title: Release of an HtrA-Like Protease from the Cell Surface of ThermophilicBrevibacillussp. WF146 via Substrate-Induced Autoprocessing of the N-terminal Membrane Anchor.
abstract: High-temperature requirement A (HtrA)-like proteases participate in protein quality control in prokaryotes and eukaryotes by degrading damaged proteins; however, little is known about HtrAs produced by thermophiles. HtrAw is an HtrA-like protease of thermophilicBrevibacillussp. WF146. The intact form of HtrAw (iHtrAw) consisting of a transmembrane segment-containing N-terminal domain, a trypsin-like protease domain, and a C-terminal PDZ domain was produced inEscherichia coli. Purified iHtrAw itself is unable to cleave the N-terminal domain, but requires protein substrates to autoprocess the N-terminal domain intermolecularly, yielding a short form (sHtrAw). Mutation at the substrate-binding site in the PDZ domain affects the conversion of iHtrAw to sHtrAw. Deletion analysis revealed that the N-terminal domain is not necessary for enzyme folding, activity, and thermostability. Compared with other known HtrAs, HtrAw contains an additional Ca2+-binding Dx[DN]xDG motif important for enzyme stability and/or activity. When produced in anhtrA/htrBdouble deletion mutant ofBacillus subtilis, iHtrAw localized predominantly to the cell pellet, and the amount of sHtrAw in the culture supernatant increased at elevated temperatures. Moreover, HtrAw increased the heat resistance of theB. subtilismutant. In strain WF146, HtrAw exists in both a cell-associated intact form and a cell-free short form; an increase in growth temperature enhanced HtrAw production and the amount of cell-free short form. Release of the short form of HtrAw from the membrane may have the advantage of allowing the enzyme to freely access and degrade damaged proteins surrounding the bacterium living at high temperatures.
authors.affiliation: State Key Laboratory of Virology, College of Life Sciences, Wuhan University Wuhan, China.
authors.forename: Fengtao
authors.initials: F
authors.lastname: Zhu
authors.affiliation: State Key Laboratory of Virology, College of Life Sciences, Wuhan University Wuhan, China.
authors.forename: Xing
authors.initials: X
authors.lastname: Yang
authors.affiliation: State Key Laboratory of Virology, College of Life Sciences, Wuhan University Wuhan, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Wu
authors.affiliation: State Key Laboratory of Virology, College of Life Sciences, Wuhan University Wuhan, China.
authors.forename: Yasi
authors.initials: Y
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Virology, College of Life Sciences, Wuhan UniversityWuhan, China; Hubei Provincial Cooperative Innovation Center of Industrial FermentationWuhan, China.
authors.forename: Xiao-Feng
authors.initials: XF
authors.lastname: Tang
authors.affiliation: State Key Laboratory of Virology, College of Life Sciences, Wuhan UniversityWuhan, China; Hubei Provincial Cooperative Innovation Center of Industrial FermentationWuhan, China.
authors.forename: Bing
authors.initials: B
authors.lastname: Tang
keywords: HtrA protease; N-terminal domain; PDZ domain; autoprocessing; calcium-binding protein; heat resistance; thermophile