Effect of phosphomimetic point mutations on the ADP-ribosylation activity of PARP16

Abstract

Introduction: Phosphomimetic residue and D-loop mutants have been created and their effect studied in the enzyme PARP16.

Background: It is known that PARP16 is phosphorylated, and where the phosphorylation sites are, but not how this affects ADP-ribosylation activity. As of yet, there are no reliable ways to obtain 100% phosphorylated PARP16, so it is instead mimicked with amino-acids with similar properties. The mutant that replaces a threonine in PARP10s D-loop structure inactivates the protein, so a similar mutation was carried out in PARP16 to discover if the effects were similar.

Aim: This study aims to determine whether phosphorylation of PARP16 does or does not decrease activity and if PARP16 is as reliant on its D-loop as PARP10.

Methods: PCR reactions were used for site-directed mutagenesis in order to create the effects desired in the project's various mutants. Recombinant protein expression in E.coli bacteria was used to grow suitable amounts of protein, which were then purified through chromatography. Western blots and densitometric analysis were used to measure and quantify activity in wild-type and mutant PARP16.

Results: The mutants all showed decreased activity compared to the wild-type protein, even the mutant expected to terminate all activity.

Conclusion: Results suggest phosphorylation does decrease ADP-ribosylation in PARP16. PARP16s D-loop has some redundancies, i.e. it can function without a previously thought to be vital residue.

Popular Abstract

Mutants of the enzyme PARP16: better, worse or just different?

Enzymes are essential to the function of life, and these biological catalysts are as varied as they are vital. In this project, a closer look was taken at one small part of one small enzyme, namely PARP16. PARP16 belongs to a larger group of proteins, Poly ADP-Ribose Polymerases (PARPs), that attach a molecule known as ADP-ribose to other biological molecules. PARPs break apart a larger molecule that occurs naturally in cells, NAD+, to use one half as the ADP-ribose. The targets can be proteins, DNA or even PARPs themselves. The combining of DNA with ADP-ribose is very important in reparation of the former. PARPs can detect damage and affix ADP-ribose, which acts like a signal for other processes to start repairs. Much research has been done on how to influence and control these enzymes, as DNA damage is a factor in many cancerous diseases.

Some parts of the PARP16s structure have been shown to have phosphate groups connected to them, much in the same way that ADP-ribose can be connected to DNA. What these groups do exactly and how they affect PARP16s ability to attach ADP-ribose is not fully understood yet, but this study aims to shine a light on how PARP16 works. What is known is where these phosphate groups connect to PARP16. It is difficult to connect every single site on PARP16 to a phosphate group, so instead mutant variants of the protein were created, with inherent substitutions that have similar properties to the phosphate groups. This was done by exchanging the part where the phosphate would connect with something that looks and acts similar to a phosphate group. The replacement is of a similar size and has a negative charge, like a phosphate group, but made of different atoms. A mimic of sorts, a phospho-mimic. Alongside these, a mutant meant to be completely inactive was created, as a comparison.

With these variants created, they could then be grown in E.coli cells, using their biological machinery to mass-produce the PARP16 protein for our purposes. Once this was done, and the protein extracted and purified from these cells, testing began. The mutants were compared with the “natural” PARP16, by letting them both attach ADP-ribose to themselves in controlled reactions. The amount of ADP-ribose they attached to themselves could be compared, and the results showed that the mutants were less effective by between 40-50% than the original version. This evidence suggests that PARP16 gets worse at attaching ADP-ribose groups when it has phosphate groups on it. The mutant meant to be inactive still attached ADP-ribose groups, suggesting some redundancy in PARP16. Many times, cells have ways of slowing down or speeding up enzyme abilities to catalyse chemical reactions. The affixment of different groups, especially phosphate groups, is a common method of doing so. To summarise, PARP16 is now a little better understood, particularly how its activity can be affected by its attachments. Better understanding of PARPs will lead to being able to design cancer treatments that target these essential enzymes.

• author: Björklöf Dolan, Noah
• supervisor: Herwig Schüler
• organization: Department of Chemistry
• course: KEML10 20232
• year: 2024
• type: M2 - Bachelor Degree
• subject: Chemistry
• keywords: ADP-ribosylation, PARP16, PCR, Phosphomimetics, Western blot Specialization: biochemistry
• language: English
• id: 9144444
• date added to LUP: 2024-01-10 13:39:02
• date last changed: 2024-01-10 13:39:02