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A pathway sensor for genome-wide screens of intracellular proteolytic cleavage.

Ketteler R, Sun Z, Kovacs KF, He WW, Seed B - Genome Biol. (2008)

Bottom Line: Protein cleavage is a central event in many regulated biological processes.GLUC exits the cell without benefit of a secretory leader peptide, but can be anchored in the cell by fusion to beta-actin.Using this assay, we have identified regulators of autophagy, apoptosis and beta-actin cleavage.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Computational and Integrative Biology, Massachusetts General Hospital, Cambridge Street, Boston, MA 02114, USA.

ABSTRACT
Protein cleavage is a central event in many regulated biological processes. We describe a system for detecting intracellular proteolysis based on non-conventional secretion of Gaussia luciferase (GLUC). GLUC exits the cell without benefit of a secretory leader peptide, but can be anchored in the cell by fusion to beta-actin. By including protease cleavage sites between GLUC and beta-actin, proteolytic cleavage can be detected. Using this assay, we have identified regulators of autophagy, apoptosis and beta-actin cleavage.

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A GLUC-based sensor to monitor autophagy. (a) Rapamycin (Rap) induces release of GLUC activity from Actin-LC3-dN. Actin-LC3-dN was transfected in 293ET cells and medium was replaced after 24 h with serum-free medium containing 200 nM rapamycin for 6 h before analysis of GLUC activity in SN. (b) ATG4B but not ATG4A induces cleavage of Actin-LC3-dN. SN of 293ET cells transiently co-transfected with Actin-dN or Actin-LC3-dN and GFP, ATG4A or ATG4B were collected after 24 h and analyzed for GLUC activity. Error bars were calculated from three independent transfections. RLU, relative light units. (c) ATG4B cleaves GFP-LC3. 293ET cells transfected with GFP-LC3 and ATG4A or ATG4B were lysed in 1% NP40, resolved by 10% SDS-PAGE and blotted with anti-GFP. Full-length GFP-LC3 (LC I) runs at 45 kDa and the cleaved product runs at 43 kDa (LC II). (d) ATG4B cleaves Actin-LC3-dN to generate a small LC3-dNGLUC fragment. 293ET cells transfected with Actin-LC3-dN and GFP or ATG4B were treated for 6 h with 10 μg/ml Brefeldin A (right panel) to block secretion of cleaved dNGLUC or left untreated (left panel) before lysis in 1% NP40. Whole cell lysates were resolved by 10% SDS PAGE and blotted with an antibody raised against dNGLUC. The protein band corresponding to full-length Actin-LC3-dN is marked with an asterisk, the cleavage product is marked with an arrowhead.
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Figure 4: A GLUC-based sensor to monitor autophagy. (a) Rapamycin (Rap) induces release of GLUC activity from Actin-LC3-dN. Actin-LC3-dN was transfected in 293ET cells and medium was replaced after 24 h with serum-free medium containing 200 nM rapamycin for 6 h before analysis of GLUC activity in SN. (b) ATG4B but not ATG4A induces cleavage of Actin-LC3-dN. SN of 293ET cells transiently co-transfected with Actin-dN or Actin-LC3-dN and GFP, ATG4A or ATG4B were collected after 24 h and analyzed for GLUC activity. Error bars were calculated from three independent transfections. RLU, relative light units. (c) ATG4B cleaves GFP-LC3. 293ET cells transfected with GFP-LC3 and ATG4A or ATG4B were lysed in 1% NP40, resolved by 10% SDS-PAGE and blotted with anti-GFP. Full-length GFP-LC3 (LC I) runs at 45 kDa and the cleaved product runs at 43 kDa (LC II). (d) ATG4B cleaves Actin-LC3-dN to generate a small LC3-dNGLUC fragment. 293ET cells transfected with Actin-LC3-dN and GFP or ATG4B were treated for 6 h with 10 μg/ml Brefeldin A (right panel) to block secretion of cleaved dNGLUC or left untreated (left panel) before lysis in 1% NP40. Whole cell lysates were resolved by 10% SDS PAGE and blotted with an antibody raised against dNGLUC. The protein band corresponding to full-length Actin-LC3-dN is marked with an asterisk, the cleavage product is marked with an arrowhead.

