P53 DNA Binding Research Articles

Mutant p53 targeting by the low molecular weight compound STIMA-1.

Reactivation of mutant p53 in human tumor cells should induce cell death by apoptosis and thus eliminate the tumor. Several small molecules that reactivate mutant p53 have been identified. Here we show that STIMA-1, a low molecular weight compound with some structural similarities to the previously identified molecule CP-31398, can stimulate mutant p53 DNA binding in vitro and induce expression of p53 target proteins and trigger apoptosis in mutant p53-expressing human tumor cells. Human diploid fibroblasts are significantly more resistant to STIMA-1 than mutant or wild type p53-carrying tumor cells. STIMA-1 may provide new insights into possible mechanisms of mutant p53 reactivation and thus facilitate the development of novel anticancer drugs that target mutant p53-carrying tumors.

Stabilization of the p53-DNA Complex by the Nuclear Protein Dmp1α

ABSTRACTWe recently reported the existence of a physical interaction between the Myb-like transcription factor Dmp1 (Dmtf1) and p53 in which Dmp1 antagonized polyubiquitination of p53 by Mdm2 and promoted its nuclear localization. Dmp1 significantly stabilized p53-DNA complexes on promoters that contained p53-consensus sequences, which were either supershifted or disrupted with antibodies to Dmp1. Lysates from mice injected with doxorubicin showed that Dmp1 bound to p21Cip1, Bbc3, and Thbs1 gene regulatory regions in a p53-dependent fashion. Our data suggest that acceleration of DNA-binding of p53 by Dmp1 is a critical process for Dmp1 to increase the p53 function in Arf-deficient cells.

Germline TP53 mutations result into a constitutive defect of p53 DNA binding and transcriptional response to DNA damage.

Li-Fraumeni Syndrome (LFS) results from heterozygous germline mutations of TP53, encoding a key transcriptional factor activated in response to DNA damage. We have recently shown, from a large LFS series, that dominant-negative missense mutations are the most clinically severe and, thanks to a new p53 functional assay in lymphocytes, that they alter the p53 transcriptional response to DNA damage more drastically than null mutations. In this study, we first confirmed this observation by performing the p53 functional assay in lymphocytes from 56 TP53 mutation carriers harbouring 35 distinct alterations. Then, to compare the impact of the different types of germline TP53 mutations on DNA binding, we performed chromatin immunoprecipitation-sequencing (ChIP-Seq) in lymphocytes exposed to doxorubicin. ChIP-Seq performed in wild-type TP53 control lymphocytes accurately mapped 1287 p53-binding sites. New p53-binding sites were validated using a functional assay in yeast. ChIP-Seq analysis of LFS lymphocytes carrying TP53 null mutations (p.P152Rfs*18 or complete deletion) or the low penetrant ‘Brazilian’ p.R337H mutation revealed a moderate decrease of p53-binding sites (949, 580 and 620, respectively) and of ChIP-Seq peak depths. In contrast, analysis of LFS lymphocytes with TP53 dominant-negative missense mutations p.R273H or p.R248W revealed only 310 and 143 p53-binding sites, respectively, and the depths of the corresponding peaks were drastically reduced. Altogether, our results show that TP53 mutation carriers exhibit a constitutive defect of the transcriptional response to DNA damage and that the clinical severity of TP53 dominant-negative missense mutations is explained by a massive and global alteration of p53 DNA binding.

Regulation of NF-\u03baB by PML and PML-RAR\u03b1

Promyelocytic Leukemia (PML) is a nuclear protein that forms sub-nuclear structures termed nuclear bodies associated with transcriptionally active genomic regions. PML is a tumour suppressor and regulator of cell differentiation. We demonstrate that PML promotes TNFα-induced transcriptional responses by promoting NF-κB activity. TNFα-treated PML−/− cells show normal IκBα degradation and NF-κB nuclear translocation but significantly reduced NF-κB DNA binding and phosphorylation of NF-κB p65. We also demonstrate that the PML retinoic acid receptor-α (PML-RARα) oncofusion protein, which causes acute promyelocytic leukemia, inhibits TNFα induced gene expression and phosphorylation of NF-κB. This study establishes PML as an important regulator of NF-κB and demonstrates that PML-RARα dysregulates NF-κB.

