Regulation of ATP-independent proteolysis of a heat-shock protease, DegP
소속 :
연사 : Seokhee Kim (Massachusetts Institute of Technology)
일시 : 2012-06-05 17:00 ~
장소 : 503동 104호
일 시 : 2012년 6월 5일, 5:00 PM
장 소 : 503동 104호
-Abstract-
Protein quality control in the periplasm of Gram-negative bacteria occurs in a compartment devoid of ATP and therefore relies on mechanisms of activity regulation different from those used in the cytoplasm or in eukaryotic organelles. DegP, a member of the highly conserved HtrA family, degrades misfolded proteins in the periplasm of Escherichia coli, is essential for high-temperature bacterial survival, and has been reported to have chaperone activity. Several biochemical and structural studies show that substrate binding triggers assembly of inactive DegP hexamers to large proteolytically active cages typically containing four or eight trimers, suggesting that these conformational changes are generally involved in activity regulation of DegP protease.
To understand the substrate interactions in detail, we identified a model substrate peptide and developed the two sensitive assays. We found that the covalent linkage of two distinct motifs on a substrate leads to the tighter binding, and thus, sensitive activation and cage assembly of DegP protease. The proteolysis of the cleavage site breaks the linkage, and therefore, results in the disassembly of active cages. This ensures that DegP is proteolytically active only when there are sufficient substrates, suggesting a substrate-dependent mechanism of activity
regulation.
In contrast to the previous suggestion that cage assembly is the mechanism of proteolytic activation, we show that they can be uncoupled. DegP variants that cannot form cages retain robust proteolytic activity, and the catalytically dead DegP mutant can still form cages. These results suggest that they are independent processes that are based on different molecular interactions, and temporally coupled only by the substrate-dependence. Although cage assembly is not required for proteolytic activation, we still observed that cage assembly has
protective effects on cells against excessive proteolysis. Therefore, we propose
that cage assembly provides a new way of activity regulation that is independently evolved for proteolysis in protein quality control.
장 소 : 503동 104호
-Abstract-
Protein quality control in the periplasm of Gram-negative bacteria occurs in a compartment devoid of ATP and therefore relies on mechanisms of activity regulation different from those used in the cytoplasm or in eukaryotic organelles. DegP, a member of the highly conserved HtrA family, degrades misfolded proteins in the periplasm of Escherichia coli, is essential for high-temperature bacterial survival, and has been reported to have chaperone activity. Several biochemical and structural studies show that substrate binding triggers assembly of inactive DegP hexamers to large proteolytically active cages typically containing four or eight trimers, suggesting that these conformational changes are generally involved in activity regulation of DegP protease.
To understand the substrate interactions in detail, we identified a model substrate peptide and developed the two sensitive assays. We found that the covalent linkage of two distinct motifs on a substrate leads to the tighter binding, and thus, sensitive activation and cage assembly of DegP protease. The proteolysis of the cleavage site breaks the linkage, and therefore, results in the disassembly of active cages. This ensures that DegP is proteolytically active only when there are sufficient substrates, suggesting a substrate-dependent mechanism of activity
regulation.
In contrast to the previous suggestion that cage assembly is the mechanism of proteolytic activation, we show that they can be uncoupled. DegP variants that cannot form cages retain robust proteolytic activity, and the catalytically dead DegP mutant can still form cages. These results suggest that they are independent processes that are based on different molecular interactions, and temporally coupled only by the substrate-dependence. Although cage assembly is not required for proteolytic activation, we still observed that cage assembly has
protective effects on cells against excessive proteolysis. Therefore, we propose
that cage assembly provides a new way of activity regulation that is independently evolved for proteolysis in protein quality control.
