Sandbox Reserved 1098

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This Sandbox is Reserved from 25/11/2019, through 30/9/2020 for use in the course "Structural Biology" taught by Bruno Kieffer at the University of Strasbourg, ESBS. This reservation includes Sandbox Reserved 1091 through Sandbox Reserved 1115.

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6HMM

The is a human poly (ADP-ribose) glycohydrolase. It is an enzyme that will catalyze the hydrolysis of glycosides, here more specifically it will produce a free ADP-ribose. This protein is only present when the DNA is damaged. It influences the damaged chromatin through a derepression on a gene promoter. Consequently this protein is quite interesting for biotechnological applications. Indeed, knowing the different pathways and protein interactions leading to DNA damage repair is a meaningful goal in research especially in new cancer therapies.

1 Structure
2 Function
- 2.1 Post-translational modifications and anthraquinone
- 2.2 The mechanism
3 Diseases and Relevance

Structure

Structural highlights

This protein has four principal domains on a : an A-domain, a catalytic domain and two substrate binding domains[1]. The first 456 amino acids of the peptide chain form the . Then, from the 610th to the 795th amino acids, is located the . This catalytic domain can bind with other proteins with (the 726th and 727th amino acids). Next is located from the 869th to the 874th amino acids. [2] As such, most of the amino acids form the A-domain and the catalytic domain and only a few amino acids (8 a.a) make links with other proteins. Moreover, the ligand 7JB can bind the protein on the . These amino acids are located on a helix and on a beta sheet. [3]. There is the amino acid distribution.

Secondary Structure

This protein is 37% and 13% , distributed . Indeed, 6HMM has 25 helices on 198 residues and 23 strands on 74 residues. It also has a few 3/10 helices. [4] Concerning the torsion angles, the backbone angles and the sidechain angles can be differentiated. Indeed, no residue in the does not respect the Ramachandran's angle, whereas only 2% of the residues on the sidechain are Ramachandran outliers due to having a non-rotameric form. [5]

Tertiary Structure

The protein PARG folds into an ADP-ribose-binding macro domain with an N-terminal extension. It also consists of a diphosphate-binding loop on one side of an ADP-ribose binding cavity. On the other side there are several amino acids matching to the specific PARG signature sequence. In the macro domain fold, a loop is inserted to welcome the Glu115 side chain protecting the active site of the PARG protein. This loop gives PARG the ability tp hydrolyze PAR. Concerning the ligand pairing with the PARG protein only a small difference can be observed for the amino acids Val226 and Phe227 ^[1].

Quaternary Structure

The Poly(ADP-ribose)glycohydrolase can interact with either PCNA or NUDT5, this gives various possible functions to the protein. When this protein binds with NUDT5 it can remodel chromatin for example [6].

Function

The protein is a complex composed of the poly (ADP-ribose) glycohydrolase (PARG) and the anthraquinone PDD00013907. The post-translational modifications of the PAR protein (poly ADP-ribose) are important for DNA stability. PDD00013907 is, as already stated, an anthraquinone which is a polycyclic aromatic hydrocarbon usually used in biopesticides as a pest repellant. Here it is considered as a free (of identification number on PDB: 7JB) that can bind to the creating the.

Post-translational modifications and anthraquinone

There are several possible post-translational modifications to stabilize DNA. Most commonly they would be phosphorylation, acetylation or methylation ^[2]. Another post-translational modification concerning the 6HMM protein is made on the poly(ADP-ribose) protein (PAR). PAR is composed of a repetition of ADP-ribose units linked through glycosidic ribose-ribose bonds ^[3]. This allows the repair of single-strand breaks on DNA ^[4]. PARG, a constituent of the 6HMM protein, will degrade PAR to allow the poly (ADP-ribose) polymerase (PARP) to free itself from the damaged, now repaired, site and completes as such reparation ^[5].

The anthraquinone PDD00013907 is a weakly active and cytotoxic anthraquinone 8a acting as a free ligand binding in the ADP-ribose binding site of the PARG. This PDD00013907 should lead to the inhibition of PARG, which is of interest in the search of novel cancer therapies ^[5].

The mechanism

As said previously, poly(ADP-ribosylation) is an important post-translational modification for DNA repair. The mechanism behind this repair relies on several factors. At first, the Poly (ADP-ribose) polymerase (PARP), more specifically the subtype PARP1, will recognize and will bind to the single-stranded break on the DNA. It will then autophosphorylate due to NAD+ and form PAR chains. These will then recruit other repair proteins to the site.

The role of PARG is the hydrolyzation of the specific ribose-ribose bonds present in PAR which leads to its degradation and as such the reparation cycle will be finished^[4]. This degradation is important because without PARG the repair cycle cannot be completed ^[6] and may lead to cell death. This is partially due to the still present PARP on the previously damaged site maintained by the non-degraded PAR ^[7].