Thiosemicarbazones Functioning as Zinc Metallochaperones to Reactivate Mutant p53.

Small-molecule restoration of wild-type structure and function to mutant p53 (so-called mutant reactivation) is a highly sought-after goal in cancer drug development. We previously discovered that small-molecule zinc chelators called zinc metallochaperones (ZMCs) reactivate mutant p53 by restoring zinc binding to zinc-deficient p53 mutants. The lead compound identified from the NCI-60 human tumor cell lines screen, NSC319726 (ZMC1), belongs to the thiosemicarbazone (TSC) class of metal ion chelators that bind iron, copper, magnesium, zinc, and other transition metals. Here, we have investigated the other TSCs, NSC319725 and NSC328784, identified in the same screen, as well as the more well studied TSC, 3-AP (Triapine), to determine whether they function as ZMCs. We measured the zinc Kd zinc ionophore activity, ability to restore zinc to purified p53 DNA binding domain (DBD), and ability to restore site-specific DNA binding to purified R175H-DBD in vitro. We tested all four TSCs in a number of cell-based assays to examine mutant p53 reactivation and the generation of reactive oxygen species (ROS). We found that NSC319725 and NSC328784 behave similarly to ZMC1 in both biophysical and cell-based assays and are heretofore named ZMC2 (NSC319725) and ZMC3 (NSC328784). 3-AP generates a ROS signal similar to ZMC1-3, but it fails to function as a ZMC both in vitro and in cells and ultimately does not reactivate p53. These findings indicate that not all TSCs function as ZMCs, and much of their activity can be predicted by their affinity for zinc.

Kaempferol ameliorates H9N2 swine influenza virus-induced acute lung injury by inactivation of TLR4/MyD88-mediated NF-κB and MAPK signaling pathways.

Kaempferol, a very common type of dietary flavonoids, has been found to exert antioxidative and anti-inflammatory properties. The purpose of our investigation was designed to reveal the effect of kaempferol on H9N2 influenza virus-induced inflammation in vivo and in vitro. In vivo, BALB/C mice were infected intranasally with H9N2 influenza virus with or without kaempferol treatment to induce acute lung injury (ALI) model. In vitro, MH-S cells were infected with H9N2 influenza virus with or without kaempferol treatment. In vivo, kaempferol treatment attenuated pulmonary edema, the W/D mass ratio, pulmonary capillary permeability, myeloperoxidase (MPO) activity, and the numbers of inflammatory cells. Kaempferol reduced ROS and Malondialdehyde (MDA) production, and increased the superoxide dismutase (SOD) activity. Kaempferol also reduced overproduction of TNF-α, IL-1β and IL-6. In addition, kaempferol decreased the H9N2 viral titre. In vitro, ROS, MDA, TNF-α, IL-1β and IL-6 was also reduced by kaempferol. Moreover, our data showed that kaempferol significantly inhibited the upregulation of toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), phosphorylation level of IκBα and nuclear factor-κB (NF-κB) p65, NF-κB p65 DNA binding activity, and phosphorylation level of MAPKs, both in vivo and in vitro. These results suggest that kaempferol exhibits a protective effect on H9N2 virus-induced inflammation via suppression of TLR4/MyD88-mediated NF-κB and MAPKs pathways, and kaempferol may be considered as an effective drug for the potential treatment of influenza virus-induced ALI.

The transient manifold structure of the p53 extreme C-terminal domain: insight into disorder, recognition, and binding promiscuity by molecular dynamics simulations.

