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PubMed1996

The unique proline-rich domain of parotid proline-rich proteins functions in secretory sorting.

When expressed in pituitary AtT-20 cells, parotid proline-rich proteins enter the regulated pathway. Because the short N-terminal domain of a basic proline-rich protein is necessary for efficient export from the ER, it has not been possible to evaluate the role of this polypeptide segment as a sorting signal for regulated secretion. We now show that addition of the six-amino acid propeptide of proparathyroid hormone to the proline-rich protein, and especially to a deletion mutant lacking the N-terminal domain, dramatically accelerates intracellular transport of these polypeptides. Under these conditions the chimeric deletion mutant is stored as effectively as the full-length protein in dense core granules. The propeptide does not function as a sorting signal in AtT-20 cells as it does not reroute a constitutively secreted reporter protein to the regulated pathway. During transit, the propeptide is cleaved from the chimeric polypeptides such that the original structures of the full-length and the deletion mutant proline-rich proteins are reestablished. We have also found that the percentage stimulated secretion of the proline-rich proteins increases incrementally (almost twofold) as their level of expression is elevated. The increase reflects an enrichment of these polypeptides in the granule pool and its incremental nature suggests that sorting of proline-rich proteins involves an aggregation-based process. Because we can now rule out contributions to sorting by both N- and C-terminal segments of the proline-rich protein, we deduce that the unique proline-rich domain is responsible for storage. Thus at least some of the determinants of sorting for regulated secretion are protein-specific rather than universal.

Animals↗

PubMed1988

Proline porters effect the utilization of proline as nutrient or osmoprotectant for bacteria.

Proline is utilized by all organisms as a protein constituent. It may also serve as a source of carbon, energy and nitrogen for growth or as an osmoprotectant. The molecular characteristics of the proline transport systems which mediate the multiple functions of proline in the Gram negative enteric bacteria, Escherichia coli and Salmonella typhimurium, are now becoming apparent. Recent research on those organisms has provided both protocols for the genetic and biochemical characterization of the enzymes mediating proline transport and molecular probes with which the degree of homology among the proline transport systems of archaebacteria, eubacteria and eukaryotes can be assessed. This review has provided a detailed summary of recent research on proline transport in E. coli and S. typhimurium; the properties of other organisms are cited primarily to illustrate the generality of those observations and to show where homologous proline transport systems might be expected to occur. The characteristics of proline transport in eukaryotic microorganisms have recently been reviewed (Horak, 1986).

Bacteria↗

PubMed1990

Plasma proline kinetics and concentrations in young men in response to dietary proline deprivation.

This study examined plasma proline concentration flux, oxidation, and endogenous biosynthesis in five healthy young men given three isocaloric, isonitrogenous diets for 1 wk [a complete egg-pattern amino acid diet (diet 1), an amino acid mixture devoid of proline (diet 2), and a diet composed solely of indispensable amino acids (diet 3)]. At the end of each dietary period, a 360-min postabsorptive, primed, continuous stable-isotope-tracer infusion of L-[1-13C]proline and L-[methyl-2H3]leucine was performed in all subjects. Plasma proline concentrations declined by 22% on diet 2 (p less than 0.02) and by 29% on diet 3 (p less than 0.01). No statistically significant (p greater than 0.2) changes were observed for proline oxidation, endogenous biosynthesis, or flux. The data suggest that the absence of proline in the human diet does not trigger changes in proline dynamics during the postabsorptive state. The metabolic significance of the reduction of plasma proline concentrations requires elucidation.

Adult↗

PubMed1994

Plasma proline and leucine kinetics: response to 4 wk with proline-free diets in young adults.

The effects of removing proline from the diet on plasma leucine and proline kinetics were investigated. After a 1-wk control period, during which young adult men received a diet containing a complete L-amino acid mixture, seven subjects were given for 4 wk a diet devoid of proline (group 1); six received a diet devoid of proline, arginine, aspartate, glutamate, and serine (group 2); and seven continued with the complete diet (group 3). At the end of the control and 4-wk periods subjects were given a continuous, (3-h fast, 5-h fed) intravenous infusion of L-[1-13C]leucine and L-[5,5-2H]proline. Plasma proline was reduced significantly, especially during the fed state, in groups 1 and 2 after the 4-wk diet periods. Small but statistically significant (P < 0.05) reductions occurred in nonoxidative leucine disappearance and leucine appearance during the fasted state in group 2. Proline fluxes decreased by approximately 50% in fasted and fed states in groups 1 and 2. Mean de novo proline synthesis during the fasted state declined markedly (P < 0.05) after 4 wk in groups 1 and 2.

