Investigating Arginine Methylation of PGC-1α, a Master Metabolic Regulator
By: Tiffany Lubrino
Schmid Senior Research Conference (SSRC)
May 1st, 2022
Objectives
01
Methods
03
Results
04
Conclusions & Future Work
05
Table of contents
Background
02
Acknowledgements
06
Background
Types of Protein Arginine Methyltransferases
Source: Feng Y, et al. (2013) Mammalian protein arginine methyltransferase 7 (PRMT7) specifically targets RXR sites in lysine- and arginine-rich regions. The Journal of biological chemistry 288(52):37010-37025.
What is PGC-1α?
PGC-1α: (PeroxisomeProliferator-activated receptor γ coactivator 1α)
is a transcriptional co-activator that plays an important role in mitochondrial biogenesis and energy.
This figures shows the full length of PGC-1α.
Source: Database, A. F. P. S. (n.d.). Alphafold protein structure database. Retrieved April 28, 2022, from https://alphafold.ebi.ac.uk/entry/Q9UBK2
PGC-1α is implicated in the musculoskeletal system
PTM
PGC-1α is a thermoregulator is implicated in muscle cell and adipose tissue to induce gene expression. Thus, this co-activator protein is correlated with mitochondrial energy levels (Tan et al., 2016).
Source: Tan, Z., Luo, X., Xiao, L., Tang, M., Bode, A. M., Dong, Z., & Cao, Y. (2016). The role of PGC1α in cancer metabolism and its therapeutic implications. Molecular Cancer Therapeutics, 15(5), 774–782.
Ex: for diabetes and obesity regulation
PGC-1α is heavily modified
Structure of PGC-1α and known PTMs. Area of interest shown: amino acid residues 481-798 and corresponding enzymes. (Adapted from Teyssier et al., 2005)
Source: Meryl Mendoza, Cal State LA.
Our Objective
Methods
Using the C-terminus of PGC-1α to test for arginine methylation.
Methylation of constructs of various lengths of PGC-1α as a substrate. Various C-terminal PGC-1α constructs were used as substrates for the methylation reactions and mass spectrometry analyses.
The human recombinant PGC-1α fragment known as G1 corresponds to amino acid residue 532 to 640 (a gift from Dr. M. Stallcup, USC).
Methylation Reactions: Enzyme PRMT7, Substrate PGC-1α
Since both PRMT7 and PGC-1α are temperature sensitive, in vitro methylation reactions were carried out at various temperatures 37, 30, 21, 18, 16, and 4 ˚C.
G1 PGC-1α construct was first used as a substrate and detected via fluorography.
Mix PGC-1α with PRMT7 methyltransferase and AdoMet (methyl donor). Incubate reactions at various temperatures in water bath for 1h.
Source: Meryl Mendoza, Cal State LA.
Two ways to detect methylated PGC-1α
Fluorography for general detection of methylated protein - a band on the fluorograph indicates methylated protein.
Mass spectrometry for identifying the location of methylated arginine residues on the protein.
Source: Meryl Mendoza, Cal State LA.
Results
Fluorography Results: PGC-1alpha is methylated by PRMT7 at various temperatures.
Source: Meryl Mendoza, Cal State LA.
Mass Spectrometry Results: PGC-1α is methylated at arginine residues 548, 568, and 570
Identification of arginine residues that received methyl groups in the C-terminus of of PGC-1α.
Enzymes used: PRMT1 (control) and PRMT7, these sites were methylated at the following temperatures: 37, 30, 21, 18, 16, and 4 ˚C.
Molecular Modeling:
Used to Determine Whether Methylation takes Place on the Surface of PGC-1α
(Left) Molecular model of the C-terminus of PGC-1α with methylated arginine groups identified in this study highlighted in red. (Right) Magnified portion of the C-terminus containing only the RNA Recognition Motif (RRM) domain with methylated arginine 753 highlighted in red. Figures were generated using Phyre2 and PyMOL. (Kelly et al., 2015).
Source: Meryl Mendoza, Cal State LA.
Spring 2022 Work
Since previous work was done using only the C-terminus, would we get similar results with full-length PGC-1α?
Source: Meryl Mendoza, Cal State LA.
