My research focuses on genetic diseases associated with altered energy metabolism in human and transgenic mice. In this context I aim to integrate systems biology, non-invasive imaging and Invivo Magnetic Resonance spectroscopy to understand disease associated changes in the brain, and skeletal muscle of transgenic mice. In the past I worked in the laboratory of Prof Roland Kanaar and Dr Jeroen Essers. There I discovered functional pathways linked to enhanced arterial aging due to reduced expression of Fibulin-4 in the aorta of transgenic mice.
High resolution multidimensional NMR spectroscopy and structure biology of DNA repair proteins is my major research expertise.For my doctorate degree I worked in the laboratory of Prof Rolf Boelens and Prof Robert Kaptein. In their group I successfully participated in protein and nucleic acid structure determination projects (PDB: 2AQ0, 2KN7,1Z00, 2JPD, 2Y4W, 2BJC).
During my MS study I worked on multiple quantum (13C-13C, 13C-1H, 1H-1H) NMR methods. Within this project I developed polarization enhancement and gradient selected spectral editing sequences. My objective was to improve signal to noise ratio (SNR) and to simplify spectral complexity observed in the single quantum (SQ) spectra. Substantial advances has been made since my initial effort. Now it is possible to detect 2-7 order HMQ transitions in a strongly coupled spin system, wherein one of the nuclei is weakly coupled to several other nuclei. There can be further extension of HMQ NMR to understand molecular chirality and temperature induced alteration in the structure of biomolecules and synthetic compounds soluble in liquid crystals.
Abstract: Human XPF/ERCC1 is a structure specific DNA endonuclease that nicks the damaged DNA
strand at the 5’ end during nucleotide excision repair. We determined the structure of the
complex of C-terminal domain of XPF with 10 nt ssDNA. A positively charged region within the
second helix of the first HhH motif contacts the ssDNA phosphate backbone. One guanine base is
flipped out of register and positioned in a pocket contacting residues from both HhH motifs of
XPF. Comparison to other HhH containing proteins indicates a one-residue deletion in the second
HhH motif of XPF that has altered the hairpin conformation permitting ssDNA interaction.
Previously we found that ERCC1 in the XPF-ERCC1 heterodimer can bind dsDNA (Tripsianes
et al., 2005). Combining the two observations gives a model that underscores the asymmetry of
the human XPF/ERCC1 heterodimer in binding at a ss/ds DNA junction.
Abstract: The human ubiquitin-conjugating enzyme Rad6 (E2), with ubiquitin ligase enzyme Rad18 (RING E3), monoubiquitinates proliferating cell nuclear antigen at stalled replication forks in DNA translesion synthesis. Here, we determine the structure of the homodimeric Rad18 RING domains by X-ray crystallography and classify it to RING-RING dimers that dimerize through helices adjacent to the RING domains and through the canonical RING domains. Using NMR spectroscopy and site-directed mutagenesis, we demonstrate that the Rad6b binding site, for the Rad18 RING domain, strongly resembles that of other E2/E3 RING/U-box complexes. We show that the homodimeric Rad18 RING domain can recruit two Rad6b E2 enzymes, whereas the full-length Rad18 homodimer binds only to a single Rad6b molecule. Such asymmetry is a common feature of RING-RING heterodimers and has been observed for the CHIP U-box homodimer. We propose that asymmetry may be a common feature of dimeric RING E3 ligases.
Abstract: Fibulin-4 is a secreted glycoprotein, which is expressed in medial layers of blood vessels. All six reported fibulin-4 patients suffer from cardiovascular complications including aortic aneurysms, arterial tortuosity and stenosis and elastin abnormalities. We have engineered fibulin-4 mouse models that express reduced levels of the fibulin-4 protein and develop aortic abnormalities similar to fibulin-4 patients. Interestingly, heterozygous fibulin-4+/R mice show mild symptoms of aneurysm formation like ballooning of the right subclavian artery and a slightly dilated aorta, while the homozygous fibulin-4R/R mice show elongated and 2-3 fold dilated ascending aorta. To get insight into the underlying molecular pathways involved in aneurysm formation and response to aortic failure, we determined the aorta proteome of fibulin-4+/R and fibulin-4R/R animals. A full un-biased qualitative MS/MS proteomic screen of the aorta protein extracts identified 192 unique proteins in fibulin-4+/R and 220 unique proteins in fibulin-4R/R mice compared to fibulin-4+/+ animals, of which 80 were commonly present in both mutants. Next, an overlay of the aorta tissue proteome and transcriptome was made to find a limited set of biomarkers that were identified by both gene and protein expression analysis of the fibulin-4+/Rand fibulin-4R/R mice. In the fibulin-4R/R mice a limited set of biomarkers pointed towards altered regulation of 17-beta-estradiol and TNF-alpha pathways. Interestingly, the 17-beta-estradiol pathway was also found in the fibulin-4+/R mice. The signaling molecule 17-beta estradiol is a metabolite known to deregulate production of reactive oxygen species (ROS) by poorly understood mechanisms. Notably both these regulatory pathways are also deregulated by oxidative stress, which is a hallmark of aging and age related cardiovascular diseases. To address altered cell signaling and cell death in the aneurismal aorta we have performed TUNEL staining, and to validate differential production of ROS and antioxidants we have indirectly quantified ROS by superoxide staining. These results uncover new regulatory pathways likely to be associated with enhanced arterial aging in aneurismal fibulin-4 mice.