Adult↗

PubMed1999

Osmoprotection of Escherichia coli by peptone is mediated by the uptake and accumulation of free proline but not of proline-containing peptides.

The effect of meat peptone type I (Sigma) on the growth of Escherichia coli cells under hyperosmotic stress has been investigated. Peptone is a complex mixture of peptides with a small content of free amino acids, which resembles nutrients found in natural environments. Our data showed that peptone enhances the growth of E. coli cells in high-osmolarity medium to levels higher than those achieved with the main compatible solute in bacteria, glycine betaine. The mechanism of osmoprotection by peptone comprises the uptake and accumulation of the compatible solute, proline. The main role of the peptides contained in peptone is the provision of nutrients rather than the intracellular accumulation of osmolytes. In contrast to Listeria monocytogenes (M. R. Amezaga, I. Davidson, D. McLaggan, A. Verheul, T. Abee, and I. R. Booth, Microbiology 141:41-49, 1995), E. coli does not accumulate exogenous peptides for osmoprotection and peptides containing proline do not lead to the accumulation of proline as a compatible solute. In late-logarithmic-phase cultures of E. coli growing at high osmolarity plus peptone, proline becomes the limiting factor for growth, and the intracellular pools of proline are not maintained. This is a consequence of the low concentration of free proline in peptone, the catabolism of proline by E. coli, and the inability of E. coli to utilize proline-containing peptides as a source of compatible solutes. Our data highlight the role that natural components in food such as peptides play in undermining food preservation regimes, such as high osmolarity, and also that the specific mechanisms of osmoprotection by these compounds differ according to the organism.

Amino Acids↗

PubMed1987

Induction of tissue-specific proline-rich protein multigene families in rat and mouse parotid glands by isoproterenol. Unusual strain differences of proline-rich protein mRNAs.

A dramatic induction of proline-rich protein mRNAs by the beta-agonist isoproterenol in the parotid and submandibular glands of both rats and mice has been demonstrated using Northern and dot-blot hybridizations and cell-free translation. Proline-rich protein mRNAs were either very low or not detectable in glands of control rats and mice. After 4 days of isoproterenol treatment, mRNAs encoding these unusual proteins comprised over 50% of the total glandular mRNAs. A 2-4-fold increase in proline-rich protein mRNAs was observed in rat parotid glands as soon as 4 h after treatment. The rat proline-rich protein multigene family encodes two groups of mRNAs with sizes ranging from 600 to 1100 bases. Cell-free translations gave about 10-12 proline-rich proteins. In glands of isoproterenol-treated mice, major species of proline-rich protein mRNAs were observed at 1050 and 1300 bases for BALB/cJ and DBA/2J mice and at 1100 and 1200 bases for CD-1 and C57BL/6J mice. Cell-free translations showed unusual differences in proteins synthesized from the four strains after isoproterenol treatment. AtT20 cells were transfected with a mouse proline-rich protein gene inserted into the plasmid pUC8 (pUMP2-BE). Transcription of proline-rich protein mRNA was induced by exposing these transfected cells to either isoproterenol or cAMP, plus theophylline.

Animals↗

PubMed1983

An assay of dipeptidyl peptidase IV activity in human serum and serum of pregnant women with glycyl-L-proline-1-naphthylamide and other glycyl-L-proline-arylamides as substrates.

