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PARP14 Interaction with Key Kinases in the Warburg Effect

Casmir, Ikechukwu LU (2025) KEMR30 20251
Department of Chemistry
Abstract
Introduction: PARP14 is a member of the PARP superfamily of enzymes, which are responsible for the ADP-ribosylation of target proteins and nucleic acids. The enzyme performs mono-ADP-ribosylation of its targets and is involved in many cellular processes, including DNA repair, cellular differentiation and apoptosis. PARP14 is specifically regarded as a writer, reader, as well as an eraser of ADP-ribosylation. Background: PARP14 has been shown to be a pro-survival protein by lowering the activity of PKM2, a key regulator of the Warburg effect. The decreased activity or inactivity of PKM2 brings about the inhibition of the pro-apoptotic protein, MAPK8, thereby, encouraging cells to evade apoptosis. This phenomenon known as Warburg effect... (More)
Introduction: PARP14 is a member of the PARP superfamily of enzymes, which are responsible for the ADP-ribosylation of target proteins and nucleic acids. The enzyme performs mono-ADP-ribosylation of its targets and is involved in many cellular processes, including DNA repair, cellular differentiation and apoptosis. PARP14 is specifically regarded as a writer, reader, as well as an eraser of ADP-ribosylation. Background: PARP14 has been shown to be a pro-survival protein by lowering the activity of PKM2, a key regulator of the Warburg effect. The decreased activity or inactivity of PKM2 brings about the inhibition of the pro-apoptotic protein, MAPK8, thereby, encouraging cells to evade apoptosis. This phenomenon known as Warburg effect describes the alternative glycolysis that cancer cells adopt to suppress apoptosis and continue proliferating. The mechanism by which PARP14 inhibits MAPK8 in the Warburg effect is significant in cancer. Aims: The aim of this study is the biochemical characterization of PARP14 and some of its binding partners, with special emphasis on MAPK8. The study also aims to achieve a PARP14-MAPK8 complex for structural elucidation and further characterization. Methods: PARP14 construct and MAPK8 WT were expressed in E. coli cells and purified by IMAC and SEC. The enzymatic activity of PARP14 was tested using Western blot, which detected ADP-ribosylation of MAPK8. The binding affinity of PARP14 to MAPK8 was evaluated by fluorescence polarization and microscale thermophoresis assays and the dissociation constant, KD determined. Different techniques were applied to produce a stable complex of PARP14 and MAPK8, including co-expression, gel filtration of purified proteins on analytical column, and chemical cross-linking. Result: The study confirmed MAPK8 as a direct target of PARP14 with a KD of ~ 500 nM. However, this study was only able to show a dynamic complex formation with the methods employed to isolate the proteins as a complex. Conclusion: The techniques used in complex formation, including analytical gel filtration and chemical cross-linking need further optimization. New techniques, such as, glutaraldehyde cross-linking in SEC column should also be applied to achieve a stable complex of PARP14 and MAPK8 for structural studies and more characterization.
Keywords: PARP14, MAPK8, Warburg effect, complex formation (Less)
Popular Abstract
PARP14 Interaction with Key Kinases in the Warburg Effect
Cancer cells are abnormal cells, that grow uncontrollably, spreading and invading other tissues. These genetic changes are most often devastating, leading to fatalities. Normal cells are programmed to die at a certain stage in a carefully controlled mechanism coordinated by enzymes. The malfunctioning or complete absence of these enzymes causes normal cells to evade cell death (apoptosis). Once this happens, the tissue or organ housing these abnormal cells develop cancer. A lot of resources are currently deployed in cancer research to discover and develop chemotherapies to fight this scourge.
The Warburg effect describes the phenomenon by which cancer cells evade apoptosis by... (More)
PARP14 Interaction with Key Kinases in the Warburg Effect
Cancer cells are abnormal cells, that grow uncontrollably, spreading and invading other tissues. These genetic changes are most often devastating, leading to fatalities. Normal cells are programmed to die at a certain stage in a carefully controlled mechanism coordinated by enzymes. The malfunctioning or complete absence of these enzymes causes normal cells to evade cell death (apoptosis). Once this happens, the tissue or organ housing these abnormal cells develop cancer. A lot of resources are currently deployed in cancer research to discover and develop chemotherapies to fight this scourge.
