Limits...
Proteins of nucleotide and base excision repair pathways interact in mitochondria to protect from loss of subcutaneous fat, a hallmark of aging.

Kamenisch Y, Fousteri M, Knoch J, von Thaler AK, Fehrenbacher B, Kato H, Becker T, Dollé ME, Kuiper R, Majora M, Schaller M, van der Horst GT, van Steeg H, Röcken M, Rapaport D, Krutmann J, Mullenders LH, Berneburg M - J. Exp. Med. (2010)

Bottom Line: We show functional increase of CSA and CSB inside mt and complex formation with mtDNA, mt human 8-oxoguanine glycosylase (mtOGG)-1, and mt single-stranded DNA binding protein (mtSSBP)-1 upon oxidative stress.MtDNA mutations are highly increased in cells from CS patients and in subcutaneous fat of aged Csb(m/m) and Csa(-/-) mice.Thus, the NER-proteins CSA and CSB localize to mt and directly interact with BER-associated human mitochondrial 8-oxoguanine glycosylase-1 to protect from aging- and stress-induced mtDNA mutations and apoptosis-mediated loss of subcutaneous fat, a hallmark of aging found in animal models, human progeroid syndromes like CS and in normal human aging.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Dermatology, Eberhard Karls University, D-72076 Tuebingen, Germany.

ABSTRACT
Defects in the DNA repair mechanism nucleotide excision repair (NER) may lead to tumors in xeroderma pigmentosum (XP) or to premature aging with loss of subcutaneous fat in Cockayne syndrome (CS). Mutations of mitochondrial (mt)DNA play a role in aging, but a link between the NER-associated CS proteins and base excision repair (BER)-associated proteins in mitochondrial aging remains enigmatic. We show functional increase of CSA and CSB inside mt and complex formation with mtDNA, mt human 8-oxoguanine glycosylase (mtOGG)-1, and mt single-stranded DNA binding protein (mtSSBP)-1 upon oxidative stress. MtDNA mutations are highly increased in cells from CS patients and in subcutaneous fat of aged Csb(m/m) and Csa(-/-) mice. Thus, the NER-proteins CSA and CSB localize to mt and directly interact with BER-associated human mitochondrial 8-oxoguanine glycosylase-1 to protect from aging- and stress-induced mtDNA mutations and apoptosis-mediated loss of subcutaneous fat, a hallmark of aging found in animal models, human progeroid syndromes like CS and in normal human aging.

Show MeSH

Related in: MedlinePlus

Mitochondrial localization of CSA and CSB in oxidatively stressed cells. Confocal laser scanning microscopy with green fluorescence for nuclear staining, blue fluorescence for mitochondrial staining, and red fluorescence staining for either CSA (A) or CSB (B). Pink fluorescence results from colocalization (white circles) of red mitochondrial CSA/CSB and blue mitochondrial staining. Pictures are representatives of at least five separate experiments. Bar, 1 µm. Exposure of cells to 25 µM H2O2 for 12 h leads to signal increase for mitochondrial CSA (C) and CSB (D). Histograms show signal intensities along the red line in the picture with simultaneous signal increment. (E) CSA-deficient fibroblasts transfected with CSA WT protein. Pictures are representatives of at least three independent experiments. Bar, 1 µm. (F) Whole-cell extracts (WCE) and mitochondrial extracts (Mt) prepared from H2O2-treated cells. Presence of mtDNA (IS) and absence of nuclear DNA (GAPDH), shown by PCR. Immunoblotting of mitochondrial ATP-synthetase β, phospho-NF-κB, S6, CSA, and CSB. Pictures are representatives of at least three independent experiments. (G) Electron microscopic image of oxidatively stressed normal human fibroblasts stained with gold-labeled (circles) CSA (top) or CSB (bottom). Bars: (top CSA image) 1.1 µm; (top CSB image) 1.6 µm; (bottom) 165 nm. Electron micrographs are representatives of at least two separate experiments. (H) Top: mt (10 µg protein per lane) isolated from H2O2-treated WT cells were incubated where indicated with 100 µg/ml proteinase K (PK) in the presence or absence of Triton X-100 (Tx-100). SDS-PAGE of mitochondrial proteins with antibodies against CSA, Tom20, Tim23, and superoxide dismutase (SOD). Bottom: mt (25 µg per lane) isolated from H2O2-treated WT cells were incubated with 25 µg/ml proteinase K (PK) in the presence or absence of Triton X-100 (Tx-100). SDS-PAGE of mitochondrial proteins with antibodies against CSB and cytochrome C. Pictures are representatives of at least two independent experiments.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC2822596&req=5

