20 research outputs found

    Transcriptional activation by mitochondrial transcription factor A involves preferential distortion of promoter DNA

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    Mitochondrial transcription factor A (mtTFA/TFAM) is a nucleus-encoded, high-mobility-group-box (HMG-box) protein that regulates transcription of the mitochondrial genome by specifically recognizing light-strand and heavy-strand promoters (LSP, HSP1). TFAM also binds mitochondrial DNA in a non-sequence specific (NSS) fashion and facilitates its packaging into nucleoid structures. However, the requirement and contribution of DNA-bending for these two different binding modes has not been addressed in detail, which prompted this comparison of binding and bending properties of TFAM on promoter and non-promoter DNA. Promoter DNA increased the stability of TFAM to a greater degree than non-promoter DNA. However, the thermodynamic properties of DNA binding for TFAM with promoter and non-specific (NS) DNA were similar to each other and to other NSS HMG-box proteins. Fluorescence resonance energy transfer assays showed that TFAM bends promoter DNA to a greater degree than NS DNA. In contrast, TFAM lacking the C-terminal tail distorted both promoter and non-promoter DNA to a significantly reduced degree, corresponding with markedly decreased transcriptional activation capacity at LSP and HSP1 in vitro. Thus, the enhanced bending of promoter DNA imparted by the C-terminal tail is a critical component of the ability of TFAM to activate promoter-specific initiation by the core mitochondrial transcription machinery

    The sea urchin mitochondrial transcription factor A binds and bends DNA efficiently despite its unusually short C-terminal tail

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    Mitochondrial transcription factor A (TFAM) is a key component for the protection and transcription of the mitochondrial genome. TFAM belongs to the high mobility group (HMG) box family of DNA binding proteins that are able to bind to and bend DNA. Human TFAM (huTFAM) contains two HMG box domains separated by a linker region, and a 26 amino acid C-terminal tail distal to the second HMG box. Previous studies on huTFAM have shown that requisites for proper DNA bending and specific binding to the mitochondrial genome are specific intercalating residues and the C-terminal tail. We have characterized TFAM from the sea urchin Paracentrotus lividus (suTFAM). Differently from human, suTFAM contains a short 9 amino acid C-terminal tail, yet it still has the ability to specifically bind to mtDNA. To provide information on the mode of binding of the protein we used fluorescence resonance energy transfer (FRET) assays and found that, in spite of the absence of a canonical C-terminal tail, suTFAM distorts DNA at a great extent and recognizes specific target with high affinity. Site directed mutagenesis showed that the two Phe residues placed in corresponding position of the two intercalating Leu of huTFAM are responsible for the strong bending and the great binding affinity of suTFAM

    Somatic loss of PIK3R1 may sensitize breast cancer to inhibitors of the MAPK pathway

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    Purpose: The PI3K pathway, which includes the PI3K catalytic subunits p110α (PIK3CA) and the PI3K regulatory subunit p85α (PIK3R1), is the most frequently altered pathway in cancer. We encountered a breast cancer patient whose tumor contained a somatic alteration in PIK3R1. Some commercial sequencing platforms suggest that somatic mutations in PIK3R1 may sensitize cancers to drugs that inhibit the mammalian target of rapamycin (mTOR). However, a review of the preclinical and clinical literature did not find evidence substantiating that hypothesis. The purpose of this study was to knock out PIK3R1 in order to determine the optimal therapeutic approach for breast cancers lacking p85α. Methods: We created an isogenic cellular system by knocking out both alleles of the PIK3R1 gene in the non-tumorigenic human breast cell line MCF-10A. Knockout cells were compared with wild-type cells by measuring growth, cellular signaling, and response to drugs. Results: We observed hyperphosphorylation of MEK in these knockouts, which sensitized PIK3R1-null cells to a MEK inhibitor, trametinib. However, they were not sensitized to the mTOR inhibitor, everolimus. Conclusions: Our findings suggest that breast cancers with loss of p85α may not respond to mTOR inhibition, but may be sensitive to MEK inhibition

    The sea urchin mitochondrial transcription factor A binds and bends DNA efficiently despite its unusually short C-terminal tail

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    Mitochondrial transcription factor A (TFAM) is a key component for the protection and transcription of the mitochondrial genome. TFAM belongs to the high mobility group (HMG) box family of DNA binding proteins that are able to bind to and bend DNA. Human TFAM (huTFAM) contains two HMG box domains separated by a linker region, and a 26 amino acid C-terminal tail distal to the second HMG box. Previous studies on huTFAM have shown that requisites for proper DNA bending and specific binding to the mitochondrial genome are specific intercalating residues and the C-terminal tail. We have characterized TFAM from the sea urchin Paracentrotus lividus (suTFAM). Differently from human, suTFAM contains a short 9 amino acid C-terminal tail, yet it still has the ability to specifically bind to mtDNA. To provide information on the mode of binding of the protein we used fluorescence resonance energy transfer (FRET) assays and found that, in spite of the absence of a canonical C-terminal tail, suTFAM distorts DNA at a great extent and recognizes specific target with high affinity. Site directed mutagenesis showed that the two Phe residues placed in corresponding position of the two intercalating Leu of huTFAM are responsible for the strong bending and the great binding affinity of suTFAM

    Investigation of the thermodynamic drivers of the interaction between the high mobility group box domain of Sox2 and bacterial lipopolysaccharide

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    Gastric cancer is associated with high mortality and is preceded by an infection with Helicobacter pylori (H. pylori). H. pylori stimulates inflammation which involves the activation of Toll-like receptor 4 by lipopolysaccharide molecules from the H. pylori. This leads to chronic inflammation that can eventually lead to gastric cancer. Sox2 is a member of the high mobility group (HMG) box family of proteins, and recent studies have shown that HMG box proteins can modulate immune response by altering signaling to Toll-like receptors. Sox2 is overexpressed in most types of cancer with the exception of gastric cancer where expression of Sox2 is decreased. Here, we demonstrate that Sox2 can bind LPS and we investigated the thermodynamic drivers of the Sox2/LPS interaction

    Mechanism of Mitochondrial Transcription Factor A Attenuation of CpG-Induced Antibody Production - Fig 2

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    <p><b>TFAM does not alter the secondary structure of CpG-B or CpG-C (A)</b> Circular dichroism (CD) analysis of CpG-B (PS) and CpG-C ODNs. CD spectra measured in millidegrees are shown for the 25mer dsLSP DNA (solid line), ssLSP ODN (dashed line), CpG-B (PS) ODN (dotted line), and CpG-C (PS) (red line) at a concentration of 20 μM. <b>(B)</b> CD analysis of CpG-B (PS), and CpG-C (PS) in the presence and absence of TFAM. The DNA concentration was 10 μM for both samples. In the CpG-B (PS) and CpG-C (PS) spectra, buffer was subtracted, whereas for the CpG-B (PS)+TFAM and CpG-C (PS)+TFAM spectra, the CD contribution both from the buffer and TFAM was subtracted. All CD experiments were conducted at 20°C in 10 mM Na-phosphate buffer and 150 mM NaCl.</p
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