Effect of proline position on the antimicrobial mechanism of buforin II

Buforin II (BF2) is a histone-derived antimicrobial peptide that causes cell death by translocating across membranes and interacting with nucleic acids. It contains one proline residue critical for its function. Previous research found that mutations replacing proline lead to decreased membrane translocation and antimicrobial activity as well as increased membrane permeabilization. This study further investigates the role of proline in BF2's antimicrobial mechanism by considering the effect of changing proline position on membrane translocation, membrane permeabilization, and antimicrobial activity. For this purpose, four mutants were made with proline substitution (P11A) or relocation (P11A/G7P, P11A/V12P, P11A/V15P). These mutations altered the amount of helical content. Although antimicrobial activity correlated with the α-helical content for the peptides containing proline, membrane translocation did not. This observation suggests that factors in BF2's bactericidal mechanism other than translocation must be altered by these mutations. To better explain these trends we also measured the nucleic acid binding and membrane permeabilization of the mutant peptides. A comparison of mutant and wild type BF2 activity revealed that BF2 relies principally on membrane translocation and nucleic acid binding for antimicrobial activity, although membrane permeabilization may play a secondary role for some BF2 variants. A better understanding of the role of proline in the BF2 antimicrobial mechanism will contribute to the further design and development of BF2 analogs. Moreover, since proline residues are prevalent among other antimicrobial peptides, this systematic characterization of BF2 provides general insights that can promote our understanding of other systems.     

Nitrogen availability impacts oilseed rape (Brassica napus L.) plant water status and proline production efficiency under water-limited conditions

Large amounts of nitrogen (N) fertilizers are used in the production of oilseed rape. However, as low-input methods of crop management are introduced crops will need to withstand temporary N deficiency. In temperate areas, oilseed rape will also be affected by frequent drought periods. Here we evaluated the physiological and metabolic impact of nitrate limitation on the oilseed rape response to water deprivation. Different amounts of N fertilizer were applied to plants at the vegetative stage, which were then deprived of water and rehydrated. Both water and N depletion accelerated leaf senescence and reduced leaf development. N-deprived plants exhibited less pronounced symptoms of wilting during drought, probably because leaves were smaller and stomata were partially closed. Efficiency of proline production, a major stress-induced diversion of nitrogen metabolism, was assessed at different positions along the whole plant axis and related to leaf developmental stage and water status indices. Proline accumulation, preferentially in younger leaves, accounted for 25-85% of the free amino acid pool. This was mainly due to a better capacity for proline synthesis in fully N-supplied plants whether they were subjected to drought or not, as deduced from the expression patterns of the proline metabolism BnP5CS and BnPDH genes. Although less proline accumulated in the oldest leaves, a significant amount was transported from senescing to emerging leaves. Moreover, during rehydration proline was readily recycled. Our results therefore suggest that proline plays a significant role in leaf N remobilization and in N use efficiency in oilseed rape.     

Proline accumulation in plants: Not only stress

In addition to its role in protein synthesis and the plant cells'' response to environmental stresses, circumstantial evidence suggest that proline may also play a role in flowering and development both as a metabolite and as a signal molecule. Although there is a growing consensus that proline is of special importance throughout the reproductive phase (from flower transition to seed development) a general agreement on the molecular and genetic mechanisms proline is involved in, is yet to be established. In this paper we shall review and critically discuss most of the evidence supporting a role for proline in plant development, paying special attention to the recently reported role of proline in flower transition.Key words: proline, flower transition, embryo development, P5CS1, P5CS2     

Mutations in the listerial proB gene leading to proline overproduction: effects on salt tolerance and murine infection

The observed sensitivity of Listeria monocytogenes to the toxic proline analogue L-azetidine-2-carboxylic acid (AZ) suggested that proline synthesis in Listeria may be regulated by feedback inhibition of gamma-glutamyl kinase (GK), the first enzyme of the proline biosynthesis pathway, encoded by the proB gene. Taking advantage of the Epicurian coli mutator strain XL1-Red, we performed random mutagenesis of the recently described proBA operon and generated three independent mutations in the listerial proB homologue, leading to proline overproduction and salt tolerance when expressed in an E. coli (DeltaproBA) background. While each of the mutations (located within a conserved 26-amino-acid region of GK) was shown to confer AZ resistance (AZ(r)) on an L. monocytogenes proBA mutant, listerial transformants failed to exhibit the salt-tolerant phenotype observed in E. coli. Since proline accumulation has previously been linked to the virulence potential of a number of pathogenic bacteria, we analyzed the effect of proline overproduction on Listeria pathogenesis. However, our results suggest that as previously described for proline auxotrophy, proline hyperproduction has no apparent impact on the virulence potential of Listeria.     