The p53 tumour suppressor is a transcription activator that signals for cell cycle arrest and apoptosis. In its active form p53 is a tetramer, with each monomer organised in domains with different degrees of structural stability, ranging from the well folded DNA-binding domain (DBD) and tetramerization domain (TET), to the intrinsically disordered transactivation domain (TAD), and extreme C-terminal domain (CTD). Compared to all other domains, the structure/function relationship of the p53-CTD within the full-length p53 tetramer is still poorly understood due to its high degree of conformational disorder. Meanwhile, the structure of p53-CTD-like peptides has been well characterized when in complex with a variety of receptors, where, as other intrinsically disordered regions (IDR), it adopts specific, while diverse, conformations. Receptor-specific folding is likely to occur upon binding, either from a random coil, or as a result of an initial recognition of a pre-formed structural motif, known as molecular recognition feature (MoRF), selected by the receptor within the conformational ensemble of the IDP in solution. In this latter case, MoRFs act as nucleation sites, favouring the initiation of the folding process within the binding site. In this work we show the results of over 20 μs of cumulative molecular dynamics (MD) simulations of a 22 residue peptide unbound in solution with sequence corresponding to the p53-CTD 367-388 section. Such extensive sampling allowed us to identify and characterize the structure of specific sets of minimal structural MoRFs within the p53-CTD peptide conformational ensemble at equilibrium. These motifs are short, involving only 3 to 4 residues, and specifically localized within the peptide sequence. Corresponding patterns of secondary structure propensity along the p53-CTD sequence are also predicted by disorder prediction calculations. Based on these findings we discuss how the structural complementarity of specific minimal structural MoRFs to the binding site of different receptors could regulate the p53-CTD binding promiscuity.

Altered S-nitrosylation of p53 is responsible for impaired antioxidant response in skeletal muscle during aging.

p53 transcriptional activity has been proposed to regulate both homeostasis and sarcopenia of skeletal muscle during aging. However, the exact molecular function of p53 remains to be clearly defined. We demonstrated a requirement of nuclear p53 S-nitrosylation in inducing a nitric oxide/PGC-1α-mediated antioxidant pathway in skeletal muscle. Importantly, mutant form of p53-DNA binding domain (C124S) did not undergo nuclear S-nitrosylation and failed in inducing the expression of antioxidant genes (i.e. SOD2 and GCLC). Moreover, we found that during aging the nuclear S-nitrosylation of p53 significantly declines in gastrocnemius/soleus leading to an impairment of redox homeostasis of skeletal muscle. We suggested that decreased level of nuclear neuronal nitric oxide synthase (nNOS)/Syntrophin complex, which we observed during aging, could be responsible for impaired nuclear S-nitrosylation. Taken together, our data indicate that altered S-nitrosylation of p53 during aging could be a contributing factor of sarcopenia condition and of other skeletal muscle pathologies associated with oxidative/nitrosative stress.

Concurrent Treatment with Taxifolin and Cilostazol on the Lowering of β-Amyloid Accumulation and Neurotoxicity via the Suppression of P-JAK2/P-STAT3/NF-κB/BACE1 Signaling Pathways

