Effects of maternal L-proline supplementation on inflammatory cytokines at the placenta and fetus interface of mice.

Dietary L-proline (proline) supplementation during gestation enhances fetal survival and placental development in mice. The objective of the present study was to test the hypothesis that this beneficial effect of proline was associated with alterations in inflammatory response at the placenta and fetus interface. Populations of immune cells present in peripheral blood mononuclear cells (PBMC) were determined by flow cytometry analysis. The concentrations of immunoglobulins in plasma, and the concentrations of cytokines in plasma, uterus, placenta, and amniotic fluid were measured using a bead-based immunoassay. The data showed that proline supplementation led to higher (P < 0.05) populations of B lymphocytes (CD3-CD19+), natural killer (NK) cells (CD3-NK1.1+), and dendritic cells (DCs, CD11c+MHCII+) in peripheral blood, as compared with the controls. Conversely, mice fed a proline-supplemented diet had a lower population of neutrophils (CD11b+F4/80-). Further study showed that proline supplementation decreased (P < 0.05) the concentrations of (1) interleukin (IL)-23, IL-1α, and IL-6 in plasma; (2) IL-6 in the uterus; and (3) tumor necrosis factor alpha (TNF-α), monocyte chemotactic protein (MCP)-1, and IL-17 in the placenta; and (4) interferon (IFN)-γ in amniotic fluid, compared with controls. Conversely, proline supplementation resulted in higher (P < 0.05) concentrations of (1) IL-10, IL-17 and granulocyte-macrophage colony-stimulating factor (GM-CSF) in plasma; (2) IL-10 and IL-1α in the uterus; and (3) IL-1α, IL-1ß, IL-10, IL-27, and IFN-ß in amniotic fluid, compared with controls. Moreover, concentrations of immunoglobulin (Ig) G2b and IgM were enhanced (P < 0.05) by proline administration. Taken together, our results reveal a regulatory effect of proline in the immunological response at the maternal-fetal interface, which is critical for embryonic development and fetal survival.

Probing the role of proline -135 on the structure, stability, and cell proliferation activity of human acidic fibroblast growth factor.

Human acidic fibroblast growth factor 1 (hFGF1) is a protein intricately involved in cell growth and tissue repair. In this study, we investigate the effect(s) of understanding the role of a conserved proline (P135), located in the heparin binding pocket, on the structure, stability, heparin binding affinity, and cell proliferation activity of hFGF1. Substitution of proline-135 with a positively charged lysine (P135K) resulted in partial destabilization of the protein; however, the overall structural integrity of the protein was maintained upon substitution of proline-135 with either a negative charge (P135E) or a polar amino acid (P135Q). Interestingly, upon heparin binding, an increase in thermal stability equivalent to that of wt-hFGF1 was observed when P135 was replaced with a positive (P135K) or a negative charge (P135E), or with a polar amino acid (P135Q). Surprisingly, introduction of negative charge in the heparin-binding pocket at position 135 (P135E) increased hFGF1's affinity for heparin by 3-fold, while the P135K mutation, did not alter the heparin-binding affinity. However, the enhanced heparin-binding affinity of mutant P135E did not translate to an increase in cell proliferation activity. Interestingly, the P135K and P135E double mutations, P135K/R136E and P135/R136E, reduced the heparin binding affinity by ∼3-fold. Furthermore, the cell proliferation activity was increased when the charge reversal mutation R136E was paired with both P135E (P135E/R136E) and P135K (P135K/R136E). Overall, the results of this study suggest that while heparin is useful for stabilizing hFGF1 on the cell surface, this interaction is not mandatory for activation of the FGF receptor.

Transcriptomic analysis comparing stay-green and senescent Sorghum bicolor lines identifies a role for proline biosynthesis in the stay-green trait.

