검색 상세

Identification and functional characterization of RIPK3-binding protein in necroptosis

  • 박한희 (아주대학교 일반대학원)

초록/요약

Necroptosis is a type of programmed cell death induced by various stress signals, such as the tumor necrosis factor (TNF) family, Toll-like receptor (TLR), interferons (IFNs), or lipopolysaccharide (LPS). Such stimuli cause the formation of necrosome complex containing receptor interaction protein kinases 1, 3 (RIPK1, RIPK3) and mixed lineage kinase domain-like (MLKL), which is essential for death to proceed. RIPK3-dependent plasma membrane localization of MLKL is necessary for necroptosis, and leads to plasma membrane disruption. In addition to MLKL, it has been identified several metabolic enzymes in screening for interactions with RIPK3 but there are not many proteins are identified. In my study, I conducted two approaches to identify novel binding partner of RIPK3. In proteome microarrays, I found that pellino E3 ubiquitin protein ligase 1 (PELI1) as a novel direct binding partner of RIPK3. PELI1 targets RIPK3 protein for proteasome-dependent degradation. I identified RIPK3 K363 as the main target for PELI1-mediated ubiquitylation by mass spectrometric analysis. PELI1 associates with, and then preferentially target, kinase-active RIPK3. Further investigation revealed that phosphorylation of RIPK3 on T182, which only occurs in kinase-active RIPK3 leads to interaction with the FHA (forkhead associated) domain of PELI1. PELI1-mediated RIPK3 degradation effectively prevents cell death triggered by RIPK3 hyperactivation. Importantly, upregulated RIPK3 expression in keratinocytes from toxic epidermal necrolysis (TEN) patients is correlated with low expression of PELI1, suggesting that loss of PELI1 may play a role in the pathogenesis of TEN. So, I propose that PELI1 may function to control the inadvertent activation of RIPK3, thus preventing aberrant cell death and maintaining cellular homeostasis. Next, in Tandem-affinity purification linked to mass spectrometry (TAP-MASS) analysis, I found Tripartite Motif Protein 28 (TRIM28) as a RIPK3 interacting protein. The co-repressor function of TRIM28 have been shown recently to be linked to the development of various cancers, such as non-small cell lung cancer, breast cancer, cervical cancer, colon cancer, gastric cancer, and ovarian cancer. In this study, I found that TRIM28 is a negative transcriptional regulator that is itself negatively regulated when cells undergoing necroptosis continue de novo synthesis of immunostimulatory cytokines. TRIM28 antagonizes NF-κB transactivation independent of p65 chromatin occupancy, but RIPK3 activation-mediated phosphorylation of TRIM28 at serine 473 facilitates its derepression. Moreover, RIPK3 activation triggers a remarkable reduction in TRIM28 binding events in chromatin that leads to increased other transcriptional factor activity, such as SOX9. These results revealed a new necroptosis-mediated transcription circuit that is modulated by RIPK3 activation-dependent de-repression of TRIM28, which provides a mechanism to promote robust anti-tumor immunity and contributes to tumor immunogenicity.

more

목차

PARTⅠPELI1 Selectively Targets Kinase-Active RIPK3 for Ubiquitylation-Dependent Proteasomal Degradation 1
Ⅰ. INTRODUCTION 2
Ⅱ. MATERIALS AND METHODS 4
1. Cell lines and culture conditions. 4
2. Antibodies and chemical reagents. 4
3. Plasmid construction, mutagenesis and transfection 5
4. siRNA, shRNA and reverse transcription-PCR (RT-PCR) 5
5. Cytotoxicity Assays 6
6. Immunoprecipitation and immunoblot analysis 6
7. In vitro ubiquitylation assay 6
8. Protein Microarray 7
9. Mass Spectrometric Analysis 8
10. Purification of recombinant proteins 8
11. In vitro kinase assays 8
12. Immunohistochemistry and Image analysis 9
13. Statistical analysis 10
Ⅲ. RESULTS 11
1. PELI1 is a novel binding partner of RIPK3 11
2. PELI1 causes proteasome-dependent degradation of RIPK3 14
3. PELI1 mediates K48-linked RIPK3 polyubiquitylation 17
4. The PELI1 RING-like domain is required for RIPK3 ubiquitylation and degradation 22
5. The PELI1 FHA domain mediates its interaction with RIPK3 25
6. A lower-mobility phospho-RIPK3 species is preferentially recognized by PELI1 29
7. RIPK3 kinase activity is required for its interaction with PELI1, but not CHIP 31
8. pT 182 is important for regulating RIPK3 kinase activity 36
9. Disruption in T182 inhibits S227 auto-phosphorylation and TNF-induced necroptosis 36
10. PELI1-mediated RIPK3 degradation abolishes necroptotic cell death 40
11. Low expression of PELI1 potentiates RIPK3 expression in TEN keratinocytes 46
Ⅳ. Discussions 49 
PARTⅡ. RIPK3 activation induces TRIM28 derepression in cancer cells and enhances the anti-tumor microenvironment 52
Ⅰ. INTRODUCTION 53
Ⅱ. MATERIALS AND METHODS 56
1. Cell lines and culture conditions 56
2. Lentiviral shRNA experiments 56
3. Primary culture and activation of BMDCs 57
4. Antibodies and chemical reagents 57
5. Plasmid construction and transfection 58
6. Cytotoxicity assays 58
7. Immunoprecipitation and immunoblot analysis 58
8. Cell fractionation assay 59
9. TAP & MASS analysis 59
10. TCGA analysis 60
11. ChIP-Seq library construction and analysis 60
12. Immunofluorescence analysis 60
13. Flow cytometry 61
14. Real-time PCR 61
15. NF-κB reporter assay 61
16. Human gastric tumor tissue preparation 62
17. Immunohistochemistry 62
18. Quantification and statistical analysis 62
Ⅲ. RESULTS 63
1. NF-κB transactivation alone is insufficient to explain necroptosis-specific cytokine production. 63
2. TRIM28 is a transcriptional regulator that interacts kinase-active RIPK3. 69
3. TRIM28 antagonizes NF-κB transactivation independent of p65 chromatin occupancy. 72
4. RIPK3 activation induces TRIM28 phosphorylation at serine 473. 75
5. RIPK3-dependent phosphorylation of TRIM28 induces enhanced transcriptional activity. 79
6. TRIM28 negatively regulates necrosome-induced cytokine production. 83
7. Derepression of TRIM28 leads to synthesis of immunostimulatory cytokines 87
Ⅳ. Discussions 91
REFERENCE 100
국문요약 111

more
반출 Meta View 목록