The regulatory mechanism for peptide delivery to the MHC class I by protein disulfide isomerase
- 주제(키워드) protein quality control
- 발행기관 고려대학교 대학원
- 지도교수 강성만
- 발행년도 2011
- 학위수여년월 2011. 8
- 학위구분 박사
- 학과 일반대학원 생명공학과
- 원문페이지 120 p
- 실제URI http://www.dcollection.net/handler/korea/000000026279
- 본문언어 영어
- 제출원본 000045669636
초록/요약
Most antigenic peptides are generated by proteasomes in the cytosol and are transported by the transporter associated with antigen processing (TAP) into the endoplasmic reticulum (ER), where they bind with nascent major histocompatibilitiy complex class I (MHC-I). Although the overall process of peptide-MHC-I complex assembly is well studied, the mechanism by which free peptides are delivered from TAP to MHC-I is unknown. In this study, I investigated the possible role of protein disulfide isomerase (PDI) as a peptide carrier between TAP and MHC-I. Analysis of PDI？peptide complexes reconstituted in vitro showed that PDI exhibits some degree of specificity for peptides corresponding to antigenic ligands of various human leukocyte antigen (HLA) alleles. Mutations of either anchor residues of the peptide ligand or the peptide-binding site of PDI inhibited the PDI？peptide interaction. The PDI？peptide interaction increased under reducing conditions, whereas binding of the peptide to PDI decreased under oxidizing conditions. TAP-associated PDI was predominantly present in the reduced form, whereas the MHC-I-associated PDI was present in the oxidized form. Furthermore, upon binding of optimal peptides, PDI was released from TAP and sequentially associated with HLA-A2.1. These results revealed a redox-regulated chaperone function of PDI in delivering antigenic peptides from TAP to MHC-I.
more목차
CONTENTS
CONTENTS I
LIST OF FIGURES IV
LIST OF ABBREVIATIONS VI
TITLE 1
ABSTRACT 2
INTRODUCTION 5
MATERIALS AND METHODS 17
1. Cell lines and antibodies 18
2. Constructs and Site-directed mutagenesis 18
3. Mal-PEG modification of cysteine residues 20
in the active sites of PDI
4. Lysozyme refolding assay 21
5. Peptide competition assay 22
CONTENTS (Continued)
6. Protein expression and purification 23
7. Peptides 23
8. Crosslinking 24
9. Metabolic labeling and re-immunoprecipitation 25
10. Coimmunoprecipitation and immunoblot analysis 26
11. Microsome purification 27
12. Peptide binding assay and UV crosslinking 28
RESULTS 30
1. PDI specifically interacts with peptides through 31
its b′ domain
2. PDI displays selectivity toward antigenic peptide 45
3. Specific interaction of PDI with peptides depends 52
on the PDI redox state
4. Interaction of endogenous PDI with peptide 64
5. Peptide-dependent sequential association of PDI 69
with TAP and HLA-A2.1
CONTENTS (Continued)
DISCUSSION 79
REFERENCES 89
ABSTRACT IN KOREAN 103
APPENDIX 106
ACKNOWLEDGEMENTS 108
