Transport of Biologically Active Ultrashort Peptides Using POT and LAT Carriers

Peptides are molecules containing from 2 to 100 amino-acid residues linked by peptide bonds. According to the number of amino-acid residues, there is a division of peptides into polypeptides (from 10 to 100 amino-acid residues) and short peptides or oligopeptides (no more than 10 amino-acid residues) [ 1 ]. According to the classification of the International Union of Pure and Applied Chemistry (IUPAC), short peptides consist of 10–20 amino-acid residues, while polypeptides consist of 20 or more amino-acid residues [ 1 , 2 ]. According to another classification, short peptides include compounds up to 40 amino-acid residues in length [ 3 ]. In addition, there is a group of ultrashort peptides (USPs) consisting, according to some data, of 2–4 amino-acid residues [ 4 ], or, according to others, of 3–8 amino-acid residues [ 5 , 6 , 7 ]. Peptides possess antioxidant, antimicrobial, antibacterial, anti-inflammatory, anticarcinogenic, antitumor and immunoregulatory properties [ 8 , 9 , 10 ]. They regulate the functions of endocrine, nervous and immune systems and are involved in cell differentiation, apoptosis [ 11 , 12 ] and proliferation [ 11 , 13 ]. Thus, peptides have a wide range of targeted biological activities [ 14 ]. This allows us to consider them as promising molecules for the development of drugs [ 15 ]. The ability of positively charged short peptides to enter cells was first discovered in human immunodeficiency virus studies. Currently, these peptides are used to transport drugs into cells [ 16 ]. The use of fluorescence and electron microscopy combined with molecular modeling revealed passive transport of positively charged peptides into HeLa cells based on cell membrane fusion induced by transported peptides [ 17 ]. There is evidence in the literature that USP can enter into the cell with the participation of carriers of proton-dependent oligopeptide cotransporters (POT). Since ultrashort peptides have a short length (from 2 to 8 amino-acid residues), it can be assumed that their transport can be realized with the participation of L-type amino-acid transporters (LATs). Di- and tripeptides, as well as various peptide-like compounds, such as β-lactam antibiotics, are absorbed into the epithelial cells of the intestine and kidneys using POT. These include PEPT1, PEPT2, PHT1, PHT2 carriers, consisting of 572–729 amino-acid residues (≈62–102 kDa). All the POT family members contain 12 transmembrane domains with N- and C-termini facing the cytosol. PHT1 and PHT2 recognize L-histidine as a substrate. PEPT1 and PEPT2 have high interspecies homology (about 80% in mice, rats, rabbits and humans), but sequence homology between these carriers for the same species is low (about 50%). Rat PHT1 and PHT2 have an amino-acid identity of about 50%, but they show insignificant sequence homology with PEPT1 and PEPT2 (less than 20%) [ 18 ]. PEPT1 and PEPT2 have a high degree of overlapping substrate specificity, being capable of amino-acid-sequence-independent transport of dipeptides and tripeptides [ 18 , 19 , 20 , 21 ]. It is unclear whether PHT1 and PHT2 proteins can transport the same range of di/tripeptides. However, the ability of PHT1/PHT2 to transport L-histidine distinguishes them from PEPT1/PEPT2 [ 19 ]. PEPT1 expression is more pronounced in the small intestine than in the kidneys. PEPT2 is predominantly expressed in the kidneys [ 22 , 23 ]. Both transporters are localized in the brush border membranes of epithelial cells. PEPT1 is a low-affinity peptide transporter, while PEPT2 is a high-affinity peptide transporter [ 18 ]. Amino-acid transporters are vital for nutrient uptake, neurotransmitter recycling, cell-redox balance and cell signaling. Essential amino acids are transported across the blood–brain barrier (BBB) using special carriers. Based on the difference in substrates, amino-acid carriers are divided into cationic, anionic and neutral. LAT are heterodimeric amino-acid transporters. LAT1 predominantly transports neutral amino acids such as leucine, tryptophan, tyrosine and phenylalanine across the BBB via a Na-independent pathway. It is also involved in the brain delivery of some other biologically active substances, including L-DOPA, melphalan, gabapentin and baclofen [ 19 ]. LAT2 exhibits broader substrate specificity, transporting smaller neutral amino acids [ 20 ]. There are only a few publications describing the mechanisms of peptide transport into the cell, and they are mainly dedicated to peptides longer than 7–10 amino-acid residues. USP transport into the cell has not been properly studied yet. Based on literature data and analysis of the structure of active centers of amino acid and peptide carriers, it can be assumed that some USPs can interact with active centers of transporters, including LAT, and be transported into the cell. There are works devoted to some USPs that refute this assumption. However, no such analysis has been carried out for the entire pool of di- and tripeptides so far

