Method Development for PKM2 Activity Analysis in Cancer-Associated Signaling Pathways

Abstract

Introduction: PARP14 belongs to the superfamily of PARPs that perform ADP-ribosylation of target proteins. The enzyme is involved in many cellular processes, including regulation of glycolysis, protein degradation, and genomic reparation.
Background: PKM2 has been shown to be regulated indirectly by PARP14 to promote the Warburg effect. This describes an alternative glycolysis that cancer cells adopt to suppress apoptosis and continue proliferating. The dimeric structure of PKM2 is significant for the ability of PKM2 to promote the Warburg effect in specific cancer cells.
Aim: This study aims to develop a coupled enzymatic assay to the activity of PKM2 under differing reaction conditions. The assay will be used to investigate a possible difference between unmodified and ADP-ribosylated PKM2 activity.
Methods: Human PKM2 WT was expressed in E. coli cells and purified with IMAC and SEC. The enzymatic activity of PARP14 was tested with a western blot, detecting ADP-ribosylation of PKM2. A PKM2-LDH coupled enzymatic assay was set up with varying conditions to optimize the method. The activity of PKM2 was tested under unmodified and PARP14-mediated ADP-ribosylated conditions. The activity change was determined by investigating the initial rate of reaction, which could be determined by extracting the slope of the linear regression of the measured data points (V0 = initial reaction rate).
Results: A method was set up but could not be fully optimized to produce reproducible results. The results, motivated by statistical analysis, showed indications of a decreased activity of ADP-ribosylated PKM2.
Conclusion: The assay needs further optimization, but it shows great potential for investigating PKM2 activity. Determining the multimeric state of the enzymatically active species of PKM2 would require other types of assays, but it could be important in investigating PARP14’s role in regulating PKM2 activity.

Popular Abstract

Enzymes play a crucial part of keeping the body and all its complicated processes running smoothly, without us having to lift a finger. Most of their hard work goes unnoticed and is usually only recognized when their mechanisms have been altered – causing disruption of finely regulated physiological processes. The altered outcome may seem minor at first, but in some cases, can contribute to severe diseases, including cancer. Luckily, a lot of research has been done to tackle this problem, helping us get closer to finding treatments for this devastating disease.

One enzyme of interest, PKM2, plays a role in the Warburg effect—an altered glycolysis pathway that helps cancer cells thrive. While previous studies suggested an indirect influence of PARP14 on PKM2 in relation to the Warburg effect, my bachelor’s thesis found signs of a direct interaction through ADP-ribosylation performed by PARP14 on PKM1, an isoform of the pyruvate kinase. This project aimed to investigate this further by developing an assay to measure PKM2 activity, which would be used to investigate possible change in activity depending on modification of PKM2.

Continuing the theme of investigating enzymes of cell metabolism, a coupled enzymatic assay was set up, where PKM2 would generate the substrate for an LDH-catalyzed reaction. As LDH converted its substrate, the concentration of the light-absorbing molecule NADH would gradually drop and not only make it possible for us to detect activity of LDH but also equate that activity to the one of PKM2. When the method had been optimized, the two conditions for PKM2 were carried out and compared: unmodified and ADP-ribosylated PKM2.

Turns out that the method would need more optimization in order to produce more trustworthy data. With some initial screenings, indications of lowered activity of ADP-ribosylated PKM2 compared to unmodified PKM2 can be identified. This will hopefully lead to further research of this very interesting phenomena – and help find solutions in eliminating non-functioning enzymes from cancer cells.

• author: Rundberg, Moa
• supervisor: Herwig Schüler
• organization: Department of Chemistry
• course: KEMP20 20251
• year: 2025
• type: L3 - Miscellaneous, Projetcs etc.
• subject: Chemistry
• keywords: PARP14, PKM2, Coupled enzymatic assay, Biochemistry
• language: English
• id: 9186867
• date added to LUP: 2025-04-02 15:05:51
• date last changed: 2025-04-02 15:05:51