Role Of Proline Research Articles

Differences in Cowpea Root Growth Triggered by Salinity and Dehydration are Associated with Oxidative Modulation Involving Types I and III Peroxidases and Apoplastic Ascorbate

The aim of this work was to investigate the balance between the activities of ascorbate peroxidase (APX) and phenol peroxidases (POD) and cowpea root growth in response to dehydration and salt stress. Root growth and indicators of oxidative response were markedly changed in response to salinity and dehydration. Salt treatment strongly inhibited root elongation, which was associated with an increase in lignin content and a significant decrease in the concentrations of apoplastic hydrogen peroxide (H2O2) and ascorbate. In conditions of extreme salinity, cytosol–APX activity was significantly decreased. In contrast, cell-wall POD activity was greatly increased, whereas lipid peroxidation was unchanged. These results indicate that POD could be involved in both H2O2 scavenging and the inhibition of root elongation under high salinity. In contrast, dehydration stimulated primary root elongation and increased lipid peroxidation and apoplastic ascorbate content, but it did not change APX and POD activities or H2O2 concentration. When cowpea roots were subjected to salinity followed by dehydration, the water and pressure potentials were decreased, and lipid peroxidation was markedly increased, highlighting the additive nature of the inhibitory effects caused by salt and dehydration. The proline concentration was markedly increased by dehydration alone, as well as by salt followed by dehydration, suggesting a possible role for proline in osmotic adjustment. Salinity and dehydration induce contrasting responses in the growth and morphology of cowpea roots. These effects are associated with different types of oxidative modulation involving cytosolic-APX and cell-wall POD activities and apoplast H2O2 and ascorbate levels.

Raw and fungal-treated olive-mill wastewater effects on selected parameters of lettuce (Lactuca sativa L.) growth – The role of proline

Olive-mill wastewater (OMW) constitutes a major agricultural waste stream for which disposal is associated with significant environmental repercussions. No data are available on the effects of biotreated OMW and of the protective role of exogenously provided proline on plant physiology. In the present study, OMW was administered, either raw or previously treated by the white-rot fungus Pleurotus ostreatus, with or without proline amendment, to lettuce plants growing in sterilized sand. Biotreated OMW and proline addition resulted in significant moderation of OMW adverse effects on plant biomass production and ascorbic acid content, while their synergistic action alleviated the severe negative impact on net photosynthetic rate, water use efficiency and photosynthetic activity (Fv/Fo) invoked by the effluent. Moreover, biotreated OMW supplemented with proline, moderated the decrease in chlorophylls exerted by raw OMW, but it did not contribute at restoring carotenoids content. Restoration of plant transpiration was complete when biotreated OMW was used (with or without proline); proline alone mitigated the negative impact of OMW on photosynthetic efficiency (Fv/Fm and Fv′/Fm′). It seems that key photosynthetic parameters could be exploited as suitable evaluators of wastewater-induced plant toxicity, while plant fertigation with biotreated and/or supplemented OMW could be an interesting prospect in valorizing this effluent.

For the Sequence YKGQ, the Turn and Extended Conformational Forms Are Separated by Small Barriers and the Turn Propensity Persists Even at High Temperatures: Implications for Protein Folding

The folding of the sequence (21)DTVKLMYKGQPMTFR(35) from staphylococcal nuclease into a β-hairpin, nucleated by the turn region YKGQP, is known to be an early folding event. With YKGQ being the shortest sequence for a β-turn model and in view of its importance to the folding of staphylococcal nuclease, we investigated the thermodynamics of turn formation at a range of temperatures from 280 to 380 K, with a regular interval of 10 K. Eleven independent molecular dynamics simulations (under NPT conditions) were performed using the GROMACS package of programs and the OPLS-AA/L all-atom force field, each for a time period of 1 μs. Turn formation is supported by enthalpy at lower temperatures, while entropy supports it at higher temperatures. There are modest free energy barriers between turn and extended conformational ensembles. The turn propensity persists even at elevated temperatures. The role of proline in driving the turn formation has been re-examined, and it is inferred that the absence of proline does not affect turn propensity.

