Systems biology approaches to study mammalian mitochondrial metabolism
Cell Biology
Final Report Abstract
In summary I have addressed the aims of this proposed research project and achieved the following results during my appointment at UCSD in the lab of Prof. Christian Metallo: 1) I established and applied techniques in the field of cell biology, molecular biology and mass spectrometry, and data analysis using bioinformatics, including CRISPR/Cas9 technology as well as metabolic flux analysis and respiration in both, intact and permeabilized cells (Aim 1 and 2). In addition to the proposed methods, I also established a system to functionally analyze transport activity using Lactobacillus lactis (L. lactis). 2) I characterized potential transporter essential for early steps for heme biosynthesis (Aim 1), engineered knockout cell lines targeting potential glycine/ serine carrier (SLC25A38) and SLC25A39 (Aim 1a), and performed metabolomics, stable isotope tracing, and respirometry (Aim 1b/c/d). 3) I processed data from metabolic screens of the SLC25 cell library (knockdown library) (Aim 2a) and decided to focus my research on SLC25A44. Follow-up studies (Aim 2b) on SLC25A44 indicated a potential role for glutamine metabolism/glutamine transport. However, research on metabolic flux analysis, respiration, and transport assays demonstrates that glutamine uptake seems to be intact upon knockout of SLC25A44 suggesting a different role of this transporter for glutamine metabolism that needs to be explored in more detail. 4) In addition to the proposed project, I engineered a SLC knockout library targeting ten mitochondrial carrier proteins. I began characterizing metabolism of SLC candidates in a discovery-based approach. Further research to annotate these mitochondrial carriers is under progress. 5) My established workflow/technologies can be applied to study other transporter of interest. I collected an intense amount of data on various SLC25 transporters that I intend to use for follow-up studies. 6) In addition to the proposed project, I’ve applied stable isotopes and analyzed metabolic fluxes in mitochondria in the context of various state diseases, including cancer, inflammation, and aging.
Publications
- (2016) Immunoresponsive gene 1 and itaconate inhibit succinate dehydrogenase to modulate intracellular succinate levels. Journal of Biological Chemistry, 291(27), 14274-14284
Cordes T., Wallace M., Michelucci A., Divakaruni A.S., Sapcariu S.C., Sousa C., ...& Metallo, C.M.
(See online at https://doi.org/10.1074/jbc.M115.685792) - (2016). Tracing insights into human metabolism using chemical engineering approaches. Current Opinion in Chemical Engineering, 14, 72-81
Cordes, T., & Metallo, C. M.
(See online at https://doi.org/10.1016/j.coche.2016.08.019) - (2017). ATF4-induced metabolic reprograming is a synthetic vulnerability of the p62-deficient tumor stroma. Cell metabolism, 26(6), 817-829
Linares, J. F., Cordes, T., Duran, A., Reina-Campos, M., Valencia, T., Ahn, C. S., ... & Diaz-Meco, M. T.
(See online at https://doi.org/10.1016/j.cmet.2017.09.001) - (2019): Quantifying Intermediary Metabolism and Lipogenesis in Cultured Mammalian Cells Using Stable Isotope Tracing and Mass Spectrometry. In: Methods in molecular biology (Clifton, N.J.) 1978, S. 219–241
Cordes, T., & Metallo, C. M.
(See online at https://doi.org/10.1007/978-1-4939-9236-2_14)