Influence of Proline Substitution on the Bioactivity of Mammalian-Derived Antimicrobial Peptide NK-2

Multidrug-resistant bacteria are emerging as a global threat, making the search for alternative compounds urgent. Antimicrobial peptides (AMPs) became a promising hotspot due to their distinct action mechanism and possibility to be used as an alternative or complement to traditional antibiotics. However, gaining a better understanding about the relationship between antimicrobial peptides structure and its bioactivity is crucial for the development of next generation of antimicrobial agents. NK-2, derived from mammalian protein NK-lysin, has potent antitumor and bactericidal abilities. As proline was considered to be an effective α-helix breaker due to its restricted conformation, to better comprehend the effects of proline in the structure-activity relationship of NK-2, we constructed two NK-2 analogs. We examined the biological activities of NK-2 and its proline substitution analogs and analyzed the resulting conformational changes. Our results showed that introducing proline into the primary sequence of NK-2 significantly decreased the antitumor, antibacterial, and cytotoxic effects, as well as DNA binding activity by changing the α-helix content. However, α-helical content was not the only determining factor, the position of proline inserted was also critical. This study will allow for clearer insight into the role of proline in structure and bioactivity of NK-2 and provide a foundation for future studies.     

逆境条件下植物体内渗透调节物质的积累与活性氧代谢

本文介绍了逆境迫下植物体内渗透调节物质的积累和作用,及其对活性氧的产生与清除的影响,阐述以脯氨酸为代表的渗透调节物质对活性氧的直接清除作用,Ca^2 ,甜菜碱等对抗氧化酶活性及抗氧化剂含量的影响,近年来人们广泛利用转基因技术合成氨酸,甜菜碱,为提高作物的抗氧化能车及培育抗逆新品种提供了一条有效途径。     

逆境条件下植物体内渗透调节物质的积累与活性氧代谢

本文介绍逆境胁迫下植物体内渗透调节物质的积累和作用,及其对活性氧的产生与清除的影响。阐述以脯氨酸为代表的渗透调节物质对活性氧的直接清除作用,Ca2+、甜菜碱等对抗氧化酶活性及抗氧化剂含量的影响。近年来人们广泛利用转基因技术合成脯氨酸、甜菜碱,为提高作物的抗氧化能力及培育抗逆新品种提供了一条有效途径。     

Medicago truncatula stress associated protein 1 gene (MtSAP1) overexpression confers tolerance to abiotic stress and impacts proline accumulation in transgenic tobacco

Stress associated proteins (SAP) have been already reported to play a role in tolerance acquisition of some abiotic stresses. In the present study, the role of MtSAP1 (Medicago truncatula) in tolerance to temperature, osmotic and salt stresses has been studied in tobacco transgenic seedlings. Compared to wild type, MtSAP1 overexpressors were less affected in their growth and development under all tested stress conditions. These results confirm that MtSAP1 is involved in the response processes to various abiotic constraints. In parallel, we have performed studies on an eventual link between MtSAP1 overexpression and proline, a major player in stress response. In an interesting way, the results for the transgenic lines did not show any increase of proline content under osmotic and salt stress, contrary to the WT which usually accumulated proline in response to stress. These data strongly suggest that MtSAP1 is not involved in signaling pathway responsible for the proline accumulation in stress conditions. This could be due to the fact that the overexpression of MtSAP1 provides sufficient tolerance to seedlings to cope with stress without requiring the free proline action. Beyond that, the processes by which the MtSAP1 overexpression lead to the suppression of proline accumulation will be discussed in relation with data from our previous study involving nitric oxide.     

