Proline Editing: A General and Practical Approach to the Synthesis of Functionally and Structurally Diverse Peptides. Analysis of Steric versus Stereoelectronic Effects of 4-Substituted Prolines on Conformation within Peptides

PubMed Central

Pandey, Anil K.; Naduthambi, Devan; Thomas, Krista M.; Zondlo, Neal J.

2013-01-01

Functionalized proline residues have diverse applications. Herein we describe a practical approach, proline editing, for the synthesis of peptides with stereospecifically modified proline residues. Peptides are synthesized by standard solid-phase-peptide-synthesis to incorporate Fmoc-Hydroxyproline (4R-Hyp). In an automated manner, the Hyp hydroxyl is protected and the remainder of the peptide synthesized. After peptide synthesis, the Hyp protecting group is orthogonally removed and Hyp selectively modified to generate substituted proline amino acids, with the peptide main chain functioning to “protect” the proline amino and carboxyl groups. In a model tetrapeptide (Ac-TYPN-NH2), 4R-Hyp was stereospecifically converted to 122 different 4-substituted prolyl amino acids, with 4R or 4S stereochemistry, via Mitsunobu, oxidation, reduction, acylation, and substitution reactions. 4-Substituted prolines synthesized via proline editing include incorporated structured amino acid mimetics (Cys, Asp/Glu, Phe, Lys, Arg, pSer/pThr), recognition motifs (biotin, RGD), electron-withdrawing groups to induce stereoelectronic effects (fluoro, nitrobenzoate), handles for heteronuclear NMR (19F:fluoro; pentafluorophenyl or perfluoro-tert-butyl ether; 4,4-difluoro; 77SePh) and other spectroscopies (fluorescence, IR: cyanophenyl ether), leaving groups (sulfonate, halide, NHS, bromoacetate), and other reactive handles (amine, thiol, thioester, ketone, hydroxylamine, maleimide, acrylate, azide, alkene, alkyne, aryl halide, tetrazine, 1,2-aminothiol). Proline editing provides access to these proline derivatives with no solution phase synthesis. All peptides were analyzed by NMR to identify stereoelectronic and steric effects on conformation. Proline derivatives were synthesized to permit bioorthogonal conjugation reactions, including azide-alkyne, tetrazinetrans-cyclooctene, oxime, reductive amination, native chemical ligation, Suzuki, Sonogashira, cross-metathesis, and Diels-Alder reactions. These proline derivatives allowed three parallel bioorthogonal reactions to be conducted in one solution. PMID:23402492

Simultaneous accumulation of proline and trehalose in industrial baker's yeast enhances fermentation ability in frozen dough.

PubMed

Sasano, Yu; Haitani, Yutaka; Hashida, Keisuke; Ohtsu, Iwao; Shima, Jun; Takagi, Hiroshi

2012-05-01

Freeze tolerance is a necessary characteristic for industrial baker's yeast because frozen-dough baking is one of the key technologies for supplying oven-fresh bakery products to consumers. Both proline and trehalose are known to function as cryoprotectants in yeast cells. In order to enhance the freeze tolerance of yeast cells, we constructed a self-cloning diploid baker's yeast strain with simultaneous accumulation of proline, by expressing the PRO1-I150T allele, encoding the proline-feedback inhibition-less sensitive γ-glutamyl kinase, and trehalose, by disrupting the NTH1 gene, encoding neutral trehalase. The resultant strain retained higher tolerance to oxidative and freezing stresses than did the single proline- or trehalose-accumulating strain. Interestingly, our results suggest that proline and trehalose protect yeast cells from short-term and long-term freezing, respectively. Simultaneous accumulation of proline and trehalose in industrial baker's yeast also enhanced the fermentation ability in the frozen dough compared with the single accumulation of proline or trehalose. These results indicate that baker's yeast that accumulates both proline and trehalose is applicable for frozen-dough baking. Copyright © 2012 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Role of proline and GABA in sexual reproduction of angiosperms

PubMed Central

Biancucci, Marco; Mattioli, Roberto; Forlani, Giuseppe; Funck, Dietmar; Costantino, Paolo; Trovato, Maurizio

2015-01-01

Two glutamate derivatives, proline and γ-aminobutyric acid (GABA), appear to play pivotal roles in different aspects of sexual reproduction in angiosperms, although their precise function in plant reproduction and the molecular basis of their action are not yet fully understood. Proline and GABA have long been regarded as pivotal amino acids in pollen vitality and fertility. Proline may constitute up to 70% of the free amino acid pool in pollen grains and it has been recently shown that Arabidopsis mutants affected in the first and rate-limiting step in proline synthesis produce aberrant and infertile pollen grains, indicating that proline synthesis is required for pollen development and fertility. Concerning GABA, a large body of evidence points to this glutamate derivative as a key determinant of post-pollination fertilization. Intriguingly, proline has also been associated with pollination, another aspect of sexual reproduction, since honeybees were reported to show a strong preference for proline-enriched nectars. In this review, we survey current knowledge on the roles of proline and GABA in plant fertility, and discuss future perspectives potentially capable to improve our understanding on the functions of these amino acids in pollen development, pollination, and pollen tube guidance. PMID:26388884

Poly-proline-based chiral stationary phases: a molecular dynamics study of triproline, tetraproline, pentaproline and hexaproline interfaces.

PubMed

Ashtari, M; Cann, N M

2012-11-23

Poly-proline chains and derivatives have been recently examined as the basis for new chiral stationary phases in high performance liquid chromatography. The selectivity of poly-proline has been measured for peptides with up to ten proline units. In this article, we employ molecular dynamics simulations to examine the interfacial structure and solvation of surface-bound poly-proline chiral selectors. Specifically, we study the interfacial structure of trimethylacetyl-terminated poly-proline chains with three-to-six prolines. The surface includes silanol groups and end-caps, to better capture the characteristics of the stationary phase, and the solvent is either a polar water/methanol or a relatively apolar n-hexane/2-propanol mixture. We begin with a comprehensive ab initio study of the conformers, their energies, and an assessment of conformer flexibility. Force fields have been developed for each poly-proline selector. Molecular dynamics simulations are employed to study the preferred backbone conformations and solvent hydrogen bonding for different poly-proline/solvent interfaces. For triproline, the effect of two different terminal groups, trimethylacetyl and t-butyl carbamate are compared. Copyright © 2012 Elsevier B.V. All rights reserved.

Proteomic analysis reveals diverse proline hydroxylation-mediated oxygen-sensing cellular pathways in cancer cells

PubMed Central

Liu, Bing; Gao, Yankun; Ruan, Hai-Bin; Chen, Yue

2016-01-01

Proline hydroxylation is a critical cellular mechanism regulating oxygen-response pathways in tumor initiation and progression. Yet, its substrate diversity and functions remain largely unknown. Here, we report a system-wide analysis to characterize proline hydroxylation substrates in cancer cells using an immunoaffinity-purification assisted proteomics strategy. We identified 562 sites from 272 proteins in HeLa cells. Bioinformatic analysis revealed that proline hydroxylation substrates are significantly enriched with mRNA processing and stress-response cellular pathways with canonical and diverse flanking sequence motifs. Structural analysis indicates a significant enrichment of proline hydroxylation participating in the secondary structure of substrate proteins. Our study identified and validated Brd4, a key transcription factor, as a novel proline hydroxylation substrate. Functional analysis showed that the inhibition of proline hydroxylation pathway significantly reduced the proline hydroxylation abundance on Brd4 and affected Brd4-mediated transcriptional activity as well as cell proliferation in AML leukemia cells. Taken together, our study identified a broad regulatory role of proline hydroxylation in cellular oxygen-sensing pathways and revealed potentially new targets that dynamically respond to hypoxia microenvironment in tumor cells. PMID:27764789

Nitrogen availability regulates proline and ethylene production and alleviates salinity stress in mustard (Brassica juncea).

PubMed

Iqbal, Noushina; Umar, Shahid; Khan, Nafees A

2015-04-15

Proline content and ethylene production have been shown to be involved in salt tolerance mechanisms in plants. To assess the role of nitrogen (N) in the protection of photosynthesis under salt stress, the effect of N (0, 5, 10, 20 mM) on proline and ethylene was studied in mustard (Brassica juncea). Sufficient N (10 mM) optimized proline production under non-saline conditions through an increase in proline-metabolizing enzymes, leading to osmotic balance and protection of photosynthesis through optimal ethylene production. Excess N (20 mM), in the absence of salt stress, inhibited photosynthesis and caused higher ethylene evolution but lower proline production compared to sufficient N. In contrast, under salt stress with an increased demand for N, excess N optimized ethylene production, which regulates the proline content resulting in recovered photosynthesis. The effect of excess N on photosynthesis under salt stress was further substantiated by the application of the ethylene biosynthesis inhibitor, 1-aminoethoxy vinylglycine (AVG), which inhibited proline production and photosynthesis. Without salt stress, AVG promoted photosynthesis in plants receiving excess N by inhibiting stress ethylene production. The results suggest that a regulatory interaction exists between ethylene, proline and N for salt tolerance. Nitrogen differentially regulates proline production and ethylene formation to alleviate the adverse effect of salinity on photosynthesis in mustard. Copyright © 2015 Elsevier GmbH. All rights reserved.

L-Proline uptake in Saccharomyces cerevisiae mitochondria can contribute to bioenergetics during nutrient stress as alternative mitochondrial fuel.

PubMed

Pallotta, Maria Luigia

2014-01-01

L-Proline (pyrrolidine-2-carboxylic acid) is a distinctive metabolite both biochemically and biotechnologically and is currently recognized to have a cardinal role in gene expression and cellular signaling pathways in stress response. Proline-fueled mitochondrial metabolism involves the oxidative conversion of L-Proline to L-Glutamate in two enzymatic steps by means of Put1p and Put2p that help Saccharomyces cerevisiae to respond to changes in the nutritional environment by initiating the breakdown of L-Proline as a source for nitrogen, carbon, and energy. Compartmentalization of L-Proline catabolic pathway implies that extensive L-Proline transport must take place between the cytosol where its biogenesis via Pro1p, Pro2p, Pro3p occurs and mitochondria. L-Proline uptake in S. cerevisiae purified and active mitochondria was investigated by swelling experiments, oxygen uptake and fluorimetric measurement of a membrane potential generation (ΔΨ). Our results strongly suggest that L-Proline uptake occurs via a carried-mediated process as demonstrated by saturation kinetics and experiments with N-ethylmaleimide, a pharmacological compound that is a cysteine-modifying reagent in hydrophobic protein domains and that inhibited mitochondrial transport. Plasticity of S. cerevisiae cell biochemistry according to background fluctuations is an important factor of adaptation to stress. Thus L-Proline → Glutamate route feeds Krebs cycle providing energy and anaplerotic carbon for yeast survival.

Effect of l-Proline on Sake Brewing and Ethanol Stress in Saccharomyces cerevisiae

PubMed Central

Takagi, Hiroshi; Takaoka, Miki; Kawaguchi, Akari; Kubo, Yoshito

2005-01-01

During the fermentation of sake, cells of Saccharomyces cerevisiae are exposed to high concentrations of ethanol, thereby damaging the cell membrane and functional proteins. l-Proline protects yeast cells from damage caused by freezing or oxidative stress. In this study, we evaluated the role of intracellular l-proline in cells of S. cerevisiae grown under ethanol stress. An l-proline-accumulating laboratory strain carries a mutant allele of PRO1, pro1D154N, which encodes the Asp154Asn mutant γ-glutamyl kinase. This mutation increases the activity of γ-glutamyl kinase and γ-glutamyl phosphate reductase, which catalyze the first two steps of l-proline synthesis and which together may form a complex in vivo. When cultured in liquid medium in the presence of 9% and 18% ethanol under static conditions, the cell viability of the l-proline-accumulating laboratory strain is greater than the cell viability of the parent strain. This result suggests that intracellular accumulation of l-proline may confer tolerance to ethanol stress. We constructed a novel sake yeast strain by disrupting the PUT1 gene, which is required for l-proline utilization, and replacing the wild-type PRO1 allele with the pro1D154N allele. The resultant strain accumulated l-proline and was more tolerant to ethanol stress than was the control strain. We used the strain that could accumulate l-proline to brew sake containing five times more l-proline than what is found in sake brewed with the control strain, without affecting the fermentation profiles. PMID:16332860

The γ-Aminobutyrate Permease GabP Serves as the Third Proline Transporter of Bacillus subtilis

PubMed Central

Zaprasis, Adrienne; Hoffmann, Tamara; Stannek, Lorena; Gunka, Katrin; Commichau, Fabian M.

2014-01-01

PutP and OpuE serve as proline transporters when this imino acid is used by Bacillus subtilis as a nutrient or as an osmostress protectant, respectively. The simultaneous inactivation of the PutP and OpuE systems still allows the utilization of proline as a nutrient. This growth phenotype pointed to the presence of a third proline transport system in B. subtilis. We took advantage of the sensitivity of a putP opuE double mutant to the toxic proline analog 3,4-dehydro-dl-proline (DHP) to identify this additional proline uptake system. DHP-resistant mutants were selected and found to be defective in the use of proline as a nutrient. Whole-genome resequencing of one of these strains provided the lead that the inactivation of the γ-aminobutyrate (GABA) transporter GabP was responsible for these phenotypes. DNA sequencing of the gabP gene in 14 additionally analyzed DHP-resistant strains confirmed this finding. Consistently, each of the DHP-resistant mutants was defective not only in the use of proline as a nutrient but also in the use of GABA as a nitrogen source. The same phenotype resulted from the targeted deletion of the gabP gene in a putP opuE mutant strain. Hence, the GabP carrier not only serves as an uptake system for GABA but also functions as the third proline transporter of B. subtilis. Uptake studies with radiolabeled GABA and proline confirmed this conclusion and provided information on the kinetic parameters of the GabP carrier for both of these substrates. PMID:24142252

Proline supplementation to parenteral nutrition results in greater rates of protein synthesis in the muscle, skin, and small intestine in neonatal Yucatan miniature piglets.

PubMed

Brunton, Janet A; Baldwin, Mark P; Hanna, Rodney A; Bertolo, Robert F

2012-06-01

Proline and arginine are each indispensable during parenteral feeding due to limited interconversion by an atrophied gut. Commercial amino acid parenteral products designed for neonates contain proline concentrations that differ by almost 4-fold. To assess the adequacy of the lowest concentration of proline provided in commercial total parenteral nutrition (TPN) products, we compared rates of tissue-specific protein synthesis and nitrogen balance in neonatal piglets provided TPN at 2 different proline concentrations. Yucatan miniature piglets (9-11 d old, n = 12) were randomized to complete isonitrogenous TPN diets with low proline (LP; L-proline as 3% of amino acids) or proline supplemented (PS; 9%). After 7 d of receiving TPN, rates of protein synthesis in liver, gastrocnemius muscle, jejunal mucosa, and skin were determined by the flooding dose technique and tissue free amino acids were measured. Nitrogen balance was assessed during the last 3 d. The LP TPN resulted in lower free proline concentrations in plasma, muscle, and skin (P < 0.05) and lower rates of protein synthesis in the jejunum (by 25%; P = 0.02), muscle (by 45%; P = 0.015), and skin (by 60%; P = 0.01); there was no difference in liver. Nitrogen retention was 20% lower in the LP group (P = 0.01). In conclusion, muscle and skin protein synthesis was profoundly sensitive to parenteral proline supply and the reduced protein synthesis in the intestine could affect intestinal integrity. Low-proline TPN solutions that are currently in wide use in neonatal care may result in impaired tissue growth.

Prediction and Biochemical Demonstration of a Catabolic Pathway for the Osmoprotectant Proline Betaine

PubMed Central

Kumar, Ritesh; Zhao, Suwen; Vetting, Matthew W.; Wood, B. McKay; Sakai, Ayano; Cho, Kyuil; Solbiati, José; Almo, Steven C.; Sweedler, Jonathan V.; Jacobson, Matthew P.; Gerlt, John A.; Cronan, John E.

2014-01-01

ABSTRACT Through the use of genetic, enzymatic, metabolomic, and structural analyses, we have discovered the catabolic pathway for proline betaine, an osmoprotectant, in Paracoccus denitrificans and Rhodobacter sphaeroides. Genetic and enzymatic analyses showed that several of the key enzymes of the hydroxyproline betaine degradation pathway also function in proline betaine degradation. Metabolomic analyses detected each of the metabolic intermediates of the pathway. The proline betaine catabolic pathway was repressed by osmotic stress and cold stress, and a regulatory transcription factor was identified. We also report crystal structure complexes of the P. denitrificans HpbD hydroxyproline betaine epimerase/proline betaine racemase with l-proline betaine and cis-hydroxyproline betaine. PMID:24520058

Unusual properties of aqueous solutions of L-proline: A molecular dynamics study

NASA Astrophysics Data System (ADS)

Civera, Monica; Sironi, Maurizio; Fornili, Sandro L.

2005-11-01

Aqueous solutions of the bioprotectant proline are simulated for solute molar fractions ranging from 2.0 × 10 -3 to 2.3 × 10 -1. Statistical analyses show that proline affects the water structure more strongly than glycine betaine and trimethylamine- N-oxide, two of the most effective bioprotectants widely diffuse in nature, and as strongly as tert-butyl alcohol, a protein denaturant which at high concentration self-aggregates. No evidence is found, however, that proline self-aggregates as it has been previously suggested to explain experimental findings on concentrated proline solutions. Nevertheless, the behavior of the diffusion coefficients of proline and water vs. solute concentration qualitatively agrees with such results.

DFR1-Mediated Inhibition of Proline Degradation Pathway Regulates Drought and Freezing Tolerance in Arabidopsis.

PubMed

Ren, Yongbing; Miao, Min; Meng, Yun; Cao, Jiasheng; Fan, Tingting; Yue, Junyang; Xiao, Fangming; Liu, Yongsheng; Cao, Shuqing

2018-06-26

Proline accumulation is one of the most important adaptation mechanisms for plants to cope with environmental stresses, such as drought and freezing. However, the molecular mechanism of proline homeostasis under these stresses is largely unknown. Here, we identified a mitochondrial protein, DFR1, involved in the inhibition of proline degradation in Arabidopsis. DFR1 was strongly induced by drought and cold stresses. The dfr1 knockdown mutants showed hypersensitivity to drought and freezing stresses, whereas the DFR1 overexpression plants exhibited enhanced tolerance, which was positively correlated with proline levels. DFR1 interacts with proline degradation enzymes PDH1/2 and P5CDH and compromises their activities. Genetic analysis showed that DFR1 acts upstream of PDH1/2 and P5CDH to positively regulate proline accumulation. Our results demonstrate a regulatory mechanism by which, under drought and freezing stresses, DFR1 interacts with PDH1/2 and P5CDH to abrogate their activities to maintain proline homeostasis, thereby conferring drought and freezing tolerance. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Exogenous proline enhances the sensitivity of Tobacco BY-2 cells to arsenate.

PubMed

Nahar, Mst Nur-E-Nazmun; Islam, Mohammad Muzahidul; Hoque, Md Anamul; Yonezawa, Anna; Prodhan, Md Yeasin; Nakamura, Toshiyuki; Nakamura, Yoshimasa; Munemasa, Shintaro; Murata, Yoshiyuki

2017-09-01

Arsenic causes physiological and structural disorders in plants. Proline is accumulated as a compatible solute in plants under various stress conditions and mitigates stresses. Here, we investigated the effects of exogenous proline on tobacco Bright Yellow-2 (BY-2) cultured cells under [Formula: see text] stress. Arsenate did not inhibit BY-2 cell growth at 40 and 50 μM but did it at 60 μM. Proline at 0.5 to 10 mM did not affect the cell growth but delayed it at 20 mM. At 40 μM [Formula: see text], neither 0.5 mM nor 1 mM proline affected the cell growth but 10 mM proline inhibited it. In the presence of [Formula: see text], 10 mM proline increased the number of Evans Blue-stained (dead) cells and decreased the number of total cells. Together, our results suggest that exogenous proline does not alleviate arsenate toxicity but enhances the sensitivity of BY-2 cells to arsenate.

Cytosolic Accumulation of L-Proline Disrupts GABA-Ergic Transmission through GAD Blockade.

PubMed

Crabtree, Gregg W; Park, Alan J; Gordon, Joshua A; Gogos, Joseph A

2016-10-04

Proline dehydrogenase (PRODH), which degrades L-proline, resides within the schizophrenia-linked 22q11.2 deletion suggesting a role in disease. Supporting this, elevated L-proline levels have been shown to increase risk for psychotic disorders. Despite the strength of data linking PRODH and L-proline to neuropsychiatric diseases, targets of disease-relevant concentrations of L-proline have not been convincingly described. Here, we show that Prodh-deficient mice with elevated CNS L-proline display specific deficits in high-frequency GABA-ergic transmission and gamma-band oscillations. We find that L-proline is a GABA-mimetic and can act at multiple GABA-ergic targets. However, at disease-relevant concentrations, GABA-mimesis is limited to competitive blockade of glutamate decarboxylase leading to reduced GABA production. Significantly, deficits in GABA-ergic transmission are reversed by enhancing net GABA production with the clinically relevant compound vigabatrin. These findings indicate that accumulation of a neuroactive metabolite can lead to molecular and synaptic dysfunction and help to understand mechanisms underlying neuropsychiatric disease. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Conformational flexibility determines selectivity and antibacterial, antiplasmodial, and anticancer potency of cationic α-helical peptides.

PubMed

Vermeer, Louic S; Lan, Yun; Abbate, Vincenzo; Ruh, Emrah; Bui, Tam T; Wilkinson, Louise J; Kanno, Tokuwa; Jumagulova, Elmira; Kozlowska, Justyna; Patel, Jayneil; McIntyre, Caitlin A; Yam, W C; Siu, Gilman; Atkinson, R Andrew; Lam, Jenny K W; Bansal, Sukhvinder S; Drake, Alex F; Mitchell, Graham H; Mason, A James

2012-10-05

We used a combination of fluorescence, circular dichroism (CD), and NMR spectroscopies in conjunction with size exclusion chromatography to help rationalize the relative antibacterial, antiplasmodial, and cytotoxic activities of a series of proline-free and proline-containing model antimicrobial peptides (AMPs) in terms of their structural properties. When compared with proline-free analogs, proline-containing peptides had greater activity against Gram-negative bacteria, two mammalian cancer cell lines, and intraerythrocytic Plasmodium falciparum, which they were capable of killing without causing hemolysis. In contrast, incorporation of proline did not have a consistent effect on peptide activity against Mycobacterium tuberculosis. In membrane-mimicking environments, structures with high α-helix content were adopted by both proline-free and proline-containing peptides. In solution, AMPs generally adopted disordered structures unless their sequences comprised more hydrophobic amino acids or until coordinating phosphate ions were added. Proline-containing peptides resisted ordering induced by either method. The roles of the angle subtended by positively charged amino acids and the positioning of the proline residues were also investigated. Careful positioning of proline residues in AMP sequences is required to enable the peptide to resist ordering and maintain optimal antibacterial activity, whereas varying the angle subtended by positively charged amino acids can attenuate hemolytic potential albeit with a modest reduction in potency. Maintaining conformational flexibility improves AMP potency and selectivity toward bacterial, plasmodial, and cancerous cells while enabling the targeting of intracellular pathogens.

Synthesis, spectral characterization and biological studies of some organotin(IV) complexes of L-proline, trans-hydroxy- L-proline and L-glutamine

NASA Astrophysics Data System (ADS)

Nath, Mala; Jairath, Ruchi; Eng, George; Song, Xueqing; Kumar, Ashok

2005-12-01

New organotin(IV) complexes of the general formula R 3Sn(L) (where R = Me, n-Bu and HL = L-proline; R = Me, Ph and HL = trans-hydroxy- L-proline and L-glutamine) and R 2Sn(L) 2 (where R = n-Bu, Ph and HL = L-proline; R = Ph, HL = trans-hydroxy- L-proline) have been synthesized by the reaction of R nSnCl 4- n (where n = 2 or 3) with sodium salt of the amino acid (HL). n-Bu 2Sn(Pro) 2 was synthesized by the reaction of n-Bu 2SnO with L-proline under azeotropic removal of water. The bonding and coordination behavior in these complexes have been discussed on the basis of IR and 119Sn Mössbauer spectroscopic studies in the solid-state. Their coordination behavior in solution has been discussed with the help of multinuclear ( 1H, 13C and 119Sn) NMR spectral studies. The 119Sn Mössbauer and IR studies indicate that L-proline and trans-hydroxy- L-proline show similar coordination behavior towards organotin(IV) compounds. Pentacoordinate trigonal-bipyramidal and hexacoordinate octahedral structures, respectively, have been proposed for the tri- and diorganotin(IV) complexes of L-proline and trans-hydroxy- L-proline, in which the carboxylate group acts as bidentate group. L-Glutamine shows different coordination behavior towards organotin(IV) compounds, it acts as monoanionic bidentate ligand coordinating through carboxylate and amino group. The triorganotin(IV) complexes of L-glutamine have been proposed to have trigonal-bipyramidal environment around tin. The newly synthesized complexes have been tested for their antiinflammatory and cardiovascular activities. Their LD 50 values are >1000 mg kg -1.

Functional Consequences of Intracellular Proline Levels Manipulation Affecting PRODH/POX-Dependent Pro-Apoptotic Pathways in a Novel in Vitro Cell Culture Model.

PubMed

Zareba, Ilona; Surazynski, Arkadiusz; Chrusciel, Marcin; Miltyk, Wojciech; Doroszko, Milena; Rahman, Nafis; Palka, Jerzy

2017-01-01

The effect of impaired intracellular proline availability for proline dehydrogenase/proline oxidase (PRODH/POX)-dependent apoptosis was studied. We generated a constitutively knocked-down PRODH/POX MCF-7 breast cancer cell line (MCF-7shPRODH/POX) as a model to analyze the functional consequences of impaired intracellular proline levels. We have used inhibitor of proline utilization in collagen biosynthesis, 2-metoxyestradiol (MOE), inhibitor of prolidase that generate proline, rapamycin (Rap) and glycyl-proline (GlyPro), substrate for prolidase. Collagen and DNA biosynthesis were evaluated by radiometric assays. Cell viability was determined using Nucleo-Counter NC-3000. The activity of prolidase was determined by colorimetric assay. Expression of proteins was assessed by Western blot and immunofluorescence bioimaging. Concentration of proline was analyzed by liquid chromatography with mass spectrometry. PRODH/POX knockdown decreased DNA and collagen biosynthesis, whereas increased prolidase activity and intracellular proline level in MCF-7shPRODH/POX cells. All studied compounds decreased cell viability in MCF-7 and MCF-7shPRODH/POX cells. DNA biosynthesis was similarly inhibited by Rap and MOE in both cell lines, but GlyPro inhibited the process only in MCF-7shPRODH/POX and MOE+GlyPro only in MCF-7 cells. All the compounds inhibited collagen biosynthesis, increased prolidase activity and cytoplasmic proline level in MCF-7shPRODH/POX cells and contributed to the induction of pro-survival mode only in MCF-7shPRODH/POX cells. In contrast, all studied compounds upregulated expression of pro-apoptotic protein only in MCF-7 cells. PRODH/POX was confirmed as a driver of apoptosis and proved the eligibility of MCF-7shPRODH/POX cell line as a highly effective model to elucidate the different mechanisms underlying proline utilization or generation in PRODH/POX-dependent pro-apoptotic pathways. © 2017 The Author(s). Published by S. Karger AG, Basel.

An evaluation of the role of a pyroglutamyl peptidase, a post-proline cleaving enzyme and a post-proline dipeptidyl amino peptidase, each purified from the soluble fraction of guinea-pig brain, in the degradation of thyroliberin in vitro.

PubMed

Browne, P; O'Cuinn, G

1983-12-01

The degradation of thyroliberin (less than Glu-His-Pro-NH2) to its component amino acids by the soluble fraction of guinea pig brain is catalysed by four enzymes namely a pyroglutamate aminopeptidase, a post-proline cleaving enzyme, a post-proline dipeptidyl aminopeptidase and a proline dipeptidase. 1. The pyroglutamate aminopeptidase was purified to over 90% homogeneity with a purification factor of 2868-fold and a yield of 5.7%. In addition to catalysing the hydrolysis of thyroliberin, acid thyroliberin and pyroglutamate-7-amido-4-methylcoumarin the pyroglutamate aminopeptidase catalysed the hydrolysis of the peptide bond adjacent to the pyroglutamic acid residue in luliberin, neurotensin bombesin, bradykinin-potentiating peptide B, the anorexogenic peptide and the dipeptides pyroglutamyl alanine and pyroglutamyl valine. Pyroglutamyl proline and eledoisin were not hydrolysed. 2. The post-proline cleaving enzyme was purified to apparent electrophoretic homogeneity with a purification factor of 2298-fold and a yield of 10.6%. The post-proline cleaving enzyme catalysed the hydrolysis of thyroliberin and N-benzyloxycarbonyl-glycylproline-7-amido-4-methylcoumarin. It did not catalyse the hydrolysis of glycylproline-7-amido-4-methylcoumarin or His-Pro-NH2. 3. The post-proline dipeptidyl aminopeptidase was partially purified with a purification factor of 301-fold and a yield of 8.9%. The post-proline dipeptidyl aminopeptidase catalysed the hydrolysis of His-Pro-NH2 and glycylproline-7-amido-4-methylcoumarin but did not exhibit any post-proline cleaving endopeptidase activity against thyroliberin or N-benzyloxycarbonyl-glycylproline-7-amido-4-methylcoumarin. 4. Studies with various functional reagents indicated that the pyroglutamate aminopeptidase could be specifically inhibited by 2-iodoacetamide (100% inhibition at an inhibitor concentration of 5 microM), the post-proline cleaving enzyme by bacitracin (IC50 = 42 microM) and the post-proline dipeptidyl aminopeptidase by puromycin (IC50 = 46 microM). Because of their specific inhibitory effects these three reagents were key elements in the elucidation of the overall pathway for the metabolism of thyroliberin by guinea pig brain tissue enzymes.

Influence of proline position upon the ion channel activity of alamethicin.

PubMed Central

Kaduk, C; Duclohier, H; Dathe, M; Wenschuh, H; Beyermann, M; Molle, G; Bienert, M

1997-01-01

Alamethicin, a 20-residue peptaibol, induces voltage-dependent ion channels in lipid bilayers according to the barrel-stave model. To study relationships between the proline-14-induced kink region and the channel-forming behavior of the peptide, a set of alamethicin analogs with proline incorporated at positions 11, 12, 13, 14, 15, 16, and 17, respectively, as well as an analog with alanine instead of proline at position 14 were synthesized. Macroscopic conductance experiments show that the voltage dependence of the peptides is conserved although slightly influenced, but the apparent mean number of monomers forming the channels is significantly reduced when proline is not located at position 14. This is confirmed in single-channel experiments. The analogs with proline next to position 14 (i.e., 13, 15, 16) show stable conductance levels, but of reduced number, which follows the order Alam-P14 > Alam-P15 > Alam-P16 > Alam-P13. This reduction in the number of levels is connected with changes in the lifetime of the channels. Analogs with proline at position 11, 12, or 17 produce erratic, extremely short-lived current events that could not be resolved. The changes in functional properties are related to structural properties as probed by circular dichroism. The results indicate that proline at position 14 results in optimal channel activity, whereas channels formed by the analogs bearing proline at different positions are considerably less stable. PMID:9129817

Penultimate proline in neuropeptides.

PubMed

Glover, Matthew S; Bellinger, Earl P; Radivojac, Predrag; Clemmer, David E

2015-08-18

A recent ion mobility spectrometry-mass spectrometry (IMS-MS) study revealed that tryptic peptide ions containing a proline residue at the second position from the N-terminus (i.e., penultimate proline) frequently adopt multiple conformations, owing to the cis-trans isomerization of Xaa(1)-Pro(2) peptide bonds [J. Am. Soc. Mass Spectrom. 2015, 26, 444]. Here, we present a statistical analysis of a neuropeptide database that illustrates penultimate proline residues are frequently found in neuropeptides. In order to probe the effect of penultimate proline on neuropeptide conformations, IMS-MS experiments were performed on two model peptides in which penultimate proline residues were known to be important for biological activity: the N-terminal region of human neuropeptide Y (NPY1-9, Tyr(1)-Pro(2)-Ser(3)-Lys(4)-Pro(5)-Asp(6)-Asn(7)-Pro(8)-Gly(9)-NH2) and a tachykinin-related peptide (CabTRP Ia, Ala(1)-Pro(2)-Ser(3)-Gly(4)-Phe(5)-Leu(6)-Gly(7)-Met(8)-Arg(9)-NH2). From these studies, it appears that penultimate prolines allow neuropeptides to populate multiple conformations arising from the cis-trans isomerization of Xaa(1)-Pro(2) peptide bonds. Although it is commonly proposed that the role of penultimate proline residues is to protect peptides from enzymatic degradation, the present results indicate that penultimate proline residues also are an important means of increasing the conformational heterogeneity of neuropeptides.

Hydroxyproline Ring Pucker Causes Frustration of Helix Parameters in the Collagen Triple Helix

NASA Astrophysics Data System (ADS)

Ying Chow, W.; Bihan, Dominique; Forman, Chris J.; Slatter, David A.; Reid, David G.; Wales, David J.; Farndale, Richard W.; Duer, Melinda J.

2015-07-01

Collagens, the most abundant proteins in mammals, are defined by their triple-helical structures and distinctive Gly-Xaa-Yaa repeating sequence, where Xaa is often proline and Yaa, hydroxyproline (Hyp/O). It is known that hydroxyproline in the Yaa position stabilises the triple helix, and that lack of proline hydroxylation in vivo leads to dysfunctional collagen extracellular matrix assembly, due to a range of factors such as a change in hydration properties. In addition, we note that in model peptides, when Yaa is unmodified proline, the Xaa proline has a strong propensity to adopt an endo ring conformation, whilst when Yaa is hydroxyproline, the Xaa proline adopts a range of endo and exo conformations. Here we use a combination of solid-state NMR spectroscopy and potential energy landscape modelling of synthetic triple-helical collagen peptides to understand this effect. We show that hydroxylation of the Yaa proline causes the Xaa proline ring conformation to become metastable, which in turn confers flexibility on the triple helix.

Hydroxyproline Ring Pucker Causes Frustration of Helix Parameters in the Collagen Triple Helix

PubMed Central

Ying Chow, W.; Bihan, Dominique; Forman, Chris J.; Slatter, David A.; Reid, David G.; Wales, David J.; Farndale, Richard W.; Duer, Melinda J.

2015-01-01

Collagens, the most abundant proteins in mammals, are defined by their triple-helical structures and distinctive Gly-Xaa-Yaa repeating sequence, where Xaa is often proline and Yaa, hydroxyproline (Hyp/O). It is known that hydroxyproline in the Yaa position stabilises the triple helix, and that lack of proline hydroxylation in vivo leads to dysfunctional collagen extracellular matrix assembly, due to a range of factors such as a change in hydration properties. In addition, we note that in model peptides, when Yaa is unmodified proline, the Xaa proline has a strong propensity to adopt an endo ring conformation, whilst when Yaa is hydroxyproline, the Xaa proline adopts a range of endo and exo conformations. Here we use a combination of solid-state NMR spectroscopy and potential energy landscape modelling of synthetic triple-helical collagen peptides to understand this effect. We show that hydroxylation of the Yaa proline causes the Xaa proline ring conformation to become metastable, which in turn confers flexibility on the triple helix. PMID:26220399

Enthalpy characteristics of L-proline dissolution in certain water-organic mixtures at 298.15 K

NASA Astrophysics Data System (ADS)

Badelin, V. G.; Smirnov, V. I.

2017-01-01

A thermochemical study of the processes of L-proline dissolution in aqueous solutions of acetonitrile, 1,4-dioxane, acetone, dimethyl sulfoxide, nitromethane and tetrahydrofuran at T = 298.15 K in the range of organic solvent concentrations x2 = 0-0.25 mole fractions is performed. Standard values of the enthalpies of solution and transfer of L-proline from water to mixed solvent, and the enthalpy coefficients of pairwise interactions between L-proline and molecules of organic solvents, are calculated. The effect the composition of a water-organic mixture and the structure of organic solvents have on the enthalpy characteristics of L-proline dissolution and transfer is examined. The effect the energy properties of intermolecular interactions between components of a mixed solvent has on the intermolecular interactions between L-proline and molecules of cosolvent is estimated. The correlation between the enthalpy characteristics of L-proline dissolution and electron-donor properties of organic cosolvent in aqueous solutions is determined.

Large structural modification with conserved conformation: analysis of delta(3)-fused aryl prolines in model beta-turns.

PubMed

Jeannotte, Guillaume; Lubell, William D

2004-11-10

For the first time, the influence of a fused Delta3-arylproline on peptide conformation has been studied by the synthesis and comparison of the conformations of peptides containing proline and pyrrolo-proline, 3 (PyPro). Pyrrolo-proline was demonstrated to be a conservative replacement for Pro in model beta-turns, 4 and 5, as shown by their similar DMSO titration curves, cis/trans-isomer populations, and NOESY spectral data. Pyrrolo-proline may thus be used for studying the structure activity relationships of Pro-containing peptides with minimal modification of secondary structures.

Vitamin B6 Generated by Obligate Symbionts Is Critical for Maintaining Proline Homeostasis and Fecundity in Tsetse Flies

PubMed Central

Michalkova, Veronika; Weiss, Brian L.; Attardo, Geoffrey M.; Aksoy, Serap

2014-01-01

The viviparous tsetse fly utilizes proline as a hemolymph-borne energy source. In tsetse, biosynthesis of proline from alanine involves the enzyme alanine-glyoxylate aminotransferase (AGAT), which requires pyridoxal phosphate (vitamin B6) as a cofactor. This vitamin can be synthesized by tsetse's obligate symbiont, Wigglesworthia glossinidia. In this study, we examined the role of Wigglesworthia-produced vitamin B6 for maintenance of proline homeostasis, specifically during the energetically expensive lactation period of the tsetse's reproductive cycle. We found that expression of agat, as well as genes involved in vitamin B6 metabolism in both host and symbiont, increases in lactating flies. Removal of symbionts via antibiotic treatment of flies (aposymbiotic) led to hypoprolinemia, reduced levels of vitamin B6 in lactating females, and decreased fecundity. Proline homeostasis and fecundity recovered partially when aposymbiotic tsetse were fed a diet supplemented with either yeast or Wigglesworthia extracts. RNA interference-mediated knockdown of agat in wild-type flies reduced hemolymph proline levels to that of aposymbiotic females. Aposymbiotic flies treated with agat short interfering RNA (siRNA) remained hypoprolinemic even upon dietary supplementation with microbial extracts or B vitamins. Flies infected with parasitic African trypanosomes display lower hemolymph proline levels, suggesting that the reduced fecundity observed in parasitized flies could result from parasite interference with proline homeostasis. This interference could be manifested by competition between tsetse and trypanosomes for vitamins, proline, or other factors involved in their synthesis. Collectively, these results indicate that the presence of Wigglesworthia in tsetse is critical for the maintenance of proline homeostasis through vitamin B6 production. PMID:25038091

Proline Dehydrogenase Regulates Redox State and Respiratory Metabolism in Trypanosoma cruzi

PubMed Central

Paes, Lisvane Silva; Suárez Mantilla, Brian; Zimbres, Flávia Menezes; Pral, Elisabeth Mieko Furusho; Diogo de Melo, Patrícia; Tahara, Erich B.; Kowaltowski, Alicia J.; Elias, Maria Carolina; Silber, Ariel Mariano

2013-01-01

Over the past three decades, L-proline has become recognized as an important metabolite for trypanosomatids. It is involved in a number of key processes, including energy metabolism, resistance to oxidative and nutritional stress and osmoregulation. In addition, this amino acid supports critical parasite life cycle processes by acting as an energy source, thus enabling host-cell invasion by the parasite and subsequent parasite differentiation. In this paper, we demonstrate that L-proline is oxidized to Δ1-pyrroline-5-carboxylate (P5C) by the enzyme proline dehydrogenase (TcPRODH, E.C. 1.5.99.8) localized in Trypanosoma cruzi mitochondria. When expressed in its active form in Escherichia coli, TcPRODH exhibits a Km of 16.58±1.69 µM and a Vmax of 66±2 nmol/min mg. Furthermore, we demonstrate that TcPRODH is a FAD-dependent dimeric state protein. TcPRODH mRNA and protein expression are strongly upregulated in the intracellular epimastigote, a stage which requires an external supply of proline. In addition, when Saccharomyces cerevisiae null mutants for this gene (PUT1) were complemented with the TcPRODH gene, diminished free intracellular proline levels and an enhanced sensitivity to oxidative stress in comparison to the null mutant were observed, supporting the hypothesis that free proline accumulation constitutes a defense against oxidative imbalance. Finally, we show that proline oxidation increases cytochrome c oxidase activity in mitochondrial vesicles. Overall, these results demonstrate that TcPRODH is involved in proline-dependant cytoprotection during periods of oxidative imbalance and also shed light on the participation of proline in energy metabolism, which drives critical processes of the T. cruzi life cycle. PMID:23894476

Contribution of proline to the pre-structuring tendency of transient helical secondary structure elements in intrinsically disordered proteins.

PubMed

Lee, Chewook; Kalmar, Lajos; Xue, Bin; Tompa, Peter; Daughdrill, Gary W; Uversky, Vladimir N; Han, Kyou-Hoon

2014-03-01

IDPs function without relying on three-dimensional structures. No clear rationale for such a behavior is available yet. PreSMos are transient secondary structures observed in the target-free IDPs and serve as the target-binding "active" motifs in IDPs. Prolines are frequently found in the flanking regions of PreSMos. Contribution of prolines to the conformational stability of the helical PreSMos in IDPs is investigated. MD simulations are performed for several IDP segments containing a helical PreSMo and the flanking prolines. To measure the influence of flanking-prolines on the structural content of a helical PreSMo calculations were done for wild type as well as for mutant segments with Pro→Asp, His, Lys, or Ala. The change in the helicity due to removal of a proline was measured both for the PreSMo region and for the flanking regions. The α-helical content in ~70% of the helical PreSMos at the early stage of simulation decreases due to replacement of an N-terminal flanking proline by other residues whereas the helix content in nearly all PreSMos increases when the same replacements occur at the C-terminal flanking region. The helix destabilizing/terminating role of the C-terminal flanking prolines is more pronounced than the helix promoting effect of the N-terminal flanking prolines. This work represents a novel example demonstrating that a proline is encoded in an IDP with a defined purpose. The helical PreSMos presage their target-bound conformations. As they most likely mediate IDP-target binding via conformational selection their helical content can be an important feature for IDP function. Copyright © 2013 Elsevier B.V. All rights reserved.

Vitamin B6 generated by obligate symbionts is critical for maintaining proline homeostasis and fecundity in tsetse flies.

PubMed

Michalkova, Veronika; Benoit, Joshua B; Weiss, Brian L; Attardo, Geoffrey M; Aksoy, Serap

2014-09-01

The viviparous tsetse fly utilizes proline as a hemolymph-borne energy source. In tsetse, biosynthesis of proline from alanine involves the enzyme alanine-glyoxylate aminotransferase (AGAT), which requires pyridoxal phosphate (vitamin B6) as a cofactor. This vitamin can be synthesized by tsetse's obligate symbiont, Wigglesworthia glossinidia. In this study, we examined the role of Wigglesworthia-produced vitamin B6 for maintenance of proline homeostasis, specifically during the energetically expensive lactation period of the tsetse's reproductive cycle. We found that expression of agat, as well as genes involved in vitamin B6 metabolism in both host and symbiont, increases in lactating flies. Removal of symbionts via antibiotic treatment of flies (aposymbiotic) led to hypoprolinemia, reduced levels of vitamin B6 in lactating females, and decreased fecundity. Proline homeostasis and fecundity recovered partially when aposymbiotic tsetse were fed a diet supplemented with either yeast or Wigglesworthia extracts. RNA interference-mediated knockdown of agat in wild-type flies reduced hemolymph proline levels to that of aposymbiotic females. Aposymbiotic flies treated with agat short interfering RNA (siRNA) remained hypoprolinemic even upon dietary supplementation with microbial extracts or B vitamins. Flies infected with parasitic African trypanosomes display lower hemolymph proline levels, suggesting that the reduced fecundity observed in parasitized flies could result from parasite interference with proline homeostasis. This interference could be manifested by competition between tsetse and trypanosomes for vitamins, proline, or other factors involved in their synthesis. Collectively, these results indicate that the presence of Wigglesworthia in tsetse is critical for the maintenance of proline homeostasis through vitamin B6 production. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Solvent and conformation dependence of amide I vibrations in peptides and proteins containing proline

NASA Astrophysics Data System (ADS)

Roy, Santanu; Lessing, Joshua; Meisl, Georg; Ganim, Ziad; Tokmakoff, Andrei; Knoester, Jasper; Jansen, Thomas L. C.

2011-12-01

We present a mixed quantum-classical model for studying the amide I vibrational dynamics (predominantly CO stretching) in peptides and proteins containing proline. There are existing models developed for determining frequencies of and couplings between the secondary amide units. However, these are not applicable to proline because this amino acid has a tertiary amide unit. Therefore, a new parametrization is required for infrared-spectroscopic studies of proteins that contain proline, such as collagen, the most abundant protein in humans and animals. Here, we construct the electrostatic and dihedral maps accounting for solvent and conformation effects on frequency and coupling for the proline unit. We examine the quality and the applicability of these maps by carrying out spectral simulations of a number of peptides with proline in D2O and compare with experimental observations.

Solvent and conformation dependence of amide I vibrations in peptides and proteins containing proline.

PubMed

Roy, Santanu; Lessing, Joshua; Meisl, Georg; Ganim, Ziad; Tokmakoff, Andrei; Knoester, Jasper; Jansen, Thomas L C

2011-12-21

We present a mixed quantum-classical model for studying the amide I vibrational dynamics (predominantly CO stretching) in peptides and proteins containing proline. There are existing models developed for determining frequencies of and couplings between the secondary amide units. However, these are not applicable to proline because this amino acid has a tertiary amide unit. Therefore, a new parametrization is required for infrared-spectroscopic studies of proteins that contain proline, such as collagen, the most abundant protein in humans and animals. Here, we construct the electrostatic and dihedral maps accounting for solvent and conformation effects on frequency and coupling for the proline unit. We examine the quality and the applicability of these maps by carrying out spectral simulations of a number of peptides with proline in D(2)O and compare with experimental observations.

Proline accumulation and its implication in cold tolerance of regenerable maize callus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Duncan, D.R.; Widholm, J.M.

1987-03-01

Embryogenic callus of maize (Zea mays L.) inbreds B37wx, H99, H99/sup 3/H95, Mo17, and Pa91 accumulated proline to levels 2.1 to 2.5 times that of control callus when subjected to mannitol-induced water stress, cool temperatures (19/sup 0/C) and abscisic acid (ABA). A combination of 0.53 molar mannitol plus 0.1 millimolar ABA induced a proline accumulation to about 4.5 times that of control callus, equivalent to approximately 0.18 millimoles proline per gram fresh weight of callus. Proline accumulation was directly related to the level of mannitol in the medium. Levels of ABA greater than 1.0 micromolar were required in the mediummore » to induce proline accumulation comparable to that induced by mannitol. Mannitol and ABA levels that induced maximum accumulation of proline also inhibited callus growth and increased tolerance to cold. Proline (12 millimolar) added to culture media also increased the tolerance of callus to 4/sup 0/C. The increased cold tolerance induced by the combination of mannitol and ABA has permitted the storage of the maize inbreds A632, A634Ht, B37wx, C103DTrf, Fr27rhm, H99, Pa91, Va35, and W117Ht at 4/sup 0/C for 90 days which is more than double the typical survival time of callus. These studies show that proline accumulation increase the cold tolerance of regenerable maize callus.« less

Evidence for a role of proline and hypothalamic astrocytes in the regulation of glucose metabolism in rats.

PubMed

Arrieta-Cruz, Isabel; Su, Ya; Knight, Colette M; Lam, Tony K T; Gutiérrez-Juárez, Roger

2013-04-01

The metabolism of lactate to pyruvate in the mediobasal hypothalamus (MBH) regulates hepatic glucose production. Because astrocytes and neurons are functionally linked by metabolic coupling through lactate transfer via the astrocyte-neuron lactate shuttle (ANLS), we reasoned that astrocytes might be involved in the hypothalamic regulation of glucose metabolism. To examine this possibility, we used the gluconeogenic amino acid proline, which is metabolized to pyruvate in astrocytes. Our results showed that increasing the availability of proline in rats either centrally (MBH) or systemically acutely lowered blood glucose. Pancreatic clamp studies revealed that this hypoglycemic effect was due to a decrease of hepatic glucose production secondary to an inhibition of glycogenolysis, gluconeogenesis, and glucose-6-phosphatase flux. The effect of proline was mimicked by glutamate, an intermediary of proline metabolism. Interestingly, proline's action was markedly blunted by pharmacological inhibition of hypothalamic lactate dehydrogenase (LDH) suggesting that metabolic flux through LDH was required. Furthermore, short hairpin RNA-mediated knockdown of hypothalamic LDH-A, an astrocytic component of the ANLS, also blunted the glucoregulatory action of proline. Thus our studies suggest not only a new role for proline in the regulation of hepatic glucose production but also indicate that hypothalamic astrocytes are involved in the regulatory mechanism as well.

Salt stress encourages proline accumulation by regulating proline biosynthesis and degradation in Jerusalem artichoke plantlets.

PubMed

Huang, Zengrong; Zhao, Long; Chen, Dandan; Liang, Mingxiang; Liu, Zhaopu; Shao, Hongbo; Long, Xiaohua

2013-01-01

Proline accumulation is an important mechanism for osmotic regulation under salt stress. In this study, we evaluated proline accumulation profiles in roots, stems and leaves of Jerusalem artichoke (Helianthus tuberosus L.) plantlets under NaCl stress. We also examined HtP5CS, HtOAT and HtPDH enzyme activities and gene expression patterns of putative HtP5CS1, HtP5CS2, HtOAT, HtPDH1, and HtPDH2 genes. The objective of our study was to characterize the proline regulation mechanisms of Jerusalem artichoke, a moderately salt tolerant species, under NaCl stress. Jerusalem artichoke plantlets were observed to accumulate proline in roots, stems and leaves during salt stress. HtP5CS enzyme activities were increased under NaCl stress, while HtOAT and HtPDH activities generally repressed. Transcript levels of HtP5CS2 increased while transcript levels of HtOAT, HtPDH1 and HtPDH2 generally decreased in response to NaCl stress. Our results supports that for Jerusalem artichoke, proline synthesis under salt stress is mainly through the Glu pathway, and HtP5CS2 is predominant in this process while HtOAT plays a less important role. Both HtPDH genes may function in proline degradation.

Chirally directed formation of nanometer-scale proline clusters.

PubMed

Myung, Sunnie; Fioroni, Marco; Julian, Ryan R; Koeniger, Stormy L; Baik, Mu-Hyun; Clemmer, David E

2006-08-23

Ion mobility measurements, combined with molecular mechanics simulations, are used to study enantiopure and racemic proline clusters formed by electrospray ionization. Broad distributions of cluster sizes and charge states are observed, ranging from clusters containing only a few proline units to clusters that contain more than 100 proline units (i.e., protonated clusters of the form [xPro + nH](n+) with x = 1 to >100 and n = 1-7). As the sizes of clusters increase, there is direct evidence for nanometer scale, chirally induced organization into specific structures. For n = 4 and 5, enantiopure clusters of approximately 50 to 100 prolines assemble into structures that are more elongated than the most compact structure that is observed from the racemic proline clusters. A molecular analogue, cis-4-hydroxy-proline, displays significantly different behavior, indicating that in addition to the rigidity of the side chain ring, intermolecular interactions are important in the formation of chirally directed clusters. This is the first case in which assemblies of chirally selective elongated structures are observed in this size range of amino acid clusters. Relationships between enantiopurity, cluster shape, and overall energetics are discussed.

Proline accumulation in baker's yeast enhances high-sucrose stress tolerance and fermentation ability in sweet dough.

PubMed

Sasano, Yu; Haitani, Yutaka; Ohtsu, Iwao; Shima, Jun; Takagi, Hiroshi

2012-01-03

During bread-making processes, yeast cells are exposed to various baking-associated stresses. High-sucrose concentrations exert severe osmotic stress that seriously damages cellular components by generation of reactive oxygen species (ROS). Previously, we found that the accumulation of proline conferred freeze-thaw stress tolerance and the baker's yeast strain that accumulated proline retained higher-level fermentation abilities in frozen doughs than the wild-type strain. In this study, we constructed self-cloning diploid baker's yeast strains that accumulate proline. These resultant strains showed higher cell viability and lower intracellular oxidation levels than that observed in the wild-type strain under high-sucrose stress condition. Proline accumulation also enhanced the fermentation ability in high-sucrose-containing dough. These results demonstrate the usefulness of proline-accumulating baker's yeast for sweet dough baking. Copyright © 2011 Elsevier B.V. All rights reserved.

Central l-proline attenuates stress-induced dopamine and serotonin metabolism in the chick forebrain.

PubMed

Hamasu, Kousuke; Shigemi, Kazutaka; Kabuki, Yusuke; Tomonaga, Shozo; Denbow, D Michael; Furuse, Mitsuhiro

2009-08-21

Using microdialysis, we investigated the effect of l-proline on monoamine release in the medio-rostral neostriatum/hyperstriatum ventrale (MNH) of freely moving and restricted chicks. A 30 min handling-stress resulted in a significant increase in extracellular homovallinic acid (HVA), a dopamine metabolite, and 5-hydroxyindoleacetic acid (5-HIAA), a serotonin metabolite, in the MNH. l-Proline, perfused through the microdialysis probe into the MNH during the stressed condition, significantly attenuated the average dialysate concentration of HVA produced by handling-stress. Handling-stress resulted in a significant increase in 5-HIAA levels in the control group, which were attenuated by profusion with l-proline. l-Proline did not significantly modify basal concentrations of HVA or 5-HIAA in the MNH during control conditions. These results show that perfusion of l-proline modified the turnover/metabolism of dopamine and serotonin in the MNH caused by handling-stress.

Proline-Dependent Regulation of Clostridium difficile Stickland Metabolism

PubMed Central

Bouillaut, Laurent; Self, William T.

2013-01-01

Clostridium difficile, a proteolytic Gram-positive anaerobe, has emerged as a significant nosocomial pathogen. Stickland fermentation reactions are thought to be important for growth of C. difficile and appear to influence toxin production. In Stickland reactions, pairs of amino acids donate and accept electrons, generating ATP and reducing power in the process. Reduction of the electron acceptors proline and glycine requires the d-proline reductase (PR) and the glycine reductase (GR) enzyme complexes, respectively. Addition of proline in the medium increases the level of PR protein but decreases the level of GR. We report the identification of PrdR, a protein that activates transcription of the PR-encoding genes in the presence of proline and negatively regulates the GR-encoding genes. The results suggest that PrdR is a central metabolism regulator that controls preferential utilization of proline and glycine to produce energy via the Stickland reactions. PMID:23222730

Proline accumulation in lemongrass (Cymbopogon flexuosus Stapf.) due to heavy metal stress.

PubMed

Handique, G K; Handique, A K

2009-03-01

Toxic heavy metals viz. lead, mercury and cadmium induced differential accumulation of proline in lemongrass (Cymbopogon flexuosus Stapf.) grown in soil amended with 50, 100, 200, 350 and 500 mg kg(-1) of the metals have been studied. Proline accumulation was found to be metal specific, organ specific and linear dose dependant. Further, proline accumulation following short term exposure (two months after transplantation) was higher than long term exposure (nine months after transplantation). Proline accumulation following short term exposure was 2.032 to 3.839 micro moles g(-1) for cadmium (50-200 mg kg(-1)); the corresponding range for mercury was 1.968 to 5.670 micro moles g(-1) and 0.830 to 4.567 micro moles g(-1) for lead (50-500 mg kg(-1) for mercury and lead). Proline accumulation was consistently higher in young tender leaf than old leaf, irrespective of the metal or duration of exposure. For cadmium treatment proline level was 2.032 to 3.839 micro moles g(-1) for young leaves while the corresponding value for old leaf was 1.728 to 2.396 micro moles g(-1) following short term exposure. The same trend was observed for the other two metals and duration of exposure. For control set proline accumulation in root was 0.425 micro moles g(-1) as against 0.805 and 0.533 micro moles g(-1) in young and old leaves respectively indicating that proline accumulation in root are lower than leaves, under both normal and stressed condition.

Positional preference of proline in alpha-helices.

PubMed Central

Kim, M. K.; Kang, Y. K.

1999-01-01

Conformational free energy calculations have been carried out for proline-containing alanine-based pentadecapeptides with the sequence Ac-(Ala)n-Pro-(Ala)m-NHMe, where n + m = 14, to figure out the positional preference of proline in alpha-helices. The relative free energy of each peptide was calculated by subtracting the free energy of the extended conformation from that of the alpha-helical one, which is used here as a measure of preference. The highest propensity is found for the peptide with proline at the N-terminus (i.e., Ncap + 1 position), and the next propensities are found at Ncap, N' (Ncap - 1), and C' (Ccap + 1) positions. These computed results are reasonably consistent with the positional propensities estimated from X-ray structures of proteins. The breaking in hydrogen bonds around proline is found to play a role in destabilizing alpha-helical conformations, which, however, provides the favored hydration of the corresponding N-H and C=O groups. The highest preference of proline at the beginning of alpha-helix appears to be due to the favored electrostatic and nonbonded energies between two residues preceding proline and the intrinsic stability of alpha-helical conformation of the proline residue itself as well as no disturbance in hydrogen bonds of alpha-helix by proline. The average free energy change for the substitution of Ala by Pro in a alpha-helix is computed to be 4.6 kcal/mol, which is in good agreement with the experimental value of approximately 4 kcal/mol estimated for an oligopeptide dimer and proteins of barnase and T4 lysozyme. PMID:10422838

Self-cloning baker's yeasts that accumulate proline enhance freeze tolerance in doughs.

PubMed

Kaino, Tomohiro; Tateiwa, Tetsuya; Mizukami-Murata, Satomi; Shima, Jun; Takagi, Hiroshi

2008-09-01

We constructed self-cloning diploid baker's yeast strains by disrupting PUT1, encoding proline oxidase, and replacing the wild-type PRO1, encoding gamma-glutamyl kinase, with a pro1(D154N) or pro1(I150T) allele. The resultant strains accumulated intracellular proline and retained higher-level fermentation abilities in the frozen doughs than the wild-type strain. These results suggest that proline-accumulating baker's yeast is suitable for frozen-dough baking.

Dual palladium- and proline-catalyzed allylic alkylation of enolizable ketones and aldehydes with allylic alcohols.

PubMed

Usui, Ippei; Schmidt, Stefan; Breit, Bernhard

2009-03-19

The dual Pd/proline-catalyzed alpha-allylation reaction of a variety of enolizable ketones and aldehydes with allylic alcohols is described. In this reaction, the choice of a large-bite angle ligand Xantphos and proline as the organocatalyst was essential for generation of the crucial pi-allyl Pd intermediate from allylic alcohol, followed by nucleophilic attack of the enamine formed in situ from the corresponding enolizable carbonyl substrate and proline.

l-Proline and RNA Duplex m-Value Temperature Dependence.

PubMed

Schwinefus, Jeffrey J; Baka, Nadia L; Modi, Kalpit; Billmeyer, Kaylyn N; Lu, Shutian; Haase, Lucas R; Menssen, Ryan J

2017-08-03

The temperature dependence of l-proline interactions with the RNA dodecamer duplex surface exposed after unfolding was quantified using thermal and isothermal titration denaturation monitored by uv-absorbance. The m-value quantifying proline interactions with the RNA duplex surface area exposed after unfolding was measured using RNA duplexes with GC content ranging between 17 and 83%. The m-values from thermal denaturation decreased with increasing GC content signifying increasingly favorable proline interactions with the exposed RNA surface area. However, m-values from isothermal titration denaturation at 25.0 °C were independent of GC content and less negative than those from thermal denaturation. The m-value from isothermal titration denaturation for a 50% GC RNA duplex decreased (became more negative) as the temperature increased and was in nearly exact agreement with the m-value from thermal denaturation. Since RNA duplex transition temperatures increased with GC content, the more favorable proline interactions with the high GC content duplex surface area observed from thermal denaturation resulted from the temperature dependence of proline interactions rather than the RNA surface chemical composition. The enthalpy contribution to the m-value was positive and small (indicating a slight increase in duplex unfolding enthalpy with proline) while the entropic contribution to the m-value was positive and increased with temperature. Our results will facilitate proline's use as a probe of solvent accessible surface area changes during biochemical reactions at different reaction temperatures.

Proline Scan of the hERG Channel S6 Helix Reveals the Location of the Intracellular Pore Gate

PubMed Central

Thouta, Samrat; Sokolov, Stanislav; Abe, Yuki; Clark, Sheldon J.; Cheng, Yen M.; Claydon, Tom W.

2014-01-01

In Shaker-like channels, the activation gate is formed at the bundle crossing by the convergence of the inner S6 helices near a conserved proline-valine-proline motif, which introduces a kink that allows for electromechanical coupling with voltage sensor motions via the S4-S5 linker. Human ether-a-go-go-related gene (hERG) channels lack the proline-valine-proline motif and the location of the intracellular pore gate and how it is coupled to S4 movement is less clear. Here, we show that proline substitutions within the S6 of hERG perturbed pore gate closure, trapping channels in the open state. Performing a proline scan of the inner S6 helix, from Ile655 to Tyr667 revealed that gate perturbation occurred with proximal (I655P-Q664P), but not distal (R665P-Y667P) substitutions, suggesting that Gln664 marks the position of the intracellular gate in hERG channels. Using voltage-clamp fluorimetry and gating current analysis, we demonstrate that proline substitutions trap the activation gate open by disrupting the coupling between the voltage-sensing unit and the pore of the channel. We characterize voltage sensor movement in one such trapped-open mutant channel and demonstrate the kinetics of what we interpret to be intrinsic hERG voltage sensor movement. PMID:24606930

Effective Phytoextraction of Cadmium (Cd) with Increasing Concentration of Total Phenolics and Free Proline in Cannabis sativa (L) Plant Under Various Treatments of Fertilizers, Plant Growth Regulators and Sodium Salt.

PubMed

Ahmad, Ayaz; Hadi, Fazal; Ali, Nasir

2015-01-01

The comparative effect of fertilizers (NPK), plant growth regulators (GA3, IAA, Zeatin) and sodium chloride (NaCl) on Cd phytoaccumulation, proline and phenolics production in Cannabis sativa was evaluated. Proline and phenolices were correlated with Cd contents in plant. Cd significantly reduced the plant growth. Fertilizers application (in combination) most significantly increased the growth (19 cm root and 47 cm shoot) on Cd contaminated soil. All treatments increased the Cd contents in plant tissues. This increase was highly significant in fertilizers treated plants (1101, 121 and 544 ppm in roots, stem and leaves respectively). Significantly positive correlation was found between Cd concentration and dry biomass of root (R2=0.7511) and leaves (R2=0.5524). All treatments significantly increased the proline and total phenolics and maximum was recorded in NaCl treated plants followed by fertilizers. Proline was higher in roots while phenolics in leaves. The correlation between proline and phenolics was positive in leaf (R2=0.8439) and root (R2=0.5191). Proline and phenolics showed positive correlation with Cd concentration in plant. Conclusively, fertilizers in combination seem to be the better option for Cd phytoextraction. Further investigation is suggested to study the role of phenolics and proline in Cd phytoextraction.

PROLINE OXIDASES IN HANSENULA SUBPELLICULOSA

PubMed Central

Ling, Chung-Mei; Hedrick, L. R.

1964-01-01

Ling, Chung-Mei (Illinois Institute of Technology, Chicago), and L. R. Hedrick. Proline oxidases in Hansenula subpelliculosa. J. Bacteriol. 87:1462–1470. 1964—Cells of Hansenula subpelliculosa can use l-proline as a carbon and a nitrogen source after a 6- to 8-hr induction period. However, they cannot use l-glutamate as both nitrogen and carbon sources unless the induction period is of several days' duration. Two l-proline oxidases were demonstrated in the mitochondrial preparation of this yeast. One forms the product Δ′-pyrroline-2-carboxylic acid (P2C), which is in equilibrium with α-keto-δ-amino-valeric acid; the other forms the product Δ′-pyrroline-5-carboxylic acid (P5C), which is in equilibrium with glutamic-γ-semialdehyde. The first-mentioned enzyme is induced when l-proline is the carbon source; the second appears to be constitutive, and is probably associated with the use of l-proline as a nitrogen source. The P2C-forming enzyme is specific for the l isomer of proline, and is inactive against l-hydroxyproline. The enzyme activity is at its peak when the mitochondria are prepared from logarithmically grown cells, and is rapidly reduced after cells reach the stationary phase of growth. Kinetic studies with varying concentrations of substrate indicate a Michaelis-Menten constant of 2.45 × 10−2m. Paper chromatographic studies, chemical tests with H2O2, sensitivity to freezing, and spectral measurements indicate that proline oxidase from H. subpelliculosa mitochondria forms a product from l-proline which is like, if not identical to, P2C formed by the action of sheep kidney d-proline oxidase upon dl-proline. The soluble portion of the cell extract contains NAD+ enzymes which use either P2C (α-keto-δ-amino-valeric acid) or P5C (glutamic-γ-semialdehyde) as substrates. No glutamic dehydrogenase activity could be detected when l-glutamic acid and the nicotinamide adenine dinucleotide (NAD+) cofactor were added to the supernatant solution with the yeast enzymes. The presence of a dehydrogenase NAD+ enzyme for activity with P2C (α-keto-δ-amino-valeric acid) has not been previously reported. PMID:14188729

Proline oxidase controls proline, glutamate, and glutamine cellular concentrations in a U87 glioblastoma cell line.

PubMed

Cappelletti, Pamela; Tallarita, Elena; Rabattoni, Valentina; Campomenosi, Paola; Sacchi, Silvia; Pollegioni, Loredano

2018-01-01

L-Proline is a multifunctional amino acid that plays an essential role in primary metabolism and physiological functions. Proline is oxidized to glutamate in the mitochondria and the FAD-containing enzyme proline oxidase (PO) catalyzes the first step in L-proline degradation pathway. Alterations in proline metabolism have been described in various human diseases, such as hyperprolinemia type I, velo-cardio-facial syndrome/Di George syndrome, schizophrenia and cancer. In particular, the mutation giving rise to the substitution Leu441Pro was identified in patients suffering of schizophrenia and hyperprolinemia type I. Here, we report on the expression of wild-type and L441P variants of human PO in a U87 glioblastoma human cell line in an attempt to assess their effect on glutamate metabolism. The subcellular localization of the flavoenzyme is not altered in the L441P variant, for which specific activity is halved compared to the wild-type PO. While this decrease in activity is significantly less than that previously proposed, an effect of the substitution on the enzyme stability is also apparent in our studies. At 24 hours of growth from transient transfection, the intracellular level of proline, glutamate, and glutamine is decreased in cells expressing the PO variants as compared to control U87 cells, reaching a similar figure at 72 h. On the other hand, the extracellular levels of the three selected amino acids show a similar time course for all clones. Furthermore, PO overexpression does not modify to a significant extent the expression of GLAST and GLT-1 glutamate transporters. Altogether, these results demonstrate that the proline pathway links cellular proline levels with those of glutamate and glutamine. On this side, PO might play a regulatory role in glutamatergic neurotransmission by affecting the cellular concentration of glutamate.

Reprogramming of proline and glutamine metabolism contributes to the proliferative and metabolic responses regulated by oncogenic transcription factor c-MYC

PubMed Central

Liu, Wei; Le, Anne; Hancock, Chad; Lane, Andrew N.; Dang, Chi V.; Fan, Teresa W.-M.; Phang, James M.

2012-01-01

In addition to glycolysis, the oncogenic transcription factor c-MYC (MYC) stimulates glutamine catabolism to fuel growth and proliferation of cancer cells through up-regulating glutaminase (GLS). Glutamine is converted to glutamate by GLS, entering the tricarboxylic acid cycle as an important energy source. Less well-recognized, glutamate can also be converted to proline through Δ1-pyrroline-5-carboxylate (P5C) and vice versa. This study suggests that some MYC-induced cellular effects are due to MYC regulation of proline metabolism. Proline oxidase, also known as proline dehydrogenase (POX/PRODH), the first enzyme in proline catabolism, is a mitochondrial tumor suppressor that inhibits proliferation and induces apoptosis. MiR-23b* mediates POX/PRODH down-regulation in human kidney tumors. MiR-23b* is processed from the same transcript as miR-23b; the latter inhibits the translation of GLS. Using MYC-inducible human Burkitt lymphoma model P493 and PC3 human prostate cancer cells, we showed that MYC suppressed POX/PRODH expression primarily through up-regulating miR-23b*. The growth inhibition in the absence of MYC was partially reversed by POX/PRODH knockdown, indicating the importance of suppression of POX/PRODH in MYC-mediated cellular effects. Interestingly, MYC not only inhibited POX/PRODH, but also markedly increased the enzymes of proline biosynthesis from glutamine, including P5C synthase and P5C reductase 1. MYC-induced proline biosynthesis from glutamine was directly confirmed using 13C,15N-glutamine as a tracer. The metabolic link between glutamine and proline afforded by MYC emphasizes the complexity of tumor metabolism. Further studies of the relationship between glutamine and proline metabolism should provide a deeper understanding of tumor metabolism while enabling the development of novel therapeutic strategies. PMID:22615405

Durum wheat dehydrin (DHN-5) confers salinity tolerance to transgenic Arabidopsis plants through the regulation of proline metabolism and ROS scavenging system.

PubMed

Saibi, Walid; Feki, Kaouthar; Ben Mahmoud, Rihem; Brini, Faiçal

2015-11-01

The wheat dehydrin (DHN-5) gives birth to salinity tolerance to transgenic Arabidopsis plants by the regulation of proline metabolism and the ROS scavenging system. Dehydrins (DHNs) are involved in plant abiotic stress tolerance. In this study, we reported that salt tolerance of transgenic Arabidopsis plants overexpressing durum wheat dehydrin (DHN-5) was closely related to the activation of the proline metabolism enzyme (P5CS) and some antioxidant biocatalysts. Indeed, DHN-5 improved P5CS activity in the transgenic plants generating a significant proline accumulation. Moreover, salt tolerance of Arabidopsis transgenic plants was accompanied by an excellent activation of antioxidant enzymes like catalase (CAT), superoxide dismutase (SOD) and peroxide dismutase (POD) and generation of a lower level of hydrogen peroxide (H2O2) in leaves compared to the wild-type plants. The enzyme activities were enhanced in these transgenic plants in the presence of exogenous proline. Nevertheless, proline accumulation was slightly reduced in transgenic plants promoting chlorophyll levels. All these results suggest the crucial role of DHN-5 in response to salt stress through the activation of enzymes implicated in proline metabolism and in ROS scavenging enzymes.

13C multiplet nuclear magnetic resonance relaxation-derived ring puckering and backbone dynamics in proline-containing glycine-based peptides.

PubMed Central

Mikhailov, D; Daragan, V A; Mayo, K H

1995-01-01

13CH2-multiplet nuclear magnetic resonance relaxation studies on proline (P)-containing glycine (G)-based peptides, GP, PG, GPG, PGG, and GPGG, provided numerous dipolar auto- and cross-correlation times for various motional model analyses of backbone and proline-ring bond rotations. Molecular dynamics simulations and bond rotation energy profiles were calculated to assess which motions could contribute most to observed relaxation phenomena. Results indicate that proline restricts backbone psi 1, psi 2, and phi 2 motions by 50% relative to those found for a polyglycine control peptide. psi 1 rotations are more restricted in the trans-proline isomer state than in the cis form. A two-state jump model best approximates proline ring puckering which in water could occur either by the C gamma endo-exo or by the C2 interconversion mechanism. The temperature dependence (5 degrees to 75 degrees C) of C beta, and C gamma, and C delta angular changes is rather flat, suggesting a near zero enthalpic contribution to the ring puckering process. In lower dielectric solvents, dimethylsulfoxide and methanol, which may mimic the hydrophobic environment within a protein, the endo-exo mechanism is preferred. PMID:7787039

Proline Can Have Opposite Effects on Fast and Slow Protein Folding Phases

PubMed Central

Osváth, Szabolcs; Gruebele, Martin

2003-01-01

Proline isomerization is well known to cause additional slow phases during protein refolding. We address a new question: does the presence of prolines significantly affect the very fast kinetics that lead to the formation of folding intermediates? We examined both the very slow (10–100 min) and very fast (4 μs–2.5 ms) folding kinetics of the two-domain enzyme yeast phosphoglycerate kinase by temperature-jump relaxation. Phosphoglycerate kinase contains a conserved cis-proline in position 204, in addition to several trans-prolines. Native cis-prolines have the largest effect on folding kinetics because the unfolded state favors trans isomerization, so we compared the kinetics of a P204H mutant with the wild-type as a proof of principle. The presence of Pro-204 causes an additional slow phase upon refolding from the cold denatured state, as reported in the literature. Contrary to this, the fast folding events are sped up in the presence of the cis-proline, probably by restriction of the conformational space accessible to the molecule. The wild-type and Pro204His mutant would be excellent models for off-lattice simulations probing the effects of conformational restriction on short timescales. PMID:12885665

Chirality transfer effects in proline-substituted coumarin compounds.

PubMed

Park, Eun-Kyung; Park, Bongjeong; Choi, Jun-Ho; Choi, Kihang; Cho, Minhaeng

2009-08-13

Conformations of proline-substituted chromophores are determined by using circular dichroism (CD) spectroscopy and quantum chemistry calculation method. Coumarin is chosen for the optical chromophore and proline amino acid is attached to its C7 position. The coumarin-proline conjugate considered contains both fluorophore and peptide linker where any polypeptides or biomolecules can be additionally connected to the free carboxyl group of the proline. Thus, the coumarin-proline is a potentially useful composite chirality-probe system for studies of protein dynamics in solution. However, detailed conformation of coumarin ring with respect to the proline ring has to be determined first. We found that there are two possible conformers, which differ from each other by the relative orientation of the coumarin ring. Comparing the measured CD spectra with the calculated ones, we directly show that only one of the two conformers is dominant in polar solvents except for water. The present study suggests that the local structure around an optical chromophore, when it is introduced to polypeptides or other biomolecules, can be studied by examining the electronic optical activity of the probe chromophore, as long as the chirality transfer from the attached amino acid to the chromophore is significantly large.

Effect of Proline Mutations on the Monomer Conformations of Amylin

PubMed Central

Chiu, Chi-cheng; Singh, Sadanand; de Pablo, Juan J.

2013-01-01

The formation of human islet amyloid polypeptide (hIAPP) is implicated in the loss of pancreatic β-cells in type II diabetes. Rat amylin, which differs from human amylin at six residues, does not lead to formation of amyloid fibrils. Pramlintide is a synthetic analog of human amylin that shares three proline substitutions with rat amylin. Pramlintide has a much smaller propensity to form amyloid aggregates and has been widely prescribed in amylin replacement treatment. It is known that the three prolines attenuate β-sheet formation. However, the detailed effects of these proline substitutions on full-length hIAPP remain poorly understood. In this work, we use molecular simulations and bias-exchange metadynamics to investigate the effect of proline substitutions on the conformation of the hIAPP monomer. Our results demonstrate that hIAPP can adopt various β-sheet conformations, some of which have been reported in experiments. The proline substitutions perturb the formation of long β-sheets and reduce their stability. More importantly, we find that all three proline substitutions of pramlintide are required to inhibit β conformations and stabilize the α-helical conformation. Fewer substitutions do not have a significant inhibiting effect. PMID:24010666

Synthesis of acrylic polymer beads for solid-supported proline-derived organocatalysts.

PubMed

Kristensen, Tor E; Vestli, Kristian; Fredriksen, Kim A; Hansen, Finn K; Hansen, Tore

2009-07-16

A completely non-chromatographic and highly large-scale adaptable synthesis of acrylic polymer beads containing proline and prolineamides has been developed. Novel monomeric proline (meth)acrylates are prepared from hydroxyproline in only one step. Free-radical copolymerization then gives solid-supported proline organocatalysts directly in as little as two steps overall, without using any prefabricated solid supports, by using either droplet or dispersion polymerization. These affordable acrylic beads have highly favorable and adjustable swelling characteristics and are excellent reusable catalysts for organocatalytic reactions.

Crystal structures of resorcin[4]arene and pyrogallol[4]arene complexes with proline: A model for proline recognition through Csbnd H···π interaction

NASA Astrophysics Data System (ADS)

Fujisawa, Ikuhide; Kitamura, Yuji; Kato, Ryo; Aoki, Katsuyuki

2018-07-01

Resorcin[4]arene (resorcinol cyclic tetramer, abbreviated as RCT) or pyrogallol[4]arene (pyrogallol cyclic tetramer, PCT) form host-guest 1:1 complexes with DL-proline (DL-Pro) or L-proline (L-Pro), [RCT·DL-Pro]·2MeOH·3.5H2O (1) and 2[PCT·L-Pro]·2EtOH·10H2O (2), whose crystal structures have been determined. In each complex, the proline ligand is incorporated into the bowl-shaped cavity of RCT or PCT host molecules through Csbnd H … π interactions between alkyl protons of the pyrrolidine ring of proline and π-rings of RCT or PCT, forming an [RCT/PCT·Pro] structural fragment. In the crystal lattice, two [RCT/PCT·Pro] fragments self-associate to form a ligand-mediated dimeric structure, [RCT·D-Pro·L-Pro·RCT] in 1 or [PCT·L-Pro·L-Pro·PCT] in 2. A 1H NMR solution study gave the host‒ligand binding constants of 10.0 ± 1.1 M-1 for the RCT-DL-Pro system and 17.3 ± 1.3 M-1 for the PCT-L-Pro system. These complexes provide a synthetic model for the recognition of the proline residue in proline-containing substrates or inhibitors by enzymes through Csbnd H … π interaction. The CSD survey revealed that the absolute value of the torsion angle N-Cα-Csbnd O1 (O1 is cis to N) about the carboxyl Cα-C bond of proline is significantly smaller than that of the Cβ-Cα-Csbnd O2 (O2 is cis to Cβ) torsion angle.

