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Cloning and characterization of a multidomain PARP14 construct

Chatzicharalampous, Constantinos LU (2023) KEMR30 20231
Department of Chemistry
Abstract
PARP14, a member of the PARP family that can modify target proteins with ADP-ribose has stimulated the interest of researchers as it is involved in a variety of diseases including cancer. While some structural and activity characterization has been conducted, it has not been yet expressed and purified as a single molecule, instead it was examined in small constructs that include several of its domains separately. It contains multiple domains, the RNA recognition motif (RRM), three tandem macrodomains, a WWE and an ART catalytic domain (ADP-ribosylation). In this study a suspected region of the protein that could be responsible for aggregation and contamination generation, was substituted (using Gibson-assembly cloning), by a soluble... (More)
PARP14, a member of the PARP family that can modify target proteins with ADP-ribose has stimulated the interest of researchers as it is involved in a variety of diseases including cancer. While some structural and activity characterization has been conducted, it has not been yet expressed and purified as a single molecule, instead it was examined in small constructs that include several of its domains separately. It contains multiple domains, the RNA recognition motif (RRM), three tandem macrodomains, a WWE and an ART catalytic domain (ADP-ribosylation). In this study a suspected region of the protein that could be responsible for aggregation and contamination generation, was substituted (using Gibson-assembly cloning), by a soluble SUMO-tag to express PARP14 as a single molecule, excluding the region starting from RRM until the N-terminal macrodomain. The SUMO-PARP14 was also studied from structure and activity perspective to approximate the characterization of the wild-type PARP14. The protein purification steps utilized were Immobilized Metal Affinity Chromatography, Anion exchange chromatography and Size Exclusion Chromatography. Negative staining was used to visually assess the protein structure in solution to confirm that it is suitable to be observed using cryo-EM in the future. Western blots and the MacroGreen plate assays were used to test whether SUMO-PARP14 preserves the ART activity, by monitoring the signal generated from ADP-ribosylated targets after treatment with an HRP-Streptavidin and an ADP-ribose binding macrodomain-GFP, respectively. SARS-CoV-2-NSP3-MAC1 inhibitory compounds were also tested against PARP14 macrodomain 1 due to structural homology, to determine if there is inhibition so that they could be used as ligands in the future for cryo-EM or crystallography. The methods used for the drug assessment were Western blot and Differential Scanning Fluorimetry. Ultimately, SUMO-PARP14 was successfully expressed with low signs of aggregation and contamination, and it was decided that it can be used after purification optimizations in cryo-EM. The protein retains the wild-type ART catalytic and macrodomain 1 (M1) glycohydrolytic activity (which was firstly experimentally confirmed in this study). As a confirmation that structural homologues of PARP14 M1 have glycohydrolytic capacity, PARP9 M1 which is its closest phylogenetic homologue, was assessed and determined that also has the same activity. Further, all compounds tested against PARP14 M1 were found to destabilize the protein structure observed through Tm shifts. Concluding, SUMO-PARP14 is a suitable construct to approximate the structure and activity of wild-type PARP14 as it can be highly pure with no significant aggregation present. (Less)
Popular Abstract
The interest of this study is on a family of proteins called PARPs (Poly (ADP-ribose) polymerases) with focus on a member of this family, PARP14. PARP14 attaches a molecule called ADP-ribose (Adenosine Diphosphate-ribose) to other proteins in a cell (this action is called ADP-ribosylation), altering its properties. PARP14 is responsible for immune system regulation and was also identified to be present in a variety of cancers and inflammatory diseases, thus there is a public health interest to study this protein. In previous studies conducted in our laboratory, attempts to isolate, and purify the entire PARP14 molecule from bacterial cells, lead to protein aggregation and contamination of the sample. The goal of this study was to use... (More)
The interest of this study is on a family of proteins called PARPs (Poly (ADP-ribose) polymerases) with focus on a member of this family, PARP14. PARP14 attaches a molecule called ADP-ribose (Adenosine Diphosphate-ribose) to other proteins in a cell (this action is called ADP-ribosylation), altering its properties. PARP14 is responsible for immune system regulation and was also identified to be present in a variety of cancers and inflammatory diseases, thus there is a public health interest to study this protein. In previous studies conducted in our laboratory, attempts to isolate, and purify the entire PARP14 molecule from bacterial cells, lead to protein aggregation and contamination of the sample. The goal of this study was to use biochemical approaches enabling researchers to prevent these issues from occurring and in parallel to be able to characterize the structure and activity (ADP-ribosylation) of PARP14. The tactic followed was to substitute a region in the protein, that was suspected to cause issues with purification, with a soluble protein sequence, called SUMO (Small Ubiquitin Like Modifier), to test whether the aggregation and contamination issues disappear. In parallel, the ADP-ribosylation activity was studied using assays that detect ADP-ribose presence on proteins.