Sorghum bicolor is an important cereal crop grown on the arid and semi-arid regions of >98 different countries. These regions are such that this crop is often subjected to low water conditions, which can compromise yields. Stay-green sorghum plants are able to retain green leaf area for longer under drought conditions and as such have higher yields than their senescent counterparts. However, the molecular and physiological basis of this drought tolerance is yet to be fully understood. Here, a transcriptomic approach was used to compare gene expression between stay-green (B35) and senescent (R16) sorghum varieties. Ontological analysis of the differentially expressed transcripts identified an enrichment of genes involved with the 'response to osmotic stress' Gene Ontology (GO) category. In particular, delta1-pyrroline-5-carboxylate synthase 2 (P5CS2) was highly expressed in the stay-green line compared with the senescent line, and this high expression was correlated with higher proline levels. Comparisons of the differentially expressed genes with those that lie in known stay-green qualitative trait loci (QTLs) revealed that P5CS2 lies within the Stg1 QTL. Polymorphisms in known cis-elements were identified in the putative promoter region of P5CS2 and these could be responsible for the differences in the expression of this gene. This study provides greater insight into the stay-green trait in sorghum. This will be greatly beneficial not only to improve our understanding of drought tolerance mechanisms in sorghum, but also to facilitate the improvement of future sorghum cultivars by marker-assisted selection (MAS).

Is proline accumulation per se correlated with stress tolerance or is proline homeostasis a more critical issue?

Proline has been recognized as a multi-functional molecule, accumulating in high concentrations in response to a variety of abiotic stresses. It is able to protect cells from damage by acting as both an osmotic agent and a radical scavenger. Proline accumulated during a stress episode is degraded to provide a supply of energy to drive growth once the stress is relieved. Proline homeostasis is important for actively dividing cells as it helps to maintain sustainable growth under long-term stress. It also underpins the importance of the expansion of the proline sink during the transition from vegetative to reproductive growth and the initiation of seed development. Its role in the reproductive tissue is to stabilize seed set and productivity. Thus, to cope with abiotic stress, it is important to develop strategies to increase the proline sink in the reproductive tissue. We give a holistic account of proline homeostasis, taking into account the regulation of proline synthesis, its catabolism, and intra- and intercellular transport, all of which are vital components of growth and development in plants challenged by stress.

Flight muscle-specific Pro-Ala-rich extension of troponin is important for maintaining the insect-type myofilament lattice integrity.

Insect flight muscle (IFM) can oscillate at frequencies up to 1000Hz, owing to its capability of stretch activation (SA). It is a highly specialized form of cross striated muscles, and its peculiar features include the IFM-specific isoform of troponin-I (troponin-H or TnH) with an unusually long Pro-Ala-rich extension at the C-terminus. Although we have shown that this extension does not directly take part in SA, questions remain as to what its real role is and why it is expressed only in IFM. Here we explored the structural role of the extension, be comparing X-ray diffraction patterns and electron micrographs of bumblebee IFM fibers before and after enzymatic removal of the extension. The removal had a dramatic effect on diffraction patterns: In IFMs in general, the equatorial 2,0 reflection is much stronger than the 1,1 reflection, but after removal, their intensities became almost equal (stronger 1,1 is a feature of vertebrate skeletal muscle). Electron micrographs revealed that a substantial fraction of the thin filaments showed a tendency to move towards the vertebrate position (the trigonal position between three thick filaments), while the rest of the thin filaments remained in their original insect position (midway between two neighboring thick filaments). Therefore, one of the roles of the extension is suggested to keep the filament lattice in the correct configuration for IFM. This insect-type lattice structure is preserved among IFMs from varied insect orders but not in body muscles, suggesting that the maintenance of this lattice structure is important for flight functions.

Prolines in ßA-sheet of neural cadherin act as a switch to control the dynamics of the equilibrium between monomer and dimer.

