Characterization of YY1 OPB Peptide for its Anticancer Activity

Abstract

Background: The oncoprotein binding (OPB) domain of Yin Yang 1 (YY1) consists of 26 amino acids between G201 and S226, and is involved in YY1 interaction with multiple oncogene products, including MDM2, AKT, EZH2 and E1A. Through the OPB domain, YY1 promotes the oncogenic or proliferative regulation of these oncoproteins in cancer cells. We previously demonstrated that a peptide with the OPB sequence blocked YY1-AKT interaction and inhibited breast cancer cell proliferation. Objective: In the current study, we characterized the OPB domain and determined a minimal region for peptide design to suppress cancer cell Methods: Using alanine-scan method, we identified that the amino acids at OPB C-terminal are essential to YY1 binding to AKT. Further studies suggested that serine and threonine residues, but not lysines, in OPB play a key role in YY1-AKT interaction. We generated GFP fusion expression vectors to express OPB peptides with serially deleted N-terminal and found that OPB1 (i.e. G201-S226) is cytoplasmic, but OPB2 (i.e. E206-S226), OPB3 (i.e. E206-S226) and control peptide were both nuclear and cytoplasmic. Results: Both OPB1 and 2 inhibited breast cancer cell proliferation and migration, but OPB3 exhibited similar effects to control. OPB1 and 2 caused cell cycle arrest at G1 phase, increased p53 and p21 expression, and reduced AKT(S473) phosphorylation in MCF-7 cells, but not in MDA-MB-231 cells. Conclusion: : Overall, the serines and threonines of OPB are essential to YY1 binding to oncoproteins, and OPB peptide can be minimized to E206-S226 that maintain inhibitory activity to YY1- promoted cell proliferation.

Disruption of YY1-EZH2 Interaction Using Synthetic Peptides Inhibits Breast Cancer Development

“…Previous studies indicated that EZH2 was recruited by YY1 to the promoters of target genes [ 41 ]. We reported that the peptide derived from the OPB sequence could block YY1 binding to several oncoproteins [ 30 , 42 ]. Thus, to evaluate the activity of the YPB peptide, we also synthesized the FITC-TAT-3×Glycine-OPB peptide (hereafter designated as the OPB peptide) and an unfused FITC-TAT peptide (designated as the Cont peptide) as positive and negative controls, respectively ( Figure 2 A).…”

“…The OPB domain of YY1 is involved in binding to at least four oncoproteins, including EZH2, MDM2, AKT, and E1A [ 30 ]. We reported that synthetic peptides based on the OPB sequence could inhibit breast cancer cell proliferation, but their effects in a mouse model were not evaluated [ 30 , 42 ]. In the current study, we mapped the binding site of YY1 on EZH2 and identified the YPB domain consisting of 27 residues.…”

Functional analysis of YY1 zinc fingers through cysteine mutagenesis

“…The C2H2 ZF structure was first discovered by Miller et al . in 1985 in the Xenopus laevis transcription factor TFIIIA . Many transcription factors, including SP1, GATA1, ZEB1, and YY1, utilize ZF domains to bind their target promoters .…”

“…Among them, C2H2 (Cys2-His2) is a classical and the most common type with a consensus zincbinding sequence of X 2 -C-X 2,4 -C-X 12 -H-X 3-5 -H (X: any amino acid) [21]. The C2H2 ZF structure was first discovered by Miller et al in 1985 in the Xenopus laevis transcription factor TFIIIA [14]. Many transcription factors, including SP1, GATA1, ZEB1, and YY1, utilize ZF domains to bind their target promoters [20].…”

“…Especially, an oncoprotein binding (OPB) domain of residues 201–226 is involved in YY1 binding to multiple oncogene products, including E1A, EZH2, MDM2, and AKT . Consistently, a peptide based on the OPB sequence could block YY1 interaction with its binding proteins and consequently inhibit breast cancer cell proliferation . YY1 can exert its function through mechanisms either dependent or independent of its transcriptional activity.…”