Analysis of Protein O-fucosyltransferase 1 regulation of Toll-like receptor signaling in macrophages

Tuesday, September 23, 2014 — Poster Session III
12:00 p.m. – 2:00 p.m.	FAES Academic Center	NIAID	IMMUNO-9

* FARE Award Winner

Authors

• J. M. Mann
• N. Li
• K. Laky
• I.D.C Fraser
• A. Nita-Lazar

Abstract

Protein O-fucosyltransferase 1 (Pofut1) post-translationally modifies the Notch receptor proteins by the addition of O-linked fucose to serine/threonine residues between the 2nd-3rd EGF repeats of the extracellular domains. This modification is critical for Notch binding to its ligands to signal in developmentalal events including neurogenesis and lymphopoiesis. Recently, studies have found cross-talk between Notch signaling pathways and pro-inflammatory signaling of the innate immune system activated through Toll-like receptors (TLR). In a genome-wide RNAi screen of lipopolysaccharide-induced TLR signaling in mouse RAW264.7 macrophages, a knockdown of Pofut1 resulted in a reduction in TNFα cytokine secretion. To further investigate a potentially novel role of Pofut1 in TLR-signaling, we evaluated IL-6, TNFα and IL-12p40 cytokine secretion in RAW264.7 Pofut1 and Notch siRNA transfected cells stimulated with different TLR-ligands. Under most circumstances, the pro-inflammatory cytokine levels were significantly reduced in absence of Pofut1 or Notch1/2. IL-12p40 secretion was unique and we further analyzed the mechanism of regulation using a ϒ-secretase inhibitor. We have also studied protein phosphorylation and expression profiling using Western blotting in these cells, as well as mass spectrometry. We are using bone marrow derived macrophages (BMDMs) from Pofut1 conditional knockout mice to further investigate this phenotype.

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