Mentions: To further explore the suitability of GLUC fusions for detection of native protein cleavage, we inserted the open reading frame of hMAP1LC3 (LC3), a marker of autophagy, between β-actin and dNGLUC. Autophagy is a tightly regulated cellular response to starvation that results in degradation of subcellular organelles. LC3 is cleaved during autophagy at the carboxyl terminus by the cellular protease hATG4B; the cleaved form is found associated with autophagosomes [18]. Amino acid starvation or treatment with rapamycin is sufficient to induce autophagy and LC3 cleavage. Upon treatment with 200 nM rapamycin, we detected a 7.2-fold increase in GLUC activity in SN in cells expressing Actin-LC3-dNGLUC but not Actin-dNGLUC (Figure 4a). In addition, co-expression of the cellular protease ATG4B, but not ATG4A or GFP, resulted in a 26.6-fold increase in extra-cellular luciferase activity (Figure 4b). Activity of ATG4B was confirmed by immunoblotting of transfected GFP-LC3. In the presence of ATG4B, the cleaved form of GFP-LC3 was visualized at 43 kDa and significantly increased in intensity compared to the full-length 45 kDa form, which was not evident in cells transfected with ATG4A (Figure 4c). In order to confirm cleavage of Actin-LC3-dNGLUC by ATG4B, we resolved whole cell lysates by SDS-PAGE and immuno-blotting using an antibody raised against dNGLUC as a probe. In the absence of ATG4B, we detected the full-length construct Actin-LC3-dNGLUC at 83 kDa and a smaller band at 23 kDa (Figure 4d). In cells cotransfected with an ATG4B expression plasmid, the full-length product at 83 kDa disappeared; whereas in cells cotransfected with a GFP expression plasmid, the 83 kDa product was readily apparent (Figure 4d). To visualize the secreted cleavage product, we treated cells with Brefeldin A for 6 hours prior to cell lysis. In the setting of ATG4B coexpression in Brefeldin A-treated cells, the band corresponding to Actin-LC3-dNGLUC at 83 kDa is not seen and the 23 kDa band corresponding to the dNGLUC cleavage product has increased intensity, consistent with the view that Actin-LC3-dNGLUC is cleaved by ATG4B.


A pathway sensor for genome-wide screens of intracellular proteolytic cleavage.

Ketteler R, Sun Z, Kovacs KF, He WW, Seed B - Genome Biol. (2008)

