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Unique Toll-Like Receptor 4 Activation by NAMPT/PBEF Induces NFκB Signaling and Inflammatory Lung Injury.

Camp SM, Ceco E, Evenoski CL, Danilov SM, Zhou T, Chiang ET, Moreno-Vinasco L, Mapes B, Zhao J, Gursoy G, Brown ME, Adyshev DM, Siddiqui SS, Quijada H, Sammani S, Letsiou E, Saadat L, Yousef M, Wang T, Liang J, Garcia JG - Sci Rep (2015)

Bottom Line: Although VILI severity is attenuated by reduced NAMPT/PBEF bioavailability, the precise contribution of NAMPT/PBEF and excessive mechanical stress to VILI pathobiology is unknown.Unlike MD-2, whose TLR4 binding alone is insufficient to initiate TLR4 signaling, NAMPT/PBEF alone produces robust TLR4 activation, likely via a protruding region of NAMPT/PBEF (S402-N412) with structural similarity to LPS.The identification of this unique mode of TLR4 activation by NAMPT/PBEF advances the understanding of innate immunity responses as well as the untoward events associated with mechanical stress-induced lung inflammation.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine and Arizona Respiratory Center, The University of Arizona.

ABSTRACT
Ventilator-induced inflammatory lung injury (VILI) is mechanistically linked to increased NAMPT transcription and circulating levels of nicotinamide phosphoribosyl-transferase (NAMPT/PBEF). Although VILI severity is attenuated by reduced NAMPT/PBEF bioavailability, the precise contribution of NAMPT/PBEF and excessive mechanical stress to VILI pathobiology is unknown. We now report that NAMPT/PBEF induces lung NFκB transcriptional activities and inflammatory injury via direct ligation of Toll-like receptor 4 (TLR4). Computational analysis demonstrated that NAMPT/PBEF and MD-2, a TLR4-binding protein essential for LPS-induced TLR4 activation, share ~30% sequence identity and exhibit striking structural similarity in loop regions critical for MD-2-TLR4 binding. Unlike MD-2, whose TLR4 binding alone is insufficient to initiate TLR4 signaling, NAMPT/PBEF alone produces robust TLR4 activation, likely via a protruding region of NAMPT/PBEF (S402-N412) with structural similarity to LPS. The identification of this unique mode of TLR4 activation by NAMPT/PBEF advances the understanding of innate immunity responses as well as the untoward events associated with mechanical stress-induced lung inflammation.

No MeSH data available.


