Skip to main content

LUP Student Papers

LUND UNIVERSITY LIBRARIES

Purification and Activity Analysis of PARP16 Wildtype and Mutant R49A

Nash, Angelica LU (2022) KEMK10 20221
Department of Chemistry
Abstract
Research in cancer is an ongoing project which not only includes finding more efficient treatment therapies, but also achieving a greater knowledge of how proteins of interest are targeted by inhibitors. Furthermore, research in this field can give us greater knowledge about the development of cancer cells if they are modified. One enzymatic protein family, the PARPs, has been widely studied for decades. The studies on PARPs indicate that inhibiting their activity has a great effect on cancer cells. Clinical trials have demonstrated the efficiency of PARP inhibition, especially in breast and ovarian cancer, as well as in pancreatic and prostate cancer. PARPs can ribosylate NAD+ to ADP onto themselves in the active site, this mechanism is... (More)
Research in cancer is an ongoing project which not only includes finding more efficient treatment therapies, but also achieving a greater knowledge of how proteins of interest are targeted by inhibitors. Furthermore, research in this field can give us greater knowledge about the development of cancer cells if they are modified. One enzymatic protein family, the PARPs, has been widely studied for decades. The studies on PARPs indicate that inhibiting their activity has a great effect on cancer cells. Clinical trials have demonstrated the efficiency of PARP inhibition, especially in breast and ovarian cancer, as well as in pancreatic and prostate cancer. PARPs can ribosylate NAD+ to ADP onto themselves in the active site, this mechanism is of interest since the activity inhibition has shown being effective in treating certain cancer types.

PARP16 is one of the smaller enzymes in the PARP family whose mechanism and enzymatic activity is not yet determined. Recent studies have shown that the crystal structure of PARP16 has an arginine at position 49 which is directed towards the active site. This could possibly indicate that arginine is an acceptor in ADP-ribosylation of PARP16. If arginine is exchanged with the neutral amino acid alanine, will this affect the activity of PARP16 when it is ADP-ribosylated? If not, what amino acids are targeted in ADP-ribosylation?

The aim of this study was to purify PARP16 wildtype and mutant R49A by using IMAC and SEC and to study if the mutant is less active or inactive, compared to the wildtype of PARP16. The activity analysis was done with Western blotting and protein overlay assay using MacroGreen and glycohydrolases. The results from those experiments indicate that arginine does not affect the enzymatic activity of PARP16. Further studies were done on PARP16 wildtype to determine what type of amino acid is affected in ADP-ribosylation, which was done with protein overlay assay using glycohydrolases and Western blotting. The results from those experiments indicate that PARP16 had less ADPr left, after using MacroD2 and ARH3, which target acidic amino acids and serine, respectively. (Less)
Popular Abstract (Swedish)
Aktivitetsanalys av enzymet PARP16 och dess koppling till cancerterapi

Cancer är ett ord som troligtvis berör på ett eller annat sätt. Vare sig om man drabbas själv eller inte, så påverkar det alla om det så bara är en närstående till en som blivit drabbad. Studier inom proteinkemin har länge varit av stor betydelse då proteiner spelar en nyckelroll i DNA reparation. En enzymfamilj som är av stort intresse är PARP, där studier visat på hur inhibering av dess aktivitet har en positiv påverkan vid cancerbehandling.

PARP är en förkortning av poly(ADP-ribos) polymeras, och är en enzymfamilj bestående av 17 olika enzymer. Deras viktigaste funktion är att katalysera additionen av en ADP-ribos till ett målprotein, vilket i sin tur... (More)
Aktivitetsanalys av enzymet PARP16 och dess koppling till cancerterapi

Cancer är ett ord som troligtvis berör på ett eller annat sätt. Vare sig om man drabbas själv eller inte, så påverkar det alla om det så bara är en närstående till en som blivit drabbad. Studier inom proteinkemin har länge varit av stor betydelse då proteiner spelar en nyckelroll i DNA reparation. En enzymfamilj som är av stort intresse är PARP, där studier visat på hur inhibering av dess aktivitet har en positiv påverkan vid cancerbehandling.

PARP är en förkortning av poly(ADP-ribos) polymeras, och är en enzymfamilj bestående av 17 olika enzymer. Deras viktigaste funktion är att katalysera additionen av en ADP-ribos till ett målprotein, vilket i sin tur kontrollerar reparationen av skadat DNA. ADP-ribos är en kemisk förening som skapas genom att koenzym NAD+ blir hydrolyserat. Några av PARP medlemmarna kan addera fler än en enhet ADP-ribos och på så sätt bilda långa kedjor eller förgreningar av ADP-ribos.