The study results show that the SUMO insertion in PARP14 significantly reduced the presence of aggregation and contaminants and that the new protein, called SUMO-PARP14, preserves the ADP-ribosylation activity. In addition, it was experimentally confirmed for the first time in literature, that another domain that it contains, called macrodomain 1, can remove ADP-ribose from proteins, thus reversing its own ADP-ribosylation effect. Concluding, SUMO-PARP14 can be used in the future to find approximately the structure of the original PARP14 protein and its interactions with other cell contents such as DNA, RNA and proteins, while also providing the opportunity to study in more extent its ADP-ribosylation addition and removal properties. (Less)
Please use this url to cite or link to this publication:
author
Chatzicharalampous, Constantinos LU
supervisor
organization
course
KEMR30 20231
year
type
H2 - Master's Degree (Two Years)
subject
keywords
ADP-ribosylation, Biochemistry, Glycohydrolase, Macrodomain, MacroGreen, PARP
language
English
id
9117022
date added to LUP
2023-05-31 09:47:31
date last changed
2023-05-31 09:47:31
@misc{9117022,
  abstract     = {{PARP14, a member of the PARP family that can modify target proteins with ADP-ribose has stimulated the interest of researchers as it is involved in a variety of diseases including cancer. While some structural and activity characterization has been conducted, it has not been yet expressed and purified as a single molecule, instead it was examined in small constructs that include several of its domains separately. It contains multiple domains, the RNA recognition motif (RRM), three tandem macrodomains, a WWE and an ART catalytic domain (ADP-ribosylation). In this study a suspected region of the protein that could be responsible for aggregation and contamination generation, was substituted (using Gibson-assembly cloning), by a soluble SUMO-tag to express PARP14 as a single molecule, excluding the region starting from RRM until the N-terminal macrodomain. The SUMO-PARP14 was also studied from structure and activity perspective to approximate the characterization of the wild-type PARP14. The protein purification steps utilized were Immobilized Metal Affinity Chromatography, Anion exchange chromatography and Size Exclusion Chromatography. Negative staining was used to visually assess the protein structure in solution to confirm that it is suitable to be observed using cryo-EM in the future. Western blots and the MacroGreen plate assays were used to test whether SUMO-PARP14 preserves the ART activity, by monitoring the signal generated from ADP-ribosylated targets after treatment with an HRP-Streptavidin and an ADP-ribose binding macrodomain-GFP, respectively. SARS-CoV-2-NSP3-MAC1 inhibitory compounds were also tested against PARP14 macrodomain 1 due to structural homology, to determine if there is inhibition so that they could be used as ligands in the future for cryo-EM or crystallography. The methods used for the drug assessment were Western blot and Differential Scanning Fluorimetry. Ultimately, SUMO-PARP14 was successfully expressed with low signs of aggregation and contamination, and it was decided that it can be used after purification optimizations in cryo-EM. The protein retains the wild-type ART catalytic and macrodomain 1 (M1) glycohydrolytic activity (which was firstly experimentally confirmed in this study). As a confirmation that structural homologues of PARP14 M1 have glycohydrolytic capacity, PARP9 M1 which is its closest phylogenetic homologue, was assessed and determined that also has the same activity. Further, all compounds tested against PARP14 M1 were found to destabilize the protein structure observed through Tm shifts. Concluding, SUMO-PARP14 is a suitable construct to approximate the structure and activity of wild-type PARP14 as it can be highly pure with no significant aggregation present.}},
  author       = {{Chatzicharalampous, Constantinos}},
  language     = {{eng}},
  note         = {{Student Paper}},
  title        = {{Cloning and characterization of a multidomain PARP14 construct}},
  year         = {{2023}},
}