Amino-acids, acyl-amino-acids, dipeptides, acyl-dipeptides, and derivatives of these compounds: Effects of irradiation with cathode rays and ultraviolet light.
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[Immunostimulating effects of muramyl dipeptide, glucosaminyl muramyl dipeptide and their synthetic derivatives in vitro].
The effect of muramyldipeptide (MDP), glucosaminylmuramyldipeptide (GMDP) and their six synthetic derivatives on production of tumor necrosis factor (TNF), interleukin-1 (IL-1) and interleukin-2 (IL-2) by murine spleen cells in vitro was studied. MDP induced insignificant TNF production and did not stimulate production of IL-1 by the murine splenocytes within a 24-hour cultivation period whereas in combination with lipopolysaccharide (LPS) it induced significant production of both the cytokins. GMDP induced marked production of TNF (54 per cent cytotoxic index) and IL-1 (stimulation index 8). Addition of LPS in an amount of 10 ng/ml increased production of TNF by the murine splenocytes under the effect of GMDP but had no effect on production of IL-1. Neither MDP nor GMDP even in combination with LPS induced production of IL-2 by splenocytes of mice DVA/2 and C57B1/6 at activation for 24 hours. All the synthetic derivatives of MDP and GMDP except the MDP polymer activated TNF production by the murine spleen cells. GMDP lysine had the highest effect: 67 per cent cytotoxic index. In combination with LPS its cytotoxic index amounted to 87 per cent. The TNF activity was always higher when LPS in an amount of 10 ng/ml was added to the glycopeptides.
Acetylmuramyl-Alanyl-Isoglutamine↗
[Immunostimulating properties of synthetic derivatives of muramyl dipeptide and glucosaminyl muramyl dipeptide in vitro].
Production of tumor necrosis factor (TNF) and interleukin-1 (IL-1) by macrophages of the spleen and peritoneal exudate of mice as well as cytotoxic factors (CFs) by murine splenocytes after in vitro activation was estimated. All the derivatives of muramyldipeptide (MDP) and glucosaminylmuramyldipeptide (GMDP) were able to induce production of TNF and CFs. In the presence of lipopolysaccharide (LPS), the effect was always higher. The response of the spleen macrophages to the effect of the preparations was higher than that of the peritoneal ones and ++non-fractionated splenocytes. GMDP and GMDP4 especially in the presence of LPS had the highest effect on induction of IL-1 by the murine peritoneal macrophages. On the contrary, MDP induced higher IL-1 synthesis by the spleen macrophages. The most active substances with respect to production of TNF, CFs and IL-1, i.e. MDP3 and GMDP4, might be recommended for immunotherapy of syngeneic tumors in animals.
Acetylmuramyl-Alanyl-Isoglutamine↗
Kinetic studies of dipeptide-based and amino acid-based peritoneal dialysis solutions.
BACKGROUND: Dipeptide-based peritoneal dialysis solutions may have potential advantages compared with the glucose or amino acid-based solutions. Dipeptides may hydrolyze in the peritoneal cavity, generating constituent amino acids and thereby increasing the osmolality of the dialysate. Dipeptides can also be a valuable source of amino acids, which are poorly soluble in water, such as tyrosine. METHODS: Dwell studies in rats were performed during four hours with dipeptide solutions containing five dipeptides (Gly-His, Ala-Tyr, Thr-Leu, Ser-Phe, Val-Lys), 8, or 16 mmol/L of each dipeptide (low or high dipeptide group). Dwell studies were also performed with a 1.1% amino acid solution (Nutrineal(R)). The model of dipeptide hydrolysis (hydrolysis rate, KH), diffusive (rate constant, KBDD) and convective transport as well as transport of constituent amino acids consisted of mass balance equations, written for each dipeptide and amino acid. RESULTS: Peritoneal volume with the amino acid solution decreased much faster than that with the high and low dipeptide solutions. KH for all dipeptides did not differ between the high and low dipeptide groups. In the low dipeptide group, KH was 0.004 +/- 0.004 mL/min (mean +/- SD) for Gly-His (the lowest) and 0.088 +/- 0.048 mL/min for Thr-Leu (the highest). KBDD was higher than KH for all dipeptides, the average being 0.2 +/- 0.05 mL/min. CONCLUSIONS: Dipeptides are hydrolyzed in the peritoneal cavity, generating constituent amino acids. However, the hydrolysis rate appears to be several times lower than the dipeptide diffusive transport rate from dialysate to blood. Due to the higher molecular weight and intraperitoneal generation of amino acids, the dipeptide-based solutions provide more sustained ultrafiltration than the amino acid solution. The plasma concentration of amino acids at 60 minutes, in relation to the dose of amino acids delivered between 0 and 60 minutes, is considerably higher during the dwells with amino acid-based solution than during dwells with the dipeptide-based solutions.