Related in: MedlinePlus

In silico modeling of NAMPT/PBEF and MD-2 interactions with TLR4.(Panel A) Murine sequence of TLR4-binding loop from MD-2 in the N-to-C order aligned with the loop from murine NAMPT/PBEF in the reverse C-to-N order. Residues in red are known to be involved in MD-2 binding to TLR420. The wider red bands indicate aligned identical residues, while the narrower bands indicate aligned residues with similar physicochemical properties. (Panel B) Corresponding structural alignment between the loop in MD-2 (purple) and the loop in NAMPT/PBEF (red). (Panel C) Structure of MD-2, where the surface of the TLR4-binding loop of MD-2 (99D-111E) is in purple. (Panel D) Structure of NAMPT/PBEF, where the surface of the aligned binding loop from NAMPT/PBEF (457L-445E) is in red. (Panel E) Signature pocket for the LPS-binding surface constructed from three diverse LPS binding proteins, each less than 64% of sequence identities with MD-2 but all exhibiting the same LPS binding mode as that of MD-2. Binding pockets of the three LPS binding proteins (red = oxygen; blue = nitrogen; green = carbon atoms) are shown along the hierarchical tree based on sequence order independent structural alignment of the binding surfaces28. The large yellow pocket at the root of the tree is the overall signature pocket constructed from the 7 LPS binding pockets. Spatially preserved signature LPS binding residues are in red. (Panel F) Signature residues in the LPS binding pocket of the MD-2-LPS complex including F119 (red); I52 (green); L74, L94 (blue); overall LPS binding pocket (cyan); TLR4-binding loop (purple). (Panel G) NAMPT/PBEF structure with residues corresponding to signature residues for LPS binding mapped in MD-2: F399 corresponding to F119 on MD-2 (red); L432 and L458 corresponding to L74 and L94, respectively (blue); I114 corresponding to I52 (green); other residues of LPS binding pocket (cyan); loop residues for TLR4-binding loop (purple). Signature residues for LPS binding exhibit greater spatial separation in NAMPT/PBEF compared to MD-2 with the prominent surface pocket for LPS binding, present in MD-2, absent in NAMPT/PBEF. Potential TLR4 activation by the LPS molecule (transparent-wheat) can be mapped to the protruding region of S402-N412 residues on NAMPT/PBEF (transparent-wheat).
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f6: In silico modeling of NAMPT/PBEF and MD-2 interactions with TLR4.(Panel A) Murine sequence of TLR4-binding loop from MD-2 in the N-to-C order aligned with the loop from murine NAMPT/PBEF in the reverse C-to-N order. Residues in red are known to be involved in MD-2 binding to TLR420. The wider red bands indicate aligned identical residues, while the narrower bands indicate aligned residues with similar physicochemical properties. (Panel B) Corresponding structural alignment between the loop in MD-2 (purple) and the loop in NAMPT/PBEF (red). (Panel C) Structure of MD-2, where the surface of the TLR4-binding loop of MD-2 (99D-111E) is in purple. (Panel D) Structure of NAMPT/PBEF, where the surface of the aligned binding loop from NAMPT/PBEF (457L-445E) is in red. (Panel E) Signature pocket for the LPS-binding surface constructed from three diverse LPS binding proteins, each less than 64% of sequence identities with MD-2 but all exhibiting the same LPS binding mode as that of MD-2. Binding pockets of the three LPS binding proteins (red = oxygen; blue = nitrogen; green = carbon atoms) are shown along the hierarchical tree based on sequence order independent structural alignment of the binding surfaces28. The large yellow pocket at the root of the tree is the overall signature pocket constructed from the 7 LPS binding pockets. Spatially preserved signature LPS binding residues are in red. (Panel F) Signature residues in the LPS binding pocket of the MD-2-LPS complex including F119 (red); I52 (green); L74, L94 (blue); overall LPS binding pocket (cyan); TLR4-binding loop (purple). (Panel G) NAMPT/PBEF structure with residues corresponding to signature residues for LPS binding mapped in MD-2: F399 corresponding to F119 on MD-2 (red); L432 and L458 corresponding to L74 and L94, respectively (blue); I114 corresponding to I52 (green); other residues of LPS binding pocket (cyan); loop residues for TLR4-binding loop (purple). Signature residues for LPS binding exhibit greater spatial separation in NAMPT/PBEF compared to MD-2 with the prominent surface pocket for LPS binding, present in MD-2, absent in NAMPT/PBEF. Potential TLR4 activation by the LPS molecule (transparent-wheat) can be mapped to the protruding region of S402-N412 residues on NAMPT/PBEF (transparent-wheat).

Mentions: The crystal structure of NAMPT/PBEF19, the TLR4 receptor, as well as the LPS- and TLR4-binding protein, MD-2, have been resolved2021. In silico protein structure analysis revealed that despite only ~30% total sequence identity between murine NAMPT/PBEF and MD-2, striking structural similarities exist between a loop region on NAMPT/PBEF and a loop region on MD-2 known to be involved in LPS binding to TLR4 and critical to subsequent TLR4 activation (Fig. 6A,B). Using a sequence order independent structural alignment method22, a loop (99D-111E, purple Fig. 6C) in the TLR4-binding region on MD-2 was well aligned in the N-to-C order with a loop residing in NAMPT/PBEF in the reverse C-to-N order (457L-445E, red, Fig. 6D, 30.3% identity). Six of the 7 MD-2 residues known to be important for TLR4 binding reside within this loop20 and this loop also contains a prominent motif consisting of a consecutive triplet of residues K109, G110, and E111, a D residue and an S/Y residue23 (Fig. 6A) fully conserved among different species. This motif is also present in the NAMPT/PBEF loop (thus indicating a potentially conserved biological function). We speculate that this loop represents the TLR4-binding region for NAMPT/PBEF is supported by protein analysis of the TLR4-binding protein, Der-p2, a house mite allergen with MD-2 homology (sequence identity ~26%)242526. The TLR4-binding regions of Der-p2 are highly conserved and similarly align to the loop in NAMPT/PBEF. Finally, residue R434, in spatial proximity of the predicted NAMPT/PBEF loop binding residues, is structurally aligned to R90 on MD-2 implicated to participate in TLR4-MD-2 interactions27. Thus, the observed similarity in the TLR4 binding environment between NAMPT/PBEF and MD-2, in conjunction with the biochemical and SPR studies, support our hypothesis that NAMPT/PBEF is a TLR4-binding protein.