Denna katalytiska mekanism har studerats i flera årtionden och visats ha effekt på cancerceller, när PARParnas enzymaktivitet inhiberas. Ett utav dessa enzym, PARP16, har ännu inte fått dess enzymatiska mekanism fastlagd. Däremot har studier visat att kristallstrukturen (se Figur 1) av PARP16 har en aminosyra, arginin, riktad in mot enzymets aktiva säte. Det är i det aktiva sätet som föreningar så som NAD+ kan fästas och katalyseras. Därav tros arginin ha en betydelse för ADP-ribosylering. Vid inhibering av PARP16 är inte längre enzymet aktivt. Detta medför att cancer-cellerna inte kan repareras då DNA reparationen inte är i gång, och denna är som tidigare nämnt kontrollerad av ADP-ribosylering. Detta kommer i sin tur kommer leda till apoptos (programmerad celldöd) av cancercellerna.

I denna studie renades och analyserades både vildtypen av PARP16 samt en mutant, där arginin har ersatts med en annan aminosyra, alanin. Målet var att undersöka huruvida aktiviteten för mutanten förändrades eller inte, eftersom den aminosyra som tros vara mål för ADP-ribosyleringen har ersatts.

Proteinerna lät odlas i E. coli och renades upp innan både vildtypen och mutanten reagerade med koenzymet NAD+, som hydrolyserades till ADP-ribos. Därefter gjordes aktivitetsmätningar för att avgöra hur mycket av PARP16 som bundit in ADP-ribos i det aktiva sätet. Resultatet visade att det inte fanns en märkbar skillnad mellan vildtypen och mutanten och slutsatsen kan dras att arginin inte är en del av ADP-ribosylering i PARP16.

Handledare: Herwig Schüler
Examensarbete 15 hp i kemi KEMK10, 2022
Kemiska institutionen, Lunds universitet (Less)
Please use this url to cite or link to this publication:
author
Nash, Angelica LU
supervisor
organization
course
KEMK10 20221
year
type
M2 - Bachelor Degree
subject
keywords
Biochemistry, Structural Biology, PARP, Cancer
language
English
id
9086271
date added to LUP
2022-06-17 09:27:28
date last changed
2022-06-17 09:27:28
@misc{9086271,
  abstract     = {{Research in cancer is an ongoing project which not only includes finding more efficient treatment therapies, but also achieving a greater knowledge of how proteins of interest are targeted by inhibitors. Furthermore, research in this field can give us greater knowledge about the development of cancer cells if they are modified. One enzymatic protein family, the PARPs, has been widely studied for decades. The studies on PARPs indicate that inhibiting their activity has a great effect on cancer cells. Clinical trials have demonstrated the efficiency of PARP inhibition, especially in breast and ovarian cancer, as well as in pancreatic and prostate cancer. PARPs can ribosylate NAD+ to ADP onto themselves in the active site, this mechanism is of interest since the activity inhibition has shown being effective in treating certain cancer types.

PARP16 is one of the smaller enzymes in the PARP family whose mechanism and enzymatic activity is not yet determined. Recent studies have shown that the crystal structure of PARP16 has an arginine at position 49 which is directed towards the active site. This could possibly indicate that arginine is an acceptor in ADP-ribosylation of PARP16. If arginine is exchanged with the neutral amino acid alanine, will this affect the activity of PARP16 when it is ADP-ribosylated? If not, what amino acids are targeted in ADP-ribosylation?

The aim of this study was to purify PARP16 wildtype and mutant R49A by using IMAC and SEC and to study if the mutant is less active or inactive, compared to the wildtype of PARP16. The activity analysis was done with Western blotting and protein overlay assay using MacroGreen and glycohydrolases. The results from those experiments indicate that arginine does not affect the enzymatic activity of PARP16. Further studies were done on PARP16 wildtype to determine what type of amino acid is affected in ADP-ribosylation, which was done with protein overlay assay using glycohydrolases and Western blotting. The results from those experiments indicate that PARP16 had less ADPr left, after using MacroD2 and ARH3, which target acidic amino acids and serine, respectively.}},
  author       = {{Nash, Angelica}},
  language     = {{eng}},
  note         = {{Student Paper}},
  title        = {{Purification and Activity Analysis of PARP16 Wildtype and Mutant R49A}},
  year         = {{2022}},
}