Amino Acids↗
Two broad-specificity dipeptide hydrolysing activities from cytoplasm of guinea pig brain, both of which contain prolinase but neither of which contain carnosinase activity.
Two dipeptide hydrolysing activities were purified from cytoplasm of guinea pig brain. Dipeptide hydrolase I has been shown to be a strict dipeptidase requiring a free amino and a free carboxy terminus, while dipeptide hydrolase II displays very low activity against Leu-Leu-Leu. Of the 41 dipeptides presented to both enzymes, 25 were hydrolysed by both enzymes, while six (including carnosine) were hydrolysed by neither. Six were hydrolysed solely by dipeptide hydrolase I and four were hydrolysed solely by Pro-Leu hydrolase II. Kinetic analysis suggested that dipeptides which were hydrolysed with unfavourable kinetics or which were not hydrolysed by one dipeptide hydrolase were generally hydrolysed with more favourable kinetics by the other dipeptide hydrolase. Dipeptide hydrolase I displays optimum activity at pH 9.0, while dipeptide hydrolase II was optimally active at pH 8.0. Both enzymes were inhibited by 1,10-phenanthroline, p-chloromercuribenzoate and bestatin. Dipeptide hydrolase II was more strongly inhibited by arphamenine B than was dipeptide hydrolase I. Dipeptide hydrolase II was also inhibited by N-ethyl maleimide, while dipeptide hydrolase I was inhibited by dithiothreitol. Native M(r) values of 70,000 and 67,000 were computed for dipeptide hydrolase I and dipeptide hydrolase II, respectively. Sodium dodecyl sulphate polyacrylamide gel electrophoresis conducted with each enzyme under denaturing conditions suggested that both enzymes were comprised of a single polypeptide chain.
Animals↗
Structural distribution of dipeptides that are identified to be determinants of intracellular protein stability.
The dipeptides that had been previously implicated as determinants of in vivo protein stability (Guruprasad, K., Reddy, B.V.B. and Pandit, M.W., 1990. Protein Eng. 4, 155-161) have been reassessed on a latest data set and about 25% dipeptide combinations (102 dipeptides) were found to play significant role in determining the intracellular protein stability. These were classified as stabilizing dipeptides (Stb), destabilizing dipeptides (Dst) and normal dipeptides (Nor). By different theoretical approaches we have investigated the global localization of these dipeptides in a set of 303 best resolved (< or = 2.0A) non-homologous X-ray defined protein structures. The Dst dipeptides are found to be more of hydrophilic combinations where as Stb dipeptides are more of hydrophobic combinations. We observed a significant difference in overall frequency of occurrence of Stb and Dst dipeptides in different secondary structural regions. The sensitive dipeptides (Stb + Dst) are less in beta-strands and more in coils. A high frequency of occurrence of Stb are observed in the regions closer to the molecular surface compared to the Dst and Nor dipeptides. A significantly high dipole interactions are observed in the Dst dipeptides. The studies indicate that though the Dst dipeptides are more of hydrophilic nature they are localized significantly more in the buried regions of protein structures, on the other hand Stb are more of hydrophobic nature but relatively more accessible to the solvent. These dipeptides therefore increasing sensitivity of the protein to external environment, any alteration in their occurrence in the sequence could increase or decrease intracellular stability of the protein. These observations are useful to select mutations to alter intracellular stability of a given protein and therefore have implications in protein engineering.
