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A novel role for 12/15-lipoxygenase in regulating autophagy.

Morgan AH, Hammond VJ, Sakoh-Nakatogawa M, Ohsumi Y, Thomas CP, Blanchet F, Piguet V, Kiselyov K, O'Donnell VB - Redox Biol (2014)

Bottom Line: Herein, we show that cells deficient in 12/15-LOX contain defective mitochondria and numerous cytoplasmic vacuoles containing electron dense material, indicating defects in autophagy or membrane processing, However, both LC3 expression and lipidation were normal both basally and on chloroquine treatment.A LOX-derived oxidized phospholipid, 12-hydroxyeicosatetraenoic acid-phosphatidylethanolamine (12-HETE-PE) was found to be a preferred substrate for yeast Atg8 lipidation, versus native PE, while both native and oxidized PE were effective substrates for LC3 lipidation.Thus, we show that oxidized phospholipids generated by 12/15-LOX can act as substrates for key proteins required for effective autophagy and that cells deficient in this enzyme show evidence of autophagic dysfunction.

View Article: PubMed Central - PubMed

Affiliation: Institute of Infection and Immunity, School of Medicine, Cardiff University, Cardiff CF14 4XN, UK.

No MeSH data available.


Macrophages from 12/15-LOX deficient mice show altered membrane structure on electron microscopy but LC3 expression is similar. Panel A. EM analysis of wild type and 12/15 LOX−/− peritoneal macrophages. Peritoneal cells from wild type mice were analyzed using TEM as described in Section “Materials and methods” at 10,000× magnification. Lower panels. Cells were analyzed by TEM at 20,000× magnification. Arrows indicate healthy mitochondria (in WT) (black), abnormal mitochondria (in 12/15-LOX−/−) (green), and numerous autophagosomes (blue), lysosomal storage bodies (red) and vacuoles (yellow). Panel B. Macrophages from 12/15-LOX−/− mice express similar LC3 levels to wild type. Macrophages were stimulated overnight using chloroquine (100 µM), before LC3-I and -II analysis using Western blot. Data are combined from three representative gels, with one shown as illustration. Each gel had n=3 for both WT and 12/15-LOX−/− mice. Relative density was determined for LC3-I and -II, then divided by the actin loading control density, thus the graph represents a combined n=9, mean±SEM.
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f0005: Macrophages from 12/15-LOX deficient mice show altered membrane structure on electron microscopy but LC3 expression is similar. Panel A. EM analysis of wild type and 12/15 LOX−/− peritoneal macrophages. Peritoneal cells from wild type mice were analyzed using TEM as described in Section “Materials and methods” at 10,000× magnification. Lower panels. Cells were analyzed by TEM at 20,000× magnification. Arrows indicate healthy mitochondria (in WT) (black), abnormal mitochondria (in 12/15-LOX−/−) (green), and numerous autophagosomes (blue), lysosomal storage bodies (red) and vacuoles (yellow). Panel B. Macrophages from 12/15-LOX−/− mice express similar LC3 levels to wild type. Macrophages were stimulated overnight using chloroquine (100 µM), before LC3-I and -II analysis using Western blot. Data are combined from three representative gels, with one shown as illustration. Each gel had n=3 for both WT and 12/15-LOX−/− mice. Relative density was determined for LC3-I and -II, then divided by the actin loading control density, thus the graph represents a combined n=9, mean±SEM.

Mentions: Peritoneal cells from naïve mice were analyzed using transmission EM. Representative macrophages from three separate pooled isolates is shown in Fig. 1A. Healthy-looking mitochondria (small, compact, and with well-defined cristae) are seen in wild type cells. In contrast, 12/15-LOX−/− macrophages are swollen and granular. 12/15-LOX−/− macrophages also demonstrate a large number of vacuoles (yellow arrows) and potential lysosomal storage bodies, visible as dark inclusions (red arrows). Some have double membranes, suggestive of autophagosomes (blue arrows). Far lower numbers of vacuoles and suspected lysosomal storage bodies are seen in wild type macrophages.