The presence of peptide transporters in the brain has aroused interest concerning their physiological role and localization in the central nervous system’s (CNS) tissues and cells. In the mammalian CNS, various amino acids, amines and USPs function as neurotransmitters and neuromodulators. They also participate in the cellular metabolism and homeostasis regulation. PEPT2 has been found in the cerebral cortex, olfactory bulbs, basal ganglia, cerebellum and medulla oblongata [ 43 ]. PEPT2 is largely expressed in choroid plexus epithelial cells and ependymal cells. Its expression is observed exclusively on the apical (facing CSF) membrane of choroid plexus cells in newborn and adult rats, which suggests that PEPT2 is involved in the peptide transport from the CSF into the blood [ 44 , 45 ]. PEPT2 was also found to be expressed in the cerebral cortex differently depending on age; moreover, its levels in the fetus and newborn tissues were significantly higher than in adults. PEPT2 expression was found in neurons in adult and newborn rats, and in astrocytes of newborn rats. At the same time, PEPT2 expression in BBB endothelial cells was not detected, which was consistent with in situ hybridization studies [ 46 ] and the lack of evidence for the penetration of dipeptides into endothelial cells of brain microvessels [ 47 ]. No signs of PEPT1 expression in the brain were detected. PEPT2 is expressed in epidermal keratinocytes [ 48 ]. The maximum concentration of PEPT2 is observed closer to the basal layer of the epidermis. This indicates that in epidermis, PEPT2 mediates the transport of oligopeptides from the basal layer [ 49 ]. PEPT2 mediates unilateral intracellular uptake of oligopeptides as an active transporter, activated by a transmembrane electrochemical proton gradient. Unlike keratinocytes, dermal fibroblasts and melanocytes do not express PEPT1 and PEPT2. The PEPT2 transporter, expressed mainly in keratinocytes, is assumed to be involved in the transcellular transport of oligopeptides and peptide-like drugs, delivering them to the underlying layers of the epidermis. PEPT1 is a nonspecific transporter. It is localized mainly on the brush border membrane of the small intestine, and to a lesser extent in the kidney proximal tubules [ 29 ]. PEPT2 is a more selective transporter, and is expressed predominantly in the apical membrane of kidney cells. In the kidney proximal tubules, PEPT1 and PEPT2 are localized differently: PEPT1 is found in the S1 segment; PEPT2 is found in the S2 and S3 segments [ 50 ]. In rats, radioactively labeled molecular probes detected PEPT1 in the renal-cortex region, and PEPT2—in the outer part of the medulla [ 51 ]. With a knocked-out PEPT2 gene, a violation of peptide absorption in kidneys and choroid plexuses was revealed in mice [ 52 , 53 ]. PEPT1 and PEPT2 may be applicable in the pharmacokinetics of b-lactam antibiotics by transporting them to target cells and promoting reabsorption in renal tubular cells after glomerular filtration [ 18 ]. Relatively little is known about the expression and distribution of PHT1 and PHT2 in various animal and human tissues. PHT1 mRNA has been found in the choroid plexus cells of the brain and retina in rats. PHT2 transcripts were expressed mainly in the lymphatic system, lungs and spleen of rats, but were practically not detected in the brain [ 54 , 55 ]. SLC15A4 and SLC15A3 transcripts, encoding PHT1 and PHT2, have been found in human- and rat-intestinal-tissue segments. Moreover, immunohistochemical analysis has shown that PHT1 was expressed in the small intestine villous epithelium [ 56 ]. Thus, PEPT1 and PEPT2 transporters are localized in cells of various organs and tissues. PEPT1 is predominantly found in kidney and intestinal cells, whereas PEPT2 is detected in CNS neurons and vascular endothelium. These data are important in the context of understanding the tissue-specific action of some USPs. It can be assumed that di- and tripeptides, being transported into tissues, depending on their structure, are capable of interacting with PEPT1 and PEPT2, and thus can exhibit their protective properties against cells of various tissues.