The authors described a micromethod for measuring dipeptidyl peptidase IV activity in human serum with glycyl-L-proline-1-naphthylamide as substrate. The method requires less than 20 microliters of serum. The pH optimum for cleaving glycyl-L-proline-1-naphthylamine by the enzyme in human serum in Tris-HCl buffer was 8.0 and Km value was established as 7.2 X 10(-4) mol/l. The advantage of this substrate is the absence of spontaneous hydrolysis during the assay of enzyme activity in contrast to glycyl-L-proline-4-nitroanilide. The Km values of the latter substrates and glycyl-L-proline-2-naphthylamide in the same buffer were 1.0 X 10(-4) mol/l and 2.4 X 10(-4) mol/l, respectively. Glycyl-D-proline-4-nitroanilide was not hydrolyzed by the dipeptidyl peptidase IV present in human serum. The activities of dipeptidyl peptidase IV in the sera from 30 healthy human subjects with glycyl-L-proline-1-naphthylamide as substrate were 176.1 +/- 32.8 nkat/l (mean +/- standard deviation; range 100.2-264.1 nkat/l of serum). In this group men had significantly (P less than 0.01) higher activity of the enzyme than women. The cleaving of glycyl-L-proline-1-naphthylamide and glycyl-L-proline-4-nitro anilide by dipeptidyl peptidase IV in human sera was closely correlated (r = 0.86). During normal pregnancy the activity of dipeptidyl peptidase IV in human serum decreases markedly in the first half of pregnancy. After delivery, the serum enzyme activity returns progressively to initial levels.

Adult↗

PubMed1993

Mechanism-based inhibition of proline dehydrogenase by proline analogues.

The inactivation of proline dehydrogenase by several L-Pro analogues was investigated with the aim to block the essential metabolic pathway of tsetse flies allowing the degradation of L-Pro to L-Glu. In vitro studies on rat liver mitochondria showed that only 4-methylene-L-proline was able to inactivate proline dehydrogenase. The inactivation kinetics agreed with a mechanism-based inhibition. The other tested analogues E- and Z-4-fluoromethylene-L-proline, and cis and trans-5-ethynyl-D,L-proline were neither substrate nor inactivator of the enzyme. In vivo 4-methylene-L-proline showed no toxicity against Drosophila flies, but was lethal for Glossina pallidipes flies. This result allows the consideration of 4-methylene-L-proline as an attractive compound molecule in the struggle against tsetse flies.

Animals↗

PubMed2000

Proline accumulation by mutation or disruption of the proline oxidase gene improves resistance to freezing and desiccation stresses in Saccharomyces cerevisiae.

We examined the role of intracellular proline under freezing and desiccation stress conditions in Saccharomyces cerevisiae. When cultured in liquid minimal medium, the proline-nonutilizing mutant containing the put1 mutation (proline oxidase-deficient) produced more intracellular proline, and increased the cell survival rate as compared to the wild-type strain after freezing and desiccation. We also constructed two PUT1 gene disruptants. PUT1-disrupted mutants in minimal medium supplemented with external proline at 0.1% accumulated higher proline levels than those of the control strains (17-22-fold). These disruptants also had a 2-5-fold increase in cell viability compared to the control strains after freezing and desiccation stresses. These results indicate that proline has a stress-protective function in yeast.

Adaptation, Physiological↗

PubMed2003

Characterization of the Vibrio vulnificus putAP operon, encoding proline dehydrogenase and proline permease, and its differential expression in response to osmotic stress.

The Vibrio vulnificus putAP genes encoding a proline dehydrogenase and a proline permease are transcribed in the same direction. Proline dehydrogenase activity and the level of putA transcript were determined to reach a maximum in exponential phase and were then repressed when growth slowed down. Northern blotting and primer extension analyses revealed that transcription of putAP genes results in two different transcripts, transcript A (putA transcript) and transcript AP (putAP transcript). Expression of putAP genes was directed by two promoters, promoter P(putA) and promoter P(putAP). A crp null mutation decreased proline dehydrogenase activity and the level of the put transcripts, indicating that transcription of putAP is under the positive control of cyclic AMP receptor protein. Proline dehydrogenase and the level of both put transcripts were increased by proline but repressed by glutamate. In contrast, the level of transcript A, not transcript AP, increased when proline dehydrogenase was induced by NaCl. Since P(putA) activity, not P(putAP) activity, was increased by NaCl, it is apparent that transcript A and transcript AP are transcribed through P(putA) and P(putAP), respectively. Cells challenged with NaCl and various hyperosmotic stresses accumulated higher levels of glutamate than control cells, indicating that glutamate is a compatible solute in V. vulnificus.