Quantifying Additive Interactions of the Osmolyte Proline with Individual Functional Groups of Proteins: Comparisons with Urea and Glycine Betaine, Interpretation of m-Values

PubMed Central

Diehl, Roger C.; Guinn, Emily J.; Capp, Michael W.; Tsodikov, Oleg V.; Record, M. Thomas

2013-01-01

To quantify interactions of the osmolyte L-proline with protein functional groups and predict its effects on protein processes, we use vapor pressure osmometry to determine chemical potential derivatives dµ2/dm3 = µ23 quantifying preferential interactions of proline (component 3) with 21 solutes (component 2) selected to display different combinations of aliphatic or aromatic C, amide, carboxylate, phosphate or hydroxyl O, and/or amide or cationic N surface. Solubility data yield µ23 values for 4 less-soluble solutes. Values of µ23 are dissected using an ASA-based analysis to test the hypothesis of additivity and obtain α-values (proline interaction potentials) for these eight surface types and three inorganic ions. Values of µ23 predicted from these α-values agree with experiment, demonstrating additivity. Molecular interpretation of α-values using the solute partitioning model yields partition coefficients (Kp) quantifying the local accumulation or exclusion of proline in the hydration water of each functional group. Interactions of proline with native protein surface and effects of proline on protein unfolding are predicted from α-values and ASA information and compared with experimental data, with results for glycine betaine and urea, and with predictions from transfer free energy analysis. We conclude that proline stabilizes proteins because of its unfavorable interactions with (exclusion from) amide oxygens and aliphatic hydrocarbon surface exposed in unfolding, and that proline is an effective in vivo osmolyte because of the osmolality increase resulting from its unfavorable interactions with anionic (carboxylate and phosphate) and amide oxygens and aliphatic hydrocarbon groups on the surface of cytoplasmic proteins and nucleic acids. PMID:23909383

Uptake of Amino Acids and Their Metabolic Conversion into the Compatible Solute Proline Confers Osmoprotection to Bacillus subtilis

PubMed Central

Zaprasis, Adrienne; Bleisteiner, Monika; Kerres, Anne; Hoffmann, Tamara

2014-01-01

The data presented here reveal a new facet of the physiological adjustment processes through which Bacillus subtilis can derive osmostress protection. We found that the import of proteogenic (Glu, Gln, Asp, Asn, and Arg) and of nonproteogenic (Orn and Cit) amino acids and their metabolic conversion into proline enhances growth under otherwise osmotically unfavorable conditions. Osmoprotection by amino acids depends on the functioning of the ProJ-ProA-ProH enzymes, but different entry points into this biosynthetic route are used by different amino acids to finally yield the compatible solute proline. Glu, Gln, Asp, and Asn are used to replenish the cellular pool of glutamate, the precursor for proline production, whereas Arg, Orn, and Cit are converted into γ-glutamic semialdehyde/Δ1-pyrroline-5-carboxylate, an intermediate in proline biosynthesis. The import of Glu, Gln, Asp, Asn, Arg, Orn, and Cit did not lead to a further increase in the size of the proline pool that is already present in osmotically stressed cells. Hence, our data suggest that osmoprotection of B. subtilis by this group of amino acids rests on the savings in biosynthetic building blocks and energy that would otherwise have to be devoted either to the synthesis of the proline precursor glutamate or of proline itself. Since glutamate is the direct biosynthetic precursor for proline, we studied its uptake and found that GltT, an Na+-coupled symporter, is the main uptake system for both glutamate and aspartate in B. subtilis. Collectively, our data show how effectively B. subtilis can exploit environmental resources to derive osmotic-stress protection through physiological means. PMID:25344233

Molecular mechanisms of cryoprotection in aqueous proline: light scattering and molecular dynamics simulations.

PubMed

Troitzsch, R Z; Vass, H; Hossack, W J; Martyna, G J; Crain, J

2008-04-10

Free proline amino acid is a natural cryoprotectant expressed by numerous organisms under low-temperature stress. Previous reports have suggested that complex assemblies underlie its functional properties. We investigate here aqueous proline solutions as a function of temperature using combinations of Raman spectroscopy, Rayleigh-Brillouin light scattering, and molecular dynamics simulations with the view to revealing the molecular origins of the mixtures' functionality as a cryoprotectant. The evolution of the Brillouin frequency shifts and line widths with temperature shows that, above a critical proline concentration, the water-like dynamics is suppressed and viscoelastic behavior emerges: Here, the Landau-Placzek ratio also shows a temperature-independent maximum arising from concentration fluctuations. Molecular dynamics simulations reveal that the water-water correlations in the mixtures depend much more weakly on temperature than does bulk water. By contrast, the water OH Raman bands exhibit strong red-shifts on cooling similar to those seen in ices; however, no evidence of ice lattice phonons is observed in the low-frequency spectrum. We attribute this primarily to enhanced proline-water hydrogen bonding. In general, the picture that emerges is that aqueous proline is a heterogeneous mixture on molecular length scales (characterized by significant concentration fluctuations rather than well-defined aggregates). Simulations reveal that proline also appears to suppress the normal dependence of water structure on temperature and preserves the ambient-temperature correlations even in very cold solutions. The water structure in cold proline solutions therefore appears to be similar to that at a higher effective temperature. This, coupled with the emergence of glassy dynamics offers a molecular explanation for the functional properties of proline as a cryoprotectant without the need to invoke previously proposed complex aggregates.

Global Metabolic Profiling Identifies a Pivotal Role of Proline and Hydroxyproline Metabolism in Supporting Hypoxic Response in Hepatocellular Carcinoma.

PubMed

Tang, Ling; Zeng, Jun; Geng, Pengyu; Fang, Chengnan; Wang, Yang; Sun, Mingju; Wang, Changsong; Wang, Jiao; Yin, Peiyuan; Hu, Chunxiu; Guo, Lei; Yu, Jane; Gao, Peng; Li, Enyou; Zhuang, Zhengping; Xu, Guowang; Liu, Yang

2018-01-15

Purpose: Metabolic reprogramming is frequently identified in hepatocellular carcinoma (HCC), which is the most common type of liver malignancy. The reprogrammed cellular metabolisms promote tumor cell survival, proliferation, angiogenesis, and metastasis. However, the mechanisms of this process remain unclear in HCC. Experimental Design: The global nontargeted metabolic study in 69 paired hepatic carcinomas and adjacent tissue specimens was performed using capillary electrophoresis-time of flight mass spectrometry-based approach. Key findings were validated by targeted metabolomic approach. Biological studies were also performed to investigate the role of proline biosynthesis in HCC pathogenesis. Results: Proline metabolism was markedly changed in HCC tumor tissue, characterized with accelerated consumption of proline and accumulation of hydroxyproline, which significantly correlated with α-fetoprotein levels and poor prognosis in HCC. In addition, we found that hydroxyproline promoted hypoxia- and HIF-dependent phenotype in HCC. Moreover, we demonstrated that hypoxia activated proline biosynthesis via upregulation of ALDH18A1 , subsequently leading to accumulation of hydroxyproline via attenuated PRODH2 activity. More importantly, we showed that glutamine, proline, and hydroxyproline metabolic axis supported HCC cell survival through modulating HIF1α stability in response to hypoxia. Finally, inhibition of proline biosynthesis significantly enhanced cytotoxicity of sorafenib in vitro and in vivo Conclusions: Our results demonstrate that hypoxic microenvironment activates proline metabolism, resulting in accumulation of hydroxyproline that promotes HCC tumor progression and sorafenib resistance through modulating HIF1α. These findings provide the proof of concept for targeting proline metabolism as a potential therapeutic strategy for HCC. Clin Cancer Res; 24(2); 474-85. ©2017 AACR . ©2017 American Association for Cancer Research.

Pinpointing proline substitution to be responsible for the loss of amyloidogenesis in IAPP.

PubMed

Chakraborty, Sandipan; Mukherjee, Barnali; Basu, Soumalee

2013-10-01

Human islet amyloid polypeptide (hIAPP) is highly amyloidogenic, whereas its homologs in rodents are non-amyloidogenic. This observed non-amyloidogenecity of rodent IAPP has been attributed to substitutions by proline in a region of IAPP that forms the core of the fibril. By employing molecular dynamics simulation, we have analyzed effects of position-specific proline substitution on amyloidogenesis of the core region of the hIAPP fibril (22-28). We depict that substitution to proline at the 25th position is primarily responsible for the loss of amyloidogenecity of the peptide. In addition, 25th and 26th double mutation to proline and valine has been observed to show significant fibril destabilizing ability. On the contrary, substitution at 28th position to proline has the least ability to destabilize the amyloid fibril. Results obtained from this study are particularly important to design variants of the existing antihyperglycemic drug with minimalistic mutation approach for use in patients with diabetes. © 2013 John Wiley & Sons A/S.

Primary afferent depolarization and changes in extracellular potassium concentration induced by L-glutamate and L-proline in the isolated spinal cord of the frog.

PubMed

Vyklický, L; Vyskocil, F; Kolaj, M; Jastreboff, P

1982-10-08

To test the hypothesis that L-proline acts as an antagonist on glutamate receptors [17, 18], the interaction between L-glutamate and L-proline was studied in the isolated spinal cord of the frog. Glutamate at concentrations of 10(-6) -5 x 10(-3) mol/l depolarized the primary afferent fibres and increased extracellular potassium concentration, [K+]e, by 0.3-4 mmol/l. Repeated applications lead to inactivation of the response. L-Proline at 5 x 10(-3) -10(-2) mol/l, also depolarized the primary afferents and increased [K+]e by 0.5-2 mmol/l, but there was only a slight decrease of the effects after repeated application. The effects were additive when the amino acids were applied simultaneously. The effect of L-proline was still present when it was applied during inactivation of the glutamate receptors. This suggests that L-glutamate and L-proline act on different receptors.

Comparative Aspects of Tissue Glutamine and Proline Metabolism

USDA-ARS?s Scientific Manuscript database

The cellular metabolism of glutamine and proline are closely interrelated since they can be interconverted with glutamate and ornithine via the mitochondrial pathway involving pyrolline-5-carboxylate (P5C). In adults, glutamine and proline are converted via P5C to citrulline in the gut, then citrul...

Cotton Effect in Copper-Proline Complexes in the Visible Region

ERIC Educational Resources Information Center

Volkov, Victor; Pfister, Rolf

2005-01-01

The electronic properties of Cu(II) complex with proline are considered to demonstrate the Cotton effect in the visible region. A series of experiments in optical rotatory dispersion spectroscopy with free D- and L-proline and their complexes with the Cu(II) ion in aqueous solution is suggested.

Seasonal acclimatization of thallus proline contents of Mastocarpus stellatus and Chondrus crispus: intertidal rhodophytes that differ in freezing tolerance.

PubMed

Harris, Jonathan P; Logan, Barry A

2018-02-17

Mastocarpus stellatus and Chondrus crispus often co-occur in the lower intertidal of Northern Atlantic rocky shorelines. At our field site along the Maine coast (USA), Mastocarpus stellatus thalli possessed greater contents of proline when compared with thalli of Chondrus crispus. In addition, M. stellatus thalli acclimated to colder growth conditions in winter/early spring by increasing proline content several fold; no seasonal acclimation in proline content was observed in C. crispus. Proline accumulates in the tissues of a broad diversity of freezing-tolerant organisms and is among the most common cryoprotectant molecules. Thus, our observations provide a basis for the previously well-documented greater freezing tolerance of Mastocarpus stellatus when compared with Chondrus crispus. © 2018 Phycological Society of America.

Evidence for a Role of Proline and Hypothalamic Astrocytes in the Regulation of Glucose Metabolism in Rats

PubMed Central

Arrieta-Cruz, Isabel; Su, Ya; Knight, Colette M.; Lam, Tony K.T.; Gutiérrez-Juárez, Roger

2013-01-01

The metabolism of lactate to pyruvate in the mediobasal hypothalamus (MBH) regulates hepatic glucose production. Because astrocytes and neurons are functionally linked by metabolic coupling through lactate transfer via the astrocyte-neuron lactate shuttle (ANLS), we reasoned that astrocytes might be involved in the hypothalamic regulation of glucose metabolism. To examine this possibility, we used the gluconeogenic amino acid proline, which is metabolized to pyruvate in astrocytes. Our results showed that increasing the availability of proline in rats either centrally (MBH) or systemically acutely lowered blood glucose. Pancreatic clamp studies revealed that this hypoglycemic effect was due to a decrease of hepatic glucose production secondary to an inhibition of glycogenolysis, gluconeogenesis, and glucose-6-phosphatase flux. The effect of proline was mimicked by glutamate, an intermediary of proline metabolism. Interestingly, proline’s action was markedly blunted by pharmacological inhibition of hypothalamic lactate dehydrogenase (LDH) suggesting that metabolic flux through LDH was required. Furthermore, short hairpin RNA–mediated knockdown of hypothalamic LDH-A, an astrocytic component of the ANLS, also blunted the glucoregulatory action of proline. Thus our studies suggest not only a new role for proline in the regulation of hepatic glucose production but also indicate that hypothalamic astrocytes are involved in the regulatory mechanism as well. PMID:23274895

Impact of proline application on cadmium accumulation, mineral nutrition and enzymatic antioxidant defense system of Olea europaea L. cv Chemlali exposed to cadmium stress.

PubMed

Zouari, Mohamed; Ben Ahmed, Chedlia; Elloumi, Nada; Bellassoued, Khaled; Delmail, David; Labrousse, Pascal; Ben Abdallah, Ferjani; Ben Rouina, Bechir

2016-06-01

Proline plays an important role in plant response to various environmental stresses. However, its involvement in mitigation of heavy metal stress in plants remains elusive. In this study, we examined the effectiveness of exogenous proline (10 and 20 mM) in alleviating cadmium induced inhibitory effects in young olive plants (Olea europaea L. cv. Chemlali) exposed to two Cd levels (10 and 30 mg CdCl2 kg(-1) soil). The Cd treatment induced substantial accumulation of Cd in both root and leaf tissues and a decrease in gas exchange, photosynthetic pigments contents, uptake of essential elements (Ca, Mg and K) and plant biomass. Furthermore, an elevation of antioxidant enzymes activities (superoxide dismutase, catalase, glutathione peroxydase) and proline content in association with relatively high amounts of hydrogen peroxide, thiobarbituric acid reactive substances and electrolyte leakage were observed. Interestingly, the application of exogenous proline alleviated the oxidative damage induced by Cd accumulation. In fact, Cd-stressed olive plants treated with proline showed an increase of antioxidant enzymes activities, photosynthetic activity, nutritional status, plant growth and oil content of olive fruit. Generally, it seems that proline supplementation alleviated the deleterious effects of young olive plants exposed to Cd stress. Copyright © 2016 Elsevier Inc. All rights reserved.

On the Split Personality of Penultimate Proline

PubMed Central

Glover, Matthew S.; Shi, Liuqing; Fuller, Daniel R.; Arnold, Randy J.; Radivojac, Predrag; Clemmer, David E.

2014-01-01

The influence of the position of the amino acid proline in polypeptide sequences is examined by a combination of ion mobility spectrometry-mass spectrometry (IMS-MS), amino acid substitutions, and molecular modeling. The results suggest that when proline exists as the second residue from the N-terminus (i.e., penultimate proline), two families of conformers are formed. We demonstrate the existence of these families by a study of a series of truncated and mutated peptides derived from the 11-residue peptide Ser1-Pro2-Glu3-Leu4-Pro5-Ser6-Pro7-Gln8-Ala9-Glu10-Lys11. We find that every peptide from this sequence with a penultimate proline residue has multiple conformations. Substitution of Ala for Pro residues indicates that multiple conformers arise from the cis- trans isomerization of Xaa1–Pro2 peptide bonds as Xaa–Ala peptide bonds are unlikely to adopt the cis isomer, and examination of spectra from a library of 58 peptides indicates that ~80% of sequences show this effect. A simple mechanism suggesting that the barrier between the cis-and trans-proline forms is lowered because of low steric impedance is proposed. This observation may have interesting biological implications as well, and we note that a number of biologically active peptides have penultimate proline residues. PMID:25503299

Effect of proline mutations on the monomer conformations of amylin.

PubMed

Chiu, Chi-cheng; Singh, Sadanand; de Pablo, Juan J

2013-09-03

The formation of human islet amyloid polypeptide (hIAPP) is implicated in the loss of pancreatic β-cells in type II diabetes. Rat amylin, which differs from human amylin at six residues, does not lead to formation of amyloid fibrils. Pramlintide is a synthetic analog of human amylin that shares three proline substitutions with rat amylin. Pramlintide has a much smaller propensity to form amyloid aggregates and has been widely prescribed in amylin replacement treatment. It is known that the three prolines attenuate β-sheet formation. However, the detailed effects of these proline substitutions on full-length hIAPP remain poorly understood. In this work, we use molecular simulations and bias-exchange metadynamics to investigate the effect of proline substitutions on the conformation of the hIAPP monomer. Our results demonstrate that hIAPP can adopt various β-sheet conformations, some of which have been reported in experiments. The proline substitutions perturb the formation of long β-sheets and reduce their stability. More importantly, we find that all three proline substitutions of pramlintide are required to inhibit β conformations and stabilize the α-helical conformation. Fewer substitutions do not have a significant inhibiting effect. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Hyperprolinemic larvae of the drosophilid fly, Chymomyza costata, survive cryopreservation in liquid nitrogen

PubMed Central

Koštál, Vladimír; Zahradníčková, Helena; Šimek, Petr

2011-01-01

The larva of the drosophilid fly, Chymomyza costata, is probably the most complex metazoan organism that can survive submergence in liquid nitrogen (-196 °C) in a fully hydrated state. We examined the associations between the physiological and biochemical parameters of differently acclimated larvae and their freeze tolerance. Entering diapause is an essential and sufficient prerequisite for attaining high levels of survival in liquid nitrogen (23% survival to adult stage), although cold acclimation further improves this capacity (62% survival). Profiling of 61 different metabolites identified proline as a prominent compound whose concentration increased from 20 to 147 mM during diapause transition and subsequent cold acclimation. This study provides direct evidence for the essential role of proline in high freeze tolerance. We increased the levels of proline in the larval tissues by feeding larvae proline-augmented diets and found that this simple treatment dramatically improved their freeze tolerance. Cell and tissue survival following exposure to liquid nitrogen was evident in proline-fed nondiapause larvae, and survival to adult stage increased from 0% to 36% in proline-fed diapause-destined larvae. A significant statistical correlation was found between the whole-body concentration of proline, either natural or artificial, and survival to the adult stage in liquid nitrogen for diapause larvae. Differential scanning calorimetry analysis suggested that high proline levels, in combination with a relatively low content of osmotically active water and freeze dehydration, increased the propensity of the remaining unfrozen water to undergo a glass-like transition (vitrification) and thus facilitated the prevention of cryoinjury. PMID:21788482

Osmoprotection of Bacillus subtilis through Import and Proteolysis of Proline-Containing Peptides

PubMed Central

Zaprasis, Adrienne; Brill, Jeanette; Thüring, Marietta; Wünsche, Guido; Heun, Magnus; Barzantny, Helena; Hoffmann, Tamara

2013-01-01

Bacillus subtilis can attain cellular protection against the detrimental effects of high osmolarity through osmotically induced de novo synthesis and uptake of the compatible solute l-proline. We have now found that B. subtilis can also exploit exogenously provided proline-containing peptides of various lengths and compositions as osmoprotectants. Osmoprotection by these types of peptides is generally dependent on their import via the peptide transport systems (Dpp, Opp, App, and DtpT) operating in B. subtilis and relies on their hydrolysis to liberate proline. The effectiveness with which proline-containing peptides confer osmoprotection varies considerably, and this can be correlated with the amount of the liberated and subsequently accumulated free proline by the osmotically stressed cell. Through gene disruption experiments, growth studies, and the quantification of the intracellular proline pool, we have identified the PapA (YqhT) and PapB (YkvY) peptidases as responsible for the hydrolysis of various types of Xaa-Pro dipeptides and Xaa-Pro-Xaa tripeptides. The PapA and PapB peptidases possess overlapping substrate specificities. In contrast, osmoprotection by peptides of various lengths and compositions with a proline residue positioned at their N terminus was not affected by defects in the PapA and PapB peptidases. Taken together, our data provide new insight into the physiology of the osmotic stress response of B. subtilis. They illustrate the flexibility of this ubiquitously distributed microorganism to effectively exploit environmental resources in its acclimatization to sustained high-osmolarity surroundings through the accumulation of compatible solutes. PMID:23144141

Hyperprolinemic larvae of the drosophilid fly, Chymomyza costata, survive cryopreservation in liquid nitrogen.

PubMed

Kostál, Vladimír; Zahradnícková, Helena; Šimek, Petr

2011-08-09

The larva of the drosophilid fly, Chymomyza costata, is probably the most complex metazoan organism that can survive submergence in liquid nitrogen (-196 °C) in a fully hydrated state. We examined the associations between the physiological and biochemical parameters of differently acclimated larvae and their freeze tolerance. Entering diapause is an essential and sufficient prerequisite for attaining high levels of survival in liquid nitrogen (23% survival to adult stage), although cold acclimation further improves this capacity (62% survival). Profiling of 61 different metabolites identified proline as a prominent compound whose concentration increased from 20 to 147 mM during diapause transition and subsequent cold acclimation. This study provides direct evidence for the essential role of proline in high freeze tolerance. We increased the levels of proline in the larval tissues by feeding larvae proline-augmented diets and found that this simple treatment dramatically improved their freeze tolerance. Cell and tissue survival following exposure to liquid nitrogen was evident in proline-fed nondiapause larvae, and survival to adult stage increased from 0% to 36% in proline-fed diapause-destined larvae. A significant statistical correlation was found between the whole-body concentration of proline, either natural or artificial, and survival to the adult stage in liquid nitrogen for diapause larvae. Differential scanning calorimetry analysis suggested that high proline levels, in combination with a relatively low content of osmotically active water and freeze dehydration, increased the propensity of the remaining unfrozen water to undergo a glass-like transition (vitrification) and thus facilitated the prevention of cryoinjury.

l-Proline Accumulation and Freeze Tolerance in Saccharomyces cerevisiae Are Caused by a Mutation in the PRO1 Gene Encoding γ-Glutamyl Kinase

PubMed Central

Morita, Yuko; Nakamori, Shigeru; Takagi, Hiroshi

2003-01-01

We previously isolated a mutant which showed a high tolerance to freezing that correlated with higher levels of intracellular l-proline derived from l-proline analogue-resistant mutants. The mutation responsible for the analogue resistance and l-proline accumulation was a single nuclear dominant mutation. By introducing the mutant-derived genomic library into a non-l-proline-utilizing strain, the mutant was found to carry an allele of the wild-type PRO1 gene encoding γ-glutamyl kinase, which resulted in a single amino acid replacement; Asp (GAC) at position 154 was replaced by Asn (AAC). Interestingly, the allele of PRO1 was shown to enhance the activities of γ-glutamyl kinase and γ-glutamyl phosphate reductase, both of which catalyze the first two steps of l-proline synthesis from l-glutamate and which together may form a complex in vivo. When cultured in liquid minimal medium, yeast cells expressing the mutated γ-glutamyl kinase were found to accumulate intracellular l-proline and showed a prominent increase in cell viability after freezing at −20°C compared to the viability of cells harboring the wild-type PRO1 gene. These results suggest that the altered γ-glutamyl kinase results in stabilization of the complex or has an indirect effect on γ-glutamyl phosphate reductase activity, which leads to an increase in l-proline production in Saccharomyces cerevisiae. The approach described in this paper could be a practical method for breeding novel freeze-tolerant yeast strains. PMID:12513997

The relationship of proline content and metabolism on the productivity of maize plants

PubMed Central

Špoljarević, Marija; Agić, Dejan; Lisjak, Miroslav; Gumze, Andrej; Wilson, Ian D; Hancock, John T

2011-01-01

The free proline content in maize ear-leaves, silk and pollen were analyzed in field grown plants which had matured to the pollination stage. Using maize hybrids PR34F02, PR35P12 and PR36B08 field trials were set up at two locations in eastern Croatia in two different years. Two enzymes of proline metabolism were analyzed in the same leaf samples and specific activities of synthetase (P5CS) and proline dehydrogenase (PDH). Plant productivity was evaluated at harvest by the estimation of total and fully developed grain number per ear and per plant, the mean single grain mass, and the mass of grain per plant. The year in which the plants were grown had a very significant effect on the free proline content in the leaf and pollen, as well as on the enzyme activities assayed. The differences between the plants from the two localities were very significant in all tested parameters of plant grain productivity. There was a significant genotype effect on proline content and P5CS total activity in leaf and on all the productivity parameters. Some of the correlations established suggest that the rate of proline synthesis and degradation in maize ear-leaf at pollination might contribute to the final grain production of the maize plant. Multiple regression analyses was used to further analyze the relationship between proline and grain productivity, but it is clear that future work should include other environmental conditions, plant species and organs such as roots. PMID:21415600

Unusual reversal of enantioselectivity in the proline-mediated alpha-amination of aldehydes induced by tertiary amine additives.

PubMed

Blackmond, Donna G; Moran, Antonio; Hughes, Matthew; Armstrong, Alan

2010-06-09

An intriguing reversal in product enantioselectivity accompanied by a change in the kinetic profile is observed in the alpha-amination of aldehydes catalyzed by proline in the presence of organic bases. Implications for the prevailing stereochemical models for proline and related aminocatalytic transformations are discussed.

HR-MAS NMR metabolomics of 'Swingle' citrumelo rootstock genetically modified to overproduce proline.

PubMed

de Oliveira, Caroline S; Carlos, Eduardo F; Vieira, Luiz G E; Lião, Luciano M; Alcantara, Glaucia B

2014-08-01

The accumulation of proline is a typical physiological response to abiotic stresses in higher plants. 'Swingle' citrumelo, an important rootstock for citrus production, has been modified with a mutated Δ(1)-pyrroline-5-carboxylate synthetase gene (VaP5CSF129A) linked to the cauliflower mosaic virus 35S promoter to induce the overproduction of free proline. This paper presents a comparative metabolomic study of nontransgenic versus transgenic 'Swingle' citrumelo plants with high endogenous proline. (1)H high-resolution magic angle spinning nuclear magnetic resonance spectroscopy and multivariate analysis showed significant differences in some metabolites between the nontransgenic and transgenic leaves and roots. The overproduction of proline has reduced the sucrose content in transgenic leaves, revealing a metabolic cost for these plants. In roots, the high level of free proline acts for the adjustment of cation-anion balance, causing the reduction of acetic acid content. The same sucrose level in roots indicates that they can be considered as sucrose sink. Similar behavior may be waited for fruits produced on transgenic rootstock. Copyright © 2014 John Wiley & Sons, Ltd.

Enhancement of anthraquinone production in Morinda citrifolia cell suspension cultures after stimulation of the proline cycle with two proline analogs.

PubMed

Quevedo, Carla V; Perassolo, María; Giulietti, Ana M; Rodríguez Talou, Julián

2012-03-01

Synthesis of anthraquinones (AQs) involves the shikimate and 2-C-methyl-D-erythritol 4-phosphate pathways. The proline cycle is linked to the pentose phosphate pathway (PPP) to generate NADPH needed in the first steps of this pathway. The effect of two proline analogs, azetidine-2-carboxylic acid (A2C) and thiazolidine-4-carboxylic acid (T4C), were evaluated in Morinda citrifolia suspension cultures. Both analogs gave higher proline accumulation after 6 and 10 days (68 and 179% after 6 days with A2C at 25 and 50 μM, respectively, and 111% with T4C added at 100 μM). Induction of the proline cycle increased the AQ content after 6 days (~40% for 50 μM A2C and 100 μM T4C). Whereas A2C (50 μM) increased only AQ production, T4C also enhanced total phenolics. However, no induction of the PPP was observed with any of the treatments. This pathway therefore does not limit the supply of carbon skeletons to secondary metabolic pathways.

Use of prolines for improving growth and other properties of plants and algae

DOEpatents

Unkefer, Pat J.; Knight, Thomas J.; Martinez, Rodolfo A.

2003-07-15

Increasing the concentration of prolines, such as 2-hydroxy-5-oxoproline, in the foliar portions of plants has been shown to cause an increase in carbon dioxide fixation, growth rate, dry weight, nutritional value (amino acids), nodulation and nitrogen fixation, photosynthetically derived chemical energy, and resistance to insect pests over the same properties for wild type plants. This can be accomplished in four ways: (1) the application of a solution of the proline directly to the foliar portions of the plant by spraying these portions; (2) applying a solution of the proline to the plant roots; (3) genetically engineering the plant and screening to produce lines that over-express glutamine synthetase in the leaves which gives rise to increased concentration of the metabolite, 2-hydroxy-5-oxoproline (this proline is also known as 2-oxoglutaramate); and (4) impairing the glutamine synthetase activity in the plant roots which causes increased glutamine synthetase activity in the leaves which gives rise to increased concentration of 2-hydroxy-5-oxoproline. Prolines have also been found to induce similar effects in algae.

Use of prolines for improving growth and other properties of plants and algae

DOEpatents

Unkefer, Pat J.; Knight, Thomas J.; Martinez, Rodolfo A.

2004-12-14

Increasing the concentration of prolines, such as 2-hydroxy-5-oxoproline, in the foliar portions of plants has been shown to cause an increase in carbon dioxide fixation, growth rate, dry weight, nutritional value (amino acids), nodulation and nitrogen fixation, photosynthetically derived chemical energy, and resistance to insect pests over the same properties for wild type plants. This can be accomplished in four ways: (1) the application of a solution of the proline directly to the foliar portions of the plant by spraying these portions; (2) applying a solution of the proline to the plant roots; (3) genetically engineering the plant and screening to produce lines that over-express glutamine synthetase in the leaves which gives rise to increased concentration of the metabolite, 2-hydroxy-5-oxoproline (this proline is also known as 2-oxoglutaramate); and (4) impairing the glutamine synthetase activity in the plant roots which causes increased glutamine synthetase activity in the leaves which gives rise to increased concentration of 2-hydroxy-5-oxoproline. Prolines have also been found to induce similar effects in algae.

Use of prolines for improving growth and other properties of plants and algae

DOEpatents

Unkefer, Pat J.; Knight, Thomas J.; Martinez, Rodolfo A.

2003-04-29

Increasing the concentration of prolines such as 2-hydroxy-5-oxoproline, in the foliar portions of plants has been shown to cause an increase in carbon dioxide fixation, growth rate, dry weight, nutritional value (amino acids), nodulation and nitrogen fixation, photosynthetically derived chemical energy, and resistance to insect pests over the same properties for wild type plants. This can be accomplished in four ways: (1) the application of a solution of the proline directly to the foliar portions of the plant by spraying these portions; (2) applying a solution of the proline to the plant roots; (3) genetically engineering the plant and screening to produce lines that overexpress glutamine synthetase in the leaves which gives rise to increased concentration of the metabolite, 2-hydroxy-5-oxoproline (this proline is also known as 2-oxoglutaramnate); and (4) impairing the glutamine synthetase activity in the plant roots which causes increased glutamine synthetase activity in the leaves which gives rise to increased concentration of 2-hydroxy-5-oxoproline. Prolines have also been found to induce similar effects in algae.

UV photodissociation of proline-containing peptide ions: insights from molecular dynamics.

PubMed

Girod, Marion; Sanader, Zeljka; Vojkovic, Marin; Antoine, Rodolphe; MacAleese, Luke; Lemoine, Jérôme; Bonacic-Koutecky, Vlasta; Dugourd, Philippe

2015-03-01

UV photodissociation of proline-containing peptide ions leads to unusual product ions. In this paper, we report laser-induced dissociation of a series of proline-containing peptides at 213 nm. We observe specific fragmentation pathways corresponding to the formation of (y-2), (a + 2) and (b + 2) fragment ions. This was not observed at 266 nm or for peptides which do not contain proline residues. In order to obtain insights into the fragmentation dynamics at 213 nm, a small peptide (RPK for arginine-proline-lysine) was studied both theoretically and experimentally. Calculations of absorption spectra and non-adiabatic molecular dynamics (MD) were made. Second and third excited singlet states, S(2) and S(3), lie close to 213 nm. Non-adiabatic MD simulation starting from S(2) and S(3) shows that these transitions are followed by C-C and C-N bond activation close to the proline residue. After this first relaxation step, consecutive rearrangements and proton transfers are required to produce unusual (y-2), (a + 2) and (b + 2) fragment ions. These fragmentation mechanisms were confirmed by H/D exchange experiments.

UV Photodissociation of Proline-containing Peptide Ions: Insights from Molecular Dynamics

NASA Astrophysics Data System (ADS)

Girod, Marion; Sanader, Zeljka; Vojkovic, Marin; Antoine, Rodolphe; MacAleese, Luke; Lemoine, Jérôme; Bonacic-Koutecky, Vlasta; Dugourd, Philippe

2015-03-01

UV photodissociation of proline-containing peptide ions leads to unusual product ions. In this paper, we report laser-induced dissociation of a series of proline-containing peptides at 213 nm. We observe specific fragmentation pathways corresponding to the formation of (y-2), (a + 2) and (b + 2) fragment ions. This was not observed at 266 nm or for peptides which do not contain proline residues. In order to obtain insights into the fragmentation dynamics at 213 nm, a small peptide (RPK for arginine-proline-lysine) was studied both theoretically and experimentally. Calculations of absorption spectra and non-adiabatic molecular dynamics (MD) were made. Second and third excited singlet states, S2 and S3, lie close to 213 nm. Non-adiabatic MD simulation starting from S2 and S3 shows that these transitions are followed by C-C and C-N bond activation close to the proline residue. After this first relaxation step, consecutive rearrangements and proton transfers are required to produce unusual (y-2), (a + 2) and (b + 2) fragment ions. These fragmentation mechanisms were confirmed by H/D exchange experiments.

Constituents of Propolis: Chrysin, Caffeic Acid, p-Coumaric Acid, and Ferulic Acid Induce PRODH/POX-Dependent Apoptosis in Human Tongue Squamous Cell Carcinoma Cell (CAL-27).

PubMed

Celińska-Janowicz, Katarzyna; Zaręba, Ilona; Lazarek, Urszula; Teul, Joanna; Tomczyk, Michał; Pałka, Jerzy; Miltyk, Wojciech

2018-01-01

Propolis evokes several therapeutic properties, including anticancer activity. These activities are attributed to the action of polyphenols. Previously it has been demonstrated, that one of the most abundant polyphenolic compounds in ethanolic extracts of propolis are chrysin, caffeic acid, p -coumaric acid, and ferulic acid. Although their pro-apoptotic activity on human tongue squamous cell carcinoma cells (CAL-27) was established previously, the detailed mechanism of this process remains unclear. Considering the crucial role of proline metabolism and proline dehydrogenase/proline oxidase (PRODH/POX) in the regulation of cancer cell survival/apoptosis, we studied these processes in polyphenol-treated CAL-27 cells. All studied polyphenols evoked anti-proliferative activity, accompanied by increased PRODH/POX, P53, active caspases-3 and -9 expressions and decreased collagen biosynthesis, prolidase activity and proline concentration in CAL-27 cells. These data suggest that polyphenols of propolis induce PRODH/POX-dependent apoptosis through up-regulation of mitochondrial proline degradation and down-regulation of proline utilization for collagen biosynthesis.

Constituents of Propolis: Chrysin, Caffeic Acid, p-Coumaric Acid, and Ferulic Acid Induce PRODH/POX-Dependent Apoptosis in Human Tongue Squamous Cell Carcinoma Cell (CAL-27)

PubMed Central

Celińska-Janowicz, Katarzyna; Zaręba, Ilona; Lazarek, Urszula; Teul, Joanna; Tomczyk, Michał; Pałka, Jerzy; Miltyk, Wojciech

2018-01-01

Propolis evokes several therapeutic properties, including anticancer activity. These activities are attributed to the action of polyphenols. Previously it has been demonstrated, that one of the most abundant polyphenolic compounds in ethanolic extracts of propolis are chrysin, caffeic acid, p-coumaric acid, and ferulic acid. Although their pro-apoptotic activity on human tongue squamous cell carcinoma cells (CAL-27) was established previously, the detailed mechanism of this process remains unclear. Considering the crucial role of proline metabolism and proline dehydrogenase/proline oxidase (PRODH/POX) in the regulation of cancer cell survival/apoptosis, we studied these processes in polyphenol-treated CAL-27 cells. All studied polyphenols evoked anti-proliferative activity, accompanied by increased PRODH/POX, P53, active caspases-3 and -9 expressions and decreased collagen biosynthesis, prolidase activity and proline concentration in CAL-27 cells. These data suggest that polyphenols of propolis induce PRODH/POX-dependent apoptosis through up-regulation of mitochondrial proline degradation and down-regulation of proline utilization for collagen biosynthesis. PMID:29681859

Intron-mediated alternative splicing of Arabidopsis P5CS1 and its association with natural variation in proline and climate adaptation

PubMed Central

Kesari, Ravi; Lasky, Jesse R.; Villamor, Joji Grace; Des Marais, David L.; Chen, Ying-Jiun C.; Liu, Tzu-Wen; Juenger, Thomas E.; Verslues, Paul E.

2012-01-01

Drought-induced proline accumulation is widely observed in plants but its regulation and adaptive value are not as well understood. Proline accumulation of the Arabidopsis accession Shakdara (Sha) was threefold less than that of Landsberg erecta (Ler) and quantitative trait loci mapping identified a reduced function allele of the proline synthesis enzyme Δ1-pyrroline-5-carboxylate synthetase1 (P5CS1) as a basis for the lower proline of Sha. Sha P5CS1 had additional TA repeats in intron 2 and a G-to-T transversion in intron 3 that were sufficient to promote alternative splicing and production of a nonfunctional transcript lacking exon 3 (exon 3-skip P5CS1). In Sha, and additional accessions with the same intron polymorphisms, the nonfunctional exon 3-skip P5CS1 splice variant constituted as much as half of the total P5CS1 transcript. In a larger panel of Arabidopsis accessions, low water potential-induced proline accumulation varied by 10-fold and variable production of exon 3-skip P5CS1 among accessions was an important, but not the sole, factor underlying variation in proline accumulation. Population genetic analyses suggest that P5CS1 may have evolved under positive selection, and more extensive correlation of exon 3-skip P5CS1 production than proline abundance with climate conditions of natural accessions also suggest a role of P5CS1 in local adaptation to the environment. These data identify a unique source of alternative splicing in plants, demonstrate a role of exon 3-skip P5CS1 in natural variation of proline metabolism, and suggest an association of P5CS1 and its alternative splicing with environmental adaptation. PMID:22615385

Production of proline-rich proteins by the parotid glands of rats is enhanced by feeding diets containing tannins from faba beans (Vicia faba L.).

PubMed

Jansman, A J; Frohlich, A A; Marquardt, R R

1994-02-01

Feeding a casein-based diet with either 400 g/kg of tannin-containing faba bean hulls (Vicia faba L.) (1.41% condensed tannins) or 60 g/kg of a tannin-rich hull extract of faba beans (1.99% condensed tannins) to rats over a period of 7 d resulted in a 2.6 and 1.5 fold increase in weight of the parotid glands, respectively, (P < 0.05) and a corresponding 5.5 and 3.7 fold increase in the level of proline-rich proteins in the glands (P < 0.05). In a dose-response experiment, increasing the level of tannin-rich hull extract in the diet (0.0, 3.8, 7.5, 15.0, 30.0 and 60.0 g/kg) resulted in a linear increase in both the relative size of parotid glands (R2 = 0.90; P < 0.05) and the quantity of proline-rich proteins in the glands (R2 = 0.89; P < 0.05). The apparent digestibility of total (R2 = 0.97) and individual amino acids (R2 varied from 0.27 to 0.99) decreased linearly (P < 0.05). The quantity of proline-rich proteins in the cecum of rats was estimated from the decrease in digestibility of proline, glycine and glutamic acid. The estimated secretions of proline-rich proteins, when calculated on the basis of the three respective amino acids, were 3.5, 3.5 and 3.9 mg of proline-rich proteins per 10 mg of additional hull extract (21.8% condensed tannins). The results indicate that tannins from faba beans stimulate the parotid glands to increase the secretion of proline-rich proteins in rats. The proline-rich proteins then interact with dietary condensed tannins to reduce their antinutritional effects.

Gas-phase conformation-specific photofragmentation of proline-containing peptide ions.

PubMed

Kim, Tae-Young; Valentine, Stephen J; Clemmer, David E; Reilly, James P

2010-08-01

Singly-protonated proline-containing peptides with N-terminal arginine are photodissociated with vacuum ultraviolet (VUV) light in an ESI linear ion trap/orthogonal-TOF (LIT/o-TOF). When proline is the nth residue from the N-terminus, unusual b(n) + 2 and a(n) + 2 ions are observed. Their formation is explained by homolytic cleavage of the C(alpha)-C bond in conjunction with a rearrangement of electrons and an amide hydrogen. The latter is facilitated by a proline-stabilized gas-phase peptide conformation. Copyright 2010 American Society for Mass Spectrometry. Published by Elsevier Inc. All rights reserved.

Does proline isomerization shape the folding funnel of the wild type and mutant staphylococcal nuclease?

NASA Astrophysics Data System (ADS)

Tsong, Tian Yow; Su, Zheng-Ding

1999-10-01

Cis/trans isomerization of proline residues is known to exhibit high activation energies. These kinetic barriers often dominate the energy landscape of protein folding. There are 6 proline residues (at positions 11, 31, 42, 47, 56 and 117) in staphylococcal nuclease (SNase) [EC 3.1.31.1]. Stopped-flow CD222nm measuring the evolution of the secondary structure of protein has detected 5 kinetic barriers in SNase folding (ΔG≠ for τfr<15, τf1 16.9, τf2 18.5, τf3 19.5, and τfs 21.8 kcal/mol) and 3 kinetic barriers in unfolding (ΔG≠ for τur<15, τu1 17.4, τus 21.6 kcal/mol). To investigate systematically how individual proline residues and 6 proline residues in toto can shape the folding funnel we have expediently constructed 7 proline mutants for study. They are 6 single-proline-substituted mutants (P11A, P31A, P42A, P47A, P56A and P117A) and 1 proline-free mutant (PallA). Study of equilibrium folding/unfolding and stopped-flow kinetics of the wildtype and the 7 mutants of SNase have allowed us to identify sources of 3 main kinetic barriers in the SNase folding. The highest barrier (ΔG≠=21.8 kcal) belongs to the cis/trans isomerization of Pro117. The next barrier (ΔG≠=19.5 kcal) involves synergetic effects of proline residues which limits the rate of folding of the oligonucleotide binding (OB) domain in all 7 proline-containing SNase. For the proline-free mutant (PallA) the OB domain folds rapidly. Furthermore, we have found that the equilibrium folding/unfolding properties of these proline mutants are remarkably similar to that of the wildtype despite their startlingly different folding/unfolding kinetics. These results lead us to conclude that while free energy of folding (ΔGF=-4.5 kcal/mol) provides the driving force, it is the activation energy that forms a conduit or shapes a kinetic funnel for SNase folding. The landscape for SNase folding is extremely rugged. Data support our previously proposed Least Activation Path (LAP) model for protein folding [Su, Z.D. et al. Proc. Natl. Acad. Sci. USA 93, 2539-2544 (1996)]. The LAP concept depicts protein folding as the movement of the unfolded population of protein along deep valleys of the energy landscape to reach the free energy minimum of the native state. An analogy for the LAP model would be flow of water from a highland over the rugged surface of a landscape to reach the lowest point of the ground. The fine features of the landscape will dictate kinetics and pathways of the flow.

Cryptic Production of trans-3-Hydroxyproline in Echinocandin B Biosynthesis.

PubMed

Mattay, Johanna; Houwaart, Stefanie; Hüttel, Wolfgang

2018-04-01

Echinocandins are antifungal nonribosomal hexapeptides produced by fungi. Two of the amino acids are hydroxy-l-prolines: trans -4-hydroxy-l-proline and, in most echinocandin structures, ( trans -2,3)-3-hydroxy-( trans -2,4)-4-methyl-l-proline. In the case of echinocandin biosynthesis by Glarea lozoyensis , both amino acids are found in pneumocandin A 0 , while in pneumocandin B 0 the latter residue is replaced by trans -3-hydroxy-l-proline (3-Hyp). We have recently reported that all three amino acids are generated by the 2-oxoglutarate-dependent proline hydroxylase GloF. In echinocandin B biosynthesis by Aspergillus species, 3-Hyp derivatives have not been reported. Here we describe the heterologous production and kinetic characterization of HtyE, the 2-oxoglutarate-dependent proline hydroxylase from the echinocandin B biosynthetic cluster in Aspergillus pachycristatus Surprisingly, l-proline hydroxylation with HtyE resulted in an even higher proportion (∼30%) of 3-Hyp than that with GloF. This suggests that the selectivity for methylated 3-Hyp in echinocandin B biosynthesis is due solely to a substrate-specific adenylation domain of the nonribosomal peptide synthetase. Moreover, we observed that one product of HtyE catalysis, 3-hydroxy-4-methyl-l-proline, is slowly further oxidized at the methyl group, giving 3-hydroxy-4-hydroxymethyl-l-proline, upon prolonged incubation with HtyE. This dihydroxylated amino acid has been reported as a building block of cryptocandin, an echinocandin produced by Cryptosporiopsis IMPORTANCE Secondary metabolites from bacteria and fungi are often produced by sets of biosynthetic enzymes encoded in distinct gene clusters. Usually, each enzyme catalyzes one biosynthetic step, but multiple reactions are also possible. Pneumocandins A 0 and B 0 are produced by the fungus Glarea lozoyensis They belong to the echinocandin family, a group of nonribosomal cyclic lipopeptides that exhibit a strong antifungal activity. Chemical derivatives are important drugs for the treatment of systemic fungal infections. We have recently shown that in the biosynthesis of pneumocandins A 0 and B 0 , three hydroxyproline building blocks are provided by one proline hydroxylase. Here we demonstrate that the proline hydroxylase from echinocandin B biosynthesis in Aspergillus pachycristatus produces the same hydroxyprolines, with an increased proportion of trans -3-hydroxyproline. However, echinocandin B biosynthesis does not require trans -3-hydroxyproline; its formation remains cryptic. While one can only speculate on the evolutionary background of this unexpected finding, proline hydroxylation in G. lozoyensis and A. pachycristatus provides an unusual insight into peptide antibiotic biosynthesis-namely, the complex interplay between the selectivity of a hydroxylase and the substrate specificity of a nonribosomal peptide synthetase. Copyright © 2018 American Society for Microbiology.

Position of Proline Mediates the Reactivity of S-Palmitoylation.

PubMed

Khanal, Neelam; Pejaver, Vikas; Li, Zhiyu; Radivojac, Predrag; Clemmer, David E; Mukhopadhyay, Suchetana

2015-11-20

Palmitoylation, a post-translational modification in which a saturated 16-carbon chain is added predominantly to a cysteine residue, participates in various biological functions. The position of proline relative to other residues being post-translationally modified has been previously reported as being important. We determined that proline is statistically enriched around cysteines known to be S-palmitoylated. The goal of this work was to determine how the position of proline influences the palmitoylation of the cysteine residue. We established a mass spectrometry-based approach to investigate time- and temperature-dependent kinetics of autopalmitoylation in vitro and to derive the thermodynamic parameters of the transition state associated with palmitoylation; to the best of our knowledge, our work is the first to study the kinetics and activation properties of the palmitoylation process. We then used these thermochemical parameters to determine if the position of proline relative to the modified cysteine is important for palmitoylation. Our results show that peptides with proline at the -1 position of cysteine in their sequence (PC) have lower enthalpic barriers and higher entropic barriers in comparison to the same peptides with proline at the +1 position of cysteine (CP); interestingly, the free-energy barriers for both pairs are almost identical. Molecular dynamics studies demonstrate that the flexibility of the cysteine backbone in the PC-containing peptide when compared to the CP-containing peptide explains the increased entropic barrier and decreased enthalpic barrier observed experimentally.

Isolation of baker's yeast mutants with proline accumulation that showed enhanced tolerance to baking-associated stresses.

PubMed

Tsolmonbaatar, Ariunzaya; Hashida, Keisuke; Sugimoto, Yukiko; Watanabe, Daisuke; Furukawa, Shuhei; Takagi, Hiroshi

2016-12-05

During bread-making processes, yeast cells are exposed to baking-associated stresses such as freeze-thaw, air-drying, and high-sucrose concentrations. Previously, we reported that self-cloning diploid baker's yeast strains that accumulate proline retained higher-level fermentation abilities in both frozen and sweet doughs than the wild-type strain. Although self-cloning yeasts do not have to be treated as genetically modified yeasts, the conventional methods for breeding baker's yeasts are more acceptable to consumers than the use of self-cloning yeasts. In this study, we isolated mutants resistant to the proline analogue azetidine-2-carboxylate (AZC) derived from diploid baker's yeast of Saccharomyces cerevisiae. Some of the mutants accumulated a greater amount of intracellular proline, and among them, 5 mutants showed higher cell viability than that observed in the parent wild-type strain under freezing or high-sucrose stress conditions. Two of them carried novel mutations in the PRO1 gene encoding the Pro247Ser or Glu415Lys variant of γ-glutamyl kinase (GK), which is a key enzyme in proline biosynthesis in S. cerevisiae. Interestingly, we found that these mutations resulted in AZC resistance of yeast cells and desensitization to proline feedback inhibition of GK, leading to intracellular proline accumulation. Moreover, baker's yeast cells expressing the PRO1 P247S and PRO1 E415K gene were more tolerant to freezing stress than cells expressing the wild-type PRO1 gene. The approach described here could be a practical method for the breeding of proline-accumulating baker's yeasts with higher tolerance to baking-associated stresses. Copyright © 2016 Elsevier B.V. All rights reserved.

Role of cis-trans proline isomerization in the function of pathogenic enterobacterial Periplasmic Binding Proteins

PubMed Central

Cortes-Hernandez, Paulina

2017-01-01

Periplasmic Binding Proteins (PBPs) trap nutrients for their internalization into bacteria by ABC transporters. Ligand binding triggers PBP closure by bringing its two domains together like a Venus flytrap. The atomic determinants that control PBP opening and closure for nutrient capture and release are not known, although it is proposed that opening and ligand release occur while in contact with the ABC transporter for concurrent substrate translocation. In this paper we evaluated the effect of the isomerization of a conserved proline, located near the binding site, on the propensity of PBPs to open and close. ArgT/LAO from Salmonella typhimurium and HisJ from Escherichia coli were studied through molecular mechanics at two different temperatures: 300 and 323 K. Eight microseconds were simulated per protein to analyze protein opening and closure in the absence of the ABC transporter. We show that when the studied proline is in trans, closed empty LAO and HisJ can open. In contrast, with the proline in cis, opening transitions were much less frequent and characterized by smaller changes. The proline in trans also renders the open trap prone to close over a ligand. Our data suggest that the isomerization of this conserved proline modulates the PBP mechanism: the proline in trans allows the exploration of conformational space to produce trap opening and closure, while in cis it restricts PBP movement and could limit ligand release until in productive contact with the ABC transporter. This is the first time that a proline isomerization has been related to the control of a large conformational change like the PBP flytrap mechanism. PMID:29190818

Investigation of the dynamics of aqueous proline solutions using neutron scattering and molecular dynamics simulations.

PubMed

Malo de Molina, Paula; Alvarez, Fernando; Frick, Bernhard; Wildes, Andrew; Arbe, Arantxa; Colmenero, Juan

2017-10-18

We applied quasielastic neutron scattering (QENS) techniques to samples with two different contrasts (deuterated solute/hydrogenated solvent and the opposite label) to selectively study the component dynamics of proline/water solutions. Results on diluted and concentrated solutions (31 and 6 water molecules/proline molecule, respectively) were analyzed in terms of the susceptibility and considering a recently proposed model for water dynamics [Arbe et al., Phys. Rev. Lett., 2016, 117, 185501] which includes vibrations and the convolution of localized motions and diffusion. We found that proline molecules not only reduce the average diffusion coefficient of water but also extend the time/frequency range of the crossover region ('cage') between the vibrations and purely diffusive behavior. For the high proline concentration we also found experimental evidence of water heterogeneous dynamics and a distribution of diffusion coefficients. Complementary molecular dynamics simulations show that water molecules start to perform rotational diffusion when they escape the cage regime but before the purely diffusive behavior is established. The rotational diffusion regime is also retarded by the presence of proline molecules. On the other hand, a strong coupling between proline and water diffusive dynamics which persists with decreasing temperature is directly observed using QENS. Not only are the temperature dependences of the diffusion coefficients of both components the same, but their absolute values also approach each other with increasing proline concentration. We compared our results with those reported using other techniques, in particular using dielectric spectroscopy (DS). A simple approach based on molecular hydrodynamics and a molecular treatment of DS allows rationalizing the a priori puzzling inconsistency between QENS and dielectric results regarding the dynamic coupling of the two components. The interpretation proposed is based on general grounds and therefore should be applicable to other biomolecular solutions.

Sinorhizobium meliloti Chemoreceptor McpU Mediates Chemotaxis toward Host Plant Exudates through Direct Proline Sensing

PubMed Central

Webb, Benjamin A.; Hildreth, Sherry; Helm, Richard F.

2014-01-01

Bacterial chemotaxis is an important attribute that aids in establishing symbiosis between rhizobia and their legume hosts. Plant roots and seeds exude a spectrum of molecules into the soil to attract their bacterial symbionts. The alfalfa symbiont Sinorhizobium meliloti possesses eight chemoreceptors to sense its environment and mediate chemotaxis toward its host. The methyl accepting chemotaxis protein McpU is one of the more abundant S. meliloti chemoreceptors and an important sensor for the potent attractant proline. We established a dominant role of McpU in sensing molecules exuded by alfalfa seeds. Mass spectrometry analysis determined that a single germinating seed exudes 3.72 nmol of proline, producing a millimolar concentration near the seed surface which can be detected by the chemosensory system of S. meliloti. Complementation analysis of the mcpU deletion strain verified McpU as the key proline sensor. A structure-based homology search identified tandem Cache (calcium channels and chemotaxis receptors) domains in the periplasmic region of McpU. Conserved residues Asp-155 and Asp-182 of the N-terminal Cache domain were determined to be important for proline sensing by evaluating mutant strains in capillary and swim plate assays. Differential scanning fluorimetry revealed interaction of the isolated periplasmic region of McpU (McpU40-284) with proline and the importance of Asp-182 in this interaction. Using isothermal titration calorimetry, we determined that proline binds with a Kd (dissociation constant) of 104 μM to McpU40-284, while binding was abolished when Asp-182 was substituted by Glu. Our results show that McpU is mediating chemotaxis toward host plants by direct proline sensing. PMID:24657863

Force-dependent isomerization kinetics of a highly conserved proline switch modulates the mechanosensing region of filamin

PubMed Central

Rognoni, Lorenz; Möst, Tobias; Žoldák, Gabriel; Rief, Matthias

2014-01-01

Proline switches, controlled by cis–trans isomerization, have emerged as a particularly effective regulatory mechanism in a wide range of biological processes. In this study, we use single-molecule mechanical measurements to develop a full kinetic and energetic description of a highly conserved proline switch in the force-sensing domain 20 of human filamin and how prolyl isomerization modulates the force-sensing mechanism. Proline isomerization toggles domain 20 between two conformations. A stable cis conformation with slow unfolding, favoring the autoinhibited closed conformation of filamin’s force-sensing domain pair 20–21, and a less stable, uninhibited conformation promoted by the trans form. The data provide detailed insight into the folding mechanisms that underpin the functionality of this binary switch and elucidate its remarkable efficiency in modulating force-sensing, thus combining two previously unconnected regulatory mechanisms, proline switches and mechanosensing. PMID:24706888

Trans−cis Switching Mechanisms in Proline Analogues and Their Relevance for the Gating of the 5-HT3 Receptor

PubMed Central

2009-01-01

Trans−cis isomerization of a proline peptide bond is a potential mechanism to open the channel of the 5-HT3 receptor. Here, we have used the metadynamics method to theoretically explore such a mechanism. We have determined the free energy surfaces in aqueous solution of a series of dipeptides of proline analogues and evaluated the free energy difference between the cis and trans isomers. These theoretical results were then compared with data from mutagenesis experiments, in which the response of the 5-HT3 receptor was measured when the proline at the apex of the M2-M3 transmembrane domain loop was mutated. The strong correlation between the experimental and the theoretical data supports the existence of a trans−cis proline switch for opening the 5-HT3 receptor ion channel. PMID:19663504

Trans-cis switching mechanisms in proline analogues and their relevance for the gating of the 5-HT3 receptor.

PubMed

Melis, Claudio; Bussi, Giovanni; Lummis, Sarah C R; Molteni, Carla

2009-09-03

Trans-cis isomerization of a proline peptide bond is a potential mechanism to open the channel of the 5-HT(3) receptor. Here, we have used the metadynamics method to theoretically explore such a mechanism. We have determined the free energy surfaces in aqueous solution of a series of dipeptides of proline analogues and evaluated the free energy difference between the cis and trans isomers. These theoretical results were then compared with data from mutagenesis experiments, in which the response of the 5-HT(3) receptor was measured when the proline at the apex of the M2-M3 transmembrane domain loop was mutated. The strong correlation between the experimental and the theoretical data supports the existence of a trans-cis proline switch for opening the 5-HT(3) receptor ion channel.

l-Proline, GABA Synthesis and Gamma Oscillations in Schizophrenia.

PubMed

Volk, David W; Gonzalez-Burgos, Guillermo; Lewis, David A

2016-12-01

Altered inhibition from parvalbumin-containing GABA neurons is thought to contribute to impaired gamma frequency oscillations and cognitive deficits in schizophrenia. Crabtree and colleagues report that proline dehydrogenase deficits produce excessive cytosolic levels of the GABA-mimetic l-proline which impairs GABA synthesis and gamma oscillations in a manner that mimics schizophrenia. Copyright © 2016 Elsevier Ltd. All rights reserved.

Expression and substrate specificity of betaine/proline transporters suggest a novel choline transport mechanism in sugar beet.

PubMed

Yamada, Nana; Sakakibara, Shota; Tsutsumi, Koichi; Waditee, Rungaroon; Tanaka, Yoshito; Takabe, Teruhiro

2011-09-15

Proline transporters (ProTs) originally described as highly selective transporters for proline, have been shown to also transport glycinebetaine (betaine). Here we examined and compared the transport properties of Bet/ProTs from betaine accumulating (sugar beet, Amaranthus, and Atriplex,) and non-accumulating (Arabidopsis) plants. Using a yeast mutant deficient for uptake of proline and betaine, it was shown that all these transporters exhibited higher affinity for betaine than proline. The uptake of betaine and proline was pH-dependent and inhibited by the proton uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP). We also investigated choline transport by using a choline transport-deficient yeast mutant. Results revealed that these transporters exhibited a higher affinity for choline uptake rather than betaine. Uptake of choline by sugar beet BvBet/ProT1 was independent of the proton gradient and the inhibition by CCCP was reduced compared with that for uptake of betaine, suggesting different proton binding properties between the transport of choline and betaine. Additionally, in situ hybridization experiments revealed the localization of sugar beet BvBet/ProT1 in phloem and xylem parenchyma cells. Copyright © 2011 Elsevier GmbH. All rights reserved.

Theoretical and NMR conformational studies of β-proline oligopeptides with alternating chirality of pyrrolidine units

NASA Astrophysics Data System (ADS)

Mantsyzov, Alexey B.; Savelyev, Oleg Y.; Ivantcova, Polina M.; Bräse, Stefan; Kudryavtsev, Konstantin V.; Polshakov, Vladimir I.

2018-03-01

Synthetic β-peptides are potential functional mimetics of native α-proteins. A recently developed, novel, synthetic approach provides an effective route to the broad group of β-proline oligomers with alternating patterns of stereogenic centers. Conformation of the pyrrolidine ring, Z/E isomerism of β-peptide bonds, and hindered rotation of the neighboring monomers determine the spatial structure of this group of β-proline oligopeptides. Preferences in structural organization and corresponding thermodynamic properties are determined by NMR spectroscopy, restrained molecular dynamics and quantum mechanics. The studied β-proline oligopeptides exist in dimethyl sulfoxide solution in a limited number of conformers, with compatible energy of formation and different spatial organization. In the β-proline tetrapeptide with alternating chirality of composing pyrrolidine units, one of three peptide bonds may exist in an E configuration. For the alternating β-proline pentapeptide, the presence of an E configuration for at least of one β-peptide bond is mandatory. In this case, three peptide bonds synchronously change their configurations. Larger polypeptides may only exist in the presence of several E configurations of β-peptide bonds forming a wave-like extended structure.

Theoretical and NMR Conformational Studies of β-Proline Oligopeptides With Alternating Chirality of Pyrrolidine Units.

PubMed

Mantsyzov, Alexey B; Savelyev, Oleg Y; Ivantcova, Polina M; Bräse, Stefan; Kudryavtsev, Konstantin V; Polshakov, Vladimir I

2018-01-01

Synthetic β-peptides are potential functional mimetics of native α-proteins. A recently developed, novel, synthetic approach provides an effective route to the broad group of β-proline oligomers with alternating patterns of stereogenic centers. Conformation of the pyrrolidine ring, Z / E isomerism of β-peptide bonds, and hindered rotation of the neighboring monomers determine the spatial structure of this group of β-proline oligopeptides. Preferences in their structural organization and corresponding thermodynamic properties are determined by NMR spectroscopy, restrained molecular dynamics and quantum mechanics. The studied β-proline oligopeptides exist in dimethyl sulfoxide solution in a limited number of conformers, with compatible energy of formation and different spatial organization. In the β-proline tetrapeptide with alternating chirality of composing pyrrolidine units, one of three peptide bonds may exist in an E configuration. For the alternating β-proline pentapeptide, the presence of an E configuration for at least of one β-peptide bond is mandatory. In this case, three peptide bonds synchronously change their configurations. Larger polypeptides may only exist in the presence of several E configurations of β-peptide bonds forming a wave-like extended structure.

Structure of the cyclic peptide [W8S]contryphan Vn: effect of the tryptophan/serine substitution on trans-cis proline isomerization.

PubMed

Nepravishta, Ridvan; Mandaliti, Walter; Melino, Sonia; Eliseo, Tommaso; Paci, Maurizio

2014-12-01

The structural characterization of [W8S]contryphan Vn, an analogue of Contryphan Vn with tryptophan 8 substituted with a serine residue (W8S), was performed by NMR spectroscopy, molecular dynamics simulations and fluorescence spectroscopy. Contryphan Vn, a bioactive cyclic peptide from the venom of the cone snail Conus ventricosus, contains an S-S bridge between two cysteines and a D-tryptophan. Like other Contryphans, [W8S]contryphan Vn has proline 7 isomerized trans, while the proline 4 has nearly equivalent populations of cis and trans configurations. The thermodynamic and kinetic parameters of the trans-cis isomerization of proline 4 were measured. The isomers of [W8S]contryphan Vn with proline 4 in cis and trans show structural differences. The absence of the salt bridge between the same Asp2 and Lys6, present in Contryphan Vn, may be attributed to the lack of the hydrophobic side chain of Trp8 where it likely protects the electrostatic interactions. These results may contribute to identifying, in these cyclic peptides, the structural determinants of the mechanism of proline trans-cis isomerization, this being also an important step in protein folding.

Pneumocandin biosynthesis: involvement of a trans-selective proline hydroxylase.

PubMed

Houwaart, Stefanie; Youssar, Loubna; Hüttel, Wolfgang

2014-11-03

Echinocandins are cyclic nonribosomal hexapeptides based mostly on nonproteinogenic amino acids and displaying strong antifungal activity. Despite previous studies on their biosynthesis by fungi, the origin of three amino acids, trans-4- and trans-3-hydroxyproline, as well as trans-3-hydroxy-4-methylproline, is still unknown. Here we describe the identification, overexpression, and characterization of GloF, the first eukaryotic α-ketoglutarate/Fe(II) -dependent proline hydroxylase from the pneumocandin biosynthesis cluster of the fungus Glarea lozoyensis ATCC 74030. In in vitro transformations with L-proline, GloF generates trans-4- and trans-3-hydroxyproline simultaneously in a ratio of 8:1; the latter reaction was previously unknown for proline hydroxylase catalysis. trans-4-Methyl-L-proline is converted into the corresponding trans-3-hydroxyproline. All three hydroxyprolines required for the biosynthesis of the echinocandins pneumocandins A0 and B0 in G. lozoyensis are thus provided by GloF. Sequence analyses revealed that GloF is not related to bacterial proline hydroxylases, and none of the putative proteins with high sequence similarity in the databases has been characterized so far. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Towards Understanding the Tandem Mass Spectra of Protonated Oligopeptides. 2: The Proline Effect in Collision-Induced Dissociation of Protonated Ala-Ala-Xxx-Pro-Ala (Xxx = Ala, Ser, Leu, Val, Phe, and Trp)

NASA Astrophysics Data System (ADS)

Bleiholder, Christian; Suhai, Sándor; Harrison, Alex G.; Paizs, Béla

2011-06-01

The product ion spectra of proline-containing peptides are commonly dominated by y n ions generated by cleavage at the N-terminal side of proline residues. This proline effect is investigated in the current work by collision-induced dissociation (CID) of protonated Ala-Ala-Xxx-Pro-Ala (Xxx includes Ala, Ser, Leu, Val, Phe, and Trp) in an electrospray/quadrupole/time-of-flight (QqTOF) mass spectrometer and by quantum chemical calculations on protonated Ala-Ala-Ala-Pro-Ala. The CID spectra of all investigated peptides show a dominant y 2 ion (Pro-Ala sequence). Our computational results show that the proline effect mainly arises from the particularly low threshold energy for the amide bond cleavage N-terminal to the proline residue, and from the high proton affinity of the proline-containing C-terminal fragment produced by this cleavage. These theoretical results are qualitatively supported by the experimentally observed y 2 / b 3 abundance ratios for protonated Ala-Ala-Xxx-Pro-Ala (Xxx = Ala, Ser, Leu, Val, Phe, and Trp). In the post-cleavage phase of fragmentation the N-terminal oxazolone fragment with the Ala-Ala-Xxx sequence and Pro-Ala compete for the ionizing proton for these peptides. As the proton affinity of the oxazolone fragment increases, the y 2 / b 3 abundance ratio decreases.

Towards understanding the tandem mass spectra of protonated oligopeptides. 2: The proline effect in collision-induced dissociation of protonated Ala-Ala-Xxx-Pro-Ala (Xxx = Ala, Ser, Leu, Val, Phe, and Trp).

PubMed

Bleiholder, Christian; Suhai, Sándor; Harrison, Alex G; Paizs, Béla

2011-06-01

The product ion spectra of proline-containing peptides are commonly dominated by y(n) ions generated by cleavage at the N-terminal side of proline residues. This proline effect is investigated in the current work by collision-induced dissociation (CID) of protonated Ala-Ala-Xxx-Pro-Ala (Xxx includes Ala, Ser, Leu, Val, Phe, and Trp) in an electrospray/quadrupole/time-of-flight (QqTOF) mass spectrometer and by quantum chemical calculations on protonated Ala-Ala-Ala-Pro-Ala. The CID spectra of all investigated peptides show a dominant y(2) ion (Pro-Ala sequence). Our computational results show that the proline effect mainly arises from the particularly low threshold energy for the amide bond cleavage N-terminal to the proline residue, and from the high proton affinity of the proline-containing C-terminal fragment produced by this cleavage. These theoretical results are qualitatively supported by the experimentally observed y(2)/b(3) abundance ratios for protonated Ala-Ala-Xxx-Pro-Ala (Xxx = Ala, Ser, Leu, Val, Phe, and Trp). In the post-cleavage phase of fragmentation the N-terminal oxazolone fragment with the Ala-Ala-Xxx sequence and Pro-Ala compete for the ionizing proton for these peptides. As the proton affinity of the oxazolone fragment increases, the y(2)/b(3) abundance ratio decreases.

Oxidation of Proline by Mitochondria Isolated from Water-Stressed Maize Shoots 1

PubMed Central

Sells, Gary D.; Koeppe, David E.

1981-01-01

Proline oxidation and coupled phosphorylation were measured in mitochondria after isolation from shoots of water-stressed, etiolated maize (Zea mays L.) seedlings. Both state III and state IV rates of proline oxidation decreased as a logarithmic function of increased seedling water stress between −5 and −10 bars. Proline oxidation rates decreased 62% (state III) and 58% (state IV) as seedling water potentials were decreased from −5 to −10 bars. By comparison, oxidation of succinate, exogenous NADH, or malate + pyruvate decreased only 10 to 15% in this stress range. These decreases were a linear function of increased stress and were comparable to oxidation rates of mitochondria subjected to varying in vitro osmotic potentials. Osmotically induced in vitro stress reduced proline oxidation rates linearly with more negative osmotic potentials, a decrease that was similar to the responses of the other substrates to more negative osmotic potentials. Some decrease in coupling, with all substrates as determined by ADP/O ratios, was observed under osmotic stress. Mitochondria were also isolated from shoot tissue that had been stressed and then rewatered. On a percentage basis, the recovery of proline oxidation was greater than that of the other substrates. The decreases in the proline oxidase activity of mitochondria after only slight stress indicate a mitochondrial sensitivity to water stress at significantly less negative water potentials than previously reported for measurements of maize membrane permeability and respiratory activity. PMID:16662051

Influence of proline upon the folding and geometry of the WALP19 transmembrane peptide.

PubMed

Thomas, Rachel; Vostrikov, Vitaly V; Greathouse, Denise V; Koeppe, Roger E

2009-12-22

The orientations, geometries, and lipid interactions of designed transmembrane (TM) peptides have attracted significant experimental and theoretical interest. Because the amino acid proline will introduce a known discontinuity into an alpha helix, we have sought to measure the extent of helix kinking caused by a single proline within the isolated TM helical domain of WALP19. For this purpose, we synthesized acetyl-GWWLALALAP(10)ALALALWWA-ethanolamide and included pairs of deuterated alanines by using 60-100% Fmoc-l-Ala-d(4) at selected sequence positions. Solid-state deuterium ((2)H) magnetic resonance spectra from oriented, hydrated samples (1/40, peptide/lipid; using several lipids) reveal signals from many of the alanine backbone C(alpha) deuterons as well as the alanine side-chain C(beta) methyl groups, whereas signals from C(alpha) deuterons generally have not been observed for similar peptides without proline. It is conceivable that altered peptide dynamics may be responsible for the apparent "unmasking" of the backbone resonances in the presence of the proline. Data analysis using the geometric analysis of labeled alanines (GALA) method reveals that the peptide helix is distorted due to the presence of the proline. To provide additional data points for evaluating the segmental tilt angles of the two halves of the peptide, we substituted selected leucines with l-Ala-d(4). Using this approach, we were able to deduce that the apparent average tilt of the C-terminal increases from approximately 4 degrees to approximately 12 degrees when Pro(10) is introduced. The segment N-terminal to proline is more complex and possibly is more dynamically flexible; Leu to Ala mutations within the N-terminal segment alter the average orientations of alanines in both segments. Nevertheless, in DOPC, we could estimate an apparent kink angle of approximately 19 degrees . Together, the results suggest that the central proline influences not only the geometry but also the dynamics of the membrane-spanning peptide. The results make up an important basis for understanding the functional role of proline in several families of membrane proteins.

Proline Residues as Switches in Conformational Changes Leading to Amyloid Fibril Formation

PubMed Central

Taler-Verčič, Ajda; Hasanbašić, Samra; Berbić, Selma; Stoka, Veronika; Turk, Dušan; Žerovnik, Eva

2017-01-01

Here we discuss studies of the structure, folding, oligomerization and amyloid fibril formation of several proline mutants of human stefin B, which is a protein inhibitor of lysosomal cysteine cathepsins and a member of the cystatin family. The structurally important prolines in stefin B are responsible for the slow folding phases and facilitate domain swapping (Pro 74) and loop swapping (Pro 79). Moreover, our findings are compared to β2-microglobulin, a protein involved in dialysis-related amyloidosis. The assessment of the contribution of proline residues to the process of amyloid fibril formation may shed new light on the critical molecular events involved in conformational disorders. PMID:28272335

Synteny between the Pro/sup +/ marker and human glutamate oxaloacetate transaminase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jones, C.

1975-01-01

Chinese hamster ovary cells with a specific auxotrophy for proline were fused with human cells from a variety of sources and the resulting hybrids analyzed for human genetic markers. Of 63 hybrid clones examined, 27 possessed both proline and cytoplasmic glutamate oxaloacetate transaminase markers; 36 had neither; and no clones were found possessing one and not the other. These results constitute evidence that the proline and glutamate oxaloacetate transaminase markers are syntenic. Evidence for absence of synteny between these and a variety of other human genes is presented. Biochemical tracer experiments established that the proline biosynthetic pathway through glutamate hasmore » been restored in the Pro/sup +/ hybrids.« less

Identification of two proline transport systems in Staphylococcus aureus and their possible roles in osmoregulation.

PubMed

Bae, J H; Miller, K J

1992-02-01

The food-borne pathogen Staphylococcus aureus is distinguished from other food-borne pathogens by its ability to grow at water activity values below 0.90. Previous studies have indicated that proline accumulation mediated by transport represents a primary osmoregulatory strategy utilized by this bacterium (C. B. Anderson and L. D. Witter, Appl. Environ, Microbiol. 43:1501-1503, 1982; I. Koujima, H. Hayashi, K. Tomochika, A. Okabe, and Y. Kanemasa, Appl. Environ. Microbiol. 35:467-470, 1978; K. J. Miller, S. C. Zelt, and J.-H. Bae, Curr. Microbiol. 23:131-137, 1991). In this study, we demonstrate the presence of two proline transport systems within whole cells of S. aureus, a high-affinity transport system (Km, 7 microM) and a low-affinity transport system (Km, 420 microM). Our results indicate that the low-affinity proline transport system is osmotically activated and is the primary system responsible for the accumulation of proline by this pathogen during growth at low water activity.

A Simplified Model of Local Structure in Aqueous Proline Amino Acid Revealed by First-Principles Molecular Dynamics Simulations

PubMed Central

Troitzsch, Raphael Z.; Tulip, Paul R.; Crain, Jason; Martyna, Glenn J.

2008-01-01

Aqueous proline solutions are deceptively simple as they can take on complex roles such as protein chaperones, cryoprotectants, and hydrotropic agents in biological processes. Here, a molecular level picture of proline/water mixtures is developed. Car-Parrinello ab initio molecular dynamics (CPAIMD) simulations of aqueous proline amino acid at the B-LYP level of theory, performed using IBM's Blue Gene/L supercomputer and massively parallel software, reveal hydrogen-bonding propensities that are at odds with the predictions of the CHARMM22 empirical force field but are in better agreement with results of recent neutron diffraction experiments. In general, the CPAIMD (B-LYP) simulations predict a simplified structural model of proline/water mixtures consisting of fewer distinct local motifs. Comparisons of simulation results to experiment are made by direct evaluation of the neutron static structure factor S(Q) from CPAIMD (B-LYP) trajectories as well as to the results of the empirical potential structure refinement reverse Monte Carlo procedure applied to the neutron data. PMID:18790850

Electron-Transfer Dynamics for a Donor-Bridge-Acceptor Complex in Ionic Liquids.

PubMed

DeVine, Jessalyn A; Labib, Marena; Harries, Megan E; Rached, Rouba Abdel Malak; Issa, Joseph; Wishart, James F; Castner, Edward W

2015-08-27

Intramolecular photoinduced electron transfer from an N,N-dimethyl-p-phenylenediamine donor bridged by a diproline spacer to a coumarin 343 acceptor was studied using time-resolved fluorescence measurements in three ionic liquids and in acetonitrile. The three ionic liquids have the bis[(trifluoromethyl)sulfonyl]amide anion paired with the tributylmethylammonium, 1-butyl-1-methylpyrrolidinium, and 1-decyl-1-methylpyrrolidinium cations. The dynamics in the two-proline donor-bridge-acceptor complex are compared to those observed for the same donor and acceptor connected by a single proline bridge, studied previously by Lee et al. (J. Phys. Chem. C 2012, 116, 5197). The increased conformational freedom afforded by the second bridging proline resulted in multiple energetically accessible conformations. The multiple conformations have significant variations in donor-acceptor electronic coupling, leading to dynamics that include both adiabatic and nonadiabatic contributions. In common with the single-proline bridged complex, the intramolecular electron transfer in the two-proline system was found to be in the Marcus inverted regime.

A simplified model of local structure in aqueous proline amino acid revealed by first-principles molecular dynamics simulations.

PubMed

Troitzsch, Raphael Z; Tulip, Paul R; Crain, Jason; Martyna, Glenn J

2008-12-01

Aqueous proline solutions are deceptively simple as they can take on complex roles such as protein chaperones, cryoprotectants, and hydrotropic agents in biological processes. Here, a molecular level picture of proline/water mixtures is developed. Car-Parrinello ab initio molecular dynamics (CPAIMD) simulations of aqueous proline amino acid at the B-LYP level of theory, performed using IBM's Blue Gene/L supercomputer and massively parallel software, reveal hydrogen-bonding propensities that are at odds with the predictions of the CHARMM22 empirical force field but are in better agreement with results of recent neutron diffraction experiments. In general, the CPAIMD (B-LYP) simulations predict a simplified structural model of proline/water mixtures consisting of fewer distinct local motifs. Comparisons of simulation results to experiment are made by direct evaluation of the neutron static structure factor S(Q) from CPAIMD (B-LYP) trajectories as well as to the results of the empirical potential structure refinement reverse Monte Carlo procedure applied to the neutron data.

Purification and Characterization of Put1p from Saccharomyces cerevisiae

PubMed Central

Wanduragala, Srimevan; Sanyal, Nikhilesh; Liang, Xinwen; Becker, Donald F.

2010-01-01

In Saccharomyces cerevisiae, the PUT1 and PUT2 genes are required for the conversion of proline to glutamate. The PUT1 gene encodes Put1p, a proline dehydrogenase (PRODH)1 enzyme localized in the mitochondrion. Put1p was expressed and purified from Escherichia coli and shown to have a UV-visible absorption spectrum that is typical of a bound flavin cofactor. A Km value of 36 mM proline and a kcat = 27 s−1 were determined for Put1p using an artificial electron acceptor. Put1p also exhibited high activity using ubiquinone-1 (CoQ1) as an electron acceptor with a kcat = 9.6 s−1 and a Km of 33 µM for CoQ1. In addition, knockout strains of the electron transfer flavoprotein (ETF) homolog in S. cerevisiae were able to grow on proline as the sole nitrogen source demonstrating that ETF is not required for proline utilization in yeast. PMID:20450881

A new charge-tagged proline-based organocatalyst for mechanistic studies using electrospray mass spectrometry

PubMed Central

Willms, J Alexander; Beel, Rita; Schmidt, Martin L; Mundt, Christian

2014-01-01

Summary A new 4-hydroxy-L-proline derivative with a charged 1-ethylpyridinium-4-phenoxy substituent has been synthesized with the aim of facilitating mechanistic studies of proline-catalyzed reactions by ESI mass spectrometry. The charged residue ensures a strongly enhanced ESI response compared to neutral unmodified proline. The connection by a rigid linker fixes the position of the charge tag far away from the catalytic center in order to avoid unwanted interactions. The use of a charged catalyst leads to significantly enhanced ESI signal abundances for every catalyst-derived species which are the ones of highest interest present in a reacting solution. The new charged proline catalyst has been tested in the direct asymmetric inverse aldol reaction between aldehydes and diethyl ketomalonate. Two intermediates in accordance with the List–Houk mechanism for enamine catalysis have been detected and characterized by gas-phase fragmentation. In addition, their temporal evolution has been followed using a microreactor continuous-flow technique. PMID:25246962

Modulating the Intrinsic Disorder in the Cytoplasmic Domain Alters the Biological Activity of the N-Methyl-d-aspartate-sensitive Glutamate Receptor*

PubMed Central

Choi, Ucheor B.; Kazi, Rashek; Stenzoski, Natalie; Wollmuth, Lonnie P.; Uversky, Vladimir N.; Bowen, Mark E.

