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NRBF2 regulates autophagy and prevents liver injury by modulating Atg14L-linked phosphatidylinositol-3 kinase III activity.

Lu J, He L, Behrends C, Araki M, Araki K, Jun Wang Q, Catanzaro JM, Friedman SL, Zong WX, Fiel MI, Li M, Yue Z - Nat Commun (2014)

Bottom Line: Here we report the identification of NRBF2 as a component in the specific PI3K-III complex and a modulator of PI3K-III activity.NRBF2-deficient cells exhibit enhanced vulnerability to endoplasmic reticulum (ER) stress that is reversed by re-introducing exogenous NRBF2.Thus, NRBF2 modulates autophagy via regulation of PI3K-III and prevents ER stress-mediated cytotoxicity and liver injury.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Neurology and Neuroscience, Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, New York, USA [2] School of Chinese Medicine, Hong Kong Baptist University, Hong Kong, China [3].

ABSTRACT
The Beclin 1-Vps34 complex, the core component of the class III phosphatidylinositol-3 kinase (PI3K-III), binds Atg14L or UVRAG to control different steps of autophagy. However, the mechanism underlying the control of PI3K-III activity remains elusive. Here we report the identification of NRBF2 as a component in the specific PI3K-III complex and a modulator of PI3K-III activity. Through its microtubule interaction and trafficking (MIT) domain, NRBF2 binds Atg14L directly and enhances Atg14L-linked Vps34 kinase activity and autophagy induction. NRBF2-deficient cells exhibit enhanced vulnerability to endoplasmic reticulum (ER) stress that is reversed by re-introducing exogenous NRBF2. NRBF2-deficient mice develop focal liver necrosis and ductular reaction, accompanied by impaired Atg14L-linked Vps34 activity and autophagy, although the mice show no increased mortality. Our data reveal a key role for NRBF2 in the assembly of the specific Atg14L-Beclin 1-Vps34-Vps15 complex for autophagy induction. Thus, NRBF2 modulates autophagy via regulation of PI3K-III and prevents ER stress-mediated cytotoxicity and liver injury.

