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PubMed1999

Proline affects oligomerization of a coiled coil by inducing a kink in a long helix.

The structural effect of a proline in a helix in trifluoroethanol (TFE)/water medium was examined on a 29-mer peptide and its proline analog derived from the leucine zipper (LZ)-like motif of gp41 (the transmembrane glycoprotein of HIV-1) by NMR and circular dichroism (CD) spectroscopies. Lower helical content was found for the proline mutant from the CD study. NMR data show that distortion of the helix by proline is local and occurs mainly on the N-terminal side of the substitution site. Molecular dynamics computation exhibits a bending of the helical axis of 30 degrees +/- 10 degrees, in agreement with X-ray diffraction results. Light-scattering experiments indicated that the average aggregation number of the proline-substituted mutant is substantially lower than that of the wild-type peptide. From the ratio of dissociation constants of the wild-type and the proline mutant peptides, the difference in free energy of trimeric formation is calculated to be 2.1 kcal/mol. Thermal stability, helicity, and the average aggregation number for the helix oligomers were found to be correlated. The structural alteration and the reduced coiled coil stability may account for the deficiency in the biological functions of the proline mutants of gp41 and in the inhibitory action of proline-substituted peptides. These effects may also be important in unraveling the roles played by proline in transmembrane proteins.

Amino Acid Motifs↗
PubMed1993

Role of ornithine as a proline precursor in healing wounds.

The successful healing of wounds requires the local synthesis of significant amounts of collagen. The amino acid pool that is the immediate precursor for collagen synthesis within wounds has not been specifically identified but is likely to include the free amino acids contained in the extracellular wound fluid. This fluid is here shown to be rich in proline and its metabolic precursors: ornithine, glutamate, and glutamine. While the metabolic origins for wound fluid glutamine and glutamate remain undetermined, ornithine is known to be synthesized locally through the catabolism of arginine by arginase. Experiments reported here tested the hypothesis that ornithine accumulating in the extracellular space of wounds serves as a precursor for proline contained in fibroblast secretory proteins. Results demonstrated that little ornithine is directly incorporated as protein-bound proline by wound-derived fibroblasts in culture and that the incorporation ornithine-derived proline into protein is markedly suppressed by preformed proline. In turn, the appearance of ornithine-derived free proline in the culture supernatants is quantitatively more important than its incorporation into protein and it is enhanced, rather than suppressed, by preformed proline. Therefore, ornithine may contribute to the synthesis of protein-bound proline in wounds by increasing the extracellular pool of free proline.

Animals↗
PubMed1980

Proline specific endo- and exopeptidases.

Peptidases which are specific for proline residues have been described and include endopeptidases (post-proline cleaving enzyme and proline specific endopeptidase), N-terminal exopeptidases (post-proline dipeptidyl aminopeptidase, proline iminopeptidase, aminopeptidase P), C-terminal exopeptidases (prolylcarboxypeptidase, and carboxypeptidase P) and dipeptidases (prolyl dipeptidase and proline dipeptidase). The properties, distinguishing charcteristics, and possible significance of these proline specific endo- and exopeptidases are discussed. In addition, reference is made to a series of enzymes which can hydrolyze proline containing peptide bonds, but which are not specific for proline.

Aminopeptidases↗
PubMed2006

A novel role for proline in plant floral nectars.

Plants offer metabolically rich floral nectar to attract visiting pollinators. The composition of nectar includes not only sugars, but also amino acids. We have examined the amino acid content of the nectar of ornamental tobacco and found that it is extremely rich (2 mM) in proline. Because insect pollinators preferentially utilize proline during the initial phases of insect flight and can reportedly taste proline, we determined whether honeybees showed a preference for synthetic nectars rich in proline. We therefore established an insect preference test and found that honeybees indeed prefer nectars rich in the amino acid proline. To determine whether this was a general phenomenon, we also examined the nectars of two insect-pollinated wild perennial species of soybean. These species also showed high levels of proline in their nectars demonstrating that plants often produce proline-rich floral nectar. Because insects such as honeybees prefer proline-rich nectars, we hypothesize that some plants offer proline-rich nectars as a mechanism to attract visiting pollinators.