2013-01-01

The NMDA-sensitive glutamate receptor is a ligand-gated ion channel that mediates excitatory synaptic transmission in the nervous system. Extracellular zinc allosterically regulates the NMDA receptor by binding to the extracellular N-terminal domain, which inhibits channel gating. Phosphorylation of the intrinsically disordered intracellular C-terminal domain alleviates inhibition by extracellular zinc. The mechanism for this functional effect is largely unknown. Proline is a hallmark of intrinsic disorder, so we used proline mutagenesis to modulate disorder in the cytoplasmic domain. Proline depletion selectively uncoupled zinc inhibition with little effect on receptor biogenesis, surface trafficking, or ligand-activated gating. Proline depletion also reduced the affinity for a PDZ domain involved in synaptic trafficking and affected small molecule binding. To understand the origin of these phenomena, we used single molecule fluorescence and ensemble biophysical methods to characterize the structural effects of proline mutagenesis. Proline depletion did not eliminate intrinsic disorder, but the underlying conformational dynamics were changed. Thus, we altered the form of intrinsic disorder, which appears sufficient to affect the biological activity. These findings suggest that conformational dynamics within the intrinsically disordered cytoplasmic domain are important for the allosteric regulation of NMDA receptor gating. PMID:23782697

Proline biosynthesis genes and their regulation under salinity stress in the euryhaline copepod Tigriopus californicus.

PubMed

Willett, Christopher S; Burton, Ronald S

2002-08-01

Diverse organisms regulate concentrations of intracellular organic osmolytes in response to changes in environmental salinity or desiccation. In marine crustaceans, accumulation of high concentrations of proline is a dominant component of response to hyperosmotic stress. In the euryhaline copepod Tigriopus californicus, synthesis of proline from its metabolic precursor glutamate is tightly regulated by changes in environmental salinity. Here, for the first time in a marine invertebrate, the genes responsible for this pathway have been cloned and characterized. The two proteins display the sequence features of homologous enzymes identified from other eukaryotes. One of the cloned genes, delta1-pyrroline-5-carboxylase reductase (P5CR), is demonstrated to have the reductase enzyme activity when expressed in proline-auxotroph bacteria, while the second, delta1-pyrroline-5-carboxylase synthase (P5CS), does not rescue proline-auxotroph bacteria. In contrast to results from higher plants, neither levels of P5CS nor P5CR mRNAs increase in response to salinity stress in T. californicus. Hence, regulation of proline synthesis during osmotic stress in T. californicus is likely mediated by some form of post-transcriptional regulation of either P5CS or P5CR. Understanding the regulation this pathway may elucidate the mechanisms limiting the salinity ranges of marine taxa. Copyright 2002 Elsevier Science Inc.

Interspecific differences in tannin intakes of forest-dwelling rodents in the wild revealed by a new method using fecal proline content.

PubMed

Shimada, Takuya; Nishii, Eriko; Saitoh, Takashi

2011-12-01

Mammalian herbivores adopt various countermeasures against dietary tannins, which are among the most widespread plant secondary metabolites. The large Japanese wood mouse Apodemus speciosus produces proline-rich salivary tannin-binding proteins in response to tannins. Proline-rich proteins (PRPs) react with tannins to form stable complexes that are excreted in the feces. Here, we developed a new method for estimating the tannin intake of free-living small rodents, by measuring fecal proline content, and applied the method to a field investigation. A feeding experiment with artificial diets containing various levels of tannic acid revealed that fecal proline content was clearly related to dietary tannin content in three species (A. speciosus, Apodemus argenteus, and Myodes rufocanus). We then used fecal proline content to estimate the tannin intakes of these three forest-dwelling species in a forest in Hokkaido. In the autumn, estimated tannin intakes increased significantly in the Apodemus species, but not in M. rufocanus. We speculated that an increase in tannin intake during autumn may result from consumption of tannin-rich acorns. This hypothesis was consistent with population fluctuation patterns of the three species, which were well-synchronized with acorn abundance for the Apodemus species but not for M. rufocanus.

Exogenous proline mediates alleviation of cadmium stress by promoting photosynthetic activity, water status and antioxidative enzymes activities of young date palm (Phoenix dactylifera L.).

PubMed

Zouari, M; Ben Ahmed, Ch; Zorrig, W; Elloumi, N; Rabhi, M; Delmail, D; Ben Rouina, B; Labrousse, P; Ben Abdallah, F

2016-06-01

The ability of exogenous compatible solutes, such as proline, to counteract cadmium (Cd) inhibitory effects in young date palm plants (Phoenix dactylifera L. cv Deglet Nour) was investigated. Two-year-old date palm plants were subjected for five months at different Cd stress levels (0, 10 and 30 mg CdCl2 kg(-1) soil) whether supplied or not with exogenous proline (20mM) added through the irrigation water. Different levels of Cd stress altered plant growth, gas exchanges and chlorophyll content as well as water status, but at different extent among them. In contrast, an increase of antioxidant enzymes activities of Cd-treated plants in association with high amounts of proline content, hydrogen peroxide (H2O2), thiobarbituric acid reactive substances (TBARS) and electrolyte leakage (EL) were observed. Interestingly, exogenous proline mitigated the adverse effects of Cd on young date palm. Indeed, it alleviated the oxidative damage induced by Cd accumulation and established better levels of plant growth, water status and photosynthetic activity. Moreover, proline-treated plants showed high antioxidant enzymes activities (superoxide dismutase, catalase and glutathione peroxydase) in roots and leaves as compared to Cd-treated plants. Copyright © 2016 Elsevier Inc. All rights reserved.

Proline oxidase promotes tumor cell survival in hypoxic tumor microenvironments

PubMed Central

Liu, Wei; Glunde, Kristine; Bhujwalla, Zaver M.; Raman, Venu; Sharma, Anit; Phang, James M.

2012-01-01

Proline is a readily released stress substrate that can be metabolized by proline oxidase (POX) to generate either reactive oxygen species to induce apoptosis or autophagy or ATP during times of nutrient stress. However, the contribution of proline metabolism to tumorigenesis in hypoxic microenvironments has not been explored. In this study, we investigated the different functions of POX under hypoxia and glucose depletion. We found that hypoxia induced POX expression in cancer cells in vitro and that POX upregulation co-localized with hypoxic tissues in vivo. In addition, the combination of hypoxia and low-glucose showed additive effects on POX expression. Similar to conditions of low glucose, hypoxia-mediated POX induction was dependent on AMP-activated protein kinase (AMPK) activation, but was independent of HIF-1α and HIF-2α. Under low-glucose and combined low-glucose and hypoxic conditions, proline catabolized by POX was used preferentially for ATP production, whereas under hypoxia, POX mediated autophagic signaling for survival by generating ROS. Although the specific mechanism was different for hypoxia and glucose deprivation, POX consistently contributed to tumor cell survival under these conditions. Together, our findings offer new insights into the metabolic reprogramming of tumor cells present within a hostile microenvironment and suggest that proline metabolism is a potential target for cancer therapeutics. PMID:22609800

Variation in relative water content, proline accumulation and stress gene expression in two cowpea landraces under drought.

PubMed

Zegaoui, Zahia; Planchais, Séverine; Cabassa, Cécile; Djebbar, Reda; Abrous Belbachir, Ouzna; Carol, Pierre

2017-11-01

Many landraces of cowpea [Vigna unguiculata (L.) Walp.] are adapted to particular geographical and climatic conditions. Here we describe two landraces grown respectively in arid and temperate areas of Algeria and assess their physiological and molecular responses to drought stress. As expected, when deprived of water cowpea plants lose water over time with a gradual reduction in transpiration rate. The landraces differed in their relative water content (RWC) and whole plant transpiration rate. The landrace from Menia, an arid area, retained more water in adult leaves. Both landraces responded to drought stress at the molecular level by increasing expression of stress-related genes in aerial parts, including proline metabolism genes. Expression of gene(s) encoding proline synthesis enzyme P5CS was up regulated and gene expression of ProDH, a proline catabolism enzyme, was down regulated. Relatively low amounts of proline accumulated in adult leaves with slight differences between the two landraces. During drought stress the most apical part of plants stayed relatively turgid with a high RWC compared to distal parts that wilted. Expression of key stress genes was higher and more proline accumulated at the apex than in distal leaves indicating that cowpea has a non-uniform stress response at the whole plant level. Our study reveals a developmental control of water stress through preferential proline accumulation in the upper tier of the cowpea plant. We also conclude that cowpea landraces display physiological adaptations to water stress suited to the arid and temperate climates in which they are cultivated. Copyright © 2017 Elsevier GmbH. All rights reserved.

Cold and desiccation stress induced changes in the accumulation and utilization of proline and trehalose in seasonal populations of Drosophila immigrans.

PubMed

Tamang, Aditya Moktan; Kalra, Bhawna; Parkash, Ravi

2017-01-01

Changes in the levels of energy metabolites can limit survival ability of Drosophila species under stressful conditions but this aspect has received less attention in wild populations collected in different seasons. We tested cold or desiccation triggered changes in the accumulation or utilization of two energy metabolites (trehalose and proline) in Drosophila immigrans flies reared under season specific environmental conditions. Such D.immigrans populations were subjected to different durations of cold (0°C) or desiccation stress (5% RH) or dual stress. We found stress induced effects of cold vs desiccation on the levels of trehalose as well as for proline. Different durations of cold stress led to accumulation of trehalose while desiccation stress durations revealed utilization of trehalose. In contrast, there was accumulation of proline under desiccation and utilization of proline with cold stress. Since accumulation levels were higher than utilization of each energy metabolite, the effects of dual stress showed additive effect. However, there was no utilization of total body lipids under cold or desiccation stress. We observed significant season specific differences in the amount of energy metabolites but the rate of metabolism did not vary across seasons. Stress triggered changes in trehalose and proline suggest possible link between desiccation and cold tolerance. Finally, stress specific (cold or desiccation) compensatory changes in the levels of trehalose and proline suggest possible energetic homeostasis in D.immigrans living under harsh climatic conditions of montane localities. Copyright © 2016 Elsevier Inc. All rights reserved.

Identification and Disruption of the proBA Locus in Listeria monocytogenes: Role of Proline Biosynthesis in Salt Tolerance and Murine Infection

PubMed Central

Sleator, Roy D.; Gahan, Cormac G. M.; Hill, Colin

2001-01-01

Intracellular accumulation of the amino acid proline has previously been linked to the salt tolerance and virulence potential of a number of bacteria. Taking advantage of the proBA mutant Escherichia coli CSH26, we identified a listerial proBA operon coding for enzymes functionally similar to the glutamyl kinase (GK) and glutamylphosphate reductase (GPR) enzyme complex which catalyzes the first and second steps of proline biosynthesis in E. coli. The first gene of the operon, proB, is predicted to encode GK, a 276-residue protein with a calculated molecular mass of 30.03 kDa and pl of 5.2. Distal to the promoter and overlapping the 3′ end of proB by 17 bp is proA, which encodes GPR, a 415-residue protein with a calculated molecular mass of 45.50 kDa (pl 5.3). Using this information, we created a chromosomal deletion mutant by allelic exchange which is auxotrophic for proline. This mutant was used to assess the contribution of proline anabolism to osmotolerance and virulence. While inactivation of proBA had no significant effect on virulence in mouse assays (either perorally or intraperitoneally), growth at low (2 to 4% NaCl) and high (>6% NaCl) salt concentrations in complex media was significantly reduced in the absence of efficient proline synthesis. We conclude that while proline biosynthesis plays little, if any, role in the intracellular life cycle and infectious nature of Listeria monocytogenes, it can play an important role in survival in osmolyte-depleted environments of elevated osmolarity. PMID:11375165

Engineering a trifunctional proline utilization A chimaera by fusing a DNA-binding domain to a bifunctional PutA.

PubMed

Arentson, Benjamin W; Hayes, Erin L; Zhu, Weidong; Singh, Harkewal; Tanner, John J; Becker, Donald F

2016-12-01

Proline utilization A (PutA) is a bifunctional flavoenzyme with proline dehydrogenase (PRODH) and Δ 1 -pyrroline-5-carboxylate (P5C) dehydrogenase (P5CDH) domains that catalyses the two-step oxidation of proline to glutamate. Trifunctional PutAs also have an N-terminal ribbon-helix-helix (RHH) DNA-binding domain and moonlight as autogenous transcriptional repressors of the put regulon. A unique property of trifunctional PutA is the ability to switch functions from DNA-bound repressor to membrane-associated enzyme in response to cellular nutritional needs and proline availability. In the present study, we attempt to construct a trifunctional PutA by fusing the RHH domain of Escherichia coli PutA (EcRHH) to the bifunctional Rhodobacter capsulatus PutA (RcPutA) in order to explore the modular design of functional switching in trifunctional PutAs. The EcRHH-RcPutA chimaera retains the catalytic properties of RcPutA while acquiring the oligomeric state, quaternary structure and DNA-binding properties of EcPutA. Furthermore, the EcRHH-RcPutA chimaera exhibits proline-induced lipid association, which is a fundamental characteristic of functional switching. Unexpectedly, RcPutA lipid binding is also activated by proline, which shows for the first time that bifunctional PutAs exhibit a limited form of functional switching. Altogether, these results suggest that the C-terminal domain (CTD), which is conserved by trifunctional PutAs and certain bifunctional PutAs, is essential for functional switching in trifunctional PutAs. © 2016 The Author(s).

Engineering a trifunctional proline utilization A chimaera by fusing a DNA-binding domain to a bifunctional PutA

PubMed Central

Arentson, Benjamin W.; Hayes, Erin L.; Zhu, Weidong; Singh, Harkewal; Tanner, John J.; Becker, Donald F.

2016-01-01

Proline utilization A (PutA) is a bifunctional flavoenzyme with proline dehydrogenase (PRODH) and Δ1-pyrroline-5-carboxylate (P5C) dehydrogenase (P5CDH) domains that catalyses the two-step oxidation of proline to glutamate. Trifunctional PutAs also have an N-terminal ribbon–helix–helix (RHH) DNA-binding domain and moonlight as autogenous transcriptional repressors of the put regulon. A unique property of trifunctional PutA is the ability to switch functions from DNA-bound repressor to membrane-associated enzyme in response to cellular nutritional needs and proline availability. In the present study, we attempt to construct a trifunctional PutA by fusing the RHH domain of Escherichia coli PutA (EcRHH) to the bifunctional Rhodobacter capsulatus PutA (RcPutA) in order to explore the modular design of functional switching in trifunctional PutAs. The EcRHH–RcPutA chimaera retains the catalytic properties of RcPutA while acquiring the oligomeric state, quaternary structure and DNA-binding properties of EcPutA. Furthermore, the EcRHH–RcPutA chimaera exhibits proline-induced lipid association, which is a fundamental characteristic of functional switching. Unexpectedly, RcPutA lipid binding is also activated by proline, which shows for the first time that bifunctional PutAs exhibit a limited form of functional switching. Altogether, these results suggest that the C-terminal domain (CTD), which is conserved by trifunctional PutAs and certain bifunctional PutAs, is essential for functional switching in trifunctional PutAs. PMID:27742866

Variation in Protein Intake Induces Variation in Spider Silk Expression

PubMed Central

Blamires, Sean J.; Wu, Chun-Lin; Tso, I-Min

2012-01-01

Background It is energetically expensive to synthesize certain amino acids. The proteins (spidroins) of spider major ampullate (MA) silk, MaSp1 and MaSp2, differ in amino acid composition. Glutamine and proline are prevalent in MaSp2 and are expensive to synthesize. Since most orb web spiders express high proline silk they might preferentially attain the amino acids needed for silk from food and shift toward expressing more MaSp1 in their MA silk when starved. Methodology/Principal Findings We fed three spiders; Argiope aetherea, Cyrtophora moluccensis and Leucauge blanda, high protein, low protein or no protein solutions. A. aetherea and L. blanda MA silks are high in proline, while C. moluccesnsis MA silks are low in proline. After 10 days of feeding we determined the amino acid compositions and mechanical properties of each species' MA silk and compared them between species and treatments with pre-treatment samples, accounting for ancestry. We found that the proline and glutamine of A. aetherea and L. blanda silks were affected by protein intake; significantly decreasing under the low and no protein intake treatments. Glutmaine composition in C. moluccensis silk was likewise affected by protein intake. However, the composition of proline in their MA silk was not significantly affected by protein intake. Conclusions Our results suggest that protein limitation induces a shift toward different silk proteins with lower glutamine and/or proline content. Contradictions to the MaSp model lie in the findings that C. moluccensis MA silks did not experience a significant reduction in proline and A. aetherea did not experience a significant reduction in serine on low/no protein. The mechanical properties of the silks could not be explained by a MaSp1 expressional shift. Factors other than MaSp expression, such as the expression of spidroin-like orthologues, may impact on silk amino acid composition and spinning and glandular processes may impact mechanics. PMID:22363691

Process optimization for enhancing production of cis-4-hydroxy-L-proline by engineered Escherichia coli.

PubMed

Chen, Kequan; Pang, Yang; Zhang, Bowen; Feng, Jiao; Xu, Sheng; Wang, Xin; Ouyang, Pingkai

2017-11-22

Understanding the bioprocess limitations is critical for the efficient design of biocatalysts to facilitate process feasibility and improve process economics. In this study, a proline hydroxylation process with recombinant Escherichia coli expressing L-proline cis-4-hydroxylase (SmP4H) was investigated. The factors that influencing the metabolism of microbial hosts and process economics were focused on for the optimization of cis-4-hydroxy-L-proline (CHOP) production. In recombinant E. coli, SmP4H synthesis limitation was observed. After the optimization of expression system, CHOP production was improved in accordance with the enhanced SmP4H synthesis. Furthermore, the effects of the regulation of proline uptake and metabolism on whole-cell catalytic activity were investigated. The improved CHOP production by repressing putA gene responsible for L-proline degradation or overexpressing L-proline transporter putP on CHOP production suggested the important role of substrate uptake and metabolism on the whole-cell biocatalyst efficiency. Through genetically modifying these factors, the biocatalyst activity was significantly improved, and CHOP production was increased by twofold. Meanwhile, to further improve process economics, a two-strain coupling whole-cell system was established to supply co-substrate (α-ketoglutarate, α-KG) with a cheaper chemical L-glutamate as a starting material, and 13.5 g/L of CHOP was successfully produced. In this study, SmP4H expression, and L-proline uptake and degradation, were uncovered as the hurdles for microbial production of CHOP. Accordingly, the whole-cell biocatalysts were metabolically engineered for enhancing CHOP production. Meanwhile, a two-strain biotransformation system for CHOP biosynthesis was developed aiming at supplying α-KG more economically. Our work provided valuable insights into the design of recombinant microorganism to improve the biotransformation efficiency that catalyzed by Fe(II)/α-KG-dependent dioxygenase.

Adaptation of an L-proline adenylation domain to use 4-propyl-L-proline in the evolution of lincosamide biosynthesis.

PubMed

Kadlčík, Stanislav; Kučera, Tomáš; Chalupská, Dominika; Gažák, Radek; Koběrská, Markéta; Ulanová, Dana; Kopecký, Jan; Kutejová, Eva; Najmanová, Lucie; Janata, Jiří

2013-01-01

Clinically used lincosamide antibiotic lincomycin incorporates in its structure 4-propyl-L-proline (PPL), an unusual amino acid, while celesticetin, a less efficient related compound, makes use of proteinogenic L-proline. Biochemical characterization, as well as phylogenetic analysis and homology modelling combined with the molecular dynamics simulation were employed for complex comparative analysis of the orthologous protein pair LmbC and CcbC from the biosynthesis of lincomycin and celesticetin, respectively. The analysis proved the compared proteins to be the stand-alone adenylation domains strictly preferring their own natural substrate, PPL or L-proline. The LmbC substrate binding pocket is adapted to accommodate a rare PPL precursor. When compared with L-proline specific ones, several large amino acid residues were replaced by smaller ones opening a channel which allowed the alkyl side chain of PPL to be accommodated. One of the most important differences, that of the residue corresponding to V306 in CcbC changing to G308 in LmbC, was investigated in vitro and in silico. Moreover, the substrate binding pocket rearrangement also allowed LmbC to effectively adenylate 4-butyl-L-proline and 4-pentyl-L-proline, substrates with even longer alkyl side chains, producing more potent lincosamides. A shift of LmbC substrate specificity appears to be an integral part of biosynthetic pathway adaptation to the PPL acquisition. A set of genes presumably coding for the PPL biosynthesis is present in the lincomycin--but not in the celesticetin cluster; their homologs are found in biosynthetic clusters of some pyrrolobenzodiazepines (PBD) and hormaomycin. Whereas in the PBD and hormaomycin pathways the arising precursors are condensed to another amino acid moiety, the LmbC protein is the first functionally proved part of a unique condensation enzyme connecting PPL to the specialized amino sugar building unit.

Stabilization of an α/β-hydrolase by introducing proline residues: salicylic binding protein 2 from tobacco

PubMed Central

Huang, Jun; Jones, Bryan J.; Kazlauskas, Romas J.

2015-01-01

α/β-Hydrolases are important enzymes for biocatalysis, but their stability often limits their application. As a model α/β-hydrolase, we investigated a plant esterase, salicylic acid binding protein 2 (SABP2). SABP2 shows typical stability to urea (unfolding free energy 6.9±1.5 kcal/mol) and to heat inactivation (T1/215 min 49.2±0.5 °C). Denaturation in urea occurs in two steps, but heat inactivation occurs in a single step. The first unfolding step in urea eliminates catalytic activity. Surprisingly, we found that the first unfolding likely corresponds to the unfolding of the larger catalytic domain. Replacing selected amino acid residues with proline stabilized SABP2. Proline restricts the flexibility of the unfolded protein, thereby shifting the equilibrium toward the folded conformation. Seven locations for proline substitution were chosen either by amino acid sequence alignment with a more stable homolog or by targeting flexible regions in SABP2. Introducing proline in the catalytic domain stabilized SABP2 to the first unfolding in urea for three of five cases: L46P (+0.2 M urea), S70P (+0.1) and E215P (+0.9). Introducing proline in the cap domain did not (two of two cases), supporting the assignment that the first unfolding corresponds to the catalytic domain. Proline substitutions in both domains stabilized SABP2 to heat inactivation: L46P (ΔT1/215 min = +6.4 °C), S70P (+5.4), S115P (+1.8), S141P (+4.9), and E215P (+4.2). Combining substitutions did not further increase the stability to urea denaturation, but dramatically increased resistance to heat inactivation: L46P-S70P ΔT1/215 min = +25.7 °C. This straightforward proline substitution approach may also stabilize other α/β-hydrolases. PMID:26110207

Ehrlichia chaffeensis Proliferation Begins with NtrY/NtrX and PutA/GlnA Upregulation and CtrA Degradation Induced by Proline and Glutamine Uptake

PubMed Central

Cheng, Zhihui; Lin, Mingqun

2014-01-01

ABSTRACT How the obligatory intracellular bacterium Ehrlichia chaffeensis begins to replicate upon entry into human monocytes is poorly understood. Here, we examined the potential role of amino acids in initiating intracellular replication. PutA converts proline to glutamate, and GlnA converts glutamate to glutamine. E. chaffeensis PutA and GlnA complemented Escherichia coli putA and glnA mutants. Methionine sulfoximine, a glutamine synthetase inhibitor, inhibited E. chaffeensis GlnA activity and E. chaffeensis infection of human cells. Incubation of E. chaffeensis with human cells rapidly induced putA and glnA expression that peaked at 24 h postincubation. E. chaffeensis took up proline and glutamine but not glutamate. Pretreatment of E. chaffeensis with a proline transporter inhibitor (protamine), a glutamine transporter inhibitor (histidine), or proline analogs inhibited E. chaffeensis infection, whereas pretreatment with proline or glutamine enhanced infection and upregulated putA and glnA faster than no treatment or glutamate pretreatment. The temporal response of putA and glnA expression was similar to that of NtrY and NtrX, a two-component system, and electrophoretic mobility shift assays showed specific binding of recombinant E. chaffeensis NtrX (rNtrX) to the promoter regions of E. chaffeensis putA and glnA. Furthermore, rNtrX transactivated E. chaffeensis putA and glnA promoter-lacZ fusions in E. coli. Growth-promoting activities of proline and glutamine were also accompanied by rapid degradation of the DNA-binding protein CtrA. Our results suggest that proline and glutamine uptake regulates putA and glnA expression through NtrY/NtrX and facilitates degradation of CtrA to initiate a new cycle of E. chaffeensis growth. PMID:25425236

Characterization of the basic charge variants of a human IgG1

PubMed Central

Lu, Franklin; Derfus, Gayle; Kluck, Brian; Nogal, Bartek; Emery, Craig; Summers, Christie; Zheng, Kai; Bayer, Robert; Amanullah, Ashraf

2011-01-01

We report a case study of an IgG1 with a unique basic charge variant profile caused by C-terminal proline amidation on either one or two heavy chains. The proline amidation was sensitive to copper ion concentration in the production media during cell culture: the higher the Cu2+ ion concentration, the higher the level of proline amidation detected. This conclusion was supported by the analysis of samples that revealed direct correlation between the proline amidation level observed from peptide maps and the level of basic peaks measured by imaged capillary isoelectric focusing and a pH gradient ion-exchange chromatography method. The importance of these observations to therapeutic antibody production is discussed. PMID:22123059

Characterization of the basic charge variants of a human IgG1: effect of copper concentration in cell culture media.

PubMed

Kaschak, Timothy; Boyd, Daniel; Lu, Franklin; Derfus, Gayle; Kluck, Brian; Nogal, Bartek; Emery, Craig; Summers, Christie; Zheng, Kai; Bayer, Robert; Amanullah, Ashraf; Yan, Boxu

2011-01-01

We report a case study of an IgG1 with a unique basic charge variant profile caused by C-terminal proline amidation on either one or two heavy chains. The proline amidation was sensitive to copper ion concentration in the production media during cell culture: the higher the Cu ( 2+) ion concentration, the higher the level of proline amidation detected. This conclusion was supported by the analysis of samples that revealed direct correlation between the proline amidation level observed from peptide maps and the level of basic peaks measured by imaged capillary isoelectric focusing and a pH gradient ion-exchange chromatography method. The importance of these observations to therapeutic antibody production is discussed.

High-Throughput Quantitation of Proline Betaine in Foods and Suitability as a Valid Biomarker for Citrus Consumption.

PubMed

Lang, Roman; Lang, Tatjana; Bader, Matthias; Beusch, Anja; Schlagbauer, Verena; Hofmann, Thomas

2017-03-01

Proline betaine has been proposed as a candidate dietary biomarker for citrus intake. To validate its suitability as a dietary biomarker and to gain insight into the range of this per-methylated amino acid in foods and beverages, a quick and accurate stable isotope dilution assay was developed for quantitative high-throughput HILIC-MS/MS screening of proline betaine in foods and urine after solvent-mediated matrix precipitation. Quantitative analysis of a variety of foods confirmed substantial amounts of proline betaine in citrus juices (140-1100 mg/L) and revealed high abundance in tubers of the vegetable Stachys affinis, also known as Chinese artichocke (∼700 mg/kg). Seafood including clams, shrimp, and lobster contained limited amounts (1-95 mg/kg), whereas only traces were detected in fish, cuttlefish, fresh meat, dairy products, fresh vegetable (<3 mg/kg), coffee, tea, beer, and wine (<7 mg/L). The human excretion profiles of proline betaine in urine were comparable when common portions of orange juice or fried Stachys tubers were consumed. Neither mussels nor beer provided enough proline betaine to detect significant differences between morning urine samples collected before and after consumption. As Stachys is a rather rare vegetable and not part of peoples' daily diet, the data reported here will help to monitor the subject's compliance in future nutritional human studies on citrus products or the exclusion of citrus products in the wash-out phase of an intervention study. Moreover, proline betaine measurement can contribute to the establishment of a toolbox of valid dietary biomarkers reflecting wider aspects of diet to assess metabolic profiles as measures of dietary exposure and indicators of dietary patterns, dietary changes, or effectiveness of dietary interventions.

Hydrophytes lack potential to exhibit cadmium stress induced enhancement in lipid peroxidation and accumulation of proline.

PubMed

Dhir, Bhupinder; Sharmila, P; Saradhi, P Pardha

2004-02-10

Investigations were carried out to evaluate if hydrophytes (viz. Ceratophyllum, Wolffia, and Hydrilla) can be used as markers to assess the level of heavy metal pollution in aquatic bodies. The potential of these hydrophytes for lipid peroxidation and accumulation of proline in response to cadmium (Cd2+) pollution was studied. Hydrophytes were raised in artificial pond water (APW) supplemented with various levels of Cd2+. Interestingly, unlike mesophytes none of the hydrophytes showed ability to accumulate proline. Infact, in response to Cd2+ pollution hydrophytes exhibited a decline in proline levels in comparison to controls but mesophytes (viz. Brassica juncea, Vigna radiata and Triticum aestivum) showed progressive increase in the level of proline with increase in the extent of Cd2+ pollution. Mesophytes showed six to nine-fold increase in the level of proline in response to 1 mM Cd2+. The potential of the above hydrophytes for lipid peroxidation was also low under Cd2+ stress. In contrast, as expected a significant enhancement in the lipid peroxidation was observed in all three mesophytes in response to their exposure to Cd2+. About two-fold increase in production of malondialdehyde (a cytotoxic product of lipid peroxidation) was recorded in mesophytes exposed to 1 mM Cd2+. However, a decline in chlorophyll (Chl a and Chl b) levels was recorded in response to Cd2+pollution both in hydrophytes as well as mesophytes. In summary, hydrophytes neither have potential to accumulate proline nor have ability to accelerate lipid peroxidation under heavy metal stress. This suggests that the adaptive mechanism(s) existing in hydrophytes to tackle heavy metal stress is distinct from that in mesophytes.

Physiological, biochemical and molecular responses in four Prunus rootstocks submitted to drought stress.

PubMed

Jiménez, Sergio; Dridi, Jihène; Gutiérrez, Diego; Moret, David; Irigoyen, Juan J; Moreno, María A; Gogorcena, Yolanda

2013-10-01

An understanding of the mechanisms that determine plant response to reduced water availability is essential to improve water-use efficiency (WUE) of stone fruit crops. The physiological, biochemical and molecular drought responses of four Prunus rootstocks (GF 677, Cadaman, ROOTPAC 20 and ROOTPAC(®) R) budded with 'Catherina' peach cultivar were studied. Trees were grown in 15-l containers and subjected to a progressive water stress for 26 days, monitoring soil moisture content by time domain reflectometry. Photosynthetic and gas exchange parameters were determined. Root and leaf soluble sugars and proline content were also measured. At the end of the experiment, stressed plants showed lower net photosynthesis rate, stomatal conductance and transpiration rate, and higher intrinsic leaf WUE (AN/gs). Soluble sugars and proline concentration changes were observed, in both root and leaf tissues, especially in an advanced state of stress. The accumulation of proline in roots and leaves with drought stress was related to the decrease in osmotic potential and increase in WUE, whereas the accumulation of sorbitol in leaves, raffinose in roots and proline in both tissues was related only to the increase in the WUE. Owing to the putative role of raffinose and proline as antioxidants and their low concentration, they could be ameliorating deleterious effects of drought-induced oxidative stress by protecting membranes and enzymes rather than acting as active osmolytes. Higher expression of P5SC gene in roots was also consistent with proline accumulation in the tolerant genotype GF 677. These results indicate that accumulation of sorbitol, raffinose and proline in different tissues and/or the increase in P5SC expression could be used as markers of drought tolerance in peach cultivars grafted on Prunus rootstocks.

Effects of Single Amino Acid Substitution on the Collision-Induced Dissociation of Intact Protein Ions: Turkey Ovomucoid Third Domain

PubMed Central

Newton, Kelly A.; Pitteri, Sharon J.; Laskowski, Michael; McLuckey, Scott A.

2005-01-01

Expanded understanding of the factors that direct polypeptide ion fragmentation can lead to improved specificity in the use of tandem mass spectrometry for the identification and characterization of proteins. Like the fragmentation of peptide cations, the dissociation of whole protein cations shows several preferred cleavages, the likelihood for which is parent ion charge dependent. While such cleavages are often observed, they are far from universally observed, despite the presence of the residues known to promote them. Furthermore, cleavages at residues not noted to be common in a variety of proteins can be dominant for a particular protein or protein ion charge state. Motivated by the ability to study a small protein, turkey ovomucoid third domain, for which a variety of single amino acid variants are available, the effects of changing the identity of one amino acid in the protein sequence on its dissociation behavior were examined. In particular, changes in amino acids associated with C-terminal aspartic acid cleavage and N-terminal proline cleavage were emphasized. Consistent with previous studies, the product ion spectra were found to be dependent upon the parent ion charge state. Furthermore, the fraction of possible C-terminal aspartic acid cleavages observed to occur for this protein was significantly larger than the fraction of possible N-terminal proline cleavages. In fact, very little N-terminal proline cleavage was noted for the wild-type protein despite the presence of three proline residues in the protein. The addition/removal of proline and aspartic acids was studied along with changes in selected residues adjacent to proline residues. Evidence for inhibition of proline cleavage by the presence of nearby basic residues was noted, particularly if the basic residue was likely to be protonated. PMID:15473693

Inactivation of neurotensin by rat brain synaptic membranes. Cleavage at the Pro10-Tyr11 bond by endopeptidase 24.11 (enkephalinase) and a peptidase different from proline-endopeptidase.

PubMed

Checler, F; Emson, P C; Vincent, J P; Kitabgi, P

1984-11-01

It was shown previously that the tridecapeptide neurotensin is inactivated by rat brain synaptic membranes and that one of the primary inactivating cleavages occurs at the Pro10-Try11 peptide bond, leading to the formation of NT1-10 and NT11-13. The present study was designed to investigate the possibility that this cleavage was catalyzed by proline endopeptidase and/or endopeptidase 24.11 (enkephalinase). Purified rat brain synaptic membranes were found to contain a N-benzyloxycarbonyl-Gly-Pro-4-methyl-coumarinyl-7-amide-hydrolyzin g activity that was markedly inhibited (93%) by the proline endopeptidase inhibitor N-benzyloxycarbonyl-Pro-Prolinal and partially blocked (25%) by an antiproline endopeptidase antiserum. In contrast, the cleavage of neurotensin at the Pro10-Tyr11 bond by synaptic membranes was not affected by N-benzyloxycarbonyl-Pro-Prolinal and the antiserum. When the conversion of NT1-10 to NT1-8 by angiotensin converting enzyme was blocked by captopril and when the processing of NT11-13 by aminopeptidase(s) was inhibited by bestatin, it was found that thiorphan, a potent endopeptidase 24.11 inhibitor, partially decreased the formation of NT1-10 and NT11-13 by synaptic membranes. (1) proline endopeptidase, although it is present in synaptic membranes, is not involved in the cleavage of neurotensin at the Pro10-Tyr11 bond; (2) endopeptidase 24.11 only partially contributes to this cleavage; (3) there exists in rat brain synaptic membranes a peptidase different from proline endopeptidase and endopeptidase 24.11 that is mainly responsible for inactivating neurotensin by cleaving at the Pro10-Tyr11 bond.

The role of proline-containing peptide triads in β-sheet formation: A kinetic study.

PubMed

Takor, Gaius A; Higashiya, Seiichiro; Sikirzhytski, Vitali K; Seeley, Jason P; Lednev, Igor K; Welch, John T

2015-06-01

The design of biomimetic materials through molecular self-assembly is a growing area of modern nanotechnology. With problems of protein folding, self-assembly, and sequence-structure relationships as essential in nanotechnology as in biology, the effect of the nucleation of β-hairpin formation by proline on the folding process has been investigated in model studies. Previously such studies were limited to investigations of the influence of proline on the formation of turns in short peptide sequences. The effect of proline-based triads on the folding of an 11-kDa amyloidogenic peptide GH6[(GA)3GY(GA)3GE]8 GAH6 (YE8) was investigated by selective substitution of the proline-substituted triads at the γ-turn sites. The folding and fibrillation of the singly proline-substituted polypeptides, e.g., GH6-[(GA)3GY(GA)3GE]7(GA)3GY(GA)3PD-GAH6 (8PD), and doubly proline-substituted polypeptides, e.g., GH6-[(GA)3GY(GA)3GE]3(GA)3GY(GA)3PD[(GA)3GY(GA)3GE]3(GA)3GY(GA)3PD-GAH6 (4,8PD), were directly monitored by circular dichroism and deep UV resonance Raman and fluorescence spectroscopies. These findings were used to identify the essential folding domains, i.e., the minimum number of β-strands necessary for stable folding. These experimental findings may be especially useful in the design and construction of peptidic materials for a wide range of applications as well as in understanding the mechanisms of folding critical to fibril formation. © 2015 Wiley Periodicals, Inc.

The use of Proline (Prothioconazole) to control pitch canker, Rhizoctonia foliage blight, and Fusiform rust in forest seedling nurseries and efforts to acquire registration

Treesearch

Tom E. Starkey; Scott A. Enebak

2011-01-01

Laboratory, greenhouse, and field trials have shown Proline® to be efficacious against three fungal pathogens that cause damage and seedling mortality in forest seedling nurseries. Disease control using Proline® has been obtained at 365 ml/ha (5 fl oz/ac) for the control of fusiform rust (Cronartium quercuum f.sp. fusiforme) on loblolly pine (Pinus taeda) in both...

Identification of Trans-4-Hydroxy-L-Proline as a Compatible Solute and Its Biosynthesis and Molecular Characterization in Halobacillus halophilus.

PubMed

Kim, Kyung Hyun; Jia, Baolei; Jeon, Che Ok

2017-01-01

Halobacillus halophilus , a moderately halophilic bacterium, accumulates a variety of compatible solutes including glycine betaine, glutamate, glutamine, proline, and ectoine to cope with osmotic stress. Non-targeted analysis of intracellular organic compounds using 1 H-NMR showed that a large amount of trans-4-hydroxy-L-proline (Hyp), which has not been reported as a compatible solute in H. halophilus , was accumulated in response to high NaCl salinity, suggesting that Hyp may be an important compatible solute in H. halophilus . Candidate genes encoding proline 4-hydroxylase (PH-4), which hydroxylates L-proline to generate Hyp, were retrieved from the genome of H. halophilus through domain searches based on the sequences of known PH-4 proteins. A gene, HBHAL_RS11735, which was annotated as a multidrug DMT transporter permease in GenBank, was identified as the PH-4 gene through protein expression analysis in Escherichia coli . The PH-4 gene constituted a transcriptional unit with a promoter and a rho-independent terminator, and it was distantly located from the proline biosynthetic gene cluster ( pro operon). Transcriptional analysis showed that PH-4 gene expression was NaCl concentration-dependent, and was specifically induced by chloride anion, similar to the pro operon. Accumulation of intracellular Hyp was also observed in other bacteria, suggesting that Hyp may be a widespread compatible solute in halophilic and halotolerant bacteria.

Identification of Trans-4-Hydroxy-L-Proline as a Compatible Solute and Its Biosynthesis and Molecular Characterization in Halobacillus halophilus

PubMed Central

Kim, Kyung Hyun; Jia, Baolei; Jeon, Che Ok

2017-01-01

Halobacillus halophilus, a moderately halophilic bacterium, accumulates a variety of compatible solutes including glycine betaine, glutamate, glutamine, proline, and ectoine to cope with osmotic stress. Non-targeted analysis of intracellular organic compounds using 1H-NMR showed that a large amount of trans-4-hydroxy-L-proline (Hyp), which has not been reported as a compatible solute in H. halophilus, was accumulated in response to high NaCl salinity, suggesting that Hyp may be an important compatible solute in H. halophilus. Candidate genes encoding proline 4-hydroxylase (PH-4), which hydroxylates L-proline to generate Hyp, were retrieved from the genome of H. halophilus through domain searches based on the sequences of known PH-4 proteins. A gene, HBHAL_RS11735, which was annotated as a multidrug DMT transporter permease in GenBank, was identified as the PH-4 gene through protein expression analysis in Escherichia coli. The PH-4 gene constituted a transcriptional unit with a promoter and a rho-independent terminator, and it was distantly located from the proline biosynthetic gene cluster (pro operon). Transcriptional analysis showed that PH-4 gene expression was NaCl concentration-dependent, and was specifically induced by chloride anion, similar to the pro operon. Accumulation of intracellular Hyp was also observed in other bacteria, suggesting that Hyp may be a widespread compatible solute in halophilic and halotolerant bacteria. PMID:29104571

Validation and application of an improved method for the rapid determination of proline in grape berries.

PubMed

Rienth, Markus; Romieu, Charles; Gregan, Rebecca; Walsh, Caroline; Torregrosa, Laurent; Kelly, Mary T

2014-04-16

A rapid and sensitive method is presented for the determination of proline in grape berries. Following acidification with formic acid, proline is derivatized by heating at 100 °C for 15 min with 3% ninhydrin in dimethyl sulfoxide, and the absorbance, which is stable for at least 60 min, is read at 520 nm. The method was statistically validated in the concentration range from 2.5 to 15 mg/L, giving a repeatability and intermediate precision of generally <3%; linearity was determined using the lack of fit test. Results obtained with this method concurred (r = 0.99) with those obtained for the same samples on an amino acid analyzer. In terms of sample preparation, a simple dilution (5-20-fold) is required, and sugars, primary amino acids, and anthocyanins were demonstrated not to interfere, as the latter are bleached by ninhydrin under the experimental conditions. The method was applied to the study of proline accumulation in the fruits of microvines grown in phytotrons, and it was established that proline accumulation and concentrations closely resemble those of field-grown macrovines.

The nature of the hydroxyapatite-binding site in salivary acidic proline-rich proteins.

PubMed

Bennick, A; Cannon, M; Madapallimattam, G

1979-10-01

Protein A and C, which are major components of the acidic proline-rich proteins in human saliva, were digested, before or after adsorption to hydroxyapatite, with alkaline phosphatase, trypsin, thermolysin and a proteinase preparation from salivary sediment. The results demonstrate that the binding site is located in the proline-poor N-terminal part of the protein, possibly between residues 3 and 25. Phosphoserine is necessary for maximal adsorption of the proteins to hydroxyapatite. When proteins A and C are adsorbed to hydroxyapatite before proteolytic digestion there is a protection of some of the susceptible bonds in the N-terminal part of the proteins and a gradual removal of the proline-rich C-terminal part. Thermolysin can cleave susceptible bonds in the part of the protein that remains bound to hydroxyapatite, but at least some of the resulting peptides are retained on the mineral. Since the ability of the proteins to inhibit hydroxyapatite formation and to bind calcium is located in the N-terminal proline-poor part, it is possible that these activities are retained after proteolytic digestion of the adsorbed proteins.

Comparison of influence of carmustine and new proline analog of nitrosourea on antioxidant system in breast carcinoma cells (MCF-7).

PubMed

Stankiewicz-Kranc, Anna; Miltyk, Wojciech; Skrzydlewska, Elzbieta

2010-01-01

The high toxicity and low selectivity of carmustine restrict its application in anticancer therapy. Therefore, proline analogs of nitrosourea have been synthesized to obtain compounds whose action on neoplastic cells is characterized by higher selectivity. The present studies have aimed at examining the influence of carmustine and a new proline analog of nitrosourea on the redox system of fibroblasts and breast cancer cells (MCF-7). Carmustine and the proline analog of nitrosourea caused an increase in hydrogen peroxide concentration both in fibroblasts and MCF-7 cells. Moreover, administration of carmustine and the new analog of nitrosourea caused a decrease in the activity of antioxidant enzymes. Observed changes in the antioxidant system correlated with an increase in concentration of dityrosine, as well as a decrease in tryptophan concentration. Changes in the antioxidant system were also accompanied by intensification of the lipid peroxidation process. In conclusion, carmustine and proline analog of nitrosourea produce similar changes in the antioxidant system in normal and cancer cells and are responsible for oxidative stress.

Purification, crystallization and preliminary crystallographic analysis of the SH2 domain of IL-2-inducible T-cell kinase.

PubMed

Joseph, Raji E; Ginder, Nathaniel D; Hoy, Julie A; Nix, Jay C; Honzatko, Richard B; Andreotti, Amy H

2011-02-01

Proline is a unique amino acid owing to the relatively small energy difference between the cis and trans conformations of its peptide bond. The X-Pro imide bond readily undergoes cis-trans isomerization in the context of short peptides as well as some proteins. However, the direct detection of cis-trans proline isomerization in folded proteins is technically challenging. NMR spectroscopy is well suited to the direct detection of proline isomerization in folded proteins. It is less clear how well X-ray crystallography can reveal this conformational exchange event in folded proteins. Conformational heterogeneity owing to cis-trans proline isomerization in the Src homology 2 (SH2) domain of the IL-2-inducible T-cell kinase (ITK) has been extensively characterized by NMR. Using the ITK SH2 domain as a test system, an attempt was made to determine whether proline isomerization could be detected in a crystal structure of the ITK SH2 domain. As a first step towards this goal, the purification, crystallization and preliminary characterization of the ITK SH2 domain are described.

Proline induced disruption of the structure and dynamics of water.

PubMed

Yu, Dehong; Hennig, Marcus; Mole, Richard A; Li, Ji Chen; Wheeler, Cheryl; Strässle, Thierry; Kearley, Gordon J

2013-12-21

We use quasi-elastic neutron scattering spectroscopy to study the diffusive motion of water molecules at ambient temperature as a function of the solute molar fraction of the amino acid, proline. We validate molecular dynamics simulations against experimental quasielastic neutron scattering data and then use the simulations to reveal, and understand, a strong dependence of the translational self-diffusion coefficient of water on the distance to the amino acid molecule. An analysis based on the juxtaposition of water molecules in the simulation shows that the rigidity of proline imposes itself on the local water structure, which disrupts the hydrogen-bond network of water leading to an increase in the mean lifetime of hydrogen bonds. The net effect is some distortion of the proline molecule and a slowing down of the water mobility.

Role of oral nitrate in the nitrosation of ( UC)proline by conventional microflora and germ-free rats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mallett, A.K.; Rowland, I.R.; Walters, D.G.

1985-11-01

The urinary excretion of N-nitroso-L-(U- UC)proline by conventional microflora and germ free rats was used to assess the role of the gut bacteria and oral nitrate in the endogenous formation of N-nitroso compounds. The formation of nitrosoproline was qualitatively similar in conventional and germfree rats suggesting no involvement of the intestinal flora in this reaction. Furthermore, nitrosamino acid production was similar following the administration of nitrate and (U- UC)proline or (U- UC)proline alone, demonstrating no involvement of exogenous nitrate under the conditions of the experiment. Dietary contamination with nitrate/nitrite was negligible. The results are consistent with the suggestion that nitrate/nitritemore » reserves in the body are important in the formation of nitrosoproline in vivo.« less

The role of COMT and plasma proline in the variable penetrance of autistic spectrum symptoms in 22q11.2 deletion syndrome.

PubMed

Hidding, E; Swaab, H; de Sonneville, L M J; van Engeland, H; Vorstman, J A S

2016-11-01

This paper examines how COMT 158 genotypes and plasma proline levels are associated with variable penetrance of social behavioural and social cognitive problems in 22q11.2 deletion syndrome (22q11DS). Severity of autistic spectrum symptoms of 45 participants with 22q11DS was assessed using the Autism Diagnostic Interview Revised. Face and facial emotion recognition was evaluated using standardized computer-based test-paradigms. Associations with COMT 158 genotypes and proline levels were examined. High proline levels and poor face recognition in individuals with the COMT MET allele, and poor facial emotion recognition, explained almost 50% of the variance in severity of autism symptomatology in individuals with 22q11DS. High proline levels and a decreased capacity to break down dopamine as a result of the COMT MET variant are both relevant in the expression of the social phenotype in patients. This epistatic interaction effect between the COMT 158 genotype and proline on the expression of social deficits in 22q11DS shows how factors other than the direct effects of the deletion itself can modulate the penetrance of associated cognitive and behavioural outcomes. These findings are not only relevant to our insight into 22q11DS, but also provide a model to better understand the phenomenon of variable penetrance in other pathogenic genetic variants. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Amino acid substrates impose polyamine, eIF5A, or hypusine requirement for peptide synthesis

PubMed Central

Shin, Byung-Sik; Katoh, Takayuki; Gutierrez, Erik; Kim, Joo-Ran; Suga, Hiroaki

2017-01-01

Abstract Whereas ribosomes efficiently catalyze peptide bond synthesis by most amino acids, the imino acid proline is a poor substrate for protein synthesis. Previous studies have shown that the translation factor eIF5A and its bacterial ortholog EF-P bind in the E site of the ribosome where they contact the peptidyl-tRNA in the P site and play a critical role in promoting the synthesis of polyproline peptides. Using misacylated Pro-tRNAPhe and Phe-tRNAPro, we show that the imino acid proline and not tRNAPro imposes the primary eIF5A requirement for polyproline synthesis. Though most proline analogs require eIF5A for efficient peptide synthesis, azetidine-2-caboxylic acid, a more flexible four-membered ring derivative of proline, shows relaxed eIF5A dependency, indicating that the structural rigidity of proline might contribute to the requirement for eIF5A. Finally, we examine the interplay between eIF5A and polyamines in promoting translation elongation. We show that eIF5A can obviate the polyamine requirement for general translation elongation, and that this activity is independent of the conserved hypusine modification on eIF5A. Thus, we propose that the body of eIF5A functionally substitutes for polyamines to promote general protein synthesis and that the hypusine modification on eIF5A is critically important for poor substrates like proline. PMID:28637321

Effect of an alpha-blocker (Nicergoline) and of a beta-blocker (Acebutolol) on the in vitro biosynthesis of vascular extracellular matrix.

PubMed

Moczar, M; Robert, A M; Jacotot, B; Robert, L

2001-05-01

The effect of an alpha-blocking agent and of a beta-blocking agent on the biosynthesis of extracellular matrix macromolecules of the arterial wall was investigated. Rabbit aorta explants were cultured up to 48 hours with radioactive proline, lysine or glucosamine. In presence of these drugs, at concentration shown to be effective for the inhibition of platelet-endothelial cell interactions (10(-7) M), the incorporation of 14C proline in total macromolecular proline was higher than in macromolecular hydroxyproline suggesting a relatively higher rate of biosynthesis of non-collagenous proteins as compared to collagens. The alpha-blocking increased the incorporation of 14C proline in collagenous and non-collagenous proteins after 18 hours of incubation. beta-blocking also increased the incorporation of proline in macromolecular proline and hydroxyproline as compared to control cultures. Both increased the incorporation of 3H glucosamine in newly synthesised glycosaminoglycans. beta-blocking increased mainly the neosynthesis of heparan sulphate, alpha-blocking that of hyaluronan. The incorporation of 14C-lysine in crosslinked, insoluble elastin was not modified. These experiments confirm that alpha and beta-blocking agents can influence not only the tonus of aortic smooth muscle cells but also the relative rates of biosynthesis of extracellular matrix macromolecules. This effect should be taken in consideration for the evaluation of the long range effect of alpha and beta-blocking drugs on the vascular wall.

Development of a simple fluorescence-based microplate method for the high-throughput analysis of proline in wine samples.

PubMed

Robert-Peillard, Fabien; Boudenne, Jean-Luc; Coulomb, Bruno

2014-05-01

This paper presents a simple, accurate and multi-sample method for the determination of proline in wines thanks to a 96-well microplate technique. Proline is the most abundant amino acid in wine and is an important parameter related to wine characteristics or maturation processes of grape. In the current study, an improved application of the general method based on sodium hypochlorite oxidation and o-phthaldialdehyde (OPA)-thiol spectrofluorometric detection is described. The main interfering compounds for specific proline detection in wines are strongly reduced by selective reaction with OPA in a preliminary step under well-defined pH conditions. Application of the protocol after a 500-fold dilution of wine samples provides a working range between 0.02 and 2.90gL(-1), with a limit of detection of 7.50mgL(-1). Comparison and validation on real wine samples by ion-exchange chromatography prove that this procedure yields accurate results. Simplicity of the protocol used, with no need for centrifugation or filtration, organic solvents or high temperature enables its full implementation in plastic microplates and efficient application for routine analysis of proline in wines. Copyright © 2013 Elsevier Ltd. All rights reserved.

Interaction of flavan-3-ol derivatives and different caseins is determined by more than proline content and number of proline repeats.

PubMed

Bohin, Maxime C; Vincken, Jean-Paul; Westphal, Adrie H; Tripp, Annelise M; Dekker, Peter; van der Hijden, Harry T W M; Gruppen, Harry

2014-09-01

Interactions of Type A and B flavan-3-ol dimers (procyanidins) and several monomeric flavan-3-ols, with α-casein and β-casein, were investigated. Binding affinities measured were related to the ligands structure, including several properties (e.g. intrinsic flexibility (number of rotatable bonds) and hydrophobicity), and to the amino-acid composition of the caseins. A monomeric flavan-3-ol esterified with gallic acid (EGCG) had a five to ten times higher affinity to caseins compared to the non-galloylated dimeric flavan-3-ols. In this case, the larger number of rotatable bonds in EGCG might be accountable for this difference. Comparing flavan-3-ol dimers, intrinsic flexibility did not consistently promote interactions, as procyanidin A1 displayed a higher affinity to α-casein than the supposedly more flexible B-type dimers investigated. Despite its higher content of proline, compared to α-casein, β-casein did not always have a higher affinity for the ligands investigated (e.g. no interaction with procyanidin A1 detected). These results suggest that more factors than proline content and the number of proline repeats govern phenolic-casein interactions. Copyright © 2014 Elsevier Ltd. All rights reserved.

Proline-catalysed asymmetric amination of alpha,alpha-disubstituted aldehydes: synthesis of configurationally stable enantioenriched alpha-aminoaldehydes.

PubMed

Vogt, Henning; Vanderheiden, Sylvia; Bräse, Stefan

2003-10-07

Proline-catalysed amination of alpha,alpha-disubstituted racemic aldehydes with azodicarboxylates proceeds smoothly to give configurationally stable scalemic aldehydes and oxazolidinones in up to 86% ee.

Amino acids of Diclidophora merlangi (Monogenea).

PubMed

Arme, C; Whyte, A

1975-02-01

The level of free amino acids in Diclidophora merlangi is high, comprising over 500 mu moles/g ethanol extracted dry weight. A single amino acid, proline, constitutes some 70% of the total pool. Analysis of parasite protein and host blood and mucus revealed low proline levels, suggesting that the high free pool content was not related to a requirement for protein systhesis or to its abundance in the diet of the worm. Experiments revealed that proline was not involved specifically in osmoregulation, and the reasons for the large amounts present in Diclidophora remain unknown.

Selective CB2 receptor agonists. Part 2: Structure-activity relationship studies and optimization of proline-based compounds.

PubMed

Riether, Doris; Zindell, Renee; Wu, Lifen; Betageri, Raj; Jenkins, James E; Khor, Someina; Berry, Angela K; Hickey, Eugene R; Ermann, Monika; Albrecht, Claudia; Ceci, Angelo; Gemkow, Mark J; Nagaraja, Nelamangala V; Romig, Helmut; Sauer, Achim; Thomson, David S

2015-02-01

Through a ligand-based pharmacophore model (S)-proline based compounds were identified as potent cannabinoid receptor 2 (CB2) agonists with high selectivity over the cannabinoid receptor 1 (CB1). Structure-activity relationship investigations for this compound class lead to oxo-proline compounds 21 and 22 which combine an impressive CB1 selectivity profile with good pharmacokinetic properties. In a streptozotocin induced diabetic neuropathy model, 22 demonstrated a dose-dependent reversal of mechanical hyperalgesia. Copyright © 2014 Elsevier Ltd. All rights reserved.

2,3-trans-3,4-trans-3,4-Dihydroxy-L-proline: An amino acid in toxic peptides of Amanita virosa mushrooms

PubMed Central

Buku, A.; Faulstich, H.; Wieland, T.; Dabrowski, J.

1980-01-01

Among the four possible stereoisomers of 3,4-dihydroxy-L-proline,2,3-trans-3,4-trans-3,4-dihydroxy-L-proline (IV) had not been found in nature previously. It has now been detected as a component of virotoxins, toxic peptides of Amanita virosa mushrooms. Because periodate failed to effect an oxidative glycol splitting reaction, the two hydroxyl groups in positions 3 and 4 were expected to be in a trans configuration. Furthermore, the formation of a 4-lactone on treatment with acids pointed to the carboxyl group and the hydroxyl group at position 4 being in a cis configuration. These results are in agreement with structure IV only. Final proof for structure IV was given by NMR spectroscopy and direct comparison with the 2,3-cis-3,4-trans-3,4-dihydroxy-L-proline isomer. PMID:16592813

Importance of the High-Expression of Proline Transporter PutP to the Adaptation of Escherichia coli to High Salinity.

PubMed

Sasaki, Hideaki; Sato, Daichi; Oshima, Akinobu

2017-01-01

　The effect of the amount of the proline transporter PutP expression on the mechanism of adaptation of E. coli cells to high salinity was analyzed. The PutP gene derived from the E. coli expression plasmid was introduced into the E. coli cell, and a high PutP expression strain was developed. At 1.2 M NaCl culture condition, the growth of normal E. coli cells was inhibited, whereas high ProP expression cells showed growth under 2.5 M NaCl conditions. The uptake of proline by E. coli as a compatible solute and substrate for metabolization was in good accordance with those seen in cell growth. These data suggested that the amount of the proline transporter PutP expression played an important role in the adaptation of E. coli cells to high saline conditions.

Dihydroxyacetone variants in the organocatalytic construction of carbohydrates: mimicking tagatose and fuculose aldolases.

PubMed

Suri, Jeff T; Mitsumori, Susumu; Albertshofer, Klaus; Tanaka, Fujie; Barbas, Carlos F

2006-05-12

Dihydroxyacetone variants have been explored as donors in organocatalytic aldol reactions with various aldehyde and ketone acceptors. The protected form of dihydroxyacetone that was chosen for in-depth study was 2,2-dimethyl-1,3-dioxan-5-one, 1. Among the catalysts surveyed here, proline proved to be superior in terms of yield and stereoselectivities in the construction of various carbohydrate scaffolds. In a fashion analogous to aldolase enzymes, the de novo preparation of L-ribulose, L-lyxose, D-ribose, D-tagatose, 1-amino-1-deoxy-D-lyxitol, and other carbohydrates was accomplished via the use of 1 and proline. In reactions using 2,2-dimethyl-1,3-dioxan-5-one 1 as a donor, (S)-proline can be used as a functional mimic of tagatose aldolase, whereas (R)-proline can be regarded as an organocatalytic mimic of fuculose aldolase.

Discovery and SAR studies of methionine-proline anilides as dengue virus NS2B-NS3 protease inhibitors.

PubMed

Zhou, Guo-Chun; Weng, Zhibing; Shao, Xiaoxia; Liu, Fang; Nie, Xin; Liu, Jinsong; Wang, Decai; Wang, Chunguang; Guo, Kai

2013-12-15

A series of methionine-proline dipeptide derivatives and their analogues were designed, synthesized and assayed against the serotype 2 dengue virus NS2B-NS3 protease, and methionine-proline anilides 1 and 2 were found to be the most active DENV 2 NS2B-NS3 competitive inhibitors with Ki values of 4.9 and 10.5 μM. The structure and activity relationship and the molecular docking revealed that L-proline, L-methionine and p-nitroaniline in 1 and 2 are the important characters in blocking the active site of NS2B-NS3 protease. Our current results suggest that the title dipeptidic scaffold represents a promising structural core to discover a new class of active NS2B-NS3 competitive inhibitors. Copyright © 2013 Elsevier Ltd. All rights reserved.

INCREASING PROTEIN STABILITY BY IMPROVING BETA-TURNS

PubMed Central

Fu, Hailong; Grimsley, Gerald R.; Razvi, Abbas; Scholtz, J. Martin; Pace, C. Nick

2009-01-01

Our goal was to gain a better understanding of how protein stability can be increased by improving β-turns. We studied 22 β-turns in nine proteins with 66 to 370 residues by replacing other residues with proline and glycine and measuring the stability. These two residues are statistically preferred in some β-turn positions. We studied: Cold shock protein B (CspB), Histidine-containing phosphocarrier protein (HPr), Ubiquitin, Ribonucleases Sa2, Sa3, T1, and HI, Tryptophan synthetase α-subunit (TSα), and Maltose binding protein (MBP). Of the fifteen single proline mutations, 11increased stability (Average = 0.8 ± 0.3; Range = 0.3 – 1.5 kcal/mol), and the stabilizing effect of double proline mutants was additive. Based on this and our previous work, we conclude that proteins can generally be stabilized by replacing non-proline residues with proline residues at the i + 1 position of Type I and II β-turns and at the i position in Type II β-turns. Other turn positions can sometimes be used if the φ angle is near −60° for the residue replaced. It is important that the side chain of the residue replaced is less than 50% buried. Identical substitutions in β-turns in related proteins give similar results. Proline substitutions increase stability mainly by decreasing the entropy of the denatured state. In contrast, the large, diverse group of proteins considered here had almost no residues in β-turns that could be replaced by Gly to increase protein stability. Improving β-turns by substituting Pro residues is a generally useful way of increasing protein stability. PMID:19626709

A Statistical Analysis of the PPII Propensity of Amino Acid Guests in Proline-Rich Peptides

PubMed Central

Moradi, Mahmoud; Babin, Volodymyr; Sagui, Celeste; Roland, Christopher

2011-01-01

There has been considerable debate about the intrinsic PPII propensity of amino-acid residues in denatured polypeptides. Experimentally, the propensity scale is based on the behavior of guest amino-acid residues placed in the middle of polyproline hosts. We have used classical molecular dynamics simulations, with state-of-the-art force fields to carry out a comprehensive analysis of the conformational equilibria of the proline-based host oligopeptides with single guests. The tracked structural characteristics include the PPII content, the cis/trans isomerization of the prolyl bonds, the puckering of the pyrrolidine rings of the proline residues, and the secondary structural motifs. We find no evidence for an intrinsic PPII propensity in any of the guest amino acids other than proline. Instead, the PPII content as derived from experiments may be explained in terms of: 1), a local correlation between the dihedral angles of the guest amino acid and the proline residue immediately preceding it; and 2), a nonlocal correlation between the cis/trans states of the peptide bonds. In terms of the latter, we find that the presence of a guest (other than proline, tyrosine, or tryptophan) increases the trans content of most of the prolyl bonds, which results in an effective increase of the peptide PPII content. With respect to the local dihedral correlations, we find that these are well described in terms of the so-called odds-ratio statistic. Expressed in terms of free energy language, the PPII content based on the odds-ratio of the relevant residues correlate well with the experimentally measured PPII content. PMID:21320454

Amino acid substrates impose polyamine, eIF5A, or hypusine requirement for peptide synthesis.

PubMed

Shin, Byung-Sik; Katoh, Takayuki; Gutierrez, Erik; Kim, Joo-Ran; Suga, Hiroaki; Dever, Thomas E

2017-08-21

Whereas ribosomes efficiently catalyze peptide bond synthesis by most amino acids, the imino acid proline is a poor substrate for protein synthesis. Previous studies have shown that the translation factor eIF5A and its bacterial ortholog EF-P bind in the E site of the ribosome where they contact the peptidyl-tRNA in the P site and play a critical role in promoting the synthesis of polyproline peptides. Using misacylated Pro-tRNAPhe and Phe-tRNAPro, we show that the imino acid proline and not tRNAPro imposes the primary eIF5A requirement for polyproline synthesis. Though most proline analogs require eIF5A for efficient peptide synthesis, azetidine-2-caboxylic acid, a more flexible four-membered ring derivative of proline, shows relaxed eIF5A dependency, indicating that the structural rigidity of proline might contribute to the requirement for eIF5A. Finally, we examine the interplay between eIF5A and polyamines in promoting translation elongation. We show that eIF5A can obviate the polyamine requirement for general translation elongation, and that this activity is independent of the conserved hypusine modification on eIF5A. Thus, we propose that the body of eIF5A functionally substitutes for polyamines to promote general protein synthesis and that the hypusine modification on eIF5A is critically important for poor substrates like proline. Published by Oxford University Press on behalf of Nucleic Acids Research 2017.

Variability in Proline-Accumulating Ability of Barley (Hordeum vulgare L.) Cultivars Induced by Vapor Pressure Deficit 1

PubMed Central

Naidu, Bodapati P.; Aspinall, Donald; Paleg, Leslie G.

1992-01-01

This work was undertaken in an effort to reconcile the conflicting proline-accumulating responses of the barley (Hordeum vulgare L.) cultivars, Excelsior and Proctor, reported by Singh et al. (1972) and Hanson et al. (1976). It deals with the effects of different vapor pressure deficits (VPD) during growth and subsequent drought stress on several barley cultivars. A higher VPD (1.2 kilopascals) during Clipper seedling growth resulted in higher solute-accumulating ability, seemingly independently of leaf water potential, than a lower VPD (0.12 kilopascals). The higher VPD during stress also resulted in higher solute contents, and this response may be more closely related to leaf water potential. When the responses of Excelsior and Proctor were examined in detail, it was found that the relative proline-accumulating ability of the two cultivars was dependent upon the VPD under which they were grown. At low VPD, Proctor accumulated significantly more proline than did Excelsior; whereas at higher VPD, Excelsior accumulated more proline than did Proctor. The crossover occurred at a VPD of about 0.72 kilopascals. This reversal of cultivar response was enhanced by multiplying seed under the two VPD extremes. Glycinebetaine accumulation did not demonstrate the crossover effect, although the concentration of this compound in all cultivars also depended on the VPD prevailing during growth and/or stress. Solute levels, in general, were more closely related to the decrease in relative water content than to a decrease in leaf water potential. It is concluded that the conflicting proline-accumulating responses of Excelsior and Proctor could be explained by these findings. PMID:16668700

Reorientation Motion and Preferential Interactions of a Peptide in Denaturants and Osmolyte.

PubMed

Jas, Gouri S; Rentchler, Eric C; Słowicka, Agnieszka M; Hermansen, John R; Johnson, Carey K; Middaugh, C Russell; Kuczera, Krzysztof

2016-03-31

Fluorescence anisotropy decay measurements and all atom molecular dynamics simulations are used to characterize the orientational motion and preferential interaction of a peptide, N-acetyl-tryptophan-amide (NATA) containing two peptide bonds, in aqueous, urea, guanidinium chloride (GdmCl), and proline solution. Anisotropy decay measurements as a function of temperature and concentration showed moderate slowing of reorientations in urea and GdmCl and very strong slowing in proline solution, relative to water. These effects deviate significantly from simple proportionality of peptide tumbling time to solvent viscosity, leading to the investigation of microscopic preferential interaction behavior through molecular dynamics simulations. Examination of the interactions of denaturants and osmolyte with the peptide backbone uncovers the presence of strongest interaction with urea, intermediate with proline, and weakest with GdmCl. In contrast, the strongest preferential solvation of the peptide side chain is by the nonpolar part of the proline zwitterion, followed by urea, and GdmCl. Interestingly, the local density of urea around the side chain is higher, but the GdmCl distribution is more organized. Thus, the computed preferential solvation of the side chain by the denaturants and osmolyte can account for the trend in reorientation rates. Analysis of water structure and its dynamics uncovered underlying differences between urea, GdmCl, and proline. Urea exerted the smallest perturbation of water behavior. GdmCl had a larger effect on water, slowing kinetics and stabilizing interactions. Proline had the largest overall interactions, exhibiting a strong stabilizing effect on both water-water and water-peptide hydrogen bonds. The results for this elementary peptide system demonstrate significant differences in microscopic behavior of the examined solvent environments. For the commonly used denaturants, urea tends to form disorganized local aggregates around the peptide groups and has little influence on water, while GdmCl only forms specific interactions with the side chain and tends to destabilize water structure. The protective osmolyte proline has the strongest and most specific interactions with the tryptophan side chain, and also stabilizes both water-water and water-peptide hydrogen bonds. Our results strongly suggest protein or peptide denaturation triggered by urea occurs by direct interaction, whereas GdmCl interacts favorably with side chains and destabilizes peptide-water hydrogen bonds. The stabilization of biopolymers by an osmolyte such as proline is governed by favorable preferential interaction with the side chains and stabilization of water.

Effect of proline kinks on the mechanical unfolding of α-helices

NASA Astrophysics Data System (ADS)

Arteca, Gustavo A.; Li, Zhiying

2004-12-01

Proteins unfold by applying an external force, although the microscopic mechanism is still not well understood. In this work, we use steered molecular dynamics to probe fundamental aspects of the stretching transition of α-helices, in particular how proline kinks and side chain dynamics would influence their ability to resist the applied force. We find that proline residues effectively 'cut' a helix in half when introduced on stable homopolymers, whereas their effect is smaller when present in helices that are more easily deformed. Our findings provide insight into the factors that may regulate the mechanical stretching of realistic protein domains.

Randomized Subspace Learning for Proline Cis-Trans Isomerization Prediction.

PubMed

Al-Jarrah, Omar Y; Yoo, Paul D; Taha, Kamal; Muhaidat, Sami; Shami, Abdallah; Zaki, Nazar

2015-01-01

Proline residues are common source of kinetic complications during folding. The X-Pro peptide bond is the only peptide bond for which the stability of the cis and trans conformations is comparable. The cis-trans isomerization (CTI) of X-Pro peptide bonds is a widely recognized rate-limiting factor, which can not only induces additional slow phases in protein folding but also modifies the millisecond and sub-millisecond dynamics of the protein. An accurate computational prediction of proline CTI is of great importance for the understanding of protein folding, splicing, cell signaling, and transmembrane active transport in both the human body and animals. In our earlier work, we successfully developed a biophysically motivated proline CTI predictor utilizing a novel tree-based consensus model with a powerful metalearning technique and achieved 86.58 percent Q2 accuracy and 0.74 Mcc, which is a better result than the results (70-73 percent Q2 accuracies) reported in the literature on the well-referenced benchmark dataset. In this paper, we describe experiments with novel randomized subspace learning and bootstrap seeding techniques as an extension to our earlier work, the consensus models as well as entropy-based learning methods, to obtain better accuracy through a precise and robust learning scheme for proline CTI prediction.

Trypanosoma brucei eflornithine transporter AAT6 is a low-affinity low-selective transporter for neutral amino acids.

PubMed

Mathieu, Christoph; González Salgado, Amaia; Wirdnam, Corina; Meier, Stefan; Grotemeyer, Marianne Suter; Inbar, Ehud; Mäser, Pascal; Zilberstein, Dan; Sigel, Erwin; Bütikofer, Peter; Rentsch, Doris

2014-10-01

Amino acid transporters are crucial for parasite survival since the cellular metabolism of parasitic protozoa depends on the up-take of exogenous amino acids. Amino acid transporters are also of high pharmacological relevance because they may mediate uptake of toxic amino acid analogues. In the present study we show that the eflornithine transporter AAT6 from Trypanosoma brucei (TbAAT6) mediates growth on neutral amino acids when expressed in Saccharomyces cerevisiae mutants. The transport was electrogenic and further analysed in Xenopus laevis oocytes. Neutral amino acids, proline analogues, eflornithine and acivicin induced inward currents. For proline, glycine and tryptophan the apparent affinities and maximal transport rates increased with more negative membrane potentials. Proline-induced currents were dependent on pH, but not on sodium. Although proline represents the primary energy source of T. brucei in the tsetse fly, down-regulation of TbAAT6-expression by RNAi showed that in culture TbAAT6 is not essential for growth of procyclic form trypanosomes in the presence of glucose or proline as energy source. TbAAT6-RNAi lines of both bloodstream and procyclic form trypanosomes showed reduced susceptibility to eflornithine, whereas the sensitivity to acivicin remained unchanged, indicating that acivicin enters the cell by more than one transporter.

Tropical Drosophila ananassae of wet-dry seasons show cross resistance to heat, drought and starvation

PubMed Central

Lambhod, Chanderkala; Pathak, Ankita; Munjal, Ashok K.

2017-01-01

ABSTRACT Plastic responses to multiple environmental stressors in wet or dry seasonal populations of tropical Drosophila species have received less attention. We tested plastic effects of heat hardening, acclimation to drought or starvation, and changes in trehalose, proline and body lipids in Drosophila ananassae flies reared under wet or dry season-specific conditions. Wet season flies revealed significant increase in heat knockdown, starvation resistance and body lipids after heat hardening. However, accumulation of proline was observed only after desiccation acclimation of dry season flies while wet season flies elicited no proline but trehalose only. Therefore, drought-induced proline can be a marker metabolite for dry-season flies. Further, partial utilization of proline and trehalose under heat hardening reflects their possible thermoprotective effects. Heat hardening elicited cross-protection to starvation stress. Stressor-specific accumulation or utilization as well as rates of metabolic change for each energy metabolite were significantly higher in wet-season flies than dry-season flies. Energy metabolite changes due to inter-related stressors (heat versus desiccation or starvation) resulted in possible maintenance of energetic homeostasis in wet- or dry-season flies. Thus, low or high humidity-induced plastic changes in energy metabolites can provide cross-protection to seasonally varying climatic stressors. PMID:29141954

Enhancement of the proline and nitric oxide synthetic pathway improves fermentation ability under multiple baking-associated stress conditions in industrial baker's yeast

PubMed Central

2012-01-01

Background During the bread-making process, industrial baker's yeast, mostly Saccharomyces cerevisiae, is exposed to baking-associated stresses, such as air-drying and freeze-thaw stress. These baking-associated stresses exert severe injury to yeast cells, mainly due to the generation of reactive oxygen species (ROS), leading to cell death and reduced fermentation ability. Thus, there is a great need for a baker's yeast strain with higher tolerance to baking-associated stresses. Recently, we revealed a novel antioxidative mechanism in a laboratory yeast strain that is involved in stress-induced nitric oxide (NO) synthesis from proline via proline oxidase Put1 and N-acetyltransferase Mpr1. We also found that expression of the proline-feedback inhibition-less sensitive mutant γ-glutamyl kinase (Pro1-I150T) and the thermostable mutant Mpr1-F65L resulted in an enhanced fermentation ability of baker's yeast in bread dough after freeze-thaw stress and air-drying stress, respectively. However, baker's yeast strains with high fermentation ability under multiple baking-associated stresses have not yet been developed. Results We constructed a self-cloned diploid baker's yeast strain with enhanced proline and NO synthesis by expressing Pro1-I150T and Mpr1-F65L in the presence of functional Put1. The engineered strain increased the intracellular NO level in response to air-drying stress, and the strain was tolerant not only to oxidative stress but also to both air-drying and freeze-thaw stresses probably due to the reduced intracellular ROS level. We also showed that the resultant strain retained higher leavening activity in bread dough after air-drying and freeze-thaw stress than that of the wild-type strain. On the other hand, enhanced stress tolerance and fermentation ability did not occur in the put1-deficient strain. This result suggests that NO is synthesized in baker's yeast from proline in response to oxidative stresses that induce ROS generation and that increased NO plays an important role in baking-associated stress tolerance. Conclusions In this work, we clarified the importance of Put1- and Mpr1-mediated NO generation from proline to the baking-associated stress tolerance in industrial baker's yeast. We also demonstrated that baker's yeast that enhances the proline and NO synthetic pathway by expressing the Pro1-I150T and Mpr1-F65L variants showed improved fermentation ability under multiple baking-associated stress conditions. From a biotechnological perspective, the enhancement of proline and NO synthesis could be promising for breeding novel baker's yeast strains. PMID:22462683

Enhancement of the proline and nitric oxide synthetic pathway improves fermentation ability under multiple baking-associated stress conditions in industrial baker's yeast.

PubMed

Sasano, Yu; Haitani, Yutaka; Hashida, Keisuke; Ohtsu, Iwao; Shima, Jun; Takagi, Hiroshi

2012-04-01

During the bread-making process, industrial baker's yeast, mostly Saccharomyces cerevisiae, is exposed to baking-associated stresses, such as air-drying and freeze-thaw stress. These baking-associated stresses exert severe injury to yeast cells, mainly due to the generation of reactive oxygen species (ROS), leading to cell death and reduced fermentation ability. Thus, there is a great need for a baker's yeast strain with higher tolerance to baking-associated stresses. Recently, we revealed a novel antioxidative mechanism in a laboratory yeast strain that is involved in stress-induced nitric oxide (NO) synthesis from proline via proline oxidase Put1 and N-acetyltransferase Mpr1. We also found that expression of the proline-feedback inhibition-less sensitive mutant γ-glutamyl kinase (Pro1-I150T) and the thermostable mutant Mpr1-F65L resulted in an enhanced fermentation ability of baker's yeast in bread dough after freeze-thaw stress and air-drying stress, respectively. However, baker's yeast strains with high fermentation ability under multiple baking-associated stresses have not yet been developed. We constructed a self-cloned diploid baker's yeast strain with enhanced proline and NO synthesis by expressing Pro1-I150T and Mpr1-F65L in the presence of functional Put1. The engineered strain increased the intracellular NO level in response to air-drying stress, and the strain was tolerant not only to oxidative stress but also to both air-drying and freeze-thaw stresses probably due to the reduced intracellular ROS level. We also showed that the resultant strain retained higher leavening activity in bread dough after air-drying and freeze-thaw stress than that of the wild-type strain. On the other hand, enhanced stress tolerance and fermentation ability did not occur in the put1-deficient strain. This result suggests that NO is synthesized in baker's yeast from proline in response to oxidative stresses that induce ROS generation and that increased NO plays an important role in baking-associated stress tolerance. In this work, we clarified the importance of Put1- and Mpr1-mediated NO generation from proline to the baking-associated stress tolerance in industrial baker's yeast. We also demonstrated that baker's yeast that enhances the proline and NO synthetic pathway by expressing the Pro1-I150T and Mpr1-F65L variants showed improved fermentation ability under multiple baking-associated stress conditions. From a biotechnological perspective, the enhancement of proline and NO synthesis could be promising for breeding novel baker's yeast strains.

