One of the defining characteristics of cancer cells is that they change the way they generate and use energy. Unlike normal cells, which burn fuel efficiently through a process called oxidative phosphorylation, cancer cells often switch to a less efficient but faster process called aerobic glycolysis - sometimes called the Warburg effect. This allows them to produce the building blocks needed for rapid growth and division.
Controlling this metabolic shift requires changes not just in which genes are active, but in how proteins are modified after they are made. These changes are called post-translational modifications (PTMs). PTMs act like molecular switches - they can turn a protein on or off, mark it for destruction, change where it goes inside the cell, or alter how strongly it binds to other molecules.
In colorectal cancer (CRC), which causes nearly 900,000 deaths worldwide each year, PTMs have been understudied compared to gene expression changes. This study set out to map three key PTMs - phosphorylation, ubiquitination, and malonylation - across the entire protein landscape of CRC tumors, creating the first comprehensive PTM atlas for this cancer.
Phosphorylation is the addition of a phosphate group to a protein, most commonly acting as an on/off switch for enzymatic activity and cellular signaling. It is the most well-known PTM and plays a central role in transmitting signals from the cell surface to the nucleus.
Ubiquitination is the attachment of a small protein called ubiquitin, which often tags proteins for destruction by the cell's recycling machinery (the proteasome). Ubiquitination can also regulate protein-protein interactions and subcellular location without triggering degradation. In cancer, abnormal ubiquitination can stabilize proteins that should be destroyed or degrade proteins that protect normal cell function.
Malonylation is a newer and less studied modification involving the addition of a malonyl group to lysine residues on a protein. It has been linked to regulation of energy metabolism, fatty acid synthesis, and mitochondrial function. In cancer cells, malonylation has been found to enhance the survival of tumor cells under nutrient-poor conditions.
The researchers analyzed paired tumor and adjacent normal tissue samples from 8 patients who had undergone surgery for CRC without prior chemotherapy or radiation. Using the same tissue samples for all three PTM analyses ensured direct comparability of the results.
Each type of PTM was enriched from the tissue proteins using specialized antibodies or chemical methods, then detected using high-resolution mass spectrometry on a Bruker timsTOF Pro instrument. This technology can identify thousands of modified protein sites in a single experiment with high sensitivity and specificity.
The resulting data were analyzed using bioinformatics tools including GO and KEGG pathway enrichment analysis (which identifies which biological processes are affected), protein-protein interaction (PPI) network construction, and three-dimensional structural mapping of PTM sites onto protein models generated by AlphaFold2. This multi-layered approach allowed the team to move from cataloguing which sites are modified to understanding what those modifications do.
The atlas identified 59 differential phosphorylation sites, 263 differential ubiquitination sites, and 64 differential malonylation sites in CRC tissues compared to matched normal tissue. Ubiquitination showed the largest number of changes and the greatest variability, suggesting it plays a particularly dynamic and heterogeneous regulatory role in CRC. Malonylation was predominantly downregulated in tumors, while ubiquitination showed both increases and decreases across different proteins.
Five proteins were modified by all three PTM types simultaneously: ACLY, ALDOA, GPI, PKM, and TALDO1. All five are enzymes involved in central energy pathways - glycolysis (the breakdown of glucose) and the pentose phosphate pathway (which generates building blocks for DNA and reduces oxidative stress). Their convergence as PTM targets suggests they are critical regulatory nodes in the metabolic reprogramming of CRC.
Site-specific analysis of PKM - a rate-limiting enzyme in glycolysis - revealed a large change at the K336 ubiquitination site (log2 fold change of 3.33), suggesting enhanced protein stability or altered signaling through this site. ALDOA showed consistent downregulation across all three PTM types, potentially reducing glycolytic flux. Meanwhile, GPI and PKM had elevated phosphorylation and ubiquitination alongside reduced malonylation, a pattern associated with increased glycolytic activity.
IDH1 (isocitrate dehydrogenase 1) is a TCA cycle enzyme that can produce an oncometabolite called 2-hydroxyglutarate when mutated. This study found that IDH1 undergoes ubiquitination in CRC, which promotes its degradation and disrupts the balance of metabolic intermediates in the TCA cycle, further promoting the shift toward glycolysis.
LDHA (lactate dehydrogenase A) converts pyruvate to lactate - the final step in aerobic glycolysis. Ubiquitination of LDHA in CRC was found to enhance lactate production and may also help tumor cells evade immune detection. This is consistent with the well-known role of lactate in suppressing immune cell activity in the tumor microenvironment.
PDHA1 (pyruvate dehydrogenase E1 alpha) is the enzyme that connects glycolysis to the TCA cycle by converting pyruvate to acetyl-CoA. Phosphorylation of PDHA1 by the kinase PDK1 inhibits this reaction, diverting pyruvate away from the TCA cycle and reinforcing the Warburg effect. GAPDH, another glycolytic enzyme, undergoes malonylation in CRC tumors, fine-tuning its enzymatic activity and contributing to the metabolic flexibility of cancer cells.
By constructing the first integrated PTM atlas of metabolic reprogramming in CRC, this study identifies specific protein modifications - rather than gene mutations or expression changes alone - as potential therapeutic targets. Drugs that inhibit the enzymes responsible for adding or removing these modifications could theoretically disrupt the metabolic machinery that CRC cells depend on for growth and survival.
For example, targeting the kinase PDK1 (which phosphorylates and inhibits PDHA1) could redirect pyruvate into the TCA cycle, reducing the Warburg effect and potentially making cancer cells more vulnerable to conventional treatments. Similarly, targeting ubiquitin ligases that stabilize LDHA could reduce lactate production and may restore immune cell function in the tumor microenvironment.
The authors acknowledge important limitations: the study used only 8 tumor-normal pairs, which limits statistical power, and mass spectrometry-based PTM detection may miss low-abundance modifications. Larger studies are needed to validate these findings. Translating PTM-targeting into clinical drugs also presents challenges around specificity - since many of these enzymes play roles in normal cells as well. This atlas nonetheless provides a valuable foundation for future research into metabolic vulnerabilities in colorectal cancer.