fig1: Mitochondrial localization of CSA and CSB in oxidatively stressed cells. Confocal laser scanning microscopy with green fluorescence for nuclear staining, blue fluorescence for mitochondrial staining, and red fluorescence staining for either CSA (A) or CSB (B). Pink fluorescence results from colocalization (white circles) of red mitochondrial CSA/CSB and blue mitochondrial staining. Pictures are representatives of at least five separate experiments. Bar, 1 µm. Exposure of cells to 25 µM H2O2 for 12 h leads to signal increase for mitochondrial CSA (C) and CSB (D). Histograms show signal intensities along the red line in the picture with simultaneous signal increment. (E) CSA-deficient fibroblasts transfected with CSA WT protein. Pictures are representatives of at least three independent experiments. Bar, 1 µm. (F) Whole-cell extracts (WCE) and mitochondrial extracts (Mt) prepared from H2O2-treated cells. Presence of mtDNA (IS) and absence of nuclear DNA (GAPDH), shown by PCR. Immunoblotting of mitochondrial ATP-synthetase β, phospho-NF-κB, S6, CSA, and CSB. Pictures are representatives of at least three independent experiments. (G) Electron microscopic image of oxidatively stressed normal human fibroblasts stained with gold-labeled (circles) CSA (top) or CSB (bottom). Bars: (top CSA image) 1.1 µm; (top CSB image) 1.6 µm; (bottom) 165 nm. Electron micrographs are representatives of at least two separate experiments. (H) Top: mt (10 µg protein per lane) isolated from H2O2-treated WT cells were incubated where indicated with 100 µg/ml proteinase K (PK) in the presence or absence of Triton X-100 (Tx-100). SDS-PAGE of mitochondrial proteins with antibodies against CSA, Tom20, Tim23, and superoxide dismutase (SOD). Bottom: mt (25 µg per lane) isolated from H2O2-treated WT cells were incubated with 25 µg/ml proteinase K (PK) in the presence or absence of Triton X-100 (Tx-100). SDS-PAGE of mitochondrial proteins with antibodies against CSB and cytochrome C. Pictures are representatives of at least two independent experiments.

Mentions: We started by asking whether the CSA and CSB proteins, known to be present predominantly in the nucleus, are also localized in mt. Immunocytostaining with antibodies against CSA or CSB and against mitochondrial marker proteins, as well as nuclear staining with subsequent confocal laser scanning microscopy, was performed in normal and CSA- and CSB-deficient fibroblasts (see Tables S1, S3, and S4 for detailed cell lines and antibodies). In normal fibroblasts, CSA (Fig. 1 A) and CSB (Fig. 1 B), proteins were virtually undetectable in mt; however, these proteins were clearly visible in the nucleus with some minor localization at the cytoplasm (Fig. 1, A and B). Confocal images of cells without nuclear staining confirmed the presence of CSA and CSB proteins in the nucleus and cytoplasm (Fig. S1 and not depicted). Confocal microscopy of CSA and CSB cells with the CSA and CSB antibodies, respectively, validated the specificity of the antibodies (Fig. 1, A and B, and Fig. S1). Note that in the absence of a functional CSB protein (CSB cells; Fig. 1 A, bottom), an increased cytoplasmic staining of CSA was observed, whereas the nuclear staining was similar in both CSB and normal cells.