The Warburg effect describes the phenomenon by which cancer cells evade apoptosis by using alternate glycolysis to produce energy. It has been postulated that this enzymatic link – PARP14-MAPK8-PKM2 - is implicated in the Warburg effect. Previous studies, including those conducted in our laboratory, have suggested a direct modification of MAPK8 by PARP14 through ADP-ribosylation, but the same cannot be said of the influence of PARP14 on PKM2. PARP14 is known to be an anti-apoptotic protein, while MAPK8 is a pro-apoptotic protein, and PKM2 is a key regulator of the Warburg effect. Understanding the biochemical interactions between these proteins will help to unravel the mechanism by which they promote the Warburg effect. The objectives of this project are to confirm MAPK8 as a direct target for PARP14 and attempt formation of a stable PARP14-MAPK8 complex for structural studies and further biochemical characterization.
Enzyme activity assay by Western blot established modification of MAPK8 by PARP14, whereas fluorescence polarization and microscale thermophoresis assays suggested a strong affinity between the two proteins. While analytical gel filtration and chemical cross-linking of the purified individual proteins showed no sign of complex formed, co-expression and analytical SEC purification of the proteins suggested complex formation, though dynamic and transient.
To achieve stable complex of the proteins, the methods used need optimization and new methods, including glutaraldehyde cross-linking in SEC column should be considered. Getting a stable PARP14-MAPK8 complex would give insights into the roles they play in cancer and help designing treatment strategies. (Less)
Please use this url to cite or link to this publication:
author
Casmir, Ikechukwu LU
supervisor
organization
course
KEMR30 20251
year
type
H2 - Master's Degree (Two Years)
subject
keywords
PARP14, MAPK8, Warburg effect, Complex formation, Biochemistry
language
English
id
9197222
date added to LUP
2025-06-12 11:50:28
date last changed
2025-06-12 11:50:28
@misc{9197222,
  abstract     = {{Introduction: PARP14 is a member of the PARP superfamily of enzymes, which are responsible for the ADP-ribosylation of target proteins and nucleic acids. The enzyme performs mono-ADP-ribosylation of its targets and is involved in many cellular processes, including DNA repair, cellular differentiation and apoptosis. PARP14 is specifically regarded as a writer, reader, as well as an eraser of ADP-ribosylation. Background: PARP14 has been shown to be a pro-survival protein by lowering the activity of PKM2, a key regulator of the Warburg effect. The decreased activity or inactivity of PKM2 brings about the inhibition of the pro-apoptotic protein, MAPK8, thereby, encouraging cells to evade apoptosis. This phenomenon known as Warburg effect describes the alternative glycolysis that cancer cells adopt to suppress apoptosis and continue proliferating. The mechanism by which PARP14 inhibits MAPK8 in the Warburg effect is significant in cancer. Aims: The aim of this study is the biochemical characterization of PARP14 and some of its binding partners, with special emphasis on MAPK8. The study also aims to achieve a PARP14-MAPK8 complex for structural elucidation and further characterization. Methods: PARP14 construct and MAPK8 WT were expressed in E. coli cells and purified by IMAC and SEC. The enzymatic activity of PARP14 was tested using Western blot, which detected ADP-ribosylation of MAPK8. The binding affinity of PARP14 to MAPK8 was evaluated by fluorescence polarization and microscale thermophoresis assays and the dissociation constant, KD determined. Different techniques were applied to produce a stable complex of PARP14 and MAPK8, including co-expression, gel filtration of purified proteins on analytical column, and chemical cross-linking. Result: The study confirmed MAPK8 as a direct target of PARP14 with a KD of ~ 500 nM. However, this study was only able to show a dynamic complex formation with the methods employed to isolate the proteins as a complex. Conclusion: The techniques used in complex formation, including analytical gel filtration and chemical cross-linking need further optimization. New techniques, such as, glutaraldehyde cross-linking in SEC column should also be applied to achieve a stable complex of PARP14 and MAPK8 for structural studies and more characterization.
 Keywords: PARP14, MAPK8, Warburg effect, complex formation}},
  author       = {{Casmir, Ikechukwu}},
  language     = {{eng}},
  note         = {{Student Paper}},
  title        = {{PARP14 Interaction with Key Kinases in the Warburg Effect}},
  year         = {{2025}},
}