Publications by authors named "Aleksandra Szwagierczak"

6 Publications

  • Page 1 of 1

Systematic analysis of the binding behaviour of UHRF1 towards different methyl- and carboxylcytosine modification patterns at CpG dyads.

PLoS One 2020 21;15(2):e0229144. Epub 2020 Feb 21.

Center for Integrated Protein Science Munich at the TUM School of Life Sciences, Technische Universität München, Freising, Germany.

The multi-domain protein UHRF1 is essential for DNA methylation maintenance and binds DNA via a base-flipping mechanism with a preference for hemi-methylated CpG sites. We investigated its binding to hemi- and symmetrically modified DNA containing either 5-methylcytosine (mC), 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), or 5-carboxylcytosine (caC). Our experimental results indicate that UHRF1 binds symmetrically carboxylated and hybrid methylated/carboxylated CpG dyads in addition to its previously reported substrates. Complementary molecular dynamics simulations provide a possible mechanistic explanation of how the protein could differentiate between modification patterns. First, we observe different local binding modes in the nucleotide binding pocket as well as the protein's NKR finger. Second, both DNA modification sites are coupled through key residues within the NKR finger, suggesting a communication pathway affecting protein-DNA binding for carboxylcytosine modifications. Our results suggest a possible additional function of the hemi-methylation reader UHRF1 through binding of carboxylated CpG sites. This opens the possibility of new biological roles of UHRF1 beyond DNA methylation maintenance and of oxidised methylcytosine derivates in epigenetic regulation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0229144PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7034832PMC
May 2020

Discovery of novel dual inhibitors against Mdm2 and Mdmx proteins by in silico approaches and binding assay.

Life Sci 2016 Jan 30;145:240-6. Epub 2015 Dec 30.

Department of Medicinal Chemistry, Faculty of Pharmacy, Drug Design and Development Research Center, Tehran University of Medical Sciences, 16 Azar Ave., Tehran, Iran. Electronic address:

Aims: The p53 protein, also called guardian of the genome, has a key role in cell cycle regulation. It is activated under stressful circumstances, such as DNA damage which results in permanent arrest or cell death. The protein is disabled in several types of human cancer due to over-expression of the two regulators, Mdm2 and Mdmx. As a result, inhibiting Mdm subtypes could reactivate p53 and bring about a promising therapeutic strategy in cancers.

Main Methods: Here a structure-based pharmacophore search and docking simulation are presented in order to filter our in-house library which contains 1035 compounds to find novel scaffolds that inhibit Mdm2 and Mdmx concomitantly. Afterwards, fluorescence polarization binding assay was used to obtain inhibition constant of final compounds.

Key Findings: Thirty two ligands were introduced to bioassay as a result of in-silico methods. Twelve of them inhibit both proteins with almost balanced Ki value ranging from 18 to 162μM for Mdm2 and 18 to 233μM for Mdmx. It was observed that all compounds fill Phe19 and Trp23 pockets of Mdm2/x binding sites and form a hydrogen bond with Trp23 pocket's neighbor amino acids in a manner similar to p53 protein. Additionally, it was concluded that Trp23 pocket of Mdmx has a bigger hydrophobic volume comparing with the one of Mdm2.

Significance: Three structure-activity relationship patterns are supposed which one of them presents usefulness features and can be used in future studies. This study presents first qualitative SAR for dual inhibitors against Mdm2/x.
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http://dx.doi.org/10.1016/j.lfs.2015.12.047DOI Listing
January 2016

Characterization of PvuRts1I endonuclease as a tool to investigate genomic 5-hydroxymethylcytosine.

Nucleic Acids Res 2011 Jul 4;39(12):5149-56. Epub 2011 Mar 4.

Ludwig Maximilians University Munich, Department of Biology and Center for Integrated Protein Science Munich, 82152 Planegg-Martinsried, Germany.

