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Cancer cell death induced by novel small molecules degrading the TACC3 protein via the ubiquitin-proteasome pathway.

Ohoka N, Nagai K, Hattori T, Okuhira K, Shibata N, Cho N, Naito M - Cell Death Dis (2014)

Bottom Line: In this study, we designed and synthesized novel small molecules called SNIPER(TACC3)s, which target the spindle regulatory protein transforming acidic coiled-coil-3 (TACC3).SNIPER(TACC3)s induce poly-ubiquitylation and proteasomal degradation of TACC3 and reduce the TACC3 protein level in cells.Mechanistic analysis indicated that the ubiquitin ligase APC/C(CDH1) mediates the SNIPER(TACC3)-induced degradation of TACC3.

View Article: PubMed Central - PubMed

Affiliation: Division of Biochemistry and Molecular Biology, National Institute of Health Science, Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan.

ABSTRACT
The selective degradation of target proteins with small molecules is a novel approach to the treatment of various diseases, including cancer. We have developed a protein knockdown system with a series of hybrid small compounds that induce the selective degradation of target proteins via the ubiquitin-proteasome pathway. In this study, we designed and synthesized novel small molecules called SNIPER(TACC3)s, which target the spindle regulatory protein transforming acidic coiled-coil-3 (TACC3). SNIPER(TACC3)s induce poly-ubiquitylation and proteasomal degradation of TACC3 and reduce the TACC3 protein level in cells. Mechanistic analysis indicated that the ubiquitin ligase APC/C(CDH1) mediates the SNIPER(TACC3)-induced degradation of TACC3. Intriguingly, SNIPER(TACC3) selectively induced cell death in cancer cells expressing a larger amount of TACC3 protein than normal cells. These results suggest that protein knockdown of TACC3 by SNIPER(TACC3) is a potential strategy for treating cancers overexpressing the TACC3 protein.

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SNIPER(TACC3) physically interacts with APC/CCDH1 and increases the TACC3–APC/CCDH1 interaction. (a) Cell lysates were mixed with 100 μM of SNIPER(TACC3)-1, Me-BS or KHS108 and analyzed by a thermal shift assay. (b) Scheme of the experimental procedure to detect the SNIPER(TACC3)-mediated interaction of TACC3 and APC/CCDH1. (c) HT1080 cells were co-transfected with the expression vector of Flag-TACC3 and Myc-CDH1. After 40 h, cells were treated with 25 μM of MG132 in the presence or absence of 30 μM of SNIPER(TACC3)-1 for 3 h. Cells were lysed in immunoprecipitated (IP) lysis buffer and Flag-TACC3 was IP with an anti-FLAG antibody. The precipitates were eluted with 2 mM of KHS108, Me-BS or 4-OHT. Total cell lysates, immunoprecipitates after elution and eluted fractions were western blotted with the indicated antibodies. DMSO, dimethyl sulfoxide; Elution, eluted fraction; lysate, total lysate; ppt, immunoprecipitate after elution; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis
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fig4: SNIPER(TACC3) physically interacts with APC/CCDH1 and increases the TACC3–APC/CCDH1 interaction. (a) Cell lysates were mixed with 100 μM of SNIPER(TACC3)-1, Me-BS or KHS108 and analyzed by a thermal shift assay. (b) Scheme of the experimental procedure to detect the SNIPER(TACC3)-mediated interaction of TACC3 and APC/CCDH1. (c) HT1080 cells were co-transfected with the expression vector of Flag-TACC3 and Myc-CDH1. After 40 h, cells were treated with 25 μM of MG132 in the presence or absence of 30 μM of SNIPER(TACC3)-1 for 3 h. Cells were lysed in immunoprecipitated (IP) lysis buffer and Flag-TACC3 was IP with an anti-FLAG antibody. The precipitates were eluted with 2 mM of KHS108, Me-BS or 4-OHT. Total cell lysates, immunoprecipitates after elution and eluted fractions were western blotted with the indicated antibodies. DMSO, dimethyl sulfoxide; Elution, eluted fraction; lysate, total lysate; ppt, immunoprecipitate after elution; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Mentions: To confirm the physical interaction of SNIPER(TACC3) and APC/CCDH1, we performed a thermal shift assay that is based on the biophysical principle of ligand-induced change of thermal sensitivity of target proteins32 (Figure 4a). Multiple aliquots of the cell lysates were mixed with each compounds (SNIPER(TACC3)-1, Me-BS or KHS108) and heated to graded temperatures. After cooling, the lysates were centrifuged to precipitate unfolded proteins, and the supernatants were analyzed by western blotting. The thermal sensitivity of APC/CCDH1 components (APC3, APC11 and CDH1) were changed by addition of SNIPER(TACC3)-1, but not by Me-BS and KHS108. On the other hand, the thermal sensitivity of CDC20 was not affected by these compounds. These results indicate the physical interaction of SNIPER(TACC3) and APC/CCDH1.


