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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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HTRA3 and 4 release GLUC activity from Actin-dNGLUC. (a) Actin-dNGLUC, DEVDG3 or DEVAG2F were co-transfected with GFP, caspase 9 or TBC1D10A and GLUC activity in SN was assayed after 30 h. TBC1D10A specifically releases dNGLUC from DEVDG3, but not DEVAG2F (RLU, relative light units). (b) HTRA3 and 4 release GLUC activity from Actin-dNGLUC. Caspase 8, caspase 9, HTRA3 and HTRA4 were co-transfected with Actin-dNGLUC and DEVDG2F and SN were analyzed for GLUC activity in SN after 30 h. HTRA3 and 4 release GLUC activity from Actin-dN and DEVDG2F, while caspases 8 and 9 released GLUC activity from DEVDG2F but not Actin.
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Figure 3: HTRA3 and 4 release GLUC activity from Actin-dNGLUC. (a) Actin-dNGLUC, DEVDG3 or DEVAG2F were co-transfected with GFP, caspase 9 or TBC1D10A and GLUC activity in SN was assayed after 30 h. TBC1D10A specifically releases dNGLUC from DEVDG3, but not DEVAG2F (RLU, relative light units). (b) HTRA3 and 4 release GLUC activity from Actin-dNGLUC. Caspase 8, caspase 9, HTRA3 and HTRA4 were co-transfected with Actin-dNGLUC and DEVDG2F and SN were analyzed for GLUC activity in SN after 30 h. HTRA3 and 4 release GLUC activity from Actin-dN and DEVDG2F, while caspases 8 and 9 released GLUC activity from DEVDG2F but not Actin.

Mentions: In order to identify novel proteins that might induce caspase-mediated cleavage, we performed a functional screen using the Origene Trueclone™ expression vector collection. We co-transfected 96-well plates with single cDNA expression vectors and DEVDG2F and measured luciferase activity in SN and cellular lysates in triplicate plates. To normalize for cellular expression and cell numbers, we determined the ratio of luciferase activity in SN over cellular lysates from the same 96-well plate. Three wells on each plate were transfected with DEVDG2F only to determine the level of background secretion. In Table 2, we summarize genes that showed more than a three-fold increase in GLUC activity released from cells expressing DEVDG2F compared to cells transfected with reporter only. The candidates found include known inducers of apoptosis, such as BAK, FADD, BAD and caspase 8, in partial validation of the approach to identify regulators of caspase activation. In addition, we identified the novel genes for ASPH, PIR121, PERP and TBC1D10A, which induced 14.2-, 12.1-, 10.4- and 5.5-fold increases in GLUC activity in SN from DEVDG2F cells, respectively (Table 2). TBC1D10A is a member of the Tre/Bub2/Cdc16 (TBC) family that exhibits GTPase activating protein (GAP) activity and, thus, is an interesting candidate gene in the context of apoptotic signaling. Since DEVDG2F harbors additional aspartate residues within the Flag peptide sequence that might serve as cleavage target sites, we also generated a construct with three DEVDG-repeats without a Flag tag, Actin-DEVDG3-dNGLUC (DEVDG3). In addition, we generated a variant reporter in which the DEVDG-motif was replaced with a DEVAG motif that is not a substrate for caspases. TBC1D10A was co-transfected with Actin-dNGLUC, DEVDG3 or DEVAG2F and the release of GLUC into SN was measured. Caspase 9 induced a 4.1-fold and TBC1D10A a 4.3-fold increase in extra-cellular GLUC activity compared to GFP, but did not release dNGLUC from Actin-dNGLUC or DEVAG2F (Figure 3a). These results are consistent with the view that the cleavage promoted by caspase 9 and TBC1D10A is specific to the caspase cleavage site introduced in the reporter substrate.


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

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

HTRA3 and 4 release GLUC activity from Actin-dNGLUC. (a) Actin-dNGLUC, DEVDG3 or DEVAG2F were co-transfected with GFP, caspase 9 or TBC1D10A and GLUC activity in SN was assayed after 30 h. TBC1D10A specifically releases dNGLUC from DEVDG3, but not DEVAG2F (RLU, relative light units). (b) HTRA3 and 4 release GLUC activity from Actin-dNGLUC. Caspase 8, caspase 9, HTRA3 and HTRA4 were co-transfected with Actin-dNGLUC and DEVDG2F and SN were analyzed for GLUC activity in SN after 30 h. HTRA3 and 4 release GLUC activity from Actin-dN and DEVDG2F, while caspases 8 and 9 released GLUC activity from DEVDG2F but not Actin.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 3: HTRA3 and 4 release GLUC activity from Actin-dNGLUC. (a) Actin-dNGLUC, DEVDG3 or DEVAG2F were co-transfected with GFP, caspase 9 or TBC1D10A and GLUC activity in SN was assayed after 30 h. TBC1D10A specifically releases dNGLUC from DEVDG3, but not DEVAG2F (RLU, relative light units). (b) HTRA3 and 4 release GLUC activity from Actin-dNGLUC. Caspase 8, caspase 9, HTRA3 and HTRA4 were co-transfected with Actin-dNGLUC and DEVDG2F and SN were analyzed for GLUC activity in SN after 30 h. HTRA3 and 4 release GLUC activity from Actin-dN and DEVDG2F, while caspases 8 and 9 released GLUC activity from DEVDG2F but not Actin.
Mentions: In order to identify novel proteins that might induce caspase-mediated cleavage, we performed a functional screen using the Origene Trueclone™ expression vector collection. We co-transfected 96-well plates with single cDNA expression vectors and DEVDG2F and measured luciferase activity in SN and cellular lysates in triplicate plates. To normalize for cellular expression and cell numbers, we determined the ratio of luciferase activity in SN over cellular lysates from the same 96-well plate. Three wells on each plate were transfected with DEVDG2F only to determine the level of background secretion. In Table 2, we summarize genes that showed more than a three-fold increase in GLUC activity released from cells expressing DEVDG2F compared to cells transfected with reporter only. The candidates found include known inducers of apoptosis, such as BAK, FADD, BAD and caspase 8, in partial validation of the approach to identify regulators of caspase activation. In addition, we identified the novel genes for ASPH, PIR121, PERP and TBC1D10A, which induced 14.2-, 12.1-, 10.4- and 5.5-fold increases in GLUC activity in SN from DEVDG2F cells, respectively (Table 2). TBC1D10A is a member of the Tre/Bub2/Cdc16 (TBC) family that exhibits GTPase activating protein (GAP) activity and, thus, is an interesting candidate gene in the context of apoptotic signaling. Since DEVDG2F harbors additional aspartate residues within the Flag peptide sequence that might serve as cleavage target sites, we also generated a construct with three DEVDG-repeats without a Flag tag, Actin-DEVDG3-dNGLUC (DEVDG3). In addition, we generated a variant reporter in which the DEVDG-motif was replaced with a DEVAG motif that is not a substrate for caspases. TBC1D10A was co-transfected with Actin-dNGLUC, DEVDG3 or DEVAG2F and the release of GLUC into SN was measured. Caspase 9 induced a 4.1-fold and TBC1D10A a 4.3-fold increase in extra-cellular GLUC activity compared to GFP, but did not release dNGLUC from Actin-dNGLUC or DEVAG2F (Figure 3a). These results are consistent with the view that the cleavage promoted by caspase 9 and TBC1D10A is specific to the caspase cleavage site introduced in the reporter substrate.

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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