Diseases and Relevance

Due to the function of the protein poly (ADP-ribose) glycohydrolase PARG to be part of post-translational processes of DNA damage repair it could be used for new treatments in cancer therapy or for ther diseases. In cancer cells the rate of DNA damaging is most probably higher than in normal cells. This could result from the considerably raised stress levels. A deficiency of PARG results in the cessing of the cell cycle and the following cell death^[4]^[5]. Consequently, the inhibition of PARG might be a solution how to destroy for example tumour cells.

For the opponent PAR protein there has already been a lot of research in this field but not for PARG. As there are no close homologues of PARG^[4], this protein provides a potential target in drug discovery.

As the protein in complex with the anthraquinone does not work properly anymore [7], the described complex shows how an inhibited PARG might act in the cell. The goal of searched therapeutics is therefore to find a way to get the protein PARG into a complex that is acting like the 6HMM complex. For now the research for anthraquinone as inhibitor has stopped as it is cytotoxic for the cell^[5].

References

↑ Slade D, Dunstan MS, Barkauskaite E, Weston R, Lafite P, Dixon N, Ahel M, Leys D, Ahel I. The structure and catalytic mechanism of a poly(ADP-ribose) glycohydrolase. Nature. 2011 Sep 4. doi: 10.1038/nature10404. PMID:21892188 doi:10.1038/nature10404
↑ Oberle C, Blattner C. Regulation of the DNA Damage Response to DSBs by Post-Translational Modifications. Curr Genomics. 2010 May;11(3):184-98. doi: 10.2174/138920210791110979. PMID:21037856 doi:http://dx.doi.org/10.2174/138920210791110979
↑ Slade D, Dunstan MS, Barkauskaite E, Weston R, Lafite P, Dixon N, Ahel M, Leys D, Ahel I. The structure and catalytic mechanism of a poly(ADP-ribose) glycohydrolase. Nature. 2011 Sep 4. doi: 10.1038/nature10404. PMID:21892188 doi:10.1038/nature10404
↑ ^4.0 ^4.1 ^4.2 ^4.3 Waszkowycz B, Smith KM, McGonagle AE, Jordan AM, Acton B, Fairweather EE, Griffiths LA, Hamilton NM, Hamilton NS, Hitchin JR, Hutton CP, James DI, Jones CD, Jones S, Mould DP, Small HF, Stowell AIJ, Tucker JA, Waddell ID, Ogilvie DJ. Cell-Active Small Molecule Inhibitors of the DNA-Damage Repair Enzyme Poly(ADP-ribose) Glycohydrolase (PARG): Discovery and Optimization of Orally Bioavailable Quinazolinedione Sulfonamides. J Med Chem. 2018 Dec 13;61(23):10767-10792. doi: 10.1021/acs.jmedchem.8b01407., Epub 2018 Nov 19. PMID:30403352 doi:http://dx.doi.org/10.1021/acs.jmedchem.8b01407
↑ ^5.0 ^5.1 ^5.2 ^5.3 James DI, Smith KM, Jordan AM, Fairweather EE, Griffiths LA, Hamilton NS, Hitchin JR, Hutton CP, Jones S, Kelly P, McGonagle AE, Small H, Stowell AI, Tucker J, Waddell ID, Waszkowycz B, Ogilvie DJ. First-in-Class Chemical Probes against Poly(ADP-ribose) Glycohydrolase (PARG) Inhibit DNA Repair with Differential Pharmacology to Olaparib. ACS Chem Biol. 2016 Oct 12. PMID:27689388 doi:http://dx.doi.org/10.1021/acschembio.6b00609
↑ Fisher AE, Hochegger H, Takeda S, Caldecott KW. Poly(ADP-ribose) polymerase 1 accelerates single-strand break repair in concert with poly(ADP-ribose) glycohydrolase. Mol Cell Biol. 2007 Aug;27(15):5597-605. doi: 10.1128/MCB.02248-06. Epub 2007 Jun, 4. PMID:17548475 doi:http://dx.doi.org/10.1128/MCB.02248-06
↑ Kim MY, Zhang T, Kraus WL. Poly(ADP-ribosyl)ation by PARP-1: 'PAR-laying' NAD+ into a nuclear signal. Genes Dev. 2005 Sep 1;19(17):1951-67. doi: 10.1101/gad.1331805. PMID:16140981 doi:http://dx.doi.org/10.1101/gad.1331805