Binding Sites↗
Uptake and metabolism of dipeptides by human red blood cells.
A function of the abundant cytoplasmic peptidases in red blood cells could be hydrolysis of oligopeptides circulating in plasma. To investigate whether human red blood cells actively transport dipeptides for this purpose, these cells were incubated with 14C-labelled glycylproline, glycylsarcosine, glycine, proline and alanine. There was uptake of each dipeptide, as indicated by their recovery as dipeptides in the cell cytoplasm. However, after a brief time (1-2 min) uptake of dipeptides abruptly ceased, while that of amino acids continued. As a result, after 30 min red blood cell uptake of amino acids was 5-13-fold greater than that of any dipeptide. Investigation of intracellular contents after 1 min of incubation revealed different metabolism for different dipeptides. The composition of intracellular radioactivity was 19-71% as intact dipeptides, 0-20% as free amino acids and 8-77% as neither dipeptides nor constituent amino acids. Investigation of the mechanism of dipeptide uptake by red blood cells showed: (1) a lack of hydrolysis by the plasma membrane, (2) no non-specific binding to the plasma membrane, and (3) a lack of saturation over a wide range of concentrations (0.05-50 mM). The data suggest that the mechanism of uptake of trace amounts of dipeptides by human red blood cells is either by simple diffusion or by a carrier system which has a very weak affinity for dipeptides. Upon entry, depending on the molecular structure, dipeptides are either hydrolysed or transformed into new compounds. The red blood cell uptake, however, does not appear to play any appreciable role in clearance of dipeptides from the plasma in the human.
Alanine↗
Conformational study on trans- and cis-N-acetyl-N'-methylamides of Pro-Xaa dipeptides.
Conformational free energy calculations using an empirical potential (ECEPP/2) and the hydration shell model were carried out on the N-acetyl-N'-methylamides of Pro-Xaa dipeptides (Xaa = Ala, Leu, Val, Gly, Cys, Met, Phe, Tyr, Asn, Asp, and Ser) with trans and cis peptide bonds preceding proline residue in the unhydrated and hydrated states. As compared with the results obtained by using the earlier version of ECEPP, the values of beta-bend probabilities are doubled. The average calculated population of cis-dipeptide is about 4%, which is close to the abundance obtained from the analysis of X-ray crystal structures of proteins. The beta-bends are the most dominant structures of cis-dipeptides. Type I, usually having intramolecular hydrogen bonds, contributes greatly to the beta-bend conformations of trans- and cis-dipeptides. However, type I beta-bends of cis-dipeptides do not have any hydrogen bonds. By including the hydration, the beta-bend probabilities for trans- and cis-dipeptides decreased, indicating that the interactions of water molecules with a backbone or side-chain may force the dipeptides to be more distorted or extended. In particular, type II is found to be a dominant beta-bend conformation of trans- and cis-Pro-Gly dipeptides in both the unhydrated and hydrated states. In general, the calculated propensities for Pro-Xaa dipeptides to adopt beta-bend conformations are reasonably consistent with available experimental data. From comparing conformations of Pro and Xaa residues in the dipeptides and single residues, we found that inter-residue interactions and hydration are of importance in determining the conformational properties of the Pro-Xaa dipeptide.
Acetates↗
Activation of mouse peritoneal adherent cells with N-acyl muramyl dipeptide derivatives.