Defect in proline synthesis: pyrroline‐5‐carboxylate reductase 1 deficiency leads to a complex clinical phenotype with collagen and elastin abnormalities

Pyrroline-5-carboxylate reductase 1 (PYCR1) catalyzes the last step in proline synthesis. Deficiency of PYCR1, caused by a defect in PYCR1, was recently described in patients with cutis laxa, intrauterine growth retardation, developmental dysplasia of the hips and mental retardation. In this paper, we describe additional six patients (ages ranging from 4 months to 55 years) from four Iranian families with clinical manifestations of a wrinkly skin disorder. All patients have distinct facial features comprising triangular face, loss of adipose tissue and thin pointed nose. Additional features are short stature, wrinkling over dorsum of hand and feet, visible veins over the chest and hyperextensible joints. Three of the patients from a large consanguineous family do not have mental retardation, while the remaining three patients from three unrelated families have mental and developmental delay. Mutation analysis revealed the presence of disease-causing variants in PYCR1, including a novel deletion of the entire PYCR1 gene in one family, and in each of the other patients the homozygous missense mutations c.616G > A (p.Gly206Arg), c.89T > A (p.Ile30Lys) and c.572G > A (p.Gly191Glu) respectively, the latter two of which are novel. Light- and electron microscopy investigations of skin biopsies showed smaller and fragmented elastic fibres, abnormal morphology of the mitochondria and their cristae, and slightly abnormal collagen fibril diameters with irregular outline and variable size. In conclusion, this study adds information on the natural course of PYCR1 deficiency and sheds light on the pathophysiology of this disorder. However, the exact pathogenesis of this new disorder and the role of proline in the development of the clinical phenotype remain to be fully explained.

Role of proline and glycinebetaine pretreatments in improving heat tolerance of sprouting sugarcane (Saccharum sp.) buds

High temperature strongly hampers the plant growth particularly at early growth stages. In this study, changes in some physiological and anatomical characteristics and possibility of mitigating the adversities of heat stress by soaking sugarcane nodal buds in 20 mM proline and glycinebetaine (GB) solutions have been explored. Heat stress reduced the rate of bud sprouting nonetheless soaking the setts in proline followed by GB was beneficial. In addition, heat stress reduced the bud fresh and dry weights, generated H2O2, reduced the tissue levels of K+ and Ca2+, while increased the osmolytes synthesis in a time course manner. Heat stress also delayed the emergence and expansion of new bud leaves, by restricting the number and area of mesophyll cells. It also caused poor and aberrant development and diffused appearance of mesophyll cells and vascular bundles in the bud leaves. However, soaking of buds in proline and GB solutions substantially reduced the H2O2 production, improved the accumulation of soluble sugars and protected the developing tissues from heat stress effects; although proline was more effective than GB. Correlations of various attributes indicated that soaking in GB and proline restricted the H2O2 generation, improved K+ and Ca2+ contents, and increased the concentrations of free proline, GB and soluble sugars eventually improving the heat tolerance of buds. Cost-benefit analysis showed that, considering increase in sprouting of buds, soaking in 20 mM solution of both osmoprotectants is economical.

Salt Tolerance in MungbeanVigna radiata[L.] Genotypes: Role of Proline and Glycinebetaine

Salinity is one of the major environmental determinants of plant growth and productivity. A pot experiment was performed using six cultivars of mungbean [Vigna radiata L.] to evaluate the difference in their response to NaCl toxicity under growth chamber conditions. The pots containing reconstituted soil were supplied with different concentration of salinity [0, 50 and 100 mM NaCl]. Increasing concentration of NaCl in the soil resulted in decreased net photosynthesis, plant dry mass and seed yield. Maximum significant reduction in growth, photosynthesis and yield were observed with 100 mM NaCl in all the cultivars. Our results indicate that the cultivar Punt Mung is found to be more tolerant to salinity stress, recording higher net photosynthesis, plant dry mass and seed yield associated with low Na+ and Cl− content and high osmolyte accumulation in the plant leaves. Cultivar T44 experienced maximum damage in terms of reduction in net photosynthesis, plant dry mass and seed yield and emerged as a sensitive cultivar. The data of tolerance index of Punt Mung were found to be higher among all tested mungbean cultivars which indicate its higher tolerance to salinity. Better coordination of osmolytes and lower values of Na+ and Cl− protected Punt mung from NaCl toxicity, whereas lesser accumulation of osmolytes and higer values of Na+ and Cl− in T44 resulted in maximum damage. Cultivars of mungbean were ranked with respect to their tolerance to salinity: Punt mung > Pusa 9531> Pusa Baisakhi > Samrat >SML 267> T44, respectively.