Taxifolin is a potent flavonoid that exerts anti-oxidative effect, and cilostazol increases intracellular cAMP levels by inhibiting phosphodiesterase 3 that shows antiinflammatory actions. BACE1 (β-site APP cleaving enzyme 1) is the rate-limiting enzyme responsible for the β-cleavage of amyloid precursor proteins to Aβ peptides. In this study, endogenous Aβ and C99 accumulation was explored in N2a Swe cells exposed to 1% FBS medium. Increased Aβ and C99 levels were significantly attenuated by taxifolin alone and in combination with cilostazol. Increased phosphorylated JAK2 at Tyr1007/1008 (P-JAK), phosphorylated STAT3 at Tyr 705 (P-STAT3) expressions and increased expressions of BACE1 mRNA and protein in the activated N2a Swe cells were significantly attenuated by taxifolin (10~50 μM), cilostazol (10~50 μM) alone and in combination at minimum concentrations. In these cells, decreased cytosol IκBα expression was elevated, and increased nuclear NF-κB p65 level and nuclear NF-κB p65 DNA binding activity were significantly reduced by taxifolin and cilostazol in a similar manner. Following STAT3 gene (~70% reduction) knockdown in N2a cells, Aβ-induced nuclear NF-κB and BACE1 expressions were not observed. Taxifolin, cilostazol, or resveratrol significantly stimulated SIRT1 protein expression. In SIRT1 gene-silenced (~50%) N2a cells, taxifolin, cilostazol, and resveratrol all failed to suppress Aβ1-42-stimulated P-STAT3 and BACE1 expression. Consequently, taxifolin and cilostazol were found to significantly decrease lipopolysaccharide (1–10 μg/ml)-induced iNOS and COX-2 expressions, and nitrite production in cultured BV-2 microglia cells and to increase N2a cell viability. In conclusion, taxifolin and cilostazol strongly inhibited amyloidogenesis in a synergistic manner by suppressing P-JAK2/P-STAT3-coupled NF-κB-linked BACE1 expression via the up-regulation of SIRT1.

Rational design, synthesis and in vitro evaluation of novel exo-methylene butyrolactone salicyloylamide as NF-κB inhibitor

(−)-Dehydroxymethylepoxyquinomicin ((−)-DHMEQ, 1) is a specific inhibitor of NF-κB. It binds to SH group in the specific cysteine residue of NF-κB components with its epoxide moiety to inhibit DNA binding. In the present research, we have designed and synthesized an epoxide-free analog called (S)-β-salicyloylamino-α-exo-methylene-ƴ-butyrolactone (SEMBL, 3). SEMBL inhibited DNA binding of NF-κB component p65 in vitro. It inhibited LPS-induced NF-κB activation, iNOS expression, and inflammatory cytokine secretions. It also inhibited NF-κB and cellular invasion in ovarian carcinoma ES-2 cells. Moreover, its stability in aqueous solution was greatly enhanced compared with (−)-DHMEQ. Thus, SEMBL has a potential to be a candidate for a new anti-inflammatory and anticancer agent.

Prevalence of TP53 Mutations in Del(17p) Myeloma: Spectrum of Mutations and Effect on Survival