Amino Acids↗
PubMed2003

Transport of proline and hydroxyproline by the neutral amino-acid exchanger ASCT1.

ASCT1 is a member of the glutamate transporter superfamily cloned from human brain and characterized as a Na(+)-dependent neutral amino-acid exchanger, which displays substrate-induced chloride-channel activity and mediates concentrative transport of alanine. Initial studies in ASCT1-expressing Xenopus laevis oocytes showed that proline did not elicit measurable currents, in contrast to what occurred with alanine, serine or cysteine, suggesting that proline was not an ASCT1 substrate, although it induced the release of alanine from preloaded oocytes. Here, we have studied the uptake of proline and hydroxyproline by ASCT1-expressing oocytes in order to investigate the ability of ASCT1 to translocate these imino acids. The results demonstrate ASCT1-mediated proline transport that is Na(+)-dependent, saturable, inhibited by the reported ASCT1 substrates as well as by hydroxyproline and can drive the imino acid against its concentration gradient. The apparent kinetic constants for the transport of alanine and the imino acids, obtained with oocytes from the same batch, showed maximal transport rate for proline and hydroxyproline to be half of that for alanine. However, K(0.5) for proline was 704 +/- 86 microM, about three times higher than alanine K(0.5) (203.3 +/- 36.4 microM), whereas hydroxyproline K(0.5) was 33.2 +/- 4.3 microM, indicating that the hydroxylation on carbon 4 of proline strongly increases the affinity of ASCT1 for this proline derivative. In summary, the present work demonstrates for the first time the ability of ASCT1 to transport proline and hydroxyproline.

Adaptation, Biological↗
PubMed1997

Abscisic acid-independent and abscisic acid-dependent regulation of proline biosynthesis following cold and osmotic stresses in Arabidopsis thaliana.

The role of the phytohormone abscisic acid (ABA) in the regulation of proline synthesis was investigated by following the expression of the At-P5S and At-P5R proline biosynthesis genes in Arabidopsis thaliana wild type, in an ABA-deficient aba1-1 mutant as well as in ABA-insensitive abi1-1 and abi2-1 mutants after ABA, cold and osmotic stress treatments. In wild-type and in ABA mutant seedlings, 50 microM ABA or osmotic stress treatment triggered expression of At-P5S, whereas At-P5R accumulation was scarcely detectable. Expression of either gene was mediated by endogenous ABA since transcript levels were similar in wild-type and in ABA-deficient mutant plants. Proline accumulated to a greater extent after osmotic stress than upon ABA or cold treatment. Thus, ABA-treated abi1-1 mutant plants accumulated less proline than the ABA-treated wild type. Upon salt stress, proline accumulated to a lesser extent in aba1-1 and abi1-1 mutant plants, suggesting an indirect role of ABA on proline accumulation during salt adaptation of the plant. These results indicate that the expression of the genes of the proline biosynthetic pathway is ABA independent upon cold and osmotic treatments, although their expression can be triggered by exogenously applied ABA. However, the endogenous ABA content may affect proline accumulation upon salt stress, suggesting post-transcriptional control of proline biosynthesis in response to NaCl.

1-Pyrroline-5-Carboxylate Dehydrogenase↗
PubMed1992

NMDA receptor-mediated depolarizing action of proline on CA1 pyramidal cells.

This study investigated the actions of proline on CA1 hippocampal pyramidal cells with use of slice preparations. Bath-applied L-proline first induced these cells to fire multiple orthodromic population spikes in response to a single stimulus and then blocked their response to both orthodromic and antidromic stimulation. These effects could be explained by postsynaptic depolarization followed by depolarization block. Grease-gap studies confirmed that L-proline depolarizes CA1 pyramidal cells. D-Proline was inactive in these tests. Excitatory amino acid antagonists reduced depolarizing responses to proline and N-methyl-D-aspartate (NMDA) in parallel. Mn2+ failed to attenuate proline-evoked depolarizations at concentrations that substantially inhibited synaptic transmission, but at a higher concentration it reduced responses to both proline and NMDA. These results suggest that proline depolarized CA1 pyramidal cells mainly by activating postsynaptic NMDA receptors. The neuroexcitatory and neurotoxic actions of proline in the hippocampus may contribute to the seizures and mental retardation associated with hyperprolinemia.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed2004

Cyclic strain stimulates L-proline transport in vascular smooth muscle cells.