Methylation of PGC-1α at 30˚C by PRMT7: Analysis by Western Blot
Antibodies:
Source: Types of western blotting equipment (cells & power supplies). BioRad. Retrieved April 28, 2022, from https://www.bio-rad.com,
Results: Optimization of Western Blot in progress
Anti-MMA
Anti- PGC-1α
260
100
50
25
15
10
Molecular Weight Ladder (kDa)
260
140
100
70
50
40
35
25
15
10
PRMT7 + G1
G1 alone
Molecular Weight Ladder (kDa)
PRMT7 alone
PRMT7 alone
G1
alone
PRMT7 + G1
Conclusions
Conclusion
•Using constructs based on the C-terminus of PGC-1α, we confirmed that PGC-1α is a substrate for PRMT7 and optimized methylation reactions mainly at room temperatures (21-30°C).
•PRMT7 methylates PGC-1α (from amino acids 573-767) at R548 at all temperatures, and R548 and R568 only methylated at 30 ˚C.
•PGC-1α is methylated in a temperature dependent manner, most likely allowing for greater functional control.
Future work: What is the role of Arginine Methylation in PGC-1α?
Source: Endotoxins – Innovative Solutions for Cell Culture Studies. Cell Sciences: Catalyst for Discovery. Retrieved April 28, 2022, from https://www.cellsciences.com, Agilent 1260 Infinity II LC System. SRA Instruments. (2018, September 5). Retrieved April 28, 2022, from https://www.srainstruments.com/p/lc-agilent-1260/
Meet the Team
*Crean College of Health and Behavioral Sciences
^Schmid College of Science and Technology
Tiffany Lubrino*
Sidney Briski^
Research Assistant:
Cal State University, Los Angeles ‘20
Poula Mansour
Research Professor
Dr. Cecilia Lopez^
Joshua Huh^
Arisbeth Mancilla-Burgos^
Acknowledgments
Former members of the Zurita-Lopez lab at Cal State LA:
Meryl Mendoza, Janielle Cuala, Immaculeta Osuji, Mariel Mendoza, Benjamin A. Garcia
Students from Cal State LA were supported by the MORE Programs Office.
Funding: NIH MARC U*STAR Program Grant #T34 GM008228, CUE Grant for Tiffany Lubrino
Thank you to my research mentor, Dr. Lopez for all of your help.
Thank you to the following: Charlie Drucker, MS Food Science, Dr. Lillian Were Lab, for installation and familiarization of HPLC. David Tranchemontagne, Customer Field Service Rep, Agilent Technologies for installation and familiarization of HPLC. Jillian Fahey and Doris Yang for cell culture training.
Thank you to Schmid for organizing this senior conference!
Concluding Words
Special thanks to:
My family: my brother, Timothy, and my parents!
Crean mentors for Health Science: Dr. Sumida, Dr. Richards
Schmid mentors: Dr. Lopez, Dr. Schwarz, Dr. Ogba, Professor Dudley
Pre-med mentor: Dr. Steven Long, MD
My research peers, you all are the best! :D
References (partial list)
Centers for Disease Control and Prevention. National Diabetes Statistics Report, 2017. Atlanta, GA: Centers for Disease Control and Prevention, U.S. Dept of Health and Human Services; 2017.
Jager S, Handschin C, St-Pierre J, & Spiegelman BM (2007) AMP-activated protein kinase (AMPK) action in skeletal muscle via direct phosphorylation of PGC-1alpha. Proceedings of the National Academy of Sciences of the United States of America 104(29):12017-12022.
Puigserver P & Spiegelman BM (2003) Peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1 alpha): transcriptional coactivator and metabolic regulator. Endocr Rev 24(1):78-90.
Rohas LM, et al. (2007) A fundamental system of cellular energy homeostasis regulated by PGC-1alpha. Proceedings of the National Academy of Sciences of the United States of America 104(19):7933-7938.
Tan, Z., Luo, X., Xiao, L., Tang, M., Bode, A. M., Dong, Z., & Cao, Y. (2016). The role of PGC1α in cancer metabolism and its therapeutic implications. Molecular Cancer Therapeutics, 15(5), 774–782.
Teyssier, C., H. Ma, R. Emter, A. Kralli, and M. Stallcup (2005) Activation of Nuclear Receptor Coactivator PGC-1 Alpha by Arginine Methylation. Genes & Development 19 (12): 1466–73. doi:10.1101/gad.1295005.
Thank you!