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Title: Isotope Tracer-Based Quantitation of Protein Scavenging in Pancreatic Cancer Cells
Authors: Zhang, Kevin
Advisors: Rabinowitz, Joshua D.
Department: Molecular Biology
Class Year: 2016
Abstract: Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer recently found to engage in protein scavenging, a process whereby cells uptake whole protein via macropinocytosis and subsequently degrade it in the lysosome to provide intracellular amino acids, supplementing nutrient stores. While qualitative evidence for protein scavenging exists, no method is currently available to quantitatively measure its magnitude. In this study, we developed a quantitative, isotope tracer-based assay that sensitively measures protein scavenging flux. Using this assay, we confirm that oncogenic Ras, one of the few genes known to stimulate macropinocytosis, greatly increases protein scavenging. We also show that a substantive proportion of PDAC intracellular amino acids can be traced to protein scavenging. To explore the genetic regulation of protein scavenging, we examined the results of a previously conducted CRISPR screen that identified genes associated with protein scavenging. Although autophagy genes were a hit in the screen, we found that autophagy deficiency does not significantly increase protein scavenging. To address recent evidence implicating mTORC1 signaling in protein scavenging, we inhibited mTORC1 and found that this slightly upregulated protein scavenging. Interestingly, by culturing cells for many generations in amino acid dropout medium supplemented with protein, we generated a cell line capable of robust protein scavenging. Cells cultured in this amino acid dropout medium engaged in significantly higher levels of protein scavenging. The protein scavenging assay presented here, along with the findings in this study, thus demonstrate the importance of protein scavenging to PDAC and characterize its regulation.
Extent: 84 pages
Type of Material: Princeton University Senior Theses
Language: en_US
Appears in Collections:Molecular Biology, 1954-2017

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