Neural cadherins dimerize through the formation of calcium-dependent strand-crossover structures. Dimerization of cadherins leads to cell-cell adhesion in multicellular organisms. Strand-crossover dimer forms exclusively between the first N-terminal extracellular modules (EC1) of the adhesive partners via swapping of their ßA-sheets and docking of tryptophan-2 in the hydrophobic pocket. In the apo-state wild-type cadherin is predominantly monomer, which indicates that the dimerization is energetically unfavorable in the absence of calcium. Addition of calcium favors dimer formation by creating strain in the monomer and lowering the energetic barrier between monomer and dimer. Dynamics of the monomer-dimer equilibrium is vital for plasticity of synapses. Prolines recurrently occur in proteins that form strand-crossover dimer and are believed to be the source of the strain in the monomer. N-cadherins have two proline residues in the ßA-sheet. We focused our studies on the role of these two prolines in calcium-dependent dimerization. Spectroscopic, electrophoretic, and chromatopgraphic studies showed that mutations of both prolines to alanines increased the dimerization affinity by ~20-fold and relieved the requirement of calcium in dimerization. The P5A and P6A mutant formed very stable dimers that required denaturation of protein to disassemble in the apo conditions. In summary, the proline residues act as a switch to control the dynamics of the equilibrium between monomer and dimer which is crucial for the plasticity of synapses.

Proline 96 of the copper ligand loop of amicyanin regulates electron transfer from methylamine dehydrogenase by positioning other residues at the protein-protein interface.

Amicyanin is a type 1 copper protein that serves as an electron acceptor for methylamine dehydrogenase (MADH). The site of interaction with MADH is a "hydrophobic patch" of amino acid residues including those that comprise a "ligand loop" that provides three of the four copper ligands. Three prolines are present in this region. Pro94 of the ligand loop was previously shown to strongly influence the redox potential of amicyanin but not affinity for MADH or mechanism of electron transfer (ET). In this study Pro96 of the ligand loop was mutated. P96A and P96G mutations did not affect the spectroscopic or redox properties of amicyanin but increased the K(d) for complex formation with MADH and altered the kinetic mechanism for the interprotein ET reaction. Values of reorganization energy (λ) and electronic coupling (H(AB)) for the ET reaction with MADH were both increased by the mutation, indicating that the true ET reaction observed with native amicyanin was now gated by or coupled to a reconfiguration of the proteins within the complex. The crystal structure of P96G amicyanin was very similar to that of native amicyanin, but notably, in addition to the change in Pro96, the side chains of residues Phe97 and Arg99 were oriented differently. These two residues were previously shown to make contacts with MADH that were important for stabilizing the amicyanin-MADH complex. The values of K(d), λ, and H(AB) for the reactions of the Pro96 mutants with MADH are remarkably similar to those obtained previously for P52G amicyanin. Mutation of this proline, also in the hydrophobic patch, caused reorientation of the side chain of Met51, another reside that interacted with MADH and caused a change in the kinetic mechanism of ET from MADH. These results show that proline residues near the copper site play key roles in positioning other amino acid residues at the amicyanin-MADH interface not only for specific binding to the redox protein partner but also to optimize the orientation of proteins for interprotein ET.

Cis-trans isomerization at a proline opens the pore of a neurotransmitter-gated ion channel.

5-hydroxytryptamine type 3 (5-HT3) receptors are members of the Cys-loop receptor superfamily. Neurotransmitter binding in these proteins triggers the opening (gating) of an ion channel by means of an as-yet-uncharacterized conformational change. Here we show that a specific proline (Pro 8*), located at the apex of the loop between the second and third transmembrane helices (M2-M3), can link binding to gating through a cis-trans isomerization of the protein backbone. Using unnatural amino acid mutagenesis, a series of proline analogues with varying preference for the cis conformer was incorporated at the 8* position. Proline analogues that strongly favour the trans conformer produced non-functional channels. Among the functional mutants there was a strong correlation between the intrinsic cis-trans energy gap of the proline analogue and the activation of the channel, suggesting that cis-trans isomerization of this single proline provides the switch that interconverts the open and closed states of the channel. Consistent with this proposal, nuclear magnetic resonance studies on an M2-M3 loop peptide reveal two distinct, structured forms. Our results thus confirm the structure of the M2-M3 loop and the critical role of Pro 8* in the 5-HT3 receptor. In addition, they suggest that a molecular rearrangement at Pro 8* is the structural mechanism that opens the receptor pore.