Unique Toll-Like Receptor 4 Activation by NAMPT/PBEF Induces NFκB Signaling and Inflammatory Lung Injury.

Camp SM, Ceco E, Evenoski CL, Danilov SM, Zhou T, Chiang ET, Moreno-Vinasco L, Mapes B, Zhao J, Gursoy G, Brown ME, Adyshev DM, Siddiqui SS, Quijada H, Sammani S, Letsiou E, Saadat L, Yousef M, Wang T, Liang J, Garcia JG - Sci Rep (2015)

In silico modeling of NAMPT/PBEF and MD-2 interactions with TLR4.(Panel A) Murine sequence of TLR4-binding loop from MD-2 in the N-to-C order aligned with the loop from murine NAMPT/PBEF in the reverse C-to-N order. Residues in red are known to be involved in MD-2 binding to TLR420. The wider red bands indicate aligned identical residues, while the narrower bands indicate aligned residues with similar physicochemical properties. (Panel B) Corresponding structural alignment between the loop in MD-2 (purple) and the loop in NAMPT/PBEF (red). (Panel C) Structure of MD-2, where the surface of the TLR4-binding loop of MD-2 (99D-111E) is in purple. (Panel D) Structure of NAMPT/PBEF, where the surface of the aligned binding loop from NAMPT/PBEF (457L-445E) is in red. (Panel E) Signature pocket for the LPS-binding surface constructed from three diverse LPS binding proteins, each less than 64% of sequence identities with MD-2 but all exhibiting the same LPS binding mode as that of MD-2. Binding pockets of the three LPS binding proteins (red = oxygen; blue = nitrogen; green = carbon atoms) are shown along the hierarchical tree based on sequence order independent structural alignment of the binding surfaces28. The large yellow pocket at the root of the tree is the overall signature pocket constructed from the 7 LPS binding pockets. Spatially preserved signature LPS binding residues are in red. (Panel F) Signature residues in the LPS binding pocket of the MD-2-LPS complex including F119 (red); I52 (green); L74, L94 (blue); overall LPS binding pocket (cyan); TLR4-binding loop (purple). (Panel G) NAMPT/PBEF structure with residues corresponding to signature residues for LPS binding mapped in MD-2: F399 corresponding to F119 on MD-2 (red); L432 and L458 corresponding to L74 and L94, respectively (blue); I114 corresponding to I52 (green); other residues of LPS binding pocket (cyan); loop residues for TLR4-binding loop (purple). Signature residues for LPS binding exhibit greater spatial separation in NAMPT/PBEF compared to MD-2 with the prominent surface pocket for LPS binding, present in MD-2, absent in NAMPT/PBEF. Potential TLR4 activation by the LPS molecule (transparent-wheat) can be mapped to the protruding region of S402-N412 residues on NAMPT/PBEF (transparent-wheat).
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f6: In silico modeling of NAMPT/PBEF and MD-2 interactions with TLR4.(Panel A) Murine sequence of TLR4-binding loop from MD-2 in the N-to-C order aligned with the loop from murine NAMPT/PBEF in the reverse C-to-N order. Residues in red are known to be involved in MD-2 binding to TLR420. The wider red bands indicate aligned identical residues, while the narrower bands indicate aligned residues with similar physicochemical properties. (Panel B) Corresponding structural alignment between the loop in MD-2 (purple) and the loop in NAMPT/PBEF (red). (Panel C) Structure of MD-2, where the surface of the TLR4-binding loop of MD-2 (99D-111E) is in purple. (Panel D) Structure of NAMPT/PBEF, where the surface of the aligned binding loop from NAMPT/PBEF (457L-445E) is in red. (Panel E) Signature pocket for the LPS-binding surface constructed from three diverse LPS binding proteins, each less than 64% of sequence identities with MD-2 but all exhibiting the same LPS binding mode as that of MD-2. Binding pockets of the three LPS binding proteins (red = oxygen; blue = nitrogen; green = carbon atoms) are shown along the hierarchical tree based on sequence order independent structural alignment of the binding surfaces28. The large yellow pocket at the root of the tree is the overall signature pocket constructed from the 7 LPS binding pockets. Spatially preserved signature LPS binding residues are in red. (Panel F) Signature residues in the LPS binding pocket of the MD-2-LPS complex including F119 (red); I52 (green); L74, L94 (blue); overall LPS binding pocket (cyan); TLR4-binding loop (purple). (Panel G) NAMPT/PBEF structure with residues corresponding to signature residues for LPS binding mapped in MD-2: F399 corresponding to F119 on MD-2 (red); L432 and L458 corresponding to L74 and L94, respectively (blue); I114 corresponding to I52 (green); other residues of LPS binding pocket (cyan); loop residues for TLR4-binding loop (purple). Signature residues for LPS binding exhibit greater spatial separation in NAMPT/PBEF compared to MD-2 with the prominent surface pocket for LPS binding, present in MD-2, absent in NAMPT/PBEF. Potential TLR4 activation by the LPS molecule (transparent-wheat) can be mapped to the protruding region of S402-N412 residues on NAMPT/PBEF (transparent-wheat).
Mentions: The crystal structure of NAMPT/PBEF19, the TLR4 receptor, as well as the LPS- and TLR4-binding protein, MD-2, have been resolved2021. In silico protein structure analysis revealed that despite only ~30% total sequence identity between murine NAMPT/PBEF and MD-2, striking structural similarities exist between a loop region on NAMPT/PBEF and a loop region on MD-2 known to be involved in LPS binding to TLR4 and critical to subsequent TLR4 activation (Fig. 6A,B). Using a sequence order independent structural alignment method22, a loop (99D-111E, purple Fig. 6C) in the TLR4-binding region on MD-2 was well aligned in the N-to-C order with a loop residing in NAMPT/PBEF in the reverse C-to-N order (457L-445E, red, Fig. 6D, 30.3% identity). Six of the 7 MD-2 residues known to be important for TLR4 binding reside within this loop20 and this loop also contains a prominent motif consisting of a consecutive triplet of residues K109, G110, and E111, a D residue and an S/Y residue23 (Fig. 6A) fully conserved among different species. This motif is also present in the NAMPT/PBEF loop (thus indicating a potentially conserved biological function). We speculate that this loop represents the TLR4-binding region for NAMPT/PBEF is supported by protein analysis of the TLR4-binding protein, Der-p2, a house mite allergen with MD-2 homology (sequence identity ~26%)242526. The TLR4-binding regions of Der-p2 are highly conserved and similarly align to the loop in NAMPT/PBEF. Finally, residue R434, in spatial proximity of the predicted NAMPT/PBEF loop binding residues, is structurally aligned to R90 on MD-2 implicated to participate in TLR4-MD-2 interactions27. Thus, the observed similarity in the TLR4 binding environment between NAMPT/PBEF and MD-2, in conjunction with the biochemical and SPR studies, support our hypothesis that NAMPT/PBEF is a TLR4-binding protein.