A novel role for 12/15-lipoxygenase in regulating autophagy.

Morgan AH, Hammond VJ, Sakoh-Nakatogawa M, Ohsumi Y, Thomas CP, Blanchet F, Piguet V, Kiselyov K, O'Donnell VB - Redox Biol (2014)

Macrophages from 12/15-LOX deficient mice show altered membrane structure on electron microscopy but LC3 expression is similar. Panel A. EM analysis of wild type and 12/15 LOX−/− peritoneal macrophages. Peritoneal cells from wild type mice were analyzed using TEM as described in Section “Materials and methods” at 10,000× magnification. Lower panels. Cells were analyzed by TEM at 20,000× magnification. Arrows indicate healthy mitochondria (in WT) (black), abnormal mitochondria (in 12/15-LOX−/−) (green), and numerous autophagosomes (blue), lysosomal storage bodies (red) and vacuoles (yellow). Panel B. Macrophages from 12/15-LOX−/− mice express similar LC3 levels to wild type. Macrophages were stimulated overnight using chloroquine (100 µM), before LC3-I and -II analysis using Western blot. Data are combined from three representative gels, with one shown as illustration. Each gel had n=3 for both WT and 12/15-LOX−/− mice. Relative density was determined for LC3-I and -II, then divided by the actin loading control density, thus the graph represents a combined n=9, mean±SEM.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4309860&req=5

f0005: Macrophages from 12/15-LOX deficient mice show altered membrane structure on electron microscopy but LC3 expression is similar. Panel A. EM analysis of wild type and 12/15 LOX−/− peritoneal macrophages. Peritoneal cells from wild type mice were analyzed using TEM as described in Section “Materials and methods” at 10,000× magnification. Lower panels. Cells were analyzed by TEM at 20,000× magnification. Arrows indicate healthy mitochondria (in WT) (black), abnormal mitochondria (in 12/15-LOX−/−) (green), and numerous autophagosomes (blue), lysosomal storage bodies (red) and vacuoles (yellow). Panel B. Macrophages from 12/15-LOX−/− mice express similar LC3 levels to wild type. Macrophages were stimulated overnight using chloroquine (100 µM), before LC3-I and -II analysis using Western blot. Data are combined from three representative gels, with one shown as illustration. Each gel had n=3 for both WT and 12/15-LOX−/− mice. Relative density was determined for LC3-I and -II, then divided by the actin loading control density, thus the graph represents a combined n=9, mean±SEM.
Mentions: Peritoneal cells from naïve mice were analyzed using transmission EM. Representative macrophages from three separate pooled isolates is shown in Fig. 1A. Healthy-looking mitochondria (small, compact, and with well-defined cristae) are seen in wild type cells. In contrast, 12/15-LOX−/− macrophages are swollen and granular. 12/15-LOX−/− macrophages also demonstrate a large number of vacuoles (yellow arrows) and potential lysosomal storage bodies, visible as dark inclusions (red arrows). Some have double membranes, suggestive of autophagosomes (blue arrows). Far lower numbers of vacuoles and suspected lysosomal storage bodies are seen in wild type macrophages.

Bottom Line: Herein, we show that cells deficient in 12/15-LOX contain defective mitochondria and numerous cytoplasmic vacuoles containing electron dense material, indicating defects in autophagy or membrane processing, However, both LC3 expression and lipidation were normal both basally and on chloroquine treatment.A LOX-derived oxidized phospholipid, 12-hydroxyeicosatetraenoic acid-phosphatidylethanolamine (12-HETE-PE) was found to be a preferred substrate for yeast Atg8 lipidation, versus native PE, while both native and oxidized PE were effective substrates for LC3 lipidation.Thus, we show that oxidized phospholipids generated by 12/15-LOX can act as substrates for key proteins required for effective autophagy and that cells deficient in this enzyme show evidence of autophagic dysfunction.

View Article: PubMed Central - PubMed

Affiliation: Institute of Infection and Immunity, School of Medicine, Cardiff University, Cardiff CF14 4XN, UK.

No MeSH data available.