BACKGROUND: The increase in vessel wall strain in hypertension contributes to arterial remodeling by stimulating vascular smooth muscle cell (SMC) proliferation and collagen synthesis. Because L-proline is essential for the synthesis of collagen and cell growth, we examined whether cyclic strain regulates the transcellular transport of L-proline by vascular SMC. METHODS: Cultured rat aortic SMCs were subjected to mechanical strain using the Flexercell 3000 Strain Unit. RESULTS: Cyclic strain increased L-proline transport in a time- and strain-degree-dependent manner that was inhibited by cycloheximide or actinomycin D. Kinetic studies indicated that cyclic strain-induced L-proline uptake was mediated by an increase in transport capacity independent of any change in the affinity for L-proline. Cyclic strain stimulated the expression of system A amino acid transporter 2 mRNA in a time-dependent fashion that paralleled the increase in L-proline transport. Cyclic strain also induced the release of transforming growth factor-beta1 in a time- and strain-dependent manner. Moreover, conditioned media from SMCs exposed to cyclic strain stimulated the transport of L-proline in control, static SMCs and this was significantly attenuated by a transforming growth factor-beta1 neutralizing antibody. CONCLUSIONS: These results demonstrate that cyclic strain stimulates L-proline transport by inducing system A amino acid transporter 2 gene expression through the autocrine release of transforming growth factor-beta1. The ability of cyclic strain to induce system A amino acid transporter 2 expression may promote arterial remodeling in hypertension by providing vascular SMCs with the necessary intracellular levels of L-proline required for collagen synthesis and cell growth.

Amino Acid Transport System A↗
PubMed2004

Oxidation of 3,4-dehydro-D-proline and other D-amino acid analogues by D-alanine dehydrogenase from Escherichia coli.

3,4-Dehydro-DL-proline is a toxic analogue of L-proline which has been useful in studying the uptake and metabolism of this key amino acid. When membrane fractions from Escherichia coli strain UMM5 (putA1::Tn5 proC24) lacking both L-proline dehydrogenase and L-Delta(1)-pyrroline-5-carboxylate reductase were incubated with 3,4-dehydro-DL-proline, pyrrole-2-carboxylate was formed. There was no enzyme activity with 3,4-dehydro-L-proline, but activity was restored after racemization of the substrate. Oxidation of 3,4-dehydro-DL-proline by membrane fractions from strain UMM5 was induced by growth in minimal medium containing D- or L-alanine, had a pH optimum of 9, and was competitively inhibited by D-alanine. An E. coli strain with no D-alanine dehydrogenase activity due to the dadA237 mutation was unable to oxidize either 3,4-dehydro-D-proline or D-alanine, as were spontaneous Dad(-) mutants of E. coli strain UMM5. Membrane fractions containing D-alanine dehydrogenase also catalyzed the oxidation of D-2-aminobutyrate, D-norvaline, D-norleucine, cis-4-hydroxy-D-proline, and DL-ethionine. These results indicate that d-alanine dehydrogenase is responsible for the residual 3,4-dehydro-DL-proline oxidation activity in putA proC mutants of E. coli and provide further evidence that this enzyme plays a general role in the metabolism of D-amino acids and their analogues.

Alanine Dehydrogenase↗
PubMed1986

A calorimetric and infrared spectroscopic study of the stabilizing solute proline.

