Effects of phosphorylation on the ADP-ribosylation activity of PARP16
(2023) KEMP30 20222Department of Chemistry
- Abstract
- PARP16/ARTD15 is an intracellular mono-ADP-ribosyltransferase that catalyzes auto- and heteromodification by transfer of a single ADP-ribose moiety. It belongs to the ADP-ribosyltransferase (ART) superfamily. ADP-ribosylation of amino acids on protein targets is critical in the regulation of cellular pathways in eukaryotes, especially involved in physiological functions, cell stress responses and disease. It has been discovered that PARP16 plays an important role in the Unfolded Protein Response (UPR) as the main activator of 2 endoplasmic reticulum (ER) stress sensors, PERK and IRE1α kinases. In addition, it was also found that these kinases phosphorylate PARP16. The aim of this project is to investigate the phosphorylation of PARP16 by... (More)
- PARP16/ARTD15 is an intracellular mono-ADP-ribosyltransferase that catalyzes auto- and heteromodification by transfer of a single ADP-ribose moiety. It belongs to the ADP-ribosyltransferase (ART) superfamily. ADP-ribosylation of amino acids on protein targets is critical in the regulation of cellular pathways in eukaryotes, especially involved in physiological functions, cell stress responses and disease. It has been discovered that PARP16 plays an important role in the Unfolded Protein Response (UPR) as the main activator of 2 endoplasmic reticulum (ER) stress sensors, PERK and IRE1α kinases. In addition, it was also found that these kinases phosphorylate PARP16. The aim of this project is to investigate the phosphorylation of PARP16 by the kinases and whether that implies alterations of its activity. To answer this question, we coexpressed Tyrosine-kinases with YopH and Serine/Threonine-kinases with Lambda from an Addgene genetic library. The kinases were purified by a 2-step elution protocol using Ni-NTA beads and imidazole. Although the samples showed signs of phosphatase contamination, we found conditions in which we could assess the phosphorylation activity of the kinases. Finally, phosphorylation and ADP-ribosylation assays were performed with PARP16 and our kinases. The results showed clear phosphorylation of PARP16 and a variation of its ADP-ribosylation activity in the presence of the modification. This discovery opens up new questions about the biological effects that the phosphorylation of PARP16 can have, especially in cancers and protein folding diseases. Therefore, these findings can be critical for therapeutic inhibition and treatment. Further investigation should address questions such as what residues from PARP16 are modified and how phosphorylation by PERK and IRE1α affect the ADP-ribosylation activity of PARP16. (Less)
- Popular Abstract
- This report covers an investigation on protein modification and enzymatic activity. Our protein of interest is known as PARP16/ARTD15 and belongs to a protein superfamily that modifies other proteins by adding ADP-ribose using NAD+ as a substrate (ADP-ribosylation). The proteins from this superfamily are involved in several physiological functions, cell stress responses, and even disease. Therefore, they have been recently identified as important therapeutic targets for cancer and for the modulation of the immune system.
PARPs present various characteristic and essential features in their structure and sequence critical for their ADP-ribosylation activity. In particular, we could highlight their catalytic domain that contains a signature... (More) - This report covers an investigation on protein modification and enzymatic activity. Our protein of interest is known as PARP16/ARTD15 and belongs to a protein superfamily that modifies other proteins by adding ADP-ribose using NAD+ as a substrate (ADP-ribosylation). The proteins from this superfamily are involved in several physiological functions, cell stress responses, and even disease. Therefore, they have been recently identified as important therapeutic targets for cancer and for the modulation of the immune system.
PARPs present various characteristic and essential features in their structure and sequence critical for their ADP-ribosylation activity. In particular, we could highlight their catalytic domain that contains a signature histidine, tyrosine and glutamate (H-Y-E) motif. However, the catalytic domain of PARP16 is composed of H-Y-Y in the catalytic triad and is only capable of performing auto and trans mono-ADP-ribosylation, unlike other PARPs that have poly-ADP-ribosylation activity.
