Poster 110 - Mechanical Insights of MSBP1 in Regulating Cytochrome P450 Enzymes through Monitoring Intracellular Calcium

Saturday, October 26, 2024

10:00 AM - 12:00 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

KL

Katelyn Leung

high school senior, intern
University of California San Diego: Institute of Engineering in Medicine
San Diego, California, United States

Co-Author(s)

CB

Christopher Bahari

high school sophomore, intern
University of California San Diego: Institute of Engineering in Medicine, United States
AL

Aspen Lee

high school sophomore, intern
University of California San Diego: Institute of Engineering in Medicine, United States
Yangyi Liu

undergraduate researcher
University of California San Diego: Department of NanoEngineering, United States
CF

Conner Feng

freshman undergraduate, mentor
University of California San Diego: Institute of Engineering in Medicine, United States

Co-Author(s)

Abigail Qiu

Student
Pacific Ridge School, United States
GN

Gregory Ngantung

high school junior, intern
University of California San Diego: Institute of Engineering in Medicine, United States

Co-Author(s)

EP

Eshan Patibandla

high school sophomore, intern
University of California San Diego: Institute of Engineering in Medicine, United States
SL

Sneha Lele

high school senior, intern
University of California San Diego: Institute of Engineering in Medicine, United States
SX

Shanhui Xu

Postdoc
University of California San Diego: Department of NanoEngineering, United States
VG

Veronica Gomez-Godinez

Scientist
University of California, San Diego
La Jolla, California, United States

Primary Investigator(s)

Linda Shi, PhD (she/her/hers)

OPALS Program Director, Project Scientist, Co-director IEM
University of California, San Diego: Institute of Engineering in Medicine
La Jolla, California, United States

Last Author(s)

YL

Yanran Li

Principal Investigator
University of California San Diego: Department of NanoEngineering, United States

Introduction: Engineered Saccharomyces Cerevisiae is a promising platform for reconstructing various biosynthetic pathways. Cytochrome p450 (CYP450) enzymes catalyze the synthesis of important metabolites in eukaryotes. Membrane steroid-binding protein (MSBP), a family of plant scaffold proteins localized in the ER membrane, improves the efficiency of CYP450 enzymes in Arabidopsis thaliana and phytochemical production in engineered yeast. This study monitored the intracellular calcium of CYP450 and MSBP expressing yeast through fluorescence microscopy.

Materials and

Methods: For bioinformatic analysis, Protein Data Bank Pairwise Comparison was used to analyze protein structure similarity of MSBP family proteins. A phylogenetic tree was constructed using Molecular Evolutionary Genetics Analysis (MEGA), and TBtools-II was utilized to determine shared motifs and domains. Yeast strains were cultured at 30 °C in yeast peptone dextrose (YPD) or synthetic defined medium (SDM) supplemented with 2% dextrose or glycerol. For fluorescence microscopy, cells were washed with phosphate-buffered saline (PBS) and then loaded with 0.1 % Tween 20, 1 µM F127, and 5 µM Fluo4. A final concentration of 5µM 2-Aminoethyl diphenylborinate (2-APB) was added while the images were captured at 4-second intervals with a 63X Zeiss Axiovert 200M microscope. Fluorescence intensity data analysis was gathered using FIJI ImageJ, followed by analysis of variance (ANOVA) and post-hoc in R.

Results, Conclusions, and Discussions: Results and

Discussion: In the presence of MSBP1, intracellular calcium levels increased significantly after adding 2-APB (Figure 1A). In its absence, intracellular calcium levels initially rose but quickly returned to a constant rate (Figure 1A). This suggests that MSBP1 plays a significant role in creating the different patterns in cellular calcium response to 2-APB. 2-APB is an inhibitor of 1P3 receptors and store-operated channels which regulate calcium transfer on the membrane. In all three experiments, MSBP1 and CYP450 expressing yeast strain samples exhibited lower levels of Fluo-4 fluorescence intensity than CYP450-only yeast strains (Figure 1A, 1B). When cultured with glycerol, yeast cells undergo respiration and its mitochondrial activity is upregulated. In glycerol SDM medium, yeast exhibit lower Fluo4 intensity levels compared to dextrose SDM medium (Figure 1B). This indicates increased mitochondrial activity in MSBP1 and CYP450 expressing yeast cells, as mitochondria actively uptake calcium. This signals enhanced mitochondrial function during aerobic respiration, resulting in lower fluorescence intensity for glycerol samples.

Conclusions: MSBP1 plays a role in the transfer of calcium involved in interactions between the ER and mitochondria. Fluo-4 intensity negatively correlates with interactions between MSBP1 and CYP450 in yeast, in addition to increased respiration. This indicates increased communication between these organelles in MSBP1 and CYP450 expressing yeast strains.

Acknowledgements (Optional): This material was based upon work supported by a gift from Beckman Laser Institute Inc. to V. G. G. & L. S. and the National Science Foundation under Award Number 2420331. The high school students (*) participated in UCSD IEM OPALS internship. Special thanks to Dr. Shu Chien from UCSD Bioengineering and Dr. Lizhu Chen from CorDx Inc. for their donations.

References (Optional):