Development of oxetane modified building blocks for peptide synthesis.

The synthesis and use of oxetane modified dipeptide building blocks in solution and solid-phase peptide synthesis (SPPS) is reported. The preparation of building blocks containing non-glycine residues at the N-terminus in a stereochemically controlled manner is challenging. Here, a practical 4-step route to such building blocks is demonstrated, through the synthesis of dipeptides containing contiguous alanine residues. The incorporation of these new derivatives at specific sites along the backbone of an alanine-rich peptide sequence containing eighteen amino acids is demonstrated via solid-phase peptide synthesis. Additionally, new methods to enable the incorporation of all 20 of the proteinogenic amino acids into such dipeptide building blocks are reported through modifications of the synthetic route (for Cys and Met) and by changes to the protecting group strategy (for His, Ser and Thr).

N-acetylaspartylglutamate (NAAG) and glutamate carboxypeptidase II: An abundant peptide neurotransmitter-enzyme system with multiple clinical applications.

N-Acetylaspartylglutamate (NAAG) is the third most prevalent neurotransmitter in the mammalian nervous system, yet its therapeutic potential is only now being fully recognized. Drugs that inhibit the inactivation of NAAG by glutamate carboxypeptidase II (GCPII) increase its extracellular concentration and its activation of its receptor, mGluR3. These drugs warrant attention, as they are effective in animal models of several clinical disorders including stroke, traumatic brain injury and schizophrenia. In inflammatory and neuropathic pain studies, GCPII inhibitors moderated both the primary and secondary pain responses when given systemically, locally or in brain regions associated with the pain perception pathway. The finding that GCPII inhibition also moderated the motor and cognitive effects of ethanol intoxication led to the discovery of their procognitive efficacy in long-term memory tests in control mice and in short-term memory in a mouse model of Alzheimer's disease. NAAG and GCPII inhibitors respectively reduce cocaine self-administration and the rewarding effects of a synthetic stimulant. Most recently, GCPII inhibition also has been reported to be efficacious in a model of inflammatory bowel disease. GCPII was first discovered as a protein expressed by and released from metastatic prostate cells where it is known as prostate specific membrane antigen (PSMA). GCPII inhibitors with high affinity for this protein have been developed as prostate imaging and radiochemical therapies for prostate cancer. Taken together, these data militate in favor of the development and application of GCPII inhibitors in more advanced preclinical research as a prelude to clinical trials.

Cadmium-induced hepatocellular injury: Modulatory effects of γ-glutamyl cysteine on the biomarkers of inflammation, DNA damage, and apoptotic cell death.

Cadmium is an extremely toxic pollutant that reaches human body through intake of the industrially polluted food and water as well as through cigarette smoking and exposure to polluted air. Cadmium accumulates in different body organs especially the liver. It induces tissue injury largely through inflammation and oxidative stress-based mechanisms. The aim of the current study was to investigate the ability of γ glutamyl cysteine (γGC) to protect against cadmium-induced hepatocellular injury employing Wistar rats as a mammalian model. The results of the current work indicated that γGC upregulated the level of the anti-inflammatory cytokine IL-10 and downregulated the levels of the pro-inflammatory cytokines (TNF-α, IL-6, and IL-1ß) in the cadmium-exposed rats. In addition, γGC reduced the liver tissues cadmium content in the cadmium-treated rats, suppressed the cadmium-induced hepatocellular apoptosis and oxidative modifications of cellular DNA, lipids, and proteins. Additionally, γGC enhanced the antioxidant potential of the liver tissues in the cadmium-treated rats as evidenced by a remarkable increase in the activity of the antioxidant enzymes superoxide dismutase and glutathione peroxidase and significant increase in the levels of the total antioxidant capacity and reduced glutathione as well as a significant reduction in oxidized to reduced glutathione (GSSG/GSH) ratio. Moreover, it effectively improved liver cell integrity in the cadmium-treated rats as demonstrated by a significant reduction in the serum activity of the liver enzymes (ALT and AST) and amelioration of the cadmium-evoked histopathological alterations. Together, these findings underscore, for the first time, the alleviating effects of γGC against cadmium-induced hepatocellular injury that is potentially mediated through reduction of liver tissue cadmium content along with modulation of both hepatocellular redox status and inflammatory cytokines.