The effect of N-acyl derivatives of muramyl dipeptide (N-acetyl muramyl-L-alanyl-D-isoglutamine) on the activation of peritoneal adherent cells (PAC) in vivo and on the stimulation of nonspecific host resistance against Escherichia coli infection was examined in comparison with the effect of 6-O-stearoyl muramyl dipeptide. N-acyl muramyl dipeptide derivatives increased the release of hydrogen peroxide (H2O2) by PAC from mice treated 1 day before upon stimulation with phorbol myristate acetate, and their activities did not depend on the chain length or kinds of fatty acids introduced. The results obtained using N-stearoyl muramyl dipeptide analogs indicated that the acyl moiety combined to muramic acid played a more important role in the ability of PAC to release H2O2 than did the peptide moiety. PAC from mice treated with N-stearoyl muramyl dipeptide, N-(3-hydroxy-2-docosylhexacosanoyl) muramyl dipeptide, and 6-O-stearoyl muramyl dipeptide 1 day before, including 20 to 42% polymorphonuclear leukocytes, released large amount of H2O2, and most of the H2O2 released was due to the attribution of polymorphonuclear leukocytes. The cytostatic activity of PAC from mice treated with these three compounds reached a maximum on day 3 after injection, and the cytolytic activity of PAC was induced by N-stearoyl muramyl dipeptide on day 3 and by 6-O-stearoyl muramyl dipeptide on day 1 after injection. In contrast to the above results, N-acyl muramyl dipeptide derivatives did not stimulate nonspecific host resistance against E. coli infection in mice when compared to 6-O-stearoyl muramyl dipeptide.
Acetylmuramyl-Alanyl-Isoglutamine↗
Preparation of platinum(IV) complexes with dipeptide and diimine. X-ray crystal structure and 195Pt NMR spectra.
We prepared platinum(IV) complexes containing dipeptide and diimine or diamine, the [PtCl(dipeptide-N,N,O)(diimine or diamine)]Cl complex, where -N,N,O means dipeptide coordinated as a tridentate chelate, dipeptide=glycylglycine (NH(2)CH(2)CON(-)CH(2)COO(-), digly, where two protons of dipeptide are detached when the dipeptide coordinates to metal ion as a tridentate chelate), glycyl-L-alanine (NH(2)CH(2)CON(-)CHCH(3)COO(-), gly-L-ala), L-alanylglycine (NH(2)CH CH(3)CON(-)CH(2)COO(-), L-alagly), or L-alanyl-L-alanine (NH(2)CHCH(3)CON(-)CHCH(3)COO(-), dil-ala), and diimine or diamine=bipyridine (bpy), ethylenediamine (en), N-methylethylenediamine (N-Me-en), or N,N'-dimethylethylenediamine (N,N'-diMe-en). In the complexes containing gly-L-ala or dil-ala, two separate peaks of the (195)Pt NMR spectra of the [PtCl(dipeptide-N,N,O)(diimine or diamine)]Cl complexes appeared in, but in the complexes containing digly or L-alagly, one peak which contained two overlapped signals appeared. One of the two complexes containing gly-L-ala and bpy, [PtCl(gly-L-ala-N,N,O)(bpy)]NO(3), crystallized and was analyzed. This complex has the monoclinic space group P2(1)2(1)2(1) with unit cell dimensions of a=9.7906(3)A, b=11.1847(2)A, c=16.6796(2)A, Z=4. The crystal data revealed that this [PtCl(gly-L-ala-N,N,O)(bpy)]NO(3) complex has the near- (Cl, CH(3)) configuration of two possible isomers. Based on elemental analysis, the other complex must have the near- (Cl, CH(3))-[PtCl(gly-L-ala-N,N,O)(bpy)]NO(3) configuration. The (195)Pt NMR chemical shifts of the near- (Cl, CH(3))-[PtCl(gly-L-ala-N,N,O)(bpy)]NO(3) complex and the far- (Cl, CH(3))-[PtCl(gly-L-ala-N,N,O)(bpy)]NO(3) complex are 0 ppm and -19 ppm, respectively (0 ppm for the Na(2)[PtCl(6)] signal). The additive property of the (195)Pt NMR chemical shift is discussed. The (195)Pt NMR chemical shifts of [PtCl(dipeptide-N,N,O)(bpy)]Cl appeared at a higher field when the H attached to the dipeptide carbon atom was replaced with a methyl group. On the other hand, the (195)Pt NMR chemicals shifts of [PtCl(dipeptide-N,N,O)(diamine)] appeared at a lower field when the H attached to the diamine nitrogen atom was replaced with a methyl group, in the order of [PtCl(digly-N,N,O)(en)]Cl, [PtCl(digly-N,N,O)(N-Me-en)]Cl, and [PtCl(digly-N,N,O)(N,N'-diMe-en)]Cl.
