Analysis of p53-Independent Functions of the Mdm2-MdmX Complex Using Data-Independent Acquisition-Based Profiling
Background: We utilized data-independent acquisition (DIA) to study the poorly understood biology of Mdm2 and MdmX in a p53-null context. Mdm2 and MdmX form an E3-ligase complex that has as its most well-studied function the negative regulation of the tumor suppressor p53; however, it is also known...
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| Language: | English |
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MDPI AG
2025-05-01
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| Series: | Proteomes |
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| Online Access: | https://www.mdpi.com/2227-7382/13/2/18 |
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| author | Anu Jain Rafaela Muniz de Queiroz Jayanta K. Chakrabarty Karl A. T. Makepeace Carol Prives Lewis M. Brown |
| author_facet | Anu Jain Rafaela Muniz de Queiroz Jayanta K. Chakrabarty Karl A. T. Makepeace Carol Prives Lewis M. Brown |
| author_sort | Anu Jain |
| collection | DOAJ |
| description | Background: We utilized data-independent acquisition (DIA) to study the poorly understood biology of Mdm2 and MdmX in a p53-null context. Mdm2 and MdmX form an E3-ligase complex that has as its most well-studied function the negative regulation of the tumor suppressor p53; however, it is also known to interact with many other proteins in a p53-independent manner. Methods: In this work, small-molecule and siRNA-based technology were used to modify Mdm2/MdmX activity in a human non-small-cell lung carcinoma cell line lacking p53 expression. Study of the proteome of these cells helped identify biological processes where Mdm2 and MdmX may play roles in a p53-independent manner. Proteins from H1299 cells, treated with the drug MEL23 or siRNA against Mdm2 or MdmX, were analyzed. Results: Protein ontology and function were analyzed, revealing which pathways are affected by modulation of the proteins that form the complex. Insights into how those functions are dependent on the activity of the complex also gained via comparisons among the three groups of samples. Conclusions: We selected a potential target from the DIA analysis and validated it by immunoblotting and qPCR, and this allows us to demonstrate a new interaction partner of the Mdm2-MdmX complex in human cells. |
| format | Article |
| id | doaj-art-9f9906cf0617475ab7c59bcddf853810 |
| institution | OA Journals |
| issn | 2227-7382 |
| language | English |
| publishDate | 2025-05-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Proteomes |
| spelling | doaj-art-9f9906cf0617475ab7c59bcddf8538102025-08-20T02:21:47ZengMDPI AGProteomes2227-73822025-05-011321810.3390/proteomes13020018Analysis of p53-Independent Functions of the Mdm2-MdmX Complex Using Data-Independent Acquisition-Based ProfilingAnu Jain0Rafaela Muniz de Queiroz1Jayanta K. Chakrabarty2Karl A. T. Makepeace3Carol Prives4Lewis M. Brown5Department of Biological Sciences, Columbia University, New York, NY 10027, USADepartment of Biological Sciences, Columbia University, New York, NY 10027, USADepartment of Biological Sciences, Columbia University, New York, NY 10027, USADepartment of Biological Sciences, Columbia University, New York, NY 10027, USADepartment of Biological Sciences, Columbia University, New York, NY 10027, USADepartment of Biological Sciences, Columbia University, New York, NY 10027, USABackground: We utilized data-independent acquisition (DIA) to study the poorly understood biology of Mdm2 and MdmX in a p53-null context. Mdm2 and MdmX form an E3-ligase complex that has as its most well-studied function the negative regulation of the tumor suppressor p53; however, it is also known to interact with many other proteins in a p53-independent manner. Methods: In this work, small-molecule and siRNA-based technology were used to modify Mdm2/MdmX activity in a human non-small-cell lung carcinoma cell line lacking p53 expression. Study of the proteome of these cells helped identify biological processes where Mdm2 and MdmX may play roles in a p53-independent manner. Proteins from H1299 cells, treated with the drug MEL23 or siRNA against Mdm2 or MdmX, were analyzed. Results: Protein ontology and function were analyzed, revealing which pathways are affected by modulation of the proteins that form the complex. Insights into how those functions are dependent on the activity of the complex also gained via comparisons among the three groups of samples. Conclusions: We selected a potential target from the DIA analysis and validated it by immunoblotting and qPCR, and this allows us to demonstrate a new interaction partner of the Mdm2-MdmX complex in human cells.https://www.mdpi.com/2227-7382/13/2/18data-independent acquisitionlabel-free proteomicsMdm2MdmXp53-independent functionscancer |
| spellingShingle | Anu Jain Rafaela Muniz de Queiroz Jayanta K. Chakrabarty Karl A. T. Makepeace Carol Prives Lewis M. Brown Analysis of p53-Independent Functions of the Mdm2-MdmX Complex Using Data-Independent Acquisition-Based Profiling Proteomes data-independent acquisition label-free proteomics Mdm2 MdmX p53-independent functions cancer |
| title | Analysis of p53-Independent Functions of the Mdm2-MdmX Complex Using Data-Independent Acquisition-Based Profiling |
| title_full | Analysis of p53-Independent Functions of the Mdm2-MdmX Complex Using Data-Independent Acquisition-Based Profiling |
| title_fullStr | Analysis of p53-Independent Functions of the Mdm2-MdmX Complex Using Data-Independent Acquisition-Based Profiling |
| title_full_unstemmed | Analysis of p53-Independent Functions of the Mdm2-MdmX Complex Using Data-Independent Acquisition-Based Profiling |
| title_short | Analysis of p53-Independent Functions of the Mdm2-MdmX Complex Using Data-Independent Acquisition-Based Profiling |
| title_sort | analysis of p53 independent functions of the mdm2 mdmx complex using data independent acquisition based profiling |
| topic | data-independent acquisition label-free proteomics Mdm2 MdmX p53-independent functions cancer |
| url | https://www.mdpi.com/2227-7382/13/2/18 |
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