Proline residues in CD28 and the Src homology (SH)3 domain of Lck are required for T cell costimulation.

The Src family tyrosine kinases Lck and Fyn are critical for signaling via the T cell receptor. However, the exact mechanism of their activation is unknown. Recent crystal structures of Src kinases suggest that an important mechanism of kinase activation is via engagement of the Src homology (SH)3 domain by proline-containing sequences. To test this hypothesis, we identified several T cell membrane proteins that contain potential SH3 ligands. Here we demonstrate that Lck and Fyn can be activated by proline motifs in the CD28 and CD2 proteins, respectively. Supporting a role for Lck in CD28 signaling, we demonstrate that CD28 signaling in both transformed and primary T cells requires Lck as well as proline residues in CD28. These data suggest that Lck plays an essential role in CD28 costimulation.

Prolyl aminopeptidase from Streptomyces thermoluteus subsp. fuscus strain NBRC14270 and synthesis of proline-containing peptides by its S144C variant.

We specifically examined an exopeptidase, prolyl aminopeptidase (PAP), as a target for synthesis of proline-containing peptides. A PAP from Streptomyces thermoluteus subsp. fuscus NBRC14270 (PAP14270) was obtained using sequence-based screening. From PAP14270, 144Ser was replaced by Cys (scPAP14270) to give aminolysis activity. In contrast to wild-type PAP14270, scPAP14270 produced a polymer of proline benzyl ester and cyclo[Pro-Pro]. The product mass was confirmed using liquid chromatography-mass spectrometry (LC/MS). Several factors affecting the reaction, such as the pH, concentration of the substrate, and reaction time, were measured to determine their effects. Furthermore, a correlation was found between substrate specificity in proline peptide synthesis and the log D value of acyl acceptors in aminolysis catalyzed by scPAP14270. Results showed that dipeptide synthesis proceeded in a weakly acidic environment and that cyclization and polymerization occurred under alkaline conditions. Furthermore, results suggest that almost all amino acid esters whose log D value is greater than 0, except hydroxyproline benzyl ester (Hyp-OBzl), can be recognized as acyl acceptors. These findings support the use of PAPs as a tool for production of physiologically active proline peptides.

Effect of glyprolines PGP, GP, and PG on homeostasis of gastric mucosa in rats with experimental ethanol-induced gastric ulcers.

The decrease in the severity of erosions and ulcer lesions after preventive treatment with PGP or PG correlated with a decrease in the content of lipid peroxidation products to a control level. Activities of SOD and catalase also returned to control values. GP produced the weakest effect on pro- and antioxidant state of the gastric mucosa. We concluded that the pronounced preventive effect of PGP and PG on the development of ethanol-induced erosions and ulcer lesions is largely determined by their antioxidant properties. Glyprolines can be considered as a promising means for prevention and treatment of stomach and duodenal ulcers.

Silicon confers cucumber resistance to salinity stress through regulation of proline and cytokinins.

Salt stress is a continuous threat to global crop production. Here, we studied the alleviation role of exogenous silicon (Si) in NaCl-stressed cucumber, with special emphasis on plant growth, proline (Pro) and hormone metabolisms. The results showed that Si supplementation ameliorated the adverse effects of NaCl on plants growth, biomass, and oxidative stress. Salt stress greatly increased the content of Pro throughout the experiment, while Si regulated Pro content in two distinct ways. Si promoted the salt-induced Pro levels after 3 and 6 days of treatment, but decreased it after 9 and 12 days of treatment. Moreover, P5CS and ProDH activities and P5CS gene play important roles in Si and salt-regulated Pro levels in different stress phase. Under stress condition, Si addition tend to revert the content of ABA, IAA, cytokinin and SA to the control levels in most cases. Further correlation analysis revealed a negative correlation between the root cytokinin and Pro content after 3 days of treatment, suggesting the interaction between cytokinin and Pro metabolism. Exogenous application of Pro and ProDH competitive inhibitor D-Lactate confirmed the possible interplay between Pro and cytokinin metabolism. Further study identified several CKX (Csa4G647490 and Csa1G589070) and IPT (Csa7G392940 and Csa3G150100) genes that may be responsible for the regulation of cytokinin accumulation by Si and/or Pro after short-term of treatment. The results suggested that Pro is a key factor in Si-induced salt tolerance, and Si-increased Pro content may participate in the regulation of cytokinin metabolism under short-term of salt stress.