Crystallography, X-Ray↗
Principles of self-assembly of helical pores from dendritic dipeptides.
The self-assembly of the dendritic dipeptides (4-3,4-3,5)nG2-CH2-Boc-L-Tyr-L-Ala-OMe and their achiral dendritic alcohol (4-3,4-3,5)nG2-CH2OH precursors, both with n = 1-16, where n represents the number of methylenic units in the alkyl groups of the dendron, are reported. All chiral dendritic dipeptides and achiral dendritic alcohols self-assemble into helical porous columns that are stable in both solution and solid state. The pore diameter (D(pore)) of the columns self-assembled from dendritic dipeptides is approximately 10 A larger than that of structures assembled from dendritic alcohols. The increase of the D(pore) at the transition from dendritic alcohol to dendritic dipeptide is accompanied by a decreased solid angle of the building block. This trend is in agreement with previous pore size-solid angle dependences observed with different protective groups of the dipeptide and primary structures of the dendron. However, within the series of dendritic alcohols and dendritic dipeptides with various n, the D(pore) increases when the solid angle increases. The results of these investigations together with those of previous studies on the role of dipeptide stereochemistry and protective groups on this self-assembly process provide the molecular principles required to program the construction of supramolecular helical pores with diameter controlled at the A level from a single dendritic dipeptide architecture. These principles are expected to be valid for libraries of dendritic dipeptides based on dendrons and dipeptides with various primary structures.
Circular Dichroism↗
Identification of histidyl and thiol groups at the active site of rabbit renal dipeptide transporter.
Active transport of dipeptides in rabbit renal brush-border membrane vesicles is energized by an inward-directed H+ gradient rather than a Na+ gradient. We examined the effects of treatment of membrane vesicles with diethylpyrocarbonate (DEP), a reagent specific for histidyl groups, on this H+ gradient-dependent dipeptide uptake. DEP inhibited the uptake of all three dipeptides studied, Gly-sarcosine, Gly-Gly, and Gly-Pro (Ki = 0.6-0.9 mM), and the inhibition was noncompetitive. The dipeptide transporter could be protected from DEP inhibition by the presence of dipeptide substrates during the treatment of the vesicles with the inhibitor, whereas leucine plus Na+ failed to offer the protection. Na+-dependent leucine uptake was also inhibited by DEP (Ki = 2.5 mM) and the amino acid transporter could be protected from the inhibition by leucine plus Na+, but not by dipeptides. Treatment of membrane vesicles with the thiol group-specific reagents, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole,3-bromopyruvate, p-chloromercuribenzenesulfonic acid, and N-ethylmaleimide, also inhibited the H+ gradient-dependent dipeptide uptake. The potency of their inhibition was in the order: 7-chloro-4-nitrobenz-2-oxa-1,3-diazol greater than p-chloromercuribenzenesulfonic acid greater than 3-bromopyruvate greater than N-ethylmaleimide. The inhibition could be reversed in some cases by treatment of the membrane vesicles with reducing agents such as 2,3-dimercaptopropanol following incubation with the inhibitors. Dipeptide substrates could protect the dipeptide transporter from the inhibition. We conclude that histidyl and thiol groups are present at or near the substrate-binding site of the rabbit renal dipeptide transporter.
4-Chloromercuribenzenesulfonate↗
The action of leucyl-leucine methyl ester on cytotoxic lymphocytes requires uptake by a novel dipeptide-specific facilitated transport system and dipeptidyl peptidase I-mediated conversion to membranolytic products.
