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A computational module assembled from different protease family motifs identifies PI PLC from Bacillus cereus as a putative prolyl peptidase with a serine protease scaffold.

Rendón-Ramírez A, Shukla M, Oda M, Chakraborty S, Minda R, Dandekar AM, Ásgeirsson B, Goñi FM, Rao BJ - PLoS ONE (2013)

Bottom Line: This was validated by protease assays, mass spectrometry and by inhibition of the native phospholipase activity of PI-PLC by the well-known serine protease inhibitor AEBSF (IC50 = 0.018 mM).Edman degradation analysis linked the specificity of the protease activity to a proline in the amino terminal, suggesting that the PI-PLC is a prolyl peptidase.Thus, we propose a computational method of extending protein families based on the spatial and electrostatic congruence of active site residues.

View Article: PubMed Central - PubMed

Affiliation: Unidad de Biofísica, Consejo Superior de Investigaciones Científicas, Universidad del Pais Vasco/Euskal Herriko Unibertsitatea and Departamento de Bioquímica, Universidad del País Vasco, Bilbao, Spain.

ABSTRACT
Proteolytic enzymes have evolved several mechanisms to cleave peptide bonds. These distinct types have been systematically categorized in the MEROPS database. While a BLAST search on these proteases identifies homologous proteins, sequence alignment methods often fail to identify relationships arising from convergent evolution, exon shuffling, and modular reuse of catalytic units. We have previously established a computational method to detect functions in proteins based on the spatial and electrostatic properties of the catalytic residues (CLASP). CLASP identified a promiscuous serine protease scaffold in alkaline phosphatases (AP) and a scaffold recognizing a β-lactam (imipenem) in a cold-active Vibrio AP. Subsequently, we defined a methodology to quantify promiscuous activities in a wide range of proteins. Here, we assemble a module which encapsulates the multifarious motifs used by protease families listed in the MEROPS database. Since APs and proteases are an integral component of outer membrane vesicles (OMV), we sought to query other OMV proteins, like phospholipase C (PLC), using this search module. Our analysis indicated that phosphoinositide-specific PLC from Bacillus cereus is a serine protease. This was validated by protease assays, mass spectrometry and by inhibition of the native phospholipase activity of PI-PLC by the well-known serine protease inhibitor AEBSF (IC50 = 0.018 mM). Edman degradation analysis linked the specificity of the protease activity to a proline in the amino terminal, suggesting that the PI-PLC is a prolyl peptidase. Thus, we propose a computational method of extending protein families based on the spatial and electrostatic congruence of active site residues.

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Confirming the protease scaffold in PI-PLC by proteolytic assays and inhibition studies.(A) Protease activity of PI-PLC. Substrate protein (UVI31+, lane 2) was incubated with PI- PLC (lane 3) overnight at 37°C, followed by sample analysis with 15% SDS-PAGE. Lane 1, molecular weight marker. (B) Control for UVI31+, with peak at 13.436 kDa. (C) UVI31+ treated with PI-PLC, showing fragmented peaks at 11.4 kDa and (D) another fragment of 2.0 kDa. (E) The inhibition of PI-PLC activity on phosphatidylinositol (PI) by trypsin inhibitor AEBSF. (F) The inhibition of PI-PLC activity on PI by trypsin inhibitor AEBSF in a mixture with phosphatidylcholine (PC), phosphatidylethanolamine (PE), and cholesterol (CH).
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pone-0070923-g002: Confirming the protease scaffold in PI-PLC by proteolytic assays and inhibition studies.(A) Protease activity of PI-PLC. Substrate protein (UVI31+, lane 2) was incubated with PI- PLC (lane 3) overnight at 37°C, followed by sample analysis with 15% SDS-PAGE. Lane 1, molecular weight marker. (B) Control for UVI31+, with peak at 13.436 kDa. (C) UVI31+ treated with PI-PLC, showing fragmented peaks at 11.4 kDa and (D) another fragment of 2.0 kDa. (E) The inhibition of PI-PLC activity on phosphatidylinositol (PI) by trypsin inhibitor AEBSF. (F) The inhibition of PI-PLC activity on PI by trypsin inhibitor AEBSF in a mixture with phosphatidylcholine (PC), phosphatidylethanolamine (PE), and cholesterol (CH).

Mentions: We tested this prediction by performing an in vitro protease assay on commercially available PI-PLC from Bacillus cereus. The protease activity of PI-PLC on the substrate protein UVI31+ [31], [32] was inhibited by the protease inhibitor leupeptin, while other inhibitors like AEBSF were unstable during a long incubation (Fig. 2A). A MALDI TOF analysis showed a clean, 13.4 kDa peak for purified UVI31+ protein (Fig. 2B), which was split into two fragments of 2.0 kDa (Fig. 2C) and 11.4 kDa (Fig. 2D) on incubation with PI-PLC. Edman degradation analysis demonstrated that the protease activity was specific for a proline following the first seven residues of the UVI31+ protein (marked by an asterisk - MAEHQLGP*IAG). This suggested that the PI-PLC is a putative prolyl peptidase. The predicted protease scaffold was tested by assaying inhibition of its phospholipase activity by the trypsin inhibitor AEBSF (IC50 = 0.018 mM). Assays were performed with the substrate in the form of large, unilamellar vesicles. The vesicles consisted of either pure phosphatidylinositol (PI) (Fig. 2E) or an equimolar mixture of PI, phosphatidylcholine (PC), phosphatidylethanolamine (PE), and cholesterol (CH) (Fig. 2F). In both cases, the maximum reaction rates decreased in a dose-dependent way in the presence of AEBSF (Fig. S1).


