Escherichia coli Requires the Protease Activity of FtsH for Growth

2000 ◽  
Vol 380 (1) ◽  
pp. 103-107 ◽  
Author(s):  
Maithri M.K. Jayasekera ◽  
Susan K. Foltin ◽  
Eric R. Olson ◽  
Tod P. Holler
Keyword(s):  
Escherichia Coli ◽  
Protease Activity

scholarly journals Role of the PDZ Domains in Escherichia coli DegP Protein

2007 ◽  
Vol 189 (8) ◽  
pp. 3176-3186 ◽  
Author(s):  
Jack Iwanczyk ◽  
Daniela Damjanovic ◽  
Joel Kooistra ◽  
Vivian Leong ◽  
Ahmad Jomaa ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protease Activity ◽  
Pdz Domain ◽  
Structural Features ◽  
Periplasmic Protein ◽  
Pdz Domains ◽  
Protease Domain ◽  
Interaction Domains ◽  
And Function

ABSTRACT PDZ domains are modular protein interaction domains that are present in metazoans and bacteria. These domains possess unique structural features that allow them to interact with the C-terminal residues of their ligands. The Escherichia coli essential periplasmic protein DegP contains two PDZ domains attached to the C-terminal end of the protease domain. In this study we examined the role of each PDZ domain in the protease and chaperone activities of this protein. Specifically, DegP mutants with either one or both PDZ domains deleted were generated and tested to determine their protease and chaperone activities, as well as their abilities to sequester unfolded substrates. We found that the PDZ domains in DegP have different roles; the PDZ1 domain is essential for protease activity and is responsible for recognizing and sequestering unfolded substrates through C-terminal tags, whereas the PDZ2 domain is mostly involved in maintaining the hexameric cage of DegP. Interestingly, neither of the PDZ domains was required for the chaperone activity of DegP. In addition, we found that the loops connecting the protease domain to PDZ1 and connecting PDZ1 to PDZ2 are also essential for the protease activity of the hexameric DegP protein. New insights into the roles of the PDZ domains in the structure and function of DegP are provided. These results imply that DegP recognizes substrate molecules targeted for degradation and substrate molecules targeted for refolding in different manners and suggest that the substrate recognition mechanisms may play a role in the protease-chaperone switch, dictating whether the substrate is degraded or refolded.

Development and application of a fast, reproducible assay to measure HCV NS3 protease activity using Escherichia coli lysate

2015 ◽  
Vol 225 ◽  
pp. 76-86 ◽  
Author(s):  
Bin Han ◽  
Hadas Dvory-Sobol ◽  
Andrew Greenstein ◽  
Joseph F. McCarville ◽  
Magdeleine Hung ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protease Activity ◽  
Ns3 Protease

scholarly journals Cytoskeletal Effects Induced by Pet, the Serine Protease Enterotoxin of Enteroaggregative Escherichia coli

1999 ◽  
Vol 67 (5) ◽  
pp. 2184-2192 ◽  
Author(s):  
Fernando Navarro-García ◽  
Cynthia Sears ◽  
Carlos Eslava ◽  
Alejandro Cravioto ◽  
James P. Nataro
Keyword(s):  
Escherichia Coli ◽  
Serine Protease ◽  
Protease Activity ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Short Circuit Current ◽  
T84 Cells ◽  
Ussing Chambers ◽  
E Coli ◽  
Cytopathic Effects

ABSTRACT We have previously described enteroaggregative Escherichia coli (EAEC) strains that induce cytotoxic effects on T84 cells, ligated rat ileal loops, and human intestine in culture. Such strains secrete a 104-kDa protein termed Pet (for plasmid-encoded toxin). We have also shown previously that the Pet toxin induces rises in short-circuit current and decreases the electrical resistance in rat jejunum mounted in an Ussing chamber. The nucleotide sequence of thepet gene revealed that Pet is a member of the autotransporter class of secreted proteins. Here we show that a concentrated supernatant of E. coli HB101 harboring the minimal pet clone pCEFN1 induces temperature-, time- and dose-dependent cytopathic effects on HEp-2 cells and HT29 C1 cells in culture. The effects were characterized by release of the cellular focal contacts from the glass substratum, followed by complete rounding of the cells and detachment from the glass. Staining of the Pet-treated cells with Live/Dead viability stain revealed that >90% of rounded cells were viable. Pet-intoxicated HEp-2 and HT29 cells stained with fluorescein-labeled phalloidin revealed contraction of the cytoskeleton and loss of actin stress fibers. However, the effects of Pet were not inhibited by cytoskeleton-altering drugs, including colchicine, taxol, cytochalasin D, and phallicidin. The Pet protein induced proteolysis in zymogram gels, and preincubation with the serine protease inhibitor phenylmethylsulfonyl fluoride resulted in complete abrogation of Pet cytopathic effects. We introduced a mutation in a predicted catalytic serine residue and found that the mutant (Pet S260I) was deficient in protease activity and did not produce cytopathic effects, cytoskeletal damage, or enterotoxic effects in Ussing chambers. These data suggest that Pet is a cytoskeleton-altering toxin and that its protease activity is involved in each of the observed phenotypes.

scholarly journals Roles of DegP in Prevention of Protein Misfolding in the Periplasm upon Overexpression of Penicillin Acylase in Escherichia coli

