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Sci Rep
2015 Oct 13;5:14626. doi: 10.1038/srep14626.
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Structural basis for recognition of Emi2 by Polo-like kinase 1 and development of peptidomimetics blocking oocyte maturation and fertilization.
Jia JL
,
Han YH
,
Kim HC
,
Ahn M
,
Kwon JW
,
Luo Y
,
Gunasekaran P
,
Lee SJ
,
Lee KS
,
Kyu Bang J
,
Kim NH
,
Namgoong S
.
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In a mammalian oocyte, completion of meiosis is suspended until fertilization by a sperm, and the cell cycle is arrested by a biochemical activity called cytostatic factor (CSF). Emi2 is one of the CSFs, and it maintains the protein level of maturation promoting factor (MPF) by inhibiting ubiquitin ligase anaphase promoting complex/cyclosome (APC/C). Degradation of Emi2 via ubiquitin-mediated proteolysis after fertilization requires phosphorylation by Polo-like kinase 1 (Plk1). Therefore, recognition and phosphorylation of Emi2 by Plk1 are crucial steps for cell cycle resumption, but the binding mode of Emi2 and Plk1 is poorly understood. Using biochemical assays and X-ray crystallography, we found that two phosphorylated threonines (Thr(152) and Thr(176)) in Emi2 are each responsible for the recruitment of one Plk1 molecule by binding to its C-terminal polo box domain (PBD). We also found that meiotic maturation and meiosis resumption via parthenogenetic activation were impaired when Emi2 interaction with Plk1-PBD was blocked by a peptidomimetic called 103-8. Because of the inherent promiscuity of kinase inhibitors, our results suggest that targeting PBD of Plk1 may be an effective strategy for the development of novel and specific contraceptive agents that block oocyte maturation and/or fertilization.
Figure 1. The presence of two Plk1-binding regions at the N terminus of mouse Emi2.(A) Diagram illustrating molecular events for cell cycle resumption after fertilization. In matured mammalian oocytes, maturation promoting factor (MPF) activity maintain high, because of ubiquitin ligase APC/C is inhibited by Emi2. After fertilization, increased Ca2+ activates calmodulin-dependent protein kinase II (CaMKII) and it phosphorylates N-terminal of Emi2. Plk1 is recruited by recognition of phosphothreonine residues in Emi2 by polo box domain (PBD) in Plk1 and subsequently phosphorylated Emi2 and phosphorylated Emi2 become substrate of another class of ubiquitin ligase, SCF and degradated by ubiquitin-proteasome. Activated APC-C can decrease MPF levels, therefore cell cycle for meiosis can be resumed. (B) Domain architecture of Emi2 and multiple-sequence alignment of Emi2 protein sequences from zebrafish (Danio rerio: Dan), Austrian ghostshark (Callorhinchus milii: Cal), Xenopus (Xenopus laevis: Xen), chicken (Gallus: Gal), Homo sapiens (hsa), pig (Sus scrofa: Sus), and mouse (Mus musculus: Mus). ZBR : Zinc Binding Region, Peptides containing a phosphorylated threonine (Emi2146–177 or Emi2169–177) are indicated with gray bars. Threonine residues that are subject to phosphorylation are indicated with a red box. (C) Binding affinity and binding stoichiometry of phosphorylated Emi2 peptides in relation to Plk1 Polo box domain (PBD). Isothermal titration calorimetry (ITC) with Emi2 peptides and Plk1-PBD was carried out as described in Materials and Methods. The dissociation constant (Kd) and the stoichiometric ratio of an Emi2 peptide to Plk1-PBD (n) is also shown. (D) Size exclusion chromatography coupled with multiangle light scattering (SEC-MALS) of PLK1-PBD·Emi2146–177 complexes. Plk1-PBD and the phosphorylated Emi2146–177 peptide were mixed at various stoichiometric ratios (molar ratio of Plk1-PBD1 to Emi2146–177: 1:1, 1:2, and 1:3, respectively) and were separated by size exclusion chromatography. The measured molecular weight of each peak is indicated with arrows.
Figure 2. Crystal structure of Plk1 Polo box domain (PBD) and phosphorylated peptides derived from Emi2.(A) Domain organization of Plk1 and Emi2. Left: location of the kinase domain (KD) and the polo box domain (PBD) in Plk1 is indicated. Right: the location of the PBD-interacting part of Emi2 is highlighted in green. The phosphorylated threonine at amino acid positions 152 or 176 in Emi2 are also presented. ZBR: zinc-binding region. (B) Crystal structure of Plk1-PBD·Emi2146–177 (left) and Plk1-PBD·Emi2169–177. Plk1-PBD (blue) is shown as a cartoon Fig. and Emi2 fragments are shown as green sticks. Note that only electron density corresponding to amino acid residues 148–154 in Emi2146–177 is visible and was modeled. (C) Electrostatic surface representation (blue: positively charged, red: negatively charged) of Plk1-PBD bound to an Emi2-derived phosphopeptide. Left: Plk1-PBD·Emi2146–177, right: Plk1-PBD·Emi2169–177. (D) The surface of Plk1-PBD is colored according to residue conservation (conservation decreases from blue to red). Note that the Emi2-peptide binding regions including tyrosine-rich hydrophobic pockets and negatively charged regions binding Emi2156–169 are conserved (colored blue).