A computational module assembled from different protease family motifs identifies PI PLC from Bacillus cereus as a putative prolyl peptidase with a serine protease scaffold.

Rendón-Ramírez A, Shukla M, Oda M, Chakraborty S, Minda R, Dandekar AM, Ásgeirsson B, Goñi FM, Rao BJ - PLoS ONE (2013)

Confirming the protease scaffold in PI-PLC by proteolytic assays and inhibition studies.(A) Protease activity of PI-PLC. Substrate protein (UVI31+, lane 2) was incubated with PI- PLC (lane 3) overnight at 37°C, followed by sample analysis with 15% SDS-PAGE. Lane 1, molecular weight marker. (B) Control for UVI31+, with peak at 13.436 kDa. (C) UVI31+ treated with PI-PLC, showing fragmented peaks at 11.4 kDa and (D) another fragment of 2.0 kDa. (E) The inhibition of PI-PLC activity on phosphatidylinositol (PI) by trypsin inhibitor AEBSF. (F) The inhibition of PI-PLC activity on PI by trypsin inhibitor AEBSF in a mixture with phosphatidylcholine (PC), phosphatidylethanolamine (PE), and cholesterol (CH).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3733634&req=5

pone-0070923-g002: Confirming the protease scaffold in PI-PLC by proteolytic assays and inhibition studies.(A) Protease activity of PI-PLC. Substrate protein (UVI31+, lane 2) was incubated with PI- PLC (lane 3) overnight at 37°C, followed by sample analysis with 15% SDS-PAGE. Lane 1, molecular weight marker. (B) Control for UVI31+, with peak at 13.436 kDa. (C) UVI31+ treated with PI-PLC, showing fragmented peaks at 11.4 kDa and (D) another fragment of 2.0 kDa. (E) The inhibition of PI-PLC activity on phosphatidylinositol (PI) by trypsin inhibitor AEBSF. (F) The inhibition of PI-PLC activity on PI by trypsin inhibitor AEBSF in a mixture with phosphatidylcholine (PC), phosphatidylethanolamine (PE), and cholesterol (CH).
Mentions: We tested this prediction by performing an in vitro protease assay on commercially available PI-PLC from Bacillus cereus. The protease activity of PI-PLC on the substrate protein UVI31+ [31], [32] was inhibited by the protease inhibitor leupeptin, while other inhibitors like AEBSF were unstable during a long incubation (Fig. 2A). A MALDI TOF analysis showed a clean, 13.4 kDa peak for purified UVI31+ protein (Fig. 2B), which was split into two fragments of 2.0 kDa (Fig. 2C) and 11.4 kDa (Fig. 2D) on incubation with PI-PLC. Edman degradation analysis demonstrated that the protease activity was specific for a proline following the first seven residues of the UVI31+ protein (marked by an asterisk - MAEHQLGP*IAG). This suggested that the PI-PLC is a putative prolyl peptidase. The predicted protease scaffold was tested by assaying inhibition of its phospholipase activity by the trypsin inhibitor AEBSF (IC50 = 0.018 mM). Assays were performed with the substrate in the form of large, unilamellar vesicles. The vesicles consisted of either pure phosphatidylinositol (PI) (Fig. 2E) or an equimolar mixture of PI, phosphatidylcholine (PC), phosphatidylethanolamine (PE), and cholesterol (CH) (Fig. 2F). In both cases, the maximum reaction rates decreased in a dose-dependent way in the presence of AEBSF (Fig. S1).

Bottom Line: This was validated by protease assays, mass spectrometry and by inhibition of the native phospholipase activity of PI-PLC by the well-known serine protease inhibitor AEBSF (IC50 = 0.018 mM).Edman degradation analysis linked the specificity of the protease activity to a proline in the amino terminal, suggesting that the PI-PLC is a prolyl peptidase.Thus, we propose a computational method of extending protein families based on the spatial and electrostatic congruence of active site residues.

View Article: PubMed Central - PubMed

Affiliation: Unidad de Biofísica, Consejo Superior de Investigaciones Científicas, Universidad del Pais Vasco/Euskal Herriko Unibertsitatea and Departamento de Bioquímica, Universidad del País Vasco, Bilbao, Spain.

ABSTRACT
Proteolytic enzymes have evolved several mechanisms to cleave peptide bonds. These distinct types have been systematically categorized in the MEROPS database. While a BLAST search on these proteases identifies homologous proteins, sequence alignment methods often fail to identify relationships arising from convergent evolution, exon shuffling, and modular reuse of catalytic units. We have previously established a computational method to detect functions in proteins based on the spatial and electrostatic properties of the catalytic residues (CLASP). CLASP identified a promiscuous serine protease scaffold in alkaline phosphatases (AP) and a scaffold recognizing a β-lactam (imipenem) in a cold-active Vibrio AP. Subsequently, we defined a methodology to quantify promiscuous activities in a wide range of proteins. Here, we assemble a module which encapsulates the multifarious motifs used by protease families listed in the MEROPS database. Since APs and proteases are an integral component of outer membrane vesicles (OMV), we sought to query other OMV proteins, like phospholipase C (PLC), using this search module. Our analysis indicated that phosphoinositide-specific PLC from Bacillus cereus is a serine protease. This was validated by protease assays, mass spectrometry and by inhibition of the native phospholipase activity of PI-PLC by the well-known serine protease inhibitor AEBSF (IC50 = 0.018 mM). Edman degradation analysis linked the specificity of the protease activity to a proline in the amino terminal, suggesting that the PI-PLC is a prolyl peptidase. Thus, we propose a computational method of extending protein families based on the spatial and electrostatic congruence of active site residues.

Show MeSH
Related in: MedlinePlus