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Proteasome-independent degradation of canonical NFkappaB complex components by the NleC protein of pathogenic Escherichia coli.

Mühlen S, Ruchaud-Sparagano MH, Kenny B - J. Biol. Chem. (2010)

Bottom Line: Many pathogens encode effector proteins that target factors regulating NFκB activity and can provide novel insights on regulatory mechanisms.This raises the prospect that mammalian cells, or other pathogens, employ a similar mechanism to modulate NFκB activity.Moreover, NleC represents a novel tool for validating NFκB as a therapeutic target and, indeed, as a possible therapeutic reagent.

View Article: PubMed Central - PubMed

Affiliation: Institute for Cell and Molecular Biosciences, Newcastle University Medical School, Newcastle-upon-Tyne NE2 4HH, United Kingdom.

ABSTRACT
The NFκB transcription factor is a key component of immune and inflammatory signaling as its activation induces the expression of antimicrobial reagents, chemokines, cytokines, and anti-apoptotic factors. Many pathogens encode effector proteins that target factors regulating NFκB activity and can provide novel insights on regulatory mechanisms. Given the link of NFκB dysfunction with inflammatory diseases and some cancers, these effectors have therapeutic potential. Here, screening enteropathogenic Escherichia coli proteins for those implicated in suppressing NFκB function revealed that eGFP-NleC, unlike eGFP, strongly inhibited basal and TNFα-induced NFκB reporter activity to prevent secretion of the chemokine, IL-8. Work involving NleC variants, chemical inhibitors, and immunoprecipitation studies support NleC being a zinc metalloprotease that degrades NFκB-IκBα complexes. The findings are consistent with features between residues 33-65 recruiting NFκB for proteasomal-independent degradation by a mechanism inhibited by metalloprotease inhibitors or disruption of a consensus zinc metalloprotease motif spanning NleC residues 183-187. This raises the prospect that mammalian cells, or other pathogens, employ a similar mechanism to modulate NFκB activity. Moreover, NleC represents a novel tool for validating NFκB as a therapeutic target and, indeed, as a possible therapeutic reagent.

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Interaction of p65 and p50 with eGFP-NleC1–237 and critical role for NleC residues 33–64. A, representative immunoblot of anti-GFP immunoprecipitate isolated from cells transfected with pEGFP, pEGFP-NleC, or pEGFP-NleC1–237 probed for p65 and p50. The data reveals that p65 and p50 can be isolated with eGFP-NleC1–237 but not eGFP or eGFP-NleC, the latter presumably due to rapid p65/p50 degradation. B, schematic of NleC N-terminal truncation variants constructed and screened in NFκB luciferase reporter assay. C, relative luciferase activity of cells expressing NleC and indicated variants, compared with control pEGFP-transfected cells. Data shown are mean (±S.D.) of three experiments done in triplicate with level of significance (Student's t test) indicated. *, p ≤ 0.05; ***, p ≤ 0.005 as compared with empty vector control. NleC33–330 but not NleC65–330 inhibits NFκB luciferase reporter activity revealing a critical role for residues between 33 and 64. D, representative immunoblot probed for GFP, p65, and actin that links loss of NFκB luciferase reporter activity of the NleC65–330 variants with a major defect in depleting p65 from the cell.
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Figure 5: Interaction of p65 and p50 with eGFP-NleC1–237 and critical role for NleC residues 33–64. A, representative immunoblot of anti-GFP immunoprecipitate isolated from cells transfected with pEGFP, pEGFP-NleC, or pEGFP-NleC1–237 probed for p65 and p50. The data reveals that p65 and p50 can be isolated with eGFP-NleC1–237 but not eGFP or eGFP-NleC, the latter presumably due to rapid p65/p50 degradation. B, schematic of NleC N-terminal truncation variants constructed and screened in NFκB luciferase reporter assay. C, relative luciferase activity of cells expressing NleC and indicated variants, compared with control pEGFP-transfected cells. Data shown are mean (±S.D.) of three experiments done in triplicate with level of significance (Student's t test) indicated. *, p ≤ 0.05; ***, p ≤ 0.005 as compared with empty vector control. NleC33–330 but not NleC65–330 inhibits NFκB luciferase reporter activity revealing a critical role for residues between 33 and 64. D, representative immunoblot probed for GFP, p65, and actin that links loss of NFκB luciferase reporter activity of the NleC65–330 variants with a major defect in depleting p65 from the cell.