The functional interplay of low molecular weight thiols in Mycobacterium tuberculosis.

BACKGROUND: Three low molecular weight thiols are synthesized by Mycobacterium tuberculosis (M.tb), namely ergothioneine (ERG), mycothiol (MSH) and gamma-glutamylcysteine (GGC). They are able to counteract reactive oxygen species (ROS) and/or reactive nitrogen species (RNS). In addition, the production of ERG is elevated in the MSH-deficient M.tb mutant, while the production of MSH is elevated in the ERG-deficient mutants. Furthermore, the production of GGC is elevated in the MSH-deficient mutant and the ERG-deficient mutants. The propensity of one thiol to be elevated in the absence of the other prompted further investigations into their interplay in M.tb. METHODS: To achieve that, we generated two M.tb mutants that are unable to produce ERG nor MSH but are able to produce a moderate (ΔegtD-mshA) or significantly high (ΔegtB-mshA) amount of GGC relative to the wild-type strain. In addition, we generated an M.tb mutant that is unable to produce GGC nor MSH but is able to produce a significantly low level of ERG (ΔegtA-mshA) relative to the wild-type strain. The susceptibilities of these mutants to various in vitro and ex vivo stress conditions were investigated and compared. RESULTS: The ΔegtA-mshA mutant was the most susceptible to cellular stress relative to its parent single mutant strains (ΔegtA and ∆mshA) and the other double mutants. In addition, it displayed a growth-defect in vitro, in mouse and human macrophages suggesting; that the complete inhibition of ERG, MSH and GGC biosynthesis is deleterious for the growth of M.tb. CONCLUSIONS: This study indicates that ERG, MSH and GGC are able to compensate for each other to maximize the protection and ensure the fitness of M.tb. This study therefore suggests that the most effective strategy to target thiol biosynthesis for anti-tuberculosis drug development would be the simultaneous inhibition of the biosynthesis of ERG, MSH and GGC.

Solution conformations of a linked construct of the Zika virus NS2B-NS3 protease.

The Zika virus presents a serious risk for global health. Crystal structures of different constructs of the Zika virus NS2B-NS3 protease (NS2B-NS3pro) have been determined with the aim to provide a basis for rational drug discovery. In these structures, the C-terminal ß-hairpin of NS2B, NS2Bc, was observed to be either disordered (open conformation) or bound to NS3pro complementing the substrate binding site (closed conformation). Enzymatically active constructs of flaviviral NS2B-NS3 proteases commonly used for inhibitor testing contain a covalent peptide linker between NS2B and NS3pro. Using a linked construct of Zika virus NS2B-NS3pro, we studied the location of NS2Bc relative to NS3pro in solution by pseudocontact shifts generated by a paramagnetic lanthanide tag attached to NS3pro. Both closed and open conformations were observed with different inhibitors. As the NS2B co-factor is involved in substrate binding of flaviviral NS2B-NS3 proteases, the destabilization of the closed conformation in the linked construct makes it an attractive tool to search for inhibitors that interfere with the formation of the enzymatically active, closed conformation.

Identification of Novel Inhibitors of Leishmania donovani γ-Glutamylcysteine Synthetase Using Structure-Based Virtual Screening, Docking, Molecular Dynamics Simulation, and in Vitro Studies.

Trypansomatids maintain their redox balance by the trypanothione-based redox system, enzymes of which exhibit differences from mammalian homologues. γ-Glutamylcysteine synthetase (Gcs) is an essential enzyme in this pathway that performs the first and rate-limiting step. l-Buthionine-(S,R)-sulfoximine (BSO), a specific inhibitor of Gcs, induces toxicity in hosts infected with Trypanosoma brucei, underlining the need for novel Gcs inhibitors. The present study reports identification of Leishmania donovani Gcs (LdGcs) inhibitors using computational approaches and their experimental validation. Analysis of inhibitor-LdGcs complexes shows modifications that could result in increased efficacy of these compounds.

Glycine release is regulated by metabotropic glutamate receptors sensitive to mGluR2/3 ligands and activated by N-acetylaspartylglutamate (NAAG).