We have studied the calorimetric and infrared spectroscopic properties of the amino acid proline which has been implicated in the stabilization of biomacromolecules during reduced water states. It has been suggested that the ability of this molecule to protect biomacromolecules during these stress states may be related to the formation of polymeric aggregates of proline monomers in solution. The structure of this aggregate is thought to be an alternates stack, forming a hydrophilic colloid-like polymer which is thought to interact with hydrophobic moieties of biomacromolecules, reducing the exposed hydrophobic area during reduced water conditions. Calorimetric data presented in this work show that in increasing concentration of proline in solution the enthalpy associated with the melting of bulk water is greatly reduced, indicating strong hydrogen bonding character of proline in aqueous solution. Proline shows two eutectic phase separations at moderate concentrations and one of these eutectics may be the proposed intermolecular state. A partial phase diagram for proline is presented. Fourier-transform infrared spectroscopic data indicate that the COO- asymmetric stretch of proline shows marked splitting with increasing proline concentration. This suggests that the carboxylate is in different environments, with the high energy vibrations representing COO- groups which are participating in the hydrogen bonding pattern associated with the formation of the intermolecular stack. Changes in the CH2 asymmetric and symmetric stretches of the pyrrolidine rings of proline are consistent with the proposed stack structure. We also suggest a possible mechanism by which these intermolecular associations may be important in the protection of biomacromolecules during reduced water states.

Calorimetry↗
PubMed2003

Proline induces oxidative stress in cerebral cortex of rats.

In the present study we investigated the in vivo and in vitro effects of proline on some parameters of oxidative stress, such as chemiluminescence, total radical-trapping antioxidant potential (TRAP) and the activity of the antioxidant enzymes catalase, glutathione peroxidase and superoxide dismutase in rat cerebral cortex. Ten-day-old rats received one subcutaneous injection of proline (12.8 micromol/g body weight), while control rats received saline in the same volumes. The animals were killed 1h after injection, the cerebral cortex was isolated and the assays immediately carried out. For the in vitro studies, homogenates from cerebral cortex of 10-day-old untreated rats were incubated for 1h at 37 degrees C with various concentrations of proline (3.0 microM-1.0mM). Results showed that proline-treated rats presented a decrease of TRAP (30%) and an increase of chemiluminescence (78%). In contrast, the activities of catalase, glutathione peroxidase and superoxide dismutase were not modified by proline acute treatment. Furthermore, the presence of proline in the medium increased chemiluminescence, decreased TRAP and the activity of superoxide dismutase at proline concentrations similar to those observed in tissues of hyperprolinemic patients (0.5-1.0mM). However, catalase and glutathione peroxidase activities were not affected by the presence of proline in the medium. The results indicate that proline induces oxidative stress in the brain, which may be related, at least in part, to the neurological dysfunction observed in hyperprolinemia.

Animals↗
PubMed1996

Structural requirements and thermodynamics of the interaction of proline peptides with profilin.

The binding to poly(L-proline) is used for the affinity purification of profilins, but little is known about the structural and thermodynamic aspects of the interaction. We used changes in the intrinsic fluorescence of profilin, CD spectroscopy, and isothermal titration calorimetry to assess how the size and composition of synthetic proline-rich peptides influence binding to Acanthamoeba and human profilins. Although a 6 residue type II poly(L-proline) helix can span the binding site, highest affinity binding is achieved by proline oligomers > or = 10 residues. Binding is stereospecific since (D-proline)11 does not bind. In 75 mM KCI the dissociation equilibrium constant for poly(L-proline) is about 10 microM proline decamer units for amoeba profilin and 20-30 microM for human profilin. Consistent with a significant hydrophobic component of the interaction, delta Cp is negative and higher salt concentrations enhance the affinity. No protons dissociate or bind during the interaction. Binding of poly(L-proline) is favored both entropically and enthalpically. Substitution of glycine in proline undecamers reduces affinity by about 1 kcal mol-1 for each substitution due to increased rotational freedom of the free peptides. Substitution of alanine has a similar effect. Disorder in the free peptides imparts an unfavorable entropic cost for immobilizing the substituted peptides on the binding site on profilin.

Acanthamoeba↗
PubMed2003

Effect of proline on creatine kinase activity in rat brain.