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NRBF2 is involved in autophagosome maturation(a) NRBF2 KO MEFs display impaired autophagosome acidification. WT and NRBF2 KO MEFs are transiently transfected with mCherry–GFP–LC3B. WT MEFs expressing mCherry–GFP–LC3B contain many red-only puncta along with yellow (presence of both red and green) puncta. The number of both red-only puncta and yellow puncta dramatically increases after rapamycin (50 μg/ml, 4 hours) or starvation (HBSS, 4 hours) treatment, suggesting the increased autophagolysosomes and nascent autophagosomes. In contrast, NRBF2 KO MEFs expressing mCherry–GFP–LC3B contain quite fewer red-only puncta and yellow puncta, even after rapamycin or starvation treatment. (b) The quantification results show that both the numbers of red-only puncta and percentage of red-only puncta (verses total puncta) in KO MEFs were dramatically reduced (Data shown as mean±SEM, P values are indicated on the figures, Mann–Whitney U test, n=20 randomly selected cells, a representative result from 3 independent repeats), indicating both the autophagolysome number and autophagosome acidification rate are reduced in KO cells. Scale bar, 20μm, or 10 μm (amplified regions). (c) Autophagolysome formation is impaired in NRBF2 KO MEFs. WT and NRBF2 KO MEFs were transiently transfected with RFP–LC3 then stained with LAMP2 antibody. The colocalization between RFP-LC3 and LAMP2 are illustrated by line profile. RFP-LC3 puncta recruit much stronger LAMP2 signal in WT MEFs than in NRBF2 KO MEFs, suggesting the impairment of autophagolysosome formation in KO cells. Scale bar, 20μm, or 10 μm (amplified regions). This is a representative image from 3 independent experiments. (d) Over-expression of NRBF2 enhances UVRAG-linked Vps34 kinase activity. HEK293T cells were co-transfected with Myc-Vps34-Vps15-His and FLAG-UVRAG, in the presence of CFP or NRBF2-CFP. UVRAG-linked Vps34 was pulled down by an anti-FLAG antibody and subjected to kinase assay. IP products and inputs are immunoblotted using the antibodies indicated. (e) Quantification of Vps34 kinase activity normalized with immunoprecipitated Vps34 shows the significant difference between 2 groups (Data shown as mean±SEM, P value is indicated on the figures, one sample t test versus hypothetical mean 1, n=5).
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Figure 4: NRBF2 is involved in autophagosome maturation(a) NRBF2 KO MEFs display impaired autophagosome acidification. WT and NRBF2 KO MEFs are transiently transfected with mCherry–GFP–LC3B. WT MEFs expressing mCherry–GFP–LC3B contain many red-only puncta along with yellow (presence of both red and green) puncta. The number of both red-only puncta and yellow puncta dramatically increases after rapamycin (50 μg/ml, 4 hours) or starvation (HBSS, 4 hours) treatment, suggesting the increased autophagolysosomes and nascent autophagosomes. In contrast, NRBF2 KO MEFs expressing mCherry–GFP–LC3B contain quite fewer red-only puncta and yellow puncta, even after rapamycin or starvation treatment. (b) The quantification results show that both the numbers of red-only puncta and percentage of red-only puncta (verses total puncta) in KO MEFs were dramatically reduced (Data shown as mean±SEM, P values are indicated on the figures, Mann–Whitney U test, n=20 randomly selected cells, a representative result from 3 independent repeats), indicating both the autophagolysome number and autophagosome acidification rate are reduced in KO cells. Scale bar, 20μm, or 10 μm (amplified regions). (c) Autophagolysome formation is impaired in NRBF2 KO MEFs. WT and NRBF2 KO MEFs were transiently transfected with RFP–LC3 then stained with LAMP2 antibody. The colocalization between RFP-LC3 and LAMP2 are illustrated by line profile. RFP-LC3 puncta recruit much stronger LAMP2 signal in WT MEFs than in NRBF2 KO MEFs, suggesting the impairment of autophagolysosome formation in KO cells. Scale bar, 20μm, or 10 μm (amplified regions). This is a representative image from 3 independent experiments. (d) Over-expression of NRBF2 enhances UVRAG-linked Vps34 kinase activity. HEK293T cells were co-transfected with Myc-Vps34-Vps15-His and FLAG-UVRAG, in the presence of CFP or NRBF2-CFP. UVRAG-linked Vps34 was pulled down by an anti-FLAG antibody and subjected to kinase assay. IP products and inputs are immunoblotted using the antibodies indicated. (e) Quantification of Vps34 kinase activity normalized with immunoprecipitated Vps34 shows the significant difference between 2 groups (Data shown as mean±SEM, P value is indicated on the figures, one sample t test versus hypothetical mean 1, n=5).

Mentions: We examined the role of NRBF2 in autophagosome maturation by using the tandem reporter mCherry-GFP-LC3B19. The dual color fluorescent LC3 becomes red when labeling mature autophagosomes due to the acidic environment, while it remains in yellow (presence of both green and red color) when localized in immature autophagosomes19, 20. Our results show that the number of red LC3 puncta per cell and red LC3 puncta percentage are both reduced in NRBF2 KO MEFs under normal conditions. The differences in red-only LC3 puncta and red-only LC3 percentage between NRBF2 KO and WT MEFs are much more robust with rapamycin treatment (or nutrient starvation) compared to that under normal medium (Fig. 4a, 4b). This finding demonstrates impaired autophagosome maturation in the absence of NRBF2, particularly under autophagy induction. Moreover, we found that RFP-LC3 puncta rarely recruited the lysosomal marker LAMP2 in NRBF2 KO MEFs expressing RFP-LC3, whereas RFP-LC3 labeled autophagosomes have extensive overlap with LAMP2 in WT MEFs (Fig. 4c).


