Yuezhou
Chen
Sponsor: David Segal, Ph.D. MED: Pharmacology Angelman Syndrome (AS) is a rare neurodevelopmental disorder that arises from a dysfunctional or missing maternal UBE3A expression in the brain. This causes severe intellectual and motor disability, and there is currently no cure or effective therapeutic. Due to a brain-specific long non-coding RNA transcript known as UBE3A-ATS, paternal UBE3A is silenced. In clinical trials of antisense oligonucleotides, brain UBE3A expression has been shown to be restored through the reactivation of the paternal allele. We have previously shown that the CRISPR-Csm complex shows greater precision than other previously described RNA targeting tools. In this study, we compared the expression levels of three different promoters that express the Csm system in lentivirus plasmids. In each HEK293T cell culture, three separate plasmids were delivered: first, a plasmid that produced the target RNA: UBE3A-ATS fused to the fluorescent protein GFP, then two plasmids that express the Csm system fused to the fluorescent protein mCherry. The Csm system was split into two plasmids due to packaging limits. By analyzing the fluorescence levels of different wavelengths, we can analyze the efficiency of the different promoters. Our results will then help with the translation of this work into human AS iPSC-derived neurons. Light-Enhancement in Catalytic Activities of PLP- Dependent Enzymes
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Yuezhou Chen
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Pyridoxal 5'-phosphate (PLP)-dependent enzymes represent about 4% of all known enzyme activities. PLP-dependent aspartate aminotransferase (AAT) was previously found to exhibit enhanced catalytic activity with L-Asp upon exposure to blue light. The light enhancement is attributed to the photoinduced deprotonation of Cα-H on the external aldimine intermediate to form the quinonoid intermediate, a partially rate-limiting step in the enzymatic catalysis. This project attempts to understand how general this phenomenon of light-enhanced catalysis is with other PLP-dependent enzymes and their reactions, since they share the similar chemistry. The five enzymatic reactions being explored include AAT with cysteine sulfinate, dialkylglycine decarboxylase (DGD) with aminoisobutyric acid (AIB), DGD with L-Ala, wild-type alanine racemase (AR) with D-Ala, and R219E AR with D-Ala. Steady-state kinetics and stopped-flow kinetics are used to observe catalytic activity increases after exposure to blue light. With the generalization of this light-enhanced kinetics, the conclusion may impact enzyme kinetics in plant biochemistry, animal biochemistry, human medicine, pharmacology, and many related applied fields due to the abundance of PLP-dependent enzymes in nature. Influence of Nitrogen Availability on Early Development in a Native and Invasive Forb Timothy Chen
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UC Davis / Chemistry / 2023
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Yuezhou Chen