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Strategies for isolation of DNA prone to biotin deficiency recombination, mitochondria-protein interaction detection and an anti-obesity screening targeting acetyl-CoA carboxylase 2
Abstract
Holocarboxylase synthetase (HLCS) is a human biotin ligase that covalently attaches biotin to carboxylases and play important role in epigenetic regulation. Evidence suggests that HLCS-mediated histone biotinylation marks are abundant in long terminal repeat (LTRs) and that biotin depletion or HLCS deficiency in cells and animal model correlate with de-repression of LTRs and genome instability, linking biotin deficiency to meiotic recombination abnormalities. A straight-forward approach to investigate biotin dependent recombination frequency is to isolate DNA containing the recombination hotspot 13-mer CCNCCNTNNCCNC, instead of expensive whole genome analysis. We demonstrated a method that selectively enriches DNA containing the 13-mer motif from human chromatin. We conclude that the capture protocol can enrich genomic sequences of interest, including degenerate, short sequences. In a second part of my thesis, we engineered a high-throughput cell death-based assay to enable researchers to investigate protein-mitochondria interactions. It has been reported that over 800 diseases have been linked to mitochondrial proteins. Putative mitochondrial proteins, e.g., carnitine palmitoyl transferase 1, acetyl-CoA carboxylase 2 (ACC2), uncoupling protein 1, and HLCS were fused to a cell death peptide {KLAKLAKKLAKLAK (KLAK)}. These constructs were expressed in human embryonic kidney HEK293 cells. The mitochondrial proteins delivered KLAK to mitochondria, thereby impairing membrane integrity and potential, and causing cell death. We have also developed an anti-obesity strategy based on preventing the anchoring of ACC2 in the mitochondrial membrane, which causes an increase in â-oxidation and fat loss. Experiments with grape materials and with an abundant compound in grapes, 3-chlorogenic acid, showed an unambiguous decrease in cell death caused by ACC2-KLAK, suggesting that compounds in grape might have anti-obesity activities. Additionally, experiments in Drosophila melanogaster brummer mutants with grape leave extracts, resveratrol-derivative piceid, and the ACC inhibitor soraphen A decreased total body fat, suggesting in vivo viability of ACC2 anti-obesity strategy. We conclude that our assay may be useful for linking the mitochondrial proteome with rare diseases, and for devising drug and nutrition based strategies for altering mitochondrial targeting of proteins. Additionally, we are in process of developing a HLCS conditional knockout mouse model to confirm roles of the HLCS-biotin-carboxylase/histone axes in genome stability.
Subject Area
Molecular biology|Biochemistry|Nutrition
Recommended Citation
Camara Teixeira, Daniel, "Strategies for isolation of DNA prone to biotin deficiency recombination, mitochondria-protein interaction detection and an anti-obesity screening targeting acetyl-CoA carboxylase 2" (2014). ETD collection for University of Nebraska-Lincoln. AAI3646913.
https://digitalcommons.unl.edu/dissertations/AAI3646913