In mammalian genomes a sixth base, 5-hydroxymethylcytosine ((hm)C), is generated by enzymatic oxidation of 5-methylcytosine ((m)C). This discovery has raised fundamental questions about the functional relevance of (hm)C in mammalian genomes. Due to their very similar chemical structure, discrimination of the rare (hm)C against the far more abundant (m)C is technically challenging and to date no methods for direct sequencing of (hm)C have been reported. Here, we report on a purified recombinant endonuclease, PvuRts1I, which selectively cleaves (hm)C-containing sequences. We determined the consensus cleavage site of PvuRts1I as (hm)CN(11-12)/N(9-10)G and show first data on its potential to interrogate (hm)C patterns in mammalian genomes.
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http://dx.doi.org/10.1093/nar/gkr118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3130283PMC
July 2011

Sensitive enzymatic quantification of 5-hydroxymethylcytosine in genomic DNA.

Nucleic Acids Res 2010 Oct 4;38(19):e181. Epub 2010 Aug 4.

Department of Biology, Center for Integrated Protein Science Munich, Ludwig Maximilians University Munich, 82152 Planegg-Martinsried, Germany.

The recent discovery of genomic 5-hydroxymethylcytosine (hmC) and mutations affecting the respective Tet hydroxylases in leukemia raises fundamental questions about this epigenetic modification. We present a sensitive method for fast quantification of genomic hmC based on specific transfer of radiolabeled glucose to hmC by a purified glucosyltransferase. We determined hmC levels in various adult tissues and differentiating embryonic stem cells and show a correlation with differential expression of tet genes.
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http://dx.doi.org/10.1093/nar/gkq684DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2965258PMC
October 2010

Structures of the arm-type binding domains of HPI and HAI7 integrases.

J Biol Chem 2009 Nov 8;284(46):31664-71. Epub 2009 Sep 8.

Max Planck Institute for Biochemistry, D-82152 Martinsried, Germany.

The structures of the N-terminal domains of two integrases of closely related but not identical asn tDNA-associated genomic islands, Yersinia HPI (high pathogenicity island; encoding siderophore yersiniabactin biosynthesis and transport) and an Erwinia carotovora genomic island with yet unknown function, HAI7, have been resolved. Both integrases utilize a novel four-stranded beta-sheet DNA-binding motif, in contrast to the known proteins that bind their DNA targets by means of three-stranded beta-sheets. Moreover, the beta-sheets in Int(HPI) and Int(HAI7) are longer than those in other integrases, and the structured helical N terminus is positioned perpendicularly to the large C-terminal helix. These differences strongly support the proposal that the integrases of the genomic islands make up a distinct evolutionary branch of the site-specific recombinases that utilize a unique DNA-binding mechanism.
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http://dx.doi.org/10.1074/jbc.M109.059261DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2797237PMC
November 2009

Molecular basis for the inhibition of p53 by Mdmx.

Cell Cycle 2007 Oct 12;6(19):2386-92. Epub 2007 Oct 12.

Max Planck Institute for Biochemistry, Martinsried, Germany.

The oncoprotein Mdm2, and the recently intensely studied, homologues protein Mdmx, are principal negative regulators of the p53 tumor suppressor. The mechanisms by which they regulate the stability and activity of p53 are not fully established. We have determined the crystal structure of the N-terminal domain of Mdmx bound to a 15-residue p53 peptide. The structure reveals that although the principle features of the Mdm2-p53 interaction are preserved in the Mdmx-p53 complex, the Mdmx hydrophobic cleft on which the p53 peptide binds is significantly altered: a part of the cleft is blocked by sidechains of Met and Tyr of the p53-binding pocket of Mdmx. Thus specific inhibitors of Mdm2-p53 would not be optimal for binding to Mdmx. Our binding assays show indeed that nutlins, the newly discovered, potent antagonists of the Mdm2-p53 interaction, are not capable to efficiently disrupt the Mdmx-p53 interaction. To achieve full activation of p53 in tumor cells, compounds that are specific for Mdmx are necessary to complement the Mdm2 specific binders.
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http://dx.doi.org/10.4161/cc.6.19.4740DOI Listing
October 2007