The presence of metabotropic glutamate receptors (mGluRs) of group II modulating glycine exocytosis from glycinergic nerve endings of mouse spinal cord was investigated. Purified synaptosomes were selectively prelabeled with [(3)H]glycine through the neuronal transporter GlyT2 and subsequently depolarized by superfusion with 12 mM KCl. The selective mGluR2/3 agonist LY379268 inhibited the K(+)-evoked overflow of [(3)H]glycine in a concentration-dependent manner (EC(50) about 0.2 nM). The effect of LY379268 was prevented by the selective mGluR2/3 antagonist LY341495 (IC(50) about 1 nM). N-acetylaspartylglutamate (NAAG) inhibited [(3)H]glycine overflow with extraordinary potency (EC(50) about 50 fmol). In contrast, glutamate was ineffective up to 0.1 nM, excluding that glutamate contamination of commercial NAAG samples is responsible for the reported activity of NAAG at mGluR3. LY341495 antagonized the NAAG inhibition of [(3)H]glycine release. The effect of a combination of maximally effective concentrations of LY379268 and NAAG exhibited no additivity. The non-hydrolysable NAAG analogue N-acetylaspartyl-ß-linked glutamate (ß-NAAG) antagonized NAAG and LY379268. In conclusion, our results show that glycinergic nerve endings in spinal cord are endowed with group II mGluRs mediating inhibition of glycine exocytosis. NAAG can activate these presynaptic receptors with extremely high affinity and with characteristics compatible with the reported mGluR3 pharmacology. This article is part of a Special Issue entitled 'Metabotropic Glutamate Receptors'.

Species-dependent uptake of glycylsarcosine but not oseltamivir in Pichia pastoris expressing the rat, mouse, and human intestinal peptide transporter PEPT1.

The purpose of this study was to determine whether glycylsarcosine (a model dipeptide) and oseltamivir (an antiviral prodrug) exhibited a species-dependent uptake in yeast Pichia pastoris expressing the rat, mouse, and human homologs of PEPT1. Experiments were performed with [(3)H]glycylsarcosine (GlySar) in yeast P. pastoris expressing human, mouse, and rat peptide transporter 1 (PEPT1), in which uptake was examined as a function of time, concentration, potential inhibitors, and the dose-response inhibition of GlySar by oseltamivir. Studies with [(14)C]oseltamivir were also performed under identical experimental conditions. We found that GlySar exhibited saturable uptake in all three species, with K(m) values for human (0.86 mM) > mouse (0.30 mM) > rat (0.16 mM). GlySar uptake in the yeast transformants was specific for peptides (glycylproline) and peptide-like drugs (cefadroxil, cephradine, and valacyclovir), but was unaffected by glycine, l-histidine, cefazolin, cephalothin, cephapirin, acyclovir, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid, tetraethylammonium, and elacridar. Although oseltamivir caused a dose-dependent inhibition of GlySar uptake [IC(50) values for human (27.4 mM) > rat (18.3 mM) > mouse (10.7 mM)], the clinical relevance of this interaction would be very low in humans. Of importance, oseltamivir was not a substrate for the intestinal PEPT1 transporter in yeast expressing the three mammalian species tested. Instead, the prodrug exhibited nonspecific binding to the yeast vector and PEPT1 transformants. Finally, the mouse appeared to be a better animal model than the rat for exploring the intestinal absorption and pharmacokinetics of peptides and peptide-like drugs in human.

Trp-His, a vasorelaxant di-peptide, can inhibit extracellular Ca2+ entry to rat vascular smooth muscle cells through blockade of dihydropyridine-like L-type Ca2+ channels.