The mechanism of toxicity for cytolytic lymphocytes of Leu-Leu-OMe and related dipeptide derivatives was examined. Selective inhibition of dipeptidyl peptidase I (DPPI), a lysosomal thiol protease highly enriched in cytotoxic lymphocytes, prevented all natural killer (NK) toxic effects of such agents. However, many DPPI substrates were found to possess no NK toxic properties. For some such agents, this lack of NK toxicity appeared to be related to the lack of uptake by lymphocytes. In this regard, Leu-Leu-OMe was found to be incorporated by lymphocytes and monocytes via a saturable facilitated transport mechanism with characteristics distinct from previously characterized mammalian dipeptide transport processes. This novel transport process was found to be specific for dipeptides composed of selective L-stereoisomer amino acids and enhanced by hydrophobic ester or amide additions to the COOH terminus of dipeptides. Maximal rates of Leu-Leu-OMe uptake by T8 and NK cell-enriched peripheral blood lymphocytes (PBL) were four- to sixfold higher than for T4-enriched PBL or PBL depleted of Leu-Leu-OMe-sensitive cytotoxic lymphocytes. All dipeptide amides or esters with NK toxic properties were found to act as competitive inhibitors of [3H]Leu-Leu-OMe uptake by PBL. However, some NK nontoxic DPPI substrates were found to be comparable with Leu-Leu-OMe in avidity for this transport process. Such agents were noted to possess one or more hydrophilic amino acid side chains and were found not to mediate red blood cell lysis when subjected to the acyl transferase activity of DPPI. Thus, uptake by a dipeptide-specific facilitated transport mechanism and conversion by DPPI to hydrophobic polymerization products with membranolytic properties were found to be common features of NK toxic dipeptide derivatives. The presence of a previously unreported dipeptide transport mechanism within blood leukocytes and the selective enrichment of the granule enzyme, DPPI, within cytotoxic effector cells of lymphoid or myeloid lineage appear to afford a unique mechanism for the targeting of immunotherapeutic reagents composed of simple dipeptide esters or amides.
Acyltransferases↗
Mechanism of hepatic assimilation of dipeptides. Transport versus hydrolysis.
To investigate dipeptide assimilation by the liver, a series of interrelated experiments were performed in rats. Partial hepatectomy prolonged the plasma half-life (min) of Gly-Ala (3.42 +/- 0.22 versus 4.90 +/- 0.35, p less than 0.05) but had no significant effect on plasma half-life of Gly-Leu, Gly-Pro, or Gly-Sar. We then investigated the rate of disappearance (mumol X (g liver X h)-1) of the above four dipeptides (initial concentration = 1 mM) from the medium during isolated liver perfusion. The order of dipeptide disappearance was: Gly-Leu (8.75 +/- 0.65) greater than Gly-Ala (3.36 +/- 0.46) greater than Gly-Pro (1.29 +/- 0.54) greater than Gly-Sar (0.35 +/- 0.12). This order of dipeptide disappearance corresponded exactly to the order of the rates of glycine accumulation in the medium during liver perfusion with the four dipeptides. Addition of glucagon had no effect on the disappearance rate of Gly-Ala from the medium, but reduced accumulation rates of glycine (3.39 +/- 0.30 versus 1.42 +/- 30, p less than 0.01) and alanine (4.42 +/- 0.66 versus 1.35 +/- 0.39, p less than 0.01). Finally, we found that hydrolysis by the liver plasma membranes and/or perfusion medium accounted for disappearance of dipeptides. In conclusion, the liver does not appear to have a transport system for dipeptides, but assimilates dipeptides by extracellular hydrolysis. Hydrolysis is achieved by enzymes either located on the plasma membranes or released from the cytosol. The amino acid residues released as the result of dipeptide hydrolysis are then taken up by the liver.
Animals↗
Proton-driven dipeptide uptake in primary cultured rabbit conjunctival epithelial cells.