Amino Acid Transport Systems, Neutral↗

PubMed2001

Proline oxidase, encoded by p53-induced gene-6, catalyzes the generation of proline-dependent reactive oxygen species.

The p53-dependent initiation of apoptosis is accompanied by the induction of proline oxidase (POX), a mitochondrial enzyme catalyzing the conversion of proline to pyrroline-5-carboxylate with the concomitant transfer of electrons to cytochrome c. However, the contribution of increased POX activity to apoptosis, if any, remains unknown. Using Adriamycin to initiate p53-dependent apoptosis, we showed that the expression of POX is up-regulated in a time- and dose-dependent manner in a human colon cancer cell line (LoVo). In cells expressing POX, the addition of proline increases reactive oxygen species (ROS) generation in a concentration-dependent manner; glutamate, a downstream product of proline oxidation, had no effect. Induction of POX was dependent on the p53 status of the cell. In the conditionally immortalized murine colonic epithelial cell line YAMC, where the p53 phenotype can be modulated by temperature, proline oxidase expression and ROS production could only be induced when the cells were phenotypically p53-positive. To confirm that the observed ROS production was not secondary to some other effect of p53, we also conditionally expressed POX in a p53-negative colon cancer line. Again, we found a proline-dependent ROS increase with POX expression. We hypothesize that proline oxidation supports the generation of ROS by donating reducing potential to an electron transport chain altered either by p53-dependent mechanisms or by overexpression of POX.

Antibiotics, Antineoplastic↗

PubMed1996

Proline pipe helix: structure of the tus proline repeat determined by 1H NMR.

The structure of a 22 amino acid peptide, TPPI [Nedved, M. L., Gottlieb, P. A., & Moe, G. R. (1994) Nucleic Acids Res. 22, 5024-5030], that is similar to the proline repeat segment of the replication arrest protein, Tus, has been determined by 1H NMR in 50% trifluroethanol. The structure is a novel left-handed helix having 5.56 residues per turn and a regular hydrogen bonding network that is limited to one side of the helix and contains a channel that runs down the helix axis. The latter feature gives the structure an overall pipe-like appearance; hence, the structure has been designated a proline pipe helix. The Tus proline pipe is also amphiphilic with one side consisting of proline and other nonpolar residues while the other side contains mostly basic and other polar residues. Tus and several other proteins that contain a similar proline repeat sequence are DNA binding proteins. It is shown here that the proline pipe helix of TPPI can be accommodated within the major grove of B-form DNA in a manner that positions nearly all of the basic residues near phosphate groups in the DNA backbone. The proline pipe helical motif may be a structural element of many other proteins including integral membrane receptor proteins.

Amino Acid Sequence↗

PubMed1987

Proline-induced germ-tube formation in Candida albicans: role of proline uptake and nitrogen metabolism.

Proline-induced germ-tube formation and cell-cell aggregation in four strains of Candida albicans were completely inhibited when the pH of the medium was 5.0 or lower, whereas morphogenesis induced by N-acetylglucosamine (GlcNAc) was unaffected even at pH 4.5. The pH sensitivity of proline-induced germ-tube formation was not caused by a modulation of proline uptake, which was unchanged over the pH range 4.5-6.5. The proline uptake system was specific, constitutive and subject to ammonium repression, and only one permease was detected, with a Km of 179 microM. Cultures deprived of nitrogen in the presence of glucose were derepressed for proline uptake but the yeast-mycelial transition could not be mediated by either proline or GlcNAc. The inhibition of morphogenesis was reversed when the nitrogen starvation was relieved by the addition of ammonium ions, proline, or certain amino acids. These results indicate that the nitrogen status of the cells is critical for the morphogenesis of C. albicans.

Acetylglucosamine↗

PubMed1996

Analysis of the critical sites for protein thermostabilization by proline substitution in oligo-1,6-glucosidase from Bacillus coagulans ATCC 7050 and the evolutionary consideration of proline residues.