Our previous findings regarding the biological activities of small peptides revealed that a di-peptide, Trp-His (WH), could play a role in the prevention of vascular lesions, including cell proliferation and atherosclerosis. Its vasoprotective effects could be associated with suppression of the vasocontraction signaling cascade, but the underlying mechanism(s) remains obscure. In this study, we attempted to elucidate the vasoprotective mechanism of WH, in opposing the proliferation of rat vascular smooth muscle cells (VSMCs). In VSMCs from 8 week-old male Wistar rat thoracic aortae, WH evoked a significant dose-dependent anti-proliferation effect, without cytotoxicity. In mitogen-stimulated cell experiments, 300 µM WH inhibited cytosolic Ca(2+) elevation in VSMCs induced by 10 µM angiotensin II (Ang II). Furthermore, WH suppressed extracellular Ca(2+) entry into CaCl(2)-stimulated VSMCs. The biological capacity of WH as an intracellular Ca(2+) ([Ca(2+)](i)) suppressor was also proven when 50 µM Bay K8644 was used to enhance Ca(2+) entry via a voltage-dependent l-type Ca(2+) channel (VDCC) and 300 µM WH elicited a 23% reduction in [Ca(2+)](i). The absence of a reduction of the [Ca(2+)](i) by the mixture of tryptophan and histidine revealed the importance of the peptide backbone in the [Ca(2+)](i) reduction effect. Furthermore, the WH-induced [Ca(2+)](i) reduction was abolished by verapamil, but not by nifedipine, indicating that WH likely binds to an extracellular site of the VDCC at a site similar to that of the dihydropyridine type-Ca(2+) channel blockers.

Cocrystal structures of primed side-extending alpha-ketoamide inhibitors reveal novel calpain-inhibitor aromatic interactions.

Calpains are intracellular cysteine proteases that catalyze the cleavage of target proteins in response to Ca(2+) signaling. When Ca(2+) homeostasis is disrupted, calpain overactivation causes unregulated proteolysis, which can contribute to diseases such as postischemic injury and cataract formation. Potent calpain inhibitors exist, but of these many cross-react with other cysteine proteases and will need modification to specifically target calpain. Here, we present crystal structures of rat calpain 1 protease core (muI-II) bound to two alpha-ketoamide-based calpain inhibitors containing adenyl and piperazyl primed-side extensions. An unexpected aromatic-stacking interaction is observed between the primed-side adenine moiety and the Trp298 side chain. This interaction increased the potency of the inhibitor toward muI-II and heterodimeric m-calpain. Moreover, stacking orients the adenine such that it can be used as a scaffold for designing novel primed-side address regions, which could be incorporated into future inhibitors to enhance their calpain specificity.

In vitro and pharmacophore-based discovery of novel hPEPT1 inhibitors.

PURPOSE: The human proton-coupled small peptide carrier (hPEPT1) is a low-affinity, high-capacity transporter with broad substrate specificity. We have taken an iterative in vitro and in silico approach to the discovery of molecules with hPEPT1 affinity. METHODS: A pharmacophore-based approach was taken to identifying hPEPT1 inhibitors. The well-characterized and relatively high affinity ligands Gly-Sar, bestatin, and enalapril were used to generate a common features (HIPHOP) pharmacophore. This consisted of two hydrophobic features, a hydrogen bond donor, acceptor, and a negative ionizable feature. RESULTS: The pharmacophore was used to search the Comprehensive Medicinal Chemistry (CMC) database of more than 8000 drug-like molecules and retrieved 145 virtual hits mapping to the pharmacophore features. The highest scoring compounds within this set were selected and tested in a stably transfected CHO-hPepT1 cell model. The antidiabetic repaglinide and HMG CoA reductase inhibitor fluvastatin were found to inhibit hPEPT1 with sub-millimolar potency (IC(50) 178 +/- 1.0 and 337 +/- 4 microM, respectively). The pharmacophore was also able to identify known hPEPT1 substrates and inhibitors in further database mining of more than 500 commonly prescribed drugs. CONCLUSIONS: This study demonstrates the potential of combining computational and in vitro approaches to determine the affinity of compounds for hPEPT1 and, in turn, provides insights into key molecular interactions with this transporter.

Amino acid ester prodrugs of the antiviral agent 2-bromo-5,6-dichloro-1-(beta-D-ribofuranosyl)benzimidazole as potential substrates of hPEPT1 transporter.