A Chemostat Study on Proline Uptake and Metabolism of Leishmania donovani

ABSTRACT Leishmania donovani grew in the chemostat on proline as its sole carbon and energy source at a maximum growth rate of 1.39 divisions per day. The efficiency of proline metabolism decreased with increasing external proline concentration. The internal concentration of proline and its intracellular metabolites was low when proline was the growth rate limiting substrate and high when proline was available in excess. In time-course experiments proline uptake leveled off after 30 min, independent of the culture conditions prior to the experiment. Proline uptake depended on the external proline concentration in a manner that is best described as the combination of an enzymatic and a diffusion component. Adaptation to different proline concentrations did not occur and no evidence was found that proline is actively transported by L. donovani.     

A chemostat study on proline uptake and metabolism of Leishmania donovani.

Leishmania donovani grew in the chemostat on proline as its sole carbon and energy source at a maximum growth rate of 1.39 divisions per day. The efficiency of proline metabolism decreased with increasing external proline concentration. The internal concentration of proline and its intracellular metabolites was low when proline was the growth rate limiting substrate and high when proline was available in excess. In time-course experiments proline uptake leveled off after 30 min, independent of the culture conditions prior to the experiment. Proline uptake depended on the external proline concentration in a manner that is best described as the combination of an enzymatic and a diffusion component. Adaptation to different proline concentrations did not occur and no evidence was found that proline is actively transported by L. donovani.     

Proline metabolism and transport in plant development

Proline fulfils diverse functions in plants. As amino acid it is a structural component of proteins, but it also plays a role as compatible solute under environmental stress conditions. Proline metabolism involves several subcellular compartments and contributes to the redox balance of the cell. Proline synthesis has been associated with tissues undergoing rapid cell divisions, such as shoot apical meristems, and appears to be involved in floral transition and embryo development. High levels of proline can be found in pollen and seeds, where it serves as compatible solute, protecting cellular structures during dehydration. The proline concentrations of cells, tissues and plant organs are regulated by the interplay of biosynthesis, degradation and intra- as well as intercellular transport processes. Among the proline transport proteins characterized so far, both general amino acid permeases and selective compatible solute transporters were identified, reflecting the versatile role of proline under stress and non-stress situations. The review summarizes our current knowledge on proline metabolism and transport in view of plant development, discussing regulatory aspects such as the influence of metabolites and hormones. Additional information from animals, fungi and bacteria is included, showing similarities and differences to proline metabolism and transport in plants.     

Comparison of proline accumulation in two mediterranean shrubs subjected to natural and experimental water deficit

The effect of water stress on proline accumulation was tested in two contrasted species of Mediterranean scrub: Halimium halimifolium (L.) Willk and Pistacia lentiscus L. Leaf water potential, stomatal resistance and proline content have been measured both in experimental and in natural water stress conditions. Both species accumulated proline in their leaves when leaf water potential dropped below a threshold value of –3.0 MPa, under natural as well as under experimental conditions. In the field, however, a time-lag between decrease of leaf water potential and proline accumulation could be observed. In Halimium halimifolium, proline accumulation appeared to be associated with severe stress conditions as most plants with high proline contents suffered irreversible wilting, especially in the greenhouse. P. lentiscus showed a different pattern, accumulating proline at two different times of the year, as a response to cold or to drought. The results of our study indicated that the role of proline in this species, rather than an osmotic agent, seems to be more related to a protective action in cases of severe stress conditions.     

Evidence that Hyperprolinemia Alters Glutamatergic Homeostasis in Rat Brain: Neuroprotector Effect of Guanosine

This study investigated the effects of acute and chronic hyperprolinemia on glutamate uptake, as well as some mechanisms underlying the proline effects on glutamatergic system in rat cerebral cortex. The protective role of guanosine on effects mediated by proline was also evaluated. Results showed that acute and chronic hyperprolinemia reduced glutamate uptake, Na(+), K(+)-ATPase activity, ATP levels and increased lipoperoxidation. GLAST and GLT-1 immunocontent were increased in acute, but not in chronic hyperprolinemic rats. Our data suggest that the effects of proline on glutamate uptake may be mediated by lipid peroxidation and disruption of Na(+), K(+)-ATPase activity, but not by decreasing in glutamate transporters. This probably induces excitotoxicity and subsequent energy deficit. Guanosine was effective to prevent most of the effects promoted by proline, reinforcing its modulator role in counteracting the glutamate toxicity. However, further studies are needed to assess the modulatory effects of guanosine on experimental hyperprolinemia.     

