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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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Assembly of Beclin 1-Vps34 complex in WT and NRBF2 mice, and schematic model for the NRBF2 function(a) NRBF2 is critical for the assembly of Vps34-Vps15 and Atg14L-Beclin 1 complex. Brain lysate from WT and NRBF2 KO mice were subjected to Co-IP by Vps34 antibody and Atg14L antibody. The IP products were subjected to in vitro lipid kinase assay and Western blotting analysis using the indicated antibodies. (b, c) Quantification results shows that Vps34-Vps15 and Atg14L-Beclin 1 complex interactions as well as Atg14L-linked Vps34 kinase activity are dramatically impaired in NRBF2 KO mice. (Data shown as mean±SEM, P values are indicated on the figures, unpaired Student’s t test, n=4 mice in each group). (d) Over-expression of NRBF2 enhances overall Vps34 kinase activity. HEK293T cells were co-transfected with Myc-Vps34-Vps15-His and CFP, NRBF2-CFP, dMIT-CFP or dCCD-CFP. Total Vps34 was pulled down by an anti-myc antibody and subjected to kinase assay. IP products and inputs are immunoblotted using the antibodies indicated. (e) Quantification of IPed Vps34 kinase activity, Vps15 protein normalized with IPed Vps34 shows that NRBF2-CFP, but not CFP, dMIT-CFP or dCCD-CFP significantly enhance Vps34 kinase activity and Vps34-Vps15 interaction. (Data shown as mean±SEM, P values are indicated on the figures. IPed Vps15 / IPed Vps34, one sample t test versus hypothetical mean 1, bonferroni correction, n=3; IPed Vps34 kinase activity / IPed Vps34, multiple t test followed by bonferroni correction, n=3). (f) Schematic model for the NRBF2 function in autophagy. Top: In WT cells, NRBF2 functions to stabilize Beclin 1-Atg14L, Vps34 and Vps15 proteins in the specific autophagy PI3K-III kinase complex. In NRBF2 KO cells, approximately 25% Atg14L remains in the complex with Vps34-Vps15; Middle and bottom: Upon autophagy induction, NRBF2-Atg14L interaction mediates the binding with other components in the cytosol and consequent assembly of a highly active PI3K-III kinase complex at the phagophore, resulting in enhanced autophagy capacity.
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Figure 8: Assembly of Beclin 1-Vps34 complex in WT and NRBF2 mice, and schematic model for the NRBF2 function(a) NRBF2 is critical for the assembly of Vps34-Vps15 and Atg14L-Beclin 1 complex. Brain lysate from WT and NRBF2 KO mice were subjected to Co-IP by Vps34 antibody and Atg14L antibody. The IP products were subjected to in vitro lipid kinase assay and Western blotting analysis using the indicated antibodies. (b, c) Quantification results shows that Vps34-Vps15 and Atg14L-Beclin 1 complex interactions as well as Atg14L-linked Vps34 kinase activity are dramatically impaired in NRBF2 KO mice. (Data shown as mean±SEM, P values are indicated on the figures, unpaired Student’s t test, n=4 mice in each group). (d) Over-expression of NRBF2 enhances overall Vps34 kinase activity. HEK293T cells were co-transfected with Myc-Vps34-Vps15-His and CFP, NRBF2-CFP, dMIT-CFP or dCCD-CFP. Total Vps34 was pulled down by an anti-myc antibody and subjected to kinase assay. IP products and inputs are immunoblotted using the antibodies indicated. (e) Quantification of IPed Vps34 kinase activity, Vps15 protein normalized with IPed Vps34 shows that NRBF2-CFP, but not CFP, dMIT-CFP or dCCD-CFP significantly enhance Vps34 kinase activity and Vps34-Vps15 interaction. (Data shown as mean±SEM, P values are indicated on the figures. IPed Vps15 / IPed Vps34, one sample t test versus hypothetical mean 1, bonferroni correction, n=3; IPed Vps34 kinase activity / IPed Vps34, multiple t test followed by bonferroni correction, n=3). (f) Schematic model for the NRBF2 function in autophagy. Top: In WT cells, NRBF2 functions to stabilize Beclin 1-Atg14L, Vps34 and Vps15 proteins in the specific autophagy PI3K-III kinase complex. In NRBF2 KO cells, approximately 25% Atg14L remains in the complex with Vps34-Vps15; Middle and bottom: Upon autophagy induction, NRBF2-Atg14L interaction mediates the binding with other components in the cytosol and consequent assembly of a highly active PI3K-III kinase complex at the phagophore, resulting in enhanced autophagy capacity.

Mentions: Although NRBF2 is important for Atg14L’s interaction with Beclin 1, Vps34 and Vps15 proteins in MEFs (Fig. 2), it is unclear whether NRBF2 affects differential Vps34 complex formation particularly in tissues. We performed Western blotting to examine the total protein levels of Vps34, Vps15, Beclin 1, UVRAG, and Atg14L in NRBF2 WT and KO mice brain lysates. We found little change in basal protein levels between NRBF2 WT and KO mice (Fig. 8a). However, using anti-Vps34 antibody to pull down Vps34 proteins, we found that Vps34 binds markedly less Atg14L in NRBF2 KO MEFs (≈75% decrease compared to WT mice). Vps34 also binds less Vps15 (≈35% reduced) and Beclin 1 (≈40% reduced) in KO mice. In contrast, Vps34-associated UVRAG are similar between NRBF2 WT and KO mice. Measurment of the activity of Vps34 IP product shows a decrease in PI(3)P production in NRBF2 KO mice (Fig. 8a, b). This result provides strong evidence that NRBF2 is required for an activated Vps34 activity by stabilizing the Vps34-Vps15-Beclin 1-Atg14L complex formation.