Background.The overall improvement in treatment outcomes and survival of multiple myeloma (MM) patients has highlighted the persistently poor outlook for patients with del(17p). Around a third of MM patients with del(17p) are also reported to have a TP53 mutation, while this mutation is rare in the absence of del(17p). The prognostic impact of TP53 mutations in the context of del(17p) is unresolved. We investigated the presence of TP53 mutations in a cohort of del(17p) patients to correlate mutation status with clinical outcome. We assessed the types of TP53 mutations, and the timing of mutations in relation to the acquisition of del(17p).Methods. CD138+ plasma cells were obtained from bone marrow of patients at diagnosis and prior to starting each new line of therapy, subjected to FISH analysis using standard probes, and surplus tumour cells banked. Between 2004-2015, we identified 52 patients who had at least one FISH result confirming del(17p) in ≥10% CD138+ cells, of whom 16 patients (30.8%) had del(17p) at diagnosis. In 22 patients, samples were available at more than one time point (2-4) for analysis. Clinical details, including treatment and disease response (2014 IMWG criteria) were collated. Adverse risk FISH was defined as t(4;14), t(14;16) and 1q+. Genomic DNA was extracted from CD138+ plasma cells using DNeasyBlood & Tissue Kit (Qiagen). 87 gDNAsamples were amplified by PCR and screened for TP53 mutations in exons 4-11 using DHPLC. TP53 mutations were verified by Sanger sequencing. Survival was estimated using Kaplan-Meier methods, and differences were assessed by log-rank test with p-values <0.05 considered significant.Results. TP53 mutations were identified in 19 (36.5%) patients, and there was no significant difference in median age at diagnosis between patients with or without mutations (61.3 vs 57.3 years, p=0.07). Of the 16 patients with del(17p) at diagnosis, 5 (31.3%) had a TP53 mutation in the diagnostic sample, while 14 (38.8%) of the 36 patients in the relapse subgroup had a mutation (p=0.8).Of the 18 TP53 variants identified, 17 were single nucleotide variants (SNV), of which 14 (82.4%) were missense mutations, 2 were nonsense mutations and 1 altered a splice site; all were documented on the IARC TP53 database. One patient had a novel in-frame indel. The majority of mutations occurred in exons 5, 6 and 8, which forms an integral part of the p53 DNA binding domain. Most mutations identified were predicted to produce a non-functional protein with less than 20% transcriptional activity, as specified by the IARC database. Eleven (57.9%) of the TP53 mutated patients also had at least one high risk genetic feature compared to 16 (48.5%) of the non-TP53 mutated patients (p=0.6).For relapse patients, median time from diagnosis to acquisition of del(17p) was 34.5 months (range 1-178), and median time from acquisition of del(17p) to TP53 mutation was 4 months (<1-45).In 3 patients with TP53 mutations detected at relapse, a preceding sample confirmed to harbour del(17p) was non-mutated, and occurrence of the mutation occurred at 24, 37 and 39 months after del(17p) was first detected. A further 2 patients had a sample preceding del(17p) acquisition, in which there was no TP53 mutation. For the remainder of patients, del(17p) and TP53 mutation were picked up simultaneously.Median overall survival (OS) from diagnosis was significantly shorter for TP53 mutated patients (23 vs 74 months, p=0.03). Median OS from time of del(17p) acquisition was also significantly shorter for TP53 mutated patients (14 vs 29 months, p=0.01), although PFS from next line of therapy was similar (7 vs 12 months, p=0.70). The median survival of TP53 mutated patients from first detection of mutant TP53 was 7 months.Summary. TP53 mutations are common in del(17p) MM with a broad range of variants, the majority of which are likely to produce non-functional variant proteins. The acquisition of TP53 mutations seems to occur after allelic loss of 17p, and to confer even worse clinical outcomes. In the era of genomic medicine, there is a case for routinely screening for TP53 mutations in these patients, to aid in management decisions and patient counselling. An urgent need exists to develop therapies to counteract the loss of this tumour suppressor pathway. [Display omitted] DisclosuresYong:Autolus Ltd: Equity Ownership, Patents & Royalties: APRIL based chimeric antigen receptor; Janssen: Research Funding.

Intra molecular interactions in the regulation of p53 pathway.

The p53 tumor suppressor is highly regulated at the level of protein degradation and transcriptional activity. The key players of the pathway, p53, MDM2, and MDMX are present at multiple conformational states that are responsive to regulation by post-translational modifications and protein-protein interactions. The structures of major functional domains of these proteins have been determined, but the mechanisms of several intrinsically disordered regions remain unclear despite their critical roles in signaling and regulation. Recent studies suggest that these disordered regions function in part by dynamic intra molecular interactions with the structured domains to regulate p53 DNA binding, MDM2 ubiquitin E3 ligase activity, and MDMX-p53 binding. These findings provide new insight on how p53 is controlled by various stress signals, and suggest potential targets for the search of allosteric regulators of the p53 pathway.

Gambogic acid inhibits the growth of ovarian cancer tumors by regulating p65 activity

Ovarian cancer patients often have poor prognosis, therefore, it is important to search for more effective therapeutic strategies to treat them. Gambogic acid (GA) exhibits an anti-tumor effect through various mechanisms, and has multiple targets in tumor cells. The present study aimed to elucidate the efficacy of GA in the treatment of ovarian cancer both in vivo and in vitro by analyzing its impact on cell survival and tumor growth through cell cycle and apoptosis analysis. GA inhibited the growth of ovarian cancer cells in a dose and time dependent manner, and arrested the cell cycle in ovarian cancer cells. Furthermore, GA increased caspase-3 and caspase-9 activity and inhibited RELA/NF-κB p65 (p65) DNA binding activity. Finally, GA suppressed tumor growth in vivo. Therefore, the current study suggests that GA inhibits the growth of ovarian cancer by regulating p65 activity, and may be developed as a novel therapeutic strategy to treat ovarian cancer.

