Two Cysteines in Raf Kinase Inhibitor Protein Make Differential Contributions to Structural Dynamics In Vitro
As a scaffolding protein, Raf kinase binding protein (RKIP) is involved in a variety of cellular pathways, including the Raf–MEK–ERK-cascade. It acts as a negative regulator by binding to its partners, making it an attractive target in the development of therapeutic strategies for cancer. Despite it...
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2025-01-01
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| author | Hyun Sang Cho Mohammad Faysal Al Mazid Eun-Young Lee Md Abu Rayhan Hyoun Sook Kim Byung Il Lee Hye Jin You |
| author_facet | Hyun Sang Cho Mohammad Faysal Al Mazid Eun-Young Lee Md Abu Rayhan Hyoun Sook Kim Byung Il Lee Hye Jin You |
| author_sort | Hyun Sang Cho |
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| description | As a scaffolding protein, Raf kinase binding protein (RKIP) is involved in a variety of cellular pathways, including the Raf–MEK–ERK-cascade. It acts as a negative regulator by binding to its partners, making it an attractive target in the development of therapeutic strategies for cancer. Despite its structural stability as a monomer, RKIP may form a dimer, resulting in the switching of binding partners. It is still unclear how RKIP switches between monomeric and dimeric forms. Here, we identified the role of cysteine 133 in RKIP structural dynamics using recombinant human RKIP (rhRKIP) proteins purified from <i>Escherichia coli</i> BL21(DE3) cells. Mutation of alanine or serine instead of cysteine in RKIP proteins did not affect the biochemical characteristics, while dynamic light scattering and liquid chromatography (LC) quadrupole time-of-flight (Q-TOF) mass spectrometry (MS) suggested distinct peaks in solution, which were identified via LC–MS/MS analyses, and further clarified the role of cysteine in RKIP dimerization. rhRKIP dimer formation was abrogated by a 32-aa peptide mimicking the region between two RKIP proteins for dimerization. In addition, the 32-aa peptide and its short derivatives were investigated for effects on cancer cell viability. Taken together, our findings suggest that it may be possible to regulate RKIP function by controlling its dynamics with reducing agents, which could aid the targeting of cancer cells. |
| format | Article |
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| institution | Kabale University |
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| language | English |
| publishDate | 2025-01-01 |
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| spelling | doaj-art-886584ca260f4dc79b4ce3c5ceabba922025-01-24T13:43:51ZengMDPI AGMolecules1420-30492025-01-0130238410.3390/molecules30020384Two Cysteines in Raf Kinase Inhibitor Protein Make Differential Contributions to Structural Dynamics In VitroHyun Sang Cho0Mohammad Faysal Al Mazid1Eun-Young Lee2Md Abu Rayhan3Hyoun Sook Kim4Byung Il Lee5Hye Jin You6Cancer Microenvironment Branch, Division of Cancer Biology, Research Institute, National Cancer Center, Goyang-si 10408, Republic of KoreaDepartment of Cancer Biomedical Science, National Cancer Center Graduate School of Cancer Science and Policy, National Cancer Center, Goyang-si 10408, Republic of KoreaCancer Microenvironment Branch, Division of Cancer Biology, Research Institute, National Cancer Center, Goyang-si 10408, Republic of KoreaDepartment of Cancer Biomedical Science, National Cancer Center Graduate School of Cancer Science and Policy, National Cancer Center, Goyang-si 10408, Republic of KoreaTargeted Therapy Branch, Division of Precision Medicine, Research Institute, National Cancer Center, Goyang-si 10408, Republic of KoreaDepartment of Cancer Biomedical Science, National Cancer Center Graduate School of Cancer Science and Policy, National Cancer Center, Goyang-si 10408, Republic of KoreaCancer Microenvironment Branch, Division of Cancer Biology, Research Institute, National Cancer Center, Goyang-si 10408, Republic of KoreaAs a scaffolding protein, Raf kinase binding protein (RKIP) is involved in a variety of cellular pathways, including the Raf–MEK–ERK-cascade. It acts as a negative regulator by binding to its partners, making it an attractive target in the development of therapeutic strategies for cancer. Despite its structural stability as a monomer, RKIP may form a dimer, resulting in the switching of binding partners. It is still unclear how RKIP switches between monomeric and dimeric forms. Here, we identified the role of cysteine 133 in RKIP structural dynamics using recombinant human RKIP (rhRKIP) proteins purified from <i>Escherichia coli</i> BL21(DE3) cells. Mutation of alanine or serine instead of cysteine in RKIP proteins did not affect the biochemical characteristics, while dynamic light scattering and liquid chromatography (LC) quadrupole time-of-flight (Q-TOF) mass spectrometry (MS) suggested distinct peaks in solution, which were identified via LC–MS/MS analyses, and further clarified the role of cysteine in RKIP dimerization. rhRKIP dimer formation was abrogated by a 32-aa peptide mimicking the region between two RKIP proteins for dimerization. In addition, the 32-aa peptide and its short derivatives were investigated for effects on cancer cell viability. Taken together, our findings suggest that it may be possible to regulate RKIP function by controlling its dynamics with reducing agents, which could aid the targeting of cancer cells.https://www.mdpi.com/1420-3049/30/2/384Raf kinase inhibitor proteindimerizationcysteinecancer |
| spellingShingle | Hyun Sang Cho Mohammad Faysal Al Mazid Eun-Young Lee Md Abu Rayhan Hyoun Sook Kim Byung Il Lee Hye Jin You Two Cysteines in Raf Kinase Inhibitor Protein Make Differential Contributions to Structural Dynamics In Vitro Molecules Raf kinase inhibitor protein dimerization cysteine cancer |
| title | Two Cysteines in Raf Kinase Inhibitor Protein Make Differential Contributions to Structural Dynamics In Vitro |
| title_full | Two Cysteines in Raf Kinase Inhibitor Protein Make Differential Contributions to Structural Dynamics In Vitro |
| title_fullStr | Two Cysteines in Raf Kinase Inhibitor Protein Make Differential Contributions to Structural Dynamics In Vitro |
| title_full_unstemmed | Two Cysteines in Raf Kinase Inhibitor Protein Make Differential Contributions to Structural Dynamics In Vitro |
| title_short | Two Cysteines in Raf Kinase Inhibitor Protein Make Differential Contributions to Structural Dynamics In Vitro |
| title_sort | two cysteines in raf kinase inhibitor protein make differential contributions to structural dynamics in vitro |
| topic | Raf kinase inhibitor protein dimerization cysteine cancer |
| url | https://www.mdpi.com/1420-3049/30/2/384 |
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