The propensity for deamidation and transamidation of peptides by transglutaminase 2 is dependent on substrate affinity and reaction conditions.

Transglutaminase 2 (TG2) catalyzes cross-linking or deamidation of glutamine residues in peptides and proteins. The in vivo deamidation of gliadin peptides plays an important role in the immunopathogenesis of celiac disease (CD). Although deamidation is considered to be a side-reaction occurring in the absence of suitable amines or at a low pH, a recent paper reported the selective deamidation of the small heat shock protein 20 (Hsp20), suggesting that deamidation could be a substrate dependent event. Here we have measured peptide deamidation and transamidation in the same reaction to reveal factors that affect the relative propensity for the two possible products. We report that the propensity for deamidation by TG2 is both substrate dependent and influenced by the reaction conditions. Direct deamidation is favored for poor substrates and at low concentrations of active TG2, while indirect deamidation (i.e. hydrolysis of transamidated product) can significantly contribute to the deamidation of good peptide substrates at higher enzyme concentrations. Further, we report for the first time that TG2 can hydrolyze iso-peptide bonds between two peptide substrates. This was observed also for gliadin peptides introducing a novel route for the generation of deamidated T cell epitopes in celiac disease.

Pinning down proline-directed phosphorylation signaling.

The reversible phosphorylation of proteins on serine or threonine residues preceding proline (Ser/Thr-Pro) is a major cellular signaling mechanism. Although it is proposed that phosphorylation regulates the function of proteins by inducing a conformational change, there are few clues about the actual conformational changes and their importance. Recent identification of the novel prolyl isomerase Pin1 that specifically isomerizes only the phosphorylated Ser/Thr-Pro bonds in certain proteins led us to propose a new signaling mechanism, whereby prolyl isomerization catalytically induces conformational changes in proteins following phosphorylation to regulate protein function. Emerging data indicate that such conformational changes have profound effects on catalytic activity, dephosphorylation, protein-protein interactions, subcellular location and/or turnover. Furthermore, this post-phosphorylation mechanism might play an important role in cell growth control and diseases such as cancer and Alzheimer's.

Stress-tolerance of baker's-yeast (Saccharomyces cerevisiae) cells: stress-protective molecules and genes involved in stress tolerance.

During the fermentation of dough and the production of baker's yeast (Saccharomyces cerevisiae), cells are exposed to numerous environmental stresses (baking-associated stresses) such as freeze-thaw, high sugar concentrations, air-drying and oxidative stresses. Cellular macromolecules, including proteins, nucleic acids and membranes, are seriously damaged under stress conditions, leading to the inhibition of cell growth, cell viability and fermentation. To avoid lethal damage, yeast cells need to acquire a variety of stress-tolerant mechanisms, for example the induction of stress proteins, the accumulation of stress protectants, changes in membrane composition and repression of translation, and by regulating the corresponding gene expression via stress-triggered signal-transduction pathways. Trehalose and proline are considered to be critical stress protectants, as is glycerol. It is known that these molecules are effective for providing protection against various types of environmental stresses. Modifications of the metabolic pathways of trehalose and proline by self-cloning methods have significantly increased tolerance to baking-associated stresses. To clarify which genes are required for stress tolerance, both a comprehensive phenomics analysis and a functional genomics analysis were carried out under stress conditions that simulated those occurring during the commercial baking process. These analyses indicated that many genes are involved in stress tolerance in yeast. In particular, it was suggested that vacuolar H+-ATPase plays important roles in yeast cells under stress conditions.

Proline as a stress protectant in the yeast Saccharomyces cerevisiae: effects of trehalose and PRO1 gene expression on stress tolerance.

