice (120 days after original transplantation of ID8 cells) pooled from 2 experiments were challenged (5 mice/group) with ID8 cells given i.p. or s.c. or with TC1 cells transplanted s.c. (C); naive mice were transplanted with tumor cells as controls (D) and their survival was recorded. Mice (6/group) with established TC1 tumors of 4-5 mm mean diameter were injected i.p. with two doses of control, anti-PD-1, anti-CD137 or anti-PD-1/CD137 mAb (0.5 mg per dose per mouse) at 4 days interval with or without coadministraation of cisplatin (10 mg/kg) at the first treatment; tumor growth was measured (E) and survival was recorded (F). Data are representative of 2 experiments for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084927#pone-0084927-g006" target="_blank">Figure 6A-D</a>.</p

Analysis of peritoneal lymphocyte subsets from mice injected with the anti-PD-1/CD137 combination.

<p>Mice (3/group) transplanted i.p. with 3 × 10⁶ ID8 cells 10 day earlier were injected i.p. twice at 4 days interval with 0.5 mg of control, anti-CD137, anti-PD-1 or anti-PD-1/CD137 mAb. Two weeks later, peritoneal lavage from treated mice was analyzed by flow cytometry for the percentage and phenotype of peritoneal lymphocytes. The percentages of CD3⁺, CD4⁺, CD8⁺ and CD19⁺ lymphocytes in peritoneal lavage and CD44⁺CD62L^- effector/memory cells in CD4⁺ and CD8⁺ T cells are shown in (A) and (B) respectively. The percentage of PD-1⁺TIM-3⁺ and PD-1^-TIM-3⁺ cells in peritoneal CD4⁺ and CD8⁺ T cells are shown in (C) with representative dotplots in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084927#pone.0084927.s002" target="_blank">Figure S2</a>. Data are presented as M±SEM from 3 mice of each group and are representative of 2 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, PD-1 or CD137 mAb compared with control mAb, PD-1/CD137 mAb compared with control and single mAb. </p

Analysis of effector CD8⁺ T lymphocyte in spleens from mice treated with mAb combinations.

<p>Mice (3/group) which had been transplanted i.p. with 3 × 10⁶ ID8 cells 10 days earlier were injected i.p. twice at 4 days interval with 0.5 mg of control, anti-CD137, anti-PD-1 or anti-PD-1/CD137 mAb. Spleens from treated mice were analyzed for phenotypes and effector functions of CD8⁺ T lymphocytes by flow cytometry 7 days after the second mAb injection. The percentages and numbers of CD44⁺CD62L^- effector/memory and CD44⁺CD62L⁺ central memory and IFN-γ- and IL-10-producing cells in the CD8⁺ T cell population are shown in (A) and representative dotplots are shown in (B). Data are presented as M±SEM from 3 mice in each group and are representative of 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, PD-1 or CD137 mAb compared with control mAb, PD-1/CD137 mAb compared with control and single mAb. </p

Analysis of lymphocyte components in spleens from mice treated with mAb combinations.

<p>Mice (3/group) transplanted i.p. with 3 × 10⁶ ID8 cells 10 days earlier were injected i.p. with 0.5 mg of control, anti-CD137, anti-PD-1 or anti-PD-1/CD137 mAb and the mAb injection was repeated 4 days later. Seven days after the second injection, spleens were harvested and single cell suspensions prepared and stained with fluorescence labeled antibodies against markers of lymphocyte subsets prior to analysis by flow cytometry. The percentages and numbers of CD3⁺, CD4⁺, CD8⁺, CD19⁺, FoxP3⁺/CD4 and GR-1⁺CD11b⁺ cells in spleens are shown in (A) and representative dotplots are shown in (B). Data are presented as M ±SEM from 3 mice/group and are representative of 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, The findings with anti- PD-1 or CD137 single mAbs are compared with control mAb, and the findings with anti- PD-1/CD137 mAbs are compared with both control and single mAb. </p

Mice injected with an anti-PD-1/CD137 mAb combination developed a tumor antigen-specific CTL response.

<p>Mice (3/group) transplanted i.p. with 3 × 10⁶ ID8 cells 10 day earlier were injected i.p. twice at 4 days interval with 0.5 mg of anti-PD-1/CD137 mAb. Seven days after the second mAb injection, pooled splenocytes (5 × 10⁶) from 3 mice were incubated with 5 × 10⁵ UV-irradiated ID8 cells for 4 days. The resultant splenocytes were then evaluated for antigen-specific CTL activity by CytoTox 96 Non-radioactive cytotoxicity assay using EL4 cells pulsed with H-2Db-restricted mesothelin or HPV-E7 peptide as target cells (A). The killing activity was also evaluated in the presence of anti-CD4, anti-CD8 or control antibody (B). Data were expressed as M±SEM of triplicate wells. </p