| 1. |
- Holmlund, Teresa
(författare)
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Molecular mechanisms of mitochondrial DNA replication
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Mitochondria are the energy producing organelles of eukaryotic cells. The organelle has its own genome, the mitochondrial DNA (mtDNA) that encodes 13 subunits of the respiratory chain (RC) complexes, two rRNAs and 22 tRNAs. Nuclear genes encode the majority of the RC subunits and all the factors required for transcription and replication of the mtDNA. Mutations in mtDNA replication factors are associated with human diseases affecting mitochondrial genome stability and maintenance. The human mtDNA replication system has been reconstituted in vitro and involves the combined actions of the DNA polymerase γ holoenzyme (POLγ), the TWINKLE helicase and the single‐stranded DNA binding protein mtSSB. The general aim of this thesis has been to further investigate the molecular mechanisms of mtDNA replication, with a major focus on the mitochondrial hexameric helicase TWINKLE, as well as the accessory B subunit of POLγ. A biochemical characterization of POLγB demonstrated that the protein blocks the exonuclease activity of the catalytic POLγA subunit. In addition, the dsDNA‐binding activity of POLγB was required for the TWINKLE‐dependant stimulation of the POLγ holoenzyme. TWINKLE displays sequence similarity to the bacteriophage T7 gene 4 protein (gp4) which contains the DNA helicase and primase activities needed at the bacteriophage replication fork. The C‐terminal domain of TWINKLE is indeed an active helicase, but there have been no reports of primase activity. The functional role of the TWINKLE N‐terminus was therefore investigated in this work. The N‐terminal domain was found to contribute to ssDNA‐binding and helicase activities of TWINKLE, and was ultimately required for full replisome activity. A structural model of TWINKLE was constructed based on homology modeling with T7 gp4. This model displayed a conserved region with significant electropositive potential, which in structurally related primases has been suggested to interact with ssDNA. Mutations in both POLγ and TWINKLE can cause autosomal dominant progressive external ophtalmoplegia (adPEO). To investigate the molecular mechanisms behind this disorder, we performed a detailed biochemical analysis on eleven different adPEO‐causing TWINKLE mutations, seven in the linker‐region and four in the N‐terminal domain. Distinct molecular phenotypes were observed, with individual consequences for multimerization, ATPase activity, helicase activity and ability to support mtDNA synthesis in vitro. The different molecular phenotypes could be interpreted using our structural model of TWINKLE. Two of the mutations in the linker region affected multimerization, whereas the N‐terminal mutations showed a striking reduction in ATPase activity and were thus proposed to impair the interplay between ssDNA‐binding and ATP hydrolysis, an essential element of the catalytic cycle of related hexameric helicases.
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| 2. |
- Holmlund, Teresa, et al.
(författare)
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Structure-function defects of the twinkle amino-terminal region in progressive external ophthalmoplegia.
- 2009
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Ingår i: Biochimica et biophysica acta. - : Elsevier BV. - 0006-3002. ; 1792:2, s. 132-9
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Tidskriftsartikel (refereegranskat)abstract
- TWINKLE is a DNA helicase needed for mitochondrial DNA replication. In lower eukaryotes the protein also harbors a primase activity, which is lost from TWINKLE encoded by mammalian cells. Mutations in TWINKLE underlie autosomal dominant progressive external ophthalmoplegia (adPEO), a disorder associated with multiple deletions in the mtDNA. Four different adPEO-causing mutations (W315L, K319T, R334Q, and P335L) are located in the N-terminal domain of TWINKLE. The mutations cause a dramatic decrease in ATPase activity, which is partially overcome in the presence of single-stranded DNA. The mutated proteins have defects in DNA helicase activity and cannot support normal levels of DNA replication. To explain the phenotypes, we use a molecular model of TWINKLE based on sequence similarities with the phage T7 gene 4 protein. The four adPEO-causing mutations are located in a region required to bind single-stranded DNA. These mutations may therefore impair an essential element of the catalytic cycle in hexameric helicases, i.e. the interplay between single-stranded DNA binding and ATP hydrolysis.
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| 3. |
- Pournara, Angeliki, et al.
(författare)
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Arsenic alters global histone modifications in lymphocytes in vitro and in vivo
- 2016
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Ingår i: Cell Biology and Toxicology. - : Springer Science and Business Media LLC. - 0742-2091 .- 1573-6822. ; 32:4, s. 275-84
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Forskningsöversikt (refereegranskat)abstract
- Arsenic, an established carcinogen and toxicant, occurs in drinking water and food and affects millions of people worldwide. Arsenic appears to interfere with gene expression through epigenetic processes, such as DNA methylation and post-translational histone modifications. We investigated the effects of arsenic on histone residues in vivo as well as in vitro. Analysis of H3K9Ac and H3K9me3 in CD4+ and CD8+ sorted blood cells from individuals exposed to arsenic through drinking water in the Argentinean Andes showed a significant decrease in global H3K9me3 in CD4+ cells, but not CD8+ cells, with increasing arsenic exposure. In vitro studies of inorganic arsenic-treated T lymphocytes (Jurkat and CCRF-CEM, 0.1, 1, and 100 μg/L) showed arsenic-related modifications of H3K9Ac and changes in the levels of the histone deacetylating enzyme HDAC2 at very low arsenic concentrations. Further, in vitro exposure of kidney HEK293 cells to arsenic (1 and 5 μM) altered the protein levels of PCNA and DNMT1, parts of a gene expression repressor complex, as well as MAML1. MAML1 co-localized and interacted with components of this complex in HEK293 cells, and in silico studies indicated that MAML1 expression correlate with HDAC2 and DNMT1 expression in kidney cells. In conclusion, our data suggest that arsenic exposure may lead to changes in the global levels of H3K9me3 and H3K9Ac in lymphocytes. Also, we show that arsenic exposure affects the expression of PCNA and DNMT1—proteins that are part of a gene expression silencing complex.
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| 4. |
- Zovko, Ana, et al.
(författare)
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Compounds from the marine sponge Cribrochalina vasculum offer a way to target IGF-1R mediated signaling in tumor cells
- 2016
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Ingår i: Oncotarget. - : Impact Journals, LLC. - 1949-2553. ; 7:31, s. 50258-50276
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Tidskriftsartikel (refereegranskat)abstract
- In this work two acetylene alcohols, compound 1 and compound 2, which were isolated and identified from the sponge Cribrochalina vasculum, and which showed antitumor effects were further studied with respect to targets and action mechanisms. Gene expression analyses suggested insulin like growth factor receptor (IGF-1R) signaling to be instrumental in controlling anti-tumor efficacy of these compounds in non-small cell lung cancer (NSCLC). Indeed compounds 1 and 2 inhibited phosphorylation of IGF-1R beta as well as reduced its target signaling molecules IRS-1 and PDK1 allowing inhibition of pro-survival signaling. In silico docking indicated that compound 1 binds to the kinase domain of IGF-1R at the same binding site as the well known tyrosine kinase inhibitor AG1024. Indeed, cellular thermal shift assay (CETSA) confirmed that C. vasculum compound 1 binds to IGF-1R but not to the membrane localized tyrosine kinase receptor EGFR. Importantly, we demonstrate that compound 1 causes IGF-1R beta but not Insulin Receptor degradation specifically in tumor cells with no effects seen in normal diploid fibroblasts. Thus, these compounds hold potential as novel therapeutic agents targeting IGF-1R signaling for anti-tumor treatment.
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