The Role of Proline in the Membrane Re-entrant Helix of Caveolin-1

Caveolin-1 has a segment of hydrophobic amino acids comprising approximately residues 103-122. We have performed an in silico analysis of the conformational preference of this segment of caveolin-1 using PepLook. We find that there is one main group of stable conformations corresponding to a hydrophobic U bent model that would not traverse the membrane. Furthermore, the calculations predict that substituting the Pro(110) residue with an Ala will change the conformation to a straight hydrophobic helix that would traverse the membrane. We have expressed the P110A mutant of caveolin-1, with a FLAG tag at the N terminus, in HEK 293 cells. We evaluate the topology of the proteins with confocal immunofluorescence microscopy in these cells. We find that FLAG tag at the N terminus of the wild type caveolin-1 is not reactive with antibodies unless the cell membrane is permeabilized with detergent. This indicates that in these cells, the hydrophobic segment of this protein is not transmembrane but takes up a bent conformation, making the protein monotopic. In contrast, the FLAG tag at the N terminus of the P110A mutant is equally exposed to antibodies, before and after membrane permeabilization. We also find that the P110A mutation causes a large reduction of endocytosis of caveolae, cellular lipid accumulation, and lipid droplet formulation. In addition, we find that this mutation markedly reduces the ability of caveolin-1 to form structures with the characteristic morphology of caveolae or to partition into the detergent-resistant membranes of these cells. Thus, the single Pro residue in the membrane-inserting segment of caveolin-1 plays an important role in both the membrane topology and localization of the protein as well as its functions.

Bentazon triggers the promotion of oxidative damage in the Portuguese ricefield cyanobacterium Anabaena cylindrica: Response of the antioxidant system

Rice fields are frequently exposed to environmental contamination by herbicides and cyanobacteria, as primary producers of these aquatic ecosystems, are adversely affected. Anabaena cylindrica is a cyanobacterium with a significantly widespread occurrence in Portuguese rice fields. This strain was studied throughout 72 h in laboratory conditions for its stress responses to sublethal concentrations (0.75-2 mM) of bentazon, a selective postemergence herbicide recommended for integrated weed management in rice, with special reference to oxidative stress, role of proline and intracellular antioxidant enzymes in herbicide-induced free radicals detoxification. Activities of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione S-transferase (GST) increased in a time- and herbicide dose-response manner and were higher than those in the control samples after 72 h. A time- and concentration-dependent increase of malondialdehyde (MDA) levels and the enhanced cell membrane leakage following bentazon exposure are indicative of lipid peroxidation, free radicals formation, and oxidative damage, while increased amounts of SOD, CAT, APX, GST, and proline indicated their involvement in free radical scavenging mechanisms. The appreciable decline in the reduced glutathione (GSH) pool after 72 h at higher bentazon concentrations could be explained by the reduction of the NADPH-dependent glutathione reductase (GR) activity. The obtained results suggested that the alterations of antioxidant systems in A. cylindrica might be useful biomarkers of bentazon exposure. As the toxic mechanism of bentazon is a complex phenomenon, this study also adds relevant findings to explain the oxidative stress pathways of bentazon promoting oxidative stress in cyanobacteria.

