Uptake of a cell permeable G7-18NATE construct into cells and binding with the Grb7-SH2 domain

Nigus Dessalew Ambaye, Reece Chih Cian Lim, Daniel John Clayton, Menachem Joseph Gunzburg, John Timothy Price, Stephanie C Pero, David Nielsen Krag, Matthew Charles James Wilce, Marie Isabel Aguilar, Patrick Perlmutter, Jacqueline Anne Wilce

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13 Citations (Scopus)

Abstract

Grb7 is an adapter protein found to be overexpressed in several breast and other cancer cell types along with ErbB2. Grb7 is normally an interaction partner with focal adhesion kinase and in cancer cells also aberrantly interacts with ErbB2. It is thus implicated in the migratory and proliferative potential of cancer cells. Previous studies have shown that the phage display-derived cyclic nonphosphorylated inhibitor peptide, G7-18NATE, when linked to PenetratinA?, is able to interfere with the interaction of Grb7 with its upstream binding partners and to impact on both cell migration and proliferation. Here we report the synthesis of a biotinylated G7-18NATE covalently attached to just the last seven residues of PenetratinA? (G7-18NATE-P-Biotin). We demonstrate that this construct is taken up efficiently into MDA-MB-468 breast cancer cells and co-localises with Grb7 in the cytoplasm. We also used isothermal titration calorimetry to determine the binding affinity of G7-18NATE-P-Biotin to the Grb7-SH2 domain, and showed that it binds with micromolar affinity (Kd=14.4 i??M), similar to the affinity of G7-18NATE (Kd= 35.4 i??M). Together this shows that this shorter G7-18NATE-P-Biotin construct is suitable for further studies of the anti-proliferative and anti-migratory potential of this inhibitor.
Original languageEnglish
Pages (from-to)181 - 188
Number of pages8
JournalJournal of Peptide Science
Volume96
Issue number2
DOIs
Publication statusPublished - 2011

Cite this

Ambaye, Nigus Dessalew ; Lim, Reece Chih Cian ; Clayton, Daniel John ; Gunzburg, Menachem Joseph ; Price, John Timothy ; Pero, Stephanie C ; Krag, David Nielsen ; Wilce, Matthew Charles James ; Aguilar, Marie Isabel ; Perlmutter, Patrick ; Wilce, Jacqueline Anne. / Uptake of a cell permeable G7-18NATE construct into cells and binding with the Grb7-SH2 domain. In: Journal of Peptide Science. 2011 ; Vol. 96, No. 2. pp. 181 - 188.
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title = "Uptake of a cell permeable G7-18NATE construct into cells and binding with the Grb7-SH2 domain",
abstract = "Grb7 is an adapter protein found to be overexpressed in several breast and other cancer cell types along with ErbB2. Grb7 is normally an interaction partner with focal adhesion kinase and in cancer cells also aberrantly interacts with ErbB2. It is thus implicated in the migratory and proliferative potential of cancer cells. Previous studies have shown that the phage display-derived cyclic nonphosphorylated inhibitor peptide, G7-18NATE, when linked to PenetratinA?, is able to interfere with the interaction of Grb7 with its upstream binding partners and to impact on both cell migration and proliferation. Here we report the synthesis of a biotinylated G7-18NATE covalently attached to just the last seven residues of PenetratinA? (G7-18NATE-P-Biotin). We demonstrate that this construct is taken up efficiently into MDA-MB-468 breast cancer cells and co-localises with Grb7 in the cytoplasm. We also used isothermal titration calorimetry to determine the binding affinity of G7-18NATE-P-Biotin to the Grb7-SH2 domain, and showed that it binds with micromolar affinity (Kd=14.4 i??M), similar to the affinity of G7-18NATE (Kd= 35.4 i??M). Together this shows that this shorter G7-18NATE-P-Biotin construct is suitable for further studies of the anti-proliferative and anti-migratory potential of this inhibitor.",
author = "Ambaye, {Nigus Dessalew} and Lim, {Reece Chih Cian} and Clayton, {Daniel John} and Gunzburg, {Menachem Joseph} and Price, {John Timothy} and Pero, {Stephanie C} and Krag, {David Nielsen} and Wilce, {Matthew Charles James} and Aguilar, {Marie Isabel} and Patrick Perlmutter and Wilce, {Jacqueline Anne}",
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journal = "Journal of Peptide Science",
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Uptake of a cell permeable G7-18NATE construct into cells and binding with the Grb7-SH2 domain. / Ambaye, Nigus Dessalew; Lim, Reece Chih Cian; Clayton, Daniel John; Gunzburg, Menachem Joseph; Price, John Timothy; Pero, Stephanie C; Krag, David Nielsen; Wilce, Matthew Charles James; Aguilar, Marie Isabel; Perlmutter, Patrick; Wilce, Jacqueline Anne.