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@@ Line 10: / Line 10: @@
 === Structural highlights ===
-This protein has four principal domains on a <scene name='82/829351/Single_chain/1'>single peptide chain</scene>: a A-domain, a catalytic domain and two substrate binding domains[http://www.uniprot.org/uniprot/Q86W56].
+This protein has four principal domains on a <scene name='82/829351/Single_chain/1'>single peptide chain</scene>: an A-domain, a catalytic domain and two substrate binding domains[http://www.uniprot.org/uniprot/Q86W56].
-The first 456 amino acids of the peptide chain form the <scene name='82/829351/A_domain/1'>A-domain</scene>. Then from 610 to 795 amino acids is located the <scene name='82/829351/Catalytic_domain/1'>catalytic domain</scene>. This catalytic domain can binding with other proteins with <scene name='82/829351/Substrat_-_catalytic_domain/1'>two amino acids</scene> (the 726 and 727 amino acids). Next <scene name='82/829351/Subtrat_jonction_domain_2/1'>the second substrate binding domain</scene> is located from the 869 th to the 874th amino acids. [http://www.uniprot.org/uniprot/Q86W56]
+The first 456 amino acids of the peptide chain form the <scene name='82/829351/A_domain/1'>A-domain</scene>. Then, from the 610th to the 795th amino acids, is located the <scene name='82/829351/Catalytic_domain/1'>catalytic domain</scene>. This catalytic domain can bind with other proteins with <scene name='82/829351/Substrat_-_catalytic_domain/1'>two amino acids</scene> (the 726th and 727th amino acids). Next <scene name='82/829351/Subtrat_jonction_domain_2/1'>the second substrate binding domain</scene> is located from the 869th to the 874th amino acids. [http://www.uniprot.org/uniprot/Q86W56]
-So, most of the amino acids form the A-domain and the catzlytic domain and only few amino acids (8 a.a) make links with other proteins. Moreover, the ligand 7JB can bind the protein on the <scene name='82/829351/Liason_7jb/1'>754, 758, 792 and 795 amino acids</scene>. These amino acids are located on a helix and on a beta sheet. [http://www.rcsb.org/pdb/explore/remediatedSequence.do?structureId=6HMM]. So the amino acids of the protein is distributed like <scene name='82/829351/Distritbution_domain/1'>this</scene>.
+As such, most of the amino acids form the A-domain and the catalytic domain and only a few amino acids (8 a.a) make links with other proteins. Moreover, the ligand 7JB can bind the protein on the <scene name='82/829351/Liason_7jb/1'>754, 758, 792 and 795 amino acids</scene>. These amino acids are located on a helix and on a beta sheet. [http://www.rcsb.org/pdb/explore/remediatedSequence.do?structureId=6HMM]. There is the <scene name='82/829351/Distritbution_domain/1'>following</scene> amino acid distribution.
 === Secondary Structure ===
-This protein is 37% <scene name='82/829351/Helix/1'>helical</scene> and 13% <scene name='82/829351/Sheet/1'>beta sheet</scene>, distributed like <scene name='82/829351/Helix_and_beta_sheet/1'>this</scene>. Indeed, it has 25 helices on 198 residues and 23 strands on 74 residues. It has also few 3/10 helices. [http://www.rcsb.org/pdb/explore/remediatedSequence.do?structureId=6HMM]
+This protein is 37% <scene name='82/829351/Helix/1'>helical</scene> and 13% <scene name='82/829351/Sheet/1'>beta sheet</scene>, distributed <scene name='82/829351/Helix_and_beta_sheet/1'>as such</scene>. Indeed, 6HMM has 25 helices on 198 residues and 23 strands on 74 residues. It also has a few 3/10 helices. [http://www.rcsb.org/pdb/explore/remediatedSequence.do?structureId=6HMM]
-Involving the torsion angles the backbone and the sidechain have to be differentiated. Indeed none residue in the <scene name='82/829351/Mainchain/1'>backbone</scene> does't respect the Ramachandran's angle, whereas in sidechains where 2% of the residues are Ramachandran outliers because they have non-rotameric sidechains. [http://files.rcsb.org/pub/pdb/validation_reports/hm/6hmm/6hmm_full_validation.pdf]
+Concerning the torsion angles, the backbone angles and the sidechain angles can be differentiated. Indeed, no residue in the <scene name='82/829351/Mainchain/1'>backbone</scene> does not respect the Ramachandran's angle, whereas only 2% of the residues on the sidechain are Ramachandran outliers due to having a non-rotameric form. [http://files.rcsb.org/pub/pdb/validation_reports/hm/6hmm/6hmm_full_validation.pdf]
 === Tertiary Structure ===
 The protein PARG folds into an ADP-ribose-binding macro domain with an N-terminal extension. It also consists of a diphosphate-binding loop on one side of an ADP-ribose binding cavity. On the other side there are several amino acids matching to the specific PARG signature sequence.
-In the macro domain fold a loop is inserted to welcome the Glu115 side chain protecting the active site of the PARG protein. This loop gives PARG the ability tp hydrolyze PAR.
+In the macro domain fold, a loop is inserted to welcome the Glu115 side chain protecting the active site of the PARG protein. This loop gives PARG the ability tp hydrolyze PAR.
 Concerning the ligand pairing with the PARG protein only a small difference can be observed for the amino acids Val226 and Phe227 <ref>PMID: 21892188</ref>.
 === Quaternary Structure ===
-Poly(ADP-ribose)glycohydrolase interact with [[PCNA]] or [[NUDT5]]. When this protein is binding with NUDT5 it can remodeling chromatin.[http://www.uniprot.org/uniprot/Q86W56]
+The Poly(ADP-ribose)glycohydrolase can interact with either [[PCNA]] or [[NUDT5]], this gives various possible functions to the protein. When this protein binds with NUDT5 it can remodel chromatin for example [http://www.uniprot.org/uniprot/Q86W56].
 == Function ==