The role of organic and inorganic solutes in the osmotic adjustment of drought-stressed Jatropha curcas plants

This study aimed to assess the accumulation of organic and inorganic solutes and their relative contribution to osmotic adjustment in roots and leaves of Jatropha curcas subjected to different water deficit intensity. Plants were grown in vermiculite 50% (control), 40%, 30%, 20% and 10% expressed in gravimetric water content. The water potential, osmotic potential and turgor potential of leaves decreased progressively in parallel to CO 2 photosynthetic assimilation, transpiration and stomatal conductance, as the water deficit increased. However, the relative water content, succulence and water content in the leaves did not show differences between the control and stressed plants, indicating osmotic adjustment associated with an efficient mechanisms to prevent water loss by transpiration through stomatal closure. The K + ions had greater quantitative participation in the osmotic adjustment in both leaves and roots followed by Na + and Cl −, while the NO 3 − ion only showed minor involvement. Of the organic solutes studied, the total soluble sugars showed the highest relative contribution to the osmotic adjustment in both organs and its concentration positively increased with more severe water deficit. The free amino acids and glycinebetaine also effectively contributed to the osmotic potential reduction of both the root and leaves. The role of proline was quantitatively insignificant in terms of osmotic adjustment, in both the control and stressed roots and leaves. Our data reveal that roots and leaves of J. curcas young plants display osmotic adjustment in response to drought stress linked with mechanisms to prevent water loss by transpiration by means of the participation of inorganic and organic solutes and stomatal closure. Of all the solutes studied, soluble sugars uniquely display a prominent drought-induced synthesis and/or accumulation in both roots and leaves.

Exogenous Proline Application Reduces Phytotoxic Effects of Selenium by Minimising Oxidative stress and Improves Growth in Bean (Phaseolus vulgaris L.) Seedlings

Bean (Phaseolus vulgaris L.) seedlings were subjected to varying selenium levels (1, 2, 4, and 6 ppm) in a hydroponic culture. The germination reached 100% in 48 h in all Se levels except 6 ppm, where it took 72 h. The root and shoot growth was stimulated at 1 and 2 ppm Se levels that was commensurate with increase in chlorophyll content, leaf water content, and cellular respiration. At 4 and 6 ppm Se levels, the growth was inhibited appreciably, which was associated with increase in stress injury measured as damage to membranes and decrease in cellular respiration, chlorophyll, and leaf water content. The oxidative injury as elevation of lipid peroxidation was larger compared to hydrogen peroxide accompanied by reduced levels of enzymatic (superoxide dismutase, catalase, ascorbate peroxidase, and glutathione reductase) and non-enzymatic (ascorbic acid and glutathione) antioxidants. Proline content was significantly higher at 1 and 2 ppm Se but diminished considerably at 4 and 6 ppm levels concomitant with the reduced growth. Exogenous application of proline (50 µM) resulted in substantiation of its endogenous levels that antagonised the toxic effects of Se by improving the growth of seedlings. The stress injury was reduced significantly with simultaneous increase in enzymatic and non-enzymatic antioxidants. Especially the components of ascorbate-glutathione cycle showed larger stimulation with proline application. The role of proline in mitigating the toxic effects of Se is discussed.

The role of proline in the membrane re‐entrant helix of caveolin‐1

Caveolin‐1 (Cav1) has a segment of hydrophobic amino acids comprising approximately residues 103 to 122. We have performed an in silico analysis of the conformational preference of this segment of Cav1 using PepLook. We find that there is one main group of stable conformations corresponding to a hydrophobic U bent model that would not traverse the membrane. Furthermore, the calculations predict that substituting the Pro110 residue with an Ala will change the conformation to a straight hydrophobic helix that would traverse the membrane. We have expressed the FLAG‐tagged P110A mutant of Cav1 in HEK 293 cells. We find that this mutation causes a large reduction of endocytosis of caveolae. We also evaluate the topology of the proteins with confocal immunofluorescence microscopy. The cells expressing Cav1 show that the hydrophobic segment of this protein is not transmembrane but takes up a bent conformation, making the protein monotopic. In contrast, the FLAG‐tagged P110A mutant is equally exposed to antibodies, before and after membrane permeabilization. Thus, the single Pro residue in the membrane‐inserting segment of Cav1 plays an important role in both the membrane topology of the protein as well as its function in the endocytosis of caveolin. (Supported by Canadian Post‐Doctoral Research Fellowships 2008–09 and by the Natural Sciences and Engineering Research Council of Canada, grant 9848)

