A simplified model depicting the mechanism how MDM2 inhibits Axin-induced p53 activation.

<p>When overexpressed, MDM2 competes the binding of both p53 and HIPK2 on Axin, and disrupts the Axin/p53/HIPK2 complex, leading to the inhibition of p53 activation and cell apoptosis.</p

Both MDM2 and MDM2 (C464A) disrupt Axin-p53 interaction by recruiting p53.

<p>(A) HEK 293T cells were transfected with 2 µg untagged Axin and increasing amounts (3 µg and 8 µg) of HA-MDM2, HA-MDM2 (C464A) or HA-MDM2Δp53. (B) HEK 293T cells were transfected with HA-Axin, FLAG-p53 and Myc-MDM2 or MDM2 (C464A) and analyzed by immunoprecipitation and western blotting. (C) In normal lysis buffer, 1 µg purified His-Axin, 1 µg His-p53 and 6 µg GST-MDM2, GST-MDM2 (C464A) or GST-MDM2Δp53 were added and p53 was immunoprecipitated following by western blotting. BSA was used to make the protein amount of each sample equal. (D) HEK 293 cells were transfected with different combinations of pCMV5-LacZ, p53-luciferase reporter, HA-Axin, and Myc-MDM2 as indicated. At 6 h after transfection, cells were treated without or with 5 µM, 10 µM and 20 µM Nutlin 3a respectively for another 24 h followed by measure of luciferase activity. Results shown are mean±s.d. of three independent experiments. (E) U2OS cells were plated in 100 mm dishes at 70% confluence, followed by treatment with 0 µM, 0.4 µM and 2.5 µM doxorubicin respectively. 24 h after treatment, cells lysates were divided into two parts, one was immunoprecipitated with anti-Axin antibody, the other part was immunoprecipitated with anti-MDM2 antibody.</p

Both MDM2 and MDM2(C464A) inhibit Axin-HIPK2 interaction.

<p>(A, B) HEK 293T cells were co-transfected with Axin, HA-HIPK2 and Myc-MDM2 or MDM2 (C464A) as indicated, followed by immunoprecipitation and western blotting analysis. (C) Both MDM2 and MDM2 (C464A) can disrupt the interaction between HIPK2Δp53 (a HIPK2 deletion mutant that fails to bind with p53) and Axin. HEK 293T cells were co-transfected with different combinations of plasmids as indicated, followed by immunoprecipitation and western blotting.</p

MDM2 Inhibits Axin-Induced p53 Activation Independently of its E3 Ligase Activity

<div><p>MDM2 plays a crucial role in negatively regulating the functions of tumor suppressor p53. Here we show that MDM2 can inhibit Axin-stimulated p53-dependent apoptosis by suppressing p53 phosphorylation at Ser 46 and apoptosis-related p53 transactivational activity. Interestingly, the ubiquitin E3 ligase activity of MDM2 is not required for this inhibitory effect. Mechanically, either wildtype MDM2 or its E3-dead mutant, disrupts the Axin-based HIPK2/p53 complex formation by blocking the binding of p53 and HIPK2 to Axin. MDM2Δp53, a deletion mutant that lacks p53 binding domain fails to exert the inhibitory effect, demonstrating that the interaction of MDM2 and p53, but not its E3 ligase activity toward p53 plays key role in suppressing Axin-stimulated p53 activation. Our results thus have revealed a novel aspect of the mechanism by which MDM2 regulates p53 activities.</p></div

MDM2 and its E3-inactivated mutant MDM2(C464A) show the similar effect on inhibition of Axin-induced p53 transcriptional activity.

<p>(A) HEK 293 cells were transfected with p53-Luc reporter, HA-Axin, Myc tagged MDM2 and its mutants in different combinations as indicated. Western blotting were performed to indicate protein expression levels (inset). All transfections were performed in duplicate and the data are means±s.d. of three independent experiments. *, <i>p</i><0.001 compared with cells transfected with HA-Axin alone (second column). Statistical analyses were done using <i>t</i> test. (B) Experiments were performed as in (A). *, <i>p</i><0.001 compared with cells transfected with HA-Axin alone (second column); ^#, <i>p</i>>0.05 compared with cells transfected with HA-Axin alone (second column).</p

MDM2 and MDM2 (C464A) inhibit Axin-induced apoptosis to the same extent.

<p>(A) H1299 cells were transfected with GFP, Myc-p53, untagged Axin, Myc-MDM2 or Myc-MDM2 (C464A) in the combinations as indicated. Cell death was quantified 24 h after transfection by Hoechst 33324 staining and results were means±s.d. of three independent experiments. *, <i>p</i><0.01 compared with cells transfected with p53 alone (second column); ^#, <i>p</i><0.01 compared with cells co-transfected with p53 and Axin (third column). Statistical analyses were done using <i>t</i> test. (B) U2OS cells were transfected with GFP, untagged Axin, Myc-MDM2 or Myc-MDM2 (C464A) in different combinations as indicated. The percentage of apoptotic cells was determined as in (A). *, <i>p</i><0.05 compared with untransfected cells (first column); ^#, <i>p</i><0.01 compared with cells transfected with Axin (second column).</p

MDM2 (C464A) dramatically inhibits p53 Ser 46 phosphorylation.

<p>(A) H1299 cells were transfected with Myc-p53, HA-Axin or HA-MDM2 (C464A) as indicated, and analyzed by immunoprecipitation and western blotting. (B) H1299 cells were co-transfected with Myc-p53, MDM2 (C464A) and pSUPER-Axin in different combinations. 24 h after transfection, cells were treated with UV (ultraviolet) of 80 J/m². At 6 h post-treatment, cells were lysed and immunoprecipitated, followed by western blotting with anti-p53 and anti-phospho-Ser 46 antibodies.</p

Data_Sheet_1_Combination of Walnut Peptide and Casein Peptide alleviates anxiety and improves memory in anxiety mices.docx

IntroductionAnxiety disorders continue to prevail as the most prevalent cluster of mental disorders following the COVID-19 pandemic, exhibiting substantial detrimental effects on individuals’ overall well-being and functioning. Even after a search spanning over a decade for novel anxiolytic compounds, none have been approved, resulting in the current anxiolytic medications being effective only for a specific subset of patients. Consequently, researchers are investigating everyday nutrients as potential alternatives to conventional medicines. Our prior study analyzed the antianxiety and memory-enhancing properties of the combination of Walnut Peptide (WP) and Casein Peptide (CP) in zebrafish.Methods and ResultsBased on this work, our current research further validates their effects in mice models exhibiting elevated anxiety levels through a combination of gavage oral administration. Our results demonstrated that at 170 + 300 mg human dose, the WP + CP combination significantly improved performances in relevant behavioral assessments related to anxiety and memory. Furthermore, our analysis revealed that the combination restores neurotransmitter dysfunction observed while monitoring Serotonin, gamma-aminobutyric acid (GABA), dopamine (DA), and acetylcholine (ACh) levels. This supplementation also elevated the expression of brain-derived neurotrophic factor mRNA, indicating protective effects against the neurological stresses of anxiety. Additionally, there were strong correlations among behavioral indicators, BDNF (brain-derived neurotrophic factor), and numerous neurotransmitters.ConclusionHence, our findings propose that the WP + CP combination holds promise as a treatment for anxiety disorder. Besides, supplementary applications are feasible when produced as powdered dietary supplements or added to common foods like powder, yogurt, or milk.</p