
==== Front
bioRxiv
BIORXIV
bioRxiv
2692-8205
Cold Spring Harbor Laboratory

10.1101/2024.08.28.609894
preprint
1
Article
Structural and systems characterization of phosphorylation on metabolic enzymes identifies sex-specific metabolic reprogramming in obesity
Tamir Tigist Y http://orcid.org/0000-0002-8194-8754

Chaudhary Shreya
Li Annie X http://orcid.org/0009-0000-7685-9389

Trojan Sonia E http://orcid.org/0000-0001-8876-6294

Flower Cameron T http://orcid.org/0000-0002-9632-9913

Vo Paula
Cui Yufei http://orcid.org/0000-0003-3014-813X

Davis Jeffrey C http://orcid.org/0000-0003-0418-9106

Mukkamala Rachit S http://orcid.org/0009-0000-8318-2257

Venditti Francesca N http://orcid.org/0009-0001-9487-4395

Hillis Alissandra L http://orcid.org/0000-0002-6667-1291

Toker Alex http://orcid.org/0000-0003-0723-4419

Vander Heiden Matthew G http://orcid.org/0000-0002-6702-4192

Spinelli Jessica B http://orcid.org/0000-0003-3657-4578

Kennedy Norman J
Davis Roger J http://orcid.org/0000-0002-0130-1652

White Forest M http://orcid.org/0000-0002-1545-1651

29 8 2024
2024.08.28.609894https://creativecommons.org/licenses/by-nc-nd/4.0/ This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which allows reusers to copy and distribute the material in any medium or format in unadapted form only, for noncommercial purposes only, and only so long as attribution is given to the creator.
http://biorxiv.org/lookup/doi/10.1101/2024.08.28.609894
nihpp-2024.08.28.609894.pdf
Abstract

Coordination of adaptive metabolism through cellular signaling networks and metabolic response is essential for balanced flow of energy and homeostasis. Post-translational modifications such as phosphorylation offer a rapid, efficient, and dynamic mechanism to regulate metabolic networks. Although numerous phosphorylation sites have been identified on metabolic enzymes, much remains unknown about their contribution to enzyme function and systemic metabolism. In this study, we stratify phosphorylation sites on metabolic enzymes based on their location with respect to functional and dimerization domains. Our analysis reveals that the majority of published phosphosites are on oxidoreductases, with particular enrichment of phosphotyrosine (pY) sites in proximity to binding domains for substrates, cofactors, active sites, or dimer interfaces. We identify phosphosites altered in obesity using a high fat diet (HFD) induced obesity model coupled to multiomics, and interrogate the functional impact of pY on hepatic metabolism. HFD induced dysregulation of redox homeostasis and reductive metabolism at the phosphoproteome and metabolome level in a sex-specific manner, which was reversed by supplementing with the antioxidant butylated hydroxyanisole (BHA). Partial least squares regression (PLSR) analysis identified pY sites that predict HFD or BHA induced changes of redox metabolites. We characterize predictive pY sites on glutathione S-transferase pi 1 (GSTP1), isocitrate dehydrogenase 1 (IDH1), and uridine monophosphate synthase (UMPS) using CRISPRi-rescue and stable isotope tracing. Our analysis revealed that sites on GSTP1 and UMPS inhibit enzyme activity while the pY site on IDH1 induces activity to promote reductive carboxylation. Overall, our approach provides insight into the convergence points where cellular signaling fine-tunes metabolism.

Summary Statement

By employing a multi-disciplinary approach we stratify structural features of phosphorylation sites on metabolic enzymes, map the systems level changes induced by obesity, identify key pathways with sex specific phosphoproteomic responses, and validate the functional role of phosphorylation sites for select enzymes.
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