Tools to investigate aspartate limited proliferation of cancer cells

Abstract

Cellular aspartate levels are tightly linked to cell proliferation rate.But how aspartate limitation decreases proliferation is unknown and could be related to its role as an amino acid as well as a substrate in multiple metabolic reactions. In this dissertation, the role of each of the metabolic fates of aspartate is interrogated. Surprisingly, it was not possible to rescue proliferation during aspartate limitation by providing the fates of aspartate, neither were there any evidence of insufficiency for protein synthesis. It is hypothesized that nucleotide imbalance is responsible and that this would elude efforts to rescue proliferation through complementation. Nevertheless, these results prompted the development of better tools to measure aspartate and the consequences of aspartate limitation. Towards that end, an aspartate biosensor was developed and tested, enabling increased throughput of non-invasive aspartate measurements under relevant growth conditions. Also, a highly accurate method for measuring tRNA aminoacylation using sequencing was developed. This tRNA-Seq method was tested on human tRNAs with a titration of charge levels, its measurements were determined to be quantitative and with enough accuracy and precision to detect small (~5%) effects on tRNA charge.