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14-3-3? and Its Modulators in Cancer.

ABSTRACT: 14-3-3? is an acidic homodimer protein with more than one hundred different protein partners associated with oncogenic signaling and cell cycle regulation. This review aims to highlight the crucial role of 14-3-3? in controlling tumor growth and apoptosis and provide a detailed discussion on the structure-activity relationship and binding interactions of the most recent 14-3-3? protein-protein interaction (PPI) modulators reported to date, which has not been reviewed previously. This includes the new fusicoccanes stabilizers (FC-NAc, DP-005), fragment stabilizers (TCF521-123, TCF521-129, AZ-003, AZ-008), phosphate-based inhibitors (IMP, PLP), peptide inhibitors (2a-d), as well as inhibitors from natural sources (85531185, 95911592). Additionally, this review will also include the discussions of the recent efforts by a different group of researchers for understanding the binding mechanisms of existing 14-3-3? PPI modulators. The strategies and state-of-the-art techniques applied by various group of researchers in the discovery of a different chemical class of 14-3-3? modulators for cancer are also briefly discussed in this review, which can be used as a guide in the development of new 14-3-3? modulators in the near future.

SUBMITTER: Aljabal G

PROVIDER: S-EPMC7761676 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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14-3-3σ and Its Modulators in Cancer.

Aljabal Ghazi G Yap Beow Keat BK

Pharmaceuticals (Basel, Switzerland) 20201203 12

14-3-3σ is an acidic homodimer protein with more than one hundred different protein partners associated with oncogenic signaling and cell cycle regulation. This review aims to highlight the crucial role of 14-3-3σ in controlling tumor growth and apoptosis and provide a detailed discussion on the structure-activity relationship and binding interactions of the most recent 14-3-3σ protein-protein interaction (PPI) modulators reported to date, which has not been reviewed previously. This includes th ...[more]

PMID: 33287252

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Project description:AimTo investigate the biological function of 14-3-3σ protein and to look for proteins that interact with 14-3-3σ protein in colon cancer stem cells.MethodsReverse transcription polymerase chain reaction was performed to amplify the 14-3-3σ gene from the mRNA of colon cancer stem cells. The gene was then cloned into the pGEM-T vector. After being sequenced, the target gene 14-3-3σ was cut from the pGEM-T vector and cloned into the pGBKT7 yeast expression plasmid. Then, the bait plasmid pGBKT7-14-3-3σ was transformed into the yeast strain AH109. After the expression of the pGBKT7-14-3-3σ fusion protein in the AH109 yeast strain was accomplished, a yeast two-hybrid screening assay was performed by mating AH109 with Y187 that contained a HeLa cDNA library plasmid. The interaction between the 14-3-3σ protein and the proteins obtained from positive colonies was further confirmed by repeating the yeast two-hybrid screen. After extracting and sequencing the plasmids from the positive colonies, we performed a bioinformatics analysis. A coimmunoprecipitation assay was performed to confirm the interaction between 14-3-3σ and the proteins obtained from the positive colonies. Finally, we constructed 14-3-3σ and potassium channel modulatory factor 1 (KCMF1) siRNA expression plasmids and transfected them into colon cancer stem cells.ResultsThe bait plasmid pGBKT7-14-3-3σ was constructed successfully, and the 14-3-3σ protein had no toxic or autonomous activation effect on the yeast. Nineteen true-positive colonies were selected and sequenced, and their full-length sequences were obtained. We searched for homologous DNA sequences for these sequences from GenBank. Among the positive colonies, four coding genes with known functions were obtained, including KCMF1, quinone oxidoreductase (NQO2), hydroxyisobutyrate dehydrogenase (HIBADH) and 14-3-3σ. For the subsequent coimmunoprecipitation assay, the plasmids PCDEF-Flag-14-3-3σ, PCDEF-Myc-KCMF1, PCDEF-Myc-NQO2 and PCDEF-Myc-HIBADH were successfully constructed, and the sequences were further confirmed by DNA sequencing. The Fugene 6 reagent was used to transfect the plasmids, and fluorescence-activated cell sorting analysis showed the transfection efficiency was 97.8% after 48 h. The HEK 293FT cells showed the stable expression of the PCDEF-Flag-14-3-3σ, PCDEF-Myc-KCMF1, PCDEF-Myc-NQO2 and PCDEF-Myc-HIBADH plasmids. After anti-Myc antibody immunoprecipitation with Myc-KCMF1, Myc-NQO2 and Myc-HIBADH from cell lysates, the presence of Flag-14-3-3σ protein in the immunoprecipitated complex was determined by western blot analysis. The knock-down expression of the 14-3-3σ and KCMF1 proteins significantly inhibited cell proliferation and colony formation of SW1116csc.ConclusionGenes of the proteins that interacted with 14-3-3σ were successfully screened from a HeLa cDNA library. KCMF1 and 14-3-3σ protein may affect the proliferation and colony formation of human colon cancer stem cells.

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14-3-3? and Its Modulators in Cancer.

Publications

14-3-3σ and Its Modulators in Cancer.

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