Isolation of cis-3-Amino-l-Proline from Cultured Mycelia of Morchella esculenta Fr

PubMed Central

Moriguchi, Mitsuaki; Sada, Shin-Ichi; Hatanaka, Shin-Ichi

1979-01-01

cis-3-Amino-l-proline, identified once as a nonprotein amino acid from the fruiting bodies of Morchella esculenta Fr., was isolated also from the growth medium and cultured mycelia of the same fungus. PMID:16345456

Synthesis of aryl azides and vinyl azides via proline-promoted CuI-catalyzed coupling reactions.

PubMed

Zhu, Wei; Ma, Dawei

2004-04-07

The coupling reaction of aryl halides or vinyl iodide with sodium azide under catalysis of CuI/L-proline works at relatively low temperature to provide aryl azides or vinyl azides in good to excellent yields.

Effect of subsoiling in fallow period on soil water storage and grain protein accumulation of dryland wheat and its regulatory effect by nitrogen application.

PubMed

Sun, Min; Gao, ZhiQiang; Zhao, WeiFeng; Deng, LianFeng; Deng, Yan; Zhao, HongMei; Ren, AiXia; Li, Gang; Yang, ZhenPing

2013-01-01

To provide a new way to increase water storage and retention of dryland wheat, a field study was conducted at Wenxi experimental site of Shanxi Agricultural University. The effect of subsoiling in fallow period on soil water storage, accumulation of proline, and formation of grain protein after anthesis were determined. Our results showed that subsoiling in fallow period could increase water storage in the 0-300 cm soil at pre-sowing stage and at anthesis stage with low or medium N application, especially for the 60-160 cm soil. However, the proline content, glutamine synthetase (GS) activity, glutamate dehydrogenase (GDH) activity in flag leaves and grains were all decreased by subsoiling in fallow period. In addition, the content of albumin, gliadin, and total protein in grains were also decreased while globulin content, Glu/Gli, protein yield, and glutelin content were increased. With N application increasing, water storage of soil layers from 20 to 200 cm was decreased at anthesis stage. High N application resulted in the increment of proline content and GS activity in grains. Besides, correlation analysis showed that soil storage in 40-160 cm soil was negatively correlated with proline content in grains; proline content in grains was positively correlated with GS and GDH activity in flag leaves. Contents of albumin, globulin and total protein in grains were positively correlated with proline content in grains and GDH activity in flag leaves. In conclusion, subsoiling in fallow period, together with N application at 150 kg·hm(-2), was beneficial to increase the protein yield and Glu/Gli in grains which improve the quality of wheat.

Ameliorating Effects of Exogenously Applied Proline on Seed Composition, Seed Oil Quality and Oil Antioxidant Activity of Maize (Zea mays L.) under Drought Stress

PubMed Central

Ali, Qasim; Anwar, Farooq; Ashraf, Muhammad; Saari, Nazamid; Perveen, Rashida

2013-01-01

This study was carried out to appraise whether or not the exogenous application of a potential osmoprotectant, proline, could ameliorate the adverse effects of drought stress on maize seed and seed oil composition, as well as oil antioxidant activity. Water stress reduced the kernel sugar, oil, protein and moisture contents and most of the seed macro- and micro-elements analyzed in both maize cultivars but it increased the contents of seed fiber and ash. Water stress increased the oil oleic acid content with a subsequent decrease in the amount of linoleic acid, resulting in an increased oil oleic/linoleic ratio for both maize cultivars. However, no variation was observed in oil stearic and palmitic acids content due to water stress. A considerable drought induced an increase in seed oil α-, γ-, δ- and total tocopherols and flavonoids were observed in both maize cultivars. However, oil phenolic and carotenoid content as well as 1,1-diphenyl-2-picryl-hydrazyl (DPPH) free radical scavenging activity decreased. Foliar-applied proline significantly increased the content of seed sugar, oil, protein, moisture, fiber and ash in both maize cultivars under well irrigated and water deficit conditions. Furthermore, exogenous application of proline increased the oil oleic and linoleic acid contents. The concentrations of antioxidant compounds namely phenolics, carotenoids, flavonoids and tocopherols estimated in the seed oil increased due to foliar-applied proline under water deficit conditions that was positively correlated with the enhanced oil DPPH free radical scavenging activity. Moreover, the increase in the contents of these antioxidant compounds and oil antioxidant activity due to the foliar application of proline was noted to be more pronounced under water deficit conditions. PMID:23344043

Vitamin D insufficiency and schizophrenia risk: evaluation of hyperprolinemia as a mediator of association.

PubMed

Clelland, James D; Read, Laura L; Drouet, Valérie; Kaon, Angela; Kelly, Alexandra; Duff, Karen E; Nadrich, Robert H; Rajparia, Amit; Clelland, Catherine L

2014-06-01

25-Hydroxyvitamin D (25(OH)D) deficits have been associated with schizophrenia susceptibility and supplementation has been recommended for those at-risk. Although the mechanism by which a deficit confers risk is unknown, vitamin D is a potent transcriptional modulator and can regulate proline dehydrogenase (PRODH) expression. PRODH maps to chromosome 22q11, a region conferring the highest known genetic risk of schizophrenia, and encodes proline oxidase, which catalyzes proline catabolism. l-Proline is a neuromodulator at glutamatergic synapses, and peripheral hyperprolinemia has been associated with decreased IQ, cognitive impairment, schizoaffective disorder, and schizophrenia. We investigated the relationship between 25(OH)D and schizophrenia, comparing fasting plasma 25(OH)D in 64 patients and 90 matched controls. We then tested for a mediating effect of hyperprolinemia on the association between 25(OH)D and schizophrenia. 25(OH)D levels were significantly lower in patients, and 25(OH)D insufficiency associated with schizophrenia (OR 2.1, adjusted p=0.044, 95% CI: 1.02-4.46). Moreover, 25(OH)D insufficient subjects had three times greater odds of hyperprolinemia than those with optimal levels (p=0.035, 95% CI: 1.08-8.91), and formal testing established that hyperprolinemia is a significantly mediating phenotype that may explain over a third of the effect of 25(OH)D insufficiency on schizophrenia risk. This study presents a mechanism by which 25(OH)D insufficiency confers risk of schizophrenia; via proline elevation due to reduced PRODH expression, and a concomitant dysregulation of neurotransmission. Although definitive causality cannot be confirmed, these findings strongly support vitamin D supplementation in patients, particularly for those with elevated proline, who may represent a large subgroup of the schizophrenia population. Copyright © 2014 Elsevier B.V. All rights reserved.

Biosynthesis of the nargenicin A1 pyrrole moiety from Nocardia sp. CS682.

PubMed

Maharjan, Sushila; Aryal, Niraj; Bhattarai, Saurabh; Koju, Dinesh; Lamichhane, Janardan; Sohng, Jae Kyung

2012-01-01

A number of structurally diverse natural products harboring pyrrole moieties possess a wide range of biological activities. Studies on biosynthesis of pyrrole ring have shown that pyrrole moieties are derived from L-proline. Nargenicin A(1), a saturated alicyclic polyketide from Nocardia sp. CS682, is a pyrrole-2-carboxylate ester of nodusmicin. We cloned and identified a set of four genes from Nocardia sp. CS682 that show sequence similarity to the respective genes involved in the biosynthesis of the pyrrole moieties of pyoluteorin in Pseudomonas fluorescens, clorobiocin in Streptomyces roseochromogenes subsp. Oscitans, coumermycin A(1) in Streptomyces rishiriensis, one of the pyrrole rings of undecylprodigiosin in Streptomyces coelicolor, and leupyrrins in Sorangium cellulosum. These genes were designated as ngnN4, ngnN5, ngnN3, and ngnN2. In this study, we presented the evidences that the pyrrole moiety of nargenicin A(1) was also derived from L-proline by the coordinated action of three proteins, NgnN4 (proline adenyltransferase), NgnN5 (proline carrier protein), and NgnN3 (flavine-dependent acyl-coenzyme A dehydrogenases). Biosynthesis of pyrrole moiety in nargenicin A(1) is initiated by NgnN4 that catalyzes ATP-dependent activation of L-proline into L-prolyl-AMP, and the latter is transferred to NgnN5 to create prolyl-S-peptidyl carrier protein (PCP). Later, NgnN3 catalyzes the two-step oxidation of prolyl-S-PCP into pyrrole-2-carboxylate. Thus, this study presents another example of a pyrrole moiety biosynthetic pathway that uses a set of three genes to convert L-proline into pyrrole-2-carboxylic acid moiety.

Biomass Yield and Steviol Glycoside Production in Callus and Suspension Culture of Stevia rebaudiana Treated with Proline and Polyethylene Glycol.

PubMed

Gupta, Pratibha; Sharma, Satyawati; Saxena, Sanjay

2015-06-01

Enhanced production of steviol glycosides (SGs) was observed in callus and suspension culture of Stevia rebaudiana treated with proline and polyethylene glycol (PEG). To study their effect, yellow-green and compact calli obtained from in vitro raised Stevia leaves were sub-cultured on MS medium supplemented with 2.0 mg l(-1) NAA and different concentrations of proline (2.5-10 mM) and PEG (2.5-10 %) for 2 weeks, and incubated at 24 ± 1 °C and 22.4 μmol m(-2) s(-1) light intensity provided by white fluorescent tubes for 16 h. Callus and suspension culture biomass (i.e. both fresh and dry weight content) was increased with 5 mM proline and 5 % PEG, while at further higher concentrations, they got reduced. Further, quantification of SGs content in callus (collected at 15th day) and suspension culture (collected at 10th and 15th day) treated with and without elicitors was analysed by HPLC. It was observed that chemical stress enhanced the production of SGs significantly. In callus, the content of SGs increased from 0.27 (control) to 1.09 and 1.83 % with 7.5 mM proline and 5 % PEG, respectively, which was about 4.0 and 7.0 times higher than control. However, in the case of suspension culture, the same concentrations of proline and polyethylene glycol enhanced the SG content from 1.36 (control) to 5.03 and 6.38 %, respectively, on 10th day which were 3.7 times and 4.7 times higher than control.

A statistical analysis of the PPII propensity of amino acid guests in proline-rich peptides.

PubMed

Moradi, Mahmoud; Babin, Volodymyr; Sagui, Celeste; Roland, Christopher

2011-02-16

There has been considerable debate about the intrinsic PPII propensity of amino-acid residues in denatured polypeptides. Experimentally, the propensity scale is based on the behavior of guest amino-acid residues placed in the middle of polyproline hosts. We have used classical molecular dynamics simulations, with state-of-the-art force fields to carry out a comprehensive analysis of the conformational equilibria of the proline-based host oligopeptides with single guests. The tracked structural characteristics include the PPII content, the cis/trans isomerization of the prolyl bonds, the puckering of the pyrrolidine rings of the proline residues, and the secondary structural motifs. We find no evidence for an intrinsic PPII propensity in any of the guest amino acids other than proline. Instead, the PPII content as derived from experiments may be explained in terms of: 1), a local correlation between the dihedral angles of the guest amino acid and the proline residue immediately preceding it; and 2), a nonlocal correlation between the cis/trans states of the peptide bonds. In terms of the latter, we find that the presence of a guest (other than proline, tyrosine, or tryptophan) increases the trans content of most of the prolyl bonds, which results in an effective increase of the peptide PPII content. With respect to the local dihedral correlations, we find that these are well described in terms of the so-called odds-ratio statistic. Expressed in terms of free energy language, the PPII content based on the odds-ratio of the relevant residues correlate well with the experimentally measured PPII content. Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Conformational dynamics of minimal elastin-like polypeptides: the role of proline revealed by molecular dynamics and nuclear magnetic resonance.

PubMed

Glaves, Rachel; Baer, Marcel; Schreiner, Eduard; Stoll, Raphael; Marx, Dominik

2008-12-22

Previous molecular dynamics studies of the elastin-like peptide (ELP) GVG(VPGVG) predict that this ELP undergoes a conformational transition from an open to a more compact closed state upon an increase in temperature. These structural changes occurring in this minimal elastin model at the so-called inverse temperature transition (ITT), which takes place when elastin is heated to temperatures of about 20-40 (omicron)C, are investigated further in this work by means of a combined theoretical and experimental approach. To do this, additional extensive classical molecular dynamics (MD) simulations of the capped octapeptide are carried out, analyzed, and compared to data obtained from homonuclear magnetic resonance (NMR) spectroscopy of the same octapeptide. Moreover, in the previous simulations, the proline residue in the ELP is found to act as a hinge, thereby allowing for the large-amplitude opening and closing conformational motion of the ITT. To explore the role of proline in such elastin repeating units, a point mutant (P5I), which replaces the proline residue with an isoleucine residue, is also investigated using the aforementioned theoretical and experimental techniques. The results show that the site-directed mutation completely alters the properties of this ELP, thus confirming the importance of the highly conserved proline residue in the ITT. Furthermore, a correlation between the two different methods employed is seen. Both methods predict the mutant ELP to be present in an unstructured form and the wild type ELP to have a beta-turn-like structure. Finally, the role of the peptidyl cis to trans isomerization of the proline hinge is assessed in detail.

Synthesis of fluorescent label, DBD-beta-proline, and the resolution efficiency for chiral amines by reversed-phase chromatography.

PubMed

Min, Jun Zhe; Toyo'oka, Toshimasa; Kato, Masaru; Fukushima, Takeshi

2005-01-01

DBD-d(and l)-beta-proline, new fluorescent chiral derivatization reagents, were synthesized from the reaction of 4-(N,N-dimethylaminosulfonyl)-7- fl uoro-2,1,3-benzoxadiazole (DBD-F) with beta-proline. The racemic mixture synthesized was separated by a chiral stationary phase (CSP) column, Chiralpak AD-H, with n-hexane-EtOH-TFA-diethylamine (70:30:0.1:0.1) as the mobile phase. The dl-forms were decided according to the results obtained from a circular dichroism (CD) detector after separation by the CSP column. The fractionated enantiomers reacted with chiral amine to produce a couple of diastereomers. The labeling proceeded in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and pyridine as the activation reagents. The reaction conditions were mild and no racemization occurred during the diastereomer formation. The resulting diastereomers fluoresced at around 570 nm (excitation at around 460 nm). Good linearity of the calibration curves was obtained in the range 1-75 pmol and the detection limits on chromatogram were less than 1 pmol. The separability of the diastereomers was compared with the diastereomers derived from DBD-d(or l)-proline. The resolution values (Rs) obtained from the diastereomers of three chiral amines with DBD-d(or l)-beta-proline were higher than those derived from DBD-d(or l)-proline, e.g. dl-phenylalanine methylester (dl-PAME), 2.23 vs 1.37; (R)(S)-1-phenylethylamine [(R)(S)-PEA], 2.09 vs 1.13; and (R)(S)-1-(1-naphthyl)ethylamines [(R)(S)-NEA], 5.19 vs 1.23. The results suggest that the position of COOH group on pyrrolidine moiety in the structures is one of the important factors for the efficient separation of a couple of the diastereomers.

Arginine and proline applied as food additives stimulate high freeze tolerance in larvae of Drosophila melanogaster.

PubMed

Koštál, Vladimír; Korbelová, Jaroslava; Poupardin, Rodolphe; Moos, Martin; Šimek, Petr

2016-08-01

The fruit fly Drosophila melanogaster is an insect of tropical origin. Its larval stage is evolutionarily adapted for rapid growth and development under warm conditions and shows high sensitivity to cold. In this study, we further developed an optimal acclimation and freezing protocol that significantly improves larval freeze tolerance (an ability to survive at -5°C when most of the freezable fraction of water is converted to ice). Using the optimal protocol, freeze survival to adult stage increased from 0.7% to 12.6% in the larvae fed standard diet (agar, sugar, yeast, cornmeal). Next, we fed the larvae diets augmented with 31 different amino compounds, administered in different concentrations, and observed their effects on larval metabolomic composition, viability, rate of development and freeze tolerance. While some diet additives were toxic, others showed positive effects on freeze tolerance. Statistical correlation revealed tight association between high freeze tolerance and high levels of amino compounds involved in arginine and proline metabolism. Proline- and arginine-augmented diets showed the highest potential, improving freeze survival to 42.1% and 50.6%, respectively. Two plausible mechanisms by which high concentrations of proline and arginine might stimulate high freeze tolerance are discussed: (i) proline, probably in combination with trehalose, could reduce partial unfolding of proteins and prevent membrane fusions in the larvae exposed to thermal stress (prior to freezing) or during freeze dehydration; (ii) both arginine and proline are exceptional among amino compounds in their ability to form supramolecular aggregates which probably bind partially unfolded proteins and inhibit their aggregation under increasing freeze dehydration. © 2016. Published by The Company of Biologists Ltd.

Effect of Subsoiling in Fallow Period on Soil Water Storage and Grain Protein Accumulation of Dryland Wheat and Its Regulatory Effect by Nitrogen Application

PubMed Central

Sun, Min; Gao, ZhiQiang; Zhao, WeiFeng; Deng, LianFeng; Deng, Yan; Zhao, HongMei; Ren, AiXia; Li, Gang; Yang, ZhenPing

2013-01-01

To provide a new way to increase water storage and retention of dryland wheat, a field study was conducted at Wenxi experimental site of Shanxi Agricultural University. The effect of subsoiling in fallow period on soil water storage, accumulation of proline, and formation of grain protein after anthesis were determined. Our results showed that subsoiling in fallow period could increase water storage in the 0–300 cm soil at pre-sowing stage and at anthesis stage with low or medium N application, especially for the 60–160 cm soil. However, the proline content, glutamine synthetase (GS) activity, glutamate dehydrogenase (GDH) activity in flag leaves and grains were all decreased by subsoiling in fallow period. In addition, the content of albumin, gliadin, and total protein in grains were also decreased while globulin content, Glu/Gli, protein yield, and glutelin content were increased. With N application increasing, water storage of soil layers from 20 to 200 cm was decreased at anthesis stage. High N application resulted in the increment of proline content and GS activity in grains. Besides, correlation analysis showed that soil storage in 40–160 cm soil was negatively correlated with proline content in grains; proline content in grains was positively correlated with GS and GDH activity in flag leaves. Contents of albumin, globulin and total protein in grains were positively correlated with proline content in grains and GDH activity in flag leaves. In conclusion, subsoiling in fallow period, together with N application at 150 kg·hm−2, was beneficial to increase the protein yield and Glu/Gli in grains which improve the quality of wheat. PMID:24098371

Mechanisms of induced susceptibility to Diplodia tip blight in drought-stressed Austrian pine.

PubMed

Sherwood, Patrick; Villari, Caterina; Capretti, Paolo; Bonello, Pierluigi

2015-05-01

Plants experiencing drought stress are frequently more susceptible to pathogens, likely via alterations in physiology that create favorable conditions for pathogens. Common plant responses to drought include the production of reactive oxygen species (ROS) and the accumulation of free amino acids (AAs), particularly proline. These same phenomena also frequently occur during pathogenic attack. Therefore, drought-induced perturbations in AA and ROS metabolism could potentially contribute to the observed enhanced susceptibility. Furthermore, nitrogen (N) availability can influence AA accumulation and affect plant resistance, but its contributions to drought-induced susceptibility are largely unexplored. Here we show that drought induces accumulation of hydrogen peroxide (H2O2) in Austrian pine (Pinus nigra Arnold) shoots, but that shoot infection by the blight and canker pathogen Diplodia sapinea (Fr.) Fuckel leads to large reductions in H2O2 levels in droughted plants. In in vitro assays, H2O2 was toxic to D. sapinea, and the fungus responded to this oxidative stress by increasing catalase and peroxidase activities, resulting in substantial H2O2 degradation. Proline increased in response to drought and infection when examined independently, but unlike all other AAs, proline further increased in infected shoots of droughted trees. In the same tissues, the proline precursor, glutamate, decreased significantly. Proline was found to protect D. sapinea from H2O2 damage, while also serving as a preferred N source in vitro. Fertilization increased constitutive and drought-induced levels of some AAs, but did not affect plant resistance. A new model integrating interactions of proline and H2O2 metabolism with drought and fungal infection of plants is proposed. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

The adsorption and reactions of the amino acid proline on rutile TiO 2(1 1 0) surfaces

NASA Astrophysics Data System (ADS)

Fleming, G. J.; Adib, K.; Rodriguez, J. A.; Barteau, M. A.; White, J. M.; Idriss, H.

2008-06-01

The reaction of the amino acid DL-proline is studied over stoichiometric and Ar-ions sputtered (reduced) TiO 2(1 1 0) single crystal surfaces by synchrotron High Resolution X-ray Photoelectron Spectroscopy (HRXPS). On the stoichiometric surface proline gives two different species at 300 K: dissociated and zwitterionic. Upon heating the zwitterionic structure is removed first from the surface followed by the dissociated form. The C1s signal for the COO function is found close to 288.5 eV for both forms while the N 1s for the dissociated form is found at 400.0 eV and that of the zwitterionic from close to 401.8 eV. From the attenuation of the Ti 2p signal the surface coverage was estimated less than ½ (about 0.35). This smaller coverage than dissociatively adsorbed carboxylic acids on this surface (usually close to ½), is attributed to lateral repulsion caused by the ring of adjacent proline molecules adsorbed on five-fold coordinated Ti cations along the [0 0 1] direction. On the reduced surface the amount of zwitterion structure is found two times higher than that on the stoichiometric surface, at 300 K, most likely due to the considerable decrease of the amount of surface oxygen available. The stability of the zwitterionic structure on this surface is however found similar to that found on the stoichiometric surface. In addition, evidence of oxidation of reduced Ti cations upon adsorption at 300 K is noticed and explained as breaking of the carbon-oxygen bond of a fraction of adsorbed proline. Variable temperature HRXPS has been collected and results indicated that proline is more stable on the reduced surface compared to the stoichiometric surface.

Effect of Azotobacter vinelandii and compatible solutes on germination wheat seeds and root concentrations of sodium and potassium under salt stress.

PubMed

Silini, A; Silini-Chérif, H; Ghoul, M

2012-02-01

The effect of plant growth-promoting Rhizobacteria (PGPR) and exogenous application of compatible solutes on seed germination and root concentrations of sodium and potassium of two wheat varieties (Triticum durum L.) were evaluated under saline stress. In this experiment, Azotobacter vinelandii strain DSM85, glycine betaine and proline were used. Inoculated seeds for each variety were placed on Whatman paper in 9 cm Petri dishes containing 15 mL of distilled water or NaCl solutions at various concentrations (control, 100, 200, 300 mM) supplemented with or without glycine betaine (GB) or proline at 5 mM. The results indicated that addition of proline (5 mM) stimulated the production of indol acetic acid and the growth of A. vinelandii at 200 and 300 mM NaCl, respectively. The germination rate index and the germination final percentage decreased significantly (p < 0.05) with increasing salinity level. The germination was significantly diminished at 300 mM with significant variation among varieties and Waha variety had higher germination percentage than Bousselam variety. Inoculation of seeds by A. vinelandii and exogenous application of proline had significantly positive effect on the germination at this concentration of NaCl. The rate of accumulation of Na+ in roots was important at 100 mM and increased at 200 mM. The concentration of K+ decreased when salinity increased. The effect of inoculation or inoculation with proline decreased the accumulation of Na' and reduced the loss of K+ under salt stress. From the present study we can conclude that the use of A. vinelandii strain DSM85 and external application of low concentrations of proline on seeds might be considered as a strategy for the protection of plants under saline stress.

Proline analogue of nitrosourea as a new cytotoxic prodrug.

PubMed

Stankiewicz-Kranc, Anna; Bielawska, Anna; Bielawski, Krzysztof; Skrzydlewska, Elzbieta

2009-11-01

Carmustine is frequently used as anticancer drug. High toxicity and low selectivity reduces the application of this drug. Though, there is a necessity to find new compounds characterized by similar therapeutic effects but a higher selectivity and safety. As a result, the proline analogue of nitrosourea, N-[N'-(2-bromophenyl)-N'-nitrosocarbamoyl]proline (AC), has been synthesized. The aim of this study was to compare the influence of carmustine and the proline analogue of nitrosourea on the antioxidant abilities of fibroblasts and leukemia cells, MOLT4. It was shown that carmustine as well as AC cause an increase in hydrogen peroxide concentration in normal and neoplastic cells. Incubation with both compounds led to a diminution of the activity of antioxidant enzymes such as superoxide dismutase, glutathione peroxidase, and reductase. Changes in activity / level of antioxidant parameters were accompanied by augmentation of lipid and oxidative protein modifications. In conclusion, carmustine and AC cause changes in the antioxidative system of normal and MOLT4 cells and are a reason of oxidative stress formation.

Polyamines induce adaptive responses in water deficit stressed cucumber roots.

PubMed

Kubiś, Jan; Floryszak-Wieczorek, Jolanta; Arasimowicz-Jelonek, Magdalena

2014-01-01

The aim of this study was to investigate the effect of exogenous polyamines (PAs) on the membrane status and proline level in roots of water stressed cucumber (Cucumis sativus cv. Dar) seedlings. It was found that water shortage resulted in an increase of membrane injury, lipoxygenase (LOX) activity, lipid peroxidation and proline concentration in cucumber roots during progressive dehydration. PA pretreatment resulted in a distinct reduction of the injury index, and this effect was reflected by a lower stress-evoked LOX activity increase and lipid peroxide levels at the end of the stress period. In contrast, PA-supplied stressed roots displayed a higher proline accumulation. The presented results suggest that exogenous PAs are able to alleviate water deficit-induced membrane permeability and diminish LOX activity. Observed changes were accompanied by an accumulation of proline, suggesting that the accumulation of this osmolyte might be another possible mode of action for PAs to attain higher membrane stability, and in this way mitigate water deficit effects in roots of cucumber seedlings.

Isolation of glycine betaine and proline betaine from human urine. Assessment of their role as osmoprotective agents for bacteria and the kidney.

PubMed Central

Chambers, S T; Kunin, C M

1987-01-01

Human urine is osmoprotective for enteric bacteria, permitting E. coli to grow with high concentrations of NaCl and other salts and even higher concentrations of sucrose and mannitol but not urea. The active material in urine is soluble in methanol and is precipitated by ammonium reineckate at acid pH. Using gel filtration and high-pressure liquid chromatography, we have identified two major osmoprotective compounds in urine. One is glycine betaine; the other is proline betaine as demonstrated by nuclear magnetic resonance, mass spectrum scanning, and chemical synthesis. Proline betaine has not been described previously to our knowledge in vertebrate tissues. It is known to be a cell volume-regulating agent for marine red algae and the euryhaline mollusk Elysia chloritica. We suggest that the presence of glycine and proline betaines in human urine may reflect an osmoprotective role for the kidney and that they protect bacteria in the urine only fortuitously. PMID:3546377

Evidence that a phosphorylated derivative of 5-oxoproline (5-OP) is an intermediate in the reaction catalyzed by 5-oxoprolinase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Seddon, A.P.; Meister, A.

1986-05-01

5-oxo-L-proline + ATP + 2 H/sub 2/O ..-->.. L-Glutamate + ADP + P/sub i/ Pseudomonas putida 5-oxoprolinase consists of 2 protein components (A and B). A catalyzes 5-OP-dependent ATPase. B, which does not appear to interact with ATP or 5-OP, is required for catalysis of the complete reaction. Application of the isotope trapping method showed that 5-OP is converted stoichiometrically to an enzyme-bound form in a process requiring ATP and its cleavage. The overall reaction is not inhibited by high concentrations of borohydride. When the reaction catalyzed by A alone is carried out in the presence of 0.1-0.6 M KBH/submore » 4/ there is catalytic formation of proline. Studies with KB/sup 3/H/sub 4/ showed incorporation of /sup 3/H into proline. Proline formation is prevented by addition of B.« less

Proline Oxidase (POX) as A Target for Cancer Therapy.

PubMed

Kononczuk, Joanna; Czyzewska, Urszula; Moczydlowska, Joanna; Surażyński, Arkadiusz; Palka, Jerzy; Miltyk, Wojciech

2015-01-01

Proline dehydrogenase/proline oxidase (PRODH/POX) is an enzyme catalyzing the first step of proline degradation, during which ROS and/or ATP is generated. POX is widely distributed in living organisms and is responsible for a number of regulatory processes such as redox homeostasis, osmotic adaptation, cell signaling and oxidative stress. Recent data provided evidence that POX plays an important role in carcinogenesis and tumor growth. POX may induce apoptosis in both intrinsic and extrinsic way. Due to ROS generation, POX may induce caspase-9 activity, which mediates mitochondrial apoptosis (intrinsic apoptosis pathway). POX can also stimulate TRAIL (tumor necrosis factorrelated apoptosis inducing ligand) and DR5 (death receptor 5) expression, resulting in cleavage of procaspase-8 and thus extrinsic apoptotic pathway. However, this tumor suppressor in certain environmental conditions may act as a prosurvival factor. Genotoxic, inflammatory and metabolic stress may switch POX from tumor growth inhibiting to tumor growth supporting factor. The potential mechanisms which may regulate switching of POX mode are discussed in this review.

Conformational Preferences of α-Substituted Proline Analogues

PubMed Central

Flores-Ortega, Alejandra; Jiménez, Ana I.; Cativiela, Carlos; Nussinov, Ruth; Alemán, Carlos; Casanovas, Jordi

2009-01-01

DFT calculations at the B3LYP/6-31+G(d,p) level have been used to investigate how the replacement of the α hydrogen by a more sterically demanding group affects the conformational preferences of proline. Specifically, the N-acetyl-N’-methylamide derivatives of L-proline, L-α-methylproline and L-α-phenylproline have been calculated, with both the cis/trans isomerism of the peptide bonds and the puckering of the pyrrolidine ring being considered. The effects of solvation have been evaluated using a Self Consistent Reaction Field model. As expected, tetrasubstitution at the α carbon destabilizes the conformers with one or more peptide bonds arranged in cis. The lowest energy minimum has been found to be identical for the three compounds investigated, but important differences are observed regarding other energetically accessible backbone conformations. The results obtained provide evidence that the distinct steric requirements of the substituent at Cα may play a significant role in modulating the conformational preferences of proline. PMID:18351745

Effect of addition of Proline, ionic liquid [Choline][Pro] on CO2 separation properties of poly(amidoamine) dendrimer / poly(ethylene glycol) hybrid membranes

NASA Astrophysics Data System (ADS)

Duan, S. H.; Kai, T.; Chowdhury, F. A.; Taniguchi, I.; Kazama, S.

2018-01-01

Poly(amidoamine) (PAMAM) dendrimers were incorporated into cross-linked poly(ethylene glycol) (PEGDMA) matrix to improve carbon dioxide (CO2) separation performance at elevated pressures. In our previous studies, PAMAM/PEGDMA hybrid membranes showed high CO2 separation properties from CO2/H2 mixed gases. In this study, proline, choline and ionic liquid [Choline][Pro] compounds were selected as rate promoters that were used to prepare PAMAM/PEGDMA hybrid membranes. The effect of addition of proline, choline, IL [Choline][Pro] on separation performance of PAMAM/PEGDMA) hybrid membranes for CO2/H2 separation was investigated. Amino acid proline, choline, and IL [Choline][Pro] were used to promote CO2 and amine reaction. With the addition of [Choline][Pro] into PAMAM/PEG membrane, CO2 permeance of PAMAM/PEG hybrid membranes are increased up to 46% without any change of selectivity of membrane for CO2.

Improved Atomistic Monte Carlo Simulations Demonstrate that Poly-L-Proline Adopts Heterogeneous Ensembles of Conformations of Semi-Rigid Segments Interrupted by Kinks

PubMed Central

Radhakrishnan, Aditya; Vitalis, Andreas; Mao, Albert H.; Steffen, Adam T.; Pappu, Rohit V.

2012-01-01

Poly-L-proline (PLP) polymers are useful mimics of biologically relevant proline-rich sequences. Biophysical and computational studies of PLP polymers in aqueous solutions are challenging because of the diversity of length scales and the slow time scales for conformational conversions. We describe an atomistic simulation approach that combines an improved ABSINTH implicit solvation model, with conformational sampling based on standard and novel Metropolis Monte Carlo moves. Refinements to forcefield parameters were guided by published experimental data for proline-rich systems. We assessed the validity of our simulation results through quantitative comparisons to experimental data that were not used in refining the forcefield parameters. Our analysis shows that PLP polymers form heterogeneous ensembles of conformations characterized by semi-rigid, rod-like segments interrupted by kinks, which result from a combination of internal cis peptide bonds, flexible backbone ψ-angles, and the coupling between ring puckering and backbone degrees of freedom. PMID:22329658

Proline adsorption on TiO 2(1 1 0) single crystal surface: A study by high resolution photoelectron spectroscopy

NASA Astrophysics Data System (ADS)

Fleming, G. J.; Adib, K.; Rodriguez, J. A.; Barteau, M. A.; Idriss, H.

2007-12-01

The surface chemistry and binding of DL-proline were investigated on the oxidised (stoichiometric) and reduced (sub-stoichiometric) TiO 2(1 1 0) single crystal surfaces. TiO 2 was chosen as the substrate as it best represents the surface of a biomedical implant, which bio-molecules interact with during the healing of bone/teeth fractures (molecular recognition). High resolution X-ray photoelectron spectroscopy (HR-XPS) studies of the C1s and N1s regions revealed that DL-proline is present in two forms (dissociated and zwitterionic) on the oxidised TiO 2 surface. On TiO 2(1 1 0) surfaces reduced by Ar + sputtering, a significant increase in the amount of zwitterionic proline at the surface was detected when compared with the oxidised surface. Study of the temperature effect showed that in both cases the zwitterionic structure was the less stable structure. The reason for its relative instability appears to be thermodynamic.

Nine of 16 stereoisomeric polyhydroxylated proline amides are potent β-N-acetylhexosaminidase inhibitors.

PubMed

Ayers, Benjamin J; Glawar, Andreas F G; Martínez, R Fernando; Ngo, Nigel; Liu, Zilei; Fleet, George W J; Butters, Terry D; Nash, Robert J; Yu, Chu-Yi; Wormald, Mark R; Nakagawa, Shinpei; Adachi, Isao; Kato, Atsushi; Jenkinson, Sarah F

2014-04-18

All 16 stereoisomeric N-methyl 5-(hydroxymethyl)-3,4-dihydroxyproline amides have been synthesized from lactones accessible from the enantiomers of glucuronolactone. Nine stereoisomers, including all eight with a (3R)-hydroxyl configuration, are low to submicromolar inhibitors of β-N-acetylhexosaminidases. A structural correlation between the proline amides is found with the ADMDP-acetamide analogues bearing an acetamidomethylpyrrolidine motif. The proline amides are generally more potent than their ADMDP-acetamide equivalents. β-N-Acetylhexosaminidase inhibition by an azetidine ADMDP-acetamide analogue is compared to an azetidine carboxylic acid amide. None of the amides are good α-N-acetylgalactosaminidase inhibitors.

21 CFR 582.5650 - Proline.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Proline. 582.5650 Section 582.5650 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements 1...

21 CFR 582.5650 - Proline.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Proline. 582.5650 Section 582.5650 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements 1...

21 CFR 582.5650 - Proline.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Proline. 582.5650 Section 582.5650 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements 1...

21 CFR 582.5650 - Proline.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Proline. 582.5650 Section 582.5650 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements 1...

21 CFR 582.5650 - Proline.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 6 2012-04-01 2012-04-01 false Proline. 582.5650 Section 582.5650 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements 1...

Prediction of cis/trans isomerization in proteins using PSI-BLAST profiles and secondary structure information.

PubMed

Song, Jiangning; Burrage, Kevin; Yuan, Zheng; Huber, Thomas

2006-03-09

The majority of peptide bonds in proteins are found to occur in the trans conformation. However, for proline residues, a considerable fraction of Prolyl peptide bonds adopt the cis form. Proline cis/trans isomerization is known to play a critical role in protein folding, splicing, cell signaling and transmembrane active transport. Accurate prediction of proline cis/trans isomerization in proteins would have many important applications towards the understanding of protein structure and function. In this paper, we propose a new approach to predict the proline cis/trans isomerization in proteins using support vector machine (SVM). The preliminary results indicated that using Radial Basis Function (RBF) kernels could lead to better prediction performance than that of polynomial and linear kernel functions. We used single sequence information of different local window sizes, amino acid compositions of different local sequences, multiple sequence alignment obtained from PSI-BLAST and the secondary structure information predicted by PSIPRED. We explored these different sequence encoding schemes in order to investigate their effects on the prediction performance. The training and testing of this approach was performed on a newly enlarged dataset of 2424 non-homologous proteins determined by X-Ray diffraction method using 5-fold cross-validation. Selecting the window size 11 provided the best performance for determining the proline cis/trans isomerization based on the single amino acid sequence. It was found that using multiple sequence alignments in the form of PSI-BLAST profiles could significantly improve the prediction performance, the prediction accuracy increased from 62.8% with single sequence to 69.8% and Matthews Correlation Coefficient (MCC) improved from 0.26 with single local sequence to 0.40. Furthermore, if coupled with the predicted secondary structure information by PSIPRED, our method yielded a prediction accuracy of 71.5% and MCC of 0.43, 9% and 0.17 higher than the accuracy achieved based on the singe sequence information, respectively. A new method has been developed to predict the proline cis/trans isomerization in proteins based on support vector machine, which used the single amino acid sequence with different local window sizes, the amino acid compositions of local sequence flanking centered proline residues, the position-specific scoring matrices (PSSMs) extracted by PSI-BLAST and the predicted secondary structures generated by PSIPRED. The successful application of SVM approach in this study reinforced that SVM is a powerful tool in predicting proline cis/trans isomerization in proteins and biological sequence analysis.

Post-Translational Modification and Secretion of Azelaic Acid Induced 1 (AZI1), a Hybrid Proline-Rich Protein from Arabidopsis

PubMed Central

Pitzschke, Andrea; Xue, Hui; Persak, Helene; Datta, Sneha; Seifert, Georg J.

2016-01-01

Arabidopsis EARLI-type hybrid proline-rich proteins (HyPRPs) consist of a putative N-terminal secretion signal, a proline-rich domain (PRD), and a characteristic eight-cysteine-motif (8-CM). They have been implicated in biotic and abiotic stress responses. AZI1 is required for systemic acquired resistance and it has recently been identified as a target of the stress-induced mitogen-activated protein kinase MPK3. AZI1 gel migration properties strongly indicate AZI1 to undergo major post-translational modifications. These occur in a stress-independent manner and are unrelated to phosphorylation by MAPKs. As revealed by transient expression of AZI1 in Nicotiana benthamiana and Tropaeolum majus, the Arabidopsis protein is similarly modified in heterologous plant species. Proline-rich regions, resembling arabinogalactan proteins point to a possible proline hydroxylation and subsequent O-glycosylation of AZI1. Consistently, inhibition of prolyl hydroxylase reduces its apparent protein size. AZI1 secretion was examined using Arabidopsis protoplasts and seedling exudates. Employing Agrobacterium-mediated leaf infiltration of N. benthamiana, we attempted to assess long-distance movement of AZI1. In summary, the data point to AZI1 being a partially secreted protein and a likely new member of the group of hydroxyproline-rich glycoproteins. Its dual location suggests AZI1 to exert both intra- and extracellular functions. PMID:26771603

Proline-poor hydrophobic domains modulate the assembly and material properties of polymeric elastin.

PubMed

Muiznieks, Lisa D; Reichheld, Sean E; Sitarz, Eva E; Miao, Ming; Keeley, Fred W

2015-10-01

Elastin is a self-assembling extracellular matrix protein that provides elasticity to tissues. For entropic elastomers such as elastin, conformational disorder of the monomer building block, even in the polymeric form, is essential for elastomeric recoil. The highly hydrophobic monomer employs a range of strategies for maintaining disorder and flexibility within hydrophobic domains, particularly involving a minimum compositional threshold of proline and glycine residues. However, the native sequence of hydrophobic elastin domain 30 is uncharacteristically proline-poor and, as an isolated polypeptide, is susceptible to formation of amyloid-like structures comprised of stacked β-sheet. Here we investigated the biophysical and mechanical properties of multiple sets of elastin-like polypeptides designed with different numbers of proline-poor domain 30 from human or rat tropoelastins. We compared the contributions of these proline-poor hydrophobic sequences to self-assembly through characterization of phase separation, and to the tensile properties of cross-linked, polymeric materials. We demonstrate that length of hydrophobic domains and propensity to form β-structure, both affecting polypeptide chain flexibility and cross-link density, play key roles in modulating elastin mechanical properties. This study advances the understanding of elastin sequence-structure-function relationships, and provides new insights that will directly support rational approaches to the design of biomaterials with defined suites of mechanical properties. © 2015 Wiley Periodicals, Inc.

Changes in Amino Acid Content of Excised Leaves During Incubation I. The Effect of Water Content of Leaves and Atmospheric Oxygen Level

PubMed Central

Thompson, John F.; Stewart, Cecil R.; Morris, Clayton J.

1966-01-01

Excised leaves were incubated at various water contents to determine the effect of water status on amino acid composition. Considerable proteolysis took place during incubation with a resultant increase in each amino acid in the non-protein fraction. However, serine, proline, γ-aminobutyric acid and methyleysteine sulfoxide were the only amino acids in which there was an accumulation (i.e., net synthesis). Serine showed a small but consistent accumulation lasting for 6 days. Proline showed a greater accumulation but this ceased after 2 days. To learn more about the control of the proline accumulation during wilting, turgid and wilted leaves were incubated under aerobic and anaerobic conditions. The amino acid analyses showed that turgid leaves did not accumulate proline and that proline and methylcysteine sulfoxide accumulation was abolished by anaerobiosis. With other amino acids, relative concentration changes between wilted and non-wilted leaves were less striking than the difference between aerobic and anaerobic conditions. Under anaerobic conditions there was an increase in alanine and a large increase in γ-aminobutyric acid which were not evident in air. Serine, aspartic acid, glutamic acid, and glutamine disappeared more rapidly and glycine disappeared less rapidly under anaerobic than under aerobic conditions. On the basis of these results, several pathways of amino acid degradation were suggested. PMID:16656443

Low-energy electron collisions with proline and pyrrolidine: A comparative study

NASA Astrophysics Data System (ADS)

Barbosa, Alessandra Souza; Freitas, Thiago Corrêa; Bettega, M. H. F.

2018-02-01

We present a comparative study on the calculated cross sections obtained for the elastic collisions of low-energy electrons with the amino acid proline (C5H9NO2) and its building block pyrrolidine (C4H9N). We employed the Schwinger multichannel method implemented with pseudopotentials to compute integral, differential, and momentum transfer cross sections in the static-exchange plus polarization approximation, for energies up to 15 eV. We report three shape resonances for proline at around 1.7 eV, 6.8 eV, and 10 eV and two shape resonances for pyrrolidine centered at 7 eV and 10.2 eV. The present resonance energies are compared with available experimental data on vertical attachment energies and dissociative electron attachment, where a good agreement is found. From the comparison of the present results with available calculated cross sections for the simplest carboxylic acid, formic acid (HCOOH), and from electronic structure calculations, we found that the first resonance of proline, at 1.7 eV, is due the presence of the carboxylic group, whereas the other two structures, at 6.8 eV and 10 eV, clearly arise from the pyrrolidine ring. A comparison between the differential cross sections for proline and pyrrolidine at some selected energies of the incident electron is also reported in this paper.

Molecular cloning and expression analysis of the gene encoding proline dehydrogenase from Jatropha curcas L.

PubMed

Wang, Haibo; Ao, Pingxing; Yang, Shuanglong; Zou, Zhurong; Wang, Shasha; Gong, Ming

2015-03-01

Proline dehydrogenase (ProDH) (EC 1.5.99.8) is a key enzyme in the catabolism of proline. The enzyme JcProDH and its complementary DNA (cDNA) were isolated from Jatropha curcas L., an important woody oil plant used as a raw material for biodiesels. It has been classified as a member of the Pro_dh superfamily based on multiple sequence alignment, phylogenetic characterization, and its role in proline catabolism. Its cDNA is 1674 bp in length with a complete open reading frame of 1485 bp, which encodes a polypeptide chain of 494 amino acids with a predicted molecular mass of 54 kD and a pI of 8.27. Phylogenetic analysis indicated that JcProDH showed high similarity with ProDH from other plants. Reverse transcription PCR (RT-PCR) analysis revealed that JcProDH was especially abundant in the seeds and flowers but scarcely present in the stems, roots, and leaves. In addition, the expression of JcProDH increased in leaves experiencing environmental stress such as cold (5 °C), heat (42 °C), salt (300 mM), and drought (30 % PEG6000). The JcProDH protein was successfully expressed in the yeast strain INVSc1 and showed high enzyme activity in proline catabolism. This result confirmed that the JcProDH gene negatively participated in the stress response.

Post-Translational Modification and Secretion of Azelaic Acid Induced 1 (AZI1), a Hybrid Proline-Rich Protein from Arabidopsis.

PubMed

Pitzschke, Andrea; Xue, Hui; Persak, Helene; Datta, Sneha; Seifert, Georg J

2016-01-12

Arabidopsis EARLI-type hybrid proline-rich proteins (HyPRPs) consist of a putative N-terminal secretion signal, a proline-rich domain (PRD), and a characteristic eight-cysteine-motif (8-CM). They have been implicated in biotic and abiotic stress responses. AZI1 is required for systemic acquired resistance and it has recently been identified as a target of the stress-induced mitogen-activated protein kinase MPK3. AZI1 gel migration properties strongly indicate AZI1 to undergo major post-translational modifications. These occur in a stress-independent manner and are unrelated to phosphorylation by MAPKs. As revealed by transient expression of AZI1 in Nicotiana benthamiana and Tropaeolum majus, the Arabidopsis protein is similarly modified in heterologous plant species. Proline-rich regions, resembling arabinogalactan proteins point to a possible proline hydroxylation and subsequent O-glycosylation of AZI1. Consistently, inhibition of prolyl hydroxylase reduces its apparent protein size. AZI1 secretion was examined using Arabidopsis protoplasts and seedling exudates. Employing Agrobacterium-mediated leaf infiltration of N. benthamiana, we attempted to assess long-distance movement of AZI1. In summary, the data point to AZI1 being a partially secreted protein and a likely new member of the group of hydroxyproline-rich glycoproteins. Its dual location suggests AZI1 to exert both intra- and extracellular functions.

Improvement of Nitrogen Assimilation and Fermentation Kinetics under Enological Conditions by Derepression of Alternative Nitrogen-Assimilatory Pathways in an Industrial Saccharomyces cerevisiae Strain

PubMed Central

Salmon, Jean-Michel; Barre, Pierre

1998-01-01

Metabolism of nitrogen compounds by yeasts affects the efficiency of wine fermentation. Ammonium ions, normally present in grape musts, reduce catabolic enzyme levels and transport activities for nonpreferred nitrogen sources. This nitrogen catabolite repression severely impairs the utilization of proline and arginine, both common nitrogen sources in grape juice that require the proline utilization pathway for their assimilation. We attempted to improve fermentation performance by genetic alteration of the regulation of nitrogen-assimilatory pathways in Saccharomyces cerevisiae. One mutant carrying a recessive allele of ure2 was isolated from an industrial S. cerevisiae strain. This mutation strongly deregulated the proline utilization pathway. Fermentation kinetics of this mutant were studied under enological conditions on simulated standard grape juices with various nitrogen levels. Mutant strains produced more biomass and exhibited a higher maximum CO2 production rate than the wild type. These differences were primarily due to the derepression of amino acid utilization pathways. When low amounts of dissolved oxygen were added, the mutants could assimilate proline. Biomass yield and fermentation rate were consequently increased, and the duration of the fermentation was substantially shortened. S. cerevisiae strains lacking URE2 function could improve alcoholic fermentation of natural media where proline and other poorly assimilated amino acids are the major potential nitrogen source, as is the case for most fruit juices and grape musts. PMID:9758807

Design of cyclic peptides featuring proline predominantly in the cis conformation under physiological conditions.

PubMed

Malešević, Miroslav; Schumann, Michael; Jahreis, Günther; Fischer, Gunter; Lücke, Christian

2012-09-24

Turns are secondary-structure elements that are omnipresent in natively folded polypeptide chains. A large variety of four-residue β-turns exist, which differ mainly in the backbone dihedral angle values of the two central residues i+1 and i+2. The βVI-type turns are of particular biological interest because the i+2 residue is always a proline in the cis conformation and might thus serve as target of peptidyl prolyl cis/trans isomerases (PPIases). We have designed cyclic hexapeptides containing two proline residues that predominantly adopt the cis conformation in aqueous solution. NMR data and MD calculations indicated that the cyclic peptide sequences c-(-DXaa-Ser-Pro-DXaa-Lys-Pro-) result in highly symmetric backbone structures when both prolines are in the cis conformation and the D-amino acids are either alanine or phenylalanine residues. Replacement of the serine residue either by phosphoserine or by tyrosine compromises this symmetry, but further increases the cis conformation content of both prolines. As a result, we obtained a cyclic hexapeptide that exists almost exclusively as the cis-Pro/cis-Pro conformer but shows no cis/trans interconversion even in the presence of the PPIase Pin1, apparently due to an energetically quite favorable but highly restricted conformational space. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Phosphorylation effects on cis/trans isomerization and the backbone conformation of serine-proline motifs: accelerated molecular dynamics analysis.

PubMed

Hamelberg, Donald; Shen, Tongye; McCammon, J Andrew

2005-02-16

The presence of serine/threonine-proline motifs in proteins provides a conformational switching mechanism of the backbone through the cis/trans isomerization of the peptidyl-prolyl (omega) bond. The reversible phosphorylation of the serine/threonine modulates this switching in regulatory proteins to alter signaling and transcription. However, the mechanism is not well understood. This is partly because cis/trans isomerization is a very slow process and, hence, difficult to study. We have used our accelerated molecular dynamics method to study the cis/trans proline isomerization, preferred backbone conformation of a serine-proline motif, and the effects of phosphorylation of the serine residue. We demonstrate that, unlike normal molecular dynamics, the accelerated molecular dynamics allows for the system to escape very easily from the trans isomer to cis isomer, and vice versa. Moreover, for both the unphosphorylated and phosphorylated peptides, the statistical thermodynamic properties are recaptured, and the results are consistent with experimental values. Isomerization of the proline omega bond is shown to be asymmetric and strongly dependent on the psi backbone angle before and after phosphorylation. The rates of escape decrease after phosphorylation. Also, the alpha-helical backbone conformation is more favored after phosphorylation. This accelerated molecular dynamics approach provides a general approach for enhancing the conformational transitions of molecular systems without having prior knowledge of the location of the minima and barriers on the potential-energy landscape.

The role of the proline-rich domain of Ssdp1 in the modular architecture of the vertebrate head organizer

PubMed Central

Enkhmandakh, Badam; Makeyev, Alexandr V.; Bayarsaihan, Dashzeveg

2006-01-01

Lim1, Ssdp1, and Ldb1 proteins are components of the Ldb1-associated transcriptional complex, which is important in the head-organizing activity during early mouse development. Depletion of each individual protein alone causes a headless phenotype. To explore in more detail the modular architecture of the complex, we have generated two different gene-trapped mouse lines that express truncated forms of Ssdp1. Embryos derived from the gene-trapped line that encodes a truncated Ssdp1 lacking the proline-rich sequence exhibit a lethal abnormal head-development phenotype, resembling mouse embryos deficient for Lim1, Ssdp1, or Otx2 genes. Embryos derived from the second gene-trapped line, in which most of the proline-rich domain of Ssdp1 is retained, did not show abnormalities in head development. Our data demonstrate that components of the Ldb1-dependent module can be subdivided further into discrete functional domains and that the proline-rich stretch of Ssdp1 is critical for embryonic head development. Furthermore, phylogenetic comparisons revealed that in Caenorhabditis elegans, a similar proline-rich sequence is absent in Ssdp but present in Ldb1. We conclude that although the overall architecture of the Ldb1-dependent module has been preserved, the genetic specification of its individual components has diversified during evolution, without compromising the function of the module. PMID:16864769

The role of the proline-rich domain of Ssdp1 in the modular architecture of the vertebrate head organizer.

PubMed

Enkhmandakh, Badam; Makeyev, Alexandr V; Bayarsaihan, Dashzeveg

2006-08-01

Lim1, Ssdp1, and Ldb1 proteins are components of the Ldb1-associated transcriptional complex, which is important in the head-organizing activity during early mouse development. Depletion of each individual protein alone causes a headless phenotype. To explore in more detail the modular architecture of the complex, we have generated two different gene-trapped mouse lines that express truncated forms of Ssdp1. Embryos derived from the gene-trapped line that encodes a truncated Ssdp1 lacking the proline-rich sequence exhibit a lethal abnormal head-development phenotype, resembling mouse embryos deficient for Lim1, Ssdp1, or Otx2 genes. Embryos derived from the second gene-trapped line, in which most of the proline-rich domain of Ssdp1 is retained, did not show abnormalities in head development. Our data demonstrate that components of the Ldb1-dependent module can be subdivided further into discrete functional domains and that the proline-rich stretch of Ssdp1 is critical for embryonic head development. Furthermore, phylogenetic comparisons revealed that in Caenorhabditis elegans, a similar proline-rich sequence is absent in Ssdp but present in Ldb1. We conclude that although the overall architecture of the Ldb1-dependent module has been preserved, the genetic specification of its individual components has diversified during evolution, without compromising the function of the module.

Proline/pipecolinic acid-promoted copper-catalyzed P-arylation.

PubMed

Huang, Cheng; Tang, Xu; Fu, Hua; Jiang, Yuyang; Zhao, Yufen

2006-06-23

We have developed a convenient and efficient approach for P-arylation of organophosphorus compounds containing P-H. Using commercially available and inexpensive proline and pipecolinic acid as the ligands greatly improved the efficiency of the coupling reactions, so the method can provide an entry to arylphosphonates, arylphosphinates and arylphosphine oxides.

An improved colorimetric method for the determination of proline in the presence of other ninhydrin-positive compounds

PubMed Central

Wren, J. J.; Wiggall, P. H.

1965-01-01

1. The conditions required for sensitive and specific colorimetric determination of proline with acidified ninhydrin were investigated. 2. A method applicable to protein samples was developed. 3. The only compound found to interfere appreciably was a hydroxyproline. PMID:14342233

The Tomato Hybrid Proline-Rich Protein regulates the abcission zone competence to respond to ethylene signals

USDA-ARS?s Scientific Manuscript database

The Tomato Hybrid Proline-Rich Protein (THyPRP) gene was specifically expressed in the tomato (Solanum lycopersicum) flower abscission zone (FAZ), and its stable antisense silencing under the control of an abscission zone (AZ)-specific promoter, Tomato Abscission Polygalacturonase4,significantly inh...

Proline-induced kink in a helix arises primarily from dihedral angle energy: a molecular dynamics simulation on alamethicin

NASA Astrophysics Data System (ADS)

Cheng, Shu-Fang; Chang, Ding-Kwo

1999-03-01

To investigate the cause of the kink in a helix induced by proline, a restrained molecular dynamics simulation was performed on alamethicin (Alm), a channel-forming peptide. Results on the linear and kinked helices indicated that the kinked form has lower energy than the linear one and the difference can be attributed largely to the dihedral angle term. The argument is strengthened by calculated results on the peptide in which proline 14 was substituted by alanine. The kinked and linear forms had virtually the same total and dihedral angle energies for the alanine analog. Possible implications for Alm voltage-gating are discussed.

Kinetic profiling of prolinate-catalyzed α-amination of aldehydes.

PubMed

Hein, Jason E; Armstrong, Alan; Blackmond, Donna G

2011-08-19

Deconvolution of the role of off-cycle species from the desired catalytic cycle leads to an optimized protocol for the prolinate-catalyzed amination of aldehydes. The scope of complex reaction networks will be greatly broadened by understanding ancillary rate processes that influence the productive catalytic pathway. © 2011 American Chemical Society

Allosteric mechanism controls traffic in the chaperone/usher pathway.

PubMed

Di Yu, Xiao; Dubnovitsky, Anatoly; Pudney, Alex F; Macintyre, Sheila; Knight, Stefan D; Zavialov, Anton V

2012-11-07

Many virulence organelles of Gram-negative bacterial pathogens are assembled via the chaperone/usher pathway. The chaperone transports organelle subunits across the periplasm to the outer membrane usher, where they are released and incorporated into growing fibers. Here, we elucidate the mechanism of the usher-targeting step in assembly of the Yersinia pestis F1 capsule at the atomic level. The usher interacts almost exclusively with the chaperone in the chaperone:subunit complex. In free chaperone, a pair of conserved proline residues at the beginning of the subunit-binding loop form a "proline lock" that occludes the usher-binding surface and blocks usher binding. Binding of the subunit to the chaperone rotates the proline lock away from the usher-binding surface, allowing the chaperone-subunit complex to bind to the usher. We show that the proline lock exists in other chaperone/usher systems and represents a general allosteric mechanism for selective targeting of chaperone:subunit complexes to the usher and for release and recycling of the free chaperone. Copyright © 2012 Elsevier Ltd. All rights reserved.

Experimental and Molecular Modeling Evaluation of the Physicochemical Properties of Proline-Based Deep Eutectic Solvents.

PubMed

van den Bruinhorst, Adriaan; Spyriouni, Theodora; Hill, Jörg-Rüdiger; Kroon, Maaike C

2018-01-11

The liquid range and applicability of deep eutectic solvents (DESs) are determined by their physicochemical properties. In this work, the physicochemical properties of glycolic acid:proline and malic acid:proline were evaluated experimentally and with MD simulations at five different ratios. Both DESs exhibited esterification upon preparation, which affected the viscosity in particular. In order to minimize oligomer formation and water release, three different experimental preparation methods were explored, but none could prevent esterification. The experimental and calculated densities of the DESs were found to be in good agreement. The measured and modeled glass transition temperature showed similar trends with composition, as did the experimental viscosity and the calculated diffusivities. The MD simulations provided additional insight at the atomistic level, showing that at acid-rich compositions, the acid-acid hydrogen bonding (HB) interactions prevail. Malic acid-based DESs show stronger acid-acid HB interactions than glycolic acid-based ones, possibly explaining its extreme viscosity. Upon the addition of proline, the interspecies interactions become predominant, confirming the formation of the widely assumed HB network between the DESs constituents in the liquid phase.

L-proline-based deep eutectic solvents (DESs) for deep catalytic oxidative desulfurization (ODS) of diesel.

PubMed

Hao, Lingwan; Wang, Meiri; Shan, Wenjuan; Deng, Changliang; Ren, Wanzhong; Shi, Zhouzhou; Lü, Hongying

2017-10-05

A series of L-proline-based DESs was prepared through an atom economic reaction between L-proline (L-Pro) and four different kinds of organic acids. The DESs were characterized by Fourier transform infrared spectroscopy (FT-IR), H nuclear magnetic resonance ( 1 HNMR), cyclic voltammogram (CV) and the Hammett method. The synthesized DESs were used for the oxidative desulfurization and the L-Pro/p-toluenesultonic acid (L-Pro/p-TsOH) system shows the highest catalytic activity that the removal of dibenzothiophene (DBT) reached 99% at 60°C in 2h, which may involve the dual activation of the L-Pro/p-TsOH. The acidity of four different L-proline-based DESs was measured and the results show that it could not simply conclude that the correlation between the acidity of DESs and desulfurization capability was positive or negative. The electrochemical measurements evidences and recycling experiment indicate a good stability performance of L-Pro/p-TsOH in desulfurization. This work will provide a novel and potential method for the deep oxidation desulfurization. Copyright © 2017 Elsevier B.V. All rights reserved.

Quantum mechanical effects in zwitterionic amino acids: The case of proline, hydroxyproline, and alanine in water

NASA Astrophysics Data System (ADS)

Ulman, Kanchan; Busch, Sebastian; Hassanali, Ali A.

2018-06-01

In this work, we use ab initio molecular dynamics simulations to elucidate the electronic properties of three hydrated zwitterionic amino acids, namely proline, hydroxyproline, and alanine, the former two forming an important constituent of collagen. In all three systems, we find a substantial amount of charge transfer between the amino acids and surrounding solvent, which, rather surprisingly, also involves the reorganization of electron density near the hydrophobic non-polar groups. Water around proline appears to be slightly more polarized, as reflected by the enhanced water dipole moment in its hydration shell. This observation is also complemented by an examination of the IR spectra of the three systems where there is a subtle red and blue shift in the O-H stretch and bend regions, respectively, for proline. We show that polarizability of these amino acids as revealed by a dipole moment analysis involves a significant enhancement from the solvent and that this also involves non-polar groups. Our results suggest that quantum mechanical effects are likely to be important in understanding the coupling between biomolecules and water in general and in hydrophobic interactions.

Increased plasma proline concentrations are associated with sarcopenia in the elderly.

PubMed

Toyoshima, Kenji; Nakamura, Marie; Adachi, Yusuke; Imaizumi, Akira; Hakamada, Tomomi; Abe, Yasuko; Kaneko, Eiji; Takahashi, Soiciro; Shimokado, Kentaro

2017-01-01

Metabolome analyses have shown that plasma amino acid profiles reflect various pathological conditions, such as cancer and diabetes mellitus. It remains unclear, however, whether plasma amino acid profiles change in patients with sarcopenia. This study therefore aimed to investigate whether sarcopenia-specific changes occur in plasma amino acid profiles. A total of 153 community-dwelling and seven institutionalized elderly individuals (56 men, 104 women; mean age, 77.7±7.0 years) were recruited for this cross-sectional analysis. We performed a comprehensive geriatric assessment, which included an evaluation of hand grip strength, gait speed, muscle mass and blood chemistry, including the concentration of 18 amino acids. Twenty-eight of the 160 participants met the criteria for sarcopenia established by the Asian Working Group on Sarcopenia in Older People. Univariate analysis revealed associations between the presence of sarcopenia and a higher plasma concentration of proline and glutamine, lower concentrations of histidine and tryptophan. Multivariable analysis revealed that a higher concentration of proline was the only variable independently associated with sarcopenia. The plasma concentration of proline may be useful for understanding the underlying pathophysiology of sarcopenia.

Prolyl hydroxylation in elastin is not random.

PubMed

Schmelzer, Christian E H; Nagel, Marcus B M; Dziomba, Szymon; Merkher, Yulia; Sivan, Sarit S; Heinz, Andrea

2016-10-01

This study aimed to investigate the prolyl and lysine hydroxylation in elastin from different species and tissues. Enzymatic digests of elastin samples from human, cattle, pig and chicken were analyzed using mass spectrometry and bioinformatics tools. It was confirmed at the protein level that elastin does not contain hydroxylated lysine residues regardless of the species. In contrast, prolyl hydroxylation sites were identified in all elastin samples. Moreover, the analysis of the residues adjacent to prolines allowed the determination of the substrate site preferences of prolyl 4-hydroxylase. It was found that elastins from all analyzed species contain hydroxyproline and that at least 20%-24% of all proline residues were partially hydroxylated. Determination of the hydroxylation degrees of specific proline residues revealed that prolyl hydroxylation depends on both the species and the tissue, however, is independent of age. The fact that the highest hydroxylation degrees of proline residues were found for elastin from the intervertebral disc and knowledge of elastin arrangement in this tissue suggest that hydroxylation plays a biomechanical role. Interestingly, a proline-rich domain of tropoelastin (domain 24), which contains several repeats of bioactive motifs, does not show any hydroxyproline residues in the mammals studied. The results show that prolyl hydroxylation is not a coincidental feature and may contribute to the adaptation of the properties of elastin to meet the functional requirements of different tissues. The study for the first time shows that prolyl hydroxylation is highly regulated in elastin. Copyright © 2016 Elsevier B.V. All rights reserved.

Preferential Osmolyte Accumulation: a Mechanism of Osmotic Stress Adaptation in Diazotrophic Bacteria

PubMed Central

Madkour, Magdy A.; Smith, Linda Tombras; Smith, Gary M.

1990-01-01

A common cellular mechanism of osmotic-stress adaptation is the intracellular accumulation of organic solutes (osmolytes). We investigated the mechanism of osmotic adaptation in the diazotrophic bacteria Azotobacter chroococcum, Azospirillum brasilense, and Klebsiella pneumoniae, which are adversely affected by high osmotic strength (i.e., soil salinity and/or drought). We used natural-abundance 13C nuclear magnetic resonance spectroscopy to identify all the osmolytes accumulating in these strains during osmotic stress generated by 0.5 M NaCl. Evidence is presented for the accumulation of trehalose and glutamate in Azotobacter chroococcum ZSM4, proline and glutamate in Azospirillum brasilense SHS6, and trehalose and proline in K. pneumoniae. Glycine betaine was accumulated in all strains grown in culture media containing yeast extract as the sole nitrogen source. Alternative nitrogen sources (e.g., NH4Cl or casamino acids) in the culture medium did not result in measurable glycine betaine accumulation. We suggest that the mechanism of osmotic adaptation in these organisms entails the accumulation of osmolytes in hyperosmotically stressed cells resulting from either enhanced uptake from the medium (of glycine betaine, proline, and glutamate) or increased net biosynthesis (of trehalose, proline, and glutamate) or both. The preferred osmolyte in Azotobacter chroococcum ZSM4 shifted from glutamate to trehalose as a consequence of a prolonged osmotic stress. Also, the dominant osmolyte in Azospirillum brasilense SHS6 shifted from glutamate to proline accumulation as the osmotic strength of the medium increased. PMID:16348295

Use of proline mutants to help solve the NMR solution structure of type III antifreeze protein.

PubMed Central

Chao, H.; Davies, P. L.; Sykes, B. D.; Sönnichsen, F. D.

1993-01-01

To help understand the structure/function relationships in antifreeze proteins (AFP), and to define the motifs required for ice binding, a Type III AFP suitable for two-dimensional (2D) NMR studies was produced in Escherichia coli. A synthetic gene for one of the Type III AFP isoforms was assembled in a T7 polymerase-directed expression vector. The 67-amino acid-long gene product differed from the natural AFP by inclusion of an N-terminal methionine but was indistinguishable in activity. The NMR spectra of this AFP were complicated by cis-trans proline isomerization from the C-terminal sequence YPPA. Substitution of this sequence by YAA eliminated isomer signals without altering the activity or structure of the mutant AFP. This variant (rQAE m1.1) was selected for sequential assignment and the secondary structure determination using 2D 1H NMR spectroscopy. Nine beta-strands are paired to form two triple-stranded antiparallel sheets and one double-stranded antiparallel sheet. Two further proline replacements, P29A and P33A, were made to delineate the role of conserved prolines in Type III AFP. These mutants were valuable in clarifying ambiguous NMR spectral assignments amongst the remaining six prolines of rQAE m1.1. In contrast to the replacement of the C-terminal prolyl residues, the exchange of P29 and P33 caused some structural changes and significantly decreased protein solubility and antifreeze activity. PMID:8401227

Proline zwitterion dynamics in solution, glass, and crystalline state.

PubMed

Kapitán, Josef; Baumruk, Vladimír; Kopecký, Vladimír; Pohl, Radek; Bour, Petr

2006-10-18

Raman and Raman optical activity spectra of L- and D-proline zwitterionic (PROZW) forms were recorded for H(2)O and D(2)O solutions in a wide frequency range and analyzed with respect to the motion of the proline ring and rotation of the carbonyl group. The solution spectra were additionally compared to Raman scattering of glass and crystalline powder proline. Solution and glass spectral band broadenings are similar and reveal information about the extent of internal molecular motion. Two distinct but equally populated flexible forms were found in the glass and the solution. The equal population is consistent with NMR data, temperature, and concentration dependencies. The molecular flexibility is reduced significantly in the crystal, however, where only one conformer is present. Consequently, the crystal bands are narrow and exhibit minor frequency shifts. The spectra were interpreted with the aid of density functional theory computations involving both continuum and explicit solvent. A two-dimensional potential energy surface pertaining to the five-member ring puckering coordinates was constructed and used for dynamical averaging of spectral properties. Comparison of the computed and experimental bandwidths suggests that the puckering is strongly correlated with the carbonyl rotation. An averaging over these two motions produces similar results. The interpretation of the Raman experiments with the aid of the simulation techniques also indicates that the environment modulates properties of the hydrophobic part of the molecule indirectly by interacting with the ionic group. Such behavior may be important for the reactivity and biological activity of proline-containing peptides and proteins.

The structure of Medicago truncatula δ 1-pyrroline-5-carboxylate reductase provides new insights into regulation of proline biosynthesis in plants

DOE PAGES

Ruszkowski, Milosz; Nocek, Boguslaw; Forlani, Giuseppe; ...

2015-10-30

The two pathways for proline biosynthesis in higher plants share the last step, the conversion of δ 1-pyrroline-5-carboxylate (P5C) to L-proline, which is catalyzed by P5C reductase (P5CR, EC 1.5.1.2) with the use of NAD(P)H as a coenzyme. There is increasing amount of evidence to suggest a complex regulation of P5CR activity at the post-translational level, yet the molecular basis of these mechanisms is unknown. Here we report the three-dimensional structure of the P5CR enzyme from the model legume Medicago truncatula (Mt). The crystal structures of unliganded MtP5CR decamer, and its complexes with the products NAD +, NADP +, andmore » L-proline were refined using x-ray diffraction data (at 1.7, 1.85, 1.95, and 2.1 Å resolution, respectively). Based on the presented structural data, the coenzyme preference for NADPH over NADH was explained, and NADPH is suggested to be the only coenzyme used by MtP5CR in vivo. Moreover, the insensitivity of MtP5CR to feed-back inhibition by proline, revealed by enzymatic analysis, was correlated with structural features. Additionally, a mechanism for the modulation of enzyme activity by chloride anions is discussed, as well as the rationale for the possible development of effective enzyme inhibitors.« less

Rational Design of Protein Stability: Effect of (2S,4R)-4-Fluoroproline on the Stability and Folding Pathway of Ubiquitin

PubMed Central

Crespo, Maria D.; Rubini, Marina

2011-01-01

Background Many strategies have been employed to increase the conformational stability of proteins. The use of 4-substituted proline analogs capable to induce pre-organization in target proteins is an attractive tool to deliver an additional conformational stability without perturbing the overall protein structure. Both, peptides and proteins containing 4-fluorinated proline derivatives can be stabilized by forcing the pyrrolidine ring in its favored puckering conformation. The fluorinated pyrrolidine rings of proline can preferably stabilize either a Cγ-exo or a Cγ-endo ring pucker in dependence of proline chirality (4R/4S) in a complex protein structure. To examine whether this rational strategy can be generally used for protein stabilization, we have chosen human ubiquitin as a model protein which contains three proline residues displaying Cγ-exo puckering. Methodology/Principal Findings While (2S,4R)-4-fluoroproline ((4R)-FPro) containing ubiquitinin can be expressed in related auxotrophic Escherichia coli strain, all attempts to incorporate (2S,4S)-4-fluoroproline ((4S)-FPro) failed. Our results indicate that (4R)-FPro is favoring the Cγ-exo conformation present in the wild type structure and stabilizes the protein structure due to a pre-organization effect. This was confirmed by thermal and guanidinium chloride-induced denaturation profile analyses, where we observed an increase in stability of −4.71 kJ·mol−1 in the case of (4R)-FPro containing ubiquitin ((4R)-FPro-ub) compared to wild type ubiquitin (wt-ub). Expectedly, activity assays revealed that (4R)-FPro-ub retained the full biological activity compared to wt-ub. Conclusions/Significance The results fully confirm the general applicability of incorporating fluoroproline derivatives for improving protein stability. In general, a rational design strategy that enforces the natural occurring proline puckering conformation can be used to stabilize the desired target protein. PMID:21625626

Role of proline and pyrroline-5-carboxylate metabolism in plant defense against invading pathogens

PubMed Central

Qamar, Aarzoo; Mysore, Kirankumar S.; Senthil-Kumar, Muthappa

2015-01-01

Pyrroline-5-carboxylate (P5C) is an intermediate product of both proline biosynthesis and catabolism. Recent evidences indicate that proline-P5C metabolism is tightly regulated in plants, especially during pathogen infection and abiotic stress. However, role of P5C and its metabolism in plants has not yet been fully understood. Studies indicate that P5C synthesized in mitochondria has a role in both resistance (R)-gene-mediated and non-host resistance against invading pathogens. Proline dehydrogenase and delta-ornithine amino transferase-encoding genes, both involved in P5C synthesis in mitochondria are implicated in defense response of Nicotiana benthamiana and Arabidopsis thaliana against bacterial pathogens. Such defense response is proposed to involve salicylic acid-dependent pathway, reactive oxygen species (ROS) and hypersensitive response (HR)-associated cell death. Recently HR, a form of programmed cell death (PCD), has been proposed to be induced by changes in mitochondrial P5C synthesis or the increase in P5C levels per se in plants inoculated with either a host pathogen carrying suitable avirulent (Avr) gene or a non-host pathogen. Consistently, A. thaliana mutant plants deficient in P5C catabolism showed HR like cell death when grown in external P5C or proline supplemented medium. Similarly, yeast and plant cells under oxidative stress were shown to increase ROS production and PCD due to increase in P5C levels. Similar mechanism has also been reported as one of the triggers for apoptosis in mammalian cells. This review critically analyzes results from various studies and enumerates the pathways for regulation of P5C levels in the plant cell, especially in mitochondria, during pathogen infection. Further, mechanisms regulating P5C- mediated defense responses, namely HR are outlined. This review also provides new insights into the differential role of proline-P5C metabolism in plants exposed to pathogen infection. PMID:26217357

Mechanical design of the third FnIII domain of tenascin-C.

PubMed

Peng, Qing; Zhuang, Shulin; Wang, Meijia; Cao, Yi; Khor, Yuanai; Li, Hongbin

2009-03-13

By combining single-molecule atomic force microscopy (AFM), proline mutagenesis and steered molecular dynamics (SMD) simulations, we investigated the mechanical unfolding dynamics and mechanical design of the third fibronectin type III domain of tenascin-C (TNfn3) in detail. We found that the mechanical stability of TNfn3 is similar to that of other constituting FnIII domains of tenascin-C, and the unfolding process of TNfn3 is an apparent two-state process. By employing proline mutagenesis to block the formation of backbone hydrogen bonds and introduce structural disruption in beta sheet, we revealed that in addition to the important roles played by hydrophobic core packing, backbone hydrogen bonds in beta hairpins are also responsible for the overall mechanical stability of TNfn3. Furthermore, proline mutagenesis revealed that the mechanical design of TNfn3 is robust and the mechanical stability of TNfn3 is very resistant to structural disruptions caused by proline substitutions in beta sheets. Proline mutant F88P is one exception, as the proline mutation at position 88 reduced the mechanical stability of TNfn3 significantly and led to unfolding forces of < 20 pN. This result suggests that Phe88 is a weak point of the mechanical resistance for TNfn3. We used SMD simulations to understand the molecular details underlying the mechanical unfolding of TNfn3. The comparison between the AFM results and SMD simulations revealed similarities and discrepancies between the two. We compared the mechanical unfolding and design of TNfn3 and its structural homologue, the tenth FnIII domain from fibronectin. These results revealed the complexity underlying the mechanical design of FnIII domains and will serve as a starting point for systematically analyzing the mechanical architecture of other FnIII domains in tenascins-C, and will help to gain a better understanding of some of the complex features observed for the stretching of native tenascin-C.

A POX on Renal Cancer Cells | Center for Cancer Research

Cancer.gov

Proline oxidase, or POX, is an enzyme responsible for metabolizing the amino acid proline. POX contributes to the regulation of cell death that occurs when cellular systems malfunction, a process called apoptosis. Previous studies have determined that levels of POX are reduced in several types of human cancer. Likewise, many cancer cells become resistant to apoptosis,

Conformational study of the proline rich peptide from bovine neurohypophysis secretory granules

NASA Astrophysics Data System (ADS)

Alieva, Irada; Velieva, Lala; Aliev, Dshavanchir; Gojayev, Niftali; Demukhamedova, Svetlana

2004-01-01

The spatial organization and conformational properties of the Proline Rich Peptide (PRP) from bovine neurohypophysis secretory granules have been established by the methods of molecular mechanics and molecular dynamics simulations in water solution. Conformational studies showed the peptide with limited conformational flexibility. Two β-type III turns are observed in PRP spatial organization.

Synthesis of cis-4-trifluoromethyl- and cis-4-difluoromethyl-l-pyroglutamic acids.

PubMed

Qiu, Xiao-Long; Qing, Feng-Ling

2003-05-02

Efforts to synthesize 4-trifluoromethyl- and 4-difluoromethyl-l-pyroglutamic acids are described. After many arduous efforts, we successfully synthesized our target molecules cis-4-trifluoromethyl-l-pyroglutamic acid 25 and cis-4-difluoromethyl-l-pyroglutamic acid 26 from trans-4-hydroxy-l-proline through oxidation of fluorinated prolinates with RuO(4).

General synthesis of C-glycosyl amino acids via proline-catalyzed direct electrophilic alpha-amination of C-glycosylalkyl aldehydes.

PubMed

Nuzzi, Andrea; Massi, Alessandro; Dondoni, Alessandro

2008-10-16

Non-natural axially and equatorially linked C-glycosyl alpha-amino acids (glycines, alanines, and CH2-serine isosteres) with either S or R alpha-configuration were prepared by D- and L-proline-catalyzed (de >95%) alpha-amination of C-glycosylalkyl aldehydes using dibenzyl azodicarboxylate as the electrophilic reagent.

Cyclopropyl glycine and proline-containing preparation noopept evoke two types of membrane potential responses in synaptoneurosomes.

PubMed

Lutsenko, V K; Vukolova, M N; Gudasheva, T A

2003-06-01

Proline, cyclo(Pro-Gly), and acyl-prolyl-containing dipeptide GVS-111 decreased synaptoneurosome membrane potential in a Ca2+-free medium. The efficiency of these preparations decreased in the following order: GVS>cyclo(Pro-Gly)>proline. Depolarization responses induced by endogenous nootropic agent cyclo(Pro-Gly) was dose-dependent and saturable; the threshold concentration of cyclo(Pro-Gly) was 10(-9) M. In a Ca2+-containing medium GVS and cyclo(Pro-Gly) induced both hyperpolarizing and depolarizing membrane responses of synaptoneurosomes. Possible mechanisms underlying changes in the membrane potential of synaptoneurosomes induced by nootropic agents are discussed. It was interesting whether modulation of electrogenesis can improve memory and potentiate the neuroprotective effect of the test nootropic agents.

Isotope Effects Reveal the Mechanism of Enamine Formation in l-Proline-Catalyzed α-Amination of Aldehydes.