Proteins of nucleotide and base excision repair pathways interact in mitochondria to protect from loss of subcutaneous fat, a hallmark of aging.

Kamenisch Y, Fousteri M, Knoch J, von Thaler AK, Fehrenbacher B, Kato H, Becker T, Dollé ME, Kuiper R, Majora M, Schaller M, van der Horst GT, van Steeg H, Röcken M, Rapaport D, Krutmann J, Mullenders LH, Berneburg M - J. Exp. Med. (2010)

Mitochondrial localization of CSA and CSB in oxidatively stressed cells. Confocal laser scanning microscopy with green fluorescence for nuclear staining, blue fluorescence for mitochondrial staining, and red fluorescence staining for either CSA (A) or CSB (B). Pink fluorescence results from colocalization (white circles) of red mitochondrial CSA/CSB and blue mitochondrial staining. Pictures are representatives of at least five separate experiments. Bar, 1 µm. Exposure of cells to 25 µM H2O2 for 12 h leads to signal increase for mitochondrial CSA (C) and CSB (D). Histograms show signal intensities along the red line in the picture with simultaneous signal increment. (E) CSA-deficient fibroblasts transfected with CSA WT protein. Pictures are representatives of at least three independent experiments. Bar, 1 µm. (F) Whole-cell extracts (WCE) and mitochondrial extracts (Mt) prepared from H2O2-treated cells. Presence of mtDNA (IS) and absence of nuclear DNA (GAPDH), shown by PCR. Immunoblotting of mitochondrial ATP-synthetase β, phospho-NF-κB, S6, CSA, and CSB. Pictures are representatives of at least three independent experiments. (G) Electron microscopic image of oxidatively stressed normal human fibroblasts stained with gold-labeled (circles) CSA (top) or CSB (bottom). Bars: (top CSA image) 1.1 µm; (top CSB image) 1.6 µm; (bottom) 165 nm. Electron micrographs are representatives of at least two separate experiments. (H) Top: mt (10 µg protein per lane) isolated from H2O2-treated WT cells were incubated where indicated with 100 µg/ml proteinase K (PK) in the presence or absence of Triton X-100 (Tx-100). SDS-PAGE of mitochondrial proteins with antibodies against CSA, Tom20, Tim23, and superoxide dismutase (SOD). Bottom: mt (25 µg per lane) isolated from H2O2-treated WT cells were incubated with 25 µg/ml proteinase K (PK) in the presence or absence of Triton X-100 (Tx-100). SDS-PAGE of mitochondrial proteins with antibodies against CSB and cytochrome C. Pictures are representatives of at least two independent experiments.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2822596&req=5