PURPOSE: To characterize proton-driven carrier-mediated dipeptide uptake in primary cultured conjunctival epithelial cells of the pigmented rabbit using beta-alanyl-L-histidine (L-carnosine) as a model dipeptide substrate. METHODS: Uptake of tritiated L-carnosine was monitored using conjunctival epithelial cells on days 6 through 8 in culture on a filter support. The structural features of dileucine stereoisomers and cephalexin contributing to interaction with the dipeptide transporter were evaluated by computer modeling and inhibition of tritiated L-carnosine uptake. RESULTS: Uptake of L-carnosine by primary cultured conjunctival epithelial cells in the presence of an inwardly directed proton gradient showed directional asymmetry (favoring apical uptake by a factor of five), temperature dependence, and saturability correlated with substrate concentration, with a Michaelis-Menten constant (Km) of 0.3 +/- 0.03 mM and a maximum uptake rate (Vmax) of 22.0 +/- 1.0 picomoles per milligram protein per minute. L-Carnosine uptake was optimal at pH 6.0 and was reduced by 60% and 35%, respectively, by 50 microM p-trifluoromethoxyphenylhydrazone (a proton ionophore) and by acid preloading with 50 mM NH4Cl. The constituent amino acids did not inhibit L-carnosine uptake. L-Carnosine uptake was inhibited, however, from 50% to 80% by other dipeptides and structurally similar drugs such as bestatin, beta-lactam antibiotics, and angiotensin-converting enzyme inhibitors. The LL, LD, or DL forms of the dipeptide Leu-Leu inhibited tritiated L-carnosine uptake by approximately 60%, 40%, and 70%, respectively. By contrast, the DD form did not inhibit uptake. Results from computer modeling suggest that an appropriate dipeptide N-terminal to C-terminal distance and a favorable orientation of the side chains may be important for substrate interaction with the conjunctival dipeptide transporter. CONCLUSIONS: Uptake of the dipeptide L-carnosine in primary cultured pigmented rabbit conjunctival epithelial cells is probably mediated by a proton-driven dipeptide transporter. This transporter may be used for optimizing the uptake of structurally similar peptidomimetic drugs.
Ammonium Chloride↗
Detection of muramyl dipeptide-sensing pathway defects in patients with Crohn's disease.
BACKGROUND AND AIMS: Crohn's disease is strongly associated with double mutations in NOD2/CARD15. Three common mutations (Arg702Trp, Gly908Arg, Leu1007fs) impair innate immune responses to bacterial muramyl dipeptide. Rare NOD2 variants occur, but it is difficult to both identify them and assess their functional effect. We assessed the true frequency of defective muramyl dipeptide sensing in Crohn's disease and developed a rapid diagnostic assay. MATERIALS AND METHODS: An ex vivo assay was established and validated based on muramyl dipeptide stimulation of peripheral blood mononuclear cell cytokine production. Muramyl dipeptide-induced enhancement of interleukin (IL)-8 secretion and synergistic increase in lipopolysaccharide-induced IL-1beta secretion were studied. Assay results were compared with NOD2 genotype status (3 common mutations and rare variants) in 91 individuals including a prospective cohort of 49 patients with Crohn's disease. RESULTS: The assay was highly sensitive and specific for detection of profound defects in muramyl dipeptide sensing caused by double NOD2 mutations (IL-8 P = 0.0002; IL-1beta P = 0.0002). Disease state, active inflammation, or concurrent use of immunosuppressive medication did not influence results. Healthy NOD2 heterozygotes had modest impairment of muramyl dipeptide induced IL-8 secretion (P = 0.003). Only 1 of 7 patients with Crohn's disease with both a common mutation and a rare variant had a profound muramyl dipeptide-sensing defect. CONCLUSIONS: Profound defects in muramyl dipeptide sensing were found in 10% of patients with Crohn's disease. Defects were caused exclusively by inherited mutations in NOD2. The ex vivo assay has multiple potential applications as a clinical diagnostic tool to distinguish patients with muramyl dipeptide-sensing defects and for research investigation.
Acetylmuramyl-Alanyl-Isoglutamine↗
Cyclic retro-inverso dipeptides with two aromatic side chains. II. Conformational analysis.