The spectrum of USPs’ biological activity is wide: they regulate the functions of the endocrine, nervous and immune systems. The mechanism of action of peptides consists in their ability to regulate gene expression and protein synthesis in plants, microorganisms, insects, birds, rodents, primates and humans [ 10 , 14 ]. USP can penetrate cell nucleus and nucleolus and interact with the nucleosome, histone proteins and single- and double-stranded DNA. DNA–peptide interactions are vital for template synthesis reactions, including sequence recognition in gene promoters, replication, transcription and repair. USPs are able to regulate the DNA methylation status, which is an epigenetic mechanism of gene activation or repression in normal, pathological and ageing conditions [ 14 ]. In this regard, it can be assumed that evolutionarily USPs, similar to the POT family peptide carriers, were one of the first signaling molecules capable of cellular homeostasis regulation. Violation of peptide bioregulation reduces the body’s resistance to external and internal destabilizing factors, which is one of the reasons for accelerated ageing and development of age-associated pathology [ 14 ]. In this regard, disruption of peptide transport may be an important link in the implementation of ageing mechanisms and pathogenesis of a wide range of diseases. PEPT1 and PEPT2 carriers transport mainly USP. Irie M. et al. (2005) studied the transport of neutral and charged USP with the participation of PEPT1. Transfer of Gly-Sar (glycyl-sarcosine) dipeptide into the cell using PEPT1 was described by methods of physicochemistry and molecular modeling [ 87 ]. Transport mechanisms of antihypertensive tripeptides LKP (Leu-Lys-Pro) and IQW (Ile-Gln-Trp) obtained from egg white were studied using the system of co-cultivation of monolayers of Caco-2 and HT29 cell lines. The results showed that the transport was significantly inhibited by Gly-Pro dipeptide, a competitive substrate of the PEPT1 peptide transporter. Transport from the apical to the basolateral side was significantly higher than in the opposite direction. These results indicated that PEPT1 was involved in the transport of LKP and IQW peptides. Moreover, siRNA was also used to knock PEPT1 expression down, and inhibited the transport significantly, suggesting that PEPT1 was involved in the transport process. Thus, an active pathway via PEPT1 transporter for the antihypertensive peptides LKP and IQW lies through monolayers of co-cultured Caco-2 and HT29 cell lines [ 88 ]. In Xenopus laevus oocytes, a new function of the β-Klotho protein was revealed, which consisted in the activity reduction for peptide transporters PEPT1 and PEPT2 [ 89 ]. As shown for the TRPV5 epithelial Ca 2+ channel [ 90 ] of amino-acid transporters [ 91 ], β-Klotho can stabilize transport proteins in the cell membrane and also suppress the action of peptide transporters. β-Klotho protein can probably affect the intestine transport of peptides. Peptide transport was activated in β-Klotho-deficient mice. It is assumed that an age-related decrease in the β-Klotho synthesis leads to a slowdown in peptide transport in intestinal cells [ 30 ]. The interaction of the antibacterial phosphonodipeptide alaphosphalin with mammalian H( + )/peptide cotransporters was studied in Caco-2 cells expressing low-affinity intestinal-type PEPT1 and SKPT cells expressing high-affinity renal-type PEPT2. Alafosfalin inhibited [(14)C]glycyl-sarcosine (Gly-Sar) uptake for PEPT1 and PEPT2, respectively. In both cell types, alaphosphalin was shown to affect only the affinity constant, but not the maximum uptake rate of glycylsarcosine (Gly-Sar). It has been established that alaphosphalin interacted with H( + )/peptide symporters and was transported through the intestinal epithelium in the H( + )-symport. Dipeptides with C-terminal carboxyl group substituted with a phosphonic function are high-affinity substrates for mammalian H( + )/peptide cotransporters. The antibacterial phosphonodipeptide alaphosphalin interacts with both mammalian H + /peptide symporters with high affinity. PEPT1 and PEPT2 do not seem to distinguish between a dipeptide and its derivative, where the C-terminal carboxyl group is replaced by a phosphonic acid. Phosphonodipeptides are of interest for studying the structural affinity bonds of PEPT1 and PEPT2 substrates [ 92 ]. There is evidence that PEPT1 may not transport all tripeptides into the cell [ 93 ]. The transepithelial transport of the VLPVPQK peptide (C peptide), which is an antioxidant and an angiotensin-converting enzyme inhibitor, via the PEPT1 transporter also remains poorly understood [ 94 ]. Thus, PEPT1 and PEPT2, as well as PHT1 and PHT2, belonging to the SLC transporters family, are the main peptide transporters in the body responsible for the proton-coupled transport of dipeptides and tripeptides. Their main function is to take up nitrogen in the small intestine (PEPT1) and reabsorb nitrogen from the g