PubMed

Ashley, Melissa A; Hirschi, Jennifer S; Izzo, Joseph A; Vetticatt, Mathew J

2016-02-17

The mechanism of l-proline-catalyzed α-amination of 3-phenylpropionaldehyde was studied using a combination of experimental kinetic isotope effects (KIEs) and theoretical calculations. Observation of a significant carbonyl (13)C KIE and a large primary α-deuterium KIE support rate-determining enamine formation. Theoretical predictions of KIEs exclude the widely accepted mechanism of enamine formation via intramolecular deprotonation of an iminium carboxylate intermediate. An E2 elimination mechanism catalyzed by a bifunctional base that directly forms an N-protonated enamine species from an oxazolidinone intermediate accounts for the experimental KIEs. These findings provide the first experimental picture of the transition-state geometry of enamine formation and clarify the role of oxazolidinones as nonparasitic intermediates in proline catalysis.

Pyrroline-5-Carboxylate Reductase in Chlorella autotrophica and Chlorella saccharophila in Relation to Osmoregulation 1

PubMed Central

Laliberté, Gilles; Hellebust, Johan A.

1989-01-01

Pyrroline-5-carboxylate (P5C) reductase (EC 1.5.1.2), which catalyzes the reduction of P5C to proline, was partially purified from two Chlorella species; Chlorella autotrophica, a euryhaline marine alga that responds to increases in salinity by accumulating proline and ions, and Chlorella saccharophila, which does not accumulate proline for osmoregulation. From the elution profile of this enzyme from an anion exchange column in Tris-HCl buffer (pH 7.6), containing sorbitol and glycine betaine, it was shown that P5C reductase from C. autotrophica was a neutral protein whereas the enzyme from C. saccharophila was negatively charged. The kinetic mechanisms of the reductase was characteristic of a ping-pong mechanism with double competitive substrate inhibition. Both enzymes showed high specificity for NADH as cofactor. The affinities of the reductases for their substrates did not change when the cells were grown at different salinities. In both algae, the apparent Km values of the reductase for P5C and NADH were 0.17 and 0.10 millimolar, respectively. A fourfold increase in maximal velocity of the reductase was observed when C. autotrophica was transferred from 50 to 150% artificial sea water. Even though the reductase was inhibited by NaCl, KCl, and proline, it still showed appreciable activity in the presence of these compounds at molar concentrations. A possible role for the regulation of proline synthesis at the step catalyzed by P5C reductase is discussed in relation to the specificity of P5C reductase for NADH and its responses to salt treatments. PMID:16667157

Increasing protein stability by improving beta-turns.

PubMed

Fu, Hailong; Grimsley, Gerald R; Razvi, Abbas; Scholtz, J Martin; Pace, C Nick

2009-11-15

Our goal was to gain a better understanding of how protein stability can be increased by improving beta-turns. We studied 22 beta-turns in nine proteins with 66-370 residues by replacing other residues with proline and glycine and measuring the stability. These two residues are statistically preferred in some beta-turn positions. We studied: Cold shock protein B (CspB), Histidine-containing phosphocarrier protein, Ubiquitin, Ribonucleases Sa2, Sa3, T1, and HI, Tryptophan synthetase alpha-subunit, and Maltose binding protein. Of the 15 single proline mutations, 11 increased stability (Average = 0.8 +/- 0.3; Range = 0.3-1.5 kcal/mol), and the stabilizing effect of double proline mutants was additive. On the basis of this and our previous work, we conclude that proteins can generally be stabilized by replacing nonproline residues with proline residues at the i + 1 position of Type I and II beta-turns and at the i position in Type II beta-turns. Other turn positions can sometimes be used if the phi angle is near -60 degrees for the residue replaced. It is important that the side chain of the residue replaced is less than 50% buried. Identical substitutions in beta-turns in related proteins give similar results. Proline substitutions increase stability mainly by decreasing the entropy of the denatured state. In contrast, the large, diverse group of proteins considered here had almost no residues in beta-turns that could be replaced by Gly to increase protein stability. Improving beta-turns by substituting Pro residues is a generally useful way of increasing protein stability. 2009 Wiley-Liss, Inc.

Resolving the cofactor-binding site in the proline biosynthetic enzyme human pyrroline-5-carboxylate reductase 1

PubMed Central

Christensen, Emily M.; Patel, Sagar M.; Korasick, David A.; Campbell, Ashley C.; Krause, Kurt L.; Becker, Donald F.; Tanner, John J.

2017-01-01

Pyrroline-5-carboxylate reductase (PYCR) is the final enzyme in proline biosynthesis, catalyzing the NAD(P)H-dependent reduction of Δ1-pyrroline-5-carboxylate (P5C) to proline. Mutations in the PYCR1 gene alter mitochondrial function and cause the connective tissue disorder cutis laxa. Furthermore, PYCR1 is overexpressed in multiple cancers, and the PYCR1 knock-out suppresses tumorigenic growth, suggesting that PYCR1 is a potential cancer target. However, inhibitor development has been stymied by limited mechanistic details for the enzyme, particularly in light of a previous crystallographic study that placed the cofactor-binding site in the C-terminal domain rather than the anticipated Rossmann fold of the N-terminal domain. To fill this gap, we report crystallographic, sedimentation-velocity, and kinetics data for human PYCR1. Structures of binary complexes of PYCR1 with NADPH or proline determined at 1.9 Å resolution provide insight into cofactor and substrate recognition. We see NADPH bound to the Rossmann fold, over 25 Å from the previously proposed site. The 1.85 Å resolution structure of a ternary complex containing NADPH and a P5C/proline analog provides a model of the Michaelis complex formed during hydride transfer. Sedimentation velocity shows that PYCR1 forms a concentration-dependent decamer in solution, consistent with the pentamer-of-dimers assembly seen crystallographically. Kinetic and mutational analysis confirmed several features seen in the crystal structure, including the importance of a hydrogen bond between Thr-238 and the substrate as well as limited cofactor discrimination. PMID:28258219

Effect of proline and glycine residues on dynamics and barriers of loop formation in polypeptide chains.

PubMed

Krieger, Florian; Möglich, Andreas; Kiefhaber, Thomas

2005-03-16

Glycine and proline residues are frequently found in turn and loop structures of proteins and are believed to play an important role during chain compaction early in folding. We investigated their effect on the dynamics of intrachain loop formation in various unstructured polypeptide chains. Loop formation is significantly slower around trans prolyl peptide bonds and faster around glycine residues compared to any other amino acid. However, short loops are formed fastest around cis prolyl bonds with a time constant of 6 ns for end-to-end contact formation in a four-residue loop. Formation of short loops encounters activation energies in the range of 15 to 30 kJ/mol. The altered dynamics around glycine and trans prolyl bonds can be mainly ascribed to their effects on the activation energy. The fast dynamics around cis prolyl bonds, in contrast, originate in a higher Arrhenius pre-exponential factor, which compensates for an increased activation energy for loop formation compared to trans isomers. All-atom simulations of proline-containing peptides indicate that the conformational space for cis prolyl isomers is largely restricted compared to trans isomers. This leads to decreased average end-to-end distances and to a smaller loss in conformational entropy upon loop formation in cis isomers. The results further show that glycine and proline residues only influence formation of short loops containing between 2 and 10 residues, which is the typical loop size in native proteins. Formation of larger loops is not affected by the presence of a single glycine or proline residue.

Coherent Experimental and Simulation Approach To Explore the Underlying Mechanism of Denaturation of Stem Bromelain in Osmolytes.

PubMed

Rani, Anjeeta; Taha, Mohamed; Venkatesu, Pannuru; Lee, Ming-Jer

2017-07-13

Characterization of a protein in the context of its environment is of crucial importance for a complete understanding of its function. Although biophysical techniques provide powerful tools for studying the stability and activity of the enzyme in the presence of various cosolvents, an approach of combining both experimental techniques and molecular dynamic (MD) simulations may lead to the mechanistic insight into the interactions governing the stability of an enzyme. The knowledge of these interactions can be further utilized for range of modifications in the wild form of an enzyme for various pharmaceutical applications. Herein, we employed florescence, UV-visible, circular dichroism (CD), dynamic light scattering (DLS) study, and MD simulations for comprehensive understanding of stem bromelain (BM) in the presence of betaine, sarcosine, arginine, and proline. The thermal stability of BM in the presence of 1 M of osmolytes is found to be in order: proline > betaine > buffer > arginine > sarcosine. BM gets more preferentially hydrated in the presence of betaine and proline than in sarcosine and arginine. Nonetheless, MD simulations suggest that betaine, sarcosine, and arginine at 1 M interact with the active site of BM through H-bonding except proline which are responsible for more disruption of active site. The distances between the catalytic site residues are 1.6, 1.9, 4.3, 5.0, and 6.2 Å for BM in proline, buffer, betaine, arginine, and sarcosine at 1 M, respectively. To the best of our knowledge, this is the first report on detailed unequivocal evidence of denaturation and deactivation of BM in the presence of methylamines and amino acids.

The ring residue proline 8 is crucial for the thermal stability of the lasso peptide caulosegnin II.

PubMed

Hegemann, Julian D; Fage, Christopher D; Zhu, Shaozhou; Harms, Klaus; Di Leva, Francesco Saverio; Novellino, Ettore; Marinelli, Luciana; Marahiel, Mohamed A

2016-04-01

Lasso peptides are fascinating natural products with a unique structural fold that can exhibit tremendous thermal stability. Here, we investigate factors responsible for the thermal stability of caulosegnin II. By employing X-ray crystallography, mutational analysis and molecular dynamics simulations, the ring residue proline 8 was proven to be crucial for thermal stability.

cis-Proline-mediated Ser(P)[superscript 5] Dephosphorylation by the RNA Polymerase II C-terminal Domain Phosphatase Ssu72

DOE Office of Scientific and Technical Information (OSTI.GOV)

Werner-Allen, Jon W.; Lee, Chul-Jin; Liu, Pengda

2012-05-16

RNA polymerase II coordinates co-transcriptional events by recruiting distinct sets of nuclear factors to specific stages of transcription via changes of phosphorylation patterns along its C-terminal domain (CTD). Although it has become increasingly clear that proline isomerization also helps regulate CTD-associated processes, the molecular basis of its role is unknown. Here, we report the structure of the Ser(P){sup 5} CTD phosphatase Ssu72 in complex with substrate, revealing a remarkable CTD conformation with the Ser(P){sup 5}-Pro{sup 6} motif in the cis configuration. We show that the cis-Ser(P){sup 5}-Pro{sup 6} isomer is the minor population in solution and that Ess1-catalyzed cis-trans-proline isomerizationmore » facilitates rapid dephosphorylation by Ssu72, providing an explanation for recently discovered in vivo connections between these enzymes and a revised model for CTD-mediated small nuclear RNA termination. This work presents the first structural evidence of a cis-proline-specific enzyme and an unexpected mechanism of isomer-based regulation of phosphorylation, with broad implications for CTD biology« less

[Agrobacterium-mediated sunflower transformation (Helianthus annuus L.) in vitro and in Planta using strain of LBA4404 harboring binary vector pBi2E with dsRNA-suppressor proline dehydrogenase gene].

PubMed

Tishchenko, E N; Komisarenko, A G; Mikhal'skaia, S I; Sergeeva, L E; Adamenko, N I; Morgun, B V; Kochetov, A V

2014-01-01

To estimate the efficiency of proline dehydrogenase gene suppression towards increasing of sunflower (Helianthus annuus L.) tolerance level to water deficit and salinity, we employed strain LBA4404 harboring pBi2E with double-stranded RNA-suppressor, which were prepared on basis arabidopsis ProDH1 gene. The techniques of Agrobacterium-mediated transformation in vitro and in planta during fertilization sunflower have been proposed. There was shown the genotype-depended integration of T-DNA in sunflower genome. PCR-analysis showed that ProDH1 presents in genome of inbred lines transformed in planta, as well as in T1- and T2-generations. In trans-genic regenerants the essential accumulation of free L-proline during early stages of in vitro cultivation under normal conditions was shown. There was established the essential accumulation of free proline in transgenic regenerants during cultivation under lethal stress pressure (0.4 M mannitol and 2.0% sea water salts) and its decline upon the recovery period. These data are declared about effectiveness of suppression of sunflower ProDH and gene participation in processes connected with osmotolerance.

Effects of proline cis-trans isomerization on TB domain secondary structure.

PubMed Central

Yuan, X.; Werner, J. M.; Knott, V.; Handford, P. A.; Campbell, I. D.; Downing, K.

1998-01-01

The transforming growth factor beta (TGF-beta) binding protein-like (TB) domain is found principally in proteins localized to extracellular matrix fibrils, including human fibrillin-1, the defective protein in the Marfan syndrome. Analysis of the nuclear magnetic resonance (NMR) data for the sixth TB module from human fibrillin-1 has revealed the existence of two stable conformers that differ in the isomerization states of two proline residues. Unusually, the two isoforms do not readily interconvert and are stable on the time scale of milliseconds. We have computed independent structures of the major and minor conformers of TB6 to assess how the domain fold adjusts to incorporate alternatively cis- or trans-prolines. Based on previous observations, it has been suggested that multiple conformers can only be accommodated in flexible regions of protein structure. In contrast, P22, which exists in trans in the major form and cis in the minor form of TB6, is in a rigid region of the domain, which is confirmed by backbone dynamics measurements. Overall, the structures of the major and minor conformers are similar. However, the secondary structure topologies of the two forms differ as a direct consequence of the changes in proline conformation. PMID:9792099

Increased plasma proline concentrations are associated with sarcopenia in the elderly

PubMed Central

Adachi, Yusuke; Imaizumi, Akira; Hakamada, Tomomi; Abe, Yasuko; Kaneko, Eiji; Takahashi, Soiciro; Shimokado, Kentaro

2017-01-01

Background and purpose Metabolome analyses have shown that plasma amino acid profiles reflect various pathological conditions, such as cancer and diabetes mellitus. It remains unclear, however, whether plasma amino acid profiles change in patients with sarcopenia. This study therefore aimed to investigate whether sarcopenia-specific changes occur in plasma amino acid profiles. Methods A total of 153 community-dwelling and seven institutionalized elderly individuals (56 men, 104 women; mean age, 77.7±7.0 years) were recruited for this cross-sectional analysis. We performed a comprehensive geriatric assessment, which included an evaluation of hand grip strength, gait speed, muscle mass and blood chemistry, including the concentration of 18 amino acids. Results Twenty-eight of the 160 participants met the criteria for sarcopenia established by the Asian Working Group on Sarcopenia in Older People. Univariate analysis revealed associations between the presence of sarcopenia and a higher plasma concentration of proline and glutamine, lower concentrations of histidine and tryptophan. Multivariable analysis revealed that a higher concentration of proline was the only variable independently associated with sarcopenia. Conclusions The plasma concentration of proline may be useful for understanding the underlying pathophysiology of sarcopenia. PMID:28934309

A conserved proline residue in the leucine zipper region of AtbZIP34 and AtbZIP61 in Arabidopsis thaliana interferes with the formation of homodimer.

PubMed

Shen, Huaishun; Cao, Kaiming; Wang, Xiping

2007-10-19

Two putative Arabidopsis E group bZIP transcript factors, AtbZIP34 and AtbZIP61, are nuclear-localized and their transcriptional activation domain is in their N-terminal region. By searching GenBank, we found other eight plant homologues of AtbZIP34 and AtbZIP61. All of them have a proline residue in the third heptad of zipper region. Yeast two-hybrid assay and EMSA showed that AtbZIP34 and AtbZIP61 could not form homodimer while their mutant forms, AtbZIP34m and AtbZIP61m, which the proline residue was replaced by an alanine residue in the zipper region, could form homodimer and bind G-box element. These results suggest that the conserved proline residue interferes with the homodimer formation. However, both AtbZIP34 and AtbZIP61 could form heterodimers with members of I group and S group transcription factors in which some members involved in vascular development. So we speculate that AtbZIP34 and AtbZIP61 may participate in plant development via interacting with other group bZIP transcription factors.

Infrared Spectroscopy of Mobility-Selected H+-Gly-Pro-Gly-Gly (GPGG)

NASA Astrophysics Data System (ADS)

Masson, Antoine; Kamrath, Michael Z.; Perez, Marta A. S.; Glover, Matthew S.; Rothlisberger, U.; Clemmer, David E.; Rizzo, Thomas R.

2015-09-01

We report the first results from a new instrument capable of acquiring infrared spectra of mobility-selected ions. This demonstration involves using ion mobility to first separate the protonated peptide Gly-Pro-Gly-Gly (GPGG) into two conformational families with collisional cross-sections of 93.8 and 96.8 Å2. After separation, each family is independently analyzed by acquiring the infrared predissociation spectrum of the H2-tagged molecules. The ion mobility and spectroscopic data combined with density functional theory (DFT) based molecular dynamics simulations confirm the presence of one major conformer per family, which arises from cis/ trans isomerization about the proline residue. We induce isomerization between the two conformers by using collisional activation in the drift tube and monitor the evolution of the ion distribution with ion mobility and infrared spectroscopy. While the cis-proline species is the preferred gas-phase structure, its relative population is smaller than that of the trans-proline species in the initial ion mobility drift distribution. This suggests that a portion of the trans-proline ion population is kinetically trapped as a higher energy conformer and may retain structural elements from solution.

Proline isomerization in the C-terminal region of HSP27.

PubMed

Alderson, T Reid; Benesch, Justin L P; Baldwin, Andrew J

2017-07-01

In mammals, small heat-shock proteins (sHSPs) typically assemble into interconverting, polydisperse oligomers. The dynamic exchange of sHSP oligomers is regulated, at least in part, by molecular interactions between the α-crystallin domain and the C-terminal region (CTR). Here we report solution-state nuclear magnetic resonance (NMR) spectroscopy investigations of the conformation and dynamics of the disordered and flexible CTR of human HSP27, a systemically expressed sHSP. We observed multiple NMR signals for residues in the vicinity of proline 194, and we determined that, while all observed forms are highly disordered, the extra resonances arise from cis-trans peptidyl-prolyl isomerization about the G193-P194 peptide bond. The cis-P194 state is populated to near 15% at physiological temperatures, and, although both cis- and trans-P194 forms of the CTR are flexible and dynamic, both states show a residual but differing tendency to adopt β-strand conformations. In NMR spectra of an isolated CTR peptide, we observed similar evidence for isomerization involving proline 182, found within the IPI/V motif. Collectively, these data indicate a potential role for cis-trans proline isomerization in regulating the oligomerization of sHSPs.

Unique Structural Features and Sequence Motifs of Proline Utilization A (PutA)

PubMed Central

Singh, Ranjan K.; Tanner, John J.

2013-01-01

Proline utilization A proteins (PutAs) are bifunctional enzymes that catalyze the oxidation of proline to glutamate using spatially separated proline dehydrogenase and pyrroline-5-carboxylate dehydrogenase active sites. Here we use the crystal structure of the minimalist PutA from Bradyrhizobium japonicum (BjPutA) along with sequence analysis to identify unique structural features of PutAs. This analysis shows that PutAs have secondary structural elements and domains not found in the related monofunctional enzymes. Some of these extra features are predicted to be important for substrate channeling in BjPutA. Multiple sequence alignment analysis shows that some PutAs have a 17-residue conserved motif in the C-terminal 20–30 residues of the polypeptide chain. The BjPutA structure shows that this motif helps seal the internal substrate-channeling cavity from the bulk medium. Finally, it is shown that some PutAs have a 100–200 residue domain of unknown function in the C-terminus that is not found in minimalist PutAs. Remote homology detection suggests that this domain is homologous to the oligomerization beta-hairpin and Rossmann fold domain of BjPutA. PMID:22201760

Prolidase is a critical enzyme for complete gliadin digestion in Tenebrio molitor larvae.

PubMed

Tereshchenkova, Valeriia F; Goptar, Irina A; Zhuzhikov, Dmitry P; Belozersky, Mikhail A; Dunaevsky, Yakov E; Oppert, Brenda; Filippova, Irina Yu; Elpidina, Elena N

2017-08-01

Prolidase is a proline-specific metallopeptidase that cleaves imidodipeptides with C-terminal Pro residue. Prolidase was purified and characterized from the Tenebrio molitor larval midgut. The enzyme was localized in the soluble fraction of posterior midgut tissues, corresponding to a predicted cytoplasmic localization of prolidase according to the structure of the mRNA transcript. Expression of genes encoding prolidase and the major digestive proline-specific peptidase (PSP)-dipeptidyl peptidase 4-were similar. The pH optimum of T. molitor prolidase was 7.5, and the enzyme was inhibited by Z-Pro, indicating that it belongs to type I prolidases. In mammals, prolidase is particularly important in the catabolism of a proline-rich protein-collagen. We propose that T. molitor larval prolidase is a critical enzyme for the final stages of digestion of dietary proline-rich gliadins, providing hydrolysis of imidodipeptides in the cytoplasm of midgut epithelial cells. We propose that the products of hydrolysis are absorbed from the luminal contents by peptide transporters, which we have annotated in the T. molitor larval gut transcriptome. The origin of prolidase substrates in the insect midgut is discussed in the context of overall success of grain feeding insects. © 2017 Wiley Periodicals, Inc.

Biochemical, physiological and molecular evaluation of rice cultivars differing in salt tolerance at the seedling stage.

PubMed

Kordrostami, Mojtaba; Rabiei, Babak; Hassani Kumleh, Hassan

2017-07-01

Changes in the antioxidant enzymes, lipid peroxidation, sodium and potassium, chlorophyll, H 2 O 2 and proline content were monitored in the leaves of 42 rice varieties which were not yet well-documented for the salinity tolerance under different salinity levels. The tolerant varieties (FL478, Hassani, Shahpasand, Gharib and Nemat) showed signs of tolerance (lower Na + /K + ratio, high proline accumulation, less membrane damage, lower H 2 O 2 production, and higher superoxide dismutase and catalase activity) very well. The positive relationship between the level of salt tolerance and the amount of proline accumulation in the rice varieties support the important role of proline under the salt stress. The varieties were genotyped for 12 microsatellite markers that were closely linked to SalTol QTL. The results of association analysis indicated that RM1287, RM8094, RM3412 and AP3206 markers had the high value of R 2 for the regression models of the studied traits. It shows the important role of SalTol in controlling physio-biochemical traits. The results can be used in the future marker assisted selection (MAS) directly, if the results are confirmed.

Evolution of plant δ 1-pyrroline-5-carboxylate reductases from phylogenetic and structural perspectives

DOE PAGES

Forlani, Giuseppe; Makarova, Kira S.; Ruszkowski, Milosz; ...

2015-08-03

Proline plays a crucial role in cell growth and stress responses, and its accumulation is essential for the tolerance of adverse environmental conditions in plants. Two routes are used to biosynthesize proline in plants. The main route uses glutamate as a precursor, while in the other route proline is derived from ornithine. The terminal step of both pathways, the conversion of δ 1-pyrroline-5-carboxylate (P5C) to L-proline, is catalyzed by P5C reductase (P5CR) using NADH or NADPH as a cofactor. Since P5CRs are important housekeeping enzymes, they are conserved across all domains of life and appear to be relatively unaffected throughoutmore » evolution. However, global analysis of these enzymes unveiled significant functional diversity in the preference for cofactors (NADPH vs. NADH), variation in metal dependence and the differences in the oligomeric state. In our study we investigated evolutionary patterns through phylogenetic and structural analysis of P5CR representatives from all kingdoms of life, with emphasis on the plant species. We attempted to correlate local sequence/structure variation among the functionally and structurally characterized members of the family.« less

A leucine-to-proline substitution causes a defective [alpha]-antichymotrypsin allele associated with familial obstructive lung disease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Poller, W.; Scholz, S.; Fischer, M.

1993-09-01

Using denaturing gradient gel electrophoresis and direct sequencing of amplified genomic DNA, the authors have identified two defective mutants of the human [alpha][sub 1]-antichymotrypsin (ACT) gene associated with chronic obstructive pulmonary disease (COPD). A leucine 55-to-proline substitution causing a defective ACT allele (Bochum-1) was observed in a family with COPD in three subsequent generations. Another mutation, proline 229-to-alanine (Bonn-1), was associated with ACT serum deficiency in four patients with a positive family history. These mutations were not detected among 100 healthy control subjects, suggesting a possible pathogenetic role of ACT gene defects in a subset of patients with COPD. 14more » refs., 1 fig., 1 tab.« less

A POX on Renal Cancer Cells | Center for Cancer Research

Cancer.gov

Proline oxidase, or POX, is an enzyme responsible for metabolizing the amino acid proline. POX contributes to the regulation of cell death that occurs when cellular systems malfunction, a process called apoptosis. Previous studies have determined that levels of POX are reduced in several types of human cancer. Likewise, many cancer cells become resistant to apoptosis, suggesting a link between POX and cancer cell survival.

Amino acids in eight species of Monogenea.

PubMed

Arme, C

1977-04-15

The ethanol-extractable amino acids of several species of Monogenea were analysed. In the Monopisthocotylea levels were high ( greater than 1,200 micronmoles/g/ethanol extracted dry wt); in the Polyopisthocotylea lower amounts were present (196-562 micronmoles). High proline levels were present in parasites of hosts from a marine environment but low in freshwater forms. No correlation between diet and proline content was observed.

In situ catalyst improvement in the proline-mediated alpha-amination of aldehydes.

PubMed

Iwamura, Hiroshi; Mathew, Suju P; Blackmond, Donna G

2004-09-29

Kinetic investigations show that the proline-mediated alpha-amination of aldehydes exhibits autoinductive rate behavior and amplification of product enantiomeric excess. Further experiments highlight the role of product, offering suggestions for the design of catalysts of improved efficiency for such transformations. The unusual characteristics exhibited by these reactions implicate amino acid catalysis in rationalizations of the origin of biological homochirality.

Control of Rhizoctonia foliar blight in forest seedling nurseries: A 3-year study

Treesearch

Tom E. Starkey; Scott A. Enebak; Ken McQuage; Kevin Barfield

2013-01-01

Laboratory and field trials have shown Proline® (prothioconazole) to be efficacious against the causal agent of Rhizoctonia foliar blight on loblolly pine (Pinus taeda). A biweekly application of Proline ® at 5 fl oz/ac in nursery field tests significantly reduced Rhizoctonia foliar blight on loblolly pine when compared to applications of Abound ® azoxystrobin (24 fl...

Unique proline-benzoquinone pigment from the colored nectar of "bird's Coca cola tree" functions in bird attractions.

PubMed

Luo, Shi-Hong; Liu, Yan; Hua, Juan; Niu, Xue-Mei; Jing, Shu-Xi; Zhao, Xu; Schneider, Bernd; Gershenzon, Jonathan; Li, Sheng-Hong

2012-08-17

The major pigment responsible for the dark brown nectar of the "bird's Coca cola tree", Leucosceptrum canum (Labiatae), was isolated and identified as a unique symmetric proline-quinone conjugate, 2,5-di-(N-(-)-prolyl)-para-benzoquinone (DPBQ). Behavioral experiments with both isolated and synthetic authentic samples indicated that DPBQ functions mainly as a color attractant to bird pollinators.

Calcium-mediated responses and glutamine synthetase expression in greater duckweed (Spirodela polyrhiza L.) under diethyl phthalate-induced stress.

PubMed

Cheng, Lee-Ju; Hung, Meng-Ju; Cheng, Yen-I; Cheng, Tai-Sheng

2013-11-15

This study was carried out to assess the influence of diethyl phthalate (DEP) alone or associated with calcium chloride (CaCl2) on greater duckweed plants, emphasizing the implications of calcium in amelioration of DEP-induced stress on plant growth. Greater duckweed were treated with DEP in variable concentrations, as 0, 0.25, 0.5, 1.0 and 2.0mM for 7 days, or treated with the same concentration either 2mM DEP or 2mM DEP plus 10mM CaCl2·2H2O in different duration 0-7 days. Treatment with 2mM DEP resulted in increasing proline content, protease activity, and ammonia accumulation in duckweed tissues. NADH-glutamate dehydrogenase (NADH-GDH; EC 1.4.1.2) and Δ(1)-pyrroline-5-carboxylate reductase (P5CR; EC 1.5.1.2), two key enzymes in the glutamate pathway of proline synthesis, showed increase in activity with DEP treatment and positively correlated with proline accumulation. No further increase in proline accumulation was observed with addition of calcium chloride to the DEP-treated cultures. However, supplementation of Ca(2+) can mitigate the adverse effect of DEP, at least in part to decrease the DEP-induced superoxide accumulation and increase in GDH activity for ammonia assimilation in duckweed fronds. In addition, effects of calcium on mitigation of DEP injury were also observed in glutamine synthetase (GS; EC 6.3.1.2) expression. Both GS1 and GS2 polypeptide accumulation and the level of total GS activity were nearly equivalent to the control. Exogenous proline protects GS2 from DEP-modulated redox damage in the chloroplast lysates but there is no remarkable protection effects on D1 (the 32kDa protein in photosystem II reaction center) degradation. In conclusion, the glutamate pathway of proline synthesis might be involved in mitigation of DEP-induced injury, and calcium plays an important role in increasing GDH, P5CR, and GS expression. Copyright © 2013 Elsevier B.V. All rights reserved.

Recessive constitutive mutant Chinese hamster ovary cells (CHO-K1) with an altered A system for amino acid transport and the mechanism of gene regulation of the A system.

PubMed Central

Moffett, J; Englesberg, E

1984-01-01

Chinese hamster ovary cells (CHO-K1) starved for 24 h for amino acids show a severalfold increase in velocity of proline transport through the A system (Vmax is five times that of unstarved cells). This increase is inhibited by cycloheximide, actinomycin D, N-methyl-alpha-amino isobutyric acid (MeAIB, a non-metabolizable specific A system amino acid analog), and by other amino acids that are generally transported by the A system. However, transport by the A system is not a prerequisite for this repression, and all compounds that have affinity for the A system do not necessarily act as "co-repressors." The addition of proline, MeAIB, or other amino acids, as described above, to derepressed cells results in a rapid decrease in A system activity. As shown with proline and MeAIB, this decrease in activity is in part due to a rapid trans-inhibition and a slow, irreversible inactivation of the A system. Neither process is inhibited by cycloheximide or actinomycin D. Alanine antagonizes the growth of CHO-K1 pro cells by preventing proline transport, and alanine-resistant mutants (alar) have been isolated (Moffett et al., Somatic Cell Genet. 9:189-213, 1983). alar2 and alar4 are partial and full constitutive mutants for the A system and have two and six times the Vmax for proline uptake by the A system, respectively. The A system in alar4 is also immune to the co-repressor-induced inactivation. Both alar2 and alar4 phenotypes are recessive. Alar3 shows an increase in Vmax and Km for proline transport through the A system, and this phenotype is codominant. All three mutants have a pleiotropic effect, producing increases in activity of the ASC and P systems of amino acid transport. This increase is not due to an increase in the Na+ gradient. The ASC and P phenotypes behave similarly to the A system in hybrids. A model has been proposed incorporating these results. PMID:6538929

Synthetic Biology of Proteins: Tuning GFPs Folding and Stability with Fluoroproline

PubMed Central

Steiner, Thomas; Hess, Petra; Bae, Jae Hyun; Wiltschi, Birgit; Moroder, Luis; Budisa, Nediljko

2008-01-01

Background Proline residues affect protein folding and stability via cis/trans isomerization of peptide bonds and by the Cγ-exo or -endo puckering of their pyrrolidine rings. Peptide bond conformation as well as puckering propensity can be manipulated by proper choice of ring substituents, e.g. Cγ-fluorination. Synthetic chemistry has routinely exploited ring-substituted proline analogs in order to change, modulate or control folding and stability of peptides. Methodology/Principal Findings In order to transmit this synthetic strategy to complex proteins, the ten proline residues of enhanced green fluorescent protein (EGFP) were globally replaced by (4R)- and (4S)-fluoroprolines (FPro). By this approach, we expected to affect the cis/trans peptidyl-proline bond isomerization and pyrrolidine ring puckering, which are responsible for the slow folding of this protein. Expression of both protein variants occurred at levels comparable to the parent protein, but the (4R)-FPro-EGFP resulted in irreversibly unfolded inclusion bodies, whereas the (4S)-FPro-EGFP led to a soluble fluorescent protein. Upon thermal denaturation, refolding of this variant occurs at significantly higher rates than the parent EGFP. Comparative inspection of the X-ray structures of EGFP and (4S)-FPro-EGFP allowed to correlate the significantly improved refolding with the Cγ-endo puckering of the pyrrolidine rings, which is favored by 4S-fluorination, and to lesser extents with the cis/trans isomerization of the prolines. Conclusions/Significance We discovered that the folding rates and stability of GFP are affected to a lesser extent by cis/trans isomerization of the proline bonds than by the puckering of pyrrolidine rings. In the Cγ-endo conformation the fluorine atoms are positioned in the structural context of the GFP such that a network of favorable local interactions is established. From these results the combined use of synthetic amino acids along with detailed structural knowledge and existing protein engineering methods can be envisioned as a promising strategy for the design of complex tailor-made proteins and even cellular structures of superior properties compared to the native forms. PMID:18301757

Abscisic acid-regulated protein degradation causes osmotic stress-induced accumulation of branched-chain amino acids in Arabidopsis thaliana.

PubMed

Huang, Tengfang; Jander, Georg

2017-10-01

Whereas proline accumulates through de novo biosynthesis in plants subjected to osmotic stress, leucine, isoleucine, and valine accumulation in drought-stressed Arabidopsis thaliana is caused by abscisic acid-regulated protein degradation. In response to several kinds of abiotic stress, plants greatly increase their accumulation of free amino acids. Although stress-induced proline increases have been studied the most extensively, the fold-increase of other amino acids, in particular branched-chain amino acids (BCAAs; leucine, isoleucine, and valine), is often higher than that of proline. In Arabidopsis thaliana (Arabidopsis), BCAAs accumulate in response to drought, salt, mannitol, polyethylene glycol, herbicide treatment, and nitrogen starvation. Plants that are deficient in abscisic acid signaling accumulate lower amounts of BCAAs, but not proline and most other amino acids. Previous bioinformatic studies had suggested that amino acid synthesis, rather than protein degradation, is responsible for the observed BCAA increase in osmotically stressed Arabidopsis. However, whereas treatment with the protease inhibitor MG132 decreased drought-induced BCAA accumulation, inhibition of BCAA biosynthesis with the acetolactate synthase inhibitors chlorsulfuron and imazapyr did not. Additionally, overexpression of BRANCHED-CHAIN AMINO ACID TRANSFERASE2 (BCAT2), which is upregulated in response to osmotic stress and functions in BCAA degradation, decreased drought-induced BCAA accumulation. Together, these results demonstrate that BCAA accumulation in osmotically stressed Arabidopsis is primarily the result of protein degradation. After relief of the osmotic stress, BCAA homeostasis is restored over time by amino acid degradation involving BCAT2. Thus, drought-induced BCAA accumulation is different from that of proline, which is accumulated due to de novo synthesis in an abscisic acid-independent manner and remains elevated for a more prolonged period of time after removal of the osmotic stress.

Ethephon increases photosynthetic-nitrogen use efficiency, proline and antioxidant metabolism to alleviate decrease in photosynthesis under salinity stress in mustard.

PubMed

Iqbal, Noushina; Umar, Shahid; Per, Tasir S; Khan, Nafees A

2017-05-04

Salinity is a serious threat to plant growth and development worldwide reducing agricultural productivity each year. Ethylene is an important phytohormone that affects plants performance under normal and abiotic stress conditions. In this study, role of ethylene was investigated in mitigating salinity stress (100 mM NaCl) effects on photosynthesis in mustard plants subjected to different nitrogen (N; 5 and 10 mM) levels. Plants under salinity stress exhibited marked increase in proline and reduced glutathione (GSH) content and activity of antioxidant enzymes. Nitrogen supplementation at 10 mM was better than 200 µl l -1 ethephon treatment under no stress. However, under salinity stress, both N and ethephon were equally effective. The combined application of 10 mM N and ethephon to salinity stressed plants produced greatest increase in photosynthesis by increasing proline and antioxidant metabolism. Ethylene evolution was high under salinity stress, but treatment of 10 mM N and 200 µl l -1 ethephon greatly decreased ethylene evolution that was equivalent to the 10 mM N treatment alone. This concentration of ethylene decreased the oxidative stress and increased the photosynthetic nitrogen use efficiency (NUE) maximally to increase photosynthesis. The use of ethylene action inhibitor, norbornadiene (NBD) showed reduction in ethylene mediated effects in alleviating salinity. Norbornadiene decreased the photosynthetic-NUE, proline and GSH content that resulted in decrease in photosynthesis under salinity stress. This study indicated that ethylene regulated the proline and antioxidant metabolism under salinity stress to increase photosynthetic functions of mustard grown with low and optimum N. The modulation of ethylene could be adopted in agricultural practices to increase photosynthesis under salinity stress.

D-Amino Acid Aberrations in Cerebrospinal Fluid and Plasma of Smokers

PubMed Central

Luykx, Jurjen J; Bakker, Steven C; van Boxmeer, Loes; Vinkers, Christiaan H; Smeenk, Hanne E; Visser, Wouter F; Verhoeven-Duif, Nanda M; Strengman, Eric; Buizer-Voskamp, Jacobine E; de Groene, Lizzy; van Dongen, Eric PA; Borgdorff, Paul; Bruins, Peter; de Koning, Tom J; Kahn, René S; Ophoff, Roel A

2013-01-01

The glutamatergic neurotransmission system and the N-methyl-D-aspartate receptor (NMDAR) have been implicated in smoking and alcohol consumption behavior. Preclinical studies have demonstrated that nicotine and ethanol influence NMDAR functionality, which may have a role in tendencies to consume these substances. Nonetheless, little is known about concentrations of NMDAR coagonists in the cerebrospinal fluid (CSF) and plasma of individuals who smoke or consume alcohol. Glycine and L- and D-stereoisomers of alanine, serine, and proline were therefore measured using ultra-high-performance liquid chromatography-tandem mass spectrometry in 403 healthy subjects. Nicotine and alcohol consumption were quantified using questionnaires. Possible differences in NMDAR coagonist concentrations in plasma and CSF were investigated using ANCOVA with age, body mass index, and storage duration as covariates. The significance threshold was Bonferroni corrected (α=0.00625). Compared with non-smokers, smokers displayed lower levels of D-proline in plasma (p=0.0027, Cohen's d=−0.41) and D-proline in CSF (p=0.0026, Cohen's d=−0.43). D-Serine in CSF was higher in smokers than in non-smokers (p=0.0052, Cohen's d=0.41). After subdividing participants based on smoking quantity, dose-dependent decreases were demonstrated in smokers for D-proline in plasma (F=5.65, p=0.0039) and D-proline in CSF (F=5.20, p=0.0060). No differences in NMDAR coagonist levels between alcohol consumption groups were detected. To our knowledge, this is the first report to implicate D-amino acids in smoking behavior of humans. Whether such concentration differences lie at the root of or result from smoking habits may be addressed in prospective studies. PMID:23615666

An N-terminal di-proline motif is essential for fatty acid–dependent degradation of Δ9-desaturase in Drosophila

PubMed Central

Murakami, Akira; Nagao, Kohjiro; Juni, Naoto; Hara, Yuji; Umeda, Masato

2017-01-01

The Δ9-fatty acid desaturase introduces a double bond at the Δ9 position of the acyl moiety of acyl-CoA and regulates the cellular levels of unsaturated fatty acids. However, it is unclear how Δ9-desaturase expression is regulated in response to changes in the levels of fatty acid desaturation. In this study, we found that the degradation of DESAT1, the sole Δ9-desaturase in the Drosophila cell line S2, was significantly enhanced when the amounts of unsaturated acyl chains of membrane phospholipids were increased by supplementation with unsaturated fatty acids, such as oleic and linoleic acids. In contrast, inhibition of DESAT1 activity remarkably suppressed its degradation. Of note, removal of the DESAT1 N-terminal domain abolished the responsiveness of DESAT1 degradation to the level of fatty acid unsaturation. Further truncation and amino acid replacement analyses revealed that two sequential prolines, the second and third residues of DESAT1, were responsible for the unsaturated fatty acid–dependent degradation. Although degradation of mouse stearoyl-CoA desaturase 1 (SCD1) was unaffected by changes in fatty acid unsaturation, introduction of the N-terminal sequential proline residues into SCD1 conferred responsiveness to unsaturated fatty acid–dependent degradation. Furthermore, we also found that the Ca2+-dependent cysteine protease calpain is involved in the sequential proline–dependent degradation of DESAT1. In light of these findings, we designated the sequential prolines at the second and third positions of DESAT1 as a “di-proline motif,” which plays a crucial role in the regulation of Δ9-desaturase expression in response to changes in the level of cellular unsaturated fatty acids. PMID:28972163

Influence of NaCl on Growth, Proline, and Phosphoenolpyruvate Carboxylase Levels in Mesembryanthemum crystallinum Suspension Cultures 1

PubMed Central

Thomas, John C.; De Armond, Richard L.; Bohnert, Hans J.

1992-01-01

The facultative halophyte Mesembryanthemum crystallinum responds to salt stress by increasing the levels of phosphoenolpyruvate carboxylase (PEPCase) and other enzymes associated with Crassulacean acid metabolism. A more common response to salt stress in sensitive and tolerant species, including M. crystallinum, is the accumulation of proline. We have established M. crystallinum suspension cultures to investigate whether both these salt-induced responses occur at the cellular level. Leaf-and root-derived cultures maintain 5% of the total soluble amino acids as proline. Cell culture growth slows upon addition of 400 millimolar NaCl, and proline levels increase to 40% of the total soluble amino acids. These results suggest a functional salt-stress and response program in Mesembryanthemum cells. Suspension cultures grown with or without 400 millimolar NaCl have PEPCase levels that compare with those from roots and unstressed leaves. The predominant protein cross-reacting with an anti-PEPCase antibody corresponds to 105 kilodaltons (apparent molecular mass), whereas a second species of approximately 110 kilodaltons is present at low levels. In salt-stressed leaves, the 110 kilodalton protein is more prevalent. Levels of mRNA for both ppc1 (salt stress induced in leaves) and ppc2 (constitutive) genes in salt-treated suspensions cultures are equal to unstressed leaves, and only twice the levels found in untreated suspension cultures. Whereas cells accumulate proline in response to NaCl, PEPCase protein amounts remain similar in salt-treated and untreated cultures. The induction upon salt stress of the 110 kilodalton PEPCase protein and other Crassulacean acid metabolism enzymes in organized tissues is not observed in cell culture and may depend on tissue-dependent or photoautotrophy-dependent programs. ImagesFigure 4Figure 5 PMID:16668687

Responses of Reactive Oxygen Scavenging Enzymes, Proline and Malondialdehyde to Water Deficits among Six Secondary Successional Seral Species in Loess Plateau

PubMed Central

Du, Feng; Shi, Huijun; Zhang, Xingchang; Xu, Xuexuan

2014-01-01

Drought can impact local vegetation dynamics in a long term. In order to predict the possible successional pathway of local community under drought, the responses of some drought resistance indices of six successional seral species in the semi-arid Loss Hilly Region of China were illustrated and compared on three levels of soil water deficits along three growing months (7, 8 and 9). The results showed that: 1) the six species had significant differences in SOD, POD activities and MDA content. The rank correlations between SOD, POD activities and the successional niche positions of the six species were positive, and the correlation between MDA content and the niche positions was negative; 2) activities of SOD, CAT and POD, and content of proline and MDA had significant differences among the three months; 3) there existed significant interactions of SOD, CAT, POD activities and MDA content between months and species. With an exception, no interaction of proline was found. Proline in leaves had a general decline in reproductive month; 4) SOD, CAT, POD activities and proline content had negative correlations with MDA content. Among which, the correlation between SOD activity and MDA content was significant. The results implied that, in arid or semiarid region, the species at later successional stage tend to have strong drought resistance than those at early stage. Anti-drought indices can partially interpret the pathway of community succession in the drought impacted area. SOD activity is more distinct and important on the scope of protecting membrane damage through the scavenging of ROS on exposure to drought. PMID:24914928

Biochemical characterization of the triticale TsPAP1, a new type of plant prolyl aminopeptidase, and its impact on proline content and flowering time in transgenic Arabidopsis plants.

PubMed

Zdunek-Zastocka, Edyta; Grabowska, Agnieszka; Branicki, Tomasz; Michniewska, Beata

2017-07-01

Proline aminopeptidase (PAP, EC 3.4.11.5) is the only enzyme that effectively releases proline from the N-termini of peptides. The amino acid sequence of the PAP from Triticosecale, TsPAP1, comprises conserved regions, characteristic of the monomeric forms of PAP found in bacteria but not yet identified in plants. Therefore, we aimed to obtain and biochemically characterize the TsPAP1 protein. The recombinant TsPAP1 protein was received through heterologous expression of the TsPAP1 coding sequence in a bacterial expression system and purified with affinity chromatography. Gel filtration chromatography and SDS electrophoresis revealed that TsPAP1 is a monomer with a molecular mass of 37.5 kDa. TsPAP1 prefers substrates with proline at the N-terminus but is also capable of hydrolyzing β-naphthylamides of hydroxyproline and alanine. Among the peptides tested, the most preferred were di- and tripeptides, especially those with glycine in the Y position. The use of diagnostic inhibitors indicated that TsPAP1 is a serine peptidase; however, further characterization revealed that the SH residues are also important for maintaining its activity. To examine the role of TsPAP1 under physiological conditions, we developed transgenic Arabidopsis plants overexpressing TsPAP1. Compared with wild-type plants, the transgenic lines accumulated more proline, flowered an average of 3.5 days earlier, and developed more siliques than did untransformed controls. Our paper is the first to describe the biochemical properties of a novel monomeric plant PAP and contributes to the functional characterization of PAP proteins in plants. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Conformational change of Sos-derived proline-rich peptide upon binding Grb2 N-terminal SH3 domain probed by NMR

NASA Astrophysics Data System (ADS)

Ogura, Kenji; Okamura, Hideyasu

2013-10-01

Growth factor receptor-bound protein 2 (Grb2) is a small adapter protein composed of a single SH2 domain flanked by two SH3 domains. The N-terminal SH3 (nSH3) domain of Grb2 binds a proline-rich region present in the guanine nucleotide releasing factor, son of sevenless (Sos). Using NMR relaxation dispersion and chemical shift analysis methods, we investigated the conformational change of the Sos-derived proline-rich peptide during the transition between the free and Grb2 nSH3-bound states. The chemical shift analysis revealed that the peptide does not present a fully random conformation but has a relatively rigid structure. The relaxation dispersion analysis detected conformational exchange of several residues of the peptide upon binding to Grb2 nSH3.

Proline-rich antimicrobial peptides: converging to a non-lytic mechanism of action.

PubMed

Scocchi, Marco; Tossi, Alessandro; Gennaro, Renato

2011-07-01

Proline-rich antimicrobial peptides are a group of cationic host defense peptides of vertebrates and invertebrates characterized by a high content of proline residues, often associated with arginine residues in repeated motifs. Those isolated from some mammalian and insect species, although not evolutionarily related, use a similar mechanism to selectively kill Gram-negative bacteria, with a low toxicity to animals. Unlike other types of antimicrobial peptides, their mode of action does not involve the lysis of bacterial membranes but entails penetration into susceptible cells, where they then act intracellularly. Some aspects of the transport system and cytoplasmic targets have been elucidated. These features make them attractive both as anti-infective lead compounds and as a new class of potential cell-penetrating peptides capable of internalising membrane-impermeant drugs into both bacterial and eukaryotic cells.

Application of L-proline derivatives as chiral shift reagents for enantiomeric recognition of carboxylic acids.

PubMed

Naziroglu, Hayriye Nevin; Durmaz, Mustafa; Bozkurt, Selahattin; Sirit, Abdulkadir

2011-07-01

Four proline-derived chiral receptors 5-8 were readily synthesized starting from L-proline. The enantiomeric recognition ability of chiral receptors was examined with a series of carboxylic acids by (1) H NMR spectroscopy. The molar ratio and the association constants of the chiral compounds with each of the enantiomers of guest molecules were determined by using Job plots and a nonlinear least-squares fitting method, respectively. The Job plots indicate that the hosts form 1:1 instantaneous complexes with all guests. The receptors exhibited different chiral recognition abilities toward the enantiomers of racemic guests. Among the chiral receptors used in this study, prolinamide 6 was found to be the best chiral shift reagent and is effective for the determination of the enantiomeric excess of chiral carboxylic acids. Copyright © 2011 Wiley-Liss, Inc.

CuI/L-proline-catalyzed coupling reactions of aryl halides with activated methylene compounds.

PubMed

Xie, Xiaoan; Cai, Guorong; Ma, Dawei

2005-10-13

[reaction: see text] The arylation of ethyl acetoacetate, ethyl benzoyl acetate, and diethyl malonate under the catalysis of CuI/L-proline in DMSO proceeds smoothly at 40-50 degrees C in the presence of Cs2CO3 to provide the 2-aryl-1,3-dicarbonyl compounds in good yields. Both aryl iodides and aryl bromides are compatible with these reaction conditions.

Alternative Strategies in Response to Saline Stress in Two Varieties of Portulaca oleracea (Purslane).

PubMed

Mulry, Kristina R; Hanson, Bryan A; Dudle, Dana A

2015-01-01

Purslane (Portulaca oleracea) is a globally-distributed plant with a long history of use in folk medicine and cooking. We have developed purslane as a model system for exploring plant responses to stress. We exposed two varieties of purslane to saline stress with the objective of identifying differences between the varieties in the plasticity of morphological and physiological traits. The varieties responded to saline stress with significantly different changes in the measured traits, which included inter alia biomass, flower counts, proline concentrations and betalain pigment concentrations. The alternative responses of the two varieties consisted of complex, simultaneous changes in multiple traits. In particular, we observed that while both varieties increased production of betalain pigments and proline under saline stress, one variety invested more in betalain pigments while the other invested more in proline. Proline and betalain pigments undoubtedly play multiple roles in plant tissues, but in this case their role as antioxidants deployed to ameliorate saline stress appears to be important. Taken holistically, our results suggest that the two varieties employ different strategies in allocating resources to cope with saline stress. This conclusion establishes purslane as a suitable model system for the study of saline stress and the molecular basis for differential responses.

PPII propensity of multiple-guest amino acids in a proline-rich environment.

PubMed

Moradi, Mahmoud; Babin, Volodymyr; Sagui, Celeste; Roland, Christopher

2011-07-07

There has been considerable debate about the intrinsic PPII propensity of amino acid residues in denatured polypeptides. Experimentally, this scale is based on the behavior of guest amino acid residues placed in the middle of proline-based hosts. We have used classical molecular dynamics simulations combined with replica-exchange methods to carry out a comprehensive analysis of the conformational equilibria of proline-based host oligopeptides with multiple guest amino acids including alanine, glutamine, valine, and asparagine. The tracked structural characteristics include the secondary structural motifs based on the Ramachandran angles and the cis/trans isomerization of the prolyl bonds. In agreement with our recent study of single amino acid guests, we did not observe an intrinsic PPII propensity in any of the guest amino acids in a multiple-guest setting. Instead, the experimental results can be explained in terms of (i) the steric restrictions imposed on the C-terminal guest amino acid that is immediately followed by a proline residue and (ii) an increase in the trans content of the prolyl bonds due to the presence of guest residues. In terms of the latter, we found that the more guests added to the system, the larger the increase in the trans content of the prolyl bonds, which results in an effective increase in the PPII content of the peptide.

Alternative Strategies in Response to Saline Stress in Two Varieties of Portulaca oleracea (Purslane)

PubMed Central

Mulry, Kristina R.; Hanson, Bryan A.; Dudle, Dana A.

2015-01-01

Purslane (Portulaca oleracea) is a globally-distributed plant with a long history of use in folk medicine and cooking. We have developed purslane as a model system for exploring plant responses to stress. We exposed two varieties of purslane to saline stress with the objective of identifying differences between the varieties in the plasticity of morphological and physiological traits. The varieties responded to saline stress with significantly different changes in the measured traits, which included inter alia biomass, flower counts, proline concentrations and betalain pigment concentrations. The alternative responses of the two varieties consisted of complex, simultaneous changes in multiple traits. In particular, we observed that while both varieties increased production of betalain pigments and proline under saline stress, one variety invested more in betalain pigments while the other invested more in proline. Proline and betalain pigments undoubtedly play multiple roles in plant tissues, but in this case their role as antioxidants deployed to ameliorate saline stress appears to be important. Taken holistically, our results suggest that the two varieties employ different strategies in allocating resources to cope with saline stress. This conclusion establishes purslane as a suitable model system for the study of saline stress and the molecular basis for differential responses. PMID:26398279

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rod, M.L. Alam, K.Y.; Cunningham, P.R.; Clark, D.P.

When grown at high osmotic pressure, some strains of Escherichia coli K-12 synthesized substantial levels of free sugar and accumulated proline if it was present in the growth medium. The sugar was identified as trehalose. Strains of E. coli K-12 could be divided into two major classes with respect of osmoregulation. Those of class A showed a large increase in trehalose levels with increasing medium osmolarity and also accumulated proline from the medium, whereas those in class B showed no accumulation of trehalose or proline. Most class A strains carried suppressor mutations which arose during their derivation from the wildmore » type, whereas the osmodefective strains of class B were suppressor free. When amber suppressor mutations at the supD, supE, or supF loci were introduced into such sup{sup o} osmodefective strains, they became osmotolerant and gained the ability to accumulate trehalose in response to elevated medium osmolarity. It appears that the original K-12 strain of E. coli carries an amber mutation in a gene affecting osmoregulation. Mutants lacking ADP-glucose synthetase (glgC) accumulated trehalose normally, whereas mutants lacking UDP-glucose synthetase (galU) did not make trehalose and grew poorly in medium of high osmolarity. Trehalose synthesis was repressed by exogenous glycine betaine but not by proline.« less

Cyclophilin A catalyzes proline isomerization by an electrostatic handle mechanism

DOE Office of Scientific and Technical Information (OSTI.GOV)

Camilloni, Carlo; Sahakyan, Aleksander B.; Holliday, Michael

2014-07-15

Proline isomerization is a ubiquitous process that plays a key role in the folding of proteins and in the regulation of their functions1-3. Different families of enzymes, known as peptidyl-prolyl isomerases (PPIases), catalyse this reaction, which involves the interconversion between the cis and trans isomers of the Nterminal amide bond of the amino acid proline2,3. A complete description of the mechanisms by which these enzymes function, however, has remained elusive. Here, we show that cyclophilin A, one of the most common PPIases4, provides a catalytic environment that acts on the substrate through an electrostatic lever mechanism. In this mechanism, themore » electrostatic field in the catalytic site turns the electric dipole associated with the carboxylic group of the amino acid preceding the proline in the substrate, thus causing the rotation of the peptide bond between the two residues. This mechanism resulted from the analysis of an ensemble of conformations populated by cyclophilin A during the enzymatic reaction using a combination of NMR measurements, molecular dynamics simulations and density functional theory calculations. We anticipate that this approach will be helpful in elucidating whether the electrostatic lever mechanism that we describe is common to other PPIases, and more generally to characterise other enzymatic processes.« less

Molecular Dynamics of the Proline Switch and Its Role in Crk Signaling

PubMed Central

2015-01-01

The Crk adaptor proteins play a central role as a molecular timer for the formation of protein complexes including various growth and differentiation factors. The loss of regulation of Crk results in many kinds of cancers. A self-regulatory mechanism for Crk was recently proposed, which involves domain–domain rearrangement. It is initiated by a cis–trans isomerization of a specific proline residue (Pro238 in chicken Crk II) and can be accelerated by Cyclophilin A. To understand how the proline switch controls the autoinhibition at the molecular level, we performed large-scale molecular dynamics and metadynamics simulations in the context of short peptides and multidomain constructs of chicken Crk II. We found that the equilibrium and kinetic properties of the macrostates are regulated not only by the local environments of specified prolines but also by the global organization of multiple domains. We observe the two macrostates (cis closed/autoinhibited and trans open/uninhibited) consistent with NMR experiments and predict barriers. We also propose an intermediate state, the trans closed state, which interestingly was reported to be a prevalent state in human Crk II. The existence of this macrostate suggests that the rate of switching off the autoinhibition by Cyp A may be limited by the relaxation rate of this intermediate state. PMID:24702481

Cyclophilin A catalyzes proline isomerization by an electrostatic handle mechanism

PubMed Central

Camilloni, Carlo; Sahakyan, Aleksandr B.; Holliday, Michael J.; Isern, Nancy G.; Zhang, Fengli; Eisenmesser, Elan Z.; Vendruscolo, Michele

2014-01-01

Proline isomerization is a ubiquitous process that plays a key role in the folding of proteins and in the regulation of their functions. Different families of enzymes, known as “peptidyl-prolyl isomerases” (PPIases), catalyze this reaction, which involves the interconversion between the cis and trans isomers of the N-terminal amide bond of the amino acid proline. However, complete descriptions of the mechanisms by which these enzymes function have remained elusive. We show here that cyclophilin A, one of the most common PPIases, provides a catalytic environment that acts on the substrate through an electrostatic handle mechanism. In this mechanism, the electrostatic field in the catalytic site turns the electric dipole associated with the carbonyl group of the amino acid preceding the proline in the substrate, thus causing the rotation of the peptide bond between the two residues. We identified this mechanism using a combination of NMR measurements, molecular dynamics simulations, and density functional theory calculations to simultaneously determine the cis-bound and trans-bound conformations of cyclophilin A and its substrate as the enzymatic reaction takes place. We anticipate that this approach will be helpful in elucidating whether the electrostatic handle mechanism that we describe here is common to other PPIases and, more generally, in characterizing other enzymatic processes. PMID:24982184

Structure and Activity of the Peptidyl-Prolyl Isomerase Domain from the Histone Chaperone Fpr4 toward Histone H3 Proline Isomerization*

PubMed Central

Monneau, Yoan R.; Soufari, Heddy; Nelson, Christopher J.; Mackereth, Cameron D.

2013-01-01

The FK506-binding protein (FKBP) family of peptidyl-prolyl isomerases (PPIases) is characterized by a common catalytic domain that binds to the inhibitors FK506 and rapamycin. As one of four FKBPs within the yeast Saccharomyces cerevisiae, Fpr4 has been described as a histone chaperone, and is in addition implicated in epigenetic function in part due to its mediation of cis-trans conversion of proline residues within histone tails. To better understand the molecular details of this activity, we have determined the solution structure of the Fpr4 C-terminal PPIase domain by using NMR spectroscopy. This canonical FKBP domain actively increases the rate of isomerization of three decapeptides derived from the N terminus of yeast histone H3, whereas maintaining intrinsic cis and trans populations. Observation of the uncatalyzed and Fpr4-catalyzed isomerization rates at equilibrium demonstrate Pro16 and Pro30 of histone H3 as the major proline targets of Fpr4, with little activity shown against Pro38. This alternate ranking of the three target prolines, as compared with affinity determination or the classical chymotrypsin-based fluorescent assay, reveals the mechanistic importance of substrate residues C-terminal to the peptidyl-prolyl bond. PMID:23888048

Parameterization of the proline analogue Aze (azetidine-2-carboxylic acid) for molecular dynamics simulations and evaluation of its effect on homo-pentapeptide conformations.

PubMed

Bessonov, Kyrylo; Vassall, Kenrick A; Harauz, George

2013-02-01

We have parameterized and evaluated the proline homologue Aze (azetidine-2-carboxylic acid) for the gromos56a3 force-field for use in molecular dynamics simulations using GROMACS. Using bi-phasic cyclohexane/water simulation systems and homo-pentapeptides, we measured the Aze solute interaction potential energies, ability to hydrogen bond with water, and overall compaction, for comparison to Pro, Gly, and Lys. Compared to Pro, Aze has a slightly higher H-bonding potential, and stronger electrostatic but weaker non-electrostatic interactions with water. The 20-ns simulations revealed the preferential positioning of Aze and Pro at the interface of the water and cyclohexane layers, with Aze spending more time in the aqueous layer. We also demonstrated through simulations of the homo-pentapeptides that Aze has a greater propensity than Pro to undergo trans→cis peptide bond isomerization, which results in a severe 180° bend in the polypeptide chain. The results provide evidence for the hypothesis that the misincorporation of Aze within proline-rich regions of proteins could disrupt the formation of poly-proline type II structures and compromise events such as recognition and binding by SH3-domains. Copyright © 2012 Elsevier Inc. All rights reserved.

Cyclophilin A catalyzes proline isomerization by an electrostatic handle mechanism.

PubMed

Camilloni, Carlo; Sahakyan, Aleksandr B; Holliday, Michael J; Isern, Nancy G; Zhang, Fengli; Eisenmesser, Elan Z; Vendruscolo, Michele

2014-07-15

Proline isomerization is a ubiquitous process that plays a key role in the folding of proteins and in the regulation of their functions. Different families of enzymes, known as "peptidyl-prolyl isomerases" (PPIases), catalyze this reaction, which involves the interconversion between the cis and trans isomers of the N-terminal amide bond of the amino acid proline. However, complete descriptions of the mechanisms by which these enzymes function have remained elusive. We show here that cyclophilin A, one of the most common PPIases, provides a catalytic environment that acts on the substrate through an electrostatic handle mechanism. In this mechanism, the electrostatic field in the catalytic site turns the electric dipole associated with the carbonyl group of the amino acid preceding the proline in the substrate, thus causing the rotation of the peptide bond between the two residues. We identified this mechanism using a combination of NMR measurements, molecular dynamics simulations, and density functional theory calculations to simultaneously determine the cis-bound and trans-bound conformations of cyclophilin A and its substrate as the enzymatic reaction takes place. We anticipate that this approach will be helpful in elucidating whether the electrostatic handle mechanism that we describe here is common to other PPIases and, more generally, in characterizing other enzymatic processes.

Molecular dynamics of the proline switch and its role in Crk signaling.

PubMed

Xia, Junchao; Levy, Ronald M

2014-05-01

The Crk adaptor proteins play a central role as a molecular timer for the formation of protein complexes including various growth and differentiation factors. The loss of regulation of Crk results in many kinds of cancers. A self-regulatory mechanism for Crk was recently proposed, which involves domain-domain rearrangement. It is initiated by a cis-trans isomerization of a specific proline residue (Pro238 in chicken Crk II) and can be accelerated by Cyclophilin A. To understand how the proline switch controls the autoinhibition at the molecular level, we performed large-scale molecular dynamics and metadynamics simulations in the context of short peptides and multidomain constructs of chicken Crk II. We found that the equilibrium and kinetic properties of the macrostates are regulated not only by the local environments of specified prolines but also by the global organization of multiple domains. We observe the two macrostates (cis closed/autoinhibited and trans open/uninhibited) consistent with NMR experiments and predict barriers. We also propose an intermediate state, the trans closed state, which interestingly was reported to be a prevalent state in human Crk II. The existence of this macrostate suggests that the rate of switching off the autoinhibition by Cyp A may be limited by the relaxation rate of this intermediate state.

Proline-linked nitrosoureas as prolidase-convertible prodrugs in human breast cancer cells.

PubMed

Bielawski, Krzysztof; Bielawska, Anna; Słodownik, Tomasz; Bołkun-Skórnicka, Urszula; Muszyńska, Anna

2008-01-01

A number of novel proline-linked nitrosoureas (1-4) were synthesized and examined for cytotoxicity and influence on DNA and collagen biosynthesis in MDA-MB-231 and MCF-7 human breast cancer cells. Evaluation of the cytotoxicity of these compounds employing a MTT assay and inhibition of [(3)H]thymidine incorporation into DNA in both MDA-MB-231 and MCF-7 breast cancer cells demonstrated that compound 2, the most active of the series, proved to be only slightly less potent than carmustine. It has also been found that carmustine did not inhibit MCF&-7 cells prolidase activity, while compounds 1-4 significantly increased its activity, when used at 50-250 microM concentrations. Proline-linked nitrosoureas (1-4) also had lower ability to inhibit collagen biosynthesis in MCF-7 cells, compared to carmustine. The expression of beta(1)-integrin receptor and phosphorylated MAPK, ERK(1) and ERK(2) was significantly decreased in MCF-7 cells incubated for 24 h with 60 microM of compounds 2 and 4 compared to the control, untreated cells, whereas under the same conditions carmustine did not evoke any changes in expression of all these signaling proteins, as shown by Western immunoblot analysis. These results indicate the proline-linked nitrosoureas (1-4), represent multifunctional inhibitors of breast cancer cell growth and metabolism.

Proline levels, oxidative metabolism and photosynthetic pigments during in vitro growth and acclimatization of Pitcairnia encholirioides L.B. Sm. (Bromeliaceae).

PubMed

Resende, C F; Braga, V F; Pereira, P F; Silva, C J; Vale, V F; Bianchetti, R E; Forzza, R C; Ribeiro, C; Peixoto, P H P

2016-02-01

This study aimed to evaluate the variation in the levels of proline, oxidative metabolism and photosynthetic pigments in plants of Pitcairnia encholirioides grown in vitro under different conditions and after acclimatization. The analyses were performed after 150 days of in vitro cultivation in MS media supplemented with 10 µM GA3 or 0.2 µM NAA, sucrose at 15 or 30 g L-1, in test tubes which allowed gas exchange or in a hermetically sealed system, and 180 days after acclimatization. The in vitro maintenance in hermetically sealed flasks, with GA3 and 15 g L-1 sucrose had adverse metabolic effects, which was demonstrated by the lower proline and photosynthetic pigments accumulation and by the increase in antioxidant enzymes activities. After acclimatization, differences for proline and photosynthetic pigments were no longer found and the enzymatic activities ranged unevenly. The results suggest that the in vitro cultivation in media with 0.2 µM NAA and 30 g L-1 sucrose, in test tubes capped with closures which allowed gas exchange, is more suitable for micropropagation of P. encholirioides, providing a prolonged maintenance of in vitro cultures and plantlets with superior quality for ex vitro development.

A concise and practical stereoselective synthesis of ipragliflozin L-proline

PubMed Central

Ma, Shuai; Liu, Zhenren; Pan, Jing; Zhang, Shunli

2017-01-01

A concise and practical stereoselective synthesis of ipragliflozin L-proline was presented starting from 2-[(5-iodo-2-fluorophenyl)methyl]-1-benzothiophene and 2,3,4,6-tetra-O-pivaloyl-α-D-glucopyranosyl bromide without catalyst via iodine–lithium–zinc exchange. The overall yield was 52% in three steps and the product purity was excellent. Two key diastereomers were prepared with efficient and direct access to the α-C-arylglucoside. PMID:28684985

Investigating MUC1/ICAM-1 Binding Induced Signaling in Breast Cancer Metastasis

DTIC Science & Technology

2011-05-01

expected that covalently linked species would remain intact. Reducing (R, + !-mercaptoethanol) and non-reducing (NR, no !-mercaptoethanol) samples were...binding site, containing both proline and arginine residues. We mutated the SH2 and/or putative SH3 binding domains on the MUC1-CFP-Fv plasmid...Structure and regulation of Src family kinases. Oncogene 2004, 23:7918- 7927. 31. Li SSC: Specificity and versatility of SH3 and other proline -recognition

UNC Center for Dynamic Combinatorial Chemistry

DTIC Science & Technology

2014-04-09

Thioester Exchange, Organic Letters (04 2010) Chung, Schiltz, Lee, and Gagne. The Effect of Gas-Phase Reactions on the Quantitation of Cyclic...Figure 6.1c) These monomers have L-tryptophan, L-leucine, L- phenylalanine , and the sequential L-proline and D-proline functions. These functions...are known as the parts of three hydrophobic side chains in the tumor suppressor protein p53 and make direct contacts deep in the MDM2 cleft (Figure

The Use of Fluoroproline in MUC1 Antigen Enables Efficient Detection of Antibodies in Patients with Prostate Cancer.

PubMed

Somovilla, Víctor J; Bermejo, Iris A; Albuquerque, Inês S; Martínez-Sáez, Nuria; Castro-López, Jorge; García-Martín, Fayna; Compañón, Ismael; Hinou, Hiroshi; Nishimura, Shin-Ichiro; Jiménez-Barbero, Jesús; Asensio, Juan L; Avenoza, Alberto; Busto, Jesús H; Hurtado-Guerrero, Ramón; Peregrina, Jesús M; Bernardes, Gonçalo J L; Corzana, Francisco

2017-12-20

A structure-based design of a new generation of tumor-associated glycopeptides with improved affinity against two anti-MUC1 antibodies is described. These unique antigens feature a fluorinated proline residue, such as a (4S)-4-fluoro-l-proline or 4,4-difluoro-l-proline, at the most immunogenic domain. Binding assays using biolayer interferometry reveal 3-fold to 10-fold affinity improvement with respect to the natural (glyco)peptides. According to X-ray crystallography and MD simulations, the fluorinated residues stabilize the antigen-antibody complex by enhancing key CH/π interactions. Interestingly, a notable improvement in detection of cancer-associated anti-MUC1 antibodies from serum of patients with prostate cancer is achieved with the non-natural antigens, which proves that these derivatives can be considered better diagnostic tools than the natural antigen for prostate cancer.

Regulation of osmoadaptation in the moderate halophile Halobacillus halophilus: chloride, glutamate and switching osmolyte strategies

PubMed Central

Saum, Stephan H; Müller, Volker

2008-01-01

The moderate halophile Halobacillus halophilus is the paradigm for chloride dependent growth in prokaryotes. Recent experiments shed light on the molecular basis of the chloride dependence that is reviewed here. In the presence of moderate salinities Halobacillus halophilus mainly accumulates glutamine and glutamate to adjust turgor. The transcription of glnA2 (encoding a glutamine synthetase) as well as the glutamine synthetase activity were identified as chloride dependent steps. Halobacillus halophilus switches its osmolyte strategy and produces proline as the main compatible solute at high salinities. Furthermore, Halobacillus halophilus also shifts its osmolyte strategy at the transition from the exponential to the stationary phase where proline is exchanged by ectoine. Glutamate was found as a “second messenger” essential for proline production. This observation leads to a new model of sensing salinity by sensing the physico-chemical properties of different anions. PMID:18442383

Thermodynamics of grape and wine tannin interaction with polyproline: implications for red wine astringency.

PubMed

McRae, Jacqui M; Falconer, Robert J; Kennedy, James A

2010-12-08

The astringency of red wine is largely due to the interaction between wine tannins and salivary proline-rich proteins and is known to change as wine ages. To further understand the mechanisms behind wine astringency change over time, thermodynamics of the interactions between poly(l-proline) (PLP) and grape seed and skin tannins (preveraison (PV) and commercially ripe) or Shiraz wine tannins (2 years old and 9-10 years old) was analyzed using isothermal titration calorimetry (ITC). The nature of these interactions varied with changes to the tannin structure that are associated with maturation. The change in enthalpy associated with hydrophobic interaction and hydrogen bonding decreased with tannin age and the stoichiometry of binding indicated that grape tannins associated with more proline residues than wine tannins, irrespective of molecular size. These results could provide an explanation for the observed change in wine astringency quality with age.

The proline-rich domain of TonB possesses an extended polyproline II-like conformation of sufficient length to span the periplasm of Gram-negative bacteria

PubMed Central

Köhler, Silvia Domingo; Weber, Annemarie; Howard, S Peter; Welte, Wolfram; Drescher, Malte

2010-01-01

TonB from Escherichia coli and its homologues are critical for the uptake of siderophores through the outer membrane of Gram-negative bacteria using chemiosmotic energy. When different models for the mechanism of TonB mediated energy transfer from the inner to the outer membrane are discussed, one of the key questions is whether TonB spans the periplasm. In this article, we use long range distance measurements by spin-label pulsed EPR (Double Electron–Electron Resonance, DEER) and CD spectroscopy to show that the proline-rich segment of TonB exists in a PPII-like conformation. The result implies that the proline-rich segment of TonB possesses a length of more than 15 nm, sufficient to span the periplasm of Gram-negative bacteria. PMID:20095050

Proline transport in Leishmania donovani amastigotes: dependence on pH gradients and membrane potential.

PubMed

Glaser, T A; Mukkada, A J

1992-03-01

Amastigotes of Leishmania donovani develop and multiply within the acidic phagolysosomes of mammalian macrophages. Isolated amastigotes are acidophilic; they catabolize substrates and synthesize macromolecules optimally at pH 5.5. Substrate transport in amastigotes has not been characterized. Here we show that amastigotes exhibit an uphill transport of proline (active transport) with an acid pH optimum (pH 5.5). It is dependent upon metabolic energy and is driven by proton motive force. Agents which selectively disturb the component forces of proton motive force, such as carbonyl cyanide chlorophenylhydrazone, nigericin and valinomycin, inhibit proline transport. Transport is sensitive to dicyclohexylcarbodiimide and insensitive to ouabain, demonstrating the involvement of a proton ATPase in the maintenance of proton motive force. It is suggested that the plasma membrane pH gradient probably makes the greatest contribution to proton motive force that drives substrate transport in the amastigote stage.

L-proline-stabilized human IgG: Privigen® 10% for intravenous use and Hizentra® 20% for subcutaneous use.

PubMed

Berger, Melvin

2011-02-01

Liquid IgG preparations are preferred over lyophilized preparations because reconstitution is not required. Formation of dimers and aggregates in liquid preparations increases adverse effects and limits the shelf life of most liquid IgG products. Improved understanding of the binding interactions in IgG dimers and aggregates led to the selection of L-proline at pH 4.8 as an excipient that would minimize their formation. CSL Behring has developed the L-proline-stabilized products Privigen®, a 10% IgG solution for intravenous use; and Hizentra®, a 20% solution for subcutaneous use. The former has the longest shelf life of any liquid IgG in the USA--36 months, and the latter is the most concentrated IgG available. These improvements, which translate into improved convenience for pharmacies and patients, were achieved with no compromise in safety, efficacy or tolerability of the products.

Frameshift Suppression in SACCHAROMYCES CEREVISIAE. V. Isolation and Genetic Properties of Nongroup-Specific Suppressors

PubMed Central

Culbertson, Michael R.; Gaber, Richard F.; Cummins, Claudia M.

1982-01-01

Two classes of frameshift suppressors distributed at 22 different loci were identified in previous studies in the yeast Saccharomyces cerevisiae. These suppressors exhibited allele-specific suppression of +1 G:C insertion mutations in either glycine or proline codons, designated as group II and group III frameshift mutations, respectively. Genes corresponding to representative suppressors of each group have been shown to encode altered glycine or proline tRNAs containing four base anticodons.—This communication reports the existence of a third class of frameshift suppressor that exhibits a wider range in specificity of suppression. The suppressors map at three loci, suf12, suf13, and suf14, which are located on chromosomes IV, XV, and XIV, respectively. The phenotypes of these suppressors suggest that suppression may be mediated by genes other than those encoding the primary structure of glycine or proline tRNAs. PMID:6757053

Dietary proline supplementation alters colonic luminal microbiota and bacterial metabolite composition between days 45 and 70 of pregnancy in Huanjiang mini-pigs.

PubMed

Ji, Yujiao; Guo, Qiuping; Yin, Yulong; Blachier, Francois; Kong, Xiangfeng

2018-01-01

Pregnancy is associated with important changes in gut microbiota composition. Dietary factors may affect the diversity, composition, and metabolic activity of the intestinal microbiota. Among amino acids, proline is known to play important roles in protein metabolism and structure, cell differentiation, conceptus growth and development, and gut microbiota re-equilibration in case of dysbiosis. Dietary supplementation with 1% proline decreased ( P  < 0.05) the amounts of Klebsiella pneumoniae , Peptostreptococcus productus , Pseudomonas , and Veillonella spp. in distal colonic contents than that in the control group. The colonic contents of Butyrivibrio fibrisolvens , Bifidobacterium sp., Clostridium coccoides , Clostridium coccoides-Eubacterium rectale , Clostridium leptum subgroup, Escherichia coli , Faecalibacterium prausnitzii , Fusobacterium prausnitzii , and Prevotella increased ( P  < 0.05) on d 70 of pregnancy as compared with those on d 45 of pregnancy. The colonic concentrations of acetate, total straight-chain fatty acid, and total short-chain fatty acids (SCFA) in the proline-supplemented group were lower ( P  < 0.05), and butyrate level ( P  = 0.06) decreased as compared with the control group. Almost all of the SCFA displayed higher ( P  < 0.05) concentrations in proximal colonic contents on d 70 of pregnancy than those on d 45 of pregnancy. The concentrations of 1,7-heptyl diamine ( P  = 0.09) and phenylethylamine ( P  < 0.05) in proximal colonic contents were higher, while those of spermidine ( P  = 0.05) and total bioamine ( P  = 0.06) tended to be lower in the proline-supplemented group than those in the control group. The concentrations of spermidine, spermine, and total bioamine in colonic contents were higher ( P  < 0.05) on d 70 of pregnancy than those measured on d 45 of pregnancy. In contrast, the concentration of phenylethylamine was lower ( P  < 0.05) on d 70 than on d 45 of pregnancy. These findings indicate that L -proline supplementation modifies both the colonic microbiota composition and the luminal concentrations of several bacterial metabolites. Furthermore, our data show that both the microbiota composition and the concentrations of bacterial metabolites are evolving in the course of pregnancy. These results are discussed in terms of possible implication in terms of luminal environment and consequences for gut physiology and health.

Proline kink angle distributions for GWALP23 in lipid bilayers of different thicknesses.

PubMed

Rankenberg, Johanna M; Vostrikov, Vitaly V; DuVall, Christopher D; Greathouse, Denise V; Koeppe, Roger E; Grant, Christopher V; Opella, Stanley J

2012-05-01

By using selected (2)H and (15)N labels, we have examined the influence of a central proline residue on the properties of a defined peptide that spans lipid bilayer membranes by solid-state nuclear magnetic resonance (NMR) spectroscopy. For this purpose, GWALP23 (acetyl-GGALW(5)LALALALALALALW(19)LAGA-ethanolamide) is a suitable model peptide that employs, for the purpose of interfacial anchoring, only one tryptophan residue on either end of a central α-helical core sequence. Because of its systematic behavior in lipid bilayer membranes of differing thicknesses [Vostrikov, V. V., et al. (2010) J. Biol. Chem. 285, 31723-31730], we utilize GWALP23 as a well-characterized framework for introducing guest residues within a transmembrane sequence; for example, a central proline yields acetyl-GGALW(5)LALALAP(12)ALALALW(19)LAGA-ethanolamide. We synthesized GWALP23-P12 with specifically placed (2)H and (15)N labels for solid-state NMR spectroscopy and examined the peptide orientation and segmental tilt in oriented DMPC lipid bilayer membranes using combined (2)H GALA and (15)N-(1)H high-resolution separated local field methods. In DMPC bilayer membranes, the peptide segments N-terminal and C-terminal to the proline are both tilted substantially with respect to the bilayer normal, by ~34 ± 5° and 29 ± 5°, respectively. While the tilt increases for both segments when proline is present, the range and extent of the individual segment motions are comparable to or smaller than those of the entire GWALP23 peptide in bilayer membranes. In DMPC, the proline induces a kink of ~30 ± 5°, with an apparent helix unwinding or "swivel" angle of ~70°. In DLPC and DOPC, on the basis of (2)H NMR data only, the kink angle and swivel angle probability distributions overlap those of DMPC, yet the most probable kink angle appears to be somewhat smaller than in DMPC. As has been described for GWALP23 itself, the C-terminal helix ends before Ala(21) in the phospholipids DMPC and DLPC yet remains intact through Ala(21) in DOPC. The dynamics of bilayer-incorporated, membrane-spanning GWALP23 and GWALP23-P12 are less extensive than those observed for WALP family peptides that have more than two interfacial Trp residues.