Role of Proline in the Folding of Conotoxins

Evaluation of the role of disulfide bridges plays an important part in understanding the concept of protein folding. We are investigating how slight changes - the presence vs. the absence of the cyclic amino acid proline in a certain position of the peptide-chain - in the sequence of small peptides influence their folding properties. The present studies focus on the folding of a group of small peptides found in Conus snails, α-conotoxins SI, SIA (found in Conus Striatus), and GI (Conus Geographus), under two different oxidizing conditions. Each peptide has two disulfide bridges leading to three possible regioisomers, only one of which is found in nature. Our results indicate that peptides containing the cyclic amino acid proline had very high selectivity for the natural isomer, suggesting that this amino acid enforces a structural rigidity on the peptides. This research has been supported by funding through NIH R15 GM074654-01A2 and NSF DUE 0525440.

Conformational dynamics of the inner pore helix of voltage-gated potassium channels

Voltage-gated potassium (Kv) channels control the electrical excitability of neurons and muscles. Despite this key role, how these channels open and close or gate is not fully understood. Gating is usually attributed to the bending and straightening of pore-lining helices at glycine and proline residues. In this work we focused on the role of proline in the Pro-Val-Pro (PVP) motif of the inner S6 helix in the Kv1.2 channel. We started by developing a simple hinged-rod model to fully explore the configurational space of bent helices and we related these configurations to the degree of pore opening. We then carried out fully atomistic simulations of the S6 helices and compared these simulations to the hinged-rod model. Both methods suggest that Kv1 channels are not tightly closed when the inner helices are straight, unlike what is seen in the non-PVP containing channels KcsA and KirBac. These results invite the possibility that the S6 helices may be kinked when Kv1 channels are closed. Our simulations indicate that the wild-type helix adopts multiple spatially distinct configurations, which is consistent with its role in adopting a closed state and an open state. The two most dominant configurational basins correspond to a 6 A movement of the helix tail accompanied by the PVP region undergoing a local alpha-helix to 3(10)-helix transition. We explored how single point mutations affect the propensity of the S6 helix to adopt particular configurations. Interestingly, mutating the first proline, P405 (P473 in Shaker), to alanine completely removed the bistable nature of the S6 helix possibly explaining why this mutation compromises the channel. Next, we considered four other mutations in the area known to affect channel gating and we saw similarly dramatic changes to the helix's dynamics and range of motion. Our results suggest a possible mechanism of helix pore closure and they suggest differences in the closed state of glycine-only channels, like KcsA, and PVP containing channels.

Investigating the Role of Proline in Buforin II Function

Buforin II (BF2) is a 21 amino acid long antimicrobial peptide. Unlike many antimicrobial peptides that induce cell death by disrupting the cellular membrane, BF2 exhibits potent antimicrobial activity without significant membrane permeabilization. A histone derivative, BF2 is hypothesized to kill bacteria by translocating across the cell membrane and binding to nucleic acids. Its membrane-penetrating property makes it a potential model for novel drug delivery systems. Pro 11 of BF2 been shown to play an important role in membrane translocation. To investigate the role played by proline, it was replaced with alanine (P11A) or reintroduced at various locations (P11A/G7P, P11A/V12P and P11A/V15P). Changing the location of the proline residue alters the peptide's overall helicity and affects the peptide's antimicrobial activity. Lipid vesicle assays showed that an optimal amount of α-helicity appears related to translocation, as increased or decreased helicity both led to reduced translocation compared to wild type BF2. However, antimicrobial activities did not correlate clearly with translocation abilities. To better understand the antimicrobial mechanism of BF2 and the role of proline, pore formation and DNA binding were investigated. The pore-forming abilities of wild type BF2 and its proline mutants were examined with a lipid vesicle dye-leakage assay. These experiments showed that increased α-helicity correlates with peptides' increased ability to cause membrane permeabilization. A fluorescent intercalator assay was used to determine the peptides' ability to bind nucleic acids. These studies revealed that while P11A/G7P exhibits a significantly stronger DNA binding ability than wild type BF2, the other mutants have similar DNA binding abilities. Together, this data helps to explain the imperfect correlation between the peptides' respective antimicrobial activities and their abilities to translocate and sheds light on the role of proline in BF2 function.

