Minimal structure of IRAK-1 to induce degradation of TRAF6 |
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| Institution: | 1. Clinical Laboratory, The Rizhao People''s Hospital Affiliated to Jining Medical University, Rizhao, Shandong, China;2. Department of Blood Transfusion, The Rizhao People''s Hospital Affiliated to Jining Medical University, Rizhao, Shandong, China;3. Department of Medical Image, The Rizhao People''s Hospital Affiliated to Jining Medical University, Rizhao, Shandong, China;4. Department of Anesthesiology, The Rizhao People''s Hospital Affiliated to Jining Medical University, Rizhao, Shandong, China;1. Department of Emergency, Hainan General Hospital, Hainan Affiliated Hospital of Hainan Medical University, Haikou 570311, Hainan Province, PR China;2. Department of Cardiology, Hainan General Hospital, Hainan Affiliated Hospital of Hainan Medical University, Haikou 570311, Hainan Province, PR China;3. Key Laboratory of Emergency and Trauma, Ministry of Education, Key Laboratory of Hainan Trauma and Disaster Rescue, The First Affiliated Hospital of Hainan Medical University, College of Emergency and Trauma, Hainan Medical University, Haikou, 571199, China;1. Department of Immunology, Military Hospital of Tunis, Montfleury – 1008, Tunis, Tunisia;2. Unit IMEC-Immunology Microbiology Environmental and Carcinogenesis, Faculty of Science of Bizerte, Tunisia;3. Department of Biology, Faculty of Science of Gafsa, University of Gafsa, Gafsa, Tunisia;4. Department of Intensive Care, Military Hospital of Tunis, Mont fleury – 1008, Tunis, Tunisia;5. Research Unit 17 DN05, Military Hospital of Tunis, Montfleury – 1008, Tunis, Tunisia;6. Faculty of Medicine, University Tunis El Manar, Tunis, Tunisia;1. Department of Emergency, The Second Affiliated Hospital of Nanchang University, Nanchang, Jiangxi 330038, China;2. Department of Gastroenterology, The Second Affiliated Hospital of Nanchang University, Nanchang, Jiangxi 330038, China;1. Viral Immunopathogenesis Laboratory, ICMR-National Institute for Research in Reproductive Health, Mumbai, Maharashtra, India;2. Department of Medicine, Grant Medical College & Sir J. J. Group of Hospitals, Mumbai, Maharashtra, India;1. Department of Biotechnology, SRM Institute of Science and Technology, Kattankulathur, Tamil Nadu 603203, India;2. Department of Clinical Pharmacology, SRM Medical College Hospital and Research Centre, Kattankulathur, Tamil Nadu 603203, India |
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| Abstract: | Excessive activation of Toll-like receptor (TLR) leads to sepsis. Inflammatory responses to various microbiological components are initiated via different TLR proteins, but all TLR signals are transmitted by TRAF6. We reported that TRAF6 associated with ubiquitinated IRAK-1 undergoes proteasome-mediated degradation, suggesting that IRAK-1 has a negative regulatory role in TLR signaling. Here, we investigated the minimal structural region of IRAK-1 needed for degradation of TRAF6. The IRAK-1 protein contains an N-terminal death domain (DD; amino acids 1–102), a serine/proline/threonine-rich ProST domain (amino acids 103–197), a central kinase domain with an activation loop (amino acids 198–522), and the C-terminal C1 and C2 domains (amino acids 523–712), which contain two and one putative TRAF6-binding (TB) sites, respectively. TRAF6 degradation was severely impaired by deletion of the DD or C1 domain, and a mutant (DC1) containing only the DD and C1 domains could induce TRAF6 degradation. IRAK-1 mutants lacking the N- or C-terminal amino acids of DD induced little degradation. Deletion or mutation of TB2 (amino acids 585–591) in the C1 domain also inhibited TRAF6 degradation. An IRAK-1 mutant possessing only DD and TB2 did not induce TRAF6 degradation, although a mutant in which a short spacer was inserted between DD and TB2 induced TRAF6 degradation, which and DC1-induced degradation were inhibited by proteasome inhibitors. All IRAK-1 mutants that induced TRAF6 degradation could be immunoprecipitated with TRAF6. Meanwhile, NF-κB activation was observed for all IRAK-1 mutants–including those that failed to induce degradation and was severely impaired only for a mutant carrying mutations in both TBs of C1. These results demonstrate that only DD and TB2 separated by an appropriate distance can induce TRAF6 degradation. Conformational analysis of this minimal structural unit may aid development of low molecular compounds that negatively regulate TLR signaling. |
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| Keywords: | IRAK-1 TRAF6 Proteasome NF-κB Death domain |
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