Mentions: To test whether NleC interacts with NFκB, GFP-Trap beads were used to isolate eGFP, eGFP-NleC, and the eGFP-NleC1–237 fusion protein that does not induce NFκB degradation. Probing immunoprecipitates revealed that p65 and p50 interact, directly or indirectly, with NleC as they can be isolated with eGFP-NleC1–237 but not eGFP (Fig. 5A). The absence of p65 or p50 from eGFP-NleC co-immunoprecipitates (Fig. 5A) presumably reflects their rapid degradation. Thus, features located between NleC residues 1 and 237 are involved in recruiting NFκB components for degradation. To define domain(s) required in this interaction process, a series of N-terminal truncations was generated and screened in the luciferase reporter assay. Fig. 5B provides an illustration of the generated variants, with screening data revealing a dispensable role for the first 32 residues (Fig. 5C). By contrast, features located between residues 33 and 64 (NleC65–330) are critical for the inhibitory process (Fig. 5C) and, thus, all remaining variants failed to inhibit luciferase activity (data not shown). Western blot analyses confirmed variant expression, with the ability of eGFP-NleC33–330 but not eGFP-NleC65–330 to induce the cellular loss of a NFκB component illustrated (Fig. 5D and data not shown). Importantly, p65 and p50 could be co-immunoprecipitate with the eGFP-NleC1–66 but not eGFP-NleC65–330 variant (see Fig. 6C) revealing that N-terminal features (apparently located between residues 33–64) play a critical and sufficient role in recruiting NFκB.


Proteasome-independent degradation of canonical NFkappaB complex components by the NleC protein of pathogenic Escherichia coli.

Mühlen S, Ruchaud-Sparagano MH, Kenny B - J. Biol. Chem. (2010)

Interaction of p65 and p50 with eGFP-NleC1–237 and critical role for NleC residues 33–64. A, representative immunoblot of anti-GFP immunoprecipitate isolated from cells transfected with pEGFP, pEGFP-NleC, or pEGFP-NleC1–237 probed for p65 and p50. The data reveals that p65 and p50 can be isolated with eGFP-NleC1–237 but not eGFP or eGFP-NleC, the latter presumably due to rapid p65/p50 degradation. B, schematic of NleC N-terminal truncation variants constructed and screened in NFκB luciferase reporter assay. C, relative luciferase activity of cells expressing NleC and indicated variants, compared with control pEGFP-transfected cells. Data shown are mean (±S.D.) of three experiments done in triplicate with level of significance (Student's t test) indicated. *, p ≤ 0.05; ***, p ≤ 0.005 as compared with empty vector control. NleC33–330 but not NleC65–330 inhibits NFκB luciferase reporter activity revealing a critical role for residues between 33 and 64. D, representative immunoblot probed for GFP, p65, and actin that links loss of NFκB luciferase reporter activity of the NleC65–330 variants with a major defect in depleting p65 from the cell.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3037621&req=5

