The temperature activated HtrA protease from pathogen <i>Chlamydia trachomatis</i> acts as both a chaperone and protease at 37 °C

Huston, Wilhelmina M.; Swedberg, Joaquim E.; Harris, Jonathan M.; Walsh, Timothy M.; Mathews, Sarah A.; Timms, Peter

doi:10.1016/j.febslet.2007.06.039

Cited by 48 publications

(63 citation statements)

References 29 publications

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“…In contrast, C. jejuni carries homologs of only HtrA and SurA and thus depends on these two chaperones for protein folding in the periplasm. A few bacterial HtrA homologs have been investigated in vitro, and these experiments have shown that HtrA is a bifunctional protein that, in addition to the chaperone activity, possesses proteolytic activity (24,28,63). In general, HtrA homologs are characterized by the presence of a trypsin-like serine protease domain and one or two PDZ domains that recognize substrates and activate the protease function (25,31).…”

mentioning

confidence: 99%

Different Contributions of HtrA Protease and Chaperone Activities to Campylobacter jejuni Stress Tolerance and Physiology

Bæk

Vegge

Skórko-Glonek

et al. 2011

Appl Environ Microbiol

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The microaerophilic bacterium Campylobacter jejuni is the most common cause of bacterial food-borne infections in the developed world. Tolerance to environmental stress relies on proteases and chaperones in the cell envelope, such as HtrA and SurA. HtrA displays both chaperone and protease activities, but little is known about how each of these activities contributes to stress tolerance in bacteria. In vitro experiments showed temperature-dependent protease and chaperone activities of C. jejuni HtrA. A C. jejuni mutant lacking only the protease activity of HtrA was used to show that the HtrA chaperone activity is sufficient for growth at high temperature or under oxidative stress, whereas the HtrA protease activity is essential only under conditions close to the growth limit for C. jejuni. However, the protease activity was required to prevent induction of the cytoplasmic heat shock response even under optimal growth conditions. Interestingly, the requirement of HtrA at high temperatures was found to depend on the oxygen level, and our data suggest that HtrA may protect oxidatively damaged proteins. Finally, protease activity stimulates HtrA production and oligomer formation, suggesting that a regulatory role depends on the protease activity of HtrA. Studying a microaerophilic organism encoding only two known periplasmic chaperones (HtrA and SurA) revealed an efficient HtrA chaperone activity and proposed multiple roles of the protease activity, increasing our understanding of HtrA in bacterial physiology.

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confidence: 99%

Different Contributions of HtrA Protease and Chaperone Activities to Campylobacter jejuni Stress Tolerance and Physiology

Bæk

Vegge

Skórko-Glonek

et al. 2011

Appl Environ Microbiol

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“…CtHtrA activity was confirmed using ␤ -casein as a substrate [Huston et al, 2007] and confirmed to be purified to homogeneity by checking the preparations on Coomassie-stained SDS-PAGE. Protease assays using recombinant wild-type and mutant CtHtrA were performed as previously outlined in Huston et al [2011].…”

Section: Protease Activity and Specificity Assaysmentioning

confidence: 99%

“…CtHtrA was heterologously expressed and purified as previously described [Huston et al, 2007]. CtHtrA activity was confirmed using ␤ -casein as a substrate [Huston et al, 2007] and confirmed to be purified to homogeneity by checking the preparations on Coomassie-stained SDS-PAGE.…”

Section: Protease Activity and Specificity Assaysmentioning

confidence: 99%

“…Analysis of laboratory models of chlamydial persistence identified high levels of HtrA (high temperature requirement A) [Belland et al, 2003]. C. trachomatis HtrA (CtHtrA) is an extracytoplasmic (likely periplasmic), serine protease with chaperone and proteolytic activity and demonstrates broad sequence specificity [Huston et al, 2007]. Chlamydia is not able to be genetically manipulated and hence in vitro strategies to understand the biochemical function of proteins from this important pathogen provide an alternative investigative means.…”

Section: Introductionmentioning

confidence: 99%

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The Active Site Residue V266 of Chlamydial HtrA Is Critical for Substrate Binding during both in vitro and in vivo Conditions

