Keiler, K.C. et al. C-terminal specific protein degradation: activity and substrate specificity of the Tsp protease. Protein Sci. 4, 1507-1515

Department of Biology, Massachusetts Institute of Technology, Cambridge 02139, USA.
Protein Science (Impact Factor: 2.85). 08/1995; 4(8):1507-15. DOI: 10.1002/pro.5560040808
Source: PubMed


The activity of Tsp, a periplasmic endoprotease of Escherichia coli, has been characterized by assaying the cleavage of protein and peptide substrates, determining the cleavage sites in several substrates, and investigating the kinetics of the cleavage reaction. Tsp efficiently cleaves substrates that have apolar residues and a free alpha-carboxylate at the C-terminus. Tsp cleaves its substrates at a discrete number of sites but with rather broad primary sequence specificity. In addition to preferences for residues at the C-terminus and cleavage sites, Tsp displays a preference for substrates that are not stably folded: unstable variants of Arc repressor are better substrates than a hyperstable mutant, and a peptide with little stable structure is cleaved more efficiently than a protein substrate. These data are consistent with a model in which Tsp cleavage of a protein substrate involves binding to the C-terminal tail of the substrate, transient denaturation of the substrate, and then recognition and hydrolysis of specific peptide bonds.

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Available from: Ioannis A. Papayannopoulos, Apr 08, 2014
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    • "We conclude that changing the residues at these two positions alters the site specificity of Tsp, or reveals another Tsp cleavage site that is usually far less favored by Tsp in cleaving wild-type TcpP. This latter possibility would be consistent with the ability of E. coli Tsp to cleave its substrates at more than one site (Keiler et al., 1995). Therefore, these residues of TcpP appear to be important for substrate recognition by Tsp to initiate degradation. "
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    ABSTRACT: Vibrio cholerae uses a multiprotein transcriptional regulatory cascade to control expression of virulence factors cholera toxin and toxin-coregulated pilus. Two proteins in this cascade are ToxR and TcpP - unusual membrane-localized transcription factors with relatively undefined periplasmic domains and transcription activator cytoplasmic domains. TcpP and ToxR function with each other and two other membrane-localized proteins, TcpH and ToxS, to activate transcription of toxT, encoding the direct activator of toxin and pilus genes. Under some conditions, TcpP is degraded in a two-step proteolytic pathway known as regulated intramembrane proteolysis (RIP), thereby inactivating the cascade. The second step in this proteolytic pathway involves the zinc metalloprotease YaeL; V. cholerae cells lacking YaeL accumulate a truncated yet active form of TcpP termed TcpP*. We hypothesized that a protease acting prior to YaeL degrades TcpP to TcpP*, which is the substrate of YaeL. In this study, we demonstrate that a C-terminal protease called Tsp degrades TcpP to form TcpP*, which is then acted upon by YaeL. We present evidence that TcpH and Tsp serve to protect full-length TcpP from spurious proteolysis by YaeL. Cleavage by Tsp occurs in the periplasmic domain of TcpP, and requires residues TcpPA172 and TcpPI174 for wild-type activity. This article is protected by copyright. All rights reserved.
    Molecular Microbiology 05/2015; 97(5). DOI:10.1111/mmi.13069 · 4.42 Impact Factor
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    • "The theoretical localizations of the other four proteins (PXO_00005, PXO_01202, PXO_ 01316, and PXO_03424) were uncertain because of ambiguous prediction results. In E. coli, however, most of the proteins with similar domain compositions, including Prc (TSP) (Keiler et al. 1995), DegP (Bass et al. 1996), and DegQ (Waller and Sauer 1996), are periplasmic proteins. To experimentally confirm the subcellular localization of Prc, we again used a recombinant strain (Prc-His 6 ) in which Prc was tagged with a C-terminal His 6 epitope for determining its localization. "
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    ABSTRACT: PDZ domain-containing proteases, also known as HtrA-family proteases, play important roles in bacterial cells by modulating disease pathogenesis and cell envelope stress responses. These proteases have diverse functions through proteolysis- and non-proteolysis-dependent modes. Here, we report that the genome of the causative agent of rice bacterial blight, Xanthomonas oryzae pv. oryzae, encodes seven PDZ domain-containing proteins. Systematic inactivation of their encoding genes identified that PXO_01122 and PXO_04290 (prc) are involved in virulence. prc encodes a putative HtrA family protease that localizes in the bacterial periplasm. Mutation of prc also resulted in susceptibility to multiple environmental stresses, including H2O2, sodium dodecylsulfate, and osmolarity stresses. Comparative subproteomic analyses showed that the amounts of 34 periplasmic proteins were lower in the prc mutant than in wild-type. These proteins were associated with proteolysis, biosynthesis of macromolecules, carbohydrate or energy metabolism, signal transduction, and protein translocation or folding. We provide in vivo and in vitro evidences to demonstrate that Prc stabilizes and directly binds to one of these proteins, DppP, a dipeptidyl peptidase contributing to the full virulence. Taken together, our results suggest that Prc contributes to bacterial virulence by acting as a periplasmic modulator of cell envelope stress responses.
    Molecular Plant-Microbe Interactions 11/2013; 27(2). DOI:10.1094/MPMI-08-13-0234-R · 3.94 Impact Factor
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    • "The transcription of DsbC is activated by the RpoE regulatory pathway in response to various cell envelope stresses including the accumulation of misfolded periplasmic proteins [44]. Tsp is a periplasmic serine protease that digests unstably folded proteins at several sites with broad primary sequence specificity [45,46]; it also recognizes and digests misfolded cytoplasmic and periplasmic proteins tagged by the SsrA protein quality control system [47]. FkpB and ppiC are both peptidyl-prolyl cis/trans-isomerases catalyzing the cis-trans-isomerization of proline-containing peptide bonds [48,49]. "
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    ABSTRACT: The overexpression of scFv antibody fragments in the periplasmic space of Escherichia coli frequently results in extensive protein misfolding and loss of cell viability. Although protein folding factors such as Skp and FkpA are often exploited to restore the solubility and functionality of recombinant protein products, their exact impact on cellular metabolism during periplasmic antibody fragment expression is not clearly understood. In this study, we expressed the scFvD1.3 antibody fragment in E. coli BL21 and evaluated the overall physiological and global gene expression changes upon Skp or FkpA co-expression. The periplasmic expression of scFvD1.3 led to a rapid accumulation of insoluble scFvD1.3 proteins and a decrease in cell viability. The co-expression of Skp and FkpA improved scFvD1.3 solubility and cell viability in a dosage-dependent manner. Through mutagenesis experiments, it was found that only the chaperone activity of FkpA, not the peptidyl-prolyl isomerase (PPIase) activity, is required for the improvement in cell viability. Global gene expression analysis of the scFvD1.3 cells over the chaperone-expressing cells showed a clear up-regulation of genes involved in heat-shock and misfolded protein stress responses. These included genes of the major HSP70 DnaK chaperone family and key proteases belonging to the Clp and Lon protease systems. Other metabolic gene expression trends include: (1) the differential regulation of several energy metabolic genes, (2) down-regulation of the central metabolic TCA cycle and transport genes, and (3) up-regulation of ribosomal genes. The simultaneous activation of multiple stress related and other metabolic genes may constitute the stress response to protein misfolding in the scFvD1.3 cells. These gene expression information could prove to be valuable for the selection and construction of reporter contructs to monitor the misfolded protein stress response during antibody fragment production.
    Microbial Cell Factories 04/2010; 9(1):22. DOI:10.1186/1475-2859-9-22 · 4.22 Impact Factor
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