Human single‐chain urokinase is activated by the omptins <scp>PgtE</scp> of <i><scp>S</scp>almonella enterica</i> and <scp>P</scp>la of <i><scp>Y</scp>ersinia pestis</i> despite mutations of active site residues

Järvinen, Hanna; Laakkonen, Liisa; Haiko, Johanna; Johansson, Tiira; Juuti, Katri; Suomalainen, Maarit; Buchrieser, Carmen; Kalkkinen, Nisse; Korhonen, Timo

doi:10.1111/mmi.12293

Cited by 17 publications

(18 citation statements)

References 52 publications

(148 reference statements)

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“…Second, the activity of CroP is not inhibited by common protease inhibitors, including pepstatin A that is a potent inhibitor of aspartic proteases (53). Altogether, these lines of evidence support the reclassification of omptins apart from aspartic proteases, as proposed by others (12,18).…”

Section: Discussionsupporting

confidence: 60%

“…The high-resolution crystal structure of Y. pestis Pla revealed the presence of a water molecule that is activated by the Asp 210 -His 212 dyad and acts as a nucleophile to attack the substrate, while the Asp 83 -Asp 85 dyad is proposed to participate in the stabilization of the catalytic intermediate (10,12,17). Together, these studies showed that omptins combine features of both serine and aspartate proteases and therefore constitute a unique family of proteases (12,18). Previous studies on omptin inhibition reported that Zn 2ϩ , Cu 2ϩ , and benzamidine are able to inhibit OmpT activity (19)(20)(21).…”

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confidence: 85%

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Inhibition of Outer Membrane Proteases of the Omptin Family by Aprotinin

et al. 2015

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Bacterial proteases are important virulence factors that inactivate host defense proteins and contribute to tissue destruction and bacterial dissemination. Outer membrane proteases of the omptin family, exemplified by Escherichia coli OmpT, are found in some Gram-negative bacteria. Omptins cleave a variety of substrates at the host-pathogen interface, including plasminogen and antimicrobial peptides. Multiple omptin substrates relevant to infection have been identified; nonetheless, an effective omptin inhibitor remains to be found. Here, we purified native CroP, the OmpT ortholog in the murine pathogen Citrobacter rodentium. Purified CroP was found to readily cleave both a synthetic fluorescence resonance energy transfer substrate and the murine cathelicidin-related antimicrobial peptide. In contrast, CroP was found to poorly activate plasminogen into active plasmin. Although classical protease inhibitors were ineffective against CroP activity, we found that the serine protease inhibitor aprotinin displays inhibitory potency in the micromolar range. Aprotinin was shown to act as a competitive inhibitor of CroP activity and to interfere with the cleavage of the murine cathelicidin-related antimicrobial peptide. Importantly, aprotinin was able to inhibit not only CroP but also Yersinia pestis Pla and, to a lesser extent, E. coli OmpT. We propose a structural model of the aprotinin-omptin complex in which Lys 15 of aprotinin forms salt bridges with conserved negatively charged residues of the omptin active site.

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Section: Discussionsupporting

confidence: 60%

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confidence: 85%

Inhibition of Outer Membrane Proteases of the Omptin Family by Aprotinin

et al. 2015

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“…This preferential cleavage of the R 7 -K 8 motif was not observed for EHEC OmpT, likely because cleavage of LL-37 by the EHEC ⌬ompT(pEHompT) strain was much faster and fully cleaved LL-37 fragments were obtained within 5 min (4). Though CRAMP also contains two dibasic motifs (R 4 -K 5 and K 15 -K 16 ), we found that most of the cleavage occurred at the K 15 -K 16 motif (Fig. 4).…”

Section: Discussionmentioning

confidence: 89%

“…Omptins are ␤-barrel proteases embedded in the OM and activated by lateral interaction with lipopolysaccharide (LPS) (12)(13)(14)(15). Omptins possess a variety of attributes reminiscent of both serine and aspartic proteases that distinguish them into their own protease class (14,16,17). The omptin catalytic residues consist of two dyads nested within the extracellular active site groove.…”

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confidence: 99%

Antimicrobial Peptide Conformation as a Structural Determinant of Omptin Protease Specificity