2003 ◽  
Vol 185 (10) ◽  
pp. 3020-3030 ◽  
Author(s):  
Kao-Lu Pan ◽  
Hsu-Chou Hsiao ◽  
Chiao-Ling Weng ◽  
Ming-Sheng Wu ◽  
C. Perry Chou
Keyword(s):  
Escherichia Coli ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Protease Activity ◽  
Protein Misfolding ◽  
Inclusion Bodies ◽  
Penicillin Acylase ◽  
Chaperone Activity ◽  
Culture Performance

ABSTRACT Enhancement of the production of soluble recombinant penicillin acylase in Escherichia coli via coexpression of a periplasmic protease/chaperone, DegP, was demonstrated. Coexpression of DegP resulted in a shift of in vivo penicillin acylase (PAC) synthesis flux from the nonproductive pathway to the productive one when pac was overexpressed. The number of inclusion bodies, which consist primarily of protein aggregates of PAC precursors in the periplasm, was highly reduced, and the specific PAC activity was highly increased. DegP was a heat shock protein induced in response to pac overexpression, suggesting that the protein could possibly suppress the physiological toxicity caused by pac overexpression. Coexpression of DegPS210A, a DegP mutant without protease activity but retaining chaperone activity, could not suppress the physiological toxicity, suggesting that DegP protease activity was primarily responsible for the suppression, possibly by degradation of abnormal proteins when pac was overexpressed. However, a shortage of periplasmic protease activity was not the only reason for the deterioration in culture performance upon pac overexpression because coexpression of a DegP-homologous periplasmic protease, DegQ or DegS, could not suppress the physiological toxicity. The chaperone activity of DegP is proposed to be another possible factor contributing to the suppression.

scholarly journals Ty1 Defect in Proteolysis at High Temperature

2002 ◽  
Vol 76 (9) ◽  
pp. 4233-4240 ◽  
Author(s):  
Joseph F. Lawler ◽  
Daniel P. Haeusser ◽  
Angie Dull ◽  
Jef D. Boeke ◽  
Jill B. Keeney
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
High Temperature ◽  
Enzymatic Activity ◽  
Reverse Transcriptase ◽  
Protease Activity ◽  
Proteolytic Processing ◽  
Temperature Sensitive ◽  
Transposition Activity ◽  
Concomitant Reduction

ABSTRACT Retrotransposition of the Ty1 element of Saccharomyces cerevisiae is temperature sensitive. Transposition activity of Ty1 is abolished at temperatures above 34°C. In this report, we show that the major block to transposition at high temperature is the inhibition of processing of the Gag-Pol-p199 polyprotein and the concomitant reduction of reverse transcriptase (RT) activity. Expression of a Ty1 protease construct in Escherichia coli shows that protease enzymatic activity is inherently temperature sensitive. In yeast, Gag processing is only partially inhibited at high temperature, while cleavage of the Gag-Pol polyprotein is completely inhibited. Sites of proteolytic processing are differentially susceptible to cleavage during growth at high temperature. Overall levels of the Gag-Pol polyprotein are reduced at high temperature, although the efficiency of the requisite +1 frameshifting event appears to be increased. RT activity is inherently relatively temperature resistant, yet no cDNA is made at high temperature and the amount of RT activity is greatly reduced in virus-like particles formed at high temperature. Taken together, these results suggest that alterations in Ty1 proteins that occur at high temperature affect both protease activity and RT activity, such that Ty1 transposition is abolished.

scholarly journals Protease Activity, Secretion, Cell Entry, Cytotoxicity, and Cellular Targets of Secreted Autotransporter Toxin of Uropathogenic Escherichia coli

2006 ◽  
Vol 74 (11) ◽  
pp. 6124-6134 ◽  
Author(s):  
Nathalie M. Maroncle ◽  
Kelsey E. Sivick ◽  
Rebecca Brady ◽  
Faye-Ellen Stokes ◽  
Harry L. T. Mobley
Keyword(s):  
Escherichia Coli ◽  
Serine Protease ◽  
Active Site ◽  
Protease Activity ◽  
Host Cells ◽  
Single Mutation ◽  
Wild Type ◽  
Passenger Domain ◽  
Hek 293

ABSTRACT The secreted autotransporter toxin (Sat), found predominantly in uropathogenic Escherichia coli, is a member of the SPATE (serine protease autotransporters of Enterobacteriaceae) family and, as such, has serine protease activity and causes cytopathic effects on various cell types. To assess the contribution of the serine protease active site to the mechanism of action of Sat, mutations were made in the first (S256I), in the second (S258A), or in both (S256I/S258A) serine residues within the active site motif. Mutations in the first or both serines reduced protease activity to background levels (P < 0.001); a single mutation in the second serine reduced activity by 60% compared to wild type (P < 0.001). After reversion of the S256I mutation to wild type (I256S), we confirmed S256 as the catalytically active serine. None of these mutations affected secretion of the mature passenger domain or release into the supernatant. The S256I mutation, however, abrogated the cytotoxicity of Sat on human bladder (UM-UC-3) and kidney (HEK 293) epithelial cells, characterized by rounding and elongation, respectively, and a high level of cell detachment. Moreover, S256 is essential for Sat to mediate cytoskeletal contraction and actin loss in host cells as well as to degrade specific membrane/cytoskeletal (fodrin and leukocyte function-associated molecule 1) and nuclear [microtubule-associated proteins, LIM domain-only protein 7, Rap GTPase-activating protein, poly(ADP-ribose) polymerase] proteins in vitro. Lastly, Sat was internalized by host cells and localized to the cytoskeletal fraction where membrane/cytoskeletal target proteins reside.

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