NRBF2 regulates autophagy and prevents liver injury by modulating Atg14L-linked phosphatidylinositol-3 kinase III activity.

Lu J, He L, Behrends C, Araki M, Araki K, Jun Wang Q, Catanzaro JM, Friedman SL, Zong WX, Fiel MI, Li M, Yue Z - Nat Commun (2014)

NRBF2 is involved in autophagosome maturation(a) NRBF2 KO MEFs display impaired autophagosome acidification. WT and NRBF2 KO MEFs are transiently transfected with mCherry–GFP–LC3B. WT MEFs expressing mCherry–GFP–LC3B contain many red-only puncta along with yellow (presence of both red and green) puncta. The number of both red-only puncta and yellow puncta dramatically increases after rapamycin (50 μg/ml, 4 hours) or starvation (HBSS, 4 hours) treatment, suggesting the increased autophagolysosomes and nascent autophagosomes. In contrast, NRBF2 KO MEFs expressing mCherry–GFP–LC3B contain quite fewer red-only puncta and yellow puncta, even after rapamycin or starvation treatment. (b) The quantification results show that both the numbers of red-only puncta and percentage of red-only puncta (verses total puncta) in KO MEFs were dramatically reduced (Data shown as mean±SEM, P values are indicated on the figures, Mann–Whitney U test, n=20 randomly selected cells, a representative result from 3 independent repeats), indicating both the autophagolysome number and autophagosome acidification rate are reduced in KO cells. Scale bar, 20μm, or 10 μm (amplified regions). (c) Autophagolysome formation is impaired in NRBF2 KO MEFs. WT and NRBF2 KO MEFs were transiently transfected with RFP–LC3 then stained with LAMP2 antibody. The colocalization between RFP-LC3 and LAMP2 are illustrated by line profile. RFP-LC3 puncta recruit much stronger LAMP2 signal in WT MEFs than in NRBF2 KO MEFs, suggesting the impairment of autophagolysosome formation in KO cells. Scale bar, 20μm, or 10 μm (amplified regions). This is a representative image from 3 independent experiments. (d) Over-expression of NRBF2 enhances UVRAG-linked Vps34 kinase activity. HEK293T cells were co-transfected with Myc-Vps34-Vps15-His and FLAG-UVRAG, in the presence of CFP or NRBF2-CFP. UVRAG-linked Vps34 was pulled down by an anti-FLAG antibody and subjected to kinase assay. IP products and inputs are immunoblotted using the antibodies indicated. (e) Quantification of Vps34 kinase activity normalized with immunoprecipitated Vps34 shows the significant difference between 2 groups (Data shown as mean±SEM, P value is indicated on the figures, one sample t test versus hypothetical mean 1, n=5).
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Figure 4: NRBF2 is involved in autophagosome maturation(a) NRBF2 KO MEFs display impaired autophagosome acidification. WT and NRBF2 KO MEFs are transiently transfected with mCherry–GFP–LC3B. WT MEFs expressing mCherry–GFP–LC3B contain many red-only puncta along with yellow (presence of both red and green) puncta. The number of both red-only puncta and yellow puncta dramatically increases after rapamycin (50 μg/ml, 4 hours) or starvation (HBSS, 4 hours) treatment, suggesting the increased autophagolysosomes and nascent autophagosomes. In contrast, NRBF2 KO MEFs expressing mCherry–GFP–LC3B contain quite fewer red-only puncta and yellow puncta, even after rapamycin or starvation treatment. (b) The quantification results show that both the numbers of red-only puncta and percentage of red-only puncta (verses total puncta) in KO MEFs were dramatically reduced (Data shown as mean±SEM, P values are indicated on the figures, Mann–Whitney U test, n=20 randomly selected cells, a representative result from 3 independent repeats), indicating both the autophagolysome number and autophagosome acidification rate are reduced in KO cells. Scale bar, 20μm, or 10 μm (amplified regions). (c) Autophagolysome formation is impaired in NRBF2 KO MEFs. WT and NRBF2 KO MEFs were transiently transfected with RFP–LC3 then stained with LAMP2 antibody. The colocalization between RFP-LC3 and LAMP2 are illustrated by line profile. RFP-LC3 puncta recruit much stronger LAMP2 signal in WT MEFs than in NRBF2 KO MEFs, suggesting the impairment of autophagolysosome formation in KO cells. Scale bar, 20μm, or 10 μm (amplified regions). This is a representative image from 3 independent experiments. (d) Over-expression of NRBF2 enhances UVRAG-linked Vps34 kinase activity. HEK293T cells were co-transfected with Myc-Vps34-Vps15-His and FLAG-UVRAG, in the presence of CFP or NRBF2-CFP. UVRAG-linked Vps34 was pulled down by an anti-FLAG antibody and subjected to kinase assay. IP products and inputs are immunoblotted using the antibodies indicated. (e) Quantification of Vps34 kinase activity normalized with immunoprecipitated Vps34 shows the significant difference between 2 groups (Data shown as mean±SEM, P value is indicated on the figures, one sample t test versus hypothetical mean 1, n=5).
Mentions: We examined the role of NRBF2 in autophagosome maturation by using the tandem reporter mCherry-GFP-LC3B19. The dual color fluorescent LC3 becomes red when labeling mature autophagosomes due to the acidic environment, while it remains in yellow (presence of both green and red color) when localized in immature autophagosomes19, 20. Our results show that the number of red LC3 puncta per cell and red LC3 puncta percentage are both reduced in NRBF2 KO MEFs under normal conditions. The differences in red-only LC3 puncta and red-only LC3 percentage between NRBF2 KO and WT MEFs are much more robust with rapamycin treatment (or nutrient starvation) compared to that under normal medium (Fig. 4a, 4b). This finding demonstrates impaired autophagosome maturation in the absence of NRBF2, particularly under autophagy induction. Moreover, we found that RFP-LC3 puncta rarely recruited the lysosomal marker LAMP2 in NRBF2 KO MEFs expressing RFP-LC3, whereas RFP-LC3 labeled autophagosomes have extensive overlap with LAMP2 in WT MEFs (Fig. 4c).

