Cloning of a lymphatic peptide/histidine transporter.
Ontology highlight
ABSTRACT:
SUBMITTER: Sakata K
PROVIDER: S-EPMC1221811 | biostudies-other | 2001 May
REPOSITORIES: biostudies-other
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Project description:PHT1 is a histidine /oligopeptide transporter with an essential role in Toll-like receptor innate immune responses. It can act as a receptor by recruiting the adaptor protein TASL which leads to type I interferon production via IRF5. Persistent stimulation of this signalling pathway is known to be involved in the pathogenesis of systemic lupus erythematosus (SLE). Understanding how PHT1 recruits TASL at the molecular level, is therefore clinically important for the development of therapeutics against SLE and other autoimmune diseases. Here we present the Cryo-EM structure of PHT1 stabilized in the outward-open conformation. By combining biochemical and structural modeling techniques we propose a model of the PHT1-TASL complex, in which the first 16 N-terminal TASL residues fold into a helical structure that bind in the central cavity of the inward-open conformation of PHT1. This work provides critical insights into the molecular basis of PHT1/TASL mediated type I interferon production.
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Project description:The peptide/histidine transporter SLC15A3 is responsible for transporting histidine, certain dipeptide and peptidomimetics from inside the lysosome to cytosol. Previous studies have indicated that SLC15A3 transcripts are mainly expressed in the lymphatic system, however, its regulation and biological role in innate immune responses and inflammatory diseases are as yet unknown. In this study, mouse peritoneal macrophages (PMs), mouse bone marrow-derived macrophages (BMDMs), the human acute monocytic leukemia cell line THP-1 and the human lung epithelial carcinoma cell line A549 were used to investigate the regulation and biological role of SLC15A3 in TLR-mediated inflammatory responses. Our results showed that SLC15A3 was upregulated by TLR2, TLR4, TLR7 and TLR9 ligands in macrophages at both the mRNA and protein levels via activation of NF-?B (nuclear factor-kappa-B), MAPK (mitogen-activated protein kinase) and IRF3 (interferon regulatory factor 3). Furthermore, knockdown or overexpression of SLC15A3 influenced the TLR4-triggered expression of proinflammatory cytokines. A reporter gene assay showed that the SLC15A3 promotor contained potential NF-?B binding sites, which were reasonable for regulating SLC15A3 by TLR-activation through NF-?B signaling. Additionally, SLC15A3 expression was increased and positively related to inflammation in mice with bacterial peritonitis. The collective findings suggest that SLC15A3 is regulated by various TLRs, and that it plays an important role in regulating TLR4-mediated inflammatory responses.
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Project description:BackgroundPreviously, we determined that four-branched histidine-lysine (HK) peptides were effective carriers of plasmids and small interfering RNA. In the present study, we compared several branched HK carriers and, in particular, two closely-related H3K4b and H3K(+H)4b peptides for their ability as carriers of mRNA. The H3K(+H)4b peptide differed from its parent analogue, H3K4b, by only a single histidine in each branch.MethodsA series of four-branched HK peptides with varied sequences was synthesized on a solid-phase peptide synthesizer. The ability of these peptides to carry mRNA expressing luciferase to MDA-MB-231 cells was investigated. With gel retardation and heparin displacement assays, the stability of HK polyplexes was examined. We determined the intracellular uptake of HK polyplexes by flow cytometry and fluorescence microscopy. The size and polydispersity index of the polyplexes in several media were measured by dynamic light scattering.ResultsMDA-MB-231 cells transfected by H3K(+H)4b-mRNA polyplexes expressed 10-fold greater levels of luciferase than H3K4b polyplexes. With gel retardation and heparin displacement assays, the H3K(+H)4b polyplexes showed greater stability than H3K4b. Intracellular uptake and co-localization of H3K(+H)4b polyplexes within acidic endosomes were also significantly increased compared to H3K4b. Similar to H3K(+H)4b, several HK analogues with an additional histidine in the second domain of their branches were effective carriers of mRNA. When combined with DOTAP liposomes, H3K(+H)4b was synergistic in delivery of mRNA.ConclusionsH3K(+H)4b was a more effective carrier of mRNA than H3K4b. Mechanistic studies suggest that H3K(+H)4b polyplexes were more stable than H3K4b polyplexes. Lipopolyplexes formed with H3K(+H)4b markedly increased mRNA transfection.
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