Gloeckl

Tyndall²,

Stansfield³

et al. 2012

Microb Physiol

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HtrA is a complex, multimeric chaperone and serine protease important for the virulence and survival of many bacteria. Chlamydia trachomatis is an obligate, intracellular bacterial pathogen that is responsible for severe disease pathology. C. trachomatis HtrA (CtHtrA) has been shown to be highly expressed in laboratory models of disease. In this study, molecular modelling of CtHtrA protein active site structure identified putative S1–S3 subsite residues I242, I265, and V266. These residues were altered by site-directed mutagenesis, and these changes were shown to considerably reduce protease activity on known substrates and resulted in a narrower and distinct range of substrates compared to wild type. Bacterial two-hybrid analysis revealed that CtHtrA is able to interact in vivo with a broad range of protein sequences with high affinity. Notably, however, the interaction was significantly altered in 35 out of 69 clones when residue V266 was mutated, indicating that this residue has an important function during substrate binding.

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“…Cells were routinely cultured on Luria Bertani broth (LB) or agar plates (when appropriate) with ampicillin at 100 μg/ml. The wild-type htrA construct was previously described [32]. It was amplified using PCR and inserted into a pET-22b expression plasmid vector containing a C-terminal 6× histidine tag.…”

Section: Molecular Modeling and Refinementmentioning

confidence: 99%

Proteolytic activation of Chlamydia trachomatis HTRA is mediated by PDZ1 domain interactions with protease domain loops L3 and LC and beta strand β5

Marsh

Lott

Tyndall

et al. 2013

Cellular and Molecular Biology Letters

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Abbreviations used: CD -circular dichroism; CtHtrA -Chlamydia trachomatis high temperature requirement A; MCA -7-methoxycoumarin-4-acetic acid; MOMP -major outer membrane protein; MRW -mean residue weight; OmpA -outer membrane protein A; PDZ -PSD-95/Dics-Large/ZO-1; PmpC -polymorphic membrane protein C; pNApara-nitroalinine; SDM -site-directed mutagenesis; SDS-PAGE -sodium dodecyl sulphate polyacrylamide gel electrophoresis Abstract: Chlamydia trachomatis is a bacterial pathogen responsible for one of the most prevalent sexually transmitted infections worldwide. Its unique development cycle has limited our understanding of its pathogenic mechanisms. However, CtHtrA has recently been identified as a potential C. trachomatis virulence factor. CtHtrA is a tightly regulated quality control protein with a monomeric structural unit comprised of a chymotrypsin-like protease domain and two PDZ domains. Activation of proteolytic activity relies on the C-terminus of the substrate allosterically binding to the PDZ1 domain, which triggers subsequent conformational change and oligomerization of the protein into 24-mers enabling proteolysis. This activation is mediated by a cascade of precise structural arrangements, but the specific CtHtrA residues and structural elements required to facilitate activation are unknown. Using in vitro analysis guided by homology modeling, we show that the mutation of residues Arg362 and Arg224, predicted to disrupt the interaction between the CtHtrA PDZ1 Unauthenticated Download Date | 5/13/18 1:07 AM CELLULAR & MOLECULAR BIOLOGY LETTERS 523 domain and loop L3, and between loop L3 and loop LD, respectively, are critical for the activation of proteolytic activity. We also demonstrate that mutation to residues Arg299 and Lys160, predicted to disrupt PDZ1 domain interactions with protease loop LC and strand β5, are also able to influence proteolysis, implying their involvement in the CtHtrA mechanism of activation. This is the first investigation of protease loop LC and strand β5 with respect to their potential interactions with the PDZ1 domain. Given their high level of conservation in bacterial HtrA, these structural elements may be equally significant in the activation mechanism of DegP and other HtrA family members.

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The temperature activated HtrA protease from pathogen Chlamydia trachomatis acts as both a chaperone and protease at 37 °C

Cited by 48 publications

References 29 publications

Different Contributions of HtrA Protease and Chaperone Activities to Campylobacter jejuni Stress Tolerance and Physiology

Different Contributions of HtrA Protease and Chaperone Activities to Campylobacter jejuni Stress Tolerance and Physiology

The Active Site Residue V266 of Chlamydial HtrA Is Critical for Substrate Binding during both in vitro and in vivo Conditions

Proteolytic activation of Chlamydia trachomatis HTRA is mediated by PDZ1 domain interactions with protease domain loops L3 and LC and beta strand β5

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