Cancer cell death induced by novel small molecules degrading the TACC3 protein via the ubiquitin-proteasome pathway.

Ohoka N, Nagai K, Hattori T, Okuhira K, Shibata N, Cho N, Naito M - Cell Death Dis (2014)

SNIPER(TACC3) physically interacts with APC/CCDH1 and increases the TACC3–APC/CCDH1 interaction. (a) Cell lysates were mixed with 100 μM of SNIPER(TACC3)-1, Me-BS or KHS108 and analyzed by a thermal shift assay. (b) Scheme of the experimental procedure to detect the SNIPER(TACC3)-mediated interaction of TACC3 and APC/CCDH1. (c) HT1080 cells were co-transfected with the expression vector of Flag-TACC3 and Myc-CDH1. After 40 h, cells were treated with 25 μM of MG132 in the presence or absence of 30 μM of SNIPER(TACC3)-1 for 3 h. Cells were lysed in immunoprecipitated (IP) lysis buffer and Flag-TACC3 was IP with an anti-FLAG antibody. The precipitates were eluted with 2 mM of KHS108, Me-BS or 4-OHT. Total cell lysates, immunoprecipitates after elution and eluted fractions were western blotted with the indicated antibodies. DMSO, dimethyl sulfoxide; Elution, eluted fraction; lysate, total lysate; ppt, immunoprecipitate after elution; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4260729&req=5

fig4: SNIPER(TACC3) physically interacts with APC/CCDH1 and increases the TACC3–APC/CCDH1 interaction. (a) Cell lysates were mixed with 100 μM of SNIPER(TACC3)-1, Me-BS or KHS108 and analyzed by a thermal shift assay. (b) Scheme of the experimental procedure to detect the SNIPER(TACC3)-mediated interaction of TACC3 and APC/CCDH1. (c) HT1080 cells were co-transfected with the expression vector of Flag-TACC3 and Myc-CDH1. After 40 h, cells were treated with 25 μM of MG132 in the presence or absence of 30 μM of SNIPER(TACC3)-1 for 3 h. Cells were lysed in immunoprecipitated (IP) lysis buffer and Flag-TACC3 was IP with an anti-FLAG antibody. The precipitates were eluted with 2 mM of KHS108, Me-BS or 4-OHT. Total cell lysates, immunoprecipitates after elution and eluted fractions were western blotted with the indicated antibodies. DMSO, dimethyl sulfoxide; Elution, eluted fraction; lysate, total lysate; ppt, immunoprecipitate after elution; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis
Mentions: To confirm the physical interaction of SNIPER(TACC3) and APC/CCDH1, we performed a thermal shift assay that is based on the biophysical principle of ligand-induced change of thermal sensitivity of target proteins32 (Figure 4a). Multiple aliquots of the cell lysates were mixed with each compounds (SNIPER(TACC3)-1, Me-BS or KHS108) and heated to graded temperatures. After cooling, the lysates were centrifuged to precipitate unfolded proteins, and the supernatants were analyzed by western blotting. The thermal sensitivity of APC/CCDH1 components (APC3, APC11 and CDH1) were changed by addition of SNIPER(TACC3)-1, but not by Me-BS and KHS108. On the other hand, the thermal sensitivity of CDC20 was not affected by these compounds. These results indicate the physical interaction of SNIPER(TACC3) and APC/CCDH1.

Bottom Line: In this study, we designed and synthesized novel small molecules called SNIPER(TACC3)s, which target the spindle regulatory protein transforming acidic coiled-coil-3 (TACC3).SNIPER(TACC3)s induce poly-ubiquitylation and proteasomal degradation of TACC3 and reduce the TACC3 protein level in cells.Mechanistic analysis indicated that the ubiquitin ligase APC/C(CDH1) mediates the SNIPER(TACC3)-induced degradation of TACC3.

View Article: PubMed Central - PubMed

Affiliation: Division of Biochemistry and Molecular Biology, National Institute of Health Science, Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan.

ABSTRACT
The selective degradation of target proteins with small molecules is a novel approach to the treatment of various diseases, including cancer. We have developed a protein knockdown system with a series of hybrid small compounds that induce the selective degradation of target proteins via the ubiquitin-proteasome pathway. In this study, we designed and synthesized novel small molecules called SNIPER(TACC3)s, which target the spindle regulatory protein transforming acidic coiled-coil-3 (TACC3). SNIPER(TACC3)s induce poly-ubiquitylation and proteasomal degradation of TACC3 and reduce the TACC3 protein level in cells. Mechanistic analysis indicated that the ubiquitin ligase APC/C(CDH1) mediates the SNIPER(TACC3)-induced degradation of TACC3. Intriguingly, SNIPER(TACC3) selectively induced cell death in cancer cells expressing a larger amount of TACC3 protein than normal cells. These results suggest that protein knockdown of TACC3 by SNIPER(TACC3) is a potential strategy for treating cancers overexpressing the TACC3 protein.

Show MeSH
Related in: MedlinePlus