Notes: Author Disclosures: D. Das, None; P.M. van Heijningen, None; D.H. Dekkers, None; M. Vermeij, None; W. Sluiter, None; R. Kanaar, None; J. Demmers, None; J. Essers, None.
Abstract: The human XPF-ERCC1 protein complex plays an essential role in nucleotide excision repair by catalysing positioned nicking of a DNA strand at the 5' side of the damage. We have recently solved the structure of the heterodimeric complex of the C-terminal domains of XPF and ERCC1 (Tripsianes et al., Structure 2005;13:1849-1858). We found that this complex comprises a pseudo twofold symmetry axis and that the helix-hairpin-helix motif of ERCC1 is required for DNA binding, whereas the corresponding domain of XPF is functioning as a scaffold for complex formation with ERCC1. Despite the functional importance of heterodimerization, the C-terminal domain of XPF can also form homodimers in vitro. We here compare the stabilities of homodimeric and heterodimeric complexes of the C-terminal domains of XPF and ERCC1. The higher stability of the XPF HhH complexes under various experimental conditions, determined using CD and NMR spectroscopy and mass spectrometry, is well explained by the structural differences that exist between the HhH domains of the two complexes. The XPF HhH homodimer has a larger interaction interface, aromatic stacking interactions, and additional hydrogen bond contacts as compared to the XPF/ERCC1 HhH complex, which accounts for its higher stability.
Abstract: Human ERCC1/XPF is a structure-specific endonuclease involved in multiple DNA repair pathways. We present the solution structure of the non-catalytic ERCC1 central domain. Although this domain shows structural homology with the catalytically active XPF nuclease domain, functional investigation reveals a completely distinct function for the ERCC1 central domain by performing interactions with both XPA and single-stranded DNA. These interactions are non-competitive and can occur simultaneously through distinct interaction surfaces. Interestingly, the XPA binding by ERCC1 and the catalytic function of XPF are dependent on a structurally homologous region of the two proteins. Although these regions are strictly conserved in each protein family, amino acid composition and surface characteristics are distinct. We discuss the possibility that after XPF gene duplication, the redundant ERCC1 central domain acquired novel functions, thereby increasing the fidelity of eukaryotic DNA repair.
Abstract: Recognition of the lac operator by the lac repressor involves specific interactions between residues in the repressor's recognition helix and bases in the DNA major groove. Tyr17 and Gln18, at positions 1 and 2 in the lac repressor recognition helix, can be exchanged for other amino acids to generate mutant repressors that display altered specificity. We have solved the solution structure of a protein-DNA complex of an altered-specificity mutant lac headpiece in which Tyr17 and Gln18 were exchanged for valine and alanine, respectively, as found in the recognition helix of the gal repressor. As previously described by Lehming et al. (EMBO J. 1987, 6, 3145-3153), this altered-specificity mutant of the lac repressor recognizes a variant lac operator that is similar to the gal operator Oe. The mutant lac headpiece showed the predicted specificity and is also able to mimic the gal repressor by recognizing and bending the natural gal operator Oe. These structural data show that, while most of the anchoring points that help the lac headpiece to assemble on the lac operator were preserved, a different network of protein-DNA interactions connecting Ala17 and Val18 to bases in the DNA major groove drives the specificity towards the altered operator.
Abstract: The human ERCC1/XPF complex is a structure-specific endonuclease with defined polarity that participates in multiple DNA repair pathways. We report the heterodimeric structure of the C-terminal domains of both proteins responsible for ERCC1/XPF complex formation. Both domains exhibit the double helix-hairpin-helix motif (HhH)2, and they are related by a pseudo-2-fold symmetry axis. In the XPF domain, the hairpin of the second motif is replaced by a short turn. The ERCC1 domain folds properly only in the presence of the XPF domain, which implies a role for XPF as a scaffold for the folding of ERCC1. The intersubunit interactions are largely hydrophobic in nature. NMR titration data show that only the ERCC1 domain of the ERCC1/XPF complex is involved in DNA binding. On the basis of these findings, we propose a model for the targeting of XPF nuclease via ERCC1-mediated interactions in the context of nucleotide excision repair.
