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

Chemical Modulation of the Human Oligopeptide Transporter 1, hPepT1

In humans, peptides derived from dietary proteins and peptide-like drugs are transported via the proton-dependent oligopeptide transporter hPepT1 (SLC15A1). hPepT1 is located across the apical membranes of the small intestine and kidney, where it serves as a high-capacity low-affinity transporter of a broad range of di- and tripeptides. hPepT1 is also overexpressed in the colon of inflammatory bowel disease (IBD) patients, where it mediates the transport of harmful peptides of bacterial origin. Therefore, hPepT1 is a drug target for prodrug substrates interacting with intracellular proteins or inhibitors blocking the transport of toxic bacterial products. In this study, we construct multiple structural models of hPepT1 representing different conformational states that occur during transport and inhibition. We then identify and characterize five ligands of hPepT1 using computational methods, such as virtual screening and QM-polarized ligand docking (QPLD), and experimental testing with uptake kinetic measurements and electrophysiological assays. Our results improve our understanding of the substrate and inhibitor specificity of hPepT1. Furthermore, the newly discovered ligands exhibit unique chemotypes, providing a framework for developing tool compounds with optimal intestinal absorption as well as future IBD therapeutics against this emerging drug target

Cumulative Compliance Frequency over Time.

<p>Cumulative Compliance Frequency over Time.</p

Cumulative Abstinence Frequency over Time.

<p>Cumulative Abstinence Frequency over Time.</p

Distribution of participants over modality and level of care.

<p>IP-In-patients; RES = Residential facility; IOP = Intensive outpatients, OP =  Outpatient; OPT = Opiate treatment programs.</p><p>Distribution of participants over modality and level of care.</p

Compliance and abstinence rates of participants across the six U.S. eastern states.

<p>MD = Maryland; ME =  Maine; NC =  North Carolina; RI =  Rhode Island: SC =  South Carolina; VT = Vermont. Comp = Compliance; Abs =  Abstinence. B = Both; F = First; L = Last.</p><p>Compliance and abstinence rates of participants across the six U.S. eastern states.</p

Distribution of participants by state.

<p>Distribution of participants by state.</p

Compliance and abstinence rates by participants according to treatment modality.

<p>Comp = Compliance; Abs = Abstinence. B = Both; F = First; L = Last.</p><p>Compliance and abstinence rates by participants according to treatment modality.</p

Patient level of care and minimum days between samples.

<p>Patient level of care and minimum days between samples.</p

Compliance and abstinence rates by participants according to level of care.

<p>IP =  In-Patient; RES = Residential facility; IOP =  Intensive Out-Patient; OP =  Outpatient; OTP =  Opiate Treatment Program. Comp = Compliance; Abs = Abstinence. B = Both; F = First; L = Last.</p><p>Compliance and abstinence rates by participants according to level of care.</p