Viral IL-10 down-regulates the “MHC-I antigen processing operon” through the NF-κB signaling pathway in nasopharyngeal carcinoma cells

The HLA-I antigen processing machinery (APM) plays a crucial role in the anticancer immune response. The loss of surface expression of HLA-I molecules is particularly important as this enables tumor cells to evade recognition and lysis by cytotoxic T-lymphocytes. Transcriptional control of the APM genes is regulated by the nuclear factor kappa B (NF-κB). BCRFl is an Epstein-Barr virus homologue of human IL-10 (hIL-10) and is known as viral IL-10 (vIL-10). vIL-10 shares many immunosuppressive effects with hIL-10 but lacks the immunostimulatory effect of hIL-10. The aim of this study was to assess whether vIL-10 inhibits APM components (TAP-1, TAP-2, LMP-2, LMP-7 and HLA-I) through the NF-κB signaling pathway in nasopharyngeal carcinoma. This work demonstrated that vIL-10 inhibited NF-κB activation by blocking IKK phosphorylation and promoting the expression of IKB. TNF-α treatment led to a strong translocation of NF-κB p65, whereas pretreatment with vIL-10 before TNF-α treatment blocked NF-κB p65 translocation. vIL-10 also inhibited TNF-α-induced DNA-binding of NF-κB p65 in the nucleus. Furthermore, chromatin immunoprecipitation analysis demonstrated that NF-κB p65 could bind to the TAP-1, TAP-2, LMP-2, LMP-7 and HLA-I gene promoters, and after TNF-α stimulation, the down-regulation of TAP-1, TAP-2, LMP-2, LMP-7 and HLA-I transcription by vIL-10 correlated with the suppression of NF-κB in CNE-2 cells. Surprisingly, vIL-10 inhibits only TAP-1 and LMP-7 transcription in CNE-1 cells. Taken together, these results suggest that the inhibition of NF-κB activity may be an important mechanism for vIL-10 suppression of APM (TAP-1, TAP-2, LMP-2, LMP-7 and HLA-I) gene transcription in CNE-2 cells.

Long noncoding RNA MEG3 activation of p53 mediates ischemic neuronal death in stroke

Maternally expressed gene 3 (MEG3) is suggested to function as a long non-coding RNA (lncRNA) and to play roles in various human cancers. However, the functional properties of MEG3 in ischemic stroke remain unknown. Here, we report that expression of MEG3 is upregulated following ischemia in adult mice. Moreover, cerebral ischemia recruits p53 into the MEG3 complex in ischemic tissues. MEG3 directly binds with the p53 DNA binding domain (DBD) consisting of amino acids 271–282 (p53-DBD271–282), which stimulates p53-mediated transactivation and mediates ischemic neuronal death. Administration of the membrane-permeable peptide inhibitor Tat-p53-DBD271–282 uncouples p53 from MEG3 in vivo and protects against cerebral ischemic insults in vitro and in vivo. Our data demonstrate that MEG3 functions as a cell death promoter in ischemia and physically and functionally interacts with p53 to mediate ischemic damage. Intervention in the MEG3–p53 interaction presents a new target for the therapeutic treatment of ischemic insults.

Aggregation tendencies in the p53 family are modulated by backbone hydrogen bonds.