Amino acid ester prodrugs of 2-bromo-5,6-dichloro-1-(beta-d-ribofuranosyl)benzimidazole (BDCRB) were synthesized and evaluated for their affinity for hPEPT1, an intestinal oligopeptide transporter. Assays of competitive inhibition of [(3)H]glycylsarcosine (Gly-Sar) uptake in HeLa/hPEPT1 cells by the amino acid ester prodrugs of BDCRB suggested their 2- to 4-fold higher affinity for hPEPT1 compared to BDCRB. Further, promoieties with hydrophobic side chains and l-configuration were preferred by the hPEPT1 transporter.

Decreased activity and expression of intestinal oligopeptide transporter PEPT1 in rats with hyperthyroidism in vivo.

PURPOSE: To examine the effect of thyroid hormone status on PEPT1 in vivo, the activity and expression of PEPT1 in the small intestine were examined in euthyroid and hyperthyroid rats. METHODS: Hyperthyroidism was induced by treating rats with L-thyroxine (12 mg/L) in the drinking water for 21 days. Transport activity was measured by everted small intestinal preparations and in situ intestinal loop technique. Expressions of PEPT1 mRNA and protein were evaluated by competitive polymerase chain reaction and Western blotting, respectively. RESULTS: The uptake of [14C]glycylsarcosine by everted small intestinal preparations was significantly decreased in hyperthyroid rats, whereas that of methyl-alpha-D-[14C(U)]-glucopyranoside was not altered. Kinetic analysis showed that the Vmax value for [14C]glycylsarcosine uptake was significantly decreased in hyperthyroid rats, whereas the Km value was not affected. The mean portal vein concentrations after intrajejunal administration of [14C]glycylsarcosine were also decreased in hyperthyroid rats. Moreover, hyperthyroidism caused a significant decrease in the expression of PEPT1 mRNA in the small intestine, whereas the expression of Na+/glucose cotransporter (SGLT1) mRNA was not changed. The level of PEPT1 protein was also decreased in the small intestine of hyperthyroid rats. CONCLUSIONS: These results indicate that in hyperthyroid rats, the activity and expression of PEPT1 were decreased in the small intestine.

Two peptidase activities decrease in treated bipolar disorder not schizophrenic patients.

BACKGROUND: Inhibition of prolyl oligopeptidase (PO) in primary neuronal cultures has been shown to reverse the effect of the common mood-stabilizers lithium, valproic acid and carbamazepine. In clinical studies, abnormal plasma PO activity has been associated with bipolar disorder (BD) and schizophrenia. However, this association is complicated by the discovery in bovine plasma of a Z-Pro-prolinal-insensitive peptidase (ZIP), a novel enzyme that cleaves the same substrate as PO. METHODS: We developed an assay to distinguish between ZIP and PO and measured both activities in plasma from 48 BD and 50 schizophrenic patients undergoing treatment and compared them with 50 control subjects. RESULTS: ZIP activity is restricted to blood plasma, whereas PO activity is present in the cytosol of lymphocytes, but can also be detected in blood plasma. Significant decreases in their plasma activities were found between treated BD (p = 0.007 and 0.03 respectively) but not schizophrenic (p > 0.05) patients and controls. CONCLUSIONS: We have found that the enzyme activity previously reported as plasma PO actually comprises two enzymes, PO and ZIP. This study shows a statistically significant decrease of both enzymes in BD patients undergoing lithium treatment. No statistically significant change in PO or ZIP activity is observed in schizophrenic patients.

Overexpression of gamma-glutamylcysteine synthetase in human malignant mesothelioma.