The conformations of cis and trans cyclic retro-inverso dipeptides--2-[(4-hydroxy)benzyl]-5-benzyl-4,6(1H,2H,3H,5H)-pyrimidinedi one (c[mTyr-gPhe]), and 2-benzyl-5-amino-5-[(4-hydroxy)benzyl]-4,6(1H,2H,3H,5H)-pyrimidinedione (c[mTyr-gPhe]), and 2-benzyl-5-amino-5-[(4-hydroxy)benzyl]-4,6(1H,2H,3H,5H)-pyrimidinedione (c[(alpha-amino)mTyr-gPhe])--and the parent cyclic dipeptides--c[tyrosyl-phenylalanine] (cis-c[L-Tyr-L-Phe]) and c[tyrosyl-D-phenylalanine] (trans-c[L-Tyr-D-Phe])--were studied by using 1H-nmr spectroscopy and semiempirical energy calculations. In the cis compounds of all the cyclic retro-inverso and parent dipeptides, the most stable conformer has both aromatic side chains sharing the space over the backbone ring in a "face-to-face" fashion. All the trans compounds predominantly assume a "sandwich" conformation in which the two aromatic rings are folded back over the backbone ring on opposite sides. However, different conformational preferences were observed for the backbones between the retro-inverso and parent cyclic dipeptides. The parent cyclic dipeptide trans-c[L-Tyr-D-Phe] adopts two types of boat structures with different side-chain orientations in almost equal amounts: one with the Tyr side chain in a pseudoaxial position and the Phe side chain in a pseudoequatorial position, the other with the Tyr side chain in a pseudoequatorial position and the Phe side chain in a pseudoaxial position. On the other hand, the cyclic retro-inverso dipeptides trans-c[mPhe-gTyr] and trans c[mTyr-gPhe] assume only one type of boat structure in which the malonyl side chain is in a pseudoequatorial and the gem-diamino side chain is in a pseudoaxial position. In addition to the preferred conformations, the conformational energies of the C alpha--C beta bonds in the malonyl and gem-diamino residues were estimated from the temperature variation of vicinal 1H--1H coupling constants for the H--C alpha--C beta--H groupings observed for the trans isomers of cyclic retro-inverso dipeptides. The energies were evaluated to be 1.1 and 1.8 kcal mol-1 for the malonyl and gem-diamino residues, respectively. Applying these energies to the parent cyclic dipeptide trans-c[L-Tyr-D-Phe], the observed fractions of three side-chain conformations are reasonably reproduced. The conformational energies as well as conformational properties of the molecules estimated in this investigation may be useful to refine force constants for both parent and retro-inverso peptides with aromatic side chains.
Amino Acid Sequence↗
Dipeptide frequency/bias analysis identifies conserved sites of nonrandomness shared by cysteine-rich motifs.
This report describes the application of a simple computational tool, AAPAIR.TAB, for the systematic analysis of the cysteine-rich EGF, Sushi, and Laminin motif/sequence families at the two-amino acid level. Automated dipeptide frequency/bias analysis detects preferences in the distribution of amino acids in established protein families, by determining which "ordered dipeptides" occur most frequently in comprehensive motif-specific sequence data sets. Graphic display of the dipeptide frequency/bias data revealed family-specific preferences for certain dipeptides, but more importantly detected a shared preference for employment of the ordered dipeptides Gly-Tyr (GY) and Gly-Phe (GF) in all three protein families. The dipeptide Asn-Gly (NG) also exhibited high-frequency and bias in the EGF and Sushi motif families, whereas Asn-Thr (NT) was distinguished in the Laminin family. Evaluation of the distribution of dipeptides identified by frequency/bias analysis subsequently revealed the highly restricted localization of the G(F/Y) and N(G/T) sequence elements at two separate sites of extreme conservation in the consensus sequence of all three sequence families. The similar employment of the high-frequency/bias dipeptides in three distinct protein sequence families was further correlated with the concurrence of these shared molecular determinants at similar positions within the distinctive scaffolds of three structurally divergent, but similarly employed, motif modules.
Amino Acid Motifs↗