fig1: Mitochondrial localization of CSA and CSB in oxidatively stressed cells. Confocal laser scanning microscopy with green fluorescence for nuclear staining, blue fluorescence for mitochondrial staining, and red fluorescence staining for either CSA (A) or CSB (B). Pink fluorescence results from colocalization (white circles) of red mitochondrial CSA/CSB and blue mitochondrial staining. Pictures are representatives of at least five separate experiments. Bar, 1 µm. Exposure of cells to 25 µM H2O2 for 12 h leads to signal increase for mitochondrial CSA (C) and CSB (D). Histograms show signal intensities along the red line in the picture with simultaneous signal increment. (E) CSA-deficient fibroblasts transfected with CSA WT protein. Pictures are representatives of at least three independent experiments. Bar, 1 µm. (F) Whole-cell extracts (WCE) and mitochondrial extracts (Mt) prepared from H2O2-treated cells. Presence of mtDNA (IS) and absence of nuclear DNA (GAPDH), shown by PCR. Immunoblotting of mitochondrial ATP-synthetase β, phospho-NF-κB, S6, CSA, and CSB. Pictures are representatives of at least three independent experiments. (G) Electron microscopic image of oxidatively stressed normal human fibroblasts stained with gold-labeled (circles) CSA (top) or CSB (bottom). Bars: (top CSA image) 1.1 µm; (top CSB image) 1.6 µm; (bottom) 165 nm. Electron micrographs are representatives of at least two separate experiments. (H) Top: mt (10 µg protein per lane) isolated from H2O2-treated WT cells were incubated where indicated with 100 µg/ml proteinase K (PK) in the presence or absence of Triton X-100 (Tx-100). SDS-PAGE of mitochondrial proteins with antibodies against CSA, Tom20, Tim23, and superoxide dismutase (SOD). Bottom: mt (25 µg per lane) isolated from H2O2-treated WT cells were incubated with 25 µg/ml proteinase K (PK) in the presence or absence of Triton X-100 (Tx-100). SDS-PAGE of mitochondrial proteins with antibodies against CSB and cytochrome C. Pictures are representatives of at least two independent experiments.
Mentions: We started by asking whether the CSA and CSB proteins, known to be present predominantly in the nucleus, are also localized in mt. Immunocytostaining with antibodies against CSA or CSB and against mitochondrial marker proteins, as well as nuclear staining with subsequent confocal laser scanning microscopy, was performed in normal and CSA- and CSB-deficient fibroblasts (see Tables S1, S3, and S4 for detailed cell lines and antibodies). In normal fibroblasts, CSA (Fig. 1 A) and CSB (Fig. 1 B), proteins were virtually undetectable in mt; however, these proteins were clearly visible in the nucleus with some minor localization at the cytoplasm (Fig. 1, A and B). Confocal images of cells without nuclear staining confirmed the presence of CSA and CSB proteins in the nucleus and cytoplasm (Fig. S1 and not depicted). Confocal microscopy of CSA and CSB cells with the CSA and CSB antibodies, respectively, validated the specificity of the antibodies (Fig. 1, A and B, and Fig. S1). Note that in the absence of a functional CSB protein (CSB cells; Fig. 1 A, bottom), an increased cytoplasmic staining of CSA was observed, whereas the nuclear staining was similar in both CSB and normal cells.

Bottom Line: We show functional increase of CSA and CSB inside mt and complex formation with mtDNA, mt human 8-oxoguanine glycosylase (mtOGG)-1, and mt single-stranded DNA binding protein (mtSSBP)-1 upon oxidative stress.MtDNA mutations are highly increased in cells from CS patients and in subcutaneous fat of aged Csb(m/m) and Csa(-/-) mice.Thus, the NER-proteins CSA and CSB localize to mt and directly interact with BER-associated human mitochondrial 8-oxoguanine glycosylase-1 to protect from aging- and stress-induced mtDNA mutations and apoptosis-mediated loss of subcutaneous fat, a hallmark of aging found in animal models, human progeroid syndromes like CS and in normal human aging.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Dermatology, Eberhard Karls University, D-72076 Tuebingen, Germany.

ABSTRACT
Defects in the DNA repair mechanism nucleotide excision repair (NER) may lead to tumors in xeroderma pigmentosum (XP) or to premature aging with loss of subcutaneous fat in Cockayne syndrome (CS). Mutations of mitochondrial (mt)DNA play a role in aging, but a link between the NER-associated CS proteins and base excision repair (BER)-associated proteins in mitochondrial aging remains enigmatic. We show functional increase of CSA and CSB inside mt and complex formation with mtDNA, mt human 8-oxoguanine glycosylase (mtOGG)-1, and mt single-stranded DNA binding protein (mtSSBP)-1 upon oxidative stress. MtDNA mutations are highly increased in cells from CS patients and in subcutaneous fat of aged Csb(m/m) and Csa(-/-) mice. Thus, the NER-proteins CSA and CSB localize to mt and directly interact with BER-associated human mitochondrial 8-oxoguanine glycosylase-1 to protect from aging- and stress-induced mtDNA mutations and apoptosis-mediated loss of subcutaneous fat, a hallmark of aging found in animal models, human progeroid syndromes like CS and in normal human aging.

Show MeSH
Related in: MedlinePlus