et al. 2015

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Bacterial proteases contribute to virulence by cleaving host or bacterial proteins to promote survival and dissemination. Omptins are a family of proteases embedded in the outer membrane of Gram-negative bacteria that cleave various substrates, including host antimicrobial peptides, with a preference for cleaving at dibasic motifs. OmpT, the enterohemorrhagic Escherichia coli (EHEC) omptin, cleaves and inactivates the human cathelicidin LL-37. Similarly, the omptin CroP, found in the murine pathogen Citrobacter rodentium, which is used as a surrogate model to study human-restricted EHEC, cleaves the murine cathelicidin-related antimicrobial peptide (CRAMP). Here, we compared the abilities of OmpT and CroP to cleave LL-37 and CRAMP. EHEC OmpT degraded LL-37 and CRAMP at similar rates. In contrast, C. rodentium CroP cleaved CRAMP more rapidly than LL-37. The different cleavage rates of LL-37 and CRAMP were independent of the bacterial background and substrate sequence specificity, as OmpT and CroP have the same preference for cleaving at dibasic sites. Importantly, LL-37 was ␣-helical and CRAMP was unstructured under our experimental conditions. By altering the ␣-helicity of LL-37 and CRAMP, we found that decreasing LL-37 ␣-helicity increased its rate of cleavage by CroP. Conversely, increasing CRAMP ␣-helicity decreased its cleavage rate. This structural basis for CroP substrate specificity highlights differences between the closely related omptins of C. rodentium and E. coli. In agreement with previous studies, this difference in CroP and OmpT substrate specificity suggests that omptins evolved in response to the substrates present in their host microenvironments. IMPORTANCEOmptins are recognized as key virulence factors for various Gram-negative pathogens. Their localization to the outer membrane, their active site facing the extracellular environment, and their unique catalytic mechanism make them attractive targets for novel therapeutic strategies. Gaining insights into similarities and variations between the different omptin active sites and subsequent substrate specificities will be critical to develop inhibitors that can target multiple omptins. Here, we describe subtle differences between the substrate specificities of two closely related omptins, CroP and OmpT. This is the first reported example of substrate conformation acting as a structural determinant for omptin activity between OmpT-like proteases. Bacterial proteases are key virulence factors involved in hostpathogen interactions. Omptins are outer membrane (OM) proteases commonly found in Gram-negative pathogens. They play important roles at the host-pathogen interface by processing or degrading a variety of host and bacterial proteins (1-10). Many omptins are found among pathogens of the Enterobacteriaceae family (11). The prototypical omptins are Escherichia coli OmpT and Yersinia pestis Pla, which inactivate antimicrobial peptides (AMPs) and activate plasminogen into plasmin, respectively (4, 6). Omptins are ␤-barrel proteases embedded ...

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“…This consideration stems from their proposed cleavage mechanism, resistance to classical protease inhibitors, neutral pH-optimum, and dependency on bound lipopolysaccharide (LPS). Very recently, we have recorded data that seems to question the well-accepted definition of omptin active site: the Pla mutants D86A, D206A, H208V are inactive in plasminogen activation, as seen before (Kukkonen et al, 2001), but are able to cleave and activate precursor of the human plasminogen activator uPA (Järvinen et al, 2013). Clearly, details of the reaction mechanism of omptins still remain unknown and need further clarification.…”

Section: Structure-function Relationships Of Plamentioning

confidence: 94%

Fibrinolytic and coagulative activities of Yersinia pestis

Korhonen¹,

Haiko²,

Laakkonen³

et al. 2013

Front. Cell. Infect. Microbiol.

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The outer membrane protease Pla belongs to the omptin protease family spread by horizontal gene transfer into Gram-negative bacteria that infect animals or plants. Pla has adapted to support the life style of the plague bacterium Yersinia pestis. Pla has a β-barrel fold with 10 membrane-spanning β strands and five surface loops, and the barrel surface contains bound lipopolysaccharide (LPS) that is critical for the conformation and the activity of Pla. The biological activity of Pla is influenced by the structure of the surface loops around the active site groove and by temperature-induced LPS modifications. Several of the putative virulence-related functions documented for Pla in vitro address control of the human hemostatic system, i.e., coagulation and fibrinolysis. Pla activates human plasminogen to the serine protease plasmin and activates the physiological plasminogen activator urokinase. Pla also inactivates the protease inhibitors alpha-2-antiplasmin and plasminogen activator inhibitor 1 (PAI-1) and prevents the activation of thrombin-activatable fibrinolysis inhibitor (TAFI). These functions enhance uncontrolled fibrinolysis which is thought to improve Y. pestis dissemination and survival in the mammalian host, and lowered fibrin(ogen) deposition has indeed been observed in mice infected with Pla-positive Y. pestis. However, Pla also inactivates an anticoagulant, the tissue factor (TF) pathway inhibitor, which should increase fibrin formation and clotting. Thus, Pla and Y. pestis have complex interactions with the hemostatic system. Y. pestis modifies its LPS upon transfer to the mammalian host and we hypothesize that the contrasting biological activities of Pla in coagulation and fibrinolysis are influenced by LPS changes during infection.

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Human single‐chain urokinase is activated by the omptins PgtE of Salmonella enterica and Pla of Yersinia pestis despite mutations of active site residues

Cited by 17 publications

References 52 publications

Inhibition of Outer Membrane Proteases of the Omptin Family by Aprotinin

Inhibition of Outer Membrane Proteases of the Omptin Family by Aprotinin

Antimicrobial Peptide Conformation as a Structural Determinant of Omptin Protease Specificity

Fibrinolytic and coagulative activities of Yersinia pestis

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