Uptake of aromatic amino acids, such as L-tryptophan, by activated lymphoid cells occurs primarily through the L1 systemic transporter, a heterodimer comprising the heavy chain of CD98 and the light chain of LAT1. Regulation of amino-acid transport through LAT1-CD98 heterodimer is associated with the activation and differentiation of T-cells [ 117 ]. CD69 has been found to associate with the LAT1-CD98 transport complex and enhance the uptake of L-tryptophan, a metabolic precursor of Ah receptor ligands, which promotes IL-22 secretion [ 118 , 119 ]. IL-22 induces keratinocyte proliferation via the PI3KAkt-mtorc37 signaling pathway. CD69 was associated with the LAT1-CD98 amino-acid transfer complex on the plasma membrane of activated T cells and controlled mTORC activation in skin cells during an autoimmune inflammatory response [ 117 ]. An elevated LAT1 expression is observed in keratinocytes and skin-infiltrating lymphocytes of psoriatic lesions in humans and mice. However, deletion of LAT1 in keratinocytes does not attenuate the inflammatory response or their proliferation, which might be supported by the increased expression of alternative amino-acid transporters LAT2 and LAT3 [ 120 ]. LAT1 expression is upregulated in a number of primary tumors and metastatic lesions from more than 20 tissues/organs [ 121 ]. Moreover, correlations between LAT1 expression and negative prognosis have been shown in various tumors, such as breast cancer [ 122 ], including its highly proliferative ER+ subtype [ 123 ], bladder cancer [ 124 ], lung adenocarcinoma, lung neuroendocrine tumor, adenocarcinoma pancreatic ducts [ 125 ] and biliary tract cancer [ 126 ]. Based on these data, LAT1 was considered as a molecular target for anticancer therapy. Several LAT1-selective inhibitors have been synthesized [ 127 , 128 , 129 , 130 ]. They have demonstrated pronounced antitumor effects in animal models [ 131 , 132 ]. LAT1 influence on the endothelial cell functions in tumors has not been elucidated. In a model of rat bladder carcinoma induced by N-butyl-N-(4-hydroxybutyl)nitrosamine, increased LAT1 expression in tumor-associated microvessels was recorded [ 133 ]. A clinical and pathological study of human glioma showed LAT1 expression in both vascular endothelial cells and tumor cells, demonstrating significant correlations of LAT1 expression with the degree of pathology and intratumoral density of microvessels [ 134 ]. A connection between LAT1-mediated amino-acid signaling and growth-factor-dependent pro-angiogenic signaling, converging on the nutrient-responsive concentrator kinase mTORC1 for angiogenesis regulation, has also been revealed. The study of LAT1 functions may be promising for the development of modern cancer treatment methods, antiangiogenic therapy in particular [ 135 ]. LAT1 is suggested to play a crucial role in tumor-associated metabolic networks, supplying tumor cells with essential amino acids [ 136 ]. Tyrosine transport in human fibroblasts has been characterized by L-transporter isoforms (LAT1, LAT2, LAT3, LAT4). This study showed that LAT1 is involved in 90% of the total tyrosine uptake, as well as 51% of alanine. Not more than 10% can be accounted for by LAT2, LAT3 and LAT4 isoforms. LAT2 appears to be weak in tyrosine uptake. Alanine inhibited tyrosine transport up to 60%. Transport of tyrosine through LAT1 isoform had a higher affinity compared to other L-system transporters. Thus, LAT1 isoform is the main tyrosine transporter in human fibroblasts. It has been shown that there is competition between tyrosine and alanine for transport via LAT1 and LAT2 isoforms [ 137 ]. LAT1 transporter is involved in the pathogenesis of neurodegenerative disorders, amyotrophic lateral sclerosis (ALS) in particular. LAT1 has been shown to be involved in the uptake of [14C]L-citrulline by motor neuron-like cells NSC-34. The level of LAT1 expression was lower in superoxide dismutase 1 (NSC-34/hSOD1 G93A ) mutant cells compared to the control. Similarly, in the spinal cord of ALS in transgenic mice, a decrease in LAT1 synthesis in the motor neurons of mice with ALS was found. However, an increase in LAT1 expression in nonmotor neurons and astrocytes was observed in the gray matter of the spinal cord of mice with ALS [ 138 ]. As mentioned above, LAT1 is responsible for the transport of large neutral L-amino acids, as well as several drugs and prodrugs, across the BBB. However, the BBB is not the only barrier preventing the effective action of drugs in the brain. Brain parenchymal cell membranes represent a secondary barrier to drugs with intracellular target sites. In a study by Huttunen et al. LAT1 expression and function were quantified in cultures of primary neurons, astrocytes and immortalized BV2 mouse microglia. LAT1 was detected in all three cell types. The most active transport involving this carrier was found in astrocytes. LAT1 provided transport of the prodrugs developed during the study into the cell. Interestingly