An improved chemical approach toward the C-terminal sequence analysis of proteins containing all natural amino acids.

An improved chemical method, capable of derivatizing all natural amino acids to their corresponding thiohydantoins, is described. This involves activation by acetyl chloride in TFA followed by derivatization with ammonium thiocyanate. Possible interference of reactive side chains was investigated by reacting N-acetylamino acids as well as several peptides with propionyl chloride instead of acetyl chloride. The products were characterized by PDMS mass spectrometry and 1H-NMR. This chemical method allows, for the first time, complete derivatization of N-acetylproline to proline thiohydantoin. Applying this chemistry to peptides with a C-terminal proline, the yields for formation of proline thiohydantoin were found to be up to 60%, depending on the peptide sequence. The previous inability to derivatize C-terminal proline to thiohydantoin was thought to stem from the fact that proline cannot form the oxazolonium ion required for efficient reaction with the thiocyanate ion. However, we have found mass spectrometric evidence for the existence of a proline oxazolonium ion, under basic as well as under acidic conditions. This improvement in derivatization of C-terminal amino acids including proline is a major step forward in the development of a general chemical C-terminal sequencing method that permits the C-terminal sequence analysis of proteins of any amino acid composition.     

Nitrogen fixation in Klebsiella pneumoniae during osmotic stress. Effect of exogenous proline or a proline overproducing plasmid

Osmotic stress, imposed by 0.5 M NaCl or other electrolytes and non-electrolytes, caused over a 100-fold reduction in the whole-cell nitrogen fixation activity of Klebsiella pneumoniae, wild-type strain M5A1. This reduction of nitrogen fixation activity could be reversed by the addition of proline to the culture medium at 0.5 mM concentration. With 0.5 M NaCl, in the presence of proline, nitrogenase activity was 47-fold greater than in the absence of proline. A mutation, originally isolated in Salmonella typhimurium, which resulted in proline over-production and enhanced osmotolerance, was transferred into K. pneumoniae by F' conjugation. Intracellular proline, synthesized at high levels because of the mutation, had similar stimulatory effects on nitrogen fixation under osmotic stress as proline provided exogenously. In the overproducing strain, the cellular level of proline is elevated as much as 125-fold during stress over that seen in the control strain. To determine the mechanism of stimulation of nitrogen fixation by proline during stress, the biosynthesis of nitrogenase polypeptides was studied. Net nitrogenase biosynthesis and the biosynthesis of other unidentified peptides, is strongly inhibited during osmotic stress; proline reverses the inhibition. The role of proline in enhancing nitrogen fixation during osmotic stress is discussed.     

脯氨酸转运相关基因的研究进展

作为植物中普遍存在的一种逆境适应机制, 脯氨酸积累一直被认为是其合成和降解调控的结果。然而越来越多的研究表明, 脯氨酸转运也可能在其积累过程中起重要作用。在植物中, 有多个氨基酸转运蛋白家族, 如氨基酸通透酶家族(AAPs)、赖氨酸组氨酸转运蛋白家族(LHTs)和脯氨酸转运蛋白家族(ProTs)参与脯氨酸在各个器官间的运输。该文对参与脯氨酸运输的基因家族成员的表达模式、生理功能及表达调控进行了综述, 以期为脯氨酸运输与积累在植物抗逆方面的研究提供参考。     

Elimination of self-association as the source of the thermodynamic nonideality in aqueous proline solutions

The effect of high concentrations of proline on the diffusion coefficient of water has been examined to assess the extent to which the resulting thermodynamic nonideality could be explained on the statistical-mechanical basis of excluded volume. In fact, such a space-filling role not only accounts for the proline concentration-dependence of the diffusion coefficient of water but it also accounts for the nonideality of proline in freezing point depression and isopiestic measurements. These findings refute the conclusion (Schobert, B. and Tschesche, H. (1978) Biochim. Biophys. Acta 541, 270–277) that the stabilization of enzyme structure by high concentrations of proline stems from self-association of the imino acid via intermolecular hydrogen bonding; and thereby support the concept that the protective effect of proline on enzyme stability must reside mainly in its action as an inert, space-filling solute.     

