Functional Characterization of Human Peptide/Histidine Transporter 1 in Stably Transfected MDCK Cells

The proton-coupled oligopeptide transporter PHT1 (SLC15A4), which facilitates cross-membrane transport of histidine and small peptides from inside the endosomes or lysosomes to cytosol, plays an important role in intracellular peptides homeostasis and innate immune responses. However, it remains a challenge to elucidate functional properties of the PHT1 transporter because of its subcellular localization. The purpose of this study was to resort hPHT1 protein from the subcellular to outer cell membrane of MDCK cells stably transfected with human PHT1 mutants, and to characterize its functional activity in these cells. Using this model, the functional activity of hPHT1 was evaluated by cellular uptake studies with d₃-l-histidine, GlySar, and the bacterial peptidoglycan products MDP and Tri-DAP. We found that the disruption of two dileucine motifs was indispensable for hPHT1 transporter being preferentially targeting to plasma membranes. hPHT1 showed high affinity for d₃-l-histidine and low affinity for GlySar, with <i>K</i>_m values of 16.3 ± 1.9 μM and 1.60 ± 0.30 mM, respectively. Moreover, the bacterial peptidoglycan components MDP and Tri-DAP were shown conclusively to be hPHT1 substrates. The uptake of MDP by hPHT1 was inhibited by di/tripeptides and peptide-like drugs, but not by glycine and acyclovir. The functional activity of hPHT1 was also pH-dependent, with an optimal cellular uptake in buffer pH 6.5. Taken together, we established a novel cell model to evaluate the function of hPHT1 <i>in vitro</i>, and confirmed that MDP and Tri-DAP were substrates of hPHT1. Our findings suggest that PHT1 may serve as a potential target for reducing the immune responses and for drug treatment of inflammatory diseases

Additional file 3 of CREB3L1 promotes tumor growth and metastasis of anaplastic thyroid carcinoma by remodeling the tumor microenvironment

Additional file 3. The list of ECM and collagen genes

Additional file 2 of CREB3L1 promotes tumor growth and metastasis of anaplastic thyroid carcinoma by remodeling the tumor microenvironment

Additional file 2: Figure S2. Effect of CREB3L1 knockdown on the differentiation of CAFs. (A-B) Flow cytometry analysis of FAP or PDGFRα positive fibroblasts after co-culture with 8505C cells, respectively. CREB3L1 was knocked down in 8505C cells to evaluate its effect on the differentiation of CAFs

Additional file 1 of CREB3L1 promotes tumor growth and metastasis of anaplastic thyroid carcinoma by remodeling the tumor microenvironment

Additional file 1: Figure S1. CREB3L1 transcriptionally regulated the expression of COL5A1 to facilitate the aggressiveness of ATC. (A) The relationship of COL5A1 expression and tumor stages in thyroidcancer was analyzed. (B) Survival analysis of COL5A1 in thyroid cancer was analyzed. (C) IHC staining was used to analyze COL5A1 expression in NT, PTC and ATC tissues. (D) Transwell invasion assay was used to analyze the metastasis ability after the silence of COL5A1 in 8505C. (E-F) The AnimalTFDB3 database was used to predict the binding sequence of CREB3L1 to the COL5A1 promoter, and the Dual-luciferase assay to detect the transcriptional activation of CREB3L1 to COL5A1 wild-type or mutant binding sequence. (G) PredictProtein database was used to analyze the nuclear localization sequence of CREB3L1. Data shown are results of three independent experiments and shown as mean ± standard deviation(SD). *P < 0.05, **P < 0.01