In: Journal of Peptide Science, Vol. 96, No. 2, 2011, p. 181 - 188.

Research output: Contribution to journalArticleResearchpeer-review

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T1 - Uptake of a cell permeable G7-18NATE construct into cells and binding with the Grb7-SH2 domain

AU - Ambaye, Nigus Dessalew

AU - Lim, Reece Chih Cian

AU - Clayton, Daniel John

AU - Gunzburg, Menachem Joseph

AU - Price, John Timothy

AU - Pero, Stephanie C

AU - Krag, David Nielsen

AU - Wilce, Matthew Charles James

AU - Aguilar, Marie Isabel

AU - Perlmutter, Patrick

AU - Wilce, Jacqueline Anne

PY - 2011

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N2 - Grb7 is an adapter protein found to be overexpressed in several breast and other cancer cell types along with ErbB2. Grb7 is normally an interaction partner with focal adhesion kinase and in cancer cells also aberrantly interacts with ErbB2. It is thus implicated in the migratory and proliferative potential of cancer cells. Previous studies have shown that the phage display-derived cyclic nonphosphorylated inhibitor peptide, G7-18NATE, when linked to PenetratinA?, is able to interfere with the interaction of Grb7 with its upstream binding partners and to impact on both cell migration and proliferation. Here we report the synthesis of a biotinylated G7-18NATE covalently attached to just the last seven residues of PenetratinA? (G7-18NATE-P-Biotin). We demonstrate that this construct is taken up efficiently into MDA-MB-468 breast cancer cells and co-localises with Grb7 in the cytoplasm. We also used isothermal titration calorimetry to determine the binding affinity of G7-18NATE-P-Biotin to the Grb7-SH2 domain, and showed that it binds with micromolar affinity (Kd=14.4 i??M), similar to the affinity of G7-18NATE (Kd= 35.4 i??M). Together this shows that this shorter G7-18NATE-P-Biotin construct is suitable for further studies of the anti-proliferative and anti-migratory potential of this inhibitor.

AB - Grb7 is an adapter protein found to be overexpressed in several breast and other cancer cell types along with ErbB2. Grb7 is normally an interaction partner with focal adhesion kinase and in cancer cells also aberrantly interacts with ErbB2. It is thus implicated in the migratory and proliferative potential of cancer cells. Previous studies have shown that the phage display-derived cyclic nonphosphorylated inhibitor peptide, G7-18NATE, when linked to PenetratinA?, is able to interfere with the interaction of Grb7 with its upstream binding partners and to impact on both cell migration and proliferation. Here we report the synthesis of a biotinylated G7-18NATE covalently attached to just the last seven residues of PenetratinA? (G7-18NATE-P-Biotin). We demonstrate that this construct is taken up efficiently into MDA-MB-468 breast cancer cells and co-localises with Grb7 in the cytoplasm. We also used isothermal titration calorimetry to determine the binding affinity of G7-18NATE-P-Biotin to the Grb7-SH2 domain, and showed that it binds with micromolar affinity (Kd=14.4 i??M), similar to the affinity of G7-18NATE (Kd= 35.4 i??M). Together this shows that this shorter G7-18NATE-P-Biotin construct is suitable for further studies of the anti-proliferative and anti-migratory potential of this inhibitor.

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JO - Journal of Peptide Science

JF - Journal of Peptide Science

SN - 1075-2617

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