Peptide inhibitors of the anaphase promoting-complex that cause sensitivity to microtubule poison

<div><p>There is an interest in identifying Anaphase Promoting-Complex/Cyclosome (APC/C) inhibitors that lead to sensitivity to microtubule poisons as a strategy for targeting cancer cells. Using budding yeast <i>Saccharomyces cerevisiae</i>, peptides derived from the Mitotic Arrest Deficient 2 (Mad2)-binding motif of Cell Division Cycle 20 (Cdc20) were observed to inhibit both Cdc20- and CDC20 Homology 1 (Cdh1)-dependent APC/C activity. Over expression of peptides <i>in vivo</i> led to sensitivity to a microtubule poison and, in a recovery from a microtubule poison arrest, delayed degradation of yeast Securin protein Precocious Dissociation of Sisters 1 (Pds1). Peptides with mutations in the Cdc20 activating KILR-motif still bound APC/C, but lost the ability to inhibit APC/C <i>in vitro</i> and lost the ability to induce sensitivity to a microtubule poison <i>in vivo</i>. Thus, an APC/C binding and activation motif that promotes mitotic progression, namely the Cdc20 KILR-motif, can also function as an APC/C inhibitor when present in excess. Another activator for mitotic progression after recovery from microtubule poison is p31^comet, where a yeast predicted open-reading frame <i>YBR296C-A</i> encoding a 39 amino acid predicted protein was identified by homology to p31^comet, and named <u>T</u>iny <u>Y</u>east <u>C</u>omet 1 (<i>TYC1</i>). Tyc1 over expression resulted in sensitivity to microtubule poison. Tyc1 inhibited both APC/C^Cdc20 and APC/C^Cdh1 activities <i>in vitro</i> and bound to APC/C. A homologous peptide derived from human p31^comet bound to and inhibited yeast APC/C demonstrating evolutionary retention of these biochemical activities. Cdc20 Mad2-binding motif peptides and Tyc1 disrupted the ability of the co-factors Cdc20 and Cdh1 to bind to APC/C, and co-over expression of both together <i>in vivo</i> resulted in an increased sensitivity to microtubule poison. We hypothesize that Cdc20 Mad2-binding motif peptides, Tyc1 and human hp31 peptide can serve as novel molecular tools for investigating APC/C inhibition that leads to sensitivity to microtubule poison <i>in vivo</i>.</p></div

Mad2 binding motif peptides bind to APC/C.

<p>A) Wild type PQ65 and DQ36 pull-downs of APC/C at 20 μM peptide. Pre-coating the Avidin beads with biotin blocked the pull-down activity, but pre-coating APC/C with biotin had no effect. The addition of an excess of non-biotinylated DQ36 peptide was able to partially block the pull-down activity. B) Mutant DQ36-mut1 maintained the ability to pull-down APC/C, which was also partially blocked by an addition of excess DQ36 peptide. Mutant DQ36-mut2 was consistently observed to have partially lost APC/C binding activity relative to wild type DQ36 (n = 3).</p

Over expression of Tyc1 induced sensitivity to the microtubule poison benomyl.

<p>A) Over expression of Tyc1 gave rise to a benomyl sensitive phenotype. B) Over expression of Tyc1 increased benomyl sensitivity in a <i>mad2Δ</i> background.</p

Functional analysis of PQ65 peptides sufficient to inhibit APC/C activity.

<p>A) A schematic diagram of the amino acid residues from the N-terminus of Cdc20 containing the region of the KILR motif and Mad2-binding motif. A 65 amino acid peptide PQ65 (Cdc20 169–232) was designed and synthesized around the most highly conserved regions within fungi. B) Phosphor-images of APC/C^Cdc20 reaction time courses in the presence of buffer only or 220 μM of peptides. A significant level of APC/C^Cdc20 inhibition was observed in the presence of the PQ65, PQ65-mut1 or PQ65-mut2 peptides (n = 3). Relative to the wild type PQ65 peptide, PQ65-mut1 peptide displayed a loss of inhibitory activity at the 30 and 60 minute time points (n = 3). C) Phosphor-image of a titration dilutions series using PQ65 where an IC₅₀ of less than 50 μM was observed. D) Phosphor-images of APC/C^Cdh1 reaction time courses in the presence of buffer or 220 μM PQ65 peptide, where a significant level of APC/C^Cdh1 inhibition was observed (n = 3).</p

The human peptide hp31 inhibits budding yeast APC/C activity and binds to yeast APC/C.