Proline Kink Angle Distributions for GWALP23 in Lipid Bilayers of Different Thickness†

PubMed Central

Rankenberg, Johanna M.; Vostrikov, Vitaly V.; DuVall, Christopher D.; Greathouse, Denise V.; Koeppe, Roger E.; Grant, Christopher V.; Opella, Stanley J.

2013-01-01

By using selected 2H and 15N labels, we have examined the influence of a central proline residue upon the properties of a defined peptide that spans lipid bilayer membranes by solid-state NMR spectroscopy. For this purpose, GWALP23 (acetyl-GGALW5LALALALALALALW19LAGA-ethanolamide) is a suitable model peptide that employs—for the purpose of interfacial anchoring—only one tryptophan residue on either end of a central alpha-helical core sequence. Because of its systematic behavior in lipid bilayer membranes of differing thickness (see J. Biol. Chem. 285, 31723), we utilize GWALP23 as a well-characterized framework for introducing guest residues within a transmembrane sequence; for example, a central proline yields acetyl-GGALW5LALALAP12ALALALW19LAGA-ethanolamide. We synthesized the GWALP23-P12 with specifically placed 2H and 15N labels for solid-state NMR spectroscopy, and examined the peptide orientation and segmental tilt in oriented DMPC lipid bilayer membranes using combined (2H)-GALA and (15N-1H) high resolution separated local field methods. In DMPC bilayer membranes, the peptide segments N-terminal and C-terminal to the proline are both tilted substantially with respect to the bilayer normal, by about 34° and 29° (± 5°), respectively. While the tilt increases for both segments when proline is present, the range and extent of the individual segment motions are comparable or less than those of the entire GWALP23 peptide in bilayer membranes. In DMPC, the proline induces a kink of about 30° (± 5°), with an apparent helix unwinding or “swivel” angle of about 70°. In DLPC and DOPC, based on 2H NMR data only, the kink angle and swivel angle probability distributions overlap those of DMPC, yet the most probable kink angle appears somewhat smaller than in DMPC. As has been described for GWALP23 itself, the C-terminal helix ends before Ala-21 in the phospholipids DMPC and DLPC, yet remains intact through Ala-21 in DOPC. The dynamics of bilayer-incorporated, membrane-spanning GWALP23 and GWALP23-P12 are less extensive than observed for WALP-family peptides that have more than two interfacial Trp residues. PMID:22489564

Analysis of the thermodynamics of binding of an SH3 domain to proline-rich peptides using a chimeric fusion protein.

PubMed

Candel, Adela M; van Nuland, Nico A J; Martin-Sierra, Francisco M; Martinez, Jose C; Conejero-Lara, Francisco

2008-03-14

A complete understanding of the thermodynamic determinants of binding between SH3 domains and proline-rich peptides is crucial to the development of rational strategies for designing ligands for these important domains. Recently we engineered a single-chain chimeric protein by fusing the alpha-spectrin Src homology region 3 (SH3) domain to the decapeptide APSYSPPPPP (p41). This chimera mimics the structural and energetic features of the interaction between SH3 domains and proline-rich peptides. Here we show that analysing the unfolding thermodynamics of single-point mutants of this chimeric fusion protein constitutes a very useful approach to deciphering the thermodynamics of SH3-ligand interactions. To this end, we investigated the contribution of each proline residue of the ligand sequence to the SH3-peptide interaction by producing six single Pro-Ala mutants of the chimeric protein and analysing their unfolding thermodynamics by differential scanning calorimetry (DSC). Structural analyses of the mutant chimeras by circular dichroism, fluorescence and NMR together with NMR-relaxation measurements indicate conformational flexibility at the binding interface, which is strongly affected by the different Pro-Ala mutations. An analysis of the DSC thermograms on the basis of a three-state unfolding model has allowed us to distinguish and separate the thermodynamic magnitudes of the interaction at the binding interface. The model assumes equilibrium between the "unbound" and "bound" states at the SH3-peptide binding interface. The resulting thermodynamic magnitudes classify the different proline residues according to their importance in the interaction as P2 approximately P7 approximately P10>P9 approximately P6>P8, which agrees well with Lim's model for the interaction between SH3 domains and proline-rich peptides. In addition, the thermodynamic signature of the interaction is the same as that usually found for this type of binding, with a strong enthalpy-entropy compensation for all the mutants. This compensation appears to derive from an increase in conformational flexibility concomitant to the weakening of the interactions at the binding interface. We conclude that our approach, based on DSC and site-directed mutagenesis analysis of chimeric fusion proteins, may serve as a suitable tool to analyse the energetics of weak biomolecular interactions such as those involving SH3 domains.

Elucidating proline dynamics in spider dragline silk fibre using 2H-13C HETCOR MAS NMR.

PubMed

Shi, Xiangyan; Yarger, Jeffery L; Holland, Gregory P

2014-05-14

(2)H-(13)C HETCOR MAS NMR is performed on (2)H/(13)C/(15)N-Pro enriched A. aurantia dragline silk. Proline dynamics are extracted from (2)H NMR line shapes and T1 in a site-specific manner to elucidate the backbone and side chain molecular dynamics for the MaSp2 GPGXX β-turn regions for spider dragline silk in the dry and wet, supercontracted states.

FCCC Institutional Breast Cancer Training Program (FCCC-IBCTP)

DTIC Science & Technology

2005-01-01

impact on cancer risk; however, in support of this possibility, missense mutations of amino acid 47 (proline to leucine) are reported in the p53...differences in cancer incidence possibility, missense mutations of amino acid 47 between Caucasian and African American (proline to leucine) are reported in...was phosphorylated by active Aurora A in vitro (Fig. 1c). We made the following mutations at that site and in some cases at an adjacent Ser (Ser298

δ(15) N from soil to wine in bulk samples and proline.

PubMed

Paolini, Mauro; Ziller, Luca; Bertoldi, Daniela; Bontempo, Luana; Larcher, Roberto; Nicolini, Giorgio; Camin, Federica

2016-09-01

The feasibility of using δ(15) N as an additional isotopic marker able to link wine to its area of origin was investigated. The whole production chain (soil-leaves-grape-wine) was considered. Moreover, the research included evaluation of the effect of the fermentation process, the use of different types of yeast and white and red vinification, the addition of nitrogen adjuvants and ultrasound lysis simulating wine ageing. The δ(15) N of grapes and wine was measured in bulk samples and compounds, specifically in proline, for the first time. Despite isotopic fractionation from soil to wine, the δ(15) N values of leaves, grapes, wine and particularly must and wine proline conserved the variability of δ(15) N in the growing soil. Fermentation and ultrasound treatment did not affect the δ(15) N values of grape must, which was therefore conserved in wine. The addition of inorganic or organic adjuvants was able to influence the δ(15) N of bulk wine, depending on the amount and the difference between the δ(15) N of must and that of the adjuvant. The δ(15) N of wine proline was not influenced by adjuvant addition and is therefore the best marker for tracing the geographical origin of wine. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Molybdenum (Mo) increases endogenous phenolics, proline and photosynthetic pigments and the phytoremediation potential of the industrially important plant Ricinus communis L. for removal of cadmium from contaminated soil.

PubMed

Hadi, Fazal; Ali, Nasir; Fuller, Michael Paul

2016-10-01

Cadmium (Cd) in agricultural soil negatively affects crops yield and compromises food safety. Remediation of polluted soil is necessary for the re-establishment of sustainable agriculture and to prevent hazards to human health and environmental pollution. Phytoremediation is a promising technology for decontamination of polluted soil. The present study investigated the effect of molybdenum (Mo) (0.5, 1.0 and 2.0 ppm) on endogenous production of total phenolics and free proline, plant biomass and photosynthetic pigments in Ricinus communis plants grown in Cd (25, 50 and 100 ppm) contaminated soils and the potential for Cd phytoextraction. Mo was applied via seed soaking, soil addition and foliar spray. Foliar sprays significantly increased plant biomass, Cd accumulation and bioconcentration. Phenolic concentrations showed significantly positive correlations with Cd accumulation in roots (R 2  = 0.793, 0.807 and 0.739) and leaves (R 2  = 0.707, 721 and 0.866). Similarly, proline was significantly positively correlated with Cd accumulation in roots (R 2  = 0.668, 0.694 and 0.673) and leaves (R 2  = 0.831, 0.964 and 0.930). Foliar application was found to be the most effective way to deliver Mo in terms of increase in plant growth, Cd accumulation and production of phenolics and proline.

Distribution of sup 3 H-proline within transseptal fibers of the rat following release of orthodontic forces

DOE Office of Scientific and Technical Information (OSTI.GOV)

Row, K.L.; Johnson, R.B.

1990-10-01

Maxillary right first molar teeth of rats were tipped mesially with an orthodontic appliance for 2 weeks (experimental group), {sup 3}H-proline was injected, and orthodontic forces were removed 6 hr later (time 0). The contralateral molar teeth of treated (internal control group) and age- and weight-matched untreated animals (external control group) were also studied. Diastemata were created between the molar teeth by the orthodontic appliance, and transseptal fibers between first and second (P less than 0.001) and second and third molars (P less than 0.005) were significantly lengthened as compared to external and internal controls at time 0. Diastemata betweenmore » molar teeth were closed 5 days after removal of orthodontic force. Transseptal fibers adjacent to the source of the orthodontic force (mesial region) had the highest mean number of {sup 3}H-proline-labeled proteins at time 0 and at all times following removal of the force (P less than 0.001), and had the highest rate of labeled protein removal (P less than 0.001). Half-lives for removal of 3H-proline-labeled transseptal fiber proteins were significantly greater in mesial and distal regions and significantly less in middle regions of experimentals than in corresponding regions of external controls (P less than 0.001).« less

Docking and molecular dynamics simulations of the Fyn-SH3 domain with free and phospholipid bilayer-associated 18.5-kDa myelin basic protein (MBP)-Insights into a noncanonical and fuzzy interaction.

PubMed

Bessonov, Kyrylo; Vassall, Kenrick A; Harauz, George

2017-07-01

The molecular details of the association between the human Fyn-SH3 domain, and the fragment of 18.5-kDa myelin basic protein (MBP) spanning residues S38-S107 (denoted as xα2-peptide, murine sequence numbering), were studied in silico via docking and molecular dynamics over 50-ns trajectories. The results show that interaction between the two proteins is energetically favorable and heavily dependent on the MBP proline-rich region (P93-P98) in both aqueous and membrane environments. In aqueous conditions, the xα2-peptide/Fyn-SH3 complex adopts a "sandwich"-like structure. In the membrane context, the xα2-peptide interacts with the Fyn-SH3 domain via the proline-rich region and the β-sheets of Fyn-SH3, with the latter wrapping around the proline-rich region in a form of a clip. Moreover, the simulations corroborate prior experimental evidence of the importance of upstream segments beyond the canonical SH3-ligand. This study thus provides a more-detailed glimpse into the context-dependent interaction dynamics and importance of the β-sheets in Fyn-SH3 and proline-rich region of MBP. Proteins 2017; 85:1336-1350. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Roles of conserved proline and glycosyltransferase motifs of EmbC in biosynthesis of lipoarabinomannan.

PubMed

Berg, Stefan; Starbuck, James; Torrelles, Jordi B; Vissa, Varalakshmi D; Crick, Dean C; Chatterjee, Delphi; Brennan, Patrick J

2005-02-18

D-Arabinans, composed of D-arabinofuranose (D-Araf), dominate the structure of mycobacterial cell walls in two settings, as part of lipoarabinomannan (LAM) and arabinogalactan, each with markedly different structures and functions. Little is known of the complexity of their biosynthesis. beta-D-Arabinofuranosyl-1-monophosphoryldecaprenol is the only known sugar donor. EmbA, EmbB, and EmbC, products of the paralogous genes embA, embB, and embC, the sites of resistance to the anti-tuberculosis drug ethambutol (EMB), are the only known implicated enzymes. EmbA and -B apparently contribute to the synthesis of arabinogalactan, whereas EmbC is reserved for the synthesis of LAM. The Emb proteins show no overall similarity to any known proteins beyond Mycobacterium and related genera. However, functional motifs, equivalent to a proline-rich motif of several bacterial polysaccharide co-polymerases and a superfamily of glycosyltransferases, were found. Site-directed mutagenesis in glycosyltransferase superfamily C resulted in complete ablation of LAM synthesis. Point mutations in three amino acids of the proline motif of EmbC resulted in marked reduction of LAM-arabinan synthesis and accumulation of an unknown intermediate and of the known precursor lipomannan. Yet the pattern of the differently linked d-Araf units observed in wild type LAM-arabinan was largely retained in the proline motif mutants. The results allow for the presentation of a unique model of arabinan synthesis.

Proline Restricts Loop I Conformation of the High Affinity WW Domain from Human Nedd4-1 to a Ligand Binding-Competent Type I β-Turn.

PubMed

Schulte, Marianne; Panwalkar, Vineet; Freischem, Stefan; Willbold, Dieter; Dingley, Andrew J

2018-04-19

Sequence alignment of the four WW domains from human Nedd4-1 (neuronal precursor cell expressed developmentally down-regulated gene 4-1) reveals that the highest sequence diversity exists in loop I. Three residues in this type I β-turn interact with the PPxY motif of the human epithelial Na + channel (hENaC) subunits, indicating that peptide affinity is defined by the loop I sequence. The third WW domain (WW3*) has the highest ligand affinity and unlike the other three hNedd4-1 WW domains or other WW domains studied contains the highly statistically preferred proline at the ( i + 1) position found in β-turns. In this report, molecular dynamics simulations and experimental data were combined to characterize loop I stability and dynamics. Exchange of the proline to the equivalent residue in WW4 (Thr) results in the presence of a predominantly open seven residue Ω loop rather than the type I β-turn conformation for the wild-type apo-WW3*. In the presence of the ligand, the structure of the mutated loop I is locked into a type I β-turn. Thus, proline in loop I ensures a stable peptide binding-competent β-turn conformation, indicating that amino acid sequence modulates local flexibility to tune binding preferences and stability of dynamic interaction motifs.

Standardized ileal digestibility of proteins and amino acids in sesame expeller and soya bean meal in weaning piglets.

PubMed

Aguilera, A; Reis de Souza, T C; Mariscal-Landín, G; Escobar, K; Montaño, S; Bernal, M G

2015-08-01

Apparent ileal digestibility (AID) of diets containing sesame expeller (SE) and soya bean meal (SBM) was determined using 15 piglets (Genetiporc(®)), weaned at 17 ± 0.4 days with average body weight of 6.4 ± 0.7 kg (Fertilis 20 × G Performance, Genetiporc(®), PIC México, Querétaro, México). Piglets were randomly assigned to three treatments: (i) a reference diet with casein as the sole protein source; (ii) a mixed diet of casein-SE; and (iii) a mixed diet of casein-SBM. The chemical composition of SE and SBM was determined, and AID and standardized ileal digestibility (SID) of crude protein (CP) and amino acids (AAs) were determined for each protein source. SE contained greater quantities of ether extract, neutral detergent fibre, phytic acid, methionine and arginine than SBM. Lysine and proline contents and trypsin inhibitor activity were higher in SBM than in SE. The AID and SID of CP and AA (except for lysine and proline) were similar in SE and SBM. The AID of lysine and proline was higher in SBM than in SE (p < 0.05), and the SID of proline was higher in SE than in SBM (p < 0.05). These findings indicate that SE is an appropriate alternative protein source for early weaned pigs. Journal of Animal Physiology and Animal Nutrition © 2014 Blackwell Verlag GmbH.

Interactions driving the collapse of islet amyloid polypeptide: Implications for amyloid aggregation

NASA Astrophysics Data System (ADS)

Cope, Stephanie M.

Human islet amyloid polypeptide (hIAPP), also known as amylin, is a 37-residue intrinsically disordered hormone involved in glucose regulation and gastric emptying. The aggregation of hIAPP into amyloid fibrils is believed to play a causal role in type 2 diabetes. To date, not much is known about the monomeric state of hIAPP or how it undergoes an irreversible transformation from disordered peptide to insoluble aggregate. IAPP contains a highly conserved disulfide bond that restricts hIAPP(1-8) into a short ring-like structure: N_loop. Removal or chemical reduction of N_loop not only prevents cell response upon binding to the CGRP receptor, but also alters the mass per length distribution of hIAPP fibers and the kinetics of fibril formation. The mechanism by which N_loop affects hIAPP aggregation is not yet understood, but is important for rationalizing kinetics and developing potential inhibitors. By measuring end-to-end contact formation rates, Vaiana et al. showed that N_loop induces collapsed states in IAPP monomers, implying attractive interactions between N_loop and other regions of the disordered polypeptide chain . We show that in addition to being involved in intra-protein interactions, the N_loop is involved in inter-protein interactions, which lead to the formation of extremely long and stable beta-turn fibers. These non-amyloid fibers are present in the 10 muM concentration range, under the same solution conditions in which hIAPP forms amyloid fibers. We discuss the effect of peptide cyclization on both intra- and inter-protein interactions, and its possible implications for aggregation. Our findings indicate a potential role of N_loop-N_loop interactions in hIAPP aggregation, which has not previously been explored. Though our findings suggest that N_loop plays an important role in the pathway of amyloid formation, other naturally occurring IAPP variants that contain this structural feature are incapable of forming amyloids. For example, hIAPP readily forms amyloid fibrils in vitro, whereas the rat variant (rIAPP), differing by six amino acids, does not. In addition to being highly soluble, rIAPP is an effective inhibitor of hIAPP fibril formation . Both of these properties have been attributed to rIAPP's three proline residues: A25P, S28P and S29P. Single proline mutants of hIAPP have also been shown to kinetically inhibit hIAPP fibril formation. Because of their intrinsic dihedral angle preferences, prolines are expected to affect conformational ensembles of intrinsically disordered proteins. The specific effect of proline substitutions on IAPP structure and dynamics has not yet been explored, as the detection of such properties is experimentally challenging due to the low molecular weight, fast reconfiguration times, and very low solubility of IAPP peptides. High-resolution techniques able to measure tertiary contact formations are needed to address this issue. We employ a nanosecond laser spectroscopy technique to measure end-to-end contact formation rates in IAPP mutants. We explore the proline substitutions in IAPP and quantify their effects in terms of intrinsic chain stiffness. We find that the three proline mutations found in rIAPP increase chain stiffness. Interestingly, we also find that residue R18 plays an important role in rIAPP's unique chain stiffness and, together with the proline residues, is a determinant for its non-amyloidogenic properties. We discuss the implications of our findings on the role of prolines in IDPs.

Parenterally fed neonatal piglets have a low rate of endogenous arginine synthesis from circulating proline.

PubMed

Urschel, Kristine L; Evans, Amanda R; Wilkinson, Craig W; Pencharz, Paul B; Ball, Ronald O

2007-03-01

Parenterally fed neonatal piglets cannot synthesize sufficient arginine to maintain arginine status, presumably due to the intestinal atrophy that occurs with parenteral feeding. Parenteral feeding-induced atrophy can be reduced by the infusion of glucagon-like peptide 2 (GLP-2). GLP-2 infusion was hypothesized to increase the rate of endogenous arginine synthesis from proline, the major arginine precursor, in parenterally fed piglets receiving an arginine-deficient diet. Male piglets, fitted with jugular vein catheters for diet and isotope infusion, and femoral vein catheters for blood sampling (d 0), were allocated to a continuous infusion of either GLP-2 (n = 5; 10 nmol x kg(-1) x d(-1)) or saline (n = 5) for 7 d. Piglets received 2 d of a complete diet, followed by 5 d of an arginine-deficient [0.60 g x kg(-1) x d(-1)] diet. Piglets received primed, constant infusions of [guanido-(14)C]arginine to measure arginine flux (d 6) and [U-(14)C]proline (d 7) to measure proline conversion to arginine. Plasma arginine concentrations and arginine fluxes indicated a similar whole-body arginine status. Piglets receiving GLP-2 showed improvements in intestinal variables, including mucosal mass (P < 0.01) and villus height (P < 0.001), and a greater rate of arginine synthesis (micromol x kg(-1) x h(-1)) from proline (11.6 vs. 6.3) (P = 0.03). Mucosal mass (R(2) = 0.71; P = 0.002) and villus height were correlated (R(2) = 0.66; P = 0.004) with arginine synthesis. This study was the first to quantitate arginine synthesis in parenterally fed neonates and showed that although GLP-2 infusion increased arginine synthesis in a manner directly related to mucosal mass, this increased arginine synthesis was insufficient to improve whole-body arginine status in piglets receiving a low arginine diet.

Structural and functional analysis of the yeast N-acetyltransferase Mpr1 involved in oxidative stress tolerance via proline metabolism

PubMed Central

Nasuno, Ryo; Hirano, Yoshinori; Itoh, Takafumi; Hakoshima, Toshio; Hibi, Takao; Takagi, Hiroshi

2013-01-01

Mpr1 (sigma1278b gene for proline-analog resistance 1), which was originally isolated as N-acetyltransferase detoxifying the proline analog l-azetidine-2-carboxylate, protects yeast cells from various oxidative stresses. Mpr1 mediates the l-proline and l-arginine metabolism by acetylating l-Δ1-pyrroline-5-carboxylate, leading to the l-arginine–dependent production of nitric oxide, which confers oxidative stress tolerance. Mpr1 belongs to the Gcn5-related N-acetyltransferase (GNAT) superfamily, but exhibits poor sequence homology with the GNAT enzymes and unique substrate specificity. Here, we present the X-ray crystal structure of Mpr1 and its complex with the substrate cis-4-hydroxy-l-proline at 1.9 and 2.3 Å resolution, respectively. Mpr1 is folded into α/β-structure with eight-stranded mixed β-sheets and six α-helices. The substrate binds to Asn135 and the backbone amide of Asn172 and Leu173, and the predicted acetyl-CoA–binding site is located near the backbone amide of Phe138 and the side chain of Asn178. Alanine substitution of Asn178, which can interact with the sulfur of acetyl-CoA, caused a large reduction in the apparent kcat value. The replacement of Asn135 led to a remarkable increase in the apparent Km value. These results indicate that Asn178 and Asn135 play an important role in catalysis and substrate recognition, respectively. Such a catalytic mechanism has not been reported in the GNAT proteins. Importantly, the amino acid substitutions in these residues increased the l-Δ1-pyrroline-5-carboxylate level in yeast cells exposed to heat stress, indicating that these residues are also crucial for its physiological functions. These studies provide some benefits of Mpr1 applications, such as the breeding of industrial yeasts and the development of antifungal drugs. PMID:23818613

Role of N-Acetyl-Seryl-Aspartyl-Lysyl-Proline in the Antifibrotic and Anti-Inflammatory Effects of the Angiotensin-Converting Enzyme Inhibitor Captopril in Hypertension

PubMed Central

Peng, Hongmei; Carretero, Oscar A.; Liao, Tang-Dong; Peterson, Edward L.; Rhaleb, Nour-Eddine

2012-01-01

Angiotensin-converting enzyme inhibitors (ACEis) are known to have antifibrotic effects on the heart and kidney in both animal models and humans. N-acetyl-seryl-aspartyl-lysyl-proline is a natural inhibitor of proliferation of hematopoietic stem cells and a natural substrate of ACEi that was reported to prevent cardiac and renal fibrosis in vivo. However, it is not clear whether N-acetyl-seryl-aspartyl-lysyl-proline participates in the antifibrotic effects of ACEi. To clarify this issue, we used a model of aldosterone-salt–induced hypertension in rats treated with the ACEi captopril either alone or combined with an anti-N-acetyl-seryl-aspartyl-lysyl-proline monoclonal antibody. These hypertensive rats had the following: (1) left ventricular and renal hypertrophy, as well as increased collagen deposition in the left ventricular and the kidney; (2) glomerular matrix expansion; and (3) increased ED1-positive cells and enhanced phosphorylated-p42/44 mitogen-activated protein kinase in the left ventricle and kidney. The ACEi alone significantly lowered systolic blood pressure (P=0.008) with no effect on organ hypertrophy; it significantly lowered left ventricular collagen content, and this effect was blocked by the monoclonal antibody as confirmed by the histological data. As expected, the ACEi significantly decreased renal collagen deposition and glomerular matrix expansion, and these effects were attenuated by the monoclonal antibody. Likewise, the ACEi significantly decreased ED1-positive cells and inhibited p42/44 mitogen-activated protein kinase phosphorylation in the left ventricle and kidney, and these effects were blocked by the monoclonal antibody. We concluded that in aldosterone-salt–induced hypertension, the antifibrotic effect of ACEi on the heart and kidney, is partially mediated by N-acetyl-seryl-aspartyl-lysyl-proline, resulting in decreased inflammatory cell infiltration and p42/44 mitogen-activated protein kinase activation. PMID:17283252

The Aspergillus nidulans Proline Permease as a Model for Understanding the Factors Determining Substrate Binding and Specificity of Fungal Amino Acid Transporters*

PubMed Central

Gournas, Christos; Evangelidis, Thomas; Athanasopoulos, Alexandros; Mikros, Emmanuel; Sophianopoulou, Vicky

2015-01-01

Amino acid uptake in fungi is mediated by general and specialized members of the yeast amino acid transporter (YAT) family, a branch of the amino acid polyamine organocation (APC) transporter superfamily. PrnB, a highly specific l-proline transporter, only weakly recognizes other Put4p substrates, its Saccharomyces cerevisiae orthologue. Taking advantage of the high sequence similarity between the two transporters, we combined molecular modeling, induced fit docking, genetic, and biochemical approaches to investigate the molecular basis of this difference and identify residues governing substrate binding and specificity. We demonstrate that l-proline is recognized by PrnB via interactions with residues within TMS1 (Gly56, Thr57), TMS3 (Glu138), and TMS6 (Phe248), which are evolutionary conserved in YATs, whereas specificity is achieved by subtle amino acid substitutions in variable residues. Put4p-mimicking substitutions in TMS3 (S130C), TMS6 (F252L, S253G), TMS8 (W351F), and TMS10 (T414S) broadened the specificity of PrnB, enabling it to recognize more efficiently l-alanine, l-azetidine-2-carboxylic acid, and glycine without significantly affecting the apparent Km for l-proline. S253G and W351F could transport l-alanine, whereas T414S, despite displaying reduced proline uptake, could transport l-alanine and glycine, a phenotype suppressed by the S130C mutation. A combination of all five Put4p-ressembling substitutions resulted in a functional allele that could also transport l-alanine and glycine, displaying a specificity profile impressively similar to that of Put4p. Our results support a model where residues in these positions determine specificity by interacting with the substrates, acting as gating elements, altering the flexibility of the substrate binding core, or affecting conformational changes of the transport cycle. PMID:25572393

Amino acid content and nectar choice by forager honeybees (Apis mellifera L.).

PubMed

Bertazzini, Michele; Medrzycki, Piotr; Bortolotti, Laura; Maistrello, Lara; Forlani, Giuseppe

2010-06-01

Dual choice feeding tests were performed to determine a preference of forager honeybees for specific amino acids. Artificial nectar containing proline was preferred over those containing only sugars. Nectar containing alanine was preferred on the first day, but preference was no longer significant thereafter. On the contrary, a negative response was found for serine. When the bees were given the choice between two nectars enriched with different compounds, proline was preferred above both alanine and serine, and alanine above serine.

Radicals initiated by gamma-rays in collagen and its main components

NASA Astrophysics Data System (ADS)

Kornacka, Ewa Maria; Przybytniak, Grażyna; Zimek, Zbigniew

2018-01-01

Radical products generated by gamma-rays were identified in collagen and in microcrystalline powders of glycyne, L-proline and L-hydroksyproline. Reagents irradiated at 77 K were studied by EPR spectroscopy in the range of 100-350 K using temperature control system. Two radical centers found in collagen were localized in proline ring at α and γ positions to the carbonyl group. There were neither terminal radicals generated by chain scission nor transient products disrupting hydrogen bonding system of irradiated collagen.

Survival of Escherichia coli O157:H7 ATCC 43895 in a Model Apple Juice Medium with Different Concentrations of Proline and Caffeic Acid

PubMed Central

Reinders, Robert D.; Biesterveld, Steef; Bijker, Peter G. H.

2001-01-01

The effects of proline and caffeic acid on the survival of Shiga toxin-producing Escherichia coli (STEC) O157:H7 strain ATCC 43895 in a model apple juice medium were studied. It is hypothesized that the inhibitory effect of caffeic acid may explain why almost all outbreaks of STEC O157:H7 infections linked to apple juice or cider have occurred in October or November. PMID:11375209

Enantioselective synthesis of syn/anti-1,3-amino alcohols via proline-catalyzed sequential alpha-aminoxylation/alpha-amination and Horner-Wadsworth-Emmons olefination of aldehydes.

PubMed

Jha, Vishwajeet; Kondekar, Nagendra B; Kumar, Pradeep

2010-06-18

A novel and general method for asymmetric synthesis of both syn/anti-1,3-amino alcohols is described. The method uses proline-catalyzed sequential alpha-aminoxylation/ alpha-amination and Horner-Wadsworth-Emmons (HWE) olefination of aldehydes as the key step. By using this method, a short synthesis of a bioactive molecule, (R)-1-((S)-1-methylpyrrolidin-2-yl)-5-phenylpentan-2-ol, is also accomplished.

Endosulfan induced biochemical changes in nitrogen-fixing cyanobacteria.

PubMed

Kumar, Satyendra; Habib, Khalid; Fatma, Tasneem

2008-09-15

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.

Structure and Thermodynamic Stability of Islet Amyloid Polypeptide Monomers and Small Aggregates

NASA Astrophysics Data System (ADS)

Chiu, Chi-Cheng; Singh, Sadanand; de Pablo, Juan

2013-03-01

Human islet amyloid polypeptide (hIAPP, also known as human amylin) is associated with the development of type II diabetes. It is known to form amyloid fibrils that are found in pancreatic islets. Pramlintide, a synthetic analog of hIAPP with three proline substitutions, is not amyloidogenic and has been applied in amylin replacement treatments. In this work, we use molecular simulations with advanced sampling techniques to examine the effect of these proline substitutions on hIAPP monomer conformations. We find that all three proline substitutions are required to attenuate the formation of β-sheets encountered in amylin. Furthermore, we investigate the formation of hIAPP dimers and trimers, and investigate how that process is affected by the presence of various additives. Our simulations show that hIAPP can form a β-sheet at the N-terminus and the C-terminus independently, in agreement with experimental observations. Our results provide valuable insights into the mechanism of hIAPP early aggregation and the design of fibril formation inhibitors.

Proline N-oxides: modulators of the 3D conformation of linear peptides through "NO-turns".

PubMed

Farahani, Majid D; Honarparvar, Bahareh; Albericio, Fernando; Maguire, Glenn E M; Govender, Thavendran; Arvidsson, Per I; Kruger, Hendrik G

2014-07-07

Small peptides are essential mediators of numerous physiological processes. Consequently, there is huge interest in the de novo design of peptides with a predictable folding and related biological activity. In this study, we investigate the possibility of modulating the secondary structure of tetrapeptides through proline N-oxide moieties and N-methylation of the peptide backbone. A series of tetrapeptides were synthesised to investigate the combined effect of Pro N-oxide and N-methylation of the amide bond on the (n + 1) residue in terms of cis- and trans-isomerization, as well as how these modifications direct potential intramolecular hydrogen bonding interactions. The right combination of both these parameters led to a trans to cis-conformational interconversion and a change in the nature of the hydrogen bonding interactions, as demonstrated by NMR spectroscopic, molecular modeling analysis and thermal coefficient studies. Proline N-oxide residues were proposed to induce turns we named as NO-γ-turns and NO-β-turns based on their similarity to traditional γ- and β-turns.

Study of the effects of proline, phenylalanine, and urea foliar application to Tempranillo vineyards on grape amino acid content. Comparison with commercial nitrogen fertilisers.

PubMed

Garde-Cerdán, T; López, R; Portu, J; González-Arenzana, L; López-Alfaro, I; Santamaría, P

2014-11-15

The aim of this work was to study the influence of foliar application of different nitrogen sources on grape amino acid content. The nitrogen sources applied to Tempranillo grapevines were proline, phenylalanine, urea, and two commercial nitrogen fertilisers, both without and with amino acids in their formulations. All treatments were applied at veraison and one week later. Proline treatment did not affect the must nitrogen composition. However, phenylalanine and urea foliar application enhanced the plants' synthesis of most of the amino acids, producing similar effects. In addition, the spray of commercial nitrogen fertilisers over leaves also induced a rise in grape amino acid concentrations regardless of the presence or absence of amino acids in their formulation. The most effective treatments were phenylalanine and urea followed by nitrogen fertilisers. This finding is of oenological interest for improved must nitrogen composition, ensuring better fermentation kinetics and most likely enhancing wine quality. Copyright © 2014 Elsevier Ltd. All rights reserved.

Contribution of polyamines metabolism and GABA shunt to chilling tolerance induced by nitric oxide in cold-stored banana fruit.

PubMed

Wang, Yansheng; Luo, Zisheng; Mao, Linchun; Ying, Tiejin

2016-04-15

Effect of exogenous nitric oxide (NO) on polyamines (PAs) catabolism, γ-aminobutyric acid (GABA) shunt, proline accumulation and chilling injury of banana fruit under cold storage was investigated. Banana fruit treated with NO sustained lower chilling injury index than the control. Notably elevated nitric oxide synthetase activity and endogenous NO level were observed in NO-treated banana fruit. PAs contents in treated fruit were significantly higher than control fruit, due to the elevated activities of arginine decarboxylase and ornithine decarboxylase. NO treatment increased the activities of diamine oxidase, polyamine oxidase and glutamate decarboxylase, while reduced GABA transaminase activity to lower levels compared with control fruit, which resulted the accumulation of GABA. Besides, NO treatment upregulated proline content and significantly enhanced the ornithine aminotransferase activity. These results indicated that the chilling tolerance induced by NO treatment might be ascribed to the enhanced catabolism of PAs, GABA and proline. Copyright © 2015 Elsevier Ltd. All rights reserved.

[The influence of low partial oxygen pressure on the biolodical process of mesenchymal stromal cells].

PubMed

Berezovskyĭ, V Ia; Plotnikova, L M; Vesel'skyĭ, S P; Litovka, I H

2014-01-01

The influence of low partial oxygen pressure (Po2) on the amino acid composition in culture medium of human mesenchymal stromal cell (MSC) lines 4BL has been studied. At 23 mm Hg (3% oxygen), a significant decrease (by 31%) in the concentration of proline and hydroxyproline was registered. Under these conditions, the concentration of serine and aspartic acid decreased by 45% compared to the control. Maximum consumption of free amino acids from the culture medium required for the synthesis of collagen (proline and hydroxyproline by 42%, serine and aspartic acid by 62%) was observed at a gas-phase Po2 of 38 mm Hg (5% O2). At Po2 76 mm Hg (10% O2), a lack of amino acids proline and hydroxyproline was only 21%, while that of glutamine and alanine amounted 12% compared to the control. This intensity ratio of consumption of amino acids may indicate that the maximum of MSC vital functions occurs at Po2 38 mm Hg.

Acceleration of protein folding by four orders of magnitude through a single amino acid substitution

PubMed Central

Roderer, Daniel J. A.; Schärer, Martin A.; Rubini, Marina; Glockshuber, Rudi

2015-01-01

Cis prolyl peptide bonds are conserved structural elements in numerous protein families, although their formation is energetically unfavorable, intrinsically slow and often rate-limiting for folding. Here we investigate the reasons underlying the conservation of the cis proline that is diagnostic for the fold of thioredoxin-like thiol-disulfide oxidoreductases. We show that replacement of the conserved cis proline in thioredoxin by alanine can accelerate spontaneous folding to the native, thermodynamically most stable state by more than four orders of magnitude. However, the resulting trans alanine bond leads to small structural rearrangements around the active site that impair the function of thioredoxin as catalyst of electron transfer reactions by more than 100-fold. Our data provide evidence for the absence of a strong evolutionary pressure to achieve intrinsically fast folding rates, which is most likely a consequence of proline isomerases and molecular chaperones that guarantee high in vivo folding rates and yields. PMID:26121966

Different states of synaptotagmin regulate evoked versus spontaneous release

PubMed Central

Bai, Hua; Xue, Renhao; Bao, Huan; Zhang, Leili; Yethiraj, Arun; Cui, Qiang; Chapman, Edwin R.

2016-01-01

The tandem C2-domains of synaptotagmin 1 (syt) function as Ca2+-binding modules that trigger exocytosis; in the absence of Ca2+, syt inhibits spontaneous release. Here, we used proline linkers to constrain and alter the relative orientation of these C2-domains. Short poly-proline helices have a period of three, so large changes in the relative disposition of the C2-domains result from changing the length of the poly-proline linker by a single residue. The length of the linker was varied one residue at a time, revealing a periodicity of three for the ability of the linker mutants to interact with anionic phospholipids and drive evoked synaptic transmission; syt efficiently drove exocytosis when its tandem C2-domains pointed in the same direction. Analysis of spontaneous release revealed a reciprocal relationship between the activation and clamping activities of the linker mutants. Hence, different structural states of syt underlie the control of distinct forms of synaptic transmission. PMID:27001899

The effect of kinetin on wheat seedlings exposed to boron.

PubMed

Eser, Ahmet; Aydemir, Tülin

2016-11-01

The objective of this study was to examine relationship between boron (B) induced oxidative stress and antioxidant system in boron sensitive and tolerant wheat cultivars Bezostaya and Kutluk, and also to investigate whether Kinetin (KN) enhances the level of antioxidant system, relative growth, concentration of hydrogen peroxide (H 2 O 2 ), malondialdehyde (MDA) and proline and chlorophyll content in both cultivars exposed to B stress. B treatments diminished growth and chlorophyll content whereas, it enhanced accumulation of H 2 O 2 , MDA and proline, and various antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (GPX) and lipoxygenase (LOX) in the shoot and root of both cultivars. However, the follow-up application of KN to the B stressed plants improved growth and chlorophyll content and further enhanced the mentioned antioxidant enzymes and level of H 2 O 2 , MDA and proline. This study thus suggests that KN improves B tolerance of the studied cultivars grown under B toxicity. Copyright © 2016. Published by Elsevier Masson SAS.

Thermodynamic Parameters of the Dissolution of 4-Hydroxy-L-Proline and L-Phenylalanine in Mixed Aqueous Solvents at 298 K

NASA Astrophysics Data System (ADS)

Smirnov, V. I.; Badelin, V. G.

2018-01-01

The enthalpies of solution of 4-hydroxy-L-proline and L-phenylalanine in binary mixed aqueous solvents containing acetonitrile (AN), 1,4-dioxane (1,4-DO), or acetone (AC) at mole fractions of 0 to 0.25 are determined at T = 298.15 K via isothermal calorimetry. The standard enthalpies of solution (Δsol H°) and transfer (Δtr H°) of 4-hydroxy-L-proline and L-phenylalanine from water to mixed aqueous solvents are calculated using the experimental calorimetric data, as are the enthalpy coefficients of paired interactions ( h xy ) between the molecules of the investigated amino acids and the organic solvents. The effects the mixed aqueous solvent composition and the structure of the organic solvent molecules have on the enthalpies of solution and transfer for the investigated amino acids are considered. The correlation between the enthalpy of solution of the amino acids and the electron-donating properties of the organic solvents in the mixed aqueous solvent systems is established.

The PE/PPE multigene family codes for virulence factors and is a possible source of mycobacterial antigenic variation: perhaps more?

PubMed

Akhter, Yusuf; Ehebauer, Matthias T; Mukhopadhyay, Sangita; Hasnain, Seyed E

2012-01-01

The PE/PPE multigene family codes for approximately 10% of the Mycobacterium tuberculosis proteome and is encoded by 176 open reading frames. These proteins possess, and have been named after, the conserved proline-glutamate (PE) or proline-proline-glutamate (PPE) motifs at their N-terminus. Their genes have a conserved structure and repeat motifs that could be a potential source of antigenic variation in M. tuberculosis. PE/PPE genes are scattered throughout the genome and PE/PPE pairs are usually encoded in bicistronic operons although this is not universally so. This gene family has evolved by specific gene duplication events. PE/PPE proteins are either secreted or localized to the cell surface. Several are thought to be virulence factors, which participate in evasion of the host immune response. This review summarizes the current knowledge about the gene family in order to better understand its biological function. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

Thermally reversible gels based on acryloyl- L-proline methyl ester as drug delivery systems

NASA Astrophysics Data System (ADS)

Martellini, Flavia; Higa, Olga Z.; Takacs, Erzsebet; Safranj, Agneza; Yoshida, Masaru; Katakai, Ryoichi; Carenza, Mario

1999-06-01

Thermally reversible hydrogels were synthesized by radiation-induced copolymerization of acryloyl- L-proline methyl ester with hydrophilic or hydrophobic monomers. The swelling behaviour was found to be affected by a proper balance of the latter. In particular, the transition temperature of the different hydrogels shifted to higher or lower values depending on the presence of hydrophilic or hydrophobic moieties in the polymer chain, respectively. Acetaminophen, an analgesic and antipyretic drug, was entrapped into some hydrogels and a wide range of release rates was obtained according to the nature of the comonomers. A novel thermoresponsive hydrogel was also prepared by radiation polymerization of acryloyl- L-proline methyl ester in the presence of 4-acryloyloxy acetanilide, an acrylic derivative of acetaminophen. Again, the swelling curves showed an inverse function of temperature. It was shown that with this hydrogel bearing the drug covalently attached to the polymer backbone, the hydrolysis process was the rate-determining process of the drug release.

Nucleotide sequence of the Saccharomyces cerevisiae PUT4 proline-permease-encoding gene: similarities between CAN1, HIP1 and PUT4 permeases.

PubMed

Vandenbol, M; Jauniaux, J C; Grenson, M

1989-11-15

The complete nucleotide (nt) sequence of the PUT4 gene, whose product is required for high-affinity proline active transport in the yeast Saccharomyces cerevisiae, is presented. The sequence contains a single long open reading frame of 1881 nt, encoding a polypeptide with a calculated Mr of 68,795. The predicted protein is strongly hydrophobic and exhibits six potential glycosylation sites. Its hydropathy profile suggests the presence of twelve membrane-spanning regions flanked by hydrophilic N- and C-terminal domains. The N terminus does not resemble signal sequences found in secreted proteins. These features are characteristic of integral membrane proteins catalyzing translocation of ligands across cellular membranes. Protein sequence comparisons indicate strong resemblance to the arginine and histidine permeases of S. cerevisiae, but no marked sequence similarity to the proline permease of Escherichia coli or to other known prokaryotic or eukaryotic transport proteins. The strong similarity between the three yeast amino acid permeases suggests a common ancestor for the three proteins.

Attenuation of Pseudomonas aeruginosa virulence by marine invertebrate-derived Streptomyces sp.

PubMed

Naik, D N; Wahidullah, S; Meena, R M

2013-03-01

The study aimed to discover quorum sensing (QS) inhibitors from marine sponge-derived actinomycetes and analyse its inhibitory activities against QS-mediated virulence factors in Pseudomonas aeruginosa. Seventy-two actinomycetes isolated from marine invertebrates collected from the western coast of India were screened against the QS indicator strain Chromobacterium violaceum CV12472. Methanol extracts of 12 actinomycetes showing inhibition of violacein production were accessed for downregulation of QS-mediated virulence factors like swarming, biofilm formation, pyocyanin, rhamnolipid and LasA production in Ps. aeruginosa ATCC 27853. The isolates NIO 10068, NIO 10058 and NIO 10090 exhibited very good anti-QS activity, with NIO 10068 being the most promising one. Mass spectrometric analysis of NIO 10068 methanol extract revealed the presence of cinnamic acid and linear dipeptides proline-glycine and N-amido-α-proline in the active extract. Detailed investigation suggested that although linear dipeptide Pro-Gly is to some extent responsible for the observed biological activity, cinnamic acid seems to be the main compound responsible for it. Marine-derived actinomycetes are a potential storehouse for QS inhibitors. This is the first report not only on marine sponge-associated Streptomyces for anti-QS in Ps. aeruginosa but also on cinnamic acid and proline-derived linear dipeptides proline-glycine as QS inhibitors. The results reveal that marine-derived actinomycetes may not only play a role in the defensive mechanism of their host but also lead to new molecules useful in the development of novel antivirulence drugs. © 2012 The Society for Applied Microbiology.

Comparative analyses of universal extraction buffers for assay of stress related biochemical and physiological parameters.

PubMed

Han, Chunyu; Chan, Zhulong; Yang, Fan

2015-01-01

Comparative efficiency of three extraction solutions, including the universal sodium phosphate buffer (USPB), the Tris-HCl buffer (UTHB), and the specific buffers, were compared for assays of soluble protein, free proline, superoxide radical (O2∙-), hydrogen peroxide (H2O2), and the antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (POD), ascorbate peroxidase (APX), glutathione peroxidase (GPX), and glutathione reductase (GR) in Populus deltoide. Significant differences for protein extraction were detected via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional electrophoresis (2-DE). Between the two universal extraction buffers, the USPB showed higher efficiency for extraction of soluble protein, CAT, GR, O2∙-, GPX, SOD, and free proline, while the UTHB had higher efficiency for extraction of APX, POD, and H2O2. When compared with the specific buffers, the USPB showed higher extraction efficiency for measurement of soluble protein, CAT, GR, and O2∙-, parallel extraction efficiency for GPX, SOD, free proline, and H2O2, and lower extraction efficiency for APX and POD, whereas the UTHB had higher extraction efficiency for measurement of POD and H2O2. Further comparisons proved that 100 mM USPB buffer showed the highest extraction efficiencies. These results indicated that USPB would be suitable and efficient for extraction of soluble protein, CAT, GR, GPX, SOD, H2O2, O2∙-, and free proline.

Cell wall-associated ROOT HAIR SPECIFIC 10, a proline-rich receptor-like kinase, is a negative modulator of Arabidopsis root hair growth

PubMed Central

Hwang, Youra; Lee, Hyodong; Lee, Young-Sook; Cho, Hyung-Taeg

2016-01-01

Plant cell growth is restricted by the cell wall, and cell wall dynamics act as signals for the cytoplasmic and nuclear events of cell growth. Among various receptor kinases, ROOT HAIR SPECIFIC 10 (RHS10) belongs to a poorly known receptor kinase subfamily with a proline-rich extracellular domain. Here, we report that RHS10 defines the root hair length of Arabidopsis thaliana by negatively regulating hair growth. RHS10 modulates the duration of root hair growth rather than the growth rate. As poplar and rice RHS10 orthologs also showed a root hair-inhibitory function, this receptor kinase-mediated function appears to be conserved in angiosperms. RHS10 showed a strong association with the cell wall, most probably through its extracellular proline-rich domain (ECD). Deletion analysis of the ECD demonstrated that a minimal extracellular part, which includes a few proline residues, is required for RHS10-mediated root hair inhibition. RHS10 suppressed the accumulation of reactive oxygen species (ROS) in the root, which are necessary for root hair growth. A yeast two-hybrid screening identified an RNase (RNS2) as a putative downstream target of RHS10. Accordingly, RHS10 overexpression decreased and RHS10 loss increased RNA levels in the hair-growing root region. Our results suggest that RHS10 mediates cell wall-associated signals to maintain proper root hair length, at least in part by regulating RNA catabolism and ROS accumulation. PMID:26884603

Impact of adenosine nucleotide translocase (ANT) proline isomerization on Ca2+-induced cysteine relative mobility/mitochondrial permeability transition pore.

PubMed

Pestana, Cezar R; Silva, Carlos H T P; Uyemura, Sérgio A; Santos, Antonio C; Curti, Carlos

2010-08-01

Mitochondrial membrane carriers containing proline and cysteine, such as adenine nucleotide translocase (ANT), are potential targets of cyclophilin D (CyP-D) and potential Ca(2+)-induced permeability transition pore (PTP) components or regulators; CyP-D, a mitochondrial peptidyl-prolyl cis-trans isomerase, is the probable target of the PTP inhibitor cyclosporine A (CsA). In the present study, the impact of proline isomerization (from trans to cis) on the mitochondrial membrane carriers containing proline and cysteine was addressed using ANT as model. For this purpose, two different approaches were used: (i) Molecular dynamic (MD) analysis of ANT-Cys(56) relative mobility and (ii) light scattering techniques employing rat liver isolated mitochondria to assess both Ca(2+)-induced ANT conformational change and mitochondrial swelling. ANT-Pro(61) isomerization increased ANT-Cys(56) relative mobility and, moreover, desensitized ANT to the prevention of this effect by ADP. In addition, Ca(2+) induced ANT "c" conformation and opened PTP; while the first effect was fully inhibited, the second was only attenuated by CsA or ADP. Atractyloside (ATR), in turn, stabilized Ca(2+)-induced ANT "c" conformation, rendering the ANT conformational change and PTP opening less sensitive to the inhibition by CsA or ADP. These results suggest that Ca(2+) induces the ANT "c" conformation, apparently associated with PTP opening, but requires the CyP-D peptidyl-prolyl cis-trans isomerase activity for sustaining both effects.

Molecular building kit of fused-proline-derived peptide mimetics allowing specific adjustment of the dihedral Psi angle.

PubMed

Einsiedel, Juergen; Lanig, Harald; Waibel, Reiner; Gmeiner, Peter

2007-11-23

Proline-derived peptide mimetics have become an area of paramount importance in peptide and protein chemistry. Since protein crystal structures frequently display Psi angles of 140-170 degrees for prolyl moieties, our intention was to design a completely novel series of 2,3-fused-proline-derived lactams covering this particular conformational space. Extending our recently described toolset of spirocyclic reverse-turn mimetics, we synthesized pyrrolidinyl-fused seven-, eight-, and nine-membered unsaturated lactam model peptides taking advantage of Grubbs' ring-closing metathesis. Investigating the seven-membered lactam 3a by means of IR and NMR spectroscopy and semiempirical molecular dynamics simulations, we could not observe a U-turn conformation; however, increasing the ring size to give eight- and nine-membered congeners revealed moderate and high type IotaIota beta-turn inducing properties. Interestingly, the conformational properties of our model systems depend on both the ring size of the fused dehydro-Freidinger lactam and the position of the endocyclic double bond. Superior reverse-turn inducing properties could be observed for the fused azacyclononenone 3e. According to diagnostic transanular NOEs, a discrete folding principle of the lactam ring strongly deviating from the regioisomeric lactams 3c,f explains the conformational behavior. Hence, we were able to establish a molecular building kit that allows adjustments of a wide range of naturally occurring proline Psi angles and thus can be exploited to probe molecular recognition and functional properties of biological systems.

Core Transmembrane Domain 6 Plays a Pivotal Role in the Transport Cycle of the Sodium/Proline Symporter PutP*

PubMed Central

Bracher, Susanne; Schmidt, Claudia C.; Dittmer, Sophie I.; Jung, Heinrich

2016-01-01

Crystal structures of transporters with a LeuT-type structural fold assign core transmembrane domain 6 (TM6′) a central role in substrate binding and translocation. Here, the function of TM6′ in the sodium/proline symporter PutP, a member of the solute/sodium symporter family, was investigated. A complete scan of TM6′ identified eight amino acids as particularly important for PutP function. Of these residues, Tyr-248, His-253, and Arg-257 impact sodium binding, whereas Arg-257 and Ala-260 may participate in interactions leading to closure of the inner gate. Furthermore, the previous suggestion of an involvement of Trp-244, Tyr-248, and Pro-252 in proline binding is further supported. In addition, substitution of Gly-245, Gly-247, and Gly-250 affects the amount of PutP in the membrane. A Cys accessibility analysis suggests an involvement of the inner half of TM6′ in the formation of a hydrophilic pathway that is open to the inside in the absence of ligands and closed in the presence of sodium and proline. In conclusion, the results demonstrate that TM6′ plays a central role in substrate binding and release on the inner side of the membrane also in PutP and extend the knowledge on functionally relevant amino acids in transporters with a LeuT-type structural fold. PMID:27793991

Simultaneous measurement of proline and related compounds in oak leaves by high-performance ligand-exchange chromatography and electrospray ionization mass spectrometry for environmental stress studies.

PubMed

Oufir, Mouhssin; Schulz, Nadine; Sha Vallikhan, Patan Shaik; Wilhelm, Eva; Burg, Kornel; Hausman, Jean-Francois; Hoffmann, Lucien; Guignard, Cedric

2009-02-13

A mass spectrometer was coupled to high-performance ligand-exchange liquid chromatography (HPLEC) for simultaneous analysis of stress associated solutes such as proline, hydroxyproline, methylproline, glycine betaine and trigonelline extracted from leaves of drought stressed oaks and an internal standard namely N-acetylproline. Methanol/chloroform/water extracts were analyzed using an Aminex HPX-87C column and specifically quantified by the positive ion mode of an electrospray ionisation-mass spectrometry (ESI-MS) in single ion monitoring (SIM) mode. The recovery of N-acetyl proline added to oak leaf extracts ranged from 85.2 to 122.1% for an intra-day study. Standard calibration curves showed good linearity in the measured range from 0.3125 to 10micromolL(-1) with the lowest correlation coefficient of 0.99961 for trigonelline. The advantages of this alternative procedure, compared to previously published methods using fluorescence or amperometric detections, are the simultaneous and direct detection of osmoprotectants in a single chromatographic run, a minimal sample preparation, a good specificity and reduced limits of quantification, ranging from 0.1 to 0.6micromolL(-1). Fifty-six days of water deficit exposure resulted in increased foliar free proline levels (2.4-fold, P<0.001, 155micromolg(-1) FW) and glycine betaine contents (2.5-fold, P<0.05, 175micromolg(-1) FW) of drought stressed oak compared to control.

Some physiological responses of wheat and bean to soil salinity at low matric suctions

NASA Astrophysics Data System (ADS)

Khatar, Mahnaz; Mohammadi, Mohammad Hossein; Shekari, Farid

2017-01-01

The effect of soil matric suction (2-33 kPa) and salinity (soil solution electrical conductivity 0.7-8 dS m-1 for bean and 2-20 dS m-1 for wheat) on some physiological characteristics of bean and wheat in a clay loam soil under greenhouse condition was investigated. The results showed that the leaf chlorophyll content index and potassium concentration decrease under salinity stress and increase with matric suction from 2 to 33 kPa suction for both plants. The wheat chlorophyll content index declines during the stress spell but bean chlorophyll content index remains nearly constant. The lowest values of the content of soluble sugars and the highest values of leaf proline content are observed at2 kPa matric suction (highest aeration stress) for bean and wheat. As matric suction increases from 2 to 6 kPa, the soluble sugars increases and proline content decreases significantly and then soluble sugars decreases and proline content increases until 10 kPa suction, and the soluble sugars remains nearly constant at the higher matric suctions for both plants. While the electrical conductivity effect on the soluble sugars is not significant, the values of proline content for both crop increase significantly with electrical conductivity. It was shown that the aeration stress can result in more considerable and rapid physiological responses, in comparison with salinity stress. There is a strong correlation between wheat and bean chlorophyll content index and potassium concentration under salinity and aeration stresses.

Novel proline-hydroxyproline glycopeptides from the dandelion (Taraxacum officinale Wigg.) flowers: de novo sequencing and biological activity.

PubMed

Astafieva, Alexandra A; Enyenihi, Atim A; Rogozhin, Eugene A; Kozlov, Sergey A; Grishin, Eugene V; Odintsova, Tatyana I; Zubarev, Roman A; Egorov, Tsezi A

2015-09-01

Two novel homologous peptides named ToHyp1 and ToHyp2 that show no similarity to any known proteins were isolated from Taraxacum officinale Wigg. flowers by multidimensional liquid chromatography. Amino acid and mass spectrometry analyses demonstrated that the peptides have unusual structure: they are cysteine-free, proline-hydroxyproline-rich and post-translationally glycosylated by pentoses, with 5 carbohydrates in ToHyp2 and 10 in ToHyp1. The ToHyp2 peptide with a monoisotopic molecular mass of 4350.3Da was completely sequenced by a combination of Edman degradation and de novo sequencing via top down multistage collision induced dissociation (CID) and higher energy dissociation (HCD) tandem mass spectrometry (MS(n)). ToHyp2 consists of 35 amino acids, contains eighteen proline residues, of which 8 prolines are hydroxylated. The peptide displays antifungal activity and inhibits growth of Gram-positive and Gram-negative bacteria. We further showed that carbohydrate moieties have no significant impact on the peptide structure, but are important for antifungal activity although not absolutely necessary. The deglycosylated ToHyp2 peptide was less active against the susceptible fungus Bipolaris sorokiniana than the native peptide. Unique structural features of the ToHyp2 peptide place it into a new family of plant defense peptides. The discovery of ToHyp peptides in T. officinale flowers expands the repertoire of molecules of plant origin with practical applications. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Probing Selection Mechanism of the Most Favorable Conformation of a Dipeptide in Chaotropic and Kosmotropic Solution.

PubMed

Jas, Gouri S; Middaugh, C Russell; Kuczera, Krzysztof

2016-07-21

Chaotropes like urea and guanidinium chloride (GdmCl) tend to destabilize, and kosmotropes like proline tend to stabilize folded structures of peptides and proteins. Here, we combine fluorescence anisotropy decay measurements and molecular dynamics simulations to gain a microscopic understanding of the molecular mechanism for shifting conformational preferences in aqueous, GdmCl, urea, and proline solutions of a simple model dipeptide, N-acetyl-tryptophan-amide (NATA). Measured anisotropy decay of NATA as a function of temperature, pH, and cosolvent concentrations showed reorientations moderately slower in GdmCl and urea and substantially slower in proline compared to those of aqueous environment. A small change in pH significantly slows orientation time in water and GdmCl and less markedly in urea. Computationally, we use molecular dynamics with dihedral restraints to separately analyze the motions and interactions of the representative NATA conformers in the four different solvent environments. This novel analysis provides a dissection of the observed overall diffusion rates into contributions from individual dipeptide conformations. The variation of rotational diffusion rates with conformation are quite large. Population-weighted averaging or using properties of the major cluster reproduces the dynamical features of the full unrestrained dynamics. Additionally, we correlate the observable diffusion rates with microscopic features of conformer size, shape, and solvation. This analysis uncovered underlying differences in detailed atomistic behavior of the three cosolvents-urea, GdmCl, and proline. For both urea and the pure water system we find good agreement with hydrodynamic theory, with diffusion rates primarily correlated with conformer size and shape. In contrast, for GdmCl and proline solutions, the variation in conformer diffusion rates was mostly determined by specific interactions with the cosolvents. We also find preferences for different molecular shapes by the three cosolvents, with increased preferential solvation of smaller and more spherical conformers by urea and larger and more elongated conformers by GdmCl and proline. Additionally, our results provide a basis for a simple approximate model of the effects of pH lowering on dipeptide conformational equilibria. The translational diffusion rates of NATA are less sensitive to conformations, but variation with solvation strength is similar to rotational diffusion. Our results, combining experiment and simulation, show that we can identify the individual peptide conformers with definite microscopic properties of shape, size, and solvation, that are responsible for producing physical observables, such as translational and orientational diffusion in the complex solvent environments of denaturants and osmolytes.

Influence of pH on the cis- trans isomerization of Valine-Proline dipeptide: An integrated NMR and theoretical investigation

NASA Astrophysics Data System (ADS)

Ivanova, Galya; Yakimova, Boryana; Angelova, Silvia; Stoineva, Ivanka; Enchev, Venelin

2010-06-01

The pH influence on the cis- trans isomerization of the Valine-Proline dipeptide was investigated by means of NMR spectral techniques and quantum chemical calculations at different computational levels. It was found that the process of isomerization is strongly pH dependent. The trans-isomer of Val-Pro is the more abundant isomer when the amino group is protonated, while an approximately equal distribution between the cis and trans-isomers for the neutral and anion forms of the dipeptide was confirmed.

Novel thiourea-amine bifunctional catalysts for asymmetric conjugate addition of ketones/aldehydes to nitroalkenes: rational structural combination for high catalytic efficiency.

PubMed

Chen, Jia-Rong; Cao, Yi-Ju; Zou, You-Quan; Tan, Fen; Fu, Liang; Zhu, Xiao-Yu; Xiao, Wen-Jing

2010-03-21

A series of thiourea-amine bifunctional catalysts have been developed by a rational combination of prolines with cinchona alkaloids, which are connected by a thiourea motif. The catalyst 3a, prepared from L-proline and cinchonidine, was found to be a highly efficient catalyst for the conjugate addition of ketones/aldehydes to a wide range of nitroalkenes (up to 98/2 dr and 96% ee). The privileged cinchonidine backbone and the thiourea motif are essential to the reaction activity and enantioselectivity.

Molecular evolution and transcriptional regulation of the oilseed rape proline dehydrogenase genes suggest distinct roles of proline catabolism during development.

PubMed

Faës, Pascal; Deleu, Carole; Aïnouche, Abdelkader; Le Cahérec, Françoise; Montes, Emilie; Clouet, Vanessa; Gouraud, Anne-Marie; Albert, Benjamin; Orsel, Mathilde; Lassalle, Gilles; Leport, Laurent; Bouchereau, Alain; Niogret, Marie-Françoise

2015-02-01

Six BnaProDH1 and two BnaProDH2 genes were identified in Brassica napus genome. The BnaProDH1 genes are mainly expressed in pollen and roots' organs while BnaProDH2 gene expression is associated with leaf vascular tissues at senescence. Proline dehydrogenase (ProDH) catalyzes the first step in the catabolism of proline. The ProDH gene family in oilseed rape (Brassica napus) was characterized and compared to other Brassicaceae ProDH sequences to establish the phylogenetic relationships between genes. Six BnaProDH1 genes and two BnaProDH2 genes were identified in the B. napus genome. Expression of the three paralogous pairs of BnaProDH1 genes and the two homoeologous BnaProDH2 genes was measured by real-time quantitative RT-PCR in plants at vegetative and reproductive stages. The BnaProDH2 genes are specifically expressed in vasculature in an age-dependent manner, while BnaProDH1 genes are strongly expressed in pollen grains and roots. Compared to the abundant expression of BnaProDH1, the overall expression of BnaProDH2 is low except in roots and senescent leaves. The BnaProDH1 paralogs showed different levels of expression with BnaA&C.ProDH1.a the most strongly expressed and BnaA&C.ProDH1.c the least. The promoters of two BnaProDH1 and two BnaProDH2 genes were fused with uidA reporter gene (GUS) to characterize organ and tissue expression profiles in transformed B. napus plants. The transformants with promoters from different genes showed contrasting patterns of GUS activity, which corresponded to the spatial expression of their respective transcripts. ProDHs probably have non-redundant functions in different organs and at different phenological stages. In terms of molecular evolution, all BnaProDH sequences appear to have undergone strong purifying selection and some copies are becoming subfunctionalized. This detailed description of oilseed rape ProDH genes provides new elements to investigate the function of proline metabolism in plant development.

Hydrogen bonds are a primary driving force for de novo protein folding

DOE PAGES

Lee, Schuyler; Wang, Chao; Liu, Haolin; ...

2017-11-10

The protein-folding mechanism remains a major puzzle in life science. Purified soluble activation-induced cytidine deaminase (AID) is one of the most difficult proteins to obtain. Starting from inclusion bodies containing a C-terminally truncated version of AID (residues 1–153; AID 153 ), an optimized in vitro folding procedure was derived to obtain large amounts of AID 153 , which led to crystals with good quality and to final structural determination. Interestingly, it was found that the final refolding yield of the protein is proline residue-dependent. The difference in the distribution of cis and trans configurations of proline residues in the proteinmore » after complete denaturation is a major determining factor of the final yield. A point mutation of one of four proline residues to an asparagine led to a near-doubling of the yield of refolded protein after complete denaturation. It was concluded that the driving force behind protein folding could not overcome the cis -to- trans proline isomerization, or vice versa , during the protein-folding process. Furthermore, it was found that successful refolding of proteins optimally occurs at high pH values, which may mimic protein folding in vivo . It was found that high pH values could induce the polarization of peptide bonds, which may trigger the formation of protein secondary structures through hydrogen bonds. It is proposed that a hydrophobic environment coupled with negative charges is essential for protein folding. Combined with our earlier discoveries on protein-unfolding mechanisms, it is proposed that hydrogen bonds are a primary driving force for de novo protein folding.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Forlani, Giuseppe; Bertazzini, Michele; Zarattini, Marco

The majority of plant species accumulate high intracellular levels of proline to cope with hyperosmotic stress conditions. Proline synthesis from glutamate is tightly regulated at both the transcriptional and the translational levels, yet little is known about the mechanisms for post-translational regulation of the enzymatic activities involved. The gene coding in rice ( Oryza sativa L.) for δ 1-pyrroline-5-carboxylate (P5C) reductase, the enzyme that catalyzes the second and final step in this pathway, was isolated and expressed in Escherichia coli. The structural and functional properties of the affinity-purified protein were characterized. As for most species, rice P5C reductase was ablemore » to use in vitro either NADH or NADPH as the electron donor. However, strikingly different effects of cations and anions were found depending on the pyridine nucleotide used, namely inhibition of NADH-dependent activity and stimulation of NADPH-dependent activity. Moreover, physiological concentrations of proline and NADP + were strongly inhibitory for the NADH-dependent reaction, whereas the NADPH-dependent activity was mildly affected. Our results suggest that only NADPH may be used in vivo and that stress-dependent variations in ion homeostasis and NADPH/NADP + ratio could modulate enzyme activity, being functional in promoting proline accumulation and potentially also adjusting NADPH consumption during the defense against hyperosmotic stress. The apparent molecular weight of the native protein observed in size exclusion chromatography indicated a high oligomerization state. We also report the first crystal structure of a plant P5C reductase at 3.40-Å resolution, showing a decameric quaternary assembly. It was possible to identify dynamic structural differences among rice, human, and bacterial enzymes.« less

Hydrogen bonds are a primary driving force for de novo protein folding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Schuyler; Wang, Chao; Liu, Haolin

The protein-folding mechanism remains a major puzzle in life science. Purified soluble activation-induced cytidine deaminase (AID) is one of the most difficult proteins to obtain. Starting from inclusion bodies containing a C-terminally truncated version of AID (residues 1–153; AID 153 ), an optimized in vitro folding procedure was derived to obtain large amounts of AID 153 , which led to crystals with good quality and to final structural determination. Interestingly, it was found that the final refolding yield of the protein is proline residue-dependent. The difference in the distribution of cis and trans configurations of proline residues in the proteinmore » after complete denaturation is a major determining factor of the final yield. A point mutation of one of four proline residues to an asparagine led to a near-doubling of the yield of refolded protein after complete denaturation. It was concluded that the driving force behind protein folding could not overcome the cis -to- trans proline isomerization, or vice versa , during the protein-folding process. Furthermore, it was found that successful refolding of proteins optimally occurs at high pH values, which may mimic protein folding in vivo . It was found that high pH values could induce the polarization of peptide bonds, which may trigger the formation of protein secondary structures through hydrogen bonds. It is proposed that a hydrophobic environment coupled with negative charges is essential for protein folding. Combined with our earlier discoveries on protein-unfolding mechanisms, it is proposed that hydrogen bonds are a primary driving force for de novo protein folding.« less

Ruthenium-Nitrosyl Complexes with Glycine, l-Alanine, l-Valine, l-Proline, d-Proline, l-Serine, l-Threonine, and l-Tyrosine: Synthesis, X-ray Diffraction Structures, Spectroscopic and Electrochemical Properties, and Antiproliferative Activity

PubMed Central

2014-01-01

The reactions of [Ru(NO)Cl5]2– with glycine (Gly), l-alanine (l-Ala), l-valine (l-Val), l-proline (l-Pro), d-proline (d-Pro), l-serine (l-Ser), l-threonine (l-Thr), and l-tyrosine (l-Tyr) in n-butanol or n-propanol afforded eight new complexes (1–8) of the general formula [RuCl3(AA–H)(NO)]−, where AA = Gly, l-Ala, l-Val, l-Pro, d-Pro, l-Ser, l-Thr, and l-Tyr, respectively. The compounds were characterized by elemental analysis, electrospray ionization mass spectrometry (ESI-MS), 1H NMR, UV–visible and ATR IR spectroscopy, cyclic voltammetry, and X-ray crystallography. X-ray crystallography studies have revealed that in all cases the same isomer type (from three theoretically possible) was isolated, namely mer(Cl),trans(NO,O)-[RuCl3(AA–H)(NO)], as was also recently reported for osmium analogues with Gly, l-Pro, and d-Pro (see Z. Anorg. Allg. Chem.2013, 639, 1590–1597). Compounds 1, 4, 5, and 8 were investigated by ESI-MS with regard to their stability in aqueous solution and reactivity toward sodium ascorbate. In addition, cell culture experiments in three human cancer cell lines, namely, A549 (nonsmall cell lung carcinoma), CH1 (ovarian carcinoma), and SW480 (colon carcinoma), were performed, and the results are discussed in conjunction with the lipophilicity of compounds. PMID:24555845

Production and characterization of two major Aspergillus oryzae secreted prolyl endopeptidases able to efficiently digest proline-rich peptides of gliadin.

PubMed

Eugster, Philippe J; Salamin, Karine; Grouzmann, Eric; Monod, Michel

2015-12-01

Prolyl endopeptidases are key enzymes in the digestion of proline-rich proteins. Fungal extracts rich in prolyl endopeptidases produced by a species such as Aspergillus oryzae used in food fermentation would be of particular interest for the development of an oral enzyme therapy product in patients affected by intolerance to gluten. Two major A. oryzae secreted prolyl endopeptidases of the MEROPS S28 peptidase family, AoS28A and AoS28B, were identified when this fungus was grown at acidic pH in a medium containing soy meal protein or wheat gliadin as the sole source of nitrogen. AoS28B was produced by 12 reference A. oryzae strains used in food fermentation. AoS28A was secreted by six of these 12 strains. This protease is the orthologue of the previously characterized Aspergillus fumigatus (AfuS28) and Aspergillus niger (AN-PEP) prolyl endopeptidases which are encoded by genes with a similar intron-exon structure. Large amounts of secreted AoS28A and AoS28B were obtained by gene overexpression in A. oryzae. AoS28A and AoS28B are endoproteases able to cleave N-terminally blocked proline substrates. Both enzymes very efficiently digested the proline-rich 33-mer of gliadin, the most representative immunotoxic peptide deriving from gliadin, with some differences in terms of specificity and optimal pH. Digestion of the gliadin peptide in short peptides with both enzymes was found to occur from its N terminus.

Ketonization of Proline Residues in the Peptide Chains of Actinomycins by a 4-Oxoproline Synthase.

PubMed

Semsary, Siamak; Crnovčić, Ivana; Driller, Ronja; Vater, Joachim; Loll, Bernhard; Keller, Ullrich

2018-04-04

X-type actinomycins (Acms) contain 4-hydroxyproline (Acm X 0 ) or 4-oxoproline (Acm X 2 ) in their β-pentapeptide lactone rings, whereas their α ring contains proline. We demonstrate that these Acms are formed through asymmetric condensation of Acm half molecules (Acm halves) containing proline with 4-hydroxyproline- or 4-oxoproline-containing Acm halves. In turn, we show-using an artificial Acm half analogue (PPL 1) with proline in its peptide chain-their conversion into the 4-hydroxyproline- and 4-oxoproline-containing Acm halves, PPL 0 and PPL 2, in mycelial suspensions of Streptomyces antibioticus. Two responsible genes of the Acm X biosynthetic gene cluster of S. antibioticus, saacmM and saacmN, encoding a cytochrome P450 monooxygenase (Cyp) and a ferredoxin were identified. After coexpression in Escherichia coli, their gene products converted PPL 1 into PPL 0 and PPL 2 in vivo as well as in situ in permeabilized cell of the transformed E. coli strain in conjunction with the host-encoded ferredoxin reductase in a NADH (NADPH)-dependent manner. saAcmM has high sequence similarity to the Cyp107Z (Ema) family of Cyps, which can convert avermectin B1 into its keto derivative, 4''-oxoavermectin B1. Determination of the structure of saAcmM reveals high similarity to the Ema structure but with significant differences in residues decorating their active sites, which defines saAcmM and its orthologues as a distinct new family of peptidylprolineketonizing Cyp. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Identification and functional characterization of an Src homology domain 3 domain-binding site on Cbl.

PubMed

Sanjay, Archana; Miyazaki, Tsuyoshi; Itzstein, Cecile; Purev, Enkhtsetseg; Horne, William C; Baron, Roland

2006-12-01

Cbl is an adaptor protein and ubiquitin ligase that binds and is phosphorylated by the nonreceptor tyrosine kinase Src. We previously showed that the primary interaction between Src and Cbl is mediated by the Src homology domain 3 (SH3) of Src binding to proline-rich sequences of Cbl. The peptide Cbl RDLPPPPPPDRP(540-551), which corresponds to residues 540-551 of Cbl, inhibited the binding of a GST-Src SH3 fusion protein to Cbl, whereas RDLAPPAPPPDR(540-551) did not, suggesting that Src binds to this site on Cbl in a class I orientation. Mutating prolines 543-548 reduced Src binding to the Cbl 479-636 fragment significantly more than mutating the prolines in the PPVPPR(494-499) motif, which was previously reported to bind Src SH3. Mutating Cbl prolines 543-548 to alanines substantially reduced Src binding to Cbl, Src-induced phosphorylation of Cbl, and the inhibition of Src kinase activity by Cbl. Expressing the mutated Cbl in osteoclasts induced a moderate reduction in bone-resorbing activity and increased amounts of Src protein. In contrast, disabling the tyrosine kinase-binding domain of full-length Cbl by mutating glycine 306 to glutamic acid, and thereby preventing the previously described binding of the tyrosine kinase-binding domain to the Src phosphotyrosine 416, had no effect on Cbl phosphorylation, the inhibition of Src activity by full-length Cbl, or bone resorption. These data indicate that the Cbl RDLPPPP(540-546) sequence is a functionally important binding site for Src.

Alanine and proline content modulate global sensitivity to discrete perturbations in disordered proteins

PubMed Central

Perez, Romel B.; Tischer, Alexander; Auton, Matthew; Whitten, Steven T.

2014-01-01

Molecular transduction of biological signals is understood primarily in terms of the cooperative structural transitions of protein macromolecules, providing a mechanism through which discrete local structure perturbations affect global macromolecular properties. The recognition that proteins lacking tertiary stability, commonly referred to as intrinsically disordered proteins, mediate key signaling pathways suggests that protein structures without cooperative intramolecular interactions may also have the ability to couple local and global structure changes. Presented here are results from experiments that measured and tested the ability of disordered proteins to couple local changes in structure to global changes in structure. Using the intrinsically disordered N-terminal region of the p53 protein as an experimental model, a set of proline and alanine to glycine substitution variants were designed to modulate backbone conformational propensities without introducing non-native intramolecular interactions. The hydrodynamic radius (Rh) was used to monitor changes in global structure. Circular dichroism spectroscopy showed that the glycine substitutions decreased polyproline II (PPII) propensities relative to the wild type, as expected, and fluorescence methods indicated that substitution-induced changes in Rh were not associated with folding. The experiments showed that changes in local PPII structure cause changes in Rh that are variable and that depend on the intrinsic chain propensities of proline and alanine residues, demonstrating a mechanism for coupling local and global structure changes. Molecular simulations that model our results were used to extend the analysis to other proteins and illustrate the generality of the observed proline and alanine effects on the structures of intrinsically disordered proteins. PMID:25244701

The proline-rich region of 18.5 kDa myelin basic protein binds to the SH3-domain of Fyn tyrosine kinase with the aid of an upstream segment to form a dynamic complex in vitro

PubMed Central

De Avila, Miguel; Vassall, Kenrick A.; Smith, Graham S. T.; Bamm, Vladimir V.; Harauz, George

2014-01-01

The intrinsically disordered 18.5 kDa classic isoform of MBP (myelin basic protein) interacts with Fyn kinase during oligodendrocyte development and myelination. It does so primarily via a central proline-rich SH3 (Src homology 3) ligand (T92–R104, murine 18.5 kDa MBP sequence numbering) that is part of a molecular switch due to its high degree of conservation and modification by MAP (mitogen-activated protein) and other kinases, especially at residues T92 and T95. Here, we show using co-transfection experiments of an early developmental oligodendroglial cell line (N19) that an MBP segment upstream of the primary ligand is involved in MBP–Fyn–SH3 association in cellula. Using solution NMR spectroscopy in vitro, we define this segment to comprise MBP residues (T62–L68), and demonstrate further that residues (V83–P93) are the predominant SH3-target, assessed by the degree of chemical shift change upon titration. We show by chemical shift index analysis that there is no formation of local poly-proline type II structure in the proline-rich segment upon binding, and by NOE (nuclear Overhauser effect) and relaxation measurements that MBP remains dynamic even while complexed with Fyn–SH3. The association is a new example first of a non-canonical SH3-domain interaction and second of a fuzzy MBP complex. PMID:25343306

Both the cis-trans equilibrium and isomerization dynamics of a single proline amide modulate β2-microglobulin amyloid assembly

PubMed Central

Torbeev, Vladimir Yu.; Hilvert, Donald

2013-01-01

The human protein β2-microglobulin (β2m) aggregates as amyloid fibrils in patients undergoing long-term hemodialysis. Isomerization of Pro32 from its native cis to a nonnative trans conformation is thought to trigger β2m misfolding and subsequent amyloid assembly. To examine this hypothesis, we systematically varied the free-energy profile of proline cis-trans isomerization by replacing Pro32 with a series of 4-fluoroprolines via total chemical synthesis. We show that β2m’s stability, (un)folding, and aggregation properties are all influenced by the rate and equilibrium of Pro32 cis-trans isomerization. As anticipated, the β2m monomer was either stabilized or destabilized by respective incorporation of (2S,4S)-fluoroproline, which favors the native cis amide bond, or the stereoisomeric (2S,4R)-fluoroproline, which disfavors this conformation. However, substitution of Pro32 with 4,4-difluoroproline, which has nearly the same cis-trans preference as proline but an enhanced isomerization rate, caused pronounced destabilization of the protein and increased oligomerization at neutral pH. More remarkably, these subtle alterations in chemical composition—incorporation of one or two fluorine atoms into a single proline residue in the 99 amino acid long protein—modulated the aggregation properties of β2m, inducing the formation of polymorphically distinct amyloid fibrils. These results highlight the importance of conformational dynamics for molecular assembly of an amyloid cross-β structure and provide insights into mechanistic aspects of Pro32 cis-trans isomerism in β2m aggregation. PMID:24262149

Both the cis-trans equilibrium and isomerization dynamics of a single proline amide modulate β2-microglobulin amyloid assembly.