Mesothelioma is a fatal tumor resistant to all treatment modalities for reasons that are still unresolved. Glutathione (GSH)-associated pathways are induced by oxidants and cytotoxic drugs, and they are also involved in the progression and resistance of some tumor cells in vitro. The rate-limiting enzyme in GSH biosynthesis is gamma-glutamylcysteine synthetase (gamma GCS). However, the expression of this enzyme has not been systematically investigated in malignant tumors, and there are no studies of gamma GCS in biopsy specimens of malignant mesothelioma. We investigated the immunohistochemical distribution and expression of both subunits of gamma GCS in healthy pleural mesothelium, pleural mesothelioma tumor biopsy samples (34 cases), and mesothelioma cells in culture (7 cell lines). Nonmalignant mesothelium showed no immunoreactivity for either subunit in any of the cases. The heavy (catalytic) subunit of gamma GCS was highly immunostained in 29 and weakly positive in 5 cases. High-moderate and weak immunoreactivity of the light (regulatory) subunit of gamma GCS was found in 15 and 7 tumors, respectively, whereas 12 cases showed no reactivity. There was no correlation with either catalytic or regulatory subunit expression and patient survival. There was, however, a significant correlation between the heavy chain and multidrug resistance protein (MRP) 2 (P =.048), whereas no correlation was observed between the light chain and MRP1 or MRP2. Treatment of cultured mesothelioma cells with buthionine sulfoximine (BSO), to inhibit gamma GCS, significantly potentiated cisplatin-induced cytotoxicity mainly by nonapoptotic mechanism when assessed by counting the living cells, TUNEL (terminal deoxytransferase-mediated dUTP nick-end labeling) assay, and caspase-3 cleavage. In conclusion, gamma GCS is highly positive in most cases of malignant mesothelioma and may play an important role in the primary drug resistance of this tumor in vivo.

Calpain activity regulates the cell surface distribution of amyloid precursor protein. Inhibition of calpains enhances endosomal generation of beta-cleaved C-terminal APP fragments.

In murine L cells, treatment with calpeptin or calpain inhibitor III increased Abeta42, but not Abeta40, secretion in a dose-dependent fashion. This correlated with an increase in the levels of amyloid precursor protein (APP) carboxyl-terminal fragments (CTFs). Immunoprecipitation with novel mAbs directed against the carboxyl-terminus of APP or specific for the beta-cleaved CTF showed that generation of both alpha- and beta-cleaved CTFs increase proportionately following inhibition of calpains. Pulse-chase metabolic labeling confirmed that inhibiting calpains increases the production of alpha- and beta-cleaved APP metabolites. Immunolabeling showed greater betaCTF signal in calpeptin-treated cells, primarily in small vesicular compartments that were shown to be predominantly endosomal by colocalization with early endosomal antigen 1. A second mAb, which recognizes an extracellular/luminal epitope found on both APP and betaCTFs, gave more cell surface labeling of calpeptin-treated cells than control cells. Quantitative binding of this antibody confirmed that inhibiting calpains caused a partial redistribution of APP to the cell surface. These results demonstrate that 1) calpain inhibition results in a partial redistribution of APP to the cell surface, 2) this redistribution leads to an increase in both alpha- and beta-cleavage without changing the ratio of alphaCTFs/betaCTFs, and 3) the bulk of the betaCTFs in the cell are within early endosomes, confirming the importance of this compartment in APP processing.

Inhibitory effect of supramolecular polyrotaxane-dipeptide conjugates on digested peptide uptake via intestinal human peptide transporter.

The effect of polyrotaxane-dipeptide (Val-Lys) conjugates on the uptake of a model dipeptide (Gly-Sar) was examined via human peptide transporter (hPEPT1) on HeLa cells. Here, Val-Lys groups are introduced to alpha-CDs, which are threaded onto a poly(ethylene oxide) chain capped with bulky end-groups (polyrotaxane). The Gly-Sar uptake via hPEPT1 was significantly inhibited in the polyrotaxane conjugates, and this inhibitory effect was not explained by the sum of interaction between hPEPT1 and alpha-CD-Val-Lys conjugates. Further, the inhibition was significantly greater than those observed in dextran-Val-Lys conjugates. Therefore, our data clearly suggests that supramolecular structure in the polyrotaxane conjugates contributes considerably to the inhibitory effect via multivalent binding of Val-Lys groups with hPEPT1.

Enhanced proteolysis of IkappaBalpha and IkappaBbeta proteins in astrocytes by Moloney murine leukemia virus (MoMuLV)-ts1 infection: a potential mechanism of NF-kappaB activation.

