Hsp70, IRF7, IKKε의 상호 작용을 통해 Type I 인터페론 분비를 저해하는 조절 기전 규명 : Negative Regulation of Type I Interferon by Hsp70 through Interaction with IRF7 and IKKε

다국어 초록 (Multilingual Abstract)

Background: When pathogens invade cells, they secrete type I interferon (type I IFN) which is the key player of innate immune response.
Interferon regulatory factor 7 (IRF7) is a major transcription factor of type I IFN, especially interferon alpha and plays an important role for preventing the spread of virus. However, uncontrolled secretion of type I IFN induces auto-immune responses and inflammation. So it is important to keep the balance of type I IFN secretion. Therefore, IRF7 must be controlled tightly. But the mechanism of IRF7 inactivation has not been thoroughly investigated yet.
Heat shock protein 70 (Hsp70) are well conserved protein families in most organisms from prokaryotes to mammalian and well known for chaperone function. Recently, many researches reveal other function related in immune responses. Especially anti-inflammatory effect and immune-suppressive roles of hsp70 were investigated intensively. However the function of hsp70 as a regulator of IFN secretory-pathway is still remained unrevealed.
Purpose: I deciphered novel function of hsp70 as a negative regulator for inactivation of IRF7 and the mechanisms of suppression.
Results: First I found that activity of IFNα6 promoter was inhibited when the temperature increases in physiological range. I hypothesized that the transcription of IFNα6 might be suppressed by inhibiting IRF7 activity.
I analyzed mass spectrometry to find candidates and found hsp70 and hsp90 as binding partners to IRF7. To investigate effect of hsp70 and hsp90 over the IRF7 activity, I measured IFNα6 promoter activity via luciferase assays with Sendai virus (SV) infection or IKKε co-transfection using 293T cells. Hsp70 overexpression inhibited both SV and IKKε-induced IFNα6 promoter activation. Next, I generated hsp70 KO cell line using CRISPR-Cas9 system to confirm the involvement of hsp70 in IFNα expression inhibition. The expression level of IFNα stimulated by SV in hsp70 KO cell line was significantly higher than parental 293T cell line.
To elucidate the molecular mechanism responsible for the hsp70-mediated inhibition of IFNα production via IRF7, I checked the phosphorylation mechanism of IRF7. I confirmed that the higher concentration of IKKε, the more activity of IFNα6 promoter was observed, but the higher concentration of hsp70, the less activity of IFNα6 promoter was observed.
Next, I generated truncates that functional domain of IRF7 and hsp70 to identify the domain within IRF7 required for its interaction with hsp70 by GST pull down assay. I found that inhibition domain (INH) of IRF7 interacted with the substrate binding domainβ (SBDβ) of hsp70.
Conclusion: In this thesis, I identified that this interaction had important role in negative regulation of IRF7 by a physical blocking the interaction between IRF7 and IKKε. In other words, this is the first identification that hsp70 regulates the expression of IFNα through the control of IRF7 phosphorylation. Hsp70 is ubiquitously expressed in whole cells, so the regulation of IRF7 to dampen the excessive response of IFNα is very interesting. The further studies on the function of hsp70 against excessive immune responses will be valuable not only in the understanding pathological mechanism of auto immune response but also applying the control of hsp70 for immuno-modulating therapies.

목차 (Table of Contents)

• Abstract i
• Contents vii
• List of Figures and Tables ix
• List of Abbreviations x
• Introduction 1
• Materials and Methods 9
• 1. Cell
• 2. Generation of IRF7 gene induction cell lines using the Flp-In TM T-Rex TM system
• 3. Generation of hsp70 knockout cell lines using CRISPR-Cas9 system
• 4. Extraction of total RNA and reverse transcription
• 5. Quantitative real-time PCR
• 5.1 PCR condition
• 5.2 Primers
• 6. Luciferase assay
• 7. ELISA (Enzyme-linked immunosorbent assays)
• 8. GST pull down assay
• 9. Immunoblotting
• 10. Mice
• 11. Isolation of murine bone marrow and generation of dendritic cells
• Results 15
• 1. Inhibition of IFNα transcription in high temperature
• 2. Screening protein interacting with IRF7 by Mass spectrometry
• 3. The effect of hsp70 and hsp90 on IRF7 activity
• 4. Inhibition of IRF7 activity on IFNα expression by hsp70
• 5. Proteosomal degration of IRF7 by hsp70
• 6. Correlation among hsp70, IRF7 and IKKε
• 7. Hsp70 interference on the IRF7 activation
• 8. Generation of IRF7 truncates and identification of binding site of IRF7 to hsp70
• 9. Identification of hsp70 binding site to IRF7
• 10. Negative effect of hsp70 in the phosphorylation of IRF7 by IKKε
• Discussion 41
• References 47
• Korean Abstract 57