Identification of Inflammatory Proteomics Networks of Toll-like Receptor 4 through Immunoprecipitation-Based Chemical Cross-Linking Proteomics
Toll-like receptor 4 (TLR4) is a receptor on an immune cell that can recognize the invasion of bacteria through their attachment with bacterial lipopolysaccharides (LPS). Hence, LPS is a pro-immune response stimulus. On the other hand, statins are lipid-lowering drugs and can also lower immune cell...
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| Format: | Article |
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MDPI AG
2022-09-01
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| Series: | Proteomes |
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| Online Access: | https://www.mdpi.com/2227-7382/10/3/31 |
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| author | A. D. A. Shahinuzzaman Abu Hena Mostafa Kamal Jayanta K. Chakrabarty Aurchie Rahman Saiful M. Chowdhury |
| author_facet | A. D. A. Shahinuzzaman Abu Hena Mostafa Kamal Jayanta K. Chakrabarty Aurchie Rahman Saiful M. Chowdhury |
| author_sort | A. D. A. Shahinuzzaman |
| collection | DOAJ |
| description | Toll-like receptor 4 (TLR4) is a receptor on an immune cell that can recognize the invasion of bacteria through their attachment with bacterial lipopolysaccharides (LPS). Hence, LPS is a pro-immune response stimulus. On the other hand, statins are lipid-lowering drugs and can also lower immune cell responses. We used human embryonic kidney (HEK 293) cells engineered to express HA-tagged TLR-4 upon treatment with LPS, statin, and both statin and LPS to understand the effect of pro- and anti-inflammatory responses. We performed a monoclonal antibody (mAb) directed co-immunoprecipitation (CO-IP) of HA-tagged TLR4 and its interacting proteins in the HEK 293 extracted proteins. We utilized an ETD cleavable chemical cross-linker to capture weak and transient interactions with TLR4 protein. We tryptic digested immunoprecipitated and cross-linked proteins on beads, followed by liquid chromatography–mass spectrometry (LC-MS/MS) analysis of the peptides. Thus, we utilized the label-free quantitation technique to measure the relative expression of proteins between treated and untreated samples. We identified 712 proteins across treated and untreated samples and performed protein network analysis using Ingenuity Pathway Analysis (IPA) software to reveal their protein networks. After filtering and evaluating protein expression, we identified macrophage myristoylated alanine-rich C kinase substrate (MARCKSL1) and creatine kinase proteins as a potential part of the inflammatory networks of TLR4. The results assumed that MARCKSL1 and creatine kinase proteins might be associated with a statin-induced anti-inflammatory response due to possible interaction with the TLR4. |
| first_indexed | 2024-03-09T22:40:42Z |
| format | Article |
| id | doaj.art-81073e4f16b84f008b13bfabb731f2b1 |
| institution | Directory Open Access Journal |
| issn | 2227-7382 |
| language | English |
| last_indexed | 2024-03-09T22:40:42Z |
| publishDate | 2022-09-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Proteomes |
| spelling | doaj.art-81073e4f16b84f008b13bfabb731f2b12023-11-23T18:39:15ZengMDPI AGProteomes2227-73822022-09-011033110.3390/proteomes10030031Identification of Inflammatory Proteomics Networks of Toll-like Receptor 4 through Immunoprecipitation-Based Chemical Cross-Linking ProteomicsA. D. A. Shahinuzzaman0Abu Hena Mostafa Kamal1Jayanta K. Chakrabarty2Aurchie Rahman3Saiful M. Chowdhury4Department of Chemistry and Biochemistry, The University of Texas at Arlington, Arlington, TX 76019, USADepartment of Chemistry and Biochemistry, The University of Texas at Arlington, Arlington, TX 76019, USADepartment of Chemistry and Biochemistry, The University of Texas at Arlington, Arlington, TX 76019, USADepartment of Chemistry and Biochemistry, The University of Texas at Arlington, Arlington, TX 76019, USADepartment of Chemistry and Biochemistry, The University of Texas at Arlington, Arlington, TX 76019, USAToll-like receptor 4 (TLR4) is a receptor on an immune cell that can recognize the invasion of bacteria through their attachment with bacterial lipopolysaccharides (LPS). Hence, LPS is a pro-immune response stimulus. On the other hand, statins are lipid-lowering drugs and can also lower immune cell responses. We used human embryonic kidney (HEK 293) cells engineered to express HA-tagged TLR-4 upon treatment with LPS, statin, and both statin and LPS to understand the effect of pro- and anti-inflammatory responses. We performed a monoclonal antibody (mAb) directed co-immunoprecipitation (CO-IP) of HA-tagged TLR4 and its interacting proteins in the HEK 293 extracted proteins. We utilized an ETD cleavable chemical cross-linker to capture weak and transient interactions with TLR4 protein. We tryptic digested immunoprecipitated and cross-linked proteins on beads, followed by liquid chromatography–mass spectrometry (LC-MS/MS) analysis of the peptides. Thus, we utilized the label-free quantitation technique to measure the relative expression of proteins between treated and untreated samples. We identified 712 proteins across treated and untreated samples and performed protein network analysis using Ingenuity Pathway Analysis (IPA) software to reveal their protein networks. After filtering and evaluating protein expression, we identified macrophage myristoylated alanine-rich C kinase substrate (MARCKSL1) and creatine kinase proteins as a potential part of the inflammatory networks of TLR4. The results assumed that MARCKSL1 and creatine kinase proteins might be associated with a statin-induced anti-inflammatory response due to possible interaction with the TLR4.https://www.mdpi.com/2227-7382/10/3/31Toll-like receptor 4human embryonic kidneyprotein interactionsproteomicsmass spectrometrymacrophage myristoylated alanine-rich C kinase substrate |
| spellingShingle | A. D. A. Shahinuzzaman Abu Hena Mostafa Kamal Jayanta K. Chakrabarty Aurchie Rahman Saiful M. Chowdhury Identification of Inflammatory Proteomics Networks of Toll-like Receptor 4 through Immunoprecipitation-Based Chemical Cross-Linking Proteomics Proteomes Toll-like receptor 4 human embryonic kidney protein interactions proteomics mass spectrometry macrophage myristoylated alanine-rich C kinase substrate |
| title | Identification of Inflammatory Proteomics Networks of Toll-like Receptor 4 through Immunoprecipitation-Based Chemical Cross-Linking Proteomics |
| title_full | Identification of Inflammatory Proteomics Networks of Toll-like Receptor 4 through Immunoprecipitation-Based Chemical Cross-Linking Proteomics |
| title_fullStr | Identification of Inflammatory Proteomics Networks of Toll-like Receptor 4 through Immunoprecipitation-Based Chemical Cross-Linking Proteomics |
| title_full_unstemmed | Identification of Inflammatory Proteomics Networks of Toll-like Receptor 4 through Immunoprecipitation-Based Chemical Cross-Linking Proteomics |
| title_short | Identification of Inflammatory Proteomics Networks of Toll-like Receptor 4 through Immunoprecipitation-Based Chemical Cross-Linking Proteomics |
| title_sort | identification of inflammatory proteomics networks of toll like receptor 4 through immunoprecipitation based chemical cross linking proteomics |
| topic | Toll-like receptor 4 human embryonic kidney protein interactions proteomics mass spectrometry macrophage myristoylated alanine-rich C kinase substrate |
| url | https://www.mdpi.com/2227-7382/10/3/31 |
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