<p>A) An alignment between budding yeast Tyc1 and the synthesized human hp31 peptide derived from human p31^comet. B) Phosphor-images show that 220 μM human hp31 peptide inhibits yeast APC/C^Cdc20. C) Phosphor-images show that 220 μM human hp31 peptide inhibits yeast APC/C^Cdh1. D) The human-derived hp31 can pull-down budding yeast APC/C at 20 μM peptide (n = 3).</p

Functional analysis of DQ36 peptides sufficient to inhibit APC/C activity.

<p>A) A schematic diagram of the amino acid residues from the N-terminus of Cdc20 containing the region of the KILR motif and Mad2-binding motif. A 36 amino acid peptide DQ36 (Cdc20 197–232) was designed and synthesized around the most highly conserved regions within fungi. B) Phosphor-images of APC/C^Cdc20 reaction time courses in the presence of buffer only or 220 μM of peptides. C) Phosphor-images of APC/C^Cdh1 reaction time courses in the presence of buffer only or 220 μM of peptides. D) Phosphor-image of a titration dilutions series using DQ36 where an IC₅₀ of about 150 μM was observed.</p

Mad2-binding motif peptides induce sensitivity to the microtubule poison benomyl when over expressed <i>in vivo</i>, which requires the KILR-motif, and leads to a delay in Pds1 degradation upon recovery from benomyl.

<p>A) Over expression of PQ65 or DQ36 <i>in vivo</i> leads to sensitivity to the microtubule poison benomyl. There was no significant difference observed in the absence of benomyl. B) The benomyl sensitivity phenotype was not observed when PQ65-mut1 or DQ36-mut1 peptides were over expressed where all 5 KILR-motif residues RILQY were changed to AAAAA. C) Over expression of DQ36 caused a delay in Pds1 degradation after cell cycle recovery from the microtubule poison benomyl as detected by Western blotting. Pds1 protein was detected using an anti-MYC antibody where the endogenous Pds1 had been epitope-tagged at the C-terminus with 13xMYC. An anti-actin antibody was used to probe for the levels of actin in each sample as a loading control.</p

Summary of the functional and APC/C inhibitor analysis on the C-box and Mad2-binding motif regions of Cdc20.

<p>A) The budding yeast KILR-motif is defined as RILQY. B) A summary of the APC/C^Cdc20 inhibition and APC/C binding data. The ability to inhibit the APC/C resides in both the N- and C-terminal regions of the PQ65 peptide, where both PL29 and SQ19 peptides contain an ability to inhibit APC/C even in the absence of the core KILR-motif region. In the DQ36-mut1 peptide, although the peptide does not fully inhibit the APC/C, the peptide retains the full ability to bind APC/C. In the DQ36-mut2 and SQ19-mut2 peptides there is a correlation between the loss of APC/C binding activity and a loss in the ability to inhibit APC/C.</p

Tyc1 inhibits APC/C activity.

<p>A) Tyc1 proteins tested for their ability to inhibit APC/C activity. Tyc1-mut1 has 4 conserved amino acid residues changed to alanine. Tyc1-mut2 has 5 conserved residues changed to alanine. B) Phosphor-images show that 220 μM Tyc1 inhibits APC/C^Cdc20 activity. Tyc1-mut1 also fully inhibited APC/C^Cdc20. Tyc1-mut2 partially lost the ability to inhibit APC/C^Cdc20. C) 220 μM Tyc1 inhibits APC/C^Cdh1 activity. Tyc1-mut1 also fully inhibited APC/C^Cdh1. Tyc1-mut2 displayed a slight loss in the ability to inhibit APC/C^Cdh1. D) Phosphor-image of a titration dilutions series using Tyc1 where an IC₅₀ of about 50 μM was observed.</p

Mad2-binding motif peptides and Tyc1 disrupt co-factor binding to APC/C and display an additive phenotype for microtubule poison sensitivity when co-ever expressed.

<p>A) ³⁵S-Methionine radio-labeled full-length Cdc20 or Cdh1 were made by IVT/T and pre-incubated with buffer only as a control, or Tyc1, DQ36 or PQ65 at a final concentration of 220 μM. Samples were mixed with enriched APC/C isolated on IgG beads and allowed to bind for 1 hour. Samples were washed and beads were boiled directly in protein sample buffer. The amount of APC/C-bound co-factor was measured by phosphor-imaging (n = 3). B) Co-over expression of PQ65 with Tyc1 <i>in vivo</i> leads to an increased sensitivity to the microtubule poison benomyl. In the presence of 12.5 μg/mL benomyl a significant decrease in the average colony number was observed (n = 5).</p