Type II Hyperprolinemia is an inherited disorder caused by a deficiency of delta1-pyrroline-5-carboxilic acid dehydrogenase, whose biochemical hallmark is proline accumulation in plasma and tissues. Although neurologic symptoms occur in most patients, the neurotoxicity of proline is still controversial. The main objective of this study was to investigate the effect of acute and chronic administration of proline on creatine kinase activity in the homogenates of cerebellum and midbrain from Wistar rats. Acute treatment was performed by subcutaneous administration of one injection of proline to 22-day-old rats. For chronic treatment, proline was administered four times a day from the 6th to the 21st postpartum day. The results showed that creatine kinase activity was significantly inhibited in the cerebellum and midbrain of rats subjected to acute proline administration. In contrast, this activity was increased in animals subjected to chronic administration. We also measured the in vitro effect of proline on creatine kinase activity in the same cerebral structures of 22-day-old nontreated rats. Proline significantly inhibited creatine kinase activity. Considering the importance of creatine kinase for the maintenance of energy homeostasis in the brain, it is conceivable that an alteration of this enzyme activity in the brain may be one of the mechanisms by which proline might be neurotoxic.

Animals↗
PubMed2003

Proline reduces creatine kinase activity in the brain cortex of rats.

Type II hyperprolinemia is an inherited disorder caused by a deficiency of delta 1-pyrroline-5-carboxilic acid dehydrogenase, whose biochemical hallmark is proline accumulation in plasma and tissues. Although neurological symptoms occur in most patients, the neurotoxicity of proline is still controversial. The main objective of the present study was to investigate the effect of acute and chronic administration of proline on creatine kinase activity of brain cortex of Wistar rats. Acute treatment was performed by subcutaneous administration of one injection of proline to 22-day-old rats. For chronic treatment, proline was administered twice a day from the 6th to the 21st postpartum day. The results showed that creatine kinase activity was significantly inhibited in the brain cortex of rats subjected to acute proline administration. In contrast, this activity was increased in animals subjected to chronic administration. We also measured the in vitro effect of proline on creatine kinase activity in cerebral cortex of 22-day-old nontreated rats. Proline significantly inhibited creatine kinase activity. Considering the importance of creatine kinase forthe maintenance of energy homeostasis in the brain, it is conceivable that an alteration of this enzyme activity in the brain may be one of the mechanisms by which proline might be neurotoxic.

Animals↗
PubMed1985

Protonmotive force-driven active transport of D-glucose and L-proline in the protozoan parasite Leishmania donovani.

Midlogarithmic phase Leishmania donovani promastigotes accumulate 2-deoxy-D-glucose (2-dGlc) and L-proline, maintaining concentration gradient factors across the surface membrane of 78.7 and 60, respectively. Cyanide (1 mM) and iodoacetate (0.5 mM) inhibited the transport of both substrates. L-proline uptake was also inhibited by 2-dGlc (10 mM). Transport of neither substrate was affected by Na+, phlorizin, or ouabain, indicating the sodium-independent transport of both systems. However, N',N'-dicyclohexylcarbodiimide (DCCD; 20 microM) significantly inhibited the transport of both 2-dGlc and L-proline (70% and 90%, respectively). The ionophores valinomycin (1 microM) and nigericin (5 microM) each partially inhibited the uptake of both substrates. In parallel experiments, nigericin and valinomycin were added concomitantly to promastigotes, each at a concentration that individually inhibited the transport of 2-dGlc and L-proline by less than 30%. Under such conditions, the transport of 2-dGlc and L-proline was inhibited by 69% and 78%, respectively. However, these ionophores had no significant effect on the promastigotes cellular ATP level. Carbonylcyanide p-(trifluoromethoxy)phenylhydrazone (FCCP; 1 microM) inhibited 2-dGlc (79%) and L-proline (85%) transport, whereas ATP levels of such cells were diminished by only 20%. Symport of D-glucose/H+ and L-proline/H+ was measured directly in cells pretreated with KCN and DCCD. Upon addition of D-glucose to such cells, a rapid movement of protons into the organisms occurred and was reversed upon addition of FCCP. Conversely, no proton movement was observed when L-glucose was added to such cells. L-proline, as D-glucose, caused a rapid influx of protons into the promastigotes, indicating that both substrates were cotransported with protons. We conclude that transport of D-glucose and L-proline in L. donovani promastigotes is protonmotive force-driven and is coupled to both delta pH and delta psi.

Animals↗
PubMed1993

The sluggish-A gene of Drosophila melanogaster is expressed in the nervous system and encodes proline oxidase, a mitochondrial enzyme involved in glutamate biosynthesis.