Proline accumulation and transcriptional regulation of proline biothesynthesis and degradation in Brassica napus

To understand the molecular mechanism underlying proline accumulation in Brassica napus, cDNAs encoding Delta(1)-pyrroline-5-carboxylate synthetase (BnP5CS), ornithine delta-aminotransferase (BnOAT) and proline dehydrogenase (BnPDH) were isolated and characterized. Southern blot analysis of BnP5CSs in B. napus and its diploid ancestors suggested a gene loss may have occurred during evolution. The expression of BnP5CS1 and BnP5CS2 was induced, while the expression of BnPDH was inhibited under salt stress, ABA treatment and dehydration, prior to proline accumulation. The upregulation of BnOAT expression was only detected during prolonged severe osmotic stress. Our results indicate that stress-induced proline accumulation in B. napus results from the reciprocal action of activated biosynthesis and inhibited proline degradation. Whether the ornithine pathway is activated depends on the severity of stress. During development, proline content was high in reproductive organs and was accompanied by markedly high expression of BnP5CS and BnPDH, suggesting possible roles of proline during flower development. [BMB reports 2009; 42(1): 28-34].

Relationship of secondary metabolism to growth in oregano ( Origanum vulgare L.) shoot cultures under nutritional stress

Micropropagation of Origanum vulgare L. by shoot buds, as a potential model system for studying carbon skeleton diversion from growth to secondary metabolism as adaptive response to nutrient deficiency, has been performed. In addition, the antioxidant phenolic compounds, produced by shoots under nutritional stress or in response to exogenously added proline, have been studied. Caffeic acid, rosmarinic acid, and lithospermic acid B have been isolated in oregano shoot cultures by reversed-phase high-performance liquid chromatography, and their structures have been elucidated by tandem mass spectrometry. Both nutritional stress, which in turn causes a moderate increase of constitutive free proline, and exogenous proline affect growth and antioxidant phenolic content of oregano shoots, compared to control. The role of proline, and the associated redox cycle, as a form of metabolic signaling based on a transfer of redox potential amongst interacting cell pathways, which in turn elicit phenolic metabolism via stimulated carbon flux through oxidative pentose phosphate pathway, is discussed. Furthermore, the potential use of oregano tissue and callus cultures as a new strategy to enable the production of useful secondary metabolites on a commercial scale is also discussed.

Endosulfan induced biochemical changes in nitrogen-fixing cyanobacteria

Pesticide contamination in aquatic ecosystem including paddy fields is a serious global environmental concern. Cyanobacteria are also affected by pesticides as non- target organism. For better exploitation of cyanobacteria as biofertiliser, it is indispensable to select tolerant strains along with understanding of their tolerance. Three cyanobacterial strains viz. Aulosira fertilissima, Anabaena variabilis and Nostoc muscorum were studied for their stress responses to an organochlorine pesticide ‘endosulfan’ with special reference to oxidative stress, role of proline and antioxidant enzymes in endosulfan induced free radical detoxification. Reduction in growth, photosynthetic pigments and carbohydrate of the test microorganisms were accompanied with increase in their total protein, proline, malondialdehye (MDA), superoxide dismutase (SOD), ascorbate peroxidase (APX) and catalase (CAT) in higher endosulfan doses. Increased amount of MDA is indicative of formation of free radicals, while increased level of CAT, APX, SOD and proline indicated their involvement in free radical scavenging mechanism. In lower concentrations, test pesticide showed increase in photosynthetic pigments. Order of tolerance was Nostoc muscorum > Anabaena variabilis > Aulosira fertilissima.