PubMed

Torbeev, Vladimir Yu; Hilvert, Donald

2013-12-10

The human protein β2-microglobulin (β2m) aggregates as amyloid fibrils in patients undergoing long-term hemodialysis. Isomerization of Pro32 from its native cis to a nonnative trans conformation is thought to trigger β2m misfolding and subsequent amyloid assembly. To examine this hypothesis, we systematically varied the free-energy profile of proline cis-trans isomerization by replacing Pro32 with a series of 4-fluoroprolines via total chemical synthesis. We show that β2m's stability, (un)folding, and aggregation properties are all influenced by the rate and equilibrium of Pro32 cis-trans isomerization. As anticipated, the β2m monomer was either stabilized or destabilized by respective incorporation of (2S,4S)-fluoroproline, which favors the native cis amide bond, or the stereoisomeric (2S,4R)-fluoroproline, which disfavors this conformation. However, substitution of Pro32 with 4,4-difluoroproline, which has nearly the same cis-trans preference as proline but an enhanced isomerization rate, caused pronounced destabilization of the protein and increased oligomerization at neutral pH. More remarkably, these subtle alterations in chemical composition--incorporation of one or two fluorine atoms into a single proline residue in the 99 amino acid long protein--modulated the aggregation properties of β2m, inducing the formation of polymorphically distinct amyloid fibrils. These results highlight the importance of conformational dynamics for molecular assembly of an amyloid cross-β structure and provide insights into mechanistic aspects of Pro32 cis-trans isomerism in β2m aggregation.

P-I class metalloproteinase from Bothrops moojeni venom is a post-proline cleaving peptidase with kininogenase activity: insights into substrate selectivity and kinetic behavior.

PubMed

Okamoto, Débora N; Kondo, Marcia Y; Oliveira, Lilian C G; Honorato, Rodrigo V; Zanphorlin, Leticia M; Coronado, Monika A; Araújo, Mariana S; da Motta, Guacyara; Veronez, Camila L; Andrade, Sheila S; Oliveira, Paulo S L; Arni, Raghuvir K; Cintra, Adelia C O; Sampaio, Suely V; Juliano, Maria A; Juliano, Luiz; Murakami, Mário T; Gouvea, Iuri E

2014-03-01

Snake venom metalloproteinases (SVMPs) belonging to P-I class are able to hydrolyze extracellular matrix proteins and coagulation factors triggering local and systemic reactions by multiple molecular mechanisms that are not fully understood. BmooMPα-I, a P-I class SMVP from Bothrops moojeni venom, was active upon neuro- and vaso-active peptides including angiotensin I, bradykinin, neurotensin, oxytocin and substance P. Interestingly, BmooMPα-I showed a strong bias towards hydrolysis after proline residues, which is unusual for most of characterized peptidases. Moreover, the enzyme showed kininogenase activity similar to that observed in plasma and cells by kallikrein. FRET peptide assays indicated a relative promiscuity at its S2-S'2 subsites, with proline determining the scissile bond. This unusual post-proline cleaving activity was confirmed by the efficient hydrolysis of the synthetic combinatorial library MCA-GXXPXXQ-EDDnp, described as resistant for canonical peptidases, only after Pro residues. Structural analysis of the tripeptide LPL complexed with BmooMPα-I, generated by molecular dynamics simulations, assisted in defining the subsites and provided the structural basis for subsite preferences such as the restriction of basic residues at the S2 subsite due to repulsive electrostatic effects and the steric impediment for large aliphatic or aromatic side chains at the S1 subsite. These new functional and structural findings provided a further understanding of the molecular mechanisms governing the physiological effects of this important class of enzymes in envenomation process. Copyright © 2014 Elsevier B.V. All rights reserved.

Association of protein kinase FA/GSK-3alpha (a proline-directed kinase and a regulator of protooncogenes) with human cervical carcinoma dedifferentiation/progression.

PubMed

Yang, S D; Yu, J S; Lee, T T; Ni, M H; Yang, C C; Ho, Y S; Tsen, T Z

1995-10-01

Computer analysis of protein phosphorylation-sites sequence revealed that most transcriptional factors and viral oncoproteins are prime targets for regulation of proline-directed protein phosphorylation, suggesting an association of proline-directed protein kinase (PDPK) family with neoplastic transformation and tumorigenesis. In this report, an immunoprecipitate activity assay of protein kinase FA/glycogen synthase kinase-3alpha (kinase FA/GSK-3alpha) (a particular member of PDPK family) has been optimized for human cervical tissue and used to demonstrate for the first time significantly increased (P < 0.001) activity in poorly differentiated cervical carcinoma (82.8 +/- 6.6 U/mg of protein), moderately differentiated carcinoma (36.2 +/- 3.4 U/mg of protein), and well-differentiated carcinoma (18.3 +/- 2.4 U/mg of protein) from 36 human cervical carcinoma samples when compared to 12 normal controls (4.9 +/- 0.6 U/mg of protein). Immunoblotting analysis further revealed that increased activity of kinase FA/GSK-3alpha in cervical carcinoma is due to overexpression of protein synthesis of the kinase. Taken together, the results provide initial evidence that overexpression of protein synthesis and cellular activity of kinase FA/GSK-3alpha may be involved in human cervical carcinoma dedifferentiation/progression, supporting an association of proline-directed protein kinase with neoplastic transformation and tumorigenesis. Since protein kinase FA/GSK-3alpha may function as a possible regulator of transcription factors/proto-oncogenes, the results further suggest that kinase FA/GSK-3alpha may play a potential role in human cervical carcinogenesis, especially in its dedifferentiation and progression.

The proline-rich region of 18.5 kDa myelin basic protein binds to the SH3-domain of Fyn tyrosine kinase with the aid of an upstream segment to form a dynamic complex in vitro.

PubMed

De Avila, Miguel; Vassall, Kenrick A; Smith, Graham S T; Bamm, Vladimir V; Harauz, George

2014-12-08

The intrinsically disordered 18.5 kDa classic isoform of MBP (myelin basic protein) interacts with Fyn kinase during oligodendrocyte development and myelination. It does so primarily via a central proline-rich SH3 (Src homology 3) ligand (T92-R104, murine 18.5 kDa MBP sequence numbering) that is part of a molecular switch due to its high degree of conservation and modification by MAP (mitogen-activated protein) and other kinases, especially at residues T92 and T95. Here, we show using co-transfection experiments of an early developmental oligodendroglial cell line (N19) that an MBP segment upstream of the primary ligand is involved in MBP-Fyn-SH3 association in cellula. Using solution NMR spectroscopy in vitro, we define this segment to comprise MBP residues (T62-L68), and demonstrate further that residues (V83-P93) are the predominant SH3-target, assessed by the degree of chemical shift change upon titration. We show by chemical shift index analysis that there is no formation of local poly-proline type II structure in the proline-rich segment upon binding, and by NOE (nuclear Overhauser effect) and relaxation measurements that MBP remains dynamic even while complexed with Fyn-SH3. The association is a new example first of a non-canonical SH3-domain interaction and second of a fuzzy MBP complex.

Survival and bioactivities of selected probiotic lactobacilli in yogurt fermentation and cold storage: New insights for developing a bi-functional dairy food.

PubMed

Rutella, Giuseppina Sefora; Tagliazucchi, Davide; Solieri, Lisa

2016-12-01

In previous work, we demonstrated that two probiotic strains, namely Lactobacillus casei PRA205 and Lactobacillus rhamnosus PRA331, produce fermented milks with potent angiotensin-converting enzyme (ACE)-inhibitory and antioxidant activities. Here, we tested these strains for the survivability and the release of antihypertensive and antioxidant peptides in yogurt fermentation and cold storage. For these purposes three yogurt batches were compared: one prepared using yogurt starters alone (Lactobacillus delbrueckii subspecies bulgaricus 1932 and Streptococcus thermophilus 99), and the remaining two containing either PRA205 or PRA331 in addition to yogurt starters. Despite the lower viable counts at the fermentation end compared to PRA331, PRA205 overcame PRA331 in survivability during refrigerated storage for 28 days, leading to viable counts (>10(8) CFU/g) higher than the minimum therapeutic threshold (10(6) CFU/g). Analyses of in vitro ACE-inhibitory and antioxidant activities of peptide fractions revealed that yogurt supplemented with PRA205 displays higher amounts of antihypertensive and antioxidant peptides than that produced with PRA331 at the end of fermentation and over storage. Two ACE-inhibitory peptides, Valine-Proline-Proline (VPP) and Isoleucine-Proline-Proline (IPP), were identified and quantified. This study demonstrated that L. casei PRA205 could be used as adjunct culture for producing bi-functional yogurt enriched in bioactive peptides and in viable cells, which bring health benefits to the host as probiotics. Copyright © 2016 Elsevier Ltd. All rights reserved.

Cadmium-cysteine coordination in the BK inner pore region and its structural and functional implications.

PubMed

Zhou, Yu; Xia, Xiao-Ming; Lingle, Christopher J

2015-04-21

To probe structure and gating-associated conformational changes in BK-type potassium (BK) channels, we examined consequences of Cd(2+) coordination with cysteines introduced at two positions in the BK inner pore. At V319C, the equivalent of valine in the conserved Kv proline-valine-proline (PVP) motif, Cd(2+) forms intrasubunit coordination with a native glutamate E321, which would place the side chains of V319C and E321 much closer together than observed in voltage-dependent K(+) (Kv) channel structures, requiring that the proline between V319C and E321 introduces a kink in the BK S6 inner helix sharper than that observed in Kv channel structures. At inner pore position A316C, Cd(2+) binds with modest state dependence, suggesting the absence of an ion permeation gate at the cytosolic side of BK channel. These results highlight fundamental structural differences between BK and Kv channels in their inner pore region, which likely underlie differences in voltage-dependent gating between these channels.

Proline-rich antimicrobial peptides: potential therapeutics against antibiotic-resistant bacteria.

PubMed

Li, Wenyi; Tailhades, Julien; O'Brien-Simpson, Neil M; Separovic, Frances; Otvos, Laszlo; Hossain, M Akhter; Wade, John D

2014-10-01

The increasing resistance of pathogens to antibiotics causes a huge clinical burden that places great demands on academic researchers and the pharmaceutical industry for resolution. Antimicrobial peptides, part of native host defense, have emerged as novel potential antibiotic alternatives. Among the different classes of antimicrobial peptides, proline-rich antimicrobial peptides, predominantly sourced from insects, have been extensively investigated to study their specific modes of action. In this review, we focus on recent developments in these peptides. They show a variety of modes of actions, including mechanism shift at high concentration, non-lytic mechanisms, as well as possessing different intracellular targets and lipopolysaccharide binding activity. Furthermore, proline-rich antimicrobial peptides display the ability to not only modulate the immune system via cytokine activity or angiogenesis but also possess properties of penetrating cell membranes and crossing the blood brain barrier suggesting a role as potential novel carriers. Ongoing studies of these peptides will likely lead to the development of more potent antimicrobial peptides that may serve as important additions to the armoury of agents against bacterial infection and drug delivery.

Analysis of TP53 codon 72 polymorphism in HPV-positive and HPV-negative penile carcinoma.

PubMed

Tornesello, Maria Lina; Duraturo, Maria Luisa; Guida, Valentina; Losito, Simona; Botti, Gerardo; Pilotti, Silvana; Stefanon, Bernardina; De Palo, Giuseppe; Buonaguro, Luigi; Buonaguro, Franco M

2008-09-28

The association of the p53 polymorphism at codon 72 and susceptibility to develop human papillomavirus (HPV)-related cancer has been investigated in several studies with controversial results. In this study, 78 penile squamous cell carcinoma biopsies (n=17 from Uganda, n=61 from Italy) and blood samples from 150 healthy controls (n=57 from Uganda, n=93 from Italy) have been analyzed for the arginine and proline allele distribution. Among Ugandan cases the heterozygous, proline homozygous and arginine homozygous genotype frequency was 41.2%, 52.9% and 5.9%, respectively, and among controls was 40.3%, 54.4%, and 5.3%, respectively (P=0.9917). Conversely, among Italian cases genotype distribution was 42.6%, 4.9%, and 52.5%, and among controls was 34.4%, 7.5%, and 58.1%, respectively (P=0.5343). No significant differences in arginine and proline allele distribution were observed when the cases were stratified by HPV status. Therefore, no evidence of association between homozygosity for p53 arginine and HPV-related or HPV-unrelated penile squamous cell carcinoma was observed neither among Ugandan nor among Italian populations.

Tuberous sclerosis: aberrant metabolism of ornithine, proline and glutamate in cultured fibroblasts.

PubMed

Tanaka, H; Nakazawa, K; Arima, M; Hayashi, A

1987-01-01

To investigate aberrant metabolism of proline (Pro) and its precursors in tuberous sclerosis (TS), 6, 7 and 5 strains of control, TS (normal skin) and TS (tumor) fibroblasts, respectively, were cultured in Eagle's MEM containing dialyzed fetal bovine serum with or without 0.1 mM ornithine (Orn). Ornithine aminotransferase (OAT) activity was decreased in TS, especially in TS (tumor) after mild sonication treatment. The yield of the OAT protein was inhibited in TS (tumor) when cultured without Orn. Free glutamate (Glu) in the medium was significantly increased in TS (tumor). Free proline (Pro) in cells was significantly decreased in TS (tumor) when cultured with Orn, but protein-bound Pro was not. The relative concentration of free Glu to glutamine (Gln) in the medium and that of free Glu to Pro in cells cultured with Orn were increased in TS (tumor). These results suggest that the requirement for Orn, increased turnover of Pro to Glu and increased elimination of Glu into the medium occur in TS (tumor). Aberrant regulation or turnover of Pro and Glu metabolism may occur in TS, especially in tumor cells.

A new method of UA_CPE coupled with spectrophotometry for the faster and cost-effective detection of proline in fruit juice, honey, and wine.

PubMed

Dağdeviren, Semahat; Altunay, Nail; Sayman, Yasin; Gürkan, Ramazan

2018-07-30

The study developed a new method for proline detection in honey, wine and fruit juice using ultrasound assisted-cloud point extraction (UA-CPE) and spectrophotometry. Initially, a quaternary complex was built, containing proline, histamine, Cu(II), and fluorescein at pH 5.5. Samples were treated with ethanol-water mixture before extraction and preconcentration, using an ultrasonic bath for 10 min at 40 °C (40 kHz, 300 W). After the optimization of variables affecting extraction efficiency, good linearity was obtained between 15 and 600 µg L -1 with sensitivity enhancement factor of 105. The limits of detection and quantification were 5.7 and 19.0 µg L -1 , respectively. The recovery percentage and relative standard deviations (RSD %) were between 95.3 and 103.3%, and 2.5 and 4.2%, respectively. The accuracy of the method was verified by the analysis of a standard reference material (SRM 2389a). Copyright © 2018 Elsevier Ltd. All rights reserved.

Neurotensin-metabolizing peptidases in rat fundus plasma membranes.

PubMed

Checler, F; Barelli, H; Kwan, C Y; Kitabgi, P; Vincent, J P

1987-08-01

The mechanisms by which neurotensin (NT) was inactivated by rat fundus plasma membranes were characterized. Primary inactivating cleavages occurred at the Arg8-Arg9, Pro10-Tyr11, and Ile12-Leu13 peptidyl bonds. Hydrolysis at the Arg8-Arg9 bond was fully abolished by the use of N-[1(R,S)-carboxy-2-phenylethyl]-alanyl-alanyl-phenylalanine-p- aminobenzoate, a result indicating the involvement at this site of a recently purified soluble metallopeptidase. Hydrolysis of the Pro10-Tyr11 bond was totally resistant to N-benzyloxycarbonyl-prolyl-prolinal and thiorphan, an observation suggesting that the peptidase responsible for this cleavage was different from proline endopeptidase and endopeptidase 24.11 and might correspond to a NT-degrading neutral metallopeptidase recently isolated from rat brain synaptic membranes. The enzyme acting at the Ile12-Leu13 bond has not yet been identified. Secondary cleavages occurring on NT degradation products were mainly generated by bestatin-sensitive aminopeptidases and post-proline dipeptidyl aminopeptidase. The content in NT-metabolizing peptidases present in rat fundus plasma membranes is compared with that previously established for purified rat brain synaptic membranes.

Inhibition of insulin fibrillation by osmolytes: Mechanistic Insights

NASA Astrophysics Data System (ADS)

Choudhary, Sinjan; Kishore, Nand; Hosur, Ramakrishna V.

2015-11-01

We have studied here using a number of biophysical tools the effects of osmolytes, betaine, citrulline, proline and sorbitol which differ significantly in terms of their physical characteristics such as, charge distribution, polarity, H-bonding abilities etc, on the fibrillation of insulin. Among these, betaine, citrulline, and proline are very effective in decreasing the extent of fibrillation. Proline also causes a substantial delay in the onset of fibrillation in the concentration range (50-250 mM) whereas such an effect is seen for citrulline only at 250 mM, and in case of betaine this effect is not seen at all in the whole concentration range. The enthalpies of interaction at various stages of fibrillation process have suggested that the preferential exclusion of the osmolyte and its polar interaction with the protein are important in inhibition. The results indicate that the osmolytes are most effective when added prior to the elongation stage of fibrillation. These observations have significant biological implications, since insulin fibrillation is known to cause injection amyloidosis and our data may help in designing lead drug molecules and development of potential therapeutic strategies.

Initial solubility & density evaluation of Non-Aqueous system of amino acid salts for CO2 capture: potassium prolinate blended with ethanol and ethylene glycol

NASA Astrophysics Data System (ADS)

Murshid, Ghulam; Garg, Sahil

2018-05-01

Amine scrubbing is the state of the art technology for CO2 capture, and solvent selection can significantly reduce the capital and energy cost of the process. Higher energy requirement for aqueous amine based CO2 removal process is still a most important downside preventive its industrial deployment. Therefore, in this study, novel non-aqueous based amino acid salt system consisting of potassium prolinate, ethanol and ethylene glycol has been studied. This work presents initial CO2 solubility study and important physical properties i.e. density of the studied solvent system. Previous work showed that non-aqueous system of potassium prolinate and ethanol has good absorption rates and requires lower energy for solvent regeneration. However, during regeneration, solvent loss issues were found due to lower boiling point of the ethanol. Therefore, ethylene glycol was added into current studied system for enhancing the overall boiling point of the system. The good initial CO2 solubility and low density of studied solvent system offers several advantages as compared to conventional amine solutions.

Study of human salivary proline-rich proteins interaction with food tannins.

PubMed

Soares, Susana; García-Estévez, Ignacio; Ferrer-Galego, Raúl; Brás, Natércia F; Brandão, Elsa; Silva, Mafalda; Teixeira, Natércia; Fonseca, Fátima; Sousa, Sérgio F; Ferreira-da-Silva, Frederico; Mateus, Nuno; de Freitas, Victor

2018-03-15

In this work, saturation transfer difference-NMR, isothermal microcalorimetry and molecular dynamics simulations have been used to study the individual interactions between basic, glycosylated and acidic proline-rich proteins (bPRPS, gPRPs, aPRPs) and P-B peptide with some representative food tannins [procyanidin B2, procyanidin B2 3'-O-gallate (B2g) and procyanidin trimer (catechin-4-8-catechin-4-8-catechin)]. Results showed that P-B peptide was in general the salivary protein (SP) with higher affinity whereas aPRPs showed lower affinity to the studied procyanidins. Moreover, B2g was the procyanidin with higher affinity for all SP. Hydrophobic and hydrogen bonds were present in all interactions but the major driving force depended on the procyanidin-SP pair. Furthermore, proline clusters or residues in their vicinity were identified as the probable sites of proteins for interaction with procyanidins. For bPRP and aPRP a significant change to less extended conformations was observed, while P-B peptide did not display any structural rearrangement upon procyanidins binding. Copyright © 2017. Published by Elsevier Ltd.

Effects of crude oil on the feeding behaviour of the zoanthid Palythoa variabilis.

PubMed

Reimer, A A

1975-01-01

Palythoa variabilis (Duerden 1898) has a well-coordinated, sterotyped feeding response similar to that described for other zoanthids. The feeding reaction can be elicited by the heterocyclic amino acid proline and by some of its analogs. The addition of an OH group (hydroxyproline) or of a glycyl group (prolylglycine) annuls the activity of the proline molecule. Substitutions (thiazolidine-4-carboxylic acid) or additions (glycylproline) to the amino group do not alter the effectivity of the activator. The size of the ring can be altered within certain limits (azetidine-2-carbocylic acid and pipecolic acid) without affecting the activity of the molecule. Feeding reactions culminating with ingestion can be elicited by Marine Diesel and Bunker-C oils. Exposure to oil affects the ability of polyps to discriminate between inert and chemically active particles for 3 to 5 days; responses to proline are not altered for at least 3 days following the exposure, but become slower and are present in fewer polyps after that period. Oil is retained in the coelenteron for several days following exposure and is periodically released in the form of timy droplets.

Coupling between amino acid and water dynamics by broadband dielectric spectroscopy

NASA Astrophysics Data System (ADS)

Saiz, Luciana; Cerveny, Silvina

2014-05-01

The dynamics of proline aqueous solution was investigated for water concentrations from 40 to 60 wt% by dielectric spectroscopy (106 - 109 Hz) in the temperature range from 230 to 300 K, where the solutions remain amorphous. We found two relaxation processes, related with the reorientation of proline and water as previously observed in a higher frequency range at room temperature [1]. We found that both dynamics are strongly coupled, as previously observed in hydrated proteins powders, in spite of the fact that a single amino acid is a molecule much simpler than a protein.

Enantioselective Organocatalytic Aminomethylation of Aldehydes: A Role for Ionic Interactions and Efficient Access to β2-Amino Acids

PubMed Central

Chi, Yonggui; Gellman, Samuel H.

2009-01-01

Organocatalytic Mannich addition of aldehydes to a formaldehyde-derived iminium species catalyzed by proline-derived chiral pyrrolidines provides β-amino aldehydes with ≥ 90% ee. Mechanistic analysis of the proline-catalyzed reactions suggests that non-hydrogen-bonded ionic interactions at the Mannich reaction transition state can influence stereochemical outcome. The β-amino aldehydes from our process bear a substituent adjacent to the carbonyl and can be efficiently converted to protected β2-amino acids, which are important building blocks for β-peptide foldamers that display useful biological activities. PMID:16719457

D-piece modifications of the hemiasterlin analog HTI-286 produce potent tubulin inhibitors.

PubMed

Zask, Arie; Birnberg, Gary; Cheung, Katherine; Kaplan, Joshua; Niu, Chuan; Norton, Emily; Yamashita, Ayako; Beyer, Carl; Krishnamurthy, Girija; Greenberger, Lee M; Loganzo, Frank; Ayral-Kaloustian, Semiramis

2004-08-16

Modifications of the D-piece carboxylic acid group of the hemiasterlin analog HTI-286 gave tubulin inhibitors which were potent cytotoxic agents in taxol resistant cell lines expressing P-glycoprotein. Amides derived from proline had potency comparable to HTI-286. Reduction of the carboxylic acid to ketones and alcohols or its conversion to acidic heterocycles also gave potent analogs. Synthetic modifications of the carboxylic acid could be carried out selectively using a wide range of synthetic reagents. Proline analog 3 was found to be effective in a human xenograft model in athymic mice.

The Influence of Osmolytes on the Antimicrobial Activity of Silver Nanoparticles

NASA Astrophysics Data System (ADS)

Reed-Jones, Neiunna L.

Silver nanoparticles (Ag-NPs) are gaining popularity as antimicrobial agents due to their broad- spectrum activity and lower propensity to develop resistance in bacteria. However, with increased frequency of use, there remains a possibility for bacteria to develop resistance or adaptive mechanisms against Ag-NPs overtime. Mechanisms used by bacteria to resist antimicrobial agents include efflux pumps, heat shock proteins (Hsp) and accumulation of compatible solutes, generally referred to as osmolytes. The latter mechanism is typically employed when bacteria are under osmotic stress they accumulate osmolytes either through de novo synthesis or exogenously. Osmolytes act to stabilize the bacterial cell membrane by maintaining the native protein structure, while at the same time, ensuring compatibility with other cellular structures and functions. The most common osmolytes accumulated by bacteria are glycine betaine, proline, carnitine, choline, trehalose and glutamate. Since Ag-NPs target the cell membrane it is conceivable that osmolytes may suppress its bactericidal activity. In the present study, we assess the antimicrobial efficacy of Ag-NPs in the presence of glycine betaine and proline. Exponential phase cultures (106 cfu/ml) of Escherichia coli O157:H7, Listeria monocytogenes, Pseudomonas aeruginosa and Staphylococcus aureus were exposed to a predetermined the minimum inhibitory concentration (MIC) of 0.4 mM of citrate-stabilized Ag-NPs and incubated at 37°C for 48 h. This was replicated with the addition of 1 mM of either glycine betaine or proline. Growth was monitored by optical density, standard plate count, resazurin assay and LIVE/DEAD analyses. The result showed that Ag-NPs had no detectable effect on osmolyte treated cells. The average plate count of cultures supplemented with either glycine betaine or proline ranged from 108 to 109 cfu/ml after 48 h. Resazurin assay also showed that there was a significant increase in the cells supplemented with glycine betaine, 90% for E.coli O157:H7 and 44.3% for L. monocytogenes. No detectable metabolic activity was observed in the cells exposed to Ag-NPs alone. These results were corroborated by LIVE/DEAD analyses, which revealed over 86% cell viability in L. monocytogenes and E.coli O157:H7 treated with the osmolytes compared to 0.3% viability in those treated with Ag-NPs alone. SDS-PAGE analysis of the periplasmic proteins of the cells treated with Ag-NP and glycine betaine showed no qualitative difference in the protein profiles compared to the control cells grown in Trypticase Soy Broth alone. However, protein bands of cells treated with proline showed detectable difference relative to the control. The present findings suggest that glycine betaine and proline can suppress the antimicrobial activity of Ag-NPs.

Functionalized multi-walled carbon nanotubes in an aldol reaction

NASA Astrophysics Data System (ADS)

Chronopoulos, D. D.; Kokotos, C. G.; Karousis, N.; Kokotos, G.; Tagmatarchis, N.

2015-01-01

The covalent functionalization of multi-walled carbon nanotubes (MWCNTs) with a proline-based derivative is reported. Initially, MWCNTs were oxidized in order to introduce a large number of carboxylic units on their tips followed by N-tert-butoxycarbonyl-2,2'(ethylenedioxy)bis-(ethylamine) conjugation through an amide bond. Then, a proline derivative bearing a carboxylic terminal moiety at the 4-position was coupled furnishing proline-modified MWCNTs. This new hybrid material was fully characterized by spectroscopic and microscopy means and its catalytic activity in the asymmetric aldol reaction between acetone and 4-nitrobenzaldehyde was evaluated for the first time, showing to proceed almost quantitatively in aqueous media. Furthermore, several amino-modified MWCNTs were prepared and examined in the particular aldol reaction. These new hybrid materials exhibited an enhanced catalytic activity in water, contrasting with the pristine MWCNTs as well as the parent organic molecule, which failed to catalyze the reaction efficiently. Furthermore, the modified MWCNTs proved to catalyze the aldol reaction even after three repetitive cycles. Overall, a green approach for the aldol reaction is presented, where water can be employed as the solvent and modified MWCNTs can be used as catalysts, which can be successfully recovered and reused, while their catalytic activity is retained.The covalent functionalization of multi-walled carbon nanotubes (MWCNTs) with a proline-based derivative is reported. Initially, MWCNTs were oxidized in order to introduce a large number of carboxylic units on their tips followed by N-tert-butoxycarbonyl-2,2'(ethylenedioxy)bis-(ethylamine) conjugation through an amide bond. Then, a proline derivative bearing a carboxylic terminal moiety at the 4-position was coupled furnishing proline-modified MWCNTs. This new hybrid material was fully characterized by spectroscopic and microscopy means and its catalytic activity in the asymmetric aldol reaction between acetone and 4-nitrobenzaldehyde was evaluated for the first time, showing to proceed almost quantitatively in aqueous media. Furthermore, several amino-modified MWCNTs were prepared and examined in the particular aldol reaction. These new hybrid materials exhibited an enhanced catalytic activity in water, contrasting with the pristine MWCNTs as well as the parent organic molecule, which failed to catalyze the reaction efficiently. Furthermore, the modified MWCNTs proved to catalyze the aldol reaction even after three repetitive cycles. Overall, a green approach for the aldol reaction is presented, where water can be employed as the solvent and modified MWCNTs can be used as catalysts, which can be successfully recovered and reused, while their catalytic activity is retained. Electronic supplementary information (ESI) available: Experimental details for the synthesis of 5, 8 and 11; 1H & 13C NMR of compounds 8 and 11; ATR-IR spectra, thermographs and TEM imaging of hybrids 10 and 13. See DOI: 10.1039/c4nr06543c

Multivariate analyses of salt stress and metabolite sensing in auto- and heterotroph Chenopodium cell suspensions.

PubMed

Wongchai, C; Chaidee, A; Pfeiffer, W

2012-01-01

Global warming increases plant salt stress via evaporation after irrigation, but how plant cells sense salt stress remains unknown. Here, we searched for correlation-based targets of salt stress sensing in Chenopodium rubrum cell suspension cultures. We proposed a linkage between the sensing of salt stress and the sensing of distinct metabolites. Consequently, we analysed various extracellular pH signals in autotroph and heterotroph cell suspensions. Our search included signals after 52 treatments: salt and osmotic stress, ion channel inhibitors (amiloride, quinidine), salt-sensing modulators (proline), amino acids, carboxylic acids and regulators (salicylic acid, 2,4-dichlorphenoxyacetic acid). Multivariate analyses revealed hirarchical clusters of signals and five principal components of extracellular proton flux. The principal component correlated with salt stress was an antagonism of γ-aminobutyric and salicylic acid, confirming involvement of acid-sensing ion channels (ASICs) in salt stress sensing. Proline, short non-substituted mono-carboxylic acids (C2-C6), lactic acid and amiloride characterised the four uncorrelated principal components of proton flux. The proline-associated principal component included an antagonism of 2,4-dichlorphenoxyacetic acid and a set of amino acids (hydrophobic, polar, acidic, basic). The five principal components captured 100% of variance of extracellular proton flux. Thus, a bias-free, functional high-throughput screening was established to extract new clusters of response elements and potential signalling pathways, and to serve as a core for quantitative meta-analysis in plant biology. The eigenvectors reorient research, associating proline with development instead of salt stress, and the proof of existence of multiple components of proton flux can help to resolve controversy about the acid growth theory. © 2011 German Botanical Society and The Royal Botanical Society of the Netherlands.

Pin1 promotes transforming growth factor-beta-induced migration and invasion.

PubMed

Matsuura, Isao; Chiang, Keng-Nan; Lai, Chen-Yu; He, Dongming; Wang, Guannan; Ramkumar, Romila; Uchida, Takafumi; Ryo, Akihide; Lu, Kunping; Liu, Fang

2010-01-15

Transforming growth factor-beta (TGF-beta) regulates a wide variety of biological activities. It induces potent growth-inhibitory responses in normal cells but promotes migration and invasion of cancer cells. Smads mediate the TGF-beta responses. TGF-beta binding to the cell surface receptors leads to the phosphorylation of Smad2/3 in their C terminus as well as in the proline-rich linker region. The serine/threonine phosphorylation sites in the linker region are followed by the proline residue. Pin1, a peptidyl-prolyl cis/trans isomerase, recognizes phosphorylated serine/threonine-proline motifs. Here we show that Smad2/3 interacts with Pin1 in a TGF-beta-dependent manner. We further show that the phosphorylated threonine 179-proline motif in the Smad3 linker region is the major binding site for Pin1. Although epidermal growth factor also induces phosphorylation of threonine 179 and other residues in the Smad3 linker region the same as TGF-beta, Pin1 is unable to bind to the epidermal growth factor-stimulated Smad3. Further analysis suggests that phosphorylation of Smad3 in the C terminus is necessary for the interaction with Pin1. Depletion of Pin1 by small hairpin RNA does not significantly affect TGF-beta-induced growth-inhibitory responses and a number of TGF-beta/Smad target genes analyzed. In contrast, knockdown of Pin1 in human PC3 prostate cancer cells strongly inhibited TGF-beta-mediated migration and invasion. Accordingly, TGF-beta induction of N-cadherin, which plays an important role in migration and invasion, is markedly reduced when Pin1 is depleted in PC3 cells. Because Pin1 is overexpressed in many cancers, our findings highlight the importance of Pin1 in TGF-beta-induced migration and invasion of cancer cells.

Functional Characterization of Four Putative δ1-Pyrroline-5-Carboxylate Reductases from Bacillus subtilis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Forlani, Giuseppe; Nocek, Boguslaw; Chakravarthy, Srinivas

In most living organisms, the amino acid proline is synthesized starting from both glutamate and ornithine. In prokaryotes, in the absence of an ornithine cyclodeaminase that has been identified to date only in a small number of soil and plant bacteria, these pathways share the last step, the reduction of delta(1)-pyrroline-5-carboxylate (P5C) catalyzed by P5C reductase (EC 1.5.1.2). In several species, multiple forms of P5C reductase have been reported, possibly reflecting the dual function of proline. Aside from its common role as a building block of proteins, proline is indeed also involved in the cellular response to osmotic and oxidativemore » stress conditions. Genome analysis of Bacillus subtilis identifies the presence of four genes (ProH, ProI, ProG, and ComER) that, based on bioinformatic and phylogenic studies, were defined as respectively coding a putative P5C reductase. Here we describe the cloning, heterologous expression, functional analysis and small-angle X-ray scattering studies of the four affinity-purified proteins. Results showed that two of them, namely ProI and ComER, lost their catalytic efficiency or underwent subfunctionalization. In the case of ComER, this could be likely explained by the loss of the ability to form a dimer, which has been previously shown to be an essential structural feature of the catalytically active P5C reductase. The properties of the two active enzymes are consistent with a constitutive role for ProG, and suggest that ProH expression may be beneficial to satisfy an increased need for proline.« less

Functional Characterization of Four Putative δ1-Pyrroline-5-Carboxylate Reductases from Bacillus subtilis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Forlani, Giuseppe; Nocek, Boguslaw; Chakravarthy, Srinivas

In most living organisms, the amino acid proline is synthesized starting from both glutamate and ornithine. In prokaryotes, in the absence of an ornithine cyclodeaminase that has been identified to date only in a small number of soil and plant bacteria, these pathways share the last step, the reduction of δ1-pyrroline-5-carboxylate (P5C) catalyzed by P5C reductase (EC 1.5.1.2). In several species, multiple forms of P5C reductase have been reported, possibly reflecting the dual function of proline. Aside from its common role as a building block of proteins, proline is indeed also involved in the cellular response to osmotic and oxidativemore » stress conditions. Genome analysis of Bacillus subtilis identifies the presence of four genes (ProH, ProI, ProG, and ComER) that, based on bioinformatic and phylogenic studies, were defined as respectively coding a putative P5C reductase. Here we describe the cloning, heterologous expression, functional analysis and small-angle X-ray scattering studies of the four affinity-purified proteins. Results showed that two of them, namely ProI and ComER, lost their catalytic efficiency or underwent subfunctionalization. In the case of ComER, this could be likely explained by the loss of the ability to form a dimer, which has been previously shown to be an essential structural feature of the catalytically active P5C reductase. The properties of the two active enzymes are consistent with a constitutive role for ProG, and suggest that ProH expression may be beneficial to satisfy an increased need for proline.« less

A Randomized, Placebo-controlled Trial of Roflumilast. Effect on Proline-Glycine-Proline and Neutrophilic Inflammation in Chronic Obstructive Pulmonary Disease.

PubMed

Wells, J Michael; Jackson, Patricia L; Viera, Liliana; Bhatt, Surya P; Gautney, Joshua; Handley, Guy; King, R Wilson; Xu, Xin; Gaggar, Amit; Bailey, William C; Dransfield, Mark T; Blalock, J Edwin

2015-10-15

Roflumilast is a therapeutic agent in the treatment of chronic obstructive pulmonary disease (COPD). It has antiinflammatory effects; however, it is not known whether it can affect a biologic pathway implicated in COPD pathogenesis and progression. The self-propagating acetyl-proline-glycine-proline (AcPGP) pathway is a novel means of neutrophilic inflammation that is pathologic in the development of COPD. AcPGP is produced by extracellular matrix collagen breakdown with prolyl endopeptidase and leukotriene A4 hydrolase serving as the enzymes responsible for its production and degradation, respectively. We hypothesized that roflumilast would decrease AcPGP, halting the feed-forward cycle of inflammation. We conducted a single-center, placebo-controlled, randomized study investigating 12 weeks of roflumilast treatment added to current therapy in moderate-to-severe COPD with chronic bronchitis. Subjects underwent sputum and blood analyses, pulmonary function testing, exercise tolerance, and quality-of-life assessment at 0, 4, and 12 weeks. Twenty-seven patients were enrolled in the intention-to-treat analysis. Roflumilast treatment decreased sputum AcPGP by more than 50% (P < 0.01) and prolyl endopeptidase by 46% (P = 0.02), without significant improvement in leukotriene A4 hydrolase activity compared with placebo. Roflumilast also reduces other inflammatory markers. There were no significant changes in lung function, quality of life, or exercise tolerance between roflumilast- and placebo-treated groups. Roflumilast reduces pulmonary inflammation through decreasing prolyl endopeptidase activity and AcPGP. As expected for lower AcPGP levels, markers of neutrophilic inflammation are blunted. Inhibiting this self-propagating pathway lessens the overall inflammatory burden, which may alter the natural history of COPD, including the risk of exacerbation. Clinical trial registered with www.clinicaltrials.gov (NCT 01572948).

Identification of N-glycosylation in prolyl endoprotease from Aspergillus niger and evaluation of the enzyme for its possible application in proteomics.

PubMed

Sebela, Marek; Rehulka, Pavel; Kábrt, Jaromír; Rehulková, Helena; Ozdian, Tomás; Raus, Martin; Franc, Vojtech; Chmelík, Josef

2009-11-01

An acidic prolyl endoprotease from Aspergillus niger was isolated from the commercial product Brewers Clarex to evaluate its possible application in proteomics. The chromatographic purification yielded a single protein band in sodium dodecyl sulfate polyacrylamide gel electrophoresis providing an apparent molecular mass of 63 kDa and a broad peak (m/z 58,061) in linear matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) indicating the glycoprotein nature of the enzyme. Indeed, a colorimetric assessment with phenol and sulfuric acid showed the presence of neutral sugars (9% of weight). The subsequent treatment with N-glycosidase F released a variety of high-mannose type N-glycans, which were successfully detected using MALDI-TOF MS. MALDI-TOF/TOF tandem MS analysis of glycopeptides from a tryptic digest of prolyl endoprotease unraveled the identity of the N-glycosylation site in the primary structure. The data obtained also show that the enzyme is present in its processed form, i.e. without putative signal and propeptide parts. Spectrophotometric measurements demonstrated optimal activity at pH 4.0-4.5 and also high thermostability for the cleavage at the C-terminal part of proline residues. In-solution digestion of standard proteins (12-200 kDa) allowed to evaluate the cleavage specificity. The enzyme acts upon proline and alanine residues, but there is an additional minor cleavage at some other residues like Gly, Leu, Arg, Ser and Tyr. The digestion of a honeybee peptide comprising six proline residues (apidaecin 1A) led to the detection of specific peptides terminated by proline as it was confirmed by MALDI postsource decay analysis. Copyright 2009 John Wiley & Sons, Ltd.

Salt stress-induced proline transporters and salt stress-repressed broad specificity amino acid permeases identified by suppression of a yeast amino acid permease-targeting mutant.

PubMed Central

Rentsch, D; Hirner, B; Schmelzer, E; Frommer, W B

1996-01-01

A yeast mutant lacking SHR3, a protein specifically required for correct targeting of plasma membrane amino acid permeases, was used to study the targeting of plant transporters and as a tool to isolate new SHR3-independent amino acid transporters. For this purpose, an shr3 mutant was transformed with an Arabidopsis cDNA library. Thirty-four clones were capable of growth under selective conditions, but none showed homology with SHR3. However, genes encoding eight different amino acid transporters belonging to three different transporter families were isolated. Five of these are members of the general amino acid permease (AAP) gene family, one is a member of the NTR family, encoding an oligopeptide transporter, and two belong to a new class of transporter genes. A functional analysis of the latter two genes revealed that they encode specific proline transporters (ProT) that are distantly related to the AAP gene family. ProT1 was found to be expressed in all organs, but highest levels were found in roots, stems, and flowers. Expression in flowers was highest in the floral stalk phloem that enters the carpels and was downregulated after fertilization, indicating a specific role in supplying the ovules with proline. ProT2 transcripts were found ubiquitously throughout the plant, but expression was strongly induced under water or salt stress, implying that ProT2 plays an important role in nitrogen distribution during water stress, unlike members of the AAP gene family whose expression was repressed under the same conditions. These results corroborate the general finding that under water stress, amino acid export is impaired whereas proline export is increased. PMID:8776904

Effect of Salt Stress on Growth, Na+ Accumulation and Proline Metabolism in Potato (Solanum tuberosum) Cultivars

PubMed Central

Jaarsma, Rinse; de Vries, Rozemarijn S. M.; de Boer, Albertus H.

2013-01-01

Potato (Solanum tuberosum) is a major crop world-wide and the productivity of currently used cultivars is strongly reduced at high soil salt levels. We compared the response of six potato cultivars to increased root NaCl concentrations. Cuttings were grown hydroponically and treated with 0 mM, 60 mM and 180 mM NaCl for one week. Growth reduction on salt was strongest for the cultivars Mozart and Mona Lisa with a severe senescence response at 180 mM NaCl and Mozart barely survived the treatment. The cultivars Desiree and Russett Burbank were more tolerant showing no senescence after salt treatment. A clear difference in Na+ homeostasis was observed between sensitive and tolerant cultivars. The salt sensitive cultivar Mozart combined low Na+ levels in root and stem with the highest leaf Na+ concentration of all cultivars, resulting in a high Na+ shoot distribution index (SDI) for Mozart as compared to Desiree. Overall, a positive correlation between salt tolerance and stem Na+ accumulation was found and the SDI for Na+ points to a role of stem Na+ accumulation in tolerance. In stem tissue, Mozart accumulated more H2O2 and less proline compared to the tolerant cultivars. Analysis of the expression of proline biosynthesis genes in Mozart and Desiree showed a clear reduction in proline dehydrogenase (PDH) expression in both cultivars and an increase in pyrroline-5-carboxylate synthetase 1 (P5CS1) gene expression in Desiree, but not in Mozart. Taken together, current day commercial cultivars show promising differences in salt tolerance and the results suggest that mechanisms of tolerance reside in the capacity of Na+ accumulation in stem tissue, resulting in reduced Na+ transport to the leaves. PMID:23533673

Isolation of amino acid activating subunit-pantetheine protein complexes: Their role in chain elongation in tyrocidine synthesis

PubMed Central

Lee, Sung G.; Lipmann, Fritz

1977-01-01

Dissociation of the multienzymes of tyrocidine synthesis by prolonged incubation of crude extracts of Bacillus brevis (Dubos strain, ATCC 8185) has yielded, on Sephadex G-100 chromatography, two fractions of amino acid activating subunits, a larger one of 70,000 daltons and a smaller one of 90,000 daltons; the latter was a complex consisting of the 70,000 dalton subunit and the pantetheine-carrying protein of about 20,000 daltons. When it dissociated, the intermediate enzyme, which activates three amino acids, contained two-thirds of the subunits in the 70,000 dalton and one-third in the 90,000 dalton fraction; the heavy enzyme, which activates six amino acids, contained five-sixths of the subunits in the former fraction and one-sixth in the latter. Both fractions showed ATP-PPi exchange with all amino acids that are activated by the respective polyenzymes. With proline as an example, the 70,000 dalton subunit exhibited a single low-affinity binding site, which should correspond to the peripheral thiol acceptor site, whereas the 90,000 dalton subunit showed both a low-affinity binding site and an additional high-affinity site for proline; the high-affinity site is attributed to the pantetheine present on the pantetheine-carrying protein, and suggests that amino acids are translocated from the peripheral SH to the pantetheine-carrying moiety during chain elongation. This was confirmed by the observation that the 90,000 dalton complex, when incubated with the light enzyme in the presence of phenylalanine and proline, produced DPhe-Pro dipeptide that cyclized into DPhe-Pro diketopiperazine, but the 70,000 dalton activating subunit, when similarly incubated, did not. After subunit dissociation, however, no further elongation occurred after the transfer from phenylalanine to proline. Images PMID:196286

Enhanced thermostability of methyl parathion hydrolase from Ochrobactrum sp. M231 by rational engineering of a glycine to proline mutation.

PubMed

Tian, Jian; Wang, Ping; Gao, Shan; Chu, Xiaoyu; Wu, Ningfeng; Fan, Yunliu

2010-12-01

Protein thermostability can be increased by some glycine to proline mutations in a target protein. However, not all glycine to proline mutations can improve protein thermostability, and this method is suitable only at carefully selected mutation sites that can accommodate structural stabilization. In this study, homology modeling and molecular dynamics simulations were used to select appropriate glycine to proline mutations to improve protein thermostability, and the effect of the selected mutations was proved by the experiments. The structure of methyl parathion hydrolase (MPH) from Ochrobactrum sp. M231 (Ochr-MPH) was constructed by homology modeling, and molecular dynamics simulations were performed on the modeled structure. A profile of the root mean square fluctuations of Ochr-MPH was calculated at the nanosecond timescale, and an eight-amino acid loop region (residues 186-193) was identified as having high conformational fluctuation. The two glycines nearest to this region were selected as mutation targets that might affect protein flexibility in the vicinity. The structures and conformational fluctuations of two single mutants (G194P and G198P) and one double mutant (G194P/G198P) were modeled and analyzed using molecular dynamics simulations. The results predicted that the mutant G194P had the decreased conformational fluctuation in the loop region and might increase the thermostability of Ochr-MPH. The thermostability and kinetic behavior of the wild-type and three mutant enzymes were measured. The results were consistent with the computational predictions, and the mutant G194P was found to have higher thermostability than the wild-type enzyme. © 2010 The Authors Journal compilation © 2010 FEBS.

Genome-Wide Analyses and Functional Classification of Proline Repeat-Rich Proteins: Potential Role of eIF5A in Eukaryotic Evolution

PubMed Central

Mandal, Ajeet; Mandal, Swati; Park, Myung Hee

2014-01-01

The eukaryotic translation factor, eIF5A has been recently reported as a sequence-specific elongation factor that facilitates peptide bond formation at consecutive prolines in Saccharomyces cerevisiae, as its ortholog elongation factor P (EF-P) does in bacteria. We have searched the genome databases of 35 representative organisms from six kingdoms of life for PPP (Pro-Pro-Pro) and/or PPG (Pro-Pro-Gly)-encoding genes whose expression is expected to depend on eIF5A. We have made detailed analyses of proteome data of 5 selected species, Escherichia coli, Saccharomyces cerevisiae, Drosophila melanogaster, Mus musculus and Homo sapiens. The PPP and PPG motifs are low in the prokaryotic proteomes. However, their frequencies markedly increase with the biological complexity of eukaryotic organisms, and are higher in newly derived proteins than in those orthologous proteins commonly shared in all species. Ontology classifications of S. cerevisiae and human genes encoding the highest level of polyprolines reveal their strong association with several specific biological processes, including actin/cytoskeletal associated functions, RNA splicing/turnover, DNA binding/transcription and cell signaling. Previously reported phenotypic defects in actin polarity and mRNA decay of eIF5A mutant strains are consistent with the proposed role for eIF5A in the translation of the polyproline-containing proteins. Of all the amino acid tandem repeats (≥3 amino acids), only the proline repeat frequency correlates with functional complexity of the five organisms examined. Taken together, these findings suggest the importance of proline repeat-rich proteins and a potential role for eIF5A and its hypusine modification pathway in the course of eukaryotic evolution. PMID:25364902

Sorption isotherms, kinetic and optimization process of amino acid proline based polymer nanocomposite for the removal of selected textile dyes from industrial wastewater.

PubMed

Raghunath, Sharista; Anand, K; Gengan, R M; Nayunigari, Mithil Kumar; Maity, Arjun

2016-12-01

In this article, adsorption and kinetic studies were carried out on three textile dyes, namely Reactive Blue 222 (RB 222), Reactive Red 195 (RR 195) and Reactive Yellow 145 (RY 145). The dyes studied in a mixture were adsorbed under various conditions onto PRO-BEN, a bentonite modified with a new cationic proline polymer (l-proline-epichlorohydrin polymer). The proline polymer was characterized by 1 H NMR, Fourier transform infrared spectroscopy (FT-IR), dynamic light scattering (DLS) and TEM. The PRO-BEN composite was characterized by FT-IR, dynamic light scattering (DLS) (zeta potential), TEM imaging, SEM/EDX and X-ray photoelectron spectroscopy (characterize the binding energy). During adsorption studies, factors involving pH, temperature, the initial concentrations of the dyes and the quantity of PRO-BEN used during adsorption were established. The results revealed that the adsorption mechanism was categorized by the Langmuir type 1 isotherm. The adsorption data followed the pseudo-second order kinetic model. The intraparticle diffusion model indicated that adsorption did not only depend on the intraparticle diffusion of the dyes. The thermodynamic parameters verified that the adsorption process was spontaneous and exothermic. The Gibbs free energy values indicated that physisorption had occurred. Successful adsorption of dyes from an industrial effluent was achieved. Desorption studies concluded that PRO-BEN desorbed the dyes better than alumina. This can thereby be viewed as a recyclable remediation material. The PRO-BEN composite could be a cost efficient alternative towards the removal of organic dyes in wastewater treatment. Copyright © 2016 Elsevier B.V. All rights reserved.

Moderate Increase of Mean Daily Temperature Adversely Affects Fruit Set of Lycopersicon esculentum by Disrupting Specific Physiological Processes in Male Reproductive Development

PubMed Central

SATO, S.; KAMIYAMA, M.; IWATA, T.; MAKITA, N.; FURUKAWA, H.; IKEDA, H.

2006-01-01

• Background and Aims Global warming is gaining significance as a threat to natural and managed ecosystems since temperature is one of the major environmental factors affecting plant productivity. Hence, the effects of moderate temperature increase on the growth and development of the tomato plant (Lycopersicon esculentum) were investigated. • Methods Plants were grown at 32/26 °C as a moderately elevated temperature stress (METS) treatment or at 28/22 °C (day/night temperatures) as a control with natural light conditions. Vegetative growth and reproductive development as well as sugar content and metabolism, proline content and translocation in the androecium were investigated. • Key Results METS did not cause a significant change in biomass, the number of flowers, or the number of pollen grains produced, but there was a significant decrease in the number of fruit set, pollen viability and the number of pollen grains released. Glucose and fructose contents in the androecium (i.e. all stamens from one flower) were generally higher in the control than METS, but sucrose was higher in METS. Coincidently, the mRNA transcript abundance of acid invertase in the androecium was decreased by METS. Proline contents in the androecium were almost the same in the control and METS, while the mRNA transcript level of proline transporter 1, which expresses specifically at the surface of microspores, was significantly decreased by METS. • Conclusions The research indicated that failure of tomato fruit set under a moderately increased temperature above optimal is due to the disruption of sugar metabolism and proline translocation during the narrow window of male reproductive development. PMID:16497700

Generation of Hypertension-Associated STK39 Polymorphism Knockin Cell Lines With the Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 System.

PubMed

Mandai, Shintaro; Mori, Takayasu; Sohara, Eisei; Rai, Tatemitsu; Uchida, Shinichi

2015-12-01

Previous genome-wide association studies identified serine threonine kinase 39 (STK39), encoding STE20/SPS1-related proline/alanine-rich kinase, as one of a limited number of hypertension susceptibility genes. A recent meta-analysis confirmed the association of STK39 intronic polymorphism rs3754777 with essential hypertension, among previously reported hypertension-associated STK39 polymorphisms. However, the biochemical function of this polymorphism in the mechanism responsible for hypertension is yet to be clarified. We generated rs3754777G>A knockin human cell lines with clustered regularly interspaced short palindromic repeats-mediated genome engineering. Homozygous (A/A) and heterozygous (G/A) knockin human embryonic kidney cell lines were generated using a double nickase, single-guide RNAs targeting STK39 intron 5 around single-nucleotide polymorphism, and a 100-bp donor single-stranded DNA oligonucleotide. Reverse transcription polymerase chain reaction with sequencing analyses revealed the identical STK39 transcripts among the wild-type and both knockin cell lines. Quantitative reverse transcription polymerase chain reaction showed increased STK39 mRNA expression, and immunoblot analysis revealed increases in total and phosphorylated STE20/SPS1-related proline/alanine-rich kinase with increased phosphorylated Na-K-Cl cotransporter isoform 1 in both knockin cell lines. The largest increases in these molecules were observed in the homozygous cell line. These findings indicated that this intronic polymorphism increases STK39 transcription, leading to activation of the STE20/SPS1-related proline/alanine-rich kinase-solute carrier family 12A signaling cascade. Increased interactions between STE20/SPS1-related proline/alanine-rich kinase and the target cation-chloride cotransporters may be responsible for hypertension susceptibility in individuals with this polymorphism. © 2015 American Heart Association, Inc.

Salt-induced modulation in inorganic nutrients, antioxidant enzymes, proline content and seed oil composition in safflower (Carthamus tinctorius L.).

PubMed

Siddiqi, Ejaz Hussain; Ashraf, Muhammad; Al-Qurainy, Fahad; Akram, Nudrat Aisha

2011-12-01

Safflower (Carthamus tinctorius L.) has gained considerable ground as a potential oil-seed crop. However, its yield and oil production are adversely affected under saline conditions. The present study was conducted to appraise the influence of salt (NaCl) stress on yield, accumulation of different inorganic elements, free proline and activities of some key antioxidant enzymes in plant tissues as well as seed oil components in safflower. Two safflower accessions differing in salt tolerance (Safflower-33 (salt sensitive) and Safflower-39 (salt tolerant)) were grown under saline (150 mmol L(-1) ) conditions and salt-induced changes in the earlier-mentioned physiological attributes were determined. Salt stress enhanced leaf and root Na(+) , Cl(-) and proline accumulation and activities of leaf superoxide dismutase, catalase and peroxidase, while it decreased K(+) , Ca(2+) and K(+) /Ca(2+) and Ca(2+) /Na(+) ratios and seed yield, 100-seed weight, number of seeds, as well as capitula, seed oil contents and oil palmitic acid. No significant effect of salt stress was observed on seed oil α-tocopherols, stearic acid, oleic acid or linoleic acid contents. Of the two safflower lines, salt-sensitive Safflower-33 was higher in leaf and root Na(+) and Cl(-) , while Safflower-39 was higher in leaf and root K(+) , K(+) /Ca(2+) and Ca(2+) /Na(+) and seed yield, 100-seed weight, catalase activity, seed oil contents, seed oil α-tocopherol and palmitic acid. Other attributes remained almost unaffected in both accessions. Overall, high salt tolerance of Safflower-39 could be attributed to Na(+) and Cl(-) exclusion, high accumulation of K(+) and free proline, enhanced CAT activity, seed oil α-tocopherols and palmitic acid contents. Copyright © 2011 Society of Chemical Industry.

Multivalent binding of formin-binding protein 21 (FBP21)-tandem-WW domains fosters protein recognition in the pre-spliceosome.

PubMed

Klippel, Stefan; Wieczorek, Marek; Schümann, Michael; Krause, Eberhard; Marg, Berenice; Seidel, Thorsten; Meyer, Tim; Knapp, Ernst-Walter; Freund, Christian

2011-11-04

The high abundance of repetitive but nonidentical proline-rich sequences in spliceosomal proteins raises the question of how these known interaction motifs recruit their interacting protein domains. Whereas complex formation of these adaptors with individual motifs has been studied in great detail, little is known about the binding mode of domains arranged in tandem repeats and long proline-rich sequences including multiple motifs. Here we studied the interaction of the two adjacent WW domains of spliceosomal protein FBP21 with several ligands of different lengths and composition to elucidate the hallmarks of multivalent binding for this class of recognition domains. First, we show that many of the proteins that define the cellular proteome interacting with FBP21-WW1-WW2 contain multiple proline-rich motifs. Among these is the newly identified binding partner SF3B4. Fluorescence resonance energy transfer (FRET) analysis reveals the tandem-WW domains of FBP21 to interact with splicing factor 3B4 (SF3B4) in nuclear speckles where splicing takes place. Isothermal titration calorimetry and NMR shows that the tandem arrangement of WW domains and the multivalency of the proline-rich ligands both contribute to affinity enhancement. However, ligand exchange remains fast compared with the NMR time scale. Surprisingly, a N-terminal spin label attached to a bivalent ligand induces NMR line broadening of signals corresponding to both WW domains of the FBP21-WW1-WW2 protein. This suggests that distinct orientations of the ligand contribute to a delocalized and semispecific binding mode that should facilitate search processes within the spliceosome.

Multivalent Binding of Formin-binding Protein 21 (FBP21)-Tandem-WW Domains Fosters Protein Recognition in the Pre-spliceosome*

PubMed Central

Klippel, Stefan; Wieczorek, Marek; Schümann, Michael; Krause, Eberhard; Marg, Berenice; Seidel, Thorsten; Meyer, Tim; Knapp, Ernst-Walter; Freund, Christian

2011-01-01

The high abundance of repetitive but nonidentical proline-rich sequences in spliceosomal proteins raises the question of how these known interaction motifs recruit their interacting protein domains. Whereas complex formation of these adaptors with individual motifs has been studied in great detail, little is known about the binding mode of domains arranged in tandem repeats and long proline-rich sequences including multiple motifs. Here we studied the interaction of the two adjacent WW domains of spliceosomal protein FBP21 with several ligands of different lengths and composition to elucidate the hallmarks of multivalent binding for this class of recognition domains. First, we show that many of the proteins that define the cellular proteome interacting with FBP21-WW1-WW2 contain multiple proline-rich motifs. Among these is the newly identified binding partner SF3B4. Fluorescence resonance energy transfer (FRET) analysis reveals the tandem-WW domains of FBP21 to interact with splicing factor 3B4 (SF3B4) in nuclear speckles where splicing takes place. Isothermal titration calorimetry and NMR shows that the tandem arrangement of WW domains and the multivalency of the proline-rich ligands both contribute to affinity enhancement. However, ligand exchange remains fast compared with the NMR time scale. Surprisingly, a N-terminal spin label attached to a bivalent ligand induces NMR line broadening of signals corresponding to both WW domains of the FBP21-WW1-WW2 protein. This suggests that distinct orientations of the ligand contribute to a delocalized and semispecific binding mode that should facilitate search processes within the spliceosome. PMID:21917930

Functional properties and structural characterization of rice δ 1-pyrroline-5-carboxylate reductase

DOE PAGES

Forlani, Giuseppe; Bertazzini, Michele; Zarattini, Marco; ...

2015-07-28

The majority of plant species accumulate high intracellular levels of proline to cope with hyperosmotic stress conditions. Proline synthesis from glutamate is tightly regulated at both the transcriptional and the translational levels, yet little is known about the mechanisms for post-translational regulation of the enzymatic activities involved. The gene coding in rice ( Oryza sativa L.) for δ 1-pyrroline-5-carboxylate (P5C) reductase, the enzyme that catalyzes the second and final step in this pathway, was isolated and expressed in Escherichia coli. The structural and functional properties of the affinity-purified protein were characterized. As for most species, rice P5C reductase was ablemore » to use in vitro either NADH or NADPH as the electron donor. However, strikingly different effects of cations and anions were found depending on the pyridine nucleotide used, namely inhibition of NADH-dependent activity and stimulation of NADPH-dependent activity. Moreover, physiological concentrations of proline and NADP + were strongly inhibitory for the NADH-dependent reaction, whereas the NADPH-dependent activity was mildly affected. Our results suggest that only NADPH may be used in vivo and that stress-dependent variations in ion homeostasis and NADPH/NADP + ratio could modulate enzyme activity, being functional in promoting proline accumulation and potentially also adjusting NADPH consumption during the defense against hyperosmotic stress. The apparent molecular weight of the native protein observed in size exclusion chromatography indicated a high oligomerization state. We also report the first crystal structure of a plant P5C reductase at 3.40-Å resolution, showing a decameric quaternary assembly. It was possible to identify dynamic structural differences among rice, human, and bacterial enzymes.« less

Conserved Allosteric Hot Spots in the Transmembrane Domains of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Channels and Multidrug Resistance Protein (MRP) Pumps*

PubMed Central

Wei, Shipeng; Roessler, Bryan C.; Chauvet, Sylvain; Guo, Jingyu; Hartman, John L.; Kirk, Kevin L.

2014-01-01

ATP-binding cassette (ABC) transporters are an ancient family of transmembrane proteins that utilize ATPase activity to move substrates across cell membranes. The ABCC subfamily of the ABC transporters includes active drug exporters (the multidrug resistance proteins (MRPs)) and a unique ATP-gated ion channel (cystic fibrosis transmembrane conductance regulator (CFTR)). The CFTR channel shares gating principles with conventional ligand-gated ion channels, but the allosteric network that couples ATP binding at its nucleotide binding domains (NBDs) with conformational changes in its transmembrane helices (TMs) is poorly defined. It is also unclear whether the mechanisms that govern CFTR gating are conserved with the thermodynamically distinct MRPs. Here we report a new class of gain of function (GOF) mutation of a conserved proline at the base of the pore-lining TM6. Multiple substitutions of this proline promoted ATP-free CFTR activity and activation by the weak agonist, 5′-adenylyl-β,γ-imidodiphosphate (AMP-PNP). TM6 proline mutations exhibited additive GOF effects when combined with a previously reported GOF mutation located in an outer collar of TMs that surrounds the pore-lining TMs. Each TM substitution allosterically rescued the ATP sensitivity of CFTR gating when introduced into an NBD mutant with defective ATP binding. Both classes of GOF mutations also rescued defective drug export by a yeast MRP (Yor1p) with ATP binding defects in its NBDs. We conclude that the conserved TM6 proline helps set the energy barrier to both CFTR channel opening and MRP-mediated drug efflux and that CFTR channels and MRP pumps utilize similar allosteric mechanisms for coupling conformational changes in their translocation pathways to ATP binding at their NBDs. PMID:24876383

Structure of aqueous proline via parallel tempering molecular dynamics and neutron diffraction.

PubMed

Troitzsch, R Z; Martyna, G J; McLain, S E; Soper, A K; Crain, J

2007-07-19

The structure of aqueous L-proline amino acid has been the subject of much debate centering on the validity of various proposed models, differing widely in the extent to which local and long-range correlations are present. Here, aqueous proline is investigated by atomistic, replica exchange molecular dynamics simulations, and the results are compared to neutron diffraction and small angle neutron scattering (SANS) data, which have been reported recently (McLain, S.; Soper, A.; Terry, A.; Watts, A. J. Phys. Chem. B 2007, 111, 4568). Comparisons between neutron experiments and simulation are made via the static structure factor S(Q) which is measured and computed from several systems with different H/D isotopic compositions at a concentration of 1:20 molar ratio. Several different empirical water models (TIP3P, TIP4P, and SPC/E) in conjunction with the CHARMM22 force field are investigated. Agreement between experiment and simulation is reasonably good across the entire Q range although there are significant model-dependent variations in some cases. In general, agreement is improved slightly upon application of approximate quantum corrections obtained from gas-phase path integral simulations. Dimers and short oligomeric chains formed by hydrogen bonds (frequently bifurcated) coexist with apolar (hydrophobic) contacts. These emerge as the dominant local motifs in the mixture. Evidence for long-range association is more equivocal: No long-range structures form spontaneously in the MD simulations, and no obvious low-Q signature is seen in the SANS data. Moreover, associations introduced artificially to replicate a long-standing proposed mesoscale structure for proline correlations as an initial condition are annealed out by parallel tempering MD simulations. However, some small residual aggregates do remain, implying a greater degree of long-range order than is apparent in the SANS data.

Biochemical characterization, homology modeling and docking studies of ornithine delta-aminotransferase--an important enzyme in proline biosynthesis of plants.

PubMed

Sekhar, P Nataraj; Amrutha, R Naga; Sangam, Shubhada; Verma, D P S; Kishor, P B Kavi

2007-11-01

Ornithine delta-aminotransferase (OAT) is an important enzyme in proline biosynthetic pathway and is implicated in salt tolerance in higher plants. OAT transaminates ornithine to pyrroline 5-carboxylate, which is further catalyzed to proline by pyrroline 5-carboxylate reductase. The Vigna aconitifolia OAT cDNA, encoding a polypeptide of 48.1 kDa, was expressed in Escherichia coli and the enzyme was partially characterized following its purification using (NH(4))(2)SO(4) precipitation and gel filtration techniques. Optimal activity of the enzyme was observed at a temperature of 25 degrees C and pH 8.0. The enzyme appeared to be a monomer and exhibited high activity at 4mM ornithine. Proline did not show any apparent effect but isoleucine, valine and serine inhibited the activity when added into the assay mixture along with ornithine. Omission of pyridoxal 5'-phosphate from the reaction mixture reduced the activity of this enzyme by 60%. To further evaluate these biochemical observations, homology modeling of the OAT was performed based on the crystal structure of the ornithine delta-aminotransferase from humans (PDB code 1OAT) by using the software MODELLER6v2. With the aid of the molecular mechanics and dynamics methods, the final model was obtained and assessed subsequently by PROCHECK and VERIFY-3D graph. With this model, a flexible docking study with the substrate and inhibitors was performed and the results indicated that Gly106 and Lys256 in OAT are the important determinant residues in binding as they have strong hydrogen bonding contacts with the substrate and inhibitors. These observations are in conformity with the results obtained from experimental investigations.

Conserved allosteric hot spots in the transmembrane domains of cystic fibrosis transmembrane conductance regulator (CFTR) channels and multidrug resistance protein (MRP) pumps.

PubMed

Wei, Shipeng; Roessler, Bryan C; Chauvet, Sylvain; Guo, Jingyu; Hartman, John L; Kirk, Kevin L

2014-07-18

ATP-binding cassette (ABC) transporters are an ancient family of transmembrane proteins that utilize ATPase activity to move substrates across cell membranes. The ABCC subfamily of the ABC transporters includes active drug exporters (the multidrug resistance proteins (MRPs)) and a unique ATP-gated ion channel (cystic fibrosis transmembrane conductance regulator (CFTR)). The CFTR channel shares gating principles with conventional ligand-gated ion channels, but the allosteric network that couples ATP binding at its nucleotide binding domains (NBDs) with conformational changes in its transmembrane helices (TMs) is poorly defined. It is also unclear whether the mechanisms that govern CFTR gating are conserved with the thermodynamically distinct MRPs. Here we report a new class of gain of function (GOF) mutation of a conserved proline at the base of the pore-lining TM6. Multiple substitutions of this proline promoted ATP-free CFTR activity and activation by the weak agonist, 5'-adenylyl-β,γ-imidodiphosphate (AMP-PNP). TM6 proline mutations exhibited additive GOF effects when combined with a previously reported GOF mutation located in an outer collar of TMs that surrounds the pore-lining TMs. Each TM substitution allosterically rescued the ATP sensitivity of CFTR gating when introduced into an NBD mutant with defective ATP binding. Both classes of GOF mutations also rescued defective drug export by a yeast MRP (Yor1p) with ATP binding defects in its NBDs. We conclude that the conserved TM6 proline helps set the energy barrier to both CFTR channel opening and MRP-mediated drug efflux and that CFTR channels and MRP pumps utilize similar allosteric mechanisms for coupling conformational changes in their translocation pathways to ATP binding at their NBDs. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Proline-coated column for the capillary electrochromatographic separation of amino acids by in-column derivatization.

PubMed

Lin, Chun-Chi; Liu, Chuen-Ying

2004-10-01

With 3-trimethoxysilylpropyl chloride as the spacer, a proline-coated capillary column was prepared for the capillary electrochromatographic (CEC) separation of amino acids by in-column derivatization. Nine standard mixtures, including aspartic acid, glutamic acid, valine, phenylalanine, alanine, isoleucine, leucine, tyrosine, and tryptophan, were injected. o-Phthalaldehyde (OPA), OPA/2-mercaptoethanol (2-ME) and OPA/N-acetylcysteine (NAC) in borate buffer were tested as the derivatizing agent. Among them, OPA (50 mM) in borate buffer (pH 9.5, 50 mM) gave the best performance. The formation of isoindole could be detected by UV detection. The sandwich-type injection was carried out in hydrostatic mode (10 cm) with the program R(10 s)S(10 s) R(10 s)W(10 min) with R, S, and W being the reagent, sample, and waiting times. Mesityl oxide, benzyl alcohol, and acetone showed some interaction with the column. A current monitoring method was used instead of the determination of the electroosmotic flow (EOF). The direction of EOF was from anode to cathode even under acidic condition lower than the pI value (6.31) of the bonded group due to some unreacted silanol groups. Some parameters including pH, nature, and concentration of the mobile phase and the effect of organic modifier with regard to the CEC separation were investigated. With the proline-coated column (75 (50) cm x 75 microm ID) the best separation was performed in phosphate buffer (pH 4.00, 100 mM) with an applied voltage of -15 kV. The established method was also compared with those precolumn derivatized prior to the separation with proline-coated column as well as with in-capillary derivatization and separation with a bare fused-silica column. Copyright 2004 WILEY-VCH Verlag GmbH & Co.

Amino acid derivatives of 5-ASA as novel prodrugs for intestinal drug delivery.

PubMed

Clerici, C; Gentili, G; Boschetti, E; Santucci, C; Aburbeh, A G; Natalini, B; Pellicciari, R; Morelli, A

1994-12-01

In an attempt to obtain site-specific delivery of 5-ASA in the intestinal tract, we have determined the extent of absorption and metabolism of a number of novel 5-ASA derivatives, namely, (N-L-glutamyl)-amino-2-salicylic acid (1), (N-L-aspartyl)-amino-2-salicylic-acid (2), 5-aminosalicyl-L-proline-L-leucine (3), and 5-(N-L-glutamyl)-aminosalicyl-L-proline-L-leucine (4), which are selectively cleaved by intestinal brush border aminopeptidase A and carboxypeptidases. These novel prodrugs, 5-ASA, and sulfasalazine were administered to adult Fisher rats (N = 30) and to animals that had undergone prior colostomy (N = 30). Urine and feces were collected at timed intervals for 48 hr and the metabolites, 5-ASA, and N-acetyl-5-ASA were measured by high-performance liquid chromatography. The absorption and metabolism of all compounds were essentially identical in colostomized and normal animals. 5-ASA exhibited a rapid proximal intestinal absorption as evidenced by the high cumulative urinary excretion (> 65%) and low fecal excretion. Sulfasalazine, as expected, exhibited a lower urinary recovery (< 35%) and higher fecal excretion of 5-ASA and its metabolite. The novel glutamate and aspartate derivatives (1 and 2) behaved similarly to sulfasalazine, while administration of the proline-leucine derivative (3) resulted in urinary and fecal recovery values intermediate with respect to those observed with 5-ASA and sulfasalazine. 5-(N-L-Glutamyl)-aminosalicyl-L-proline-L-leucine yielded the highest fecal recovery of 5-ASA and its N-acetyl derivative, indicating a more efficient delivery to the distal bowel. Amino acid derivatives of 5-ASA appear to be potentially useful prodrugs for the site-specific delivery of 5-ASA to different regions of the intestinal tract.

Synthesis, kinetic evaluation, and utilization of a biotinylated dipeptide proline diphenyl phosphonate for the disclosure of dipeptidyl peptidase IV-like serine proteases.

PubMed

Gilmore, Brendan F; Carson, Louise; McShane, Laura L; Quinn, Derek; Coulter, Wilson A; Walker, Brian

2006-08-18

In this study, we report on the synthesis, kinetic characterisation, and application of a novel biotinylated and active site-directed inactivator of dipeptidyl peptidase IV (DPP-IV). Thus, the dipeptide-derived proline diphenyl phosphonate NH(2)-Glu(biotinyl-PEG)-Pro(P)(OPh)(2) has been prepared by a combination of classical solution- and solid-phase methodologies and has been shown to be an irreversible inhibitor of porcine DPP-IV, exhibiting an over all second-order rate constant (k(i)/K(i)) for inhibition of 1.57 x 10(3) M(-1) min(-1). This value compares favourably with previously reported rates of inactivation of DPP-IV by dipeptides containing a P(1) proline diphenyl phosphonate grouping [B. Boduszek, J. Oleksyszyn, C.M. Kam, J. Selzler, R.E. Smith, J.C. Powers, Dipeptide phophonates as inhibitors of dipeptidyl peptidase IV, J. Med. Chem. 37 (1994) 3969-3976; B.F. Gilmore, J.F. Lynas, C.J. Scott, C. McGoohan, L. Martin, B. Walker, Dipeptide proline diphenyl phosphonates are potent, irreversible inhibitors of seprase (FAPalpha), Biochem, Biophys. Res. Commun. 346 (2006) 436-446.], thus demonstrating that the incorporation of the side-chain modified (N-biotinyl-3-(2-(2-(3-aminopropyloxy)-ethoxy)-ethoxy)-propyl) glutamic acid residue at the P(2) position is compatible with inhibitor efficacy. The utilisation of this probe for the detection of both purified dipeptidyl peptidase IV and the disclosure of a dipeptidyl peptidase IV-like activity from a clinical isolate of Porphyromonas gingivalis, using established electrophoretic and Western blotting techniques previously developed by our group, is also demonstrated.

Biochemical response of hybrid black poplar tissue culture (Populus × canadensis) on water stress.

PubMed

Popović, B M; Štajner, D; Ždero-Pavlović, R; Tari, I; Csiszár, J; Gallé, Á; Poór, P; Galović, V; Trudić, B; Orlović, S

2017-05-01

In this study, poplar tissue culture (hybrid black poplar, M1 genotype) was subjected to water stress influenced by polyethyleneglycol 6000 (100 and 200 mOsm PEG 6000). The aim of the research was to investigate the biochemical response of poplar tissue culture on water deficit regime. Antioxidant status was analyzed including antioxidant enzymes, superoxide-dismutase (SOD), catalase (CAT), guiacol-peroxidase (GPx), glutathione-peroxidase (GSH-Px), glutathione-reductase, reduced glutathione, total phenol content, Ferric reducing antioxidant power and DPPH radical antioxidant power. Polyphenol oxidase and phenylalanine-ammonium-lyase were determined as enzymatic markers of polyphenol metabolism. Among oxidative stress parameters lipid peroxidation, carbonyl-proteins, hydrogen-peroxide, reactive oxygen species, nitric-oxide and peroxynitrite were determined. Proline, proline-dehydrogenase and glycinebetaine were measured also as parameters of water stress. Cell viability is finally determined as a biological indicator of osmotic stress. It was found that water stress induced reactive oxygen and nitrogen species and lipid peroxidation in leaves of hybrid black poplar and reduced cell viability. Antioxidant enzymes including SOD, GPx, CAT and GSH-Px were induced but total phenol content and antioxidant capacity were reduced by PEG 6000 mediated osmotic stress. The highest biochemical response and adaptive reaction was the increase of proline and GB especially by 200 mOsm PEG. While long term molecular analysis will be necessary to fully address the poplar potentials for water stress adaptation, our results on hybrid black poplar suggest that glycine-betaine, proline and PDH enzyme might be the most important markers of poplar on water stress and that future efforts should be focused on these markers and strategies to enhance their concentration in poplar.

Peptidases in dog-ileum circular and longitudinal smooth-muscle plasma membranes. Their relative contribution to the metabolism of neurotensin.

PubMed

Checler, F; Ahmad, S; Kostka, P; Barelli, H; Kitabgi, P; Fox, J A; Kwan, C Y; Daniel, E E; Vincent, J P

1987-07-15

We established the content in neuropeptide-metabolizing peptidases present in highly purified plasma membranes prepared from the circular and longitudinal muscles of dog ileum. Activities were measured by the use of fluorigenic substrates and the identities of enzymes were confirmed by the use of specific peptidase inhibitors. Endopeptidase 24.11, angiotensin-converting enzyme, post-proline dipeptidyl aminopeptidase and aminopeptidases were found in both membrane preparations. Proline endopeptidase was only detected in circular smooth muscle plasma membranes while pyroglutamyl-peptide hydrolase was not observed in either tissue. The relative contribution of these peptidases to the inactivation of neurotensin was assessed. The enzymes involved in the primary inactivating cleavages occurring on the neurotensin molecule were as follows. In both membrane preparations, endopeptidase 24.11 was responsible for the formation of neurotensin-(1-11) and contributed to the formation of neurotensin-(1-10); a recently purified neurotensin-degrading neutral metallopeptidase was also involved in the formation of neurotensin-(1-10). A carboxypeptidase-like activity hydrolysed neurotensin at the Ile12-Leu13 peptide bond, leading to the formation of neurotensin-(1-12). Proline endopeptidase and endopeptidase 24.15 only occurred in circular muscle plasma membranes, yielding neurotensin-(1-7) and neurotensin-(1-8), respectively. In addition, the secondary processing of neurotensin degradation products was catalyzed by the following peptidases. In circular and longitudinal muscle membranes, angiotensin-converting enzyme converted neurotensin-(1-10) into neurotensin-(1-8) and tyrosine resulted from the rapid hydrolysis of neurotensin-(11-13) by bestatin-sensitive aminopeptidases. A post-proline dipeptidyl aminopeptidase activity converted neurotensin-(9-13) into neurotensin-(11-13) in circular muscle plasma membranes. The mechanism of neurotensin inactivation occurring in these membranes will be compared to that previously established for membranes from central origin.

Minoxidil specifically decreases the expression of lysine hydroxylase in cultured human skin fibroblasts.