Certain gene mutations in Drosophila melanogaster cause sluggish motor activity. We have localized the transcription unit of the sluggish-A gene to a 14.7-kb region at the base of the X chromosome and have cloned corresponding cDNAs. The predicted protein product has significant sequence similarity to Saccharomyces cerevisiae proline oxidase (EC 1.5.99.8), a mitochondrial enzyme which catalyzes the first step in the conversion of proline to glutamate. In the mutant fly, mitochondrial proline oxidase activity is reduced and has kinetic properties different from those of the wild type, providing further evidence that the gene encodes proline oxidase. Indeed, the free proline level in mutant flies is elevated. When the mutant is rescued by transformation, the proline oxidase and free proline levels, as well as the motor and phototactic behavior, are restored to normal. During embryonic development the sluggish-A transcript is predominantly expressed in the nervous system. Significantly, it has previously been reported that a mouse mutant, PRO/Re, which has reduced proline oxidase activity and elevated free proline levels, also exhibits sluggish behavior.

Amino Acid Sequence↗
PubMed1999

Critical role of conserved proline residues in the transmembrane segment 4 voltage sensor function and in the gating of L-type calcium channels.

The fourth transmembrane segment (S4) has been shown to function as a voltage sensor in voltage-gated channels. On membrane depolarization, a stretch of S4 moves outward and initiates a number of conformational changes that ultimately lead to channel opening. Conserved proline residues are in the middle of the S4 of motifs I and III in voltage-dependent Ca2+ channels. Because proline often introduces a "kink" into a helical structure of proteins, these residues might have an intrinsic function in the voltage sensor. Here, we report that the removal of S4 prolines results in a dramatic shortening of channel open time whereas the introduction of extra prolines to the corresponding positions in motif IIS4 and IVS4 lengthens channel open time. The number of S4s with a proline residue showed a clear positive correlation with the mean open time of the channel. The mean open time was >11-fold longer for a channel mutagenized to have prolines in all four S4s compared with a channel that had no prolines in the S4 region. Additionally, prolines in the S4s slowed activation kinetics and shifted the voltage dependence of activation and inactivation in a hyperpolarized direction. Our results strongly suggest that proline residues in the S4s are critical for stabilizing the open state of the channel. Moreover, it is suggested that motif IS4 and IIIS4 contribute to the channel opening more efficiently than motif IIS4 and IVS4.

Amino Acid Sequence↗
PubMed1998

Effect of ascorbic acid dose taken with a meal on nitrosoproline excretion in subjects ingesting nitrate and proline.

We determined the dose of ascorbic acid (ASC) given to subjects with a standard 400-calorie meal that inhibited N-nitrosoproline (NPRO) formation when we gave 400 mg of nitrate one hour before and 500 mg of L-proline with the standard meal. Volunteers consumed their normal US diets but restricted their intakes of nitrate, proline, NPRO, and ASC. NPRO and N-nitrososarcosine (NSAR) were determined in the 18-hour urines by methylation followed by gas chromatography-thermal energy analysis. Mean NPRO yields were 10.7, 41.9, 33.2, 22.3, and 23.1 nmol for groups of 9-25 subjects taking proline alone, proline + nitrate, and proline + nitrate + 120, 240, and 480 mg of ASC, respectively. There was a significant trend to lower NPRO yields as the ASC dose was raised. These results correspond to inhibitions by ASC of 28%, 62%, and 60%, respectively. Pairwise comparison showed that each group taking ASC formed significantly less NPRO than the group given only proline + nitrate. Mean NSAR yields were 9.0 nmol when proline alone was taken and 16.9-24.0 nmol when proline + nitrate + ASC was taken, with no trend to increase as the ASC dose was raised. However, NPRO and NSAR yields in individual urines were correlated with each other. We concluded that 120 mg of ASC taken with each meal (360 mg/day) would significantly reduce in vivo nitrosamine formation, similar to tests by Leaf and co-workers (Carcinogenesis 8, 791-795, 1987) in which the reactants were taken between meals. The inhibitory dose of ASC may be < 120 mg/meal when doses of nitrate and proline are not taken.

Adult↗