Glucose-induced Remodeling of Intermediary and Energy Metabolism in Procyclic Trypanosoma brucei

The procyclic form of Trypanosoma brucei is a parasitic protozoan that normally dwells in the midgut of its insect vector. In vitro, this parasite prefers d-glucose to l -proline as a carbon source, although this amino acid is the main carbon source available in its natural habitat. Here, we investigated how l -proline is metabolized in glucose-rich and glucose-depleted conditions. Analysis of the excreted end products of (13)C-enriched l -proline metabolism showed that the amino acid is converted into succinate or l -alanine depending on the presence or absence of d-glucose, respectively. The fact that the pathway of l -proline metabolism was truncated in glucose-rich conditions was confirmed by the analysis of 13 separate RNA interference-harboring or knock-out cell lines affecting different steps of this pathway. For instance, RNA interference studies revealed the loss of succinate dehydrogenase activity to be conditionally lethal only in the absence of d-glucose, confirming that in glucose-depleted conditions, l -proline needs to be converted beyond succinate. In addition, depletion of the F(0)/F(1)-ATP synthase activity by RNA interference led to cell death in glucose-depleted medium, but not in glucose-rich medium. This implies that, in the presence of d-glucose, the importance of the F(0)/F(1)-ATP synthase is diminished and ATP is produced by substrate level phosphorylation. We conclude that trypanosomes develop an elaborate adaptation of their energy production pathways in response to carbon source availability.

Characterization of a Helicobacter hepaticus putA Mutant Strain in Host Colonization and Oxidative Stress

Helicobacter hepaticus is a gram-negative, spiral-shaped microaerophilic bacterium associated with chronic intestinal infection leading to hepatitis and colonic and hepatic carcinomas in susceptible strains of mice. In the closely related human pathogen Helicobacter pylori, L-proline is a preferred respiratory substrate and is found at significantly high levels in the gastric juice of infected patients. A previous study of the proline catabolic PutA flavoenzymes from H. pylori and H. hepaticus revealed that Helicobacter PutA generates reactive oxygen species during proline oxidation by transferring electrons from reduced flavin to molecular oxygen. We further explored the preference for proline as a respiratory substrate and the potential impact of proline metabolism on the redox environment in Helicobacter species during host infection by disrupting the putA gene in H. hepaticus. The resulting putA knockout mutant strain was characterized by oxidative stress analysis and mouse infection studies. The putA mutant strain of H. hepaticus exhibited increased proline levels and resistance to oxidative stress relative to that of the wild-type strain, consistent with proline's role as an antioxidant. The significant increase in stress resistance was attributed to higher proline content, as no upregulation of antioxidant genes was observed for the putA mutant strain. The wild-type and putA mutant H. hepaticus strains displayed similar levels of infection in mice, but in mice challenged with the putA mutant strain, significantly reduced inflammation was observed, suggesting a role for proline metabolism in H. hepaticus pathogenicity in vivo.

Another role of proline: stabilization interactions in proteins and protein complexes concerning proline and tryptophane

Proline-tryptophan complexes derived from experimental structures are investigated by quantum chemical procedures known to properly describe the London dispersion energy. We study two geometrical arrangements: the "L-shaped", stabilized by an H-bond, and the "stacked-like", where the two residues are in parallel orientation without any H-bond. Interestingly, the interaction energies in both cases are comparable and very large ( approximately 7 kcal mol(-1)). The strength of stabilization in the stacked arrangement is rather surprising considering the fact that only one partner has an aromatic character. The interaction energy decomposition using the SAPT method further demonstrates the very important role of dispersion energy in such arrangement. To elucidate the structural features responsible for this unexpectedly large stabilization we examined the role of the nitrogen heteroatom and the importance of the cyclicity of the proline residue. We show that the electrostatic interaction due to the presence of the dipole, caused by the nitrogen heteroatom, contributes largely to the strength of the interaction. Nevertheless, the cyclic arrangement of proline, which allows for the largest amount of dispersive contact with the aromatic partner, also has a notable-effect. Geometry optimizations carried out for the "stacked-like" complexes show that the arrangements derived from protein structure are close to their gas phase optimum geometry, suggesting that the environment has only a minor effect on the geometry of the interaction. We conclude that the strength of proline non-covalent interactions, combined with this residue's rigidity, might be the explanation for its prominent role in protein stabilization and recognition processes.