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The Lipid-Modifying Enzyme SMPDL3B Negatively Regulates Innate Immunity.

Heinz LX, Baumann CL, Köberlin MS, Snijder B, Gawish R, Shui G, Sharif O, Aspalter IM, Müller AC, Kandasamy RK, Breitwieser FP, Pichlmair A, Bruckner M, Rebsamen M, Blüml S, Karonitsch T, Fauster A, Colinge J, Bennett KL, Knapp S, Wenk MR, Superti-Furga G - Cell Rep (2015)

Bottom Line: Lipid metabolism and receptor-mediated signaling are highly intertwined processes that cooperate to fulfill cellular functions and safeguard cellular homeostasis.Increased cellular responses could be reverted by re-introducing affected ceramides, functionally linking membrane lipid composition and innate immune signaling.Taken together, our results identify the membrane-modulating enzyme SMPDL3B as a negative regulator of TLR signaling that functions at the interface of membrane biology and innate immunity.

View Article: PubMed Central - PubMed

Affiliation: CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, 1090 Vienna, Austria.

No MeSH data available.


Related in: MedlinePlus

Identification of SMPDL3B as GPI-Anchored TLR Interactor(A) Average spectral counts (av spc) and average % sequence coverage (av%sc) of SMPDL3B detected by mass spectrometry in endosomal TLR tandem affinity purifications.(B) Domain organization of SMPDL3B, SMPDL3A, and SMPD1/ASM. Gray triangles indicate predicted or validated N-linked glycosylation sites; black triangles indicate conserved motifs in metal coordination/substrate binding.(C) HA-specific FACS analysis of HEK293T cells stably expressing murine SMPDL3B or a deletion mutant lacking the C-terminal GPI signal.(D) Cells were lysed using 1% NP-40 or subjected to TX-114 phase separation. Proteins were analyzed by western blot for SMPDL3B, CD14, and IκBα. I, detergent-insoluble proteins; D, detergent phase, amphiphilic integral membrane proteins; A, aqueous phase, hydrophilic proteins.(E) Cells were lysed with TX-114; lysates were divided in two and treated or not with PI-PLC. Proteins were subjected to phase separation, and fractions were analyzed by western blot for SMPDL3B, CD14, and IκBα.(F) HEK293T cells were transfected with SMPDL3B and V5-tagged TLRs as indicated. Immunoprecipitates and extracts were analyzed by western blot using SMPDL3B- and V5-specific antibodies.(C–F) Data are representative of at least two independent experiments. See also Figure S1.
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fig1: Identification of SMPDL3B as GPI-Anchored TLR Interactor(A) Average spectral counts (av spc) and average % sequence coverage (av%sc) of SMPDL3B detected by mass spectrometry in endosomal TLR tandem affinity purifications.(B) Domain organization of SMPDL3B, SMPDL3A, and SMPD1/ASM. Gray triangles indicate predicted or validated N-linked glycosylation sites; black triangles indicate conserved motifs in metal coordination/substrate binding.(C) HA-specific FACS analysis of HEK293T cells stably expressing murine SMPDL3B or a deletion mutant lacking the C-terminal GPI signal.(D) Cells were lysed using 1% NP-40 or subjected to TX-114 phase separation. Proteins were analyzed by western blot for SMPDL3B, CD14, and IκBα. I, detergent-insoluble proteins; D, detergent phase, amphiphilic integral membrane proteins; A, aqueous phase, hydrophilic proteins.(E) Cells were lysed with TX-114; lysates were divided in two and treated or not with PI-PLC. Proteins were subjected to phase separation, and fractions were analyzed by western blot for SMPDL3B, CD14, and IκBα.(F) HEK293T cells were transfected with SMPDL3B and V5-tagged TLRs as indicated. Immunoprecipitates and extracts were analyzed by western blot using SMPDL3B- and V5-specific antibodies.(C–F) Data are representative of at least two independent experiments. See also Figure S1.

Mentions: The discovery of proteins modulating the activity of TLRs is crucial for the understanding of TLR-dependent immune responses. The characterization of TLR-associated protein complexes by affinity purification followed by mass spectrometry has previously led to the identification of CD14 as co-receptor for nucleic acid recognition by the endosomal TLRs 7 and 9 (Baumann et al., 2010). In the same screening campaign, SMPDL3B (UniProt: P58242, entry name: ASM3B_MOUSE) was consistently identified as membrane protein co-purifying with endosomal TLRs 3, 7, 8, and 9 with robust sequence coverage from RAW264.7 macrophages suggesting association with the same membrane compartments harboring TLRs (Figure 1A).


