Release of an HtrA-Like Protease from the Cell Surface of Thermophilic Brevibacillus sp. WF146 via Substrate-Induced Autoprocessing of the N-terminal Membrane Anchor

Fengtao Zhu,Yasi Wang,Xiao-Feng Tang,Xing Yang,Bing Tang,Yan Wu

doi:10.3389/fmicb.2017.00481

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 thermophilic Brevibacillus sp. 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 in Escherichia 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 an htrA/htrB double deletion mutant of Bacillus 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 the B. subtilis mutant. 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.

Highlights

The serine protease high-temperature requirement A (HtrA) plays a pivotal role in protein quality control in prokaryotes and eukaryotes
The 36-kDa protein was detected with the anti-His-tag antibody, and was primarily located in the periplasmic fraction (Figure 1B), indicating that it was a short form of intact form of HtrAw (iHtrAw)
An active-site variant (S249A) of HtrAw was primarily localized in the insoluble cellular fraction as the intact form (Figure 1A), suggesting that the active site is involved in the conversion of iHtrAw to sHtrAw

Summary

Introduction

The serine protease high-temperature requirement A (HtrA) plays a pivotal role in protein quality control in prokaryotes and eukaryotes. Characterization of a Thermostable HtrA Protein are synthesized as a precursor containing an N-terminal signal peptide, a trypsin-like protease domain, and two C-terminal PDZ domains (Clausen et al, 2002; Hansen and Hilgenfeld, 2013). Under harsh conditions such as elevated temperatures, the inner membrane-anchored DegS is activated via binding of misfolded/unfolded outer membrane proteins (OMPs) in the periplasm, thereby hydrolyzing the anti-σ factor RseA to induce σE-dependent transcription of stress genes including degP (Danese and Silhavy, 1997; Clausen et al, 2002; Walsh et al, 2003). While DegS (lacking its N-terminal TMS) forms stable trimers, DegP and DegQ are able to adopt multiple oligomeric states (e.g., trimer, hexamer, 12-mer, and 24-mer), which determine the active state of the enzyme (Wilken et al, 2004; Krojer et al, 2008b; Jiang et al, 2008; Bai et al, 2011; Sawa et al, 2011)

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Conclusion