PARP16 has been found to have an important role in the Unfolded Protein Response (UPR) since it is required for activation of two ER stress sensors, PERK and IRE1α. These are so-called kinases and play critical roles in disease development related with protein folding and cancer. Additionally, the same study showed that these two proteins modify PARP16 by adding phosphate residues (phosphorylation).
Aiming to understand this biological mechanism, and due to difficulties to produce PERK and IRE1α, we designed various experiments in which we expressed, purified, and tested several kinases from an Addgene genetic library called ``an open library of human kinase domain constructs for Automated Bacterial expression´´. It contained 72 kinases and 2 phosphatases. The phosphatases are of great relevance because they allow a non-toxic overexpression of the kinases.
The experimentation began with the coexpression of the phosphatases and kinases followed by purification of the kinases with Ni-NTA beads. Next, we tested the autophosphorylation activity and transphosphorylation activity to PARP16 of the kinases in presence of ATP. This showed that the kinases were active and we identified several kinases that phosphorylated PARP16. After the results of both assays, ADP-ribosylation assays were designed in order to investigate the effects that phosphorylation has on the catalytic activity of PARP16. The results showed a variation of the ADP-ribosylation activity of PARP16. It is remarkable that 2 of the 4 kinases tested in this last assay have the same specificity as PERK and IRE1α and reduce the activity of PARP16.
This discovery can be key to develop therapies to prevent or treat cancer and genetic disease development. However, further investigation is indispensable to elucidate which residues of PARP16 are modified by the kinases and how PERK and IRE1α behave in the presence of PARP16. (Less)
Please use this url to cite or link to this publication:
http://lup.lub.lu.se/student-papers/record/9106715
- author
- Martín Cristóbal, Virginia LU
- supervisor
- organization
- course
- KEMP30 20222
- year
- 2023
- type
- M2 - Bachelor Degree
- subject
- keywords
- ADP-ribosylation, Kinases, PARP16, Phosphorylation, UPR biochemistry, Biochemistry
- language
- English
- id
- 9106715
- date added to LUP
- 2023-01-26 09:26:59
- date last changed
- 2023-01-26 09:26:59
@misc{9106715,
abstract = {{PARP16/ARTD15 is an intracellular mono-ADP-ribosyltransferase that catalyzes auto- and heteromodification by transfer of a single ADP-ribose moiety. It belongs to the ADP-ribosyltransferase (ART) superfamily. ADP-ribosylation of amino acids on protein targets is critical in the regulation of cellular pathways in eukaryotes, especially involved in physiological functions, cell stress responses and disease. It has been discovered that PARP16 plays an important role in the Unfolded Protein Response (UPR) as the main activator of 2 endoplasmic reticulum (ER) stress sensors, PERK and IRE1α kinases. In addition, it was also found that these kinases phosphorylate PARP16. The aim of this project is to investigate the phosphorylation of PARP16 by the kinases and whether that implies alterations of its activity. To answer this question, we coexpressed Tyrosine-kinases with YopH and Serine/Threonine-kinases with Lambda from an Addgene genetic library. The kinases were purified by a 2-step elution protocol using Ni-NTA beads and imidazole. Although the samples showed signs of phosphatase contamination, we found conditions in which we could assess the phosphorylation activity of the kinases. Finally, phosphorylation and ADP-ribosylation assays were performed with PARP16 and our kinases. The results showed clear phosphorylation of PARP16 and a variation of its ADP-ribosylation activity in the presence of the modification. This discovery opens up new questions about the biological effects that the phosphorylation of PARP16 can have, especially in cancers and protein folding diseases. Therefore, these findings can be critical for therapeutic inhibition and treatment. Further investigation should address questions such as what residues from PARP16 are modified and how phosphorylation by PERK and IRE1α affect the ADP-ribosylation activity of PARP16.}},
author = {{Martín Cristóbal, Virginia}},
language = {{eng}},
note = {{Student Paper}},
title = {{Effects of phosphorylation on the ADP-ribosylation activity of PARP16}},
year = {{2023}},
}