Bottom Line: Although VILI severity is attenuated by reduced NAMPT/PBEF bioavailability, the precise contribution of NAMPT/PBEF and excessive mechanical stress to VILI pathobiology is unknown.Unlike MD-2, whose TLR4 binding alone is insufficient to initiate TLR4 signaling, NAMPT/PBEF alone produces robust TLR4 activation, likely via a protruding region of NAMPT/PBEF (S402-N412) with structural similarity to LPS.The identification of this unique mode of TLR4 activation by NAMPT/PBEF advances the understanding of innate immunity responses as well as the untoward events associated with mechanical stress-induced lung inflammation.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine and Arizona Respiratory Center, The University of Arizona.

ABSTRACT
Ventilator-induced inflammatory lung injury (VILI) is mechanistically linked to increased NAMPT transcription and circulating levels of nicotinamide phosphoribosyl-transferase (NAMPT/PBEF). Although VILI severity is attenuated by reduced NAMPT/PBEF bioavailability, the precise contribution of NAMPT/PBEF and excessive mechanical stress to VILI pathobiology is unknown. We now report that NAMPT/PBEF induces lung NFκB transcriptional activities and inflammatory injury via direct ligation of Toll-like receptor 4 (TLR4). Computational analysis demonstrated that NAMPT/PBEF and MD-2, a TLR4-binding protein essential for LPS-induced TLR4 activation, share ~30% sequence identity and exhibit striking structural similarity in loop regions critical for MD-2-TLR4 binding. Unlike MD-2, whose TLR4 binding alone is insufficient to initiate TLR4 signaling, NAMPT/PBEF alone produces robust TLR4 activation, likely via a protruding region of NAMPT/PBEF (S402-N412) with structural similarity to LPS. The identification of this unique mode of TLR4 activation by NAMPT/PBEF advances the understanding of innate immunity responses as well as the untoward events associated with mechanical stress-induced lung inflammation.

No MeSH data available.


Related in: MedlinePlus