Co-immunoprecipitation for Deciphering Protein Interactomes

Smith, Bradley J.; Cassoli, Juliana S.; Guest, Paul C.; Martins-de-Souza, Daniel

doi:10.1007/978-3-319-52479-5_19

Cited by 8 publications

(5 citation statements)

References 9 publications

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“…Experiments were conducted according to protocols (Smith et al, 2017). NRCMs or pre-chopped heart tissues were lysated by IP lysis buffer (Nantong, Jiangsu, China).…”

Section: Co-immunoprecipitationmentioning

confidence: 99%

PRMT5 Prevents Cardiomyocyte Hypertrophy via Symmetric Dimethylating HoxA9 and Repressing HoxA9 Expression

Cai¹,

Liu²,

Wang³

et al. 2020

Front. Pharmacol.

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The present study reveals a link between protein arginine methyltransferase 5 (PRMT5) and Homebox A9 (HoxA9) in the regulation of cardiomyocyte hypertrophy. In cardiomyocyte hypertrophy induced by β-adrenergic receptor agonist isoprenaline (ISO), PRMT5 expression was decreased while HoxA9 was upregulated. Silencing of PRMT5 or inhibition of PRMT5 by its pharmacological inhibitor EPZ augmented the expressions of cardiomyocyte hypertrophic genes brain natriuretic peptide (BNP) and β-Myosin Heavy Chain (β-MHC), whereas overexpression of PRMT5 inhibited ISO-induced cardiomyocyte hypertrophy, suggesting that PRMT5 ameliorates cardiomyocyte hypertrophy. On the contrary, HoxA9 promoted cardiomyocyte hypertrophy, as implied by the gain-of-function and loss-of-function experiments. HoxA9 was involved in the regulation of PRMT5 in cardiomyocyte hypertrophy, since HoxA9 knockdown prevented si-RPMT5-induced cardiomyocyte hypertrophy, and HoxA9 expression impaired the anti-hypertrophic effect of PRMT5. Co-immunoprecipitation experiments revealed that there were physical interactions between PRMT5 and HoxA9. The symmetric dimethylation level of HoxA9 was decreased by ISO or EPZ treatment, suggesting that HoxA9 is methylated by PRMT5. Additionally, PRMT5 repressed the expression of HoxA9. Chromatin immunoprecipitation (ChIP) assay demonstrated that HoxA9 could bind to the promoter of BNP, and that this binding affinity was further enhanced by ISO or EPZ. In conclusion, this study suggests that PRMT5 symmetric dimethylates HoxA9 and represses HoxA9 expression, thus impairing its binding to BNP promoter and ultimately protecting against cardiomyocyte hypertrophy. These findings provide a novel insight of the mechanism underlying the cardiac protective effect of PRMT5, and suggest potential therapeutic strategies of PRMT5 activation or HoxA9 inhibition in treatment of cardiac hypertrophy.

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“…Experiments were conducted according to protocols (Smith et al, 2017). NRCMs or pre-chopped heart tissues were lysated by IP lysis buffer (Nantong, Jiangsu, China).…”

Section: Co-immunoprecipitationmentioning

confidence: 99%

PRMT5 Prevents Cardiomyocyte Hypertrophy via Symmetric Dimethylating HoxA9 and Repressing HoxA9 Expression

Cai¹,

Liu²,

Wang³

et al. 2020

Front. Pharmacol.

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“…For additional verification of binding of ZINC10157406 to the TCTP-p53 interaction site, we conducted a co-immunoprecipitation experiment. This is a powerful standard technique for the detection of highly sensitive protein-protein interactions [ 46 , 47 ]. TCTP dissolved in protein solution derived from lysed MOLT-4 cells upon ZINC10157406 treatment was magnetically precipitated and subjected to Western blot detection.…”

Section: Discussionmentioning

confidence: 99%

A novel ligand of the translationally controlled tumor protein (TCTP) identified by virtual drug screening for cancer differentiation therapy

et al. 2021

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SummaryIntroduction Differentiation therapy is a promising strategy for cancer treatment. The translationally controlled tumor protein (TCTP) is an encouraging target in this context. By now, this field of research is still at its infancy, which motivated us to perform a large-scale screening for the identification of novel ligands of TCTP. We studied the binding mode and the effect of TCTP blockade on the cell cycle in different cancer cell lines. Methods Based on the ZINC-database, we performed virtual screening of 2,556,750 compounds to analyze the binding of small molecules to TCTP. The in silico results were confirmed by microscale thermophoresis. The effect of the new ligand molecules was investigated on cancer cell survival, flow cytometric cell cycle analysis and protein expression by Western blotting and co-immunoprecipitation in MOLT-4, MDA-MB-231, SK-OV-3 and MCF-7 cells. Results Large-scale virtual screening by PyRx combined with molecular docking by AutoDock4 revealed five candidate compounds. By microscale thermophoresis, ZINC10157406 (6-(4-fluorophenyl)-2-[(8-methoxy-4-methyl-2-quinazolinyl)amino]-4(3H)-pyrimidinone) was identified as TCTP ligand with a KD of 0.87 ± 0.38. ZINC10157406 revealed growth inhibitory effects and caused G0/G1 cell cycle arrest in MOLT-4, SK-OV-3 and MCF-7 cells. ZINC10157406 (2 × IC50) downregulated TCTP expression by 86.70 ± 0.44% and upregulated p53 expression by 177.60 ± 12.46%. We validated ZINC10157406 binding to the p53 interaction site of TCTP and replacing p53 by co-immunoprecipitation. Discussion ZINC10157406 was identified as potent ligand of TCTP by in silico and in vitro methods. The compound bound to TCTP with a considerably higher affinity compared to artesunate as known TCTP inhibitor. We were able to demonstrate the effect of TCTP blockade at the p53 binding site, i.e. expression of TCTP decreased, whereas p53 expression increased. This effect was accompanied by a dose-dependent decrease of CDK2, CDK4, CDK, cyclin D1 and cyclin D3 causing a G0/G1 cell cycle arrest in MOLT-4, SK-OV-3 and MCF-7 cells. Our findings are supposed to stimulate further research on TCTP-specific small molecules for differentiation therapy in oncology.

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“…Immunoprecipitation was performed as described [29].In order to detect FOXO3 ubiquitination, PC-3 cells were treated with Sinularin for 24 h followed by Bortezomib for 8 h. Anti-FOXO3 antibody (#2497, Cell Signaling Technology, USA), anti-14-3-3 antibody (sc-1657, Santa Cruz Biotechnology, USA) and Protein A/G PLUS-Agarose beads (sc-2003, SANTA CRUZ BIOTECHNOLOGY, USA) were used.…”

Section: Immunoprecipitationmentioning

confidence: 99%

Sinularin stabilizes FOXO3 protein to trigger prostate cancer cell intrinsic apoptosis

Meng,

Wang,

et al. 2024

Biochemical Pharmacology

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Co-immunoprecipitation for Deciphering Protein Interactomes

Cited by 8 publications

References 9 publications

PRMT5 Prevents Cardiomyocyte Hypertrophy via Symmetric Dimethylating HoxA9 and Repressing HoxA9 Expression

PRMT5 Prevents Cardiomyocyte Hypertrophy via Symmetric Dimethylating HoxA9 and Repressing HoxA9 Expression

A novel ligand of the translationally controlled tumor protein (TCTP) identified by virtual drug screening for cancer differentiation therapy

Sinularin stabilizes FOXO3 protein to trigger prostate cancer cell intrinsic apoptosis

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