To identify the critical sites for protein thermostabilization by proline substitution, the gene for oligo-1,6- glucosidase from a thermophilic Bacillus coagulans strain, ATCC 7050, was cloned as a 2.4-kb DNA fragment and sequenced. In spite of a big difference in their thermostabilities, B. coagulans oligo-1,6-glucosidase had a large number of points in its primary structure identical to respective points in the same enzymes from a mesophilic Bacillus cereus strain, ATCC 7064 (57%), and an obligately thermophilic Bacillus thermoglucosidasius strain, KP1006 (59%). The number of prolines (19 for B. cereus oligo-1,6-glucosidase, 24 for B. coagulans enzyme, and 32 for B. thermoglucosidasius enzyme) was observed to increase with the rise in thermostabilities of the oligo-1,6-glucosidases. Classification of proline residues in light of the amino acid sequence alignment and the protein structure revealed by X-ray crystallographic analysis also supported this tendency. Judging from proline residues occurring in B. coagulans oligo-1,6-glucosidase and the structural requirement for proline substitution (second site of the beta turn and first turn of the alpha helix) (K. Watanabe, T. Masuda, H. Ohashi, H. Mihara, and Y. Suzuki, Eur. J. Biochem. 226:277-283, 1994), the critical sites for thermostabilization were found to be Lys-121, Glu-290, Lys-457, and Glu-487 in B. cereus oligo-1,6-glucosidase. With regard to protein evolution, the oligo-1,6-glucosidases very likely follow the neutral theory. The adaptive mutations of the oligo-1,6-glucosidases that appear to increase thermostability are consistent with the substitution of proline residues for neutrally occurring residues. It is concluded that proline substitution is an important factor for the selection of thermostability in oligo-1,6-glucosidases.

Amino Acid Sequence↗

PubMed1988

Mechanism of proline-specific proteinases: (I) Substrate specificity of dipeptidyl peptidase IV from pig kidney and proline-specific endopeptidase from Flavobacterium meningosepticum.

The substrate specificity of dipeptidyl peptidase IV (dipeptidyl peptide hydrolase, EC 3.4.14.5) from pig kidney and proline-specific endopeptidase from Flavobacterium meningosepticum, was investigated with a series of N-terminal unprotected (dipeptidyl peptidases IV) and succinylated dipeptidyl-p-nitroanilides (proline-specific endopeptidase). Both enzymes are specific for the S configuration of the amino-acid residue in P1 and P2 position if the penultimate residue is proline. In the case of alanine substrates (Ala in P1, dipeptidyl peptidase IV hydrolyzes such compounds where the configuration of the P2 residue is R. The penultimate residue with dipeptidyl peptidase IV can be, beside proline and alanine, dehydroproline, hydroxyproline and pipecolic acid. Proline substrates (Pro in P1) with an R configuration in P2 are inhibitors of the hydrolysis of proline substrates with an S,S configuration in an uncompetitive (dipeptidyl peptide IV) or mixed inhibition type (proline-specific endopeptidase). Derivatives of Gly-Pro-pNA where the N-terminal amino group is methylated are hydrolyzed by dipeptidyl peptidase IV.

Amino Acid Sequence↗

PubMed1997

Identification and initial characterisation of a N-benzyloxycarbonyl-prolyl-prolinal (Z-Pro-prolinal)-insensitive 7-(N-benzyloxycarbonyl-glycyl-prolyl-amido)-4-methylcoumarin (Z-Gly-Pro-NH-Mec)-hydrolysing peptidase in bovine serum.

