c-IAP1 Binds and Processes PCSK9 Protein: Linking the c-IAP1 in a TNF-α Pathway to PCSK9-Mediated LDLR Degradation Pathway
Abstract
:1. Introduction
2. Results and Discussion
2.1. Identification of Potential PCSK9 Binding Proteins through Affinity Purification and Shotgun LC-MS/MS Analysis
Gene Symbols | Gene Names | Experiment 1 | Experiment 2 | Experiment 3 | |||
---|---|---|---|---|---|---|---|
emp | exp | emp | exp | emp | exp | ||
PCSK9 | proprotein convertase subtilisin/kexin type 9 | 3 | 1466 | 2 | 1171 | 4 | 1128 |
UGGT1 | UDP-glucose glycoprotein glucosyltransferase 1 | 0 | 9 | 0 | 12 | 0 | 15 |
PDIA4 (ERP72) | protein disulfide isomerase family A, member 4 | 0 | 7 | 0 | 5 | 0 | 7 |
SLC25A1 | solute carrier family 25 member 1 | 0 | 6 | 0 | 2 | 0 | 6 |
DNAJA1 | DnaJ (Hsp40) homolog, subfamily A, member 1 | 0 | 12 | 0 | 10 | 0 | 7 |
CLGN | Calmegin | 0 | 2 | 0 | 2 | 0 | 2 |
DNAJA2 | DnaJ (Hsp40) homolog, subfamily A, member 2 | 0 | 2 | 0 | 3 | 0 | 4 |
c-IAP1 (BIRC2) | cellular inhibitor of apoptosis protein 1 | 0 | 7 | 0 | 9 | 0 | 8 |
TBB6 | tubulin, beta 6 | 0 | 5 | 0 | 2 | 0 | 3 |
TRAF2 | TNF receptor-associated factor 2 | 0 | 4 | 0 | 3 | 0 | 3 |
SLC25A10 | solute carrier family 25. member 10 | 0 | 6 | 0 | 3 | 0 | 6 |
DNAJB11 | DnaJ (Hsp40) homolog, subfamily B, member 11 | 0 | 5 | 0 | 4 | 0 | 3 |
CDIPT | CDP-diacylglycerol-inositol 3-phosphatidyltransferase | 0 | 3 | 0 | 6 | 0 | 4 |
DNAJC10 | DnaJ (Hsp40) homolog, subfamily C, member 10 | 0 | 6 | 0 | 3 | 0 | 6 |
DNAJA3 | DnaJ (Hsp40) homolog, subfamily A, member 3 | 0 | 2 | 0 | 3 | 0 | 3 |
RHOT1 | ras homolog gene family, member T1 | 0 | 2 | 0 | 3 | 0 | 4 |
AGK | Acylglycerol kinase lipid kinase | 0 | 2 | 0 | 3 | 0 | 2 |
HSPB1 | heat shock 27kDa protein 1 | 0 | 4 | 0 | 5 | 0 | 2 |
RCN1 | reticulocalbin 1 | 0 | 3 | 0 | 4 | 0 | 5 |
Ribosomal L28 | partial ribosomal protein L28 variant | 0 | 2 | 0 | 6 | 0 | 5 |
Stub1 | STIP1 homology and U-box containing protein 1 | 0 | 2 | 0 | 3 | 0 | 2 |
PGRC1 | progesterone receptor membrane component 1 | 0 | 2 | 0 | 2 | 0 | 2 |
SLC25A12 | Solute carrier family 25 member 12 | 0 | 2 | 0 | 5 | 0 | 4 |
2.2. c-IAP1/TRAF2 Complex Binds PCSK9
2.3. c-IAP1 Knock-Down
2.4. Ubiquitination of PCSK9 by c-IAP1
2.5. Discussion
3. Experimental
3.1. Cell Culture and Immunofluorescence Staining and Image Analysis
3.2. DNA Constructs, Transfections and Western Blot Assays
3.3. Generation of Stable Cell Lines, Immunoprecipitation (IP) and Protein Purification
3.4. Shotgun Analysis of the FLAG-Tagged PCSK9 and Associated Proteins Complex Samples
3.5. RNA Interference
3.6. Mouse PCSK9 Immunoassay
3.7. In Vivo Ubiquitination Analysis
3.8. In Vitro Ubiquitination Assay
3.9. Mass Spectrometric Analysis of Ploy-Ubiquitinated PCSK9
4. Conclusions
Supplementary Materials
Acknowledgements
Authors’ Contributions
- Sample Availability: Samples are available from the corresponding author, Dr. Weiming Xu, Email: [email protected].
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Xu, W.; Liu, L.; Hornby, D. c-IAP1 Binds and Processes PCSK9 Protein: Linking the c-IAP1 in a TNF-α Pathway to PCSK9-Mediated LDLR Degradation Pathway. Molecules 2012, 17, 12086-12101. https://doi.org/10.3390/molecules171012086
Xu W, Liu L, Hornby D. c-IAP1 Binds and Processes PCSK9 Protein: Linking the c-IAP1 in a TNF-α Pathway to PCSK9-Mediated LDLR Degradation Pathway. Molecules. 2012; 17(10):12086-12101. https://doi.org/10.3390/molecules171012086
Chicago/Turabian StyleXu, Weiming, Lizhi Liu, and David Hornby. 2012. "c-IAP1 Binds and Processes PCSK9 Protein: Linking the c-IAP1 in a TNF-α Pathway to PCSK9-Mediated LDLR Degradation Pathway" Molecules 17, no. 10: 12086-12101. https://doi.org/10.3390/molecules171012086