A GLUC-based sensor to monitor autophagy. (a) Rapamycin (Rap) induces release of GLUC activity from Actin-LC3-dN. Actin-LC3-dN was transfected in 293ET cells and medium was replaced after 24 h with serum-free medium containing 200 nM rapamycin for 6 h before analysis of GLUC activity in SN. (b) ATG4B but not ATG4A induces cleavage of Actin-LC3-dN. SN of 293ET cells transiently co-transfected with Actin-dN or Actin-LC3-dN and GFP, ATG4A or ATG4B were collected after 24 h and analyzed for GLUC activity. Error bars were calculated from three independent transfections. RLU, relative light units. (c) ATG4B cleaves GFP-LC3. 293ET cells transfected with GFP-LC3 and ATG4A or ATG4B were lysed in 1% NP40, resolved by 10% SDS-PAGE and blotted with anti-GFP. Full-length GFP-LC3 (LC I) runs at 45 kDa and the cleaved product runs at 43 kDa (LC II). (d) ATG4B cleaves Actin-LC3-dN to generate a small LC3-dNGLUC fragment. 293ET cells transfected with Actin-LC3-dN and GFP or ATG4B were treated for 6 h with 10 μg/ml Brefeldin A (right panel) to block secretion of cleaved dNGLUC or left untreated (left panel) before lysis in 1% NP40. Whole cell lysates were resolved by 10% SDS PAGE and blotted with an antibody raised against dNGLUC. The protein band corresponding to full-length Actin-LC3-dN is marked with an asterisk, the cleavage product is marked with an arrowhead.
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Figure 4: A GLUC-based sensor to monitor autophagy. (a) Rapamycin (Rap) induces release of GLUC activity from Actin-LC3-dN. Actin-LC3-dN was transfected in 293ET cells and medium was replaced after 24 h with serum-free medium containing 200 nM rapamycin for 6 h before analysis of GLUC activity in SN. (b) ATG4B but not ATG4A induces cleavage of Actin-LC3-dN. SN of 293ET cells transiently co-transfected with Actin-dN or Actin-LC3-dN and GFP, ATG4A or ATG4B were collected after 24 h and analyzed for GLUC activity. Error bars were calculated from three independent transfections. RLU, relative light units. (c) ATG4B cleaves GFP-LC3. 293ET cells transfected with GFP-LC3 and ATG4A or ATG4B were lysed in 1% NP40, resolved by 10% SDS-PAGE and blotted with anti-GFP. Full-length GFP-LC3 (LC I) runs at 45 kDa and the cleaved product runs at 43 kDa (LC II). (d) ATG4B cleaves Actin-LC3-dN to generate a small LC3-dNGLUC fragment. 293ET cells transfected with Actin-LC3-dN and GFP or ATG4B were treated for 6 h with 10 μg/ml Brefeldin A (right panel) to block secretion of cleaved dNGLUC or left untreated (left panel) before lysis in 1% NP40. Whole cell lysates were resolved by 10% SDS PAGE and blotted with an antibody raised against dNGLUC. The protein band corresponding to full-length Actin-LC3-dN is marked with an asterisk, the cleavage product is marked with an arrowhead.
Mentions: To further explore the suitability of GLUC fusions for detection of native protein cleavage, we inserted the open reading frame of hMAP1LC3 (LC3), a marker of autophagy, between β-actin and dNGLUC. Autophagy is a tightly regulated cellular response to starvation that results in degradation of subcellular organelles. LC3 is cleaved during autophagy at the carboxyl terminus by the cellular protease hATG4B; the cleaved form is found associated with autophagosomes [18]. Amino acid starvation or treatment with rapamycin is sufficient to induce autophagy and LC3 cleavage. Upon treatment with 200 nM rapamycin, we detected a 7.2-fold increase in GLUC activity in SN in cells expressing Actin-LC3-dNGLUC but not Actin-dNGLUC (Figure 4a). In addition, co-expression of the cellular protease ATG4B, but not ATG4A or GFP, resulted in a 26.6-fold increase in extra-cellular luciferase activity (Figure 4b). Activity of ATG4B was confirmed by immunoblotting of transfected GFP-LC3. In the presence of ATG4B, the cleaved form of GFP-LC3 was visualized at 43 kDa and significantly increased in intensity compared to the full-length 45 kDa form, which was not evident in cells transfected with ATG4A (Figure 4c). In order to confirm cleavage of Actin-LC3-dNGLUC by ATG4B, we resolved whole cell lysates by SDS-PAGE and immuno-blotting using an antibody raised against dNGLUC as a probe. In the absence of ATG4B, we detected the full-length construct Actin-LC3-dNGLUC at 83 kDa and a smaller band at 23 kDa (Figure 4d). In cells cotransfected with an ATG4B expression plasmid, the full-length product at 83 kDa disappeared; whereas in cells cotransfected with a GFP expression plasmid, the 83 kDa product was readily apparent (Figure 4d). To visualize the secreted cleavage product, we treated cells with Brefeldin A for 6 hours prior to cell lysis. In the setting of ATG4B coexpression in Brefeldin A-treated cells, the band corresponding to Actin-LC3-dNGLUC at 83 kDa is not seen and the 23 kDa band corresponding to the dNGLUC cleavage product has increased intensity, consistent with the view that Actin-LC3-dNGLUC is cleaved by ATG4B.

Bottom Line: Protein cleavage is a central event in many regulated biological processes.GLUC exits the cell without benefit of a secretory leader peptide, but can be anchored in the cell by fusion to beta-actin.Using this assay, we have identified regulators of autophagy, apoptosis and beta-actin cleavage.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Computational and Integrative Biology, Massachusetts General Hospital, Cambridge Street, Boston, MA 02114, USA.

ABSTRACT
Protein cleavage is a central event in many regulated biological processes. We describe a system for detecting intracellular proteolysis based on non-conventional secretion of Gaussia luciferase (GLUC). GLUC exits the cell without benefit of a secretory leader peptide, but can be anchored in the cell by fusion to beta-actin. By including protease cleavage sites between GLUC and beta-actin, proteolytic cleavage can be detected. Using this assay, we have identified regulators of autophagy, apoptosis and beta-actin cleavage.

Show MeSH
Related in: MedlinePlus