The Lipid-Modifying Enzyme SMPDL3B Negatively Regulates Innate Immunity.

Heinz LX, Baumann CL, Köberlin MS, Snijder B, Gawish R, Shui G, Sharif O, Aspalter IM, Müller AC, Kandasamy RK, Breitwieser FP, Pichlmair A, Bruckner M, Rebsamen M, Blüml S, Karonitsch T, Fauster A, Colinge J, Bennett KL, Knapp S, Wenk MR, Superti-Furga G - Cell Rep (2015)

Identification of SMPDL3B as GPI-Anchored TLR Interactor(A) Average spectral counts (av spc) and average % sequence coverage (av%sc) of SMPDL3B detected by mass spectrometry in endosomal TLR tandem affinity purifications.(B) Domain organization of SMPDL3B, SMPDL3A, and SMPD1/ASM. Gray triangles indicate predicted or validated N-linked glycosylation sites; black triangles indicate conserved motifs in metal coordination/substrate binding.(C) HA-specific FACS analysis of HEK293T cells stably expressing murine SMPDL3B or a deletion mutant lacking the C-terminal GPI signal.(D) Cells were lysed using 1% NP-40 or subjected to TX-114 phase separation. Proteins were analyzed by western blot for SMPDL3B, CD14, and IκBα. I, detergent-insoluble proteins; D, detergent phase, amphiphilic integral membrane proteins; A, aqueous phase, hydrophilic proteins.(E) Cells were lysed with TX-114; lysates were divided in two and treated or not with PI-PLC. Proteins were subjected to phase separation, and fractions were analyzed by western blot for SMPDL3B, CD14, and IκBα.(F) HEK293T cells were transfected with SMPDL3B and V5-tagged TLRs as indicated. Immunoprecipitates and extracts were analyzed by western blot using SMPDL3B- and V5-specific antibodies.(C–F) Data are representative of at least two independent experiments. See also Figure S1.
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fig1: Identification of SMPDL3B as GPI-Anchored TLR Interactor(A) Average spectral counts (av spc) and average % sequence coverage (av%sc) of SMPDL3B detected by mass spectrometry in endosomal TLR tandem affinity purifications.(B) Domain organization of SMPDL3B, SMPDL3A, and SMPD1/ASM. Gray triangles indicate predicted or validated N-linked glycosylation sites; black triangles indicate conserved motifs in metal coordination/substrate binding.(C) HA-specific FACS analysis of HEK293T cells stably expressing murine SMPDL3B or a deletion mutant lacking the C-terminal GPI signal.(D) Cells were lysed using 1% NP-40 or subjected to TX-114 phase separation. Proteins were analyzed by western blot for SMPDL3B, CD14, and IκBα. I, detergent-insoluble proteins; D, detergent phase, amphiphilic integral membrane proteins; A, aqueous phase, hydrophilic proteins.(E) Cells were lysed with TX-114; lysates were divided in two and treated or not with PI-PLC. Proteins were subjected to phase separation, and fractions were analyzed by western blot for SMPDL3B, CD14, and IκBα.(F) HEK293T cells were transfected with SMPDL3B and V5-tagged TLRs as indicated. Immunoprecipitates and extracts were analyzed by western blot using SMPDL3B- and V5-specific antibodies.(C–F) Data are representative of at least two independent experiments. See also Figure S1.
Mentions: The discovery of proteins modulating the activity of TLRs is crucial for the understanding of TLR-dependent immune responses. The characterization of TLR-associated protein complexes by affinity purification followed by mass spectrometry has previously led to the identification of CD14 as co-receptor for nucleic acid recognition by the endosomal TLRs 7 and 9 (Baumann et al., 2010). In the same screening campaign, SMPDL3B (UniProt: P58242, entry name: ASM3B_MOUSE) was consistently identified as membrane protein co-purifying with endosomal TLRs 3, 7, 8, and 9 with robust sequence coverage from RAW264.7 macrophages suggesting association with the same membrane compartments harboring TLRs (Figure 1A).

Bottom Line: Lipid metabolism and receptor-mediated signaling are highly intertwined processes that cooperate to fulfill cellular functions and safeguard cellular homeostasis.Increased cellular responses could be reverted by re-introducing affected ceramides, functionally linking membrane lipid composition and innate immune signaling.Taken together, our results identify the membrane-modulating enzyme SMPDL3B as a negative regulator of TLR signaling that functions at the interface of membrane biology and innate immunity.

View Article: PubMed Central - PubMed

Affiliation: CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, 1090 Vienna, Austria.

No MeSH data available.


Related in: MedlinePlus