Role of conserved Arg40 and Arg117 in the Na+/proline transporter of Escherichia coli.

The Na+/proline transporter of Escherichia coli (PutP) is a member of a large family of Na+/solute symporters. To investigate the role of Arg residues which are conserved within this family, Arg40 at the cytoplasmic end of transmembrane domain (TM) II and Arg117 in cytoplasmic loop 4 of PutP are subjected to amino acid substitution analysis. Removal of the positive charge at position 40 (PutP-R40C, Q, E) leads to a dramatic decrease of the V(max) of Na(+)-coupled proline uptake (1-10% of PutP-wild-type). The reduced transport rates are accompanied by decreased apparent affinities of the transporter for Na+ and Li+ while the apparent affinity for proline is only slightly altered. Furthermore, single Cys PutP-R40C reacts with N-ethylmaleimide (NEM), and this reaction is partially inhibited by proline and more efficiently by Na+ ions. Remarkably, NEM modification of Cys40 inhibits Na(+)-driven proline uptake almost completely while facilitated influx of proline into deenergized cells is stimulated by this reaction, suggesting an at least partially uncoupled phenotype under these conditions. These results suggest that Arg40 is located close to the site of ion binding and is important for the coupling of ion and proline transport. The observations confirm the functional importance of TM II described in earlier studies [M. Quick and H. Jung (1997) Biochemistry 36, 4631-4636]. In contrast to Arg40, Arg117 is apparently not important for function of the mature protein. The low transport rates observed upon substitution of Arg117 (PutP-R117C, K, Q) can at least partially be attributed to reduced amounts of PutP in the membrane. However, once inserted into the membrane, PutP containing Arg117 replacements shows a stability comparable to the wild-type as indicated by pulse-chase experiments. These observations suggest that Arg117 plays a crucial role at a stage prior to complete functional insertion of PutP into the membrane, i. e., by stabilizing a folding intermediate.     

Evidence for a sodium-dependent proline and glycine-betaine uptake in the cyanobacterium Nostoc muscorum

The cyanobacterium Nostoc muscorum is able to utilized proline and glycine-betaine as a nitrogen source under unstressed growth conditions. This cyanobacterium when grow in modified Chu No. 10 medium (without Na+) unable to utilized proline and glycine-betaine as a nitrogen source. Spontaneously occurring mutant clones defective in Na+ transport (Na+-R) were isolated and analyzed for proline and glycine-betaine utilization. The mutant phenotype showed normal heterocyst frequency and nitrogenase activity even in the medium containing 1 mM proline or 1 mM glycine-betaine, indicates the role of Na+ for proline/glycine-betaine uptake. The Na+-R mutant showed 100% survival at pH 11 and was simultaneously able to uptake and utilize proline/glycine-betaine at higher alkaline pH. This indicates that proline and glycine-betaine uptake systems are more efficient at higher alkaline pH. Since, the hypersaline environments are rich in Na+ contents and have alkaline pH, therefore it is suggested that the origin and evolution of specific compatible solutes may not depend only on the osmoregulatory role they play, but also on the other ecological factors operating simultaneously in the organism's niche.     

Alterations in the activities of the enzymes of proline metabolism in Ragi (Eleusine coracana) leaves during water stress

Free proline content in Ragi (Eleusine coracana) leaves increased markedly (6 to 85 fold) as the degree of water stress, created by polyethylene gylcol treatment, was prolonged There was also a marginal increase in soluble proteins in the stressed leaves as compared to that in the controls. Water stress stimulated the activities of ornithine aminotransferase and pyrroline-5-carboxylate reductase, the enzymes of proline biosynthesis and markedly inhibited the enzymes involved in proline degradation viz., proline oxidase and pyrroline-5-carboxylate dehydrogenase. These results suggest that increase in free proline content of Ragi leaves could be due to enhanced activities of the enzymes synthesizing proline but more importantly due to severe inhibition of the enzymes degrading proline. These observations establish for the first time, the pathway of proline metabolism in plants by way of detection of the activities of all the enzymes involved and also highlight the role of these enzymes in proline accumulation during water stress.