Proline is considered to be a stress protectant in the yeast Saccharomyces cerevisiae, as is trehalose. In this study, we constructed yeast strains that accumulate proline but not trehalose and that increase proline levels in response to stress. Our results suggest that proline does not complement the function of trehalose, and that moderate expression of the PRO1 gene encoding gamma-glutamyl kinase is important to stress tolerance of yeast cells.

Understanding regulatory networks and engineering for enhanced drought tolerance in plants.

Drought stress is one of the major limitations to crop productivity. To develop crop plants with enhanced tolerance of drought stress, a basic understanding of physiological, biochemical and gene regulatory networks is essential. Various functional genomics tools have helped to advance our understanding of stress signal perception and transduction, and of the associated molecular regulatory network. These tools have revealed several stress-inducible genes and various transcription factors that regulate the drought-stress-inducible systems. Translational genomics of these candidate genes using model plants provided encouraging results, but the field testing of transgenic crop plants for better performance and yield is still minimal. Better understanding of the specific roles of various metabolites in crop stress tolerance will give rise to a strategy for the metabolic engineering of crop tolerance of drought.

Proline accumulation in plants: a review.

Proline (Pro) accumulation is a common physiological response in many plants in response to a wide range of biotic and abiotic stresses. Controversy has surrounded the possible role(s) of proline accumulation. In this review, knowledge on the regulation of Pro metabolism during development and stress, results of genetic manipulation of Pro metabolism and current debate on Pro toxicity in plants are presented.

The role of proline residues in the structure and function of human MT2 melatonin receptor.

Melatonin functions as an essential regulator of various physiological processes in all vertebrate species. In mammals, two G protein-coupled melatonin receptors (GPCR) mediate some melatonin's actions: MT1 and MT2. Transmembrane domains (TM) of most GPCRs contain a set of highly conserved proline residues that presumably play important structural and functional roles. As TM segments of MT2 receptor display several interesting differences in expression of specific proline residues compared to other rhodopsin-like receptors (rGPCRs), we investigated the role of proline residues in the structure and function of this receptor. All prolines in TM segments of MT2 receptor were individually replaced with alanine and/or glycine. In addition, the unusual NAxxY motif located in TM7 was mutated to generate highly conserved NPxxY motif found in the majority of rGPCR proteins. Following transient expression in CHO-K1 cells, binding properties of the mutant receptors and their ability to transduce signals were analyzed using (125)I-mel- and [(35)S]GTPgammaS-binding assays, respectively. The impact of the performed mutations on the receptor structure was assessed by molecular dynamic simulations of MT2 receptors embedded in the fully hydrated phospholipid bilayer. Our results indicate that residues P174, P212 and P266 are important for the ligand binding and/or signaling of the human MT2 receptor. We also show that changes within the unusual NAxxY sequence in the TM7 (mutations A305P and A305V) produce defective MT2 receptors indicating an important role of this motif in the function of melatonin receptors.

Proline provides site-specific flexibility for in vivo collagen.

Fibrillar collagens have mechanical and biological roles, providing tissues with both tensile strength and cell binding sites which allow molecular interactions with cell-surface receptors such as integrins. A key question is: how do collagens allow tissue flexibility whilst maintaining well-defined ligand binding sites? Here we show that proline residues in collagen glycine-proline-hydroxyproline (Gly-Pro-Hyp) triplets provide local conformational flexibility, which in turn confers well-defined, low energy molecular compression-extension and bending, by employing two-dimensional 13C-13C correlation NMR spectroscopy on 13C-labelled intact ex vivo bone and in vitro osteoblast extracellular matrix. We also find that the positions of Gly-Pro-Hyp triplets are highly conserved between animal species, and are spatially clustered in the currently-accepted model of molecular ordering in collagen type I fibrils. We propose that the Gly-Pro-Hyp triplets in fibrillar collagens provide fibril "expansion joints" to maintain molecular ordering within the fibril, thereby preserving the structural integrity of ligand binding sites.