Figure 3. Testing the interactions of Emi2 with Plk1 based on Emi2 degradation after pathernogenetic activation of oocyte.(A)Scheme of Emi21–300-mCherry reporter constructs used in the study. It contains Plk1 binding sites and degron required by recognition of SCF-mediated ubiquitinylation. Thr152, Phe169 and Thr176 mutated as alanine are indicated as red. After in vitro transcriptions, cRNAs were injected into MII oocyte and subjected to SrCl2-mediated parthenogenetic activation and decrease of fluorescence from mCherry was monitored by time-lapse imaging of oocytes. (B) Time-lapse microscopy of parthenogenetically activated oocyte injected with Emi21–300-mCherry cRNAs. WT: wild type Emi21–300-mCherry cRNAs; T152A: site-directed mutant Emi21–300-mCherry containing Threonine152 to Alanine substitution; F169A: Phenylalanine169 to Alanine substitution; T176A: Threone176 to Alanine substitution; Representative figures of each groups corresponding 5, 25, 45, 65, 85 and 105 min after SrCl2 treatment were shown. (C) Quantification of fluorescence from oocyte injected with Emi21–300-mCherry cRNAs. Normalized fluorescence from oocytes injected with wild type (Black, n = 7), T152A (Green, n = 6), F169A (Blue, n = 4) and T176A (Red, n = 5) at each time points (5 min interval) were measured and average of each time points were plotted. Error bars indicate standard error of mean (S.E.M).
Figure 4. Peptidomimetics emulating the Plk1-PBD·Emi2 complex inhibit maturation of mouse oocytes.(A) Peptidomimetics resemble Plk1-PBD·Emi2 and bind to the Polo box domain (PBD) of Plk1. The compounds 103, 103-2, 103-5, 103-8, 103-10, 103-12, 103-13, 103-15 and 103-16 have been described previously34, and 699, 700, 701, 702, 703, 704, 705, 706 and 707 were characterized in this study. (B) Assessment of inhibitory activity of peptidomimetics on in vitro maturation (IVM) of mouse oocytes. Peptidomimetics were microinjected into immature mouse oocytes, which were subjected to IVM. GV: germinal vesicle, GVBD: germinal vesicle breakdown, MI: metaphase I, MII: metaphase II. Time (hr) required for maturation of an oocyte is indicated at the top of an arrow. Maturation status of mouse oocytes 12 hr after injection of a peptidomimetic was evaluated. At least three independent experiments were carried out and the ratios of oocytes at each developmental stage to all oocytes were plotted. Significant differences between the treatment groups and the control group are indicated by asterisk (***P ≤ 0.001, **P ≤ 0.01, *P ≤ 0.05). (C) Crystal structure of the Plk1-PBD·702 complex. PBD domain is shown in blue and peptidomimetic 702 is shown in green. In the enlarged circle, the structures of Emi2169–177 (violet) and peptidomimetic 702 (Green) are superimposed. Note that occupation of tyrosine-rich pockets by an alkylated imidazole ring (702) or a phenylalanine ring (Emi2169–177).
Figure 5. The peptidomimetic 103-8 impairs oocyte maturation and delays parthenogenetic activation of mouse oocytes.(A) Effects of 103-8 injections on oocyte maturation. Control (oocytes injected with unphosphorylated BG34) and 103-8-injected oocytes were stained for PLK1 (red), α-tubulin (green), and DNA (blue). Bar = 20 μm. (B) Effects of 103-8 injections on parthenogenetic activation of mouse oocytes. Mature metaphase II (MII) oocytes were microinjected with a control compound (orange) or 103-8 (blue), and subjected to parthenogenetic activation by SrCl2. The progression of parthenogenetic activation was assessed by the formation of a pronucleus (examination under an inverted microscope). Statistical significance was tested by chi-square test (**P ≤ 0.01, *P ≤ 0.05, and N.S. P > 0.05). (C) Time-lapse microscopy of parthenogenetically activated oocyte injected with Emi21–300-mCherry cRNAs. Control: wild type Emi21–300-mCherry cRNAs injected with control BG34 control; 103-8: Emi21–300-mCherry cRNAs injected with 103-8 peptidomimetic; BI2536: Emi21–300-mCherry cRNAs injected and subjected to SrCl2 treatment in the presence of 100 nM BI2536. Bar = 40 μm. (D) Normalized fluorescence from oocytes injected with control (Black, n = 9), 103-8 (Red, n = 12) and BI2536 (Blue, n = 3) at each time points (5 min interval) were measured and average of each time points were plotted. Error bars indicate standard error of mean (S.E.M).
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