Bottom Line: Here we report the identification of NRBF2 as a component in the specific PI3K-III complex and a modulator of PI3K-III activity.NRBF2-deficient cells exhibit enhanced vulnerability to endoplasmic reticulum (ER) stress that is reversed by re-introducing exogenous NRBF2.Thus, NRBF2 modulates autophagy via regulation of PI3K-III and prevents ER stress-mediated cytotoxicity and liver injury.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Neurology and Neuroscience, Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, New York, USA [2] School of Chinese Medicine, Hong Kong Baptist University, Hong Kong, China [3].

ABSTRACT
The Beclin 1-Vps34 complex, the core component of the class III phosphatidylinositol-3 kinase (PI3K-III), binds Atg14L or UVRAG to control different steps of autophagy. However, the mechanism underlying the control of PI3K-III activity remains elusive. Here we report the identification of NRBF2 as a component in the specific PI3K-III complex and a modulator of PI3K-III activity. Through its microtubule interaction and trafficking (MIT) domain, NRBF2 binds Atg14L directly and enhances Atg14L-linked Vps34 kinase activity and autophagy induction. NRBF2-deficient cells exhibit enhanced vulnerability to endoplasmic reticulum (ER) stress that is reversed by re-introducing exogenous NRBF2. NRBF2-deficient mice develop focal liver necrosis and ductular reaction, accompanied by impaired Atg14L-linked Vps34 activity and autophagy, although the mice show no increased mortality. Our data reveal a key role for NRBF2 in the assembly of the specific Atg14L-Beclin 1-Vps34-Vps15 complex for autophagy induction. Thus, NRBF2 modulates autophagy via regulation of PI3K-III and prevents ER stress-mediated cytotoxicity and liver injury.

Show MeSH
Related in: MedlinePlus