Due to their high biological activity and low immunogenicity, USPs are suitable for the pharmacotherapy of many pathological conditions. USPs are convenient for synthesis, are biocompatible, are accessible for chemical modifications, and demonstrate molecular selectivity and specific interaction with various types of biological systems [ 179 , 180 , 181 ]. Physiological properties of biologically active peptides imply their direct effect on target tissues and organs; that is, when administered orally, peptides must be absorbed into the bloodstream through the intestinal barrier in their active forms. It is important to study the absorption mechanism of bioactive peptides for a better understanding of their biological effects [ 182 , 183 ]. Di- and tripeptide transporters are integral proteins of the plasma membrane. PEPT1, PEPT2, PHT1, PHT2 transporters are members of the proton-coupled oligopeptide transporter (POT) family; LAT1, LAT2, LAT3, LAT4 constitute the L system. Apparently, these transporter systems provide extra- and intracellular transport of USPs consisting of 2–4 amino-acid residues. PEPT1 is expressed in the small intestine; PEPT2 is expressed mainly in the brain and kidneys, as well as in the gastrointestinal tract, liver and lungs during late embryogenesis. This suggests that PEPT1 may serve as a peptide transporter in the embryonic period. Tissue localization of PHT1 and PHT2 transporters is currently poorly understood, although it is known that they are expressed in the brain. PEPT1 is believed to provide transport of di- and tripeptides, as well as some peptidomimetics and prodrugs, through the apical membrane of enterocytes, where they are hydrolyzed. After that, other amino acid and/or peptide transporters (probably LAT) deliver these substances into the blood. In kidneys, PEPT2 is responsible for reabsorption of these substances. Dipeptides, tripeptides, beta-lactams, ACE inhibitors and prodrugs are absorbed by cells against a concentration gradient by PEPT1 and PEPT2 carriers, whose activity is associated with an electrochemical proton gradient. Some di- and tripeptides undergo a rapid intracellular hydrolysis, after which amino acids are transported out of the cell via basolateral transporters. Hydrolysis-resistant substrates, which include most peptidomimetics, are released into the blood via a yet-to-be-identified basolateral peptide carrier and/or via other drug-transport systems. It is very important that not all USPs undergo a rapid intracellular hydrolysis [ 184 , 185 ]. It has been established that the dipeptides formed during the hydrolysis of the N-terminal fragments of the polypeptide chain by Asp, Gly and Pro or the hydrolysis of the C-terminal fragments by Pro, Ser, Thr and Asp are slightly hydrolysable. Their stability and biological activity have been confirmed experimentally [ 182 ]. A study on the hydrolysis of AEDG, KE, KEDW, KEDA peptides in saline, Ringtger’s solution, HCl in stomach homogenates, small and large intestines, and other organs (liver, kidneys and spleen) has been conducted. Tissue homogenate hydrolysis was assessed by cleavage of the terminal amino acid from each peptide using L-aminopeptidase. KEDW and KEDA peptides were found to be resistant to hydrolysis for 3–4 h in physiological saline, Ringtger’s solution, HCl and tissue homogenates [ 186 ]. KE peptide (to a greater extent) and AEDG peptide (to a lesser extent) were hydrolyzed in solutions and partially in tissues. For AEDG peptide, it remains unclear whether only the terminal amino acid was cleaved, or whether the remaining tripeptide was cleaved as well. These data may indicate a different degree of USP hydrolyzability and the presence of hydrolysis-resistant ones among them. Competitive inhibition of PEPT1/2, regulation of PEPT1/2 transcription and maintenance of the proton gradient are the factors for the regulation of transport via these carriers [ 186 , 187 ]. LAT1 and LAT2 carriers are involved in the pathogenesis of some diseases. In particular, their role in the tumor onset and development is being actively studied. LAT1 expression has been described as an important indicator of poor outcome in various human cancers [ 126 , 188 , 189 , 190 ]. The role of LAT1 and LAT2 amino-acid carriers in drug transport is being intensively studied. LAT1 preferentially transports large neutral amino acids, while LAT2 exhibits a broader substrate specificity, also transporting some smaller neutral amino acids. The exact localization of LAT1 and LAT2 on cell membranes is not always clear, while data on their distribution in tissues is sometimes contradictory. LAT1 is expressed in many tissues under normal conditions, as well as in a number of tumors. LAT2 is expressed in the kidney, colon and intestine. The potential effect of LAT1 and LAT2 on pharmacokinetics is limited by passive diffusion, competitive inhibition due to high levels of endogenous amino acids and relatively low drug affinity to LAT pro