A group of enzymes exists that specifically recognises proline within proteins and peptides. Prolyl endopeptidase is one such enzyme, which cleaves on the carboxyl side of proline within peptide substrates. Its broad specificity towards bioactive peptides has led to its implication in various disease states including neurodegenerative and psychiatric disorders. This association has been based primarily on the abnormal levels of activity observed following the enzymes detection with the reportedly specific fluorimetric substrate, 7-(N-benzyloxycarbonyl-glycyl-prolyl-amido)-4-methylcoumarin (Z-Gly-Pro-NH-Mec). In this study, we report the discovery and preliminary characterisation of a Z-Gly-Pro-NH-Mec-hydrolysing activity that is distinct from prolyl oligopeptidase (prolyl endopeptidase). Following the production of serum from bovine whole blood, Z-Gly-Pro-NH-Mec hydrolysis in serum was determined to be 7.2 U/mg protein. In the presence of 350 nM Z-Pro-prolinal, a specific inhibitor of prolyl endopeptidase, residual Z-Gly-Pro-NH-Mec hydrolysis of 2.6 U/mg protein was observed. This residual activity was resistant to inhibition by Z-Pro-prolinal at concentrations in excess of 200 times its reported Ki value for prolyl endopeptidase and could not be inhibited under conditions of prolonged incubation with the inhibitor. Following cation-exchange chromatography, Z-Gly-Pro-NH-Mec-hydrolysing activity was resolved into two distinct entities. The first of these activities was inhibited by Z-Pro-prolinal, demonstrated activity towards the thyroliberin analogue, 7-(pyroglutamyl-histidyl-prolyl)-4-methylcoumarin (Glp-His-Pro-NH-Mec), and was catalytically enhanced under reduced assay conditions. This activity was subsequently designated prolyl endopeptidase. The second activity was totally resistant to Z-Pro-prolinal inhibition, demonstrated no activity towards Glp-His-Pro-NH-Mec, and was unaffected when assayed under reduced conditions. It was subsequently designated Z-Pro-prolinal-insensitive Z-Gly-Pro-NH-Mec-hydrolysing peptidase (ZIP).

Animals↗

PubMed2006

Overexpression of proline oxidase induces proline-dependent and mitochondria-mediated apoptosis.

Proline oxidase (POX), a mitochondrial inner-membrane protein, catalyzes the rate-limiting oxidation of proline to pyrroline- 5-carboxylate (P5C). Previously we showed that overexpression of POX is associated with generation of reactive oxygen species (ROS) and apoptosis in POX-inducible colorectal cancer cells, DLD-1.POX. We also showed expression of mitochondrial MnSOD partially blunts POX-induced ROS generation and apoptosis. To further investigate the molecular basis of POX-induced apoptosis, we utilized the DLD-1.POX cells to show that cells overproducing POX exhibit an L-proline-dependent apoptotic response. The apoptotic effect is specific for L-proline, detectable at 0.2 mM, maximal at 1 mM, and occurs during 48-72 h following the addition of L-proline to cells with maximally induced POX. The apoptotic response is mitochondria-mediated with release of cytochrome c, activation of caspase-9, chromatin condensation/DNA fragmentation, and cell shrinkage. We conclude that in the presence of proline, high POX activity is sufficient to induce mitochondria-mediated apoptosis.

Apoptosis↗

PubMed1991

Investigation of cis/trans proline isomerism in a multiply occurring peptide fragment from human salivary proline-rich glycoprotein.

The solution-state conformations of eight proline-containing peptide fragments found in human salivary proline-rich glycoprotein (PRG) were investigated in 2 x distilled water (treated with metal ion chelating resin) using 13C-nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy. The peptide sequences and acronyms were as follows: PRG9-2 = NH2-G(1)-P(2)-CONH2, PRG9-3 = NH2-G(1)P(2)-P(3)-CONH2, PRG9-4 = NH2-G(1)-P(2)-P(3)-P(4)-CONH2, PRG9-5 = NH2-G(1)-P(2)-P(3)-P(4)-H(5)-CONH2, PRG9-6 = NH2-G(1)-P(2)-P(3)-P(4)-H(5)-P(6)-CONH2, PRG9-7 = NH2-G(1)-P(2)-P(3)-P(4)-H(5)-P(6)-G(7)-CONH2, PRG9-8 = NH2-G(1)-P(2)-P(3)-P(4)-H(5)-P(6)-G(7)-K(8)-CONH2 and PRG9-9 = NH2-G(1)-P(2)-P(3)-P(4)-H(5)-P(6)-G(7)-K(8)-P(9)-CONH2. Sequence-specific resonance assignments from the 13C-NMR spectra indicated that the trans proline isomer dominated the conformations of the peptides. CD results clearly showed the presence of the poly-L-proline II helix as the major conformation in PRG9-3----PRG9-5, supplemented by beta- and/or gamma-turns in PRG9-6----PRG9-9. These data suggest that in "metal free" water, native PRG could contain several small poly-L-proline II helices along with beta- and/or gamma-turns. Since proline is the major amino acid present in native PRG, these localized conformations may contribute to PRG's global conformation and act as a primary force in determining its biological activities.

Circular Dichroism↗