The p53 family of proteins is comprised of p53, p63 and p73. Because the p53 DNA binding domain (DBD) is naturally unstable and possesses an amyloidogenic sequence, it is prone to form amyloid fibrils, causing loss of functions. To develop p53 therapies, it is necessary to understand the molecular basis of p53 instability and aggregation. Light scattering, thioflavin T (ThT) and high hydrostatic pressure (HHP) assays showed that p53 DBD aggregates faster and to a greater extent than p63 and p73 DBDs, and was more susceptible to denaturation. The aggregation tendencies of p53, p63, and p73 DBDs were strongly correlated with their thermal stabilities. Molecular Dynamics (MD) simulations indicated specific regions of structural heterogeneity unique to p53, which may be promoted by elevated incidence of exposed backbone hydrogen bonds (BHBs). The results indicate regions of structural vulnerability in the p53 DBD, suggesting new targetable sites for modulating p53 stability and aggregation, a potential approach to cancer therapy.

Abstract 084: Sodium Butyrate and Retinoic Acid Attenuate Renal Inflammation and Fibrosis in Npr1 Gene-targeted Haplotype Mice: Role of NF-κB Signaling

The objective of the present study was to determine the effect of a hybrid drug of sodium butyrate (NaBu), a histone deacetylase (HDAC) inhibitor and all-trans retinoic acid (ATRA) on the attenuation of renal inflammation and fibrosis in Npr1 gene-disrupted mutant mice. Adult (18-20 week old) male Npr1 gene-disrupted heterozygous (1-copy; Npr1 +/- ) wild-type (2-copy; Npr1 +/+ ), and gene-duplicated (3-copy; Npr1 ++/+ ) mice were treated with ATRA-NaBu hybrid drug (1.0 mg/kg/day) by intraperitoneal injections for 2-weeks. A significant decrease in systolic blood pressure was observed in ATRA-NaBu-treated haplotype Npr1 +/- mice compared with untreated controls (treated, 113.3 ± 1.5 vs. control, 130.4 ± 1.9; p &lt; 0.01). After treatment with ATRA-NaBu, a marked reduction in tubulo-interstitial fibrosis (50%, p &lt; 0.001) and decreased renal collagen type I alpha 2 expression by 55% (treated, 25.9 ± 1.2 vs. control, 57.2 ± 2.9, p &lt; 0.001) was observed in Npr1 +/- mice. Treatment with ATRA-NaBu also increased creatinine clearance (ml/24 h) in Npr1 +/- mice (245.7 ± 24.3 vs. control, 83.8 ± 3.9). Similarly, higher urinary albumin to creatinine ratio was detected in Npr1 +/- mice (0.84 ± 0.03) vs. control (0.35 ± 0.03; p &lt; 0.01) and a complete reversal was observed in drug-treated Npr1 +/- mice (0.39 ± 0.05). Significant decreases were observed in renal (pg/mg protein) tumor necrosis factor-alpha (TNF-α) (4.1 ± 0.7 vs. control, 27.2 ± 2.6; p &lt; 0.01) and interleukin (IL)-6 (11.8 ± 0.8 vs. control, 59.9 ± 3.6; p &lt; 0.01) in ATRA-NaBu-treated Npr1 +/- mice. Western blot analyses showed significant reduction in renal TNF-α and IL-6 protein expression by 54%, (p &lt; 0.001) and 61%, (p &lt; 0.01), respectively, in ATRA-NaBu-treated Npr1 +/- mice. There was 49% increase in renal NF-κB (p65) DNA binding activity in Npr1 +/- mice and 51% lower activity in Npr1 +/++ mice compared with wild-type mice. Western blot analysis revealed distinctly higher levels of renal NF-κB (p65) protein expression in Npr1 +/- mice and reduced levels in Npr1 +/++ mice compared with wild-type controls. The present results provide direct evidence that ATRA-NaBu hybrid drug acts as a potent anti-inflammatory agent, which will have important implications in the pathophysiology of renal injury and hypertension.

