Using chemical biology as a tool to probe the mechanism of the HDL receptor
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2013.
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| Format: | Thesis |
| Language: | eng |
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Massachusetts Institute of Technology
2014
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| Online Access: | http://hdl.handle.net/1721.1/83782 |
| _version_ | 1811098308290019328 |
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| author | Yu, Miao, Ph. D. Massachusetts Institute of Technology |
| author2 | Monty Krieger. |
| author_facet | Monty Krieger. Yu, Miao, Ph. D. Massachusetts Institute of Technology |
| author_sort | Yu, Miao, Ph. D. Massachusetts Institute of Technology |
| collection | MIT |
| description | Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2013. |
| first_indexed | 2024-09-23T17:13:00Z |
| format | Thesis |
| id | mit-1721.1/83782 |
| institution | Massachusetts Institute of Technology |
| language | eng |
| last_indexed | 2024-09-23T17:13:00Z |
| publishDate | 2014 |
| publisher | Massachusetts Institute of Technology |
| record_format | dspace |
| spelling | mit-1721.1/837822019-04-10T19:00:13Z Using chemical biology as a tool to probe the mechanism of the HDL receptor Using chemical biology as a tool to probe the mechanism of the high density lipoprotein receptor Yu, Miao, Ph. D. Massachusetts Institute of Technology Monty Krieger. Massachusetts Institute of Technology. Department of Biology. Massachusetts Institute of Technology. Department of Biology. Biology. Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2013. Cataloged from PDF version of thesis. Includes bibliographical references. The high density lipoprotein (HDL) receptor, scavenger receptor, class B, type I (SR-BI), is a cell surface glycoprotein that controls HDL structure and metabolism by mediating cellular selective uptake of lipids from HDL. The mechanism underlying SR-BI-mediated lipid transfer involves a two-step process (binding followed by lipid transport) that is poorly understood. Our previous structure/activity analysis of the small-molecule inhibitor, BLT-1, established that the sulfur in the thiosemicarbazone moiety of BLT-1 was essential for its inhibitory activity in lipid transport. This thesis demonstrates that BLT-1 is an irreversible inhibitor of SR-BI, raising the possibility that cysteine(s) in SR-BI interact with BLT-1. Mass spectrometric analysis of purified SR-BI showed two of its six exoplasmic cysteines have free thiol groups (Cys251 and Cys384), and the other four are connected by two disulfide bonds within the conserved Cys321-Pro322-Cys323 (CPC) motif and between Cys280 and Cys334. Converting Cys384 (but not Cys251) to serine resulted in a complete loss of BLT-1 sensitivity. In addition, single amino acid substitution at position 384 can either enhance or weaken SR-BI-mediated HDL binding and lipid uptake depending on the size of the side chain, suggesting that Cys384 plays an important role in SR-BI receptor activity. The second part of this thesis explores the contributions of disulfide bonds to SR-BI-mediated HDL binding and lipid uptake. Effects of CPC mutations on activity were context dependent. Full activity required Pro322 and Cys323 only when Cys321 was present. Apparently, a free thiol at position 321 that cannot form an intra-CPC disulfide bond with Cys323 is deleterious, perhaps because of aberrant disulfide bond formation. Pro322 may stabilize an otherwise strained CPC disulfide bond, thus supporting full activity, but this disulfide bond is not absolutely required for activity. In fact, replacement of 6 out of the 8 total cysteines in SR-BI, by either serine or glycine, does not affect the overall receptor activity, but any further substitutions that disrupt the Cys280-Cys334 disulfide bond result in a complete loss of receptor activity. Together, this thesis demonstrates that a small-molecule screening approach provides a powerful springboard into the analysis of the structure and mechanism of SR-BI. Identification of additional small molecules may shed new lights on the mechanistic study of SR-BI, and represent a novel lead for further optimization prior to use in in vivo studies. by Miao Yu. Ph.D. 2014-01-09T19:54:26Z 2014-01-09T19:54:26Z 2013 Thesis http://hdl.handle.net/1721.1/83782 865078522 eng M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission. http://dspace.mit.edu/handle/1721.1/7582 226 pages application/pdf Massachusetts Institute of Technology |
| spellingShingle | Biology. Yu, Miao, Ph. D. Massachusetts Institute of Technology Using chemical biology as a tool to probe the mechanism of the HDL receptor |
| title | Using chemical biology as a tool to probe the mechanism of the HDL receptor |
| title_full | Using chemical biology as a tool to probe the mechanism of the HDL receptor |
| title_fullStr | Using chemical biology as a tool to probe the mechanism of the HDL receptor |
| title_full_unstemmed | Using chemical biology as a tool to probe the mechanism of the HDL receptor |
| title_short | Using chemical biology as a tool to probe the mechanism of the HDL receptor |
| title_sort | using chemical biology as a tool to probe the mechanism of the hdl receptor |
| topic | Biology. |
| url | http://hdl.handle.net/1721.1/83782 |
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