Moloney murine leukemia virus (MoMuLV)-ts1-mediated neuronal degeneration in mice is likely due to loss of glial support and release of inflammatory cytokines and neurotoxins from surrounding ts1-infected glial cells including astrocytes. NF-kappaB is a transcription factor that participates in the transcriptional activation of a variety of immune and inflammatory genes. We investigated whether ts1 activates NF-kappaB in astrocytes and examined the mechanism(s) responsible for the activation of NF-kappaB by ts1 infection in vitro. Here we present evidence that ts1 infection of astrocytes in vitro activates NF-kappaB by enhanced proteolysis of the NF-kappaB inhibitors, IkappaBalpha and IkappaBbeta. In in vitro studies using protease inhibitors, IkappaBalpha proteolysis in ts1-infected astrocytes was significantly blocked by a specific calpain inhibitor calpeptin but not by MG-132, a specific proteasome inhibitor, whereas rapid IkappaBbeta proteolysis was blocked by MG-132. Furthermore, treatment with MG-132 increased levels of multiubiquitinated IkappaBbeta protein in ts1-infected astrocytes. These results indicate that the calpain proteolysis is a major mechanism of IkappaBalpha proteolysis in ts1-infected astrocytes. Additionally, ts1 infection of astrocytes in vitro increased expression of inducible nitric oxide synthase (iNOS), a NF-kappaB-dependent gene product. Our results suggest that NF-kappaB activation in ts1-infected astrocytes is mediated by enhanced proteolysis of IkappaBalpha and IkappaBbeta through two different proteolytic pathways, the calpain and ubiquitin-proteasome pathways, resulting in increased expression of iNOS, a NF-kappaB-dependent gene.

PEPT2-mediated uptake of neuropeptides in rat choroid plexus.

PURPOSE: The peptide transporter PEPT2 was recently shown to be functionally active in rat choroid plexus, suggesting that it may play a role in neuropeptide homeostasis in the cerebrospinal fluid. This study, therefore, examined the role of PEPT2 in mediating neuropeptide uptake into choroid plexus. METHODS: Whole-tissue rat choroid plexus uptake studies were performed on GlySar in the absence and presence of neuropeptides and on carnosine. RESULTS: The neuropeptides NAAG, CysGly, GlyGln, kyotorphin, and carnosine inhibited the uptake of radiolabeled GlySar at 1.0 mM concentrations. In contrast, TRH, [D-Arg2]-kyotorphin, glutathione, and homocarnosine did not inhibit GlySar uptake. Kyotorphin, an analgesic, was a competitive inhibitor of GlySar with a Ki of 8.0 microM. The direct uptake of carnosine was also shown to be mediated by PEPT2 in isolated choroid plexus (Km = 39.3 microM; Vmax = 73.9 pmol/mg/min). Radiolabeled carnosine uptake was inhibited by 1.0 mM concentrations of GlySar or carnosine but not homocarnosine, L-histidine, or beta-alanine. CONCLUSIONS: These findings indicate that PEPT2 mediates the uptake of a diverse group of neuropeptides in choroid plexus, and suggests a role for PEPT2 in the regulation of neuropeptides, peptide fragments, and peptidomimetics in cerebrospinal fluid.

Significance of substrate hydrophobicity for recognition by an oligopeptide transporter (PEPT1).

Our previous paper [(1999) Bioconjugate Chem. 10, 24-31] pointed out that hydrophobicity of substrates/inhibitors plays an important role in the recognition by an oligopeptide transporter (PEPT1) expressed in the human intestinal epithelial cell line Caco-2. To determine the significance of that hydrophobicity, we have now synthesized dipeptide analogues conjugating the epsilon-amino group of Lys in Val-Lys with aliphatic carboxylic acids: acetic acid (C2), propanoic acid (C3), pentanoic acid (C5), hexanoic acid (C6), and decanoic acid (C10). The affinities of these conjugates were estimated by their inhibition of the accumulation rate of Gly-Sar, a well-established substrate for PEPT1. With the increase in length of the hydrocarbon chain of the conjugates, i.e., in the hydrophobicity of the conjugates, the inhibition strengthened. Dixon-Webb plot analysis of the inhibition by the C10-conjugated dipeptide showed competitive inhibition. The trans-stimulation effect of Val-Lys conjugated to C10 or C5 on the uptake of Ceftibuten was observed using rat brush border membrane vesicles. This findings showed that these conjugates are transportable substrates. These results confirmed that the hydrophobicity of substrates/inhibitor is one of the factors in the recognition by PEPT1.