Figure 5: Interaction of p65 and p50 with eGFP-NleC1–237 and critical role for NleC residues 33–64. A, representative immunoblot of anti-GFP immunoprecipitate isolated from cells transfected with pEGFP, pEGFP-NleC, or pEGFP-NleC1–237 probed for p65 and p50. The data reveals that p65 and p50 can be isolated with eGFP-NleC1–237 but not eGFP or eGFP-NleC, the latter presumably due to rapid p65/p50 degradation. B, schematic of NleC N-terminal truncation variants constructed and screened in NFκB luciferase reporter assay. C, relative luciferase activity of cells expressing NleC and indicated variants, compared with control pEGFP-transfected cells. Data shown are mean (±S.D.) of three experiments done in triplicate with level of significance (Student's t test) indicated. *, p ≤ 0.05; ***, p ≤ 0.005 as compared with empty vector control. NleC33–330 but not NleC65–330 inhibits NFκB luciferase reporter activity revealing a critical role for residues between 33 and 64. D, representative immunoblot probed for GFP, p65, and actin that links loss of NFκB luciferase reporter activity of the NleC65–330 variants with a major defect in depleting p65 from the cell.
Mentions: To test whether NleC interacts with NFκB, GFP-Trap beads were used to isolate eGFP, eGFP-NleC, and the eGFP-NleC1–237 fusion protein that does not induce NFκB degradation. Probing immunoprecipitates revealed that p65 and p50 interact, directly or indirectly, with NleC as they can be isolated with eGFP-NleC1–237 but not eGFP (Fig. 5A). The absence of p65 or p50 from eGFP-NleC co-immunoprecipitates (Fig. 5A) presumably reflects their rapid degradation. Thus, features located between NleC residues 1 and 237 are involved in recruiting NFκB components for degradation. To define domain(s) required in this interaction process, a series of N-terminal truncations was generated and screened in the luciferase reporter assay. Fig. 5B provides an illustration of the generated variants, with screening data revealing a dispensable role for the first 32 residues (Fig. 5C). By contrast, features located between residues 33 and 64 (NleC65–330) are critical for the inhibitory process (Fig. 5C) and, thus, all remaining variants failed to inhibit luciferase activity (data not shown). Western blot analyses confirmed variant expression, with the ability of eGFP-NleC33–330 but not eGFP-NleC65–330 to induce the cellular loss of a NFκB component illustrated (Fig. 5D and data not shown). Importantly, p65 and p50 could be co-immunoprecipitate with the eGFP-NleC1–66 but not eGFP-NleC65–330 variant (see Fig. 6C) revealing that N-terminal features (apparently located between residues 33–64) play a critical and sufficient role in recruiting NFκB.

Bottom Line: Many pathogens encode effector proteins that target factors regulating NFκB activity and can provide novel insights on regulatory mechanisms.This raises the prospect that mammalian cells, or other pathogens, employ a similar mechanism to modulate NFκB activity.Moreover, NleC represents a novel tool for validating NFκB as a therapeutic target and, indeed, as a possible therapeutic reagent.

View Article: PubMed Central - PubMed

Affiliation: Institute for Cell and Molecular Biosciences, Newcastle University Medical School, Newcastle-upon-Tyne NE2 4HH, United Kingdom.

ABSTRACT
The NFκB transcription factor is a key component of immune and inflammatory signaling as its activation induces the expression of antimicrobial reagents, chemokines, cytokines, and anti-apoptotic factors. Many pathogens encode effector proteins that target factors regulating NFκB activity and can provide novel insights on regulatory mechanisms. Given the link of NFκB dysfunction with inflammatory diseases and some cancers, these effectors have therapeutic potential. Here, screening enteropathogenic Escherichia coli proteins for those implicated in suppressing NFκB function revealed that eGFP-NleC, unlike eGFP, strongly inhibited basal and TNFα-induced NFκB reporter activity to prevent secretion of the chemokine, IL-8. Work involving NleC variants, chemical inhibitors, and immunoprecipitation studies support NleC being a zinc metalloprotease that degrades NFκB-IκBα complexes. The findings are consistent with features between residues 33-65 recruiting NFκB for proteasomal-independent degradation by a mechanism inhibited by metalloprotease inhibitors or disruption of a consensus zinc metalloprotease motif spanning NleC residues 183-187. This raises the prospect that mammalian cells, or other pathogens, employ a similar mechanism to modulate NFκB activity. Moreover, NleC represents a novel tool for validating NFκB as a therapeutic target and, indeed, as a possible therapeutic reagent.

Show MeSH
Related in: MedlinePlus