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)

Assembly of Beclin 1-Vps34 complex in WT and NRBF2 mice, and schematic model for the NRBF2 function(a) NRBF2 is critical for the assembly of Vps34-Vps15 and Atg14L-Beclin 1 complex. Brain lysate from WT and NRBF2 KO mice were subjected to Co-IP by Vps34 antibody and Atg14L antibody. The IP products were subjected to in vitro lipid kinase assay and Western blotting analysis using the indicated antibodies. (b, c) Quantification results shows that Vps34-Vps15 and Atg14L-Beclin 1 complex interactions as well as Atg14L-linked Vps34 kinase activity are dramatically impaired in NRBF2 KO mice. (Data shown as mean±SEM, P values are indicated on the figures, unpaired Student’s t test, n=4 mice in each group). (d) Over-expression of NRBF2 enhances overall Vps34 kinase activity. HEK293T cells were co-transfected with Myc-Vps34-Vps15-His and CFP, NRBF2-CFP, dMIT-CFP or dCCD-CFP. Total Vps34 was pulled down by an anti-myc antibody and subjected to kinase assay. IP products and inputs are immunoblotted using the antibodies indicated. (e) Quantification of IPed Vps34 kinase activity, Vps15 protein normalized with IPed Vps34 shows that NRBF2-CFP, but not CFP, dMIT-CFP or dCCD-CFP significantly enhance Vps34 kinase activity and Vps34-Vps15 interaction. (Data shown as mean±SEM, P values are indicated on the figures. IPed Vps15 / IPed Vps34, one sample t test versus hypothetical mean 1, bonferroni correction, n=3; IPed Vps34 kinase activity / IPed Vps34, multiple t test followed by bonferroni correction, n=3). (f) Schematic model for the NRBF2 function in autophagy. Top: In WT cells, NRBF2 functions to stabilize Beclin 1-Atg14L, Vps34 and Vps15 proteins in the specific autophagy PI3K-III kinase complex. In NRBF2 KO cells, approximately 25% Atg14L remains in the complex with Vps34-Vps15; Middle and bottom: Upon autophagy induction, NRBF2-Atg14L interaction mediates the binding with other components in the cytosol and consequent assembly of a highly active PI3K-III kinase complex at the phagophore, resulting in enhanced autophagy capacity.
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Figure 8: Assembly of Beclin 1-Vps34 complex in WT and NRBF2 mice, and schematic model for the NRBF2 function(a) NRBF2 is critical for the assembly of Vps34-Vps15 and Atg14L-Beclin 1 complex. Brain lysate from WT and NRBF2 KO mice were subjected to Co-IP by Vps34 antibody and Atg14L antibody. The IP products were subjected to in vitro lipid kinase assay and Western blotting analysis using the indicated antibodies. (b, c) Quantification results shows that Vps34-Vps15 and Atg14L-Beclin 1 complex interactions as well as Atg14L-linked Vps34 kinase activity are dramatically impaired in NRBF2 KO mice. (Data shown as mean±SEM, P values are indicated on the figures, unpaired Student’s t test, n=4 mice in each group). (d) Over-expression of NRBF2 enhances overall Vps34 kinase activity. HEK293T cells were co-transfected with Myc-Vps34-Vps15-His and CFP, NRBF2-CFP, dMIT-CFP or dCCD-CFP. Total Vps34 was pulled down by an anti-myc antibody and subjected to kinase assay. IP products and inputs are immunoblotted using the antibodies indicated. (e) Quantification of IPed Vps34 kinase activity, Vps15 protein normalized with IPed Vps34 shows that NRBF2-CFP, but not CFP, dMIT-CFP or dCCD-CFP significantly enhance Vps34 kinase activity and Vps34-Vps15 interaction. (Data shown as mean±SEM, P values are indicated on the figures. IPed Vps15 / IPed Vps34, one sample t test versus hypothetical mean 1, bonferroni correction, n=3; IPed Vps34 kinase activity / IPed Vps34, multiple t test followed by bonferroni correction, n=3). (f) Schematic model for the NRBF2 function in autophagy. Top: In WT cells, NRBF2 functions to stabilize Beclin 1-Atg14L, Vps34 and Vps15 proteins in the specific autophagy PI3K-III kinase complex. In NRBF2 KO cells, approximately 25% Atg14L remains in the complex with Vps34-Vps15; Middle and bottom: Upon autophagy induction, NRBF2-Atg14L interaction mediates the binding with other components in the cytosol and consequent assembly of a highly active PI3K-III kinase complex at the phagophore, resulting in enhanced autophagy capacity.
Mentions: Although NRBF2 is important for Atg14L’s interaction with Beclin 1, Vps34 and Vps15 proteins in MEFs (Fig. 2), it is unclear whether NRBF2 affects differential Vps34 complex formation particularly in tissues. We performed Western blotting to examine the total protein levels of Vps34, Vps15, Beclin 1, UVRAG, and Atg14L in NRBF2 WT and KO mice brain lysates. We found little change in basal protein levels between NRBF2 WT and KO mice (Fig. 8a). However, using anti-Vps34 antibody to pull down Vps34 proteins, we found that Vps34 binds markedly less Atg14L in NRBF2 KO MEFs (≈75% decrease compared to WT mice). Vps34 also binds less Vps15 (≈35% reduced) and Beclin 1 (≈40% reduced) in KO mice. In contrast, Vps34-associated UVRAG are similar between NRBF2 WT and KO mice. Measurment of the activity of Vps34 IP product shows a decrease in PI(3)P production in NRBF2 KO mice (Fig. 8a, b). This result provides strong evidence that NRBF2 is required for an activated Vps34 activity by stabilizing the Vps34-Vps15-Beclin 1-Atg14L complex formation.

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