A tryptamine-paeonol hybridization compound inhibits LPS-mediated inflammation in BV2 cells

In the present study, we synthesized and evaluated the anti-inflammatory effects of three tryptamine (Trm) hybrid compounds, HBU-375, HBU-376 and HBU-379. The Click reaction between the azido-Trm and 2- or 4-propazylated paeonol moiety resulted in HBU-376 and HBU-375, respectively. HBU-379 was generated by hybridizing Trm with propazylated acetyl-syringic acid. HBU-376 and HBU-375 dose-dependently inhibited LPS and caused nitric oxide (NO) generation in BV2 cells, whereas HBU-379 minimally inhibited NO generation, indicating that the paeonol unit plays an important role in the anti-inflammatory effect of Trm hybrid compounds. Although HBU-375 and HBU-376 demonstrated a similar inhibitory effect on LPS-induced NO generation, HBU-376 resulted in less cellular toxicity presumably due to the free phenolic hydroxyl group of paeonol. Therefore, HBU-376 may be a promising anti-inflammatory agent conferring minimal cytotoxicity. HBU-376 significantly and dose-dependently inhibited LPS-induced NO products, NO synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin-6, MCP-1 and interleukin-1β mRNA expressions and iNOS and COX-2 protein expressions. However, at the same concentrations, Trm or paeonol individually did not inhibit LPS-mediated production of inflammatory molecules. HBU-376 inhibited both LPS-induced STAT-3 phosphorylation and nuclear factor-kappa B (NF-κB) activation. Furthermore, LPS-mediated DNA binding of c-Rel, p50 and p52 to the NF-κB binding site of the iNOS promoter was inhibited by HBU-376, whereas Trm and paeonol did not inhibit LPS-induced NF-κB activation and DNA binding of c-Rel, p50 and p52. Overall, our data suggest that the Trm-paeonol hybrid compound down-regulates inflammatory responses by inhibiting NF-κB and NF-κB-dependent gene expression. This suggests that it is a potential therapeutic agent for inflammatory diseases of the central nervous system.

The double benefit of Spalax p53: surviving underground hypoxia while defying lung cancer cells in vitro via autophagy and caspase-dependent cell death.

The blind subterranean mole rat, Spalax ehrenbergi, is a model organism for hypoxia tolerance. This superspecies have adapted to severe environment by altering an array of hypoxia-mediated genes, among which an alteration in the p53 DNA binding domain (corresponding to R174K in humans) that hinders its transcriptional activity towards apoptotic genes. It is well accepted that apoptosis is not the only form of programmed cell death and that mechanisms that depend on autophagy are also involved. In the current work we have extended our research and investigated the possibility that Spalax p53 can activate autophagy. Using two complementary assays, we have established that over-expression of the Spalax p53 in p53-null cells (human lung cancer cells, H1299), potently induces autophagy. As Spalax is considered highly resistant to cancer, we further studied the relative contribution of autophagy on the outcome of H1299 cells, following transfection with Spalax p53. Results indicate that Spalax p53 acts as a tumor suppressor in lung cancer cells, inducing cell death that involves autophagy and caspases and inhibiting cell number, which is exclusively caspase-dependent. To conclude, the Spalax p53 protein was evolutionary adapted to survive severe underground hypoxia while retaining the ability to defy lung cancer.

Reactivation of mutant p53: Constraints on mechanism highlighted by principal component analysis of the DNA binding domain.

Most p53 mutations associated with cancer are located in its DNA binding domain (DBD). Many structures (X-ray and NMR) of this domain are available in the protein data bank (PDB) and a vast conformational heterogeneity characterizes the various free and complexed states. The major difference between the apo and the holo-complexed states appears to lie in the L1 loop. In particular, the conformations of this loop appear to depend intimately on the sequence of DNA to which it binds. This conclusion builds upon recent observations that implicate the tetramerization and the C-terminal domains (respectively TD and Cter) in DNA binding specificity. Detailed PCA analysis of the most recent collection of DBD structures from the PDB have been carried out. In contrast to recommendations that small molecules/drugs stabilize the flexible L1 loop to rescue mutant p53, our study highlights a need to retain the flexibility of the p53 DNA binding surface (DBS). It is the adaptability of this region that enables p53 to engage in the diverse interactions responsible for its functionality. Proteins 2016; 84:1443-1461. © 2016 Wiley Periodicals, Inc.