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Revealing, Illuminating, and Modifying Proteins in Human Diseases using Noncanonical Amino Acids

Citation

Lu, Ying Ying (2014) Revealing, Illuminating, and Modifying Proteins in Human Diseases using Noncanonical Amino Acids. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/NPBC-3N67. https://resolver.caltech.edu/CaltechTHESIS:08232013-210427528

Abstract

To better understand human diseases, much recent work has focused on proteins to either identify disease targets through proteomics or produce therapeutics via protein engineering. Noncanonical amino acids (ncAAs) are tools for altering the chemical and physical properties of proteins, providing a facile strategy not only to label proteins but also to engineer proteins with novel properties. My thesis research has focused on the development and applications of noncanonical amino acids in identifying, imaging, and engineering proteins for studying human diseases. Chapter 1 introduces the concept of ncAAs and reveals insights to how I chose my thesis projects.

ncAAs have been incorporated to tag and enrich newly synthesized proteins for mass spectrometry through a method termed BONCAT, or bioorthogonal noncanonical amino acid tagging. Chapter 2 describes the investigation of the proteomic response of human breast cancer cells to induced expression of tumor suppressor microRNA miR-126 by combining BONCAT with another proteomic method, SILAC or stable isotope labeling by amino acids in cell culture. This proteomic analysis led to the discovery of a direct target of miR-126, shedding new light on its role in suppressing cancer metastasis.

In addition to mass spectrometry, ncAAs can also be utilized to fluorescently label proteins. Chapter 3 details the synthesis of a set of cell-permeant cyclooctyne probes and demonstration of selective labeling of newly synthesized proteins in live mammalian cells using azidohomoalanine. Similar to live cell imaging, the ability to selectively label a particular cell type within a mixed cell population is important to interrogating many biological systems, such as tumor microenvironments. By taking advantage of the metabolic differences between cancer and normal cells, Chapter 5 discusses efforts to develop selective labeling of cancer cells using a glutamine analogue.

Furthermore, Chapter 4 describes the first demonstration of global replacement at polar amino acid positions and its application in developing an alternative PEGylation strategy for therapeutic proteins. Polar amino acids typically occupy solvent-exposed positions on the protein surface, and incorporation of noncanonical amino acids at these positions should allow easier modification and cause less perturbation compared to replacements at the interior positions of proteins.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:noncanonical; amino acids; proteins; cancer; microRNAs
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Chemistry
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Tirrell, David A.
Thesis Committee:
  • Dougherty, Dennis A. (chair)
  • Grubbs, Robert H.
  • Shan, Shu-ou
  • Tirrell, David A.
Defense Date:22 August 2013
Record Number:CaltechTHESIS:08232013-210427528
Persistent URL:https://resolver.caltech.edu/CaltechTHESIS:08232013-210427528
DOI:10.7907/NPBC-3N67
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:7938
Collection:CaltechTHESIS
Deposited By: Ying Lu
Deposited On:12 Nov 2015 22:50
Last Modified:04 Oct 2019 00:02

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