PubMed Central

Hautala, T; Heikkinen, J; Kivirikko, K I; Myllylä, R

1992-01-01

The levels of lysine hydroxylase protein and the levels of the mRNAs for lysine hydroxylase and the alpha- and beta-subunits of proline 4-hydroxylase were measured in cultured human skin fibroblasts treated with 1 mM-minoxidil. The data demonstrate that minoxidil decreases the amount of lysine hydroxylase protein, this being due to a decrease in the level of lysine hydroxylase mRNA. The effect of minoxidil appears to be highly specific, as no changes were observed in the amounts of mRNAs for the alpha- and beta-subunits of proline 4-hydroxylase. Images Fig. 1. Fig. 2. Fig. 3. PMID:1314568

Asymmetric Michael Addition Mediated by Chiral Ionic Liquids.

PubMed

Suzuki, Yumiko

2018-06-01

Chiral ionic liquids with a focus on their applications in asymmetric Michael additions and related reactions were reviewed. The examples were classified on the basis of the mode of asymmetric induction (e.g., external induction/non-covalent interaction or internal induction/covalent bond formation), the roles in reactions (as a solvent or catalyst), and their structural features (e.g., imidazolium-based chiral cations, other chiral oniums; proline derivatives). Most of the reactions with high chiral induction are Michael addition of ketones or aldehydes to chalcones or nitrostyrenes where proline-derived chiral ionic liquids catalyze the reaction through enamine/ iminium formation. Many reports demonstrate the recyclability of ionic liquid-tagged pyrrolidines.

Regulation of tumor cell migration by protein tyrosine phosphatase (PTP)-proline-, glutamate-, serine-, and threonine-rich sequence (PEST)

PubMed Central

Zheng, Yanhua; Lu, Zhimin

2013-01-01

Protein tyrosine phosphatase (PTP)–proline-, glutamate-, serine-, and threonine-rich sequence (PEST) is ubiquitously expressed and is a critical regulator of cell adhesion and migration. PTP-PEST activity can be regulated transcriptionally via gene deletion or mutation in several types of human cancers or via post-translational modifications, including phosphorylation, oxidation, and caspase-dependent cleavage. PTP-PEST interacts with and dephosphorylates cytoskeletal and focal adhesion-associated proteins. Dephosphorylation of PTP-PEST substrates regulates their enzymatic activities and/or their interaction with other proteins and plays an essential role in the tumor cell migration process. PMID:23237212

Diastereoselective synthesis of furanose and pyranose substituted glycine and alanine derivatives via proline-catalyzed asymmetric α-amination of aldehydes.

PubMed

Petakamsetty, Ramu; Ansari, Anas; Ramapanicker, Ramesh

2016-11-29

A concise organocatalytic route toward the synthesis of furanose and pyranose substituted glycine and alanine derivatives is reported. These compounds are core structural units of some of the naturally available antibiotics and antifungal agents. Proline-catalyzed asymmetric α-amination of aldehydes derived from sugars is used as the key reaction to synthesize twelve sugar amino acid derivatives. The asymmetric transformations proceeded in good yields and with good to excellent diastereoselectivity. The application of the synthesized amino acids is demonstrated by synthesizing a tripeptide containing one of them. Copyright © 2016 Elsevier Ltd. All rights reserved.

Double mimicry evades tRNA synthetase editing by toxic vegetable-sourced non-proteinogenic amino acid.

PubMed

Song, Youngzee; Zhou, Huihao; Vo, My-Nuong; Shi, Yi; Nawaz, Mir Hussain; Vargas-Rodriguez, Oscar; Diedrich, Jolene K; Yates, John R; Kishi, Shuji; Musier-Forsyth, Karin; Schimmel, Paul

2017-12-22

Hundreds of non-proteinogenic (np) amino acids (AA) are found in plants and can in principle enter human protein synthesis through foods. While aminoacyl-tRNA synthetase (AARS) editing potentially provides a mechanism to reject np AAs, some have pathological associations. Co-crystal structures show that vegetable-sourced azetidine-2-carboxylic acid (Aze), a dual mimic of proline and alanine, is activated by both human prolyl- and alanyl-tRNA synthetases. However, it inserts into proteins as proline, with toxic consequences in vivo. Thus, dual mimicry increases odds for mistranslation through evasion of one but not both tRNA synthetase editing systems.

Physiological and Biochemical Responses in Two Ornamental Shrubs to Drought Stress.

PubMed

Toscano, Stefania; Farieri, Elisa; Ferrante, Antonio; Romano, Daniela

2016-01-01

Drought stress is one of the most important abiotic stress limiting the plant survival and growth in the Mediterranean environment. In this work, two species typically grown in Mediterranean areas with different drought responses were used. Two shrubs, with slow (Photinia × fraseri Dress 'Red Robin') or fast (Eugenia uniflora L. 'Etna Fire') adaptation ability to drought, were subjected to three water regimes: well-watered (WW), moderate (MD), and severe (SD) drought stress conditions for 30 days. Net photosynthetic rate, stomatal conductance, maximum quantum efficiency of PSII photochemistry (Fv/Fm), relative water content (RWC), chlorophyll content, proline, malondialdehyde (MDA), and antioxidant enzyme activities (superoxide dismutase, catalase, and peroxidase) were measured. Results showed that RWC and proline were higher in Eugenia than in Photinia, demonstrating the greater tolerance of the latter to the water stress. The drought stress levels applied did not compromise photosynthetic efficiency through stomatal regulation, while a reduction of Fv/Fm ratio was observed at the end of the experimental period. MDA significantly increased after 30 days in both species. The antioxidant enzyme activities showed different responses to water stress conditions. In both species, the water stress scores showed positive, while proline content showed negative correlations with all physiological parameters.

First evidences of interaction between pyranoanthocyanins and salivary proline-rich proteins.

PubMed

García-Estévez, Ignacio; Cruz, Luís; Oliveira, Joana; Mateus, Nuno; de Freitas, Victor; Soares, Susana

2017-08-01

The contribution of other classes of polyphenol compounds besides tannins to the overall perception of astringency is still poorly understood. So, this work aimed to study the interaction between a family of salivary proline-rich proteins (aPRPs) and representative pyranoanthocyanins in red wines [pyranomalvidin-3-glucoside (vitisin B), pyranomalvidin-3-glucoside-catechol, and pyranomalvidin-3-glucoside-epicatechin] using saturation transfer difference-NMR and MALDI-TOF. For vitisin B K D was of 1.74mM; for pyranomalvidin-3-glucoside-catechol was 1.17mM and for pyranomalvidin-3-glucoside-epicatechin it was 0.87mM. The presence of the flavanol structural unit in the pyranoanthocyanins led to an increase in their interaction with aPRPs. Further, it is also interesting that the values obtained were in the range of K D obtained previously reported for the interaction between the human saliva proline-rich peptides (IB7 14 and IB9 37 ) and procyanidins. Overall, the results obtained suggest that, along with tannins, other polyphenols present in red wine, namely pyranoanthocyanins, could actively contribute to red wine global astringency. Copyright © 2017 Elsevier Ltd. All rights reserved.

Evaluation of the influence of proline, hydroxyproline or pyrrolidine in the presence of sodium nitrite on N-nitrosamine formation when heating cured meat.

PubMed

Drabik-Markiewicz, G; Dejaegher, B; De Mey, E; Impens, S; Kowalska, T; Paelinck, H; Vander Heyden, Y

2010-01-11

N-nitrosamines are meant to be probable or possible carcinogenic components, possibly formed out of a reaction between nitrite and N-containing substances such as amino acids and secondary amines. Nitrite is often used for processing meat products because of its colouring and antimicrobial properties. During this experimental setup, the influence of proline, hydroxyproline or pyrrolidine on N-nitrosamine formation in meat samples was evaluated. The N-nitrosamines concentrations were measured with gas chromatography-thermal energy analyzer. Only the concentrations of N-nitrosodimethylamine and N-nitrosopyrrolidine were found above the limit of detection in a number of tested experimental conditions. The concentration of these two N-nitrosamines was modelled as a function of temperature and nitrite concentration for different situations (presence or absence of added natural N-containing meat components). It could be concluded that proline and pyrrolidine promoted the formation of N-nitrosopyrrolidine. It could also be confirmed that the higher the temperature of the meat processing procedure and the higher the sodium nitrite amounts added, the higher were the yields of the respective N-nitrosamines.

Responses of antioxidant enzymes, photosynthetic pigments and carbohydrates in micropropagated Pitcairnia encholirioides L.B. Sm. (Bromeliaceae) under ex vitro water deficit and after rehydration.

PubMed

Resende, C F; Pacheco, V S; Dornellas, F F; Oliveira, A M S; Freitas, J C E; Peixoto, P H P

2018-03-22

In this study, the activities of antioxidant enzymes, photosynthetic pigments, proline and carbohydrate contents in Pitcairnia encholirioides under ex vitro conditions of water deficit were evaluated. Results show that plants under progressive water stress, previously in vitro cultured in media supplemented with 30 g L-1 sucrose and GA3, accumulated more proline and increased peroxidase (POD) activity and the contents of photosynthetic pigments and carbohydrates. For plants previously in vitro cultured with 15 g L-1 sucrose and NAA, no differences were found for proline content and there were reductions in activities of peroxidase (POD), catalase (CAT) and poliphenoloxidase (PPO), and in contents of carbohydrates, with progress of ex vitro water deficit. After rehydration, plants showed physiological recovery, with enzymatic activities and contents of metabolites similar to those found in the controls not submitted to dehydration, regardless of the previous in vitro culture conditions. These results show that micropropagated P. encholirioides has high tolerance to dehydration once in ex vitro conditions, which can ensure the survival of plants from tissue culture when transferred to its natural environment, emphasizing the importance of such biotechnology for the propagation of endangered species.

Unraveling the role of water in the stereoselective step of aqueous proline-catalyzed aldol reactions.

PubMed

Ribas-Arino, Jordi; Carvajal, Maria Angels; Chaumont, Alain; Masia, Marco

2012-12-03

A multiscale computational study was performed with the aim of tracing the source of stereoselectivity and disclosing the role of water in the stereoselective step of propionaldehyde aldol self-condensation catalyzed by proline amide in water, a reaction that serves as a model for aqueous organocatalytic aldol condensations. Solvent mixing and hydration behavior were assessed by classical molecular dynamics simulations, which show that the reaction between propanal and the corresponding enamine takes place in a fully hydrated environment. First-principles molecular dynamics simulations were used to study the free-energy profile of four possible reaction paths, each of which yields a different stereoisomer, and high-level static first-principles calculations were employed to characterize the transition states for microsolvated species. The first solvation shell of the oxygen atom of the electrophilic aldehyde at the transition states contains two water molecules, each of which donates one hydrogen bond to the nascent alkoxide and thereby largely stabilizes its excess electron density. The stereoselectivity originates in an extra hydrogen bond donated by the amido group of proline amide in two reaction paths. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Molecular dynamics simulation of the interactions between EHD1 EH domain and multiple peptides.

PubMed

Yu, Hua; Wang, Mao-jun; Xuan, Nan-xia; Shang, Zhi-cai; Wu, Jun

2015-10-01

To provide essential information for peptide inhibitor design, the interactions of Eps15 homology domain of Eps15 homology domain-containing protein 1 (EHD1 EH domain) with three peptides containing NPF (asparagine-proline-phenylalanine), DPF (aspartic acid-proline-phenylalanine), and GPF (glycine-proline-phenylalanine) motifs were deciphered at the atomic level. The binding affinities and the underlying structure basis were investigated. Molecular dynamics (MD) simulations were performed on EHD1 EH domain/peptide complexes for 60 ns using the GROMACS package. The binding free energies were calculated and decomposed by molecular mechanics/generalized Born surface area (MM/GBSA) method using the AMBER package. The alanine scanning was performed to evaluate the binding hot spot residues using FoldX software. The different binding affinities for the three peptides were affected dominantly by van der Waals interactions. Intermolecular hydrogen bonds provide the structural basis of contributions of van der Waals interactions of the flanking residues to the binding. van der Waals interactions should be the main consideration when we design peptide inhibitors of EHD1 EH domain with high affinities. The ability to form intermolecular hydrogen bonds with protein residues can be used as the factor for choosing the flanking residues.

Structures and unimolecular chemistry of M(Pro2-H)(+) (M = Mg, Ca, Sr, Ba, Mn, Fe, Co, Ni, Cu, Zn) by IRMPD spectroscopy, SORI-CID, and theoretical studies.

PubMed

Jami-Alahmadi, Yasaman; Fridgen, Travis D

2016-01-21

M(Pro2-H)(+) complexes were electrosprayed and isolated in an FTICR cell where their unimolecular chemistries and structures were explored using SORI-CID and IRMPD spectroscopy. These experiments were augmented by computational methods such as electronic structure, simulated annealing, and atoms in molecules (AIM) calculations. The unimolecular chemistries of the larger metal cation (Ca(2+), Sr(2+) and Ba(2+)) complexes predominantly involve loss of neutral proline whereas the complexes involving the smaller Mg(2+) and transition metal dications tend to lose small neutral molecules such as water and carbon dioxide. Interestingly, all complexes involving transition metal dications except for Cu(Pro2-H)(+) lose H2 upon collisional or IRMPD activation. IRMPD spectroscopy shows that the intact proline in the transition metal complexes and Cu(Pro2-H)(+) is predominantly canonical (charge solvated) while for the Ca(2+), Sr(2+), and Ba(2+) complexes, proline is in its zwitterionic form. The IRMPD spectra for both Mg(Pro2-H)(+) and Mn(Pro2-H)(+) are concluded to have contributions from both charge-solvated and canonical structures.

A chiral sensor based on weak measurement for the determination of Proline enantiomers in diverse measuring circumstances.

PubMed

Li, Dongmei; Guan, Tian; He, Yonghong; Liu, Fang; Yang, Anping; He, Qinghua; Shen, Zhiyuan; Xin, Meiguo

2018-07-01

A new chiral sensor based on weak measurement to accurately measure the optical rotation (OR) has been developed for the estimation of a trace amount of chiral molecule. With the principle of optical weak measurement in frequency domain, the central wavelength shift of output spectra is quantitatively relative to the angle of preselected polarization. Hence, a chiral molecule (e.g., L-amino acid, or D-amino acid) can be enantioselectively determined by modifying the preselection angle with the OR, which will cause the rotation of a polarization plane. The concentration of the chiral sample, corresponding to its optical activity, is quantitatively analyzed with the central wavelength shift of output spectra, which can be collected in real time. Immune to the refractive index change, the proposed chiral sensor is valid in complicated measuring circumstance. The detections of Proline enantiomer concentration in different solvents were implemented. The results demonstrated that weak measurement acted as a reliable method to chiral recognition of Proline enantiomers in diverse circumstance with the merits of high precision and good robustness. In addition, this real-time monitoring approach plays a crucial part in asymmetric synthesis and biological systems. Copyright © 2018. Published by Elsevier B.V.

Characterisation of secretory calcium-binding phosphoprotein-proline-glutamine-rich 1: a novel basal lamina component expressed at cell-tooth interfaces.

PubMed

Moffatt, Pierre; Wazen, Rima M; Dos Santos Neves, Juliana; Nanci, Antonio

2014-12-01

Functional genomic screening of the rat enamel organ (EO) has led to the identification of a number of secreted proteins expressed during the maturation stage of amelogenesis, including amelotin (AMTN) and odontogenic ameloblast-associated (ODAM). In this study, we characterise the gene, protein and pattern of expression of a related protein called secretory calcium-binding phosphoprotein-proline-glutamine-rich 1 (SCPPPQ1). The Scpppq1 gene resides within the secretory calcium-binding phosphoprotein (Scpp) cluster. SCPPPQ1 is a highly conserved, 75-residue, secreted protein rich in proline, leucine, glutamine and phenylalanine. In silico data mining has revealed no correlation to any known sequences. Northern blotting of various rat tissues suggests that the expression of Scpppq1 is restricted to tooth and associated tissues. Immunohistochemical analyses show that the protein is expressed during the late maturation stage of amelogenesis and in the junctional epithelium where it localises to an atypical basal lamina at the cell-tooth interface. This discrete localisation suggests that SCPPPQ1, together with AMTN and ODAM, participates in structuring the basal lamina and in mediating attachment of epithelia cells to mineralised tooth surfaces.

Natural Proline-Rich Cyclopolypeptides from Marine Organisms: Chemistry, Synthetic Methodologies and Biological Status.

PubMed

Fang, Wan-Yin; Dahiya, Rajiv; Qin, Hua-Li; Mourya, Rita; Maharaj, Sandeep

2016-10-26

Peptides have gained increased interest as therapeutics during recent years. More than 60 peptide drugs have reached the market for the benefit of patients and several hundreds of novel therapeutic peptides are in preclinical and clinical development. The key contributor to this success is the potent and specific, yet safe, mode of action of peptides. Among the wide range of biologically-active peptides, naturally-occurring marine-derived cyclopolypeptides exhibit a broad range of unusual and potent pharmacological activities. Because of their size and complexity, proline-rich cyclic peptides (PRCPs) occupy a crucial chemical space in drug discovery that may provide useful scaffolds for modulating more challenging biological targets, such as protein-protein interactions and allosteric binding sites. Diverse pharmacological activities of natural cyclic peptides from marine sponges, tunicates and cyanobacteria have encouraged efforts to develop cyclic peptides with well-known synthetic methods, including solid-phase and solution-phase techniques of peptide synthesis. The present review highlights the natural resources, unique structural features and the most relevant biological properties of proline-rich peptides of marine-origin, focusing on the potential therapeutic role that the PRCPs may play as a promising source of new peptide-based novel drugs.

Myotonia-related mutations in the distal C-terminus of ClC-1 and ClC-0 chloride channels affect the structure of a poly-proline helix

PubMed Central

Macías, María J.; Teijido, Oscar; Zifarelli, Giovanni; Martin, Pau; Ramirez-Espain, Ximena; Zorzano, Antonio; Palacín, Manuel; Pusch, Michael; Estévez, Raúl

2006-01-01

Myotonia is a state of hyperexcitability of skeletal-muscle fibres. Mutations in the ClC-1 Cl− channel cause recessive and dominant forms of this disease. Mutations have been described throughout the protein-coding region, including three sequence variations (A885P, R894X and P932L) in a distal C-terminal stretch of residues [CTD (C-terminal domain) region] that are not conserved between CLC proteins. We show that surface expression of these mutants is reduced in Xenopus oocytes compared with wild-type ClC-1. Functional, biochemical and NMR spectroscopy studies revealed that the CTD region encompasses a segment conserved in most voltage-dependent CLC channels that folds with a secondary structure containing a short type II poly-proline helix. We found that the myotonia-causing mutation A885P disturbs this structure by extending the poly-proline helix. We hypothesize that this structural modification results in the observed alteration of the common gate that acts on both pores of the channel. We provide the first experimental investigation of structural changes resulting from myotonia-causing mutations. PMID:17107341

The role for IGF-1-derived small neuropeptides as a therapeutic target for neurological disorders.

PubMed

Guan, Jian; Harris, Paul; Brimble, Margaret; Lei, Yang; Lu, Jun; Yang, Yang; Gunn, Alistair J

2015-06-01

Exogenous IGF-1 protects the brain from ischemic injury and improves function. However, its clinical application to neurological disorders is limited by its large molecular size, poor central uptake and mitogenic potential. In this review, the authors have discussed the efficacy, pharmacokinetics and mechanisms of IGF-1 derivatives on protecting acute brain injury, preventing memory impairment and improving recovery from neurological degenerative conditions evaluated in various animal models. We have included natural metabolites of IGF-1, glycine-proline-glutamate (GPE), cleaved from N-terminal IGF-1 and cyclic glycine-proline (cGP) as well as the structural analogues of GPE and cGP, glycine-2-methyl-proline-glutamate and cyclo-l-glycyl-l-2-allylproline, respectively. In addition, the regulatory role for cGP in bioavailability of IGF-1 has also been discussed. These small neuropeptides provide effective neuroprotection by offering an improved pharmacokinetic profile and more practical route of administration compared with IGF-1 administration. Developing modified neuropeptides to overcome the limitations of their endogenous counterparts represents a novel strategy of pharmaceutical discovery for neurological disorders. The mechanism of action may involve a regulation of IGF-1 bioavailability.

Signal transduction in artichoke [Cynara cardunculus L. subsp. scolymus (L.) Hayek] callus and cell suspension cultures under nutritional stress.

PubMed

Lattanzio, Vincenzo; Caretto, Sofia; Linsalata, Vito; Colella, Giovanni; Mita, Giovanni

2018-06-01

Stimulated production of secondary phenolic metabolites and proline was studied by using cell cultures of artichoke [Cynara cardunculus L. subsp. scolymus (L.) Hayek] submitted to nutritional stress. Artichoke cell cultures accumulated phenolic secondary metabolites in a pattern similar to that seen in artichoke leaves and heads (capitula). This paper shows that both callus and cell suspension cultures under nutritional stress accumulated phenolic compounds and proline, at the same time their biomass production was negatively affected by nutrient deficiency. The results obtained strongly suggest that plant tissues respond to nutrient deprivation by a defensive costly mechanism, which determines the establishment of a mechanism of trade-off between growth and adaptive response. Furthermore, the results of this research suggest that perception of abiotic stress and increased phenolic metabolites are linked by a sequence of biochemical processes that also involves the intracellular free proline and the oxidative pentose phosphate pathway. The main conclusion of this paper is that, once calli and cell suspension cultures respond to nutrient deficiency, in acclimated cells the establishment of a negative correlation between primary metabolism (growth) and secondary metabolism (defence compounds) is observed. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Natural Proline-Rich Cyclopolypeptides from Marine Organisms: Chemistry, Synthetic Methodologies and Biological Status

PubMed Central

Fang, Wan-Yin; Dahiya, Rajiv; Qin, Hua-Li; Mourya, Rita; Maharaj, Sandeep

2016-01-01

Peptides have gained increased interest as therapeutics during recent years. More than 60 peptide drugs have reached the market for the benefit of patients and several hundreds of novel therapeutic peptides are in preclinical and clinical development. The key contributor to this success is the potent and specific, yet safe, mode of action of peptides. Among the wide range of biologically-active peptides, naturally-occurring marine-derived cyclopolypeptides exhibit a broad range of unusual and potent pharmacological activities. Because of their size and complexity, proline-rich cyclic peptides (PRCPs) occupy a crucial chemical space in drug discovery that may provide useful scaffolds for modulating more challenging biological targets, such as protein-protein interactions and allosteric binding sites. Diverse pharmacological activities of natural cyclic peptides from marine sponges, tunicates and cyanobacteria have encouraged efforts to develop cyclic peptides with well-known synthetic methods, including solid-phase and solution-phase techniques of peptide synthesis. The present review highlights the natural resources, unique structural features and the most relevant biological properties of proline-rich peptides of marine-origin, focusing on the potential therapeutic role that the PRCPs may play as a promising source of new peptide-based novel drugs. PMID:27792168

Molecular dynamics simulations of trans- and cis- N-acetyl- N'-methylamides of XaaPro dipeptides

NASA Astrophysics Data System (ADS)

Hoon Choi, Seung; Yun Yu, Jeong; Kwang Shin, Jae; Shik Jhon, Mu

1994-07-01

The occurrence of cis imide bonds in proteins is much higher than that of cis amide bonds due to the unique properties of proline. In order to examine the relationship between the high occurrence of these cis imide bonds and the residues preceding the proline, we perform molecular dynamics simulations of trans- and cis- N-acetyl- N'-methylamides of XaaPro dipeptides (AcXaaProNHMe). We investigate the conformational energies and structures of trans- and cis-AcXaa where Xaa has 12 amino acids in the vacuum state and 5 amino acids in the solution state. It is found that the occurrence of the cis imide bonds is strongly affected by the residue preceding the proline, and the dihedral angles (φ,ψ) of the backbone in AcXaaProNHMe are influenced by the configuration of the imide bond. We also find that the equilibrium properties of XaaPro in solution simulations are more similar to the statistics of X-ray crystallographic data than are those in vacuum simulations and solvation causes a remarkable change in the conformation of the pyrrolidine ring from the endo to the exo form.

Biochemical and standard toxic effects of acetaminophen on the macrophyte species Lemna minor and Lemna gibba.

PubMed

Nunes, Bruno; Pinto, Glória; Martins, Liliana; Gonçalves, Fernando; Antunes, Sara C

2014-09-01

Acetaminophen is globally one of the most prescribed drugs due to its antipyretic and analgesic properties. However, it is highly toxic when the dosage surpasses the detoxification capability of an exposed organism, with involvement of an already described oxidative stress pathway. To address the issue of the ecotoxicity of acetaminophen, we performed acute exposures of two aquatic plant species, Lemna gibba and Lemna minor, to this compound. The selected biomarkers were number of fronds, biomass, chlorophyll content, lipid peroxidation (TBARS assay), and proline content. Our results showed marked differences between the two species. Acetaminophen caused a significant decrease in the number of fronds (EC50 = 446.6 mg/L), and the establishment of a dose-dependent peroxidative damage in L. minor, but not in L. gibba. No effects were reported in both species for the indicative parameters chlorophyll content and total biomass. However, the proline content in L. gibba was substantially reduced. The overall conclusions point to the occurrence of an oxidative stress scenario more prominent for L. minor. However, the mechanisms that allowed L. gibba to cope with acetaminophen exposure were distinct from those reported for L. minor, with the likely involvement of proline as antioxidant.

Antimicrobial susceptibility and biochemical patterns of Neisseria gonorrhoeae strains in Vejle area, Denmark.

PubMed

Lis-Tønder, J; Cybulski, Z

2009-10-01

Strains of Neisseria gonorrhoeae resistant to penicillin and ciprofloxacin have been isolated worldwide. Increasing number of N. gonorrhoeae that lack the enzyme proline aminopeptidase (proA-negative N. gonorrhoeae) have been detected in many countries all over the world. This study aims to assess the resistance profiles of N. gonorrhoeae isolates sent to the Department of Clinical Microbiology in Vejle, Denmark, between 2003 and 2007, and to analyse their biochemical patterns. Sixty-two strains of N. gonorrhoeae were retrospectively analysed for their susceptibility to penicillin, ciprofloxacin and ceftriaxone. The identification of isolated strains was confirmed using both biochemical and immunological tests. Twenty-one (33.9%) N. gonorrhoeae isolates were resistant to penicillin and 30 (48.4%) were resistant to ciprofloxacin. All strains were susceptible to ceftriaxone. Fifty-six (90.3%) N. gonorrhoeae strains showed API NH biochemical code 10,010 (produced acid from glucose and proline aminopeptidase). Six strains showed code 10,000 that lack the enzyme proline aminopeptidase (proA-negative N. gonorrhoeae). Ceftriaxone should be used as the first-line treatment of gonorrhoea in Vejle community area, Denmark, both for infections with proA-producing and proA-negative N. gonorrhoeae isolates, which circulate in the region.

pH titration monitored by quantum cascade laser-based vibrational circular dichroism.

PubMed

Rüther, Anja; Pfeifer, Marcel; Lórenz-Fonfría, Víctor A; Lüdeke, Steffen

2014-04-10

Vibrational circular dichroism (VCD) spectra of aqueous solutions of proline were recorded in the course of titrations from basic to acidic pH using a spectrometer equipped with a quantum cascade laser (QCL) as an infrared light source in the spectral range from 1320 to 1220 cm(-1). The pH-dependent spectra were analyzed by singular value decomposition and global fitting of a two-pK Henderson-Hasselbalch model. The analysis delivered relative fractions of the three different protonation species. Their agreement with the relative fractions obtained from performing the same analysis on pH-dependent Fourier transform infrared (FT-IR) and QCL-IR spectra validates the quantitative results from QCL-VCD. Global fitting of the pH-dependent VCD spectra of L-proline allowed for extraction of pure spectra corresponding to anionic, zwitterionic, and cationic L-proline. From a static experiment, only pure spectra of the zwitterion would be accessible in a straightforward way. A comparison to VCD spectra calculated for all three species led to assignment of vibrational modes that are characteristic for the respective protonation states. The study demonstrates the applicability of QCL-VCD both for quantitative evaluation and for qualitative interpretation of dynamic processes in aqueous solutions.

Assessing the impact of minimizing arginine conversion in fully defined SILAC culture medium in human embryonic stem cells.

PubMed

Scheerlinck, Ellen; Van Steendam, Katleen; Daled, Simon; Govaert, Elisabeth; Vossaert, Liesbeth; Meert, Paulien; Van Nieuwerburgh, Filip; Van Soom, Ann; Peelman, Luc; De Sutter, Petra; Heindryckx, Björn; Dhaenens, Maarten; Deforce, Dieter

2016-10-01

We present a fully defined culture system (adapted Essential8 TM [E8 TM ] medium in combination with vitronectin) for human embryonic stem cells that can be used for SILAC purposes. Although a complete incorporation of the labels was observed after 4 days in culture, over 90% of precursors showed at least 10% conversion. To reduce this arginine conversion, E8 TM medium was modified by adding (1) l-proline, (2) l-ornithine, (3) N ω -hydroxy-nor-l-arginine acetate, or by (4) lowering the arginine concentration. Reduction of arginine conversion was best obtained by adding 5 mM l-ornithine, followed by 3.5 mM l-proline and by lowering the arginine concentration in the medium to 99.5 μM. No major changes in pluripotency and cell amount could be observed for the adapted E8 TM media with ornithine and proline. However, our subsequent ion mobility assisted data-independent acquisition (high-definition MS) proteome analysis cautions for ongoing changes in the proteome when aiming at longer term suppression of arginine conversion. © 2016 The Authors. Proteomics Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Ab initio computational study of reaction mechanism of peptide bond formation on HF/6-31G(d,p) level

NASA Astrophysics Data System (ADS)

Siahaan, P.; Lalita, M. N. T.; Cahyono, B.; Laksitorini, M. D.; Hildayani, S. Z.

2017-02-01

Peptide plays an important role in modulation of various cell functions. Therefore, formation reaction of the peptide is important for chemical reactions. One way to probe the reaction of peptide synthesis is a computational method. The purpose of this research is to determine the reaction mechanism for peptide bond formation on Ac-PV-NH2 and Ac-VP-NH2 synthesis from amino acid proline and valine by ab initio computational approach. The calculations were carried out by theory and basis set HF/6-31G(d,p) for four mechanisms (path 1 to 4) that proposed in this research. The results show that the highest of the rate determining step between reactant and transition state (TS) for path 1, 2, 3, and 4 are 163.06 kJ.mol-1, 1868 kJ.mol-1, 5685 kJ.mol-1, and 1837 kJ.mol-1. The calculation shows that the most preferred reaction of Ac-PV-NH2 and Ac-VP-NH2 synthesis from amino acid proline and valine are on the path 1 (initiated with the termination of H+ in proline amino acid) that produce Ac-PV-NH2.

Neuropeptide-hydrolysing activities in synaptosomal fractions from dog ileum myenteric, deep muscular and submucous plexi. Their participation in neurotensin inactivation.

PubMed

Barelli, H; Ahmad, S; Kostka, P; Fox, J A; Daniel, E E; Vincent, J P; Checler, F

1989-01-01

The mapping of neuropeptidases in synaptosomal fractions prepared from dog ileum myenteric, deep muscular and submucous plexus was established by means of fluorigenic substrates and specific inhibitors. Endopeptidase 24.11, angiotensin-converting enzyme and aminopeptidases were found in all tissues, the highest amounts being recovered in the submucous preparation. Post-proline dipeptidyl aminopeptidase was obtained in high quantities whatever the tissue source while proline endopeptidase was detected in low amounts and pyroglutamyl-peptide hydrolase was never detectable. The above peptidases were examined for their putative participation in the inactivation of neurotensin by monitoring the effect of specific inhibitors on the formation of the metabolites of labeled neurotensin separated by HPLC. Endopeptidases 24.11, 24.15 and 24.16 were respectively responsible for the formation of neurotensin(1-11), neurotensin(1-8) and neurotensin(1-10) that are devoid of biological activity. The secondary attacks occurring on neurotensin degradation products were the following: cleavage of neurotensin(1-10) into neurotensin(1-8) by angiotensin-converting enzyme; conversion of neurotensin(9-13) into neurotensin(11-13) by post-proline dipeptidyl aminopeptidase; hydrolysis of neurotensin(11-13) into free tyrosine by aminopeptidase(s).

Isolation of a new class of cysteine–glycine–proline-rich beta-proteins (beta-keratins) and their expression in snake epidermis

PubMed Central

Dalla Valle, Luisa; Nardi, Alessia; Alibardi, Lorenzo

2010-01-01

Scales of snakes contain hard proteins (beta-keratins), now referred to as keratin-associated beta-proteins. In the present study we report the isolation, sequencing, and expression of a new group of these proteins from snake epidermis, designated cysteine–glycine–proline-rich proteins. One deduced protein from expressed mRNAs contains 128 amino acids (12.5 kDa) with a theoretical pI at 7.95, containing 10.2% cysteine and 15.6% glycine. The sequences of two more snake cysteine–proline-rich proteins have been identified from genomic DNA. In situ hybridization shows that the messengers for these proteins are present in the suprabasal and early differentiating beta-cells of the renewing scale epidermis. The present study shows that snake scales, as previously seen in scales of lizards, contain cysteine-rich beta-proteins in addition to glycine-rich beta-proteins. These keratin-associated beta-proteins mix with intermediate filament keratins (alpha-keratins) to produce the resistant corneous layer of snake scales. The specific proportion of these two subfamilies of proteins in different scales can determine various degrees of hardness in scales. PMID:20070430

Isolation of a new class of cysteine-glycine-proline-rich beta-proteins (beta-keratins) and their expression in snake epidermis.

PubMed

Dalla Valle, Luisa; Nardi, Alessia; Alibardi, Lorenzo

2010-03-01

Scales of snakes contain hard proteins (beta-keratins), now referred to as keratin-associated beta-proteins. In the present study we report the isolation, sequencing, and expression of a new group of these proteins from snake epidermis, designated cysteine-glycine-proline-rich proteins. One deduced protein from expressed mRNAs contains 128 amino acids (12.5 kDa) with a theoretical pI at 7.95, containing 10.2% cysteine and 15.6% glycine. The sequences of two more snake cysteine-proline-rich proteins have been identified from genomic DNA. In situ hybridization shows that the messengers for these proteins are present in the suprabasal and early differentiating beta-cells of the renewing scale epidermis. The present study shows that snake scales, as previously seen in scales of lizards, contain cysteine-rich beta-proteins in addition to glycine-rich beta-proteins. These keratin-associated beta-proteins mix with intermediate filament keratins (alpha-keratins) to produce the resistant corneous layer of snake scales. The specific proportion of these two subfamilies of proteins in different scales can determine various degrees of hardness in scales.

Rapid desensitization of the rat α7 nAChR is facilitated by the presence of a proline residue in the outer β-sheet

PubMed Central

McCormack, Thomas J; Melis, Claudio; Colón, José; Gay, Elaine A; Mike, Arpad; Karoly, Robert; Lamb, Patricia W; Molteni, Carla; Yakel, Jerrel L

2010-01-01

The rat α7 nicotinic acetylcholine receptor (nAChR) has a proline residue near the middle of the β9 strand. The replacement of this proline residue at position 180 (P180) by either threonine (α7-P180T) or serine (α7-P180S) slowed the onset of desensitization dramatically, with half-times of ∼930 and 700 ms, respectively, compared to 90 ms for the wild-type receptor. To investigate the importance of the hydroxyl group on the position 180 side-chains, the mutant receptors α7-P180Y and α7-P180F were studied and showed half-times of desensitization of 650 and 160 ms, respectively. While a position 180 side-chain OH group may contribute to the slow desensitization rates, α7-P180S and α7-P180V resulted in receptors with similar desensitization rates, suggesting that increased backbone to backbone H bonding expected in the absence of proline at position 180 would likely exert a great effect on desensitization. Single channel recordings indicated that for the α7-P180T receptor there was a significantly reduced closed time without any change in single channel conductance (as compared to wild-type). Kinetic simulations indicated that all changes observed for the mutant channel behaviour were reproduced by decreasing the rate of desensitization, and increasing the microscopic affinity to resting receptors. Molecular dynamics (MD) simulations on a homology model were used to provide insight into likely H bond interactions within the outer β-sheet that occur when the P180 residue is mutated. All mutations analysed increased about twofold the predicted number of H bonds between the residue at position 180 and the backbone of the β10 strand. Moreover, the α7-P180T and α7-P180S mutations also formed some intrastrand H bonds along the β9 strand, although H bonding of the OH groups of the threonine or serine side-chains was predicted to be infrequent. Our results indicate that rapid desensitization of the wild-type rat α7 nAChR is facilitated by the presence of the proline residue within the β9 strand. PMID:20837638

Rapid desensitization of the rat α7 nAChR is facilitated by the presence of a proline residue in the outer β-sheet.

PubMed

McCormack, Thomas J; Melis, Claudio; Colón, José; Gay, Elaine A; Mike, Arpad; Karoly, Robert; Lamb, Patricia W; Molteni, Carla; Yakel, Jerrel L

2010-11-15

The rat α7 nicotinic acetylcholine receptor (nAChR) has a proline residue near the middle of the β9 strand. The replacement of this proline residue at position 180 (P180) by either threonine (α7-P180T) or serine (α7-P180S) slowed the onset of desensitization dramatically, with half-times of ~930 and 700 ms, respectively, compared to 90 ms for the wild-type receptor. To investigate the importance of the hydroxyl group on the position 180 side-chains, the mutant receptors α7-P180Y and α7-P180F were studied and showed half-times of desensitization of 650 and 160 ms, respectively. While a position 180 side-chain OH group may contribute to the slow desensitization rates, α7-P180S and α7-P180V resulted in receptors with similar desensitization rates, suggesting that increased backbone to backbone H bonding expected in the absence of proline at position 180 would likely exert a great effect on desensitization. Single channel recordings indicated that for the α7-P180T receptor there was a significantly reduced closed time without any change in single channel conductance (as compared to wild-type). Kinetic simulations indicated that all changes observed for the mutant channel behaviour were reproduced by decreasing the rate of desensitization, and increasing the microscopic affinity to resting receptors. Molecular dynamics (MD) simulations on a homology model were used to provide insight into likely H bond interactions within the outer β-sheet that occur when the P180 residue is mutated. All mutations analysed increased about twofold the predicted number of H bonds between the residue at position 180 and the backbone of the β10 strand. Moreover, the α7-P180T and α7-P180S mutations also formed some intrastrand H bonds along the β9 strand, although H bonding of the OH groups of the threonine or serine side-chains was predicted to be infrequent. Our results indicate that rapid desensitization of the wild-type rat α7 nAChR is facilitated by the presence of the proline residue within the β9 strand.

Asymmetric Michael Addition Mediated by Chiral Ionic Liquids

PubMed Central

Suzuki, Yumiko

2018-01-01

Chiral ionic liquids with a focus on their applications in asymmetric Michael additions and related reactions were reviewed. The examples were classified on the basis of the mode of asymmetric induction (e.g., external induction/non-covalent interaction or internal induction/covalent bond formation), the roles in reactions (as a solvent or catalyst), and their structural features (e.g., imidazolium-based chiral cations, other chiral oniums; proline derivatives). Most of the reactions with high chiral induction are Michael addition of ketones or aldehydes to chalcones or nitrostyrenes where proline-derived chiral ionic liquids catalyze the reaction through enamine/ iminium formation. Many reports demonstrate the recyclability of ionic liquid-tagged pyrrolidines. PMID:29861702

[Changes in proline-specific peptidase activity in experimental model of retrograde amnesia].

PubMed

Nazarova, G A; Zolotov, N N; Krupina, N A; Kraĭneva, V A; Garibova, T L; Voronina, T A

2007-01-01

Changes in proline-specific peptidase activity in the frontal cortex and hippocampus were studied using the experimental model of retrograde amnesia in rats. In one group, the amnesia was produced by a single injection of M-cholinergic antagonist scopolamine and the other group received the maximal electroconvulsive stimulation (MES). The amnesic effect was evaluated in passive avoidance test. In the amnesia models under consideration, the activity of prolylendopeptidase was significantly increased in both frontal cortex and hippocampus. The activity of dipeptidyl peptidase IV was significantly decreased in the cortex, whereas in the hippocampus it remained unchanged. Pyracetam inhibited prolylendopeptidase in the cortex and hippocampus, whereas dipeptidyl peptidase IV activity remained unchanged.

Effects of hydrostatic pressure and temperature on the uptake and respiration of amino acids by a facultatively psychrophilic marine bacterium.

NASA Technical Reports Server (NTRS)

Paul, K. L.; Morita, R. Y.

1971-01-01

Studies of pressure and temperature effects on glutamic acid transport and utilization indicated that hydrostatic pressure and low temperature inhibit glutamate transport more than glutamate respiration. The effects of pressure on transport were reduced at temperatures near the optimum. Similar results were obtained for glycine, phenylalanine, and proline. Pressure effects on the transport systems of all four amino acids were reversible to some degree. Both proline and glutamic acid were able to protect their transport proteins against pressure damage. The data presented indicate that the uptake of amino acids by cells under pressure is inhibited, which is the cause of their inability to grow under pressure.

Response to copper and sodium chloride excess in Spirulina sp. (cyanobacteria).

PubMed

Deniz, F; Saygideger, S D; Karaman, S

2011-07-01

Physiological responses of the cyanobacterium, Spirulina sp., were evaluated following exposure to copper (0.1 and 1.0 mg/L) and sodium chloride (0.2 and 0.4 mol/L) for 7 days. Growth and chlorophyll a content exhibited decreases at most exposure levels, while increases occurred for malondialdehyde at all exposure levels. Proline content was increased at the higher exposure levels. Carotenoid levels of Spirulina sp. were not significantly changed. Increased amounts of malondialdehyde were indicative of free radical formation in Spirulina sp. under the stress, while increasing levels of proline pointed to the occurrence of a scavenging mechanism. Concentrations of copper in Spirulina sp. decreased with increasing concentrations of NaCl.

Bioremediation of adverse impact of cadmium toxicity on Cassia italica Mill by arbuscular mycorrhizal fungi

PubMed Central

Hashem, Abeer; Abd_Allah, E.F.; Alqarawi, A.A.; Egamberdieva, Dilfuza

2015-01-01

Cassia italica Mill is an important medicinal plant within the family Fabaceae. Pot experiment was conducted to evaluate cadmium stress induced changes in physiological and biochemical attributes in C. italica with and without arbuscular mycorrhizal fungi (AMF). Cadmium stressed plant showed reduced chlorophyll pigment and protein content while AMF inoculation enhanced the chlorophyll and protein content considerably. AMF also ameliorated the cadmium stress induced reduction in total chlorophyll and protein contents by 19.30% and 38.29%, respectively. Cadmium stress enhanced lipid peroxidation while AMF inoculation reduced lipid peroxidation considerably. Increase in proline and phenol content was observed due to cadmium stress and AMF inoculation caused a further increase in proline and phenol content ensuring better growth under stressed conditions. AMF alone also enhanced proline and phenol content. Activity of antioxidant enzymes enhanced under cadmium treatment and AMF inoculation further enhanced their activity thereby strengthening the antioxidant system. Enhanced activities of antioxidants and increased accumulation of osmolytes help plants to avoid damaging impact of oxidative damage. The research has shown that AMF inoculation mitigated the negative impact of stress by reducing the lipid peroxidation and enhancing the antioxidant activity. The present study strongly supports employing AMF as the biological mean for enhancing the cadmium stress tolerance of C. italica. PMID:26858537

Salivary proline-rich proteins and gluten: Do structural similarities suggest a role in celiac disease?

PubMed

Tian, Na; Messana, Irene; Leffler, Daniel A; Kelly, Ciaran P; Hansen, Joshua; Cabras, Tiziana; D'Alessandro, Alfredo; Schuppan, Detlef; Castagnola, Massimo; Helmerhorst, Eva J

2015-10-01

Gluten proteins, the culprits in celiac disease (CD), show striking similarities in primary structure with human salivary proline-rich proteins (PRPs). Both are enriched in proline and glutamine residues that often occur consecutively in their sequences. We investigated potential differences in the spectrum of salivary PRPs in health and CD. Stimulated salivary secretions were collected from CD patients, patients with refractory CD, patients with gastrointestinal complaints but no CD, and healthy controls. PRP isoforms/peptides were characterized by anionic and SDS-PAGE, PCR, and LC-ESI-MS. The gene frequencies of the acidic PRP isoforms PIF, Db, Pa, PRP1, and PRP2 did not differ between groups. At the protein level, PRPs peptides showed minor group differences, but these could not differentiate the CD and/or refractory CDs groups from the controls. This extensive study established that salivary PRPs, despite similarity to gluten proteins, show no apparent correlation with CD and thus will not serve as diagnostic markers for the disease. The structural basis for the tolerance to the gluten-like PRP proteins in CD is worthy of further exploration and may lead to the development of gluten-like analogs lacking immunogenicity that could be used therapeutically. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Fluorescent Labeling of Collagen Production by Cells for Noninvasive Imaging of Extracellular Matrix Deposition.

PubMed

Bardsley, Katie; Yang, Ying; El Haj, Alicia J

2017-04-01

Extracellular matrix (ECM) is an essential component of tissues and provides both integrity and biological cues for cells. Collagen is one of the major proteins found within the ECM and therefore is an essential component of all engineered tissues. Therefore, in this article, we present a method for the online real-time monitoring of collagen deposition in three-dimensional engineered constructs. This method revolves around modification of collagen through the addition of azide-L-proline to cell culture media. The incorporation of azide-L-proline into the neocollagen produced by cells can then be detected by reaction with 10 mM of a Click-IT Alexa Fluor 488 DIBO Alkyne. The reaction was shown as being specific to the collagen as little background staining was observed in cultures, which did not contain the modified proline, and the staining was also depleted after treatment with collagenase and colocalization of collagen type I staining by immunochemistry assay. Real-time online staining of collagen deposition was observed under different culture conditions without affecting proliferation. Collagen deposition was observed to be increased under mechanical stimulation; however, the localization varied across stimulation regimes. This is a new technique for real-time monitoring of cell-produced collagen and will be a valuable addition to the tissue engineering field.

Apoptosis-like death was involved in freeze-drying-preserved fungus Mucor rouxii and can be inhibited by L-proline.

PubMed

Wang, Xiaoyun; Wang, Youzhi

2016-02-01

Freeze-drying is one of the most effective methods to preserve fungi for an extended period. However, it is associated with a loss of viability and shortened storage time in some fungi. This study evaluated the stresses that led to the death of freeze-dried Mucor rouxii by using cell apoptotic methods. The results showed there were apoptosis-inducing stresses, such as the generation of obvious intracellular reactive oxygen species (ROS) and metacaspase activation. Moreover, nuclear condensation and a delayed cell death peak were determined after rehydration and 24 h incubation in freeze-dried M. rouxii via a propidium iodide (PI) assay, which is similar to the phenomenon of cryopreservation-induced delayed-onset cell death (CIDOCD). Then, several protective agents were tested to decrease the apoptosis-inducing stresses and to improve the viability. Finally, it was found that 1.6 mM L-proline can effectively decrease the nuclear condensation rate and increase the survival rate in freeze-dried M. rouxii. (1) apoptosis-inducing factors occur in freeze-dried M. rouxii. (2) ROS and activated metacaspases lead to death in freeze-dried M. rouxii. (3)L-proline increases the survival rate of freeze-dried M. rouxii. Copyright © 2015 Elsevier Inc. All rights reserved.

Sequence-specific unusual (1-->2)-type helical turns in alpha/beta-hybrid peptides.

PubMed

Prabhakaran, Panchami; Kale, Sangram S; Puranik, Vedavati G; Rajamohanan, P R; Chetina, Olga; Howard, Judith A K; Hofmann, Hans-Jörg; Sanjayan, Gangadhar J

2008-12-31

This article describes novel conformationally ordered alpha/beta-hybrid peptides consisting of repeating l-proline-anthranilic acid building blocks. These oligomers adopt a compact, right-handed helical architecture determined by the intrinsic conformational preferences of the individual amino acid residues. The striking feature of these oligomers is their ability to display an unusual periodic pseudo beta-turn network of nine-membered hydrogen-bonded rings formed in the forward direction of the sequence by 1-->2 amino acid interactions both in solid-state and in solution. Conformational investigations of several of these oligomers by single-crystal X-ray diffraction, solution-state NMR, and ab initio MO theory suggest that the characteristic steric and dihedral angle restraints exerted by proline are essential for stabilizing the unusual pseudo beta-turn network found in these oligomers. Replacing proline by the conformationally flexible analogue alanine (Ala) or by the conformationally more constrained alpha-amino isobutyric acid (Aib) had an adverse effect on the stabilization of this structural architecture. These findings increase the potential to design novel secondary structure elements profiting from the steric and dihedral angle constraints of the amino acid constituents and help to augment the conformational space available for synthetic oligomer design with diverse backbone structures.

Aging of Escherichia coli

PubMed Central

Clifton, C. E.

1966-01-01

Clifton, C. E. (Stanford University, Stanford, Calif.). Aging of Escherichia coli. J. Bacteriol. 92:905–912. 1966.—The rates of endogenous and exogenous (glucose) respiration decreased much more rapidly than did the viable count during the first 24 hr of aging of washed, C14-labeled cells of Escherichia coli K-12 suspended in a basal salt medium devoid of ammonium salts. The rates of decrease of respiration and of death approached each other as the age of the cells increased, but death was not the only factor involved in decreased respiratory activity of the suspensions. The greatest decrease in cellular contents with aging was noted in the ribonucleic acid fraction, of which the ribose appeared to be oxidized, while uracil accumulated in the suspension medium. The viable count and respiratory activities remained higher in glucose-fed than in nonfed suspensions. Proline-labeled cells fed glucose tended to lose more of their proline and to convert more proline into C14O2 than in unfed controls. On the other hand, uracil-labeled cells fed glucose retained more of the uracil than did nonfed cells, but glucose elicited some oxidation of uracil. An exogenous energy source such as glucose aided in the maintenance of a population, but it was not the only factor needed for such maintenance. PMID:5332874

Low levels of serum serotonin and amino acids identified in migraine patients.

PubMed

Ren, Caixia; Liu, Jia; Zhou, Juntuo; Liang, Hui; Wang, Yayun; Sun, Yinping; Ma, Bin; Yin, Yuxin

2018-02-05

Migraine is a highly disabling primary headache associated with a high socioeconomic burden and a generally high prevalence. The clinical management of migraine remains a challenge. This study was undertaken to identify potential serum biomarkers of migraine. Using Liquid Chromatography coupled to Mass Spectrometry (LC-MS), the metabolomic profile of migraine was compared with healthy individuals. Principal component analysis (PCA) and Orthogonal partial least squares-discriminant analysis (orthoPLS-DA) showed the metabolomic profile of migraine is distinguishable from controls. Volcano plot analysis identified 10 serum metabolites significantly decreased during migraine. One of these was serotonin, and the other 9 were amino acids. Pathway analysis and enrichment analysis showed tryptophan metabolism (serotonin metabolism), arginine and proline metabolism, and aminoacyl-tRNA biosynthesis are the three most prominently altered pathways in migraine. ROC curve analysis indicated Glycyl-l-proline, N-Methyl-dl-Alanine and l-Methionine are potential sensitive and specific biomarkers for migraine. Our results show Glycyl-l-proline, N-Methyl-dl-Alanine and l-Methionine may be as specific or more specific for migraine than serotonin which is the traditional biomarker of migraine. We propose that therapeutic manipulation of these metabolites or metabolic pathways may be helpful in the prevention and treatment of migraine. Copyright © 2018 Elsevier Inc. All rights reserved.

Gene Mining for Proline Based Signaling Proteins in Cell Wall of Arabidopsis thaliana

PubMed Central

Ihsan, Muhammad Z.; Ahmad, Samina J. N.; Shah, Zahid Hussain; Rehman, Hafiz M.; Aslam, Zubair; Ahuja, Ishita; Bones, Atle M.; Ahmad, Jam N.

2017-01-01

The cell wall (CW) as a first line of defense against biotic and abiotic stresses is of primary importance in plant biology. The proteins associated with cell walls play a significant role in determining a plant's sustainability to adverse environmental conditions. In this work, the genes encoding cell wall proteins (CWPs) in Arabidopsis were identified and functionally classified using geneMANIA and GENEVESTIGATOR with published microarrays data. This yielded 1605 genes, out of which 58 genes encoded proline-rich proteins (PRPs) and glycine-rich proteins (GRPs). Here, we have focused on the cellular compartmentalization, biological processes, and molecular functioning of proline-rich CWPs along with their expression at different plant developmental stages. The mined genes were categorized into five classes on the basis of the type of PRPs encoded in the cell wall of Arabidopsis thaliana. We review the domain structure and function of each class of protein, many with respect to the developmental stages of the plant. We have then used networks, hierarchical clustering and correlations to analyze co-expression, co-localization, genetic, and physical interactions and shared protein domains of these PRPs. This has given us further insight into these functionally important CWPs and identified a number of potentially new cell-wall related proteins in A. thaliana. PMID:28289422

Leucine-Enriched Essential Amino Acids Augment Mixed Protein Synthesis, But Not Collagen Protein Synthesis, in Rat Skeletal Muscle after Downhill Running

PubMed Central

Kato, Hiroyuki; Suzuki, Hiromi; Inoue, Yoshiko; Suzuki, Katsuya; Kobayashi, Hisamine

2016-01-01

Mixed and collagen protein synthesis is elevated for as many as 3 days following exercise. Immediately after exercise, enhanced amino acid availability increases synthesis of mixed muscle protein, but not muscle collagen protein. However, the potential for synergic effects of amino acid ingestion with exercise on both mixed and collagen protein synthesis remains unclear. We investigated muscle collagen protein synthesis in rats following post-exercise ingestion of leucine-enriched essential amino acids. We determined fractional protein synthesis rates (FSR) at different time points following exercise. Mixed protein and collagen protein FSRs in skeletal muscle were determined by measuring protein-bound enrichments of hydroxyproline and proline, and by measuring the intracellular enrichment of proline, using injections of flooding d3-proline doses. A leucine-enriched mixture of essential amino acids (or distilled water as a control) was administrated 30 min or 1 day post-exercise. The collagen protein synthesis in the vastus lateralis was elevated for 2 days after exercise. Although amino acid administration did not increase muscle collagen protein synthesis, it did lead to augmented mixed muscle protein synthesis 1 day following exercise. Thus, contrary to the regulation of mixed muscle protein synthesis, muscle collagen protein synthesis is not affected by amino acid availability after damage-inducing exercise. PMID:27367725

Molecular dynamics simulation of the interactions between EHD1 EH domain and multiple peptides* #

PubMed Central

Yu, Hua; Wang, Mao-Jun; Xuan, Nan-Xia; Shang, Zhi-Cai; Wu, Jun

2015-01-01

Objective: To provide essential information for peptide inhibitor design, the interactions of Eps15 homology domain of Eps15 homology domain-containing protein 1 (EHD1 EH domain) with three peptides containing NPF (asparagine-proline-phenylalanine), DPF (aspartic acid-proline-phenylalanine), and GPF (glycine-proline-phenylalanine) motifs were deciphered at the atomic level. The binding affinities and the underlying structure basis were investigated. Methods: Molecular dynamics (MD) simulations were performed on EHD1 EH domain/peptide complexes for 60 ns using the GROMACS package. The binding free energies were calculated and decomposed by molecular mechanics/generalized Born surface area (MM/GBSA) method using the AMBER package. The alanine scanning was performed to evaluate the binding hot spot residues using FoldX software. Results: The different binding affinities for the three peptides were affected dominantly by van der Waals interactions. Intermolecular hydrogen bonds provide the structural basis of contributions of van der Waals interactions of the flanking residues to the binding. Conclusions: van der Waals interactions should be the main consideration when we design peptide inhibitors of EHD1 EH domain with high affinities. The ability to form intermolecular hydrogen bonds with protein residues can be used as the factor for choosing the flanking residues. PMID:26465136

Development of a pharmaceutical cocrystal with solution crystallization technology: Preparation, characterization, and evaluation of myricetin-proline cocrystals.

PubMed

Liu, Mingyu; Hong, Chao; Yao, Yashu; Shen, Hongyi; Ji, Guang; Li, Guowen; Xie, Yan

2016-10-01

Myricetin shows low oral bioavailability (<10%) in rats due to poor aqueous solubility, although it has demonstrated various pharmacological activities such as those related to anticancer, anti-diabetes, and hepatic protection. To overcome this issue, in this study, pharmaceutical cocrystals were designed to efficiently deliver myricetin by oral administration. A 1:2 stoichiometric cocrystal of myricetin with proline was prepared successfully by solution crystallization based on the ternary phase diagram (TPD) principle, and it is presented as a new sphericity-like crystalline phase characterized by differential scanning calorimetry (DSC), powder X-ray diffraction (PXRD), and scanning electron microscopy (SEM). The formation of myricetin-proline cocrystals was a spontaneous and exothermic process, probably due to the supramolecular interactions between themselves, which were determined by Fourier transform-infrared spectroscopy (FT-IR). Consequently, the dissolution efficiency of myricetin from cocrystals was increased 7.69-fold compared with that of coarse myricetin, and the oral bioavailability of myricetin cocrystals in rats was enhanced by approximately 3.03 times compared with that of pure myricetin. The present study provides useful information for the potential application of cocrystal technology for water-insoluble drugs, especially flavonoid compounds. Copyright © 2016 Elsevier B.V. All rights reserved.

Physiological and Biochemical Responses in Two Ornamental Shrubs to Drought Stress

PubMed Central

Toscano, Stefania; Farieri, Elisa; Ferrante, Antonio; Romano, Daniela

2016-01-01

Drought stress is one of the most important abiotic stress limiting the plant survival and growth in the Mediterranean environment. In this work, two species typically grown in Mediterranean areas with different drought responses were used. Two shrubs, with slow (Photinia × fraseri Dress ‘Red Robin’) or fast (Eugenia uniflora L. ‘Etna Fire’) adaptation ability to drought, were subjected to three water regimes: well-watered (WW), moderate (MD), and severe (SD) drought stress conditions for 30 days. Net photosynthetic rate, stomatal conductance, maximum quantum efficiency of PSII photochemistry (Fv/Fm), relative water content (RWC), chlorophyll content, proline, malondialdehyde (MDA), and antioxidant enzyme activities (superoxide dismutase, catalase, and peroxidase) were measured. Results showed that RWC and proline were higher in Eugenia than in Photinia, demonstrating the greater tolerance of the latter to the water stress. The drought stress levels applied did not compromise photosynthetic efficiency through stomatal regulation, while a reduction of Fv/Fm ratio was observed at the end of the experimental period. MDA significantly increased after 30 days in both species. The antioxidant enzyme activities showed different responses to water stress conditions. In both species, the water stress scores showed positive, while proline content showed negative correlations with all physiological parameters. PMID:27242846

Bio-mimicking of Proline-Rich Motif Applied to Carbon Nanotube Reveals Unexpected Subtleties Underlying Nanoparticle Functionalization

NASA Astrophysics Data System (ADS)

Zhang, Yuanzhao; Jimenez-Cruz, Camilo A.; Wang, Jian; Zhou, Bo; Yang, Zaixing; Zhou, Ruhong

2014-11-01

Here, we report computational studies of the SH3 protein domain interacting with various single-walled carbon nanotubes (SWCNT) either bare or functionalized by mimicking the proline-rich motif (PRM) ligand (PPPVPPRR) and compare it to the SH3-PRM complex binding. With prolines or a single arginine attached, the SWCNT gained slightly on specificity when compared with the bare control, whereas with multi-arginine systems the specificity dropped dramatically to our surprise. Although the electrostatic interaction provided by arginines is crucial in the recognition between PRM and SH3 domain, our results suggest that attaching multiple arginines to the SWCNT has a detrimental effect on the binding affinity. Detailed analysis of the MD trajectories found two main factors that modulate the specificity of the binding: the existence of competing acidic patches at the surface of SH3 that leads to ``trapping and clamping'' by the arginines, and the rigidity of the SWCNT introducing entropic penalties in the proper binding. Further investigation revealed that the same ``clamping'' phenomenon exits in the PRM-SH3 system, which has not been reported in previous literature. The competing effects between nanoparticle and its functionalization components revealed by our model system should be of value to current and future nanomedicine designs.

Profilin Is Required for Optimal Actin-Dependent Transcription of Respiratory Syncytial Virus Genome RNA

PubMed Central

Burke, Emily; Mahoney, Nicole M.; Almo, Steven C.; Barik, Sailen

2000-01-01

Transcription of human respiratory syncytial virus (RSV) genome RNA exhibited an obligatory need for the host cytoskeletal protein actin. Optimal transcription, however, required the participation of another cellular protein that was characterized as profilin by a number of criteria. The amino acid sequence of the protein, purified on the basis of its transcription-optimizing activity in vitro, exactly matched that of profilin. RSV transcription was inhibited 60 to 80% by antiprofilin antibody or poly-l-proline, molecules that specifically bind profilin. Native profilin, purified from extracts of lung epithelial cells by affinity binding to a poly-l-proline matrix, stimulated the actin-saturated RSV transcription by 2.5- to 3-fold. Recombinant profilin, expressed in bacteria, stimulated viral transcription as effectively as the native protein and was also inhibited by poly-l-proline. Profilin alone, in the absence of actin, did not activate viral transcription. It is estimated that at optimal levels of transcription, every molecule of viral genomic RNA associates with approximately the following number of protein molecules: 30 molecules of L, 120 molecules of phosphoprotein P, and 60 molecules each of actin and profilin. Together, these results demonstrated for the first time a cardinal role for profilin, an actin-modulatory protein, in the transcription of a paramyxovirus RNA genome. PMID:10623728

Diplodiatoxin, chaetoglobosins, and diplonine associated with a field outbreak of Stenocarpella ear rot in Illinois.

PubMed

Rogers, Kristina D; Cannistra, Joseph C; Gloer, James B; Wicklow, Donald T

2014-05-01

Stenocarpella maydis causes a fungal dry-rot of maize ears and is associated with diplodiosis, a neuromycotoxicosis in cattle grazing harvested maize fields in southern Africa and Argentina. There have been no reports of Stenocarpella metabolites in maize crop residues. Chemical investigations of S. maydis-infected grain from ears exhibiting different levels of ear rot severity following a 2010 field outbreak of Stenocarpella ear rot in Illinois led to the detection of diplodiatoxin and chaetoglobosins M and O as major components in the ethyl acetate extracts by LC-MS. Following post-harvest moist incubation of the S. maydis-infected grain, the amounts of each compound increased (approx. tenfold) and chaetoglobosin K was detected as a dominant toxin. In separate (1)H NMR-based analyses, the neurotoxin diplonine was detected as a minor component in methanol extracts of S. maydis-infected grain as well as cultures of S. maydis isolates from Midwest corn. Proline betaine (=stachydrine) and glycine betaine were also detected in these extracts as major components. This constitutes the first report of chaetoglobosin M, chaetoglobosin O, proline betaine, or glycine betaine from S. maydis, and the first record of diplodiatoxin, diplonine, proline betaine, glycine betaine, or chaetoglobosins M, O, or K being associated with a natural field outreak of S. maydis ear rot.

The docking of chiral analytes on proline-based chiral stationary phases: A molecular dynamics study of selectivity.

PubMed

Ashtari, M; Cann, N M

2015-08-28

Molecular dynamics simulations are employed to examine the selectivity of four proline-based chiral stationary phases in two solvent environments, a relatively apolar n-hexane/2-propanol solvent and a polar water/methanol solvent. The four chiral surfaces are based on a BOC-terminated diproline, a TMA-terminated diproline, a TMA-terminated triproline and a TMA-terminated hexaproline. This range of chiral selectors allows an analysis of the impact of oligomer length and terminal group on selectivity while the two solvent environments indicate the impact of solvent hydrogen bonding and polarity. The selector-analyte interactions are examined for six closely related analytes that each have an aromatic moiety, a hydrogen, and an alcohol group directly bonded to the stereocenter. The analytes differ in the nature of the aromatic group (phenyl or anthracyl), in the attachment point (to the central ring or a side ring in the anthracyl), and in the fourth group bonded to the carbon (CH3, CF3, or C2H5). For each of the 48 solvent+selector+analyte systems, selectivity factors are calculated and, when possible, compared to experiment. The docking mode for these proline-based selectors is analyzed. Copyright © 2015 Elsevier B.V. All rights reserved.

Effects of osmolytes on Pelodiscus sinensis creatine kinase: a study on thermal denaturation and aggregation.

PubMed

Wang, Wei; Lee, Jinhyuk; Jin, Qin-Xin; Fang, Nai-Yun; Si, Yue-Xiu; Yin, Shang-Jun; Qian, Guo-Ying; Park, Yong-Doo

2013-09-01

The protective effect of osmolytes on the thermal denaturation and aggregation of Pelodiscus sinensis muscle creatine kinase (PSCK) was investigated by a combination of spectroscopic methods and thermodynamic analysis. Our results demonstrated that the addition of osmolytes, such as glycine and proline, could prevent thermal denaturation and aggregation of PSCK in a concentration-dependent manner. When the concentration of glycine and proline increased in the denatured system, the relative activation was significantly enhanced; meanwhile, the aggregation of PSCK during thermal denaturation was decreased. Spectrofluorometer results showed that glycine and proline significantly decreased the tertiary structural changes of PSCK and that thermal denaturation directly induced PSCK aggregation. In addition, we also built the 3D structure of PSCK and osmolytes by homology models. The results of computational docking simulations showed that the docking energy was relatively low and that the clustering groups were spread to the surface of PSCK, indicating that osmolytes directly protect the surface of the protein. P. sinensis are poikilothermic and quite sensitive to the change of ambient temperature; however, there were few studies on the thermal denaturation of reptile CK. Our study provides important insight into the protective effects of osmolytes on thermal denaturation and aggregation of PSCK. Copyright © 2013 Elsevier B.V. All rights reserved.

Bio-mimicking of Proline-Rich Motif Applied to Carbon Nanotube Reveals Unexpected Subtleties Underlying Nanoparticle Functionalization

PubMed Central

Zhang, Yuanzhao; Jimenez-Cruz, Camilo A.; Wang, Jian; Zhou, Bo; Yang, Zaixing; Zhou, Ruhong

2014-01-01

Here, we report computational studies of the SH3 protein domain interacting with various single-walled carbon nanotubes (SWCNT) either bare or functionalized by mimicking the proline-rich motif (PRM) ligand (PPPVPPRR) and compare it to the SH3-PRM complex binding. With prolines or a single arginine attached, the SWCNT gained slightly on specificity when compared with the bare control, whereas with multi-arginine systems the specificity dropped dramatically to our surprise. Although the electrostatic interaction provided by arginines is crucial in the recognition between PRM and SH3 domain, our results suggest that attaching multiple arginines to the SWCNT has a detrimental effect on the binding affinity. Detailed analysis of the MD trajectories found two main factors that modulate the specificity of the binding: the existence of competing acidic patches at the surface of SH3 that leads to “trapping and clamping” by the arginines, and the rigidity of the SWCNT introducing entropic penalties in the proper binding. Further investigation revealed that the same “clamping” phenomenon exits in the PRM-SH3 system, which has not been reported in previous literature. The competing effects between nanoparticle and its functionalization components revealed by our model system should be of value to current and future nanomedicine designs. PMID:25427563

Engineering of Glarea lozoyensis for Exclusive Production of the Pneumocandin B0 Precursor of the Antifungal Drug Caspofungin Acetate

PubMed Central

Chen, Li; Yue, Qun; Li, Yan; Niu, Xuemei; Xiang, Meichun; Wang, Wenzhao; Bills, Gerald F.

2014-01-01

Pneumocandins produced by the fungus Glarea lozoyensis are acylated cyclic hexapeptides of the echinocandin family. Pneumocandin B0 is the starting molecule for the first semisynthetic echinocandin antifungal drug, caspofungin acetate. In the wild-type strain, pneumocandin B0 is a minor fermentation product, and its industrial production was achieved by a combination of extensive mutation and medium optimization. The pneumocandin biosynthetic gene cluster was previously elucidated by a whole-genome sequencing approach. Knowledge of the biosynthetic cluster suggested an alternative way to produce exclusively pneumocandin B0. Disruption of GLOXY4, encoding a nonheme, α-ketoglutarate-dependent oxygenase, confirmed its involvement in l-leucine cyclization to form 4S-methyl-l-proline. The absence of 4S-methyl-l-proline abolishes pneumocandin A0 production, and 3S-hydroxyl-l-proline occupies the hexapeptide core's position 6, resulting in exclusive production of pneumocandin B0. Retrospective analysis of the GLOXY4 gene in a previously isolated pneumocandin B0-exclusive mutant (ATCC 74030) indicated that chemical mutagenesis disrupted the GLOXY4 gene function by introducing two amino acid mutations in GLOXY4. This one-step genetic manipulation can rationally engineer a high-yield production strain. PMID:25527531

Effect of saline irrigation water on gas exchange and proline metabolism in ber (Ziziphus).

PubMed

Bagdi, D L; Bagri, G K

2016-09-01

An experiment was conducted in pots of 25 kg capacity to study the effect of saline irrigation (EC 0,5,10,15 and 20 dSm-1) prepared by mixing NaCl, NaSO4, CaCl and MgCl2 in 3:1 ratio of chloride and sulphate on gas exchange traits, membrane stability, chlorophyll stability index and osmolytic defense mechanism in Ziziphus rotundifolia and Ziziphus nummularia species of Indian jujube (Z.mauritiana). Result showed that net photosynthetic rate (PN), transpiration (e) and stomatal conductance were comparatively lower in Ziziphus nummularia, which further declined with increasing level of saline irrigation water. Chlorophyll stability and membrane stability also declined significantly in salt stress, with higher magnitude in Ziziphus nummularia. The activity of proline anabolic enzymes; Δ1-Pyrrolline-5-carboxylate reductase, Δ1-Pyrrolline-5-carboxylate synthetase and Ornithine-δ-aminotransferase were recorded higher in Ziziphus rotundifolia with decrease in proline dehydrogenase. The sodium content was observed higher in roots of Ziziphus rotundifolia and leaves of Ziziphus nummularia. Therefore, it is suggested that salt tolerance mechanism was more efficiently operative in Ziziphus rotundifolia owing to better management of physiological attributes, osmolytic defense mechanism and restricted translocation of sodium from root to leaves along with larger accumulation of potassium in its leaves.

Dose- and Glucose-Dependent Effects of Amino Acids on Insulin Secretion from Isolated Mouse Islets and Clonal INS-1E Beta-Cells

PubMed Central

Liu, Zhenping; Jeppesen, Per B.; Gregersen, Søren; Chen, Xiaoping; Hermansen, Kjeld

2008-01-01

BACKGROUND: The influence of glucose and fatty acids on beta-cell function is well established whereas little is known about the role of amino acids (AAs). METHODS: Islets isolated from NMRI mice were incubated overnight. After preincubation, isolated islets as well as clonal INS-1E beta-cells were incubated for 60 min in a modified Krebs Ringer buffer containing glucose and AAs. RESULTS: At 16.7 mmol/l (mM) glucose, L-arginine, L-lysine, L-alanine, L-proline, L-leucine, and L-glutamine potentiated glucose-stimulated insulin secretion dose-dependently, while DL-homocysteine inhibited insulin secretion. Maximal insulin stimulation was obtained at 20 mM L-proline, L-lysine, L-alanine, L-arginine (islets: 2.5 to 6.7 fold increase; INS-1E cells: 1.6 to 2.2 fold increase). L-glutamine and L-leucine only increased glucose-stimulated (16.7 mM) insulin secretion (INS-1E cells: 1.5 and 1.3 fold, respectively) at an AA concentration of 20 mM. Homocysteine inhibited insulin secretion both at 5.6 mM and 16.7 mM glucose. At glucose levels ranging from 1.1 to 25 mM, the equimolar concentration of 10 mM, L-proline, L-lysine, L-arginine increased insulin secretion from mouse islets and INS-1E cells at all glucose levels applied, with a maximal effect obtained at 25 mM glucose. At a concentration of 10 mM, L-arginine and L-lysine had the highest insulinotropic potency among the AAs investigated. CONCLUSION: L-arginine, L-lysine, L-alanine, L-proline, L-leucine and L-glutamine acutely stimulate insulin secretion from mouse islets and INS-1E cells in a dose- and glucose-dependent manner, whereas DL-homocysteine inhibits insulin release. PMID:19290384

Production in Pichia pastoris of complementary protein-based polymers with heterodimer-forming WW and PPxY domains.

PubMed

Domeradzka, Natalia E; Werten, Marc W T; de Vries, Renko; de Wolf, Frits A

2016-06-10

Specific coupling of de novo designed recombinant protein polymers for the construction of precisely structured nanomaterials is of interest for applications in biomedicine, pharmaceutics and diagnostics. An attractive coupling strategy is to incorporate specifically interacting peptides into the genetic design of the protein polymers. An example of such interaction is the binding of particular proline-rich ligands by so-called WW-domains. In this study, we investigated whether these domains can be produced in the yeast Pichia pastoris as part of otherwise non-interacting protein polymers, and whether they bring about polymer coupling upon mixing. We constructed two variants of a highly hydrophilic protein-based polymer that differ only in their C-terminal extensions. One carries a C-terminal WW domain, and the other a C-terminal proline-rich ligand (PPxY). Both polymers were produced in P. pastoris with a purified protein yield of more than 2 g L(-1) of cell-free broth. The proline-rich module was found to be O-glycosylated, and uncommonly a large portion of the attached oligosaccharides was phosphorylated. Glycosylation was overcome by introducing a Ser → Ala mutation in the PPxY peptide. Tryptophan fluorescence monitored during titration of the polymer containing the WW domain with either the glycosylated or nonglycosylated PPxY-containing polymer revealed binding. The complementary polymers associated with a Kd of ~3 µM, regardless of glycosylation state of the PPxY domain. Binding was confirmed by isothermal titration calorimetry, with a Kd of ~9 µM. This article presents a blueprint for the production in P. pastoris of protein polymers that can be coupled using the noncovalent interaction between WW domains and proline-rich ligands. The availability of this highly specific coupling tool will hereafter allow us to construct various supramolecular structures and biomaterials.

Water-Deficit Tolerance in Sweet Potato [Ipomoea batatas (L.) Lam.] by Foliar Application of Paclobutrazol: Role of Soluble Sugar and Free Proline.

PubMed

Yooyongwech, Suravoot; Samphumphuang, Thapanee; Tisarum, Rujira; Theerawitaya, Cattarin; Cha-Um, Suriyan

2017-01-01

The objective of this study was to elevate water deficit tolerance by improving soluble sugar and free proline accumulation, photosynthetic pigment stabilization, photosynthetic abilities, growth performance and storage root yield in sweet potato cv. 'Tainung 57' using a foliar application of paclobutrazol (PBZ). The experiment followed a Completely Randomized Block Design with four concentrations of PBZ: 0 (control), 17, 34, and 51 μM before exposure to 47.5% (well irrigation), 32.3% (mild water deficit) or 17.5% (severe water deficit) soil water content. A sweet potato cultivar, 'Japanese Yellow', with water deficit tolerance attributes was the positive check in this study. Total soluble sugar content (sucrose, glucose, and fructose) increased by 3.96-folds in 'Tainung 57' plants treated with 34 μM PBZ grown under 32.3% soil water content (SWC) compared to the untreated plants, adjusting osmotic potential in the leaves and controlling stomatal closure (represented by stomatal conductance and transpiration rate). In addition, under the same treatment, free proline content (2.15 μmol g -1 FW) increased by 3.84-folds when exposed to 17.5% SWC. PBZ had an improved effect on leaf size, vine length, photosynthetic pigment stability, chlorophyll fluorescence, and net photosynthetic rate; hence, delaying wilting symptoms and maintaining storage root yield (26.93 g plant -1 ) at the harvesting stage. A positive relationship between photon yield of PSII (Φ PSII ) and net photosynthetic rate was demonstrated ( r 2 = 0.73). The study concludes that soluble sugar and free proline enrichment in PBZ-pretreated plants may play a critical role as major osmoprotectant to control leaf osmotic potential and stomatal closure when plants were subjected to low soil water content, therefore, maintaining the physiological and morphological characters as well as storage root yield.

Ineffective Degradation of Immunogenic Gluten Epitopes by Currently Available Digestive Enzyme Supplements

PubMed Central

Janssen, George; Christis, Chantal; Kooy-Winkelaar, Yvonne; Edens, Luppo; Smith, Drew

2015-01-01

Background Due to the high proline content of gluten molecules, gastrointestinal proteases are unable to fully degrade them leaving large proline-rich gluten fragments intact, including an immunogenic 33-mer from α-gliadin and a 26-mer from γ-gliadin. These latter peptides can trigger pro-inflammatory T cell responses resulting in tissue remodeling, malnutrition and a variety of other complications. A strict lifelong gluten-free diet is currently the only available treatment to cope with gluten intolerance. Post-proline cutting enzymes have been shown to effectively degrade the immunogenic gluten peptides and have been proposed as oral supplements. Several existing digestive enzyme supplements also claim to aid in gluten degradation. Here we investigate the effectiveness of such existing enzyme supplements in comparison with a well characterized post-proline cutting enzyme, Prolyl EndoPeptidase from Aspergillus niger (AN-PEP). Methods Five commercially available digestive enzyme supplements along with purified digestive enzymes were subjected to 1) enzyme assays and 2) mass spectrometric identification. Gluten epitope degradation was monitored by 1) R5 ELISA, 2) mass spectrometric analysis of the degradation products and 3) T cell proliferation assays. Findings The digestive enzyme supplements showed comparable proteolytic activities with near neutral pH optima and modest gluten detoxification properties as determined by ELISA. Mass spectrometric analysis revealed the presence of many different enzymes including amylases and a variety of different proteases with aminopeptidase and carboxypeptidase activity. The enzyme supplements leave the nine immunogenic epitopes of the 26-mer and 33-mer gliadin fragments largely intact. In contrast, the pure enzyme AN-PEP effectively degraded all nine epitopes in the pH range of the stomach at much lower dose. T cell proliferation assays confirmed the mass spectrometric data. Conclusion Currently available digestive enzyme supplements are ineffective in degrading immunogenic gluten epitopes. PMID:26030273

Testis-specific glyceraldehyde-3-phosphate dehydrogenase: origin and evolution

PubMed Central

2011-01-01

Background Glyceraldehyde-3-phosphate dehydrogenase (GAPD) catalyses one of the glycolytic reactions and is also involved in a number of non-glycolytic processes, such as endocytosis, DNA excision repair, and induction of apoptosis. Mammals are known to possess two homologous GAPD isoenzymes: GAPD-1, a well-studied protein found in all somatic cells, and GAPD-2, which is expressed solely in testis. GAPD-2 supplies energy required for the movement of spermatozoa and is tightly bound to the sperm tail cytoskeleton by the additional N-terminal proline-rich domain absent in GAPD-1. In this study we investigate the evolutionary history of GAPD and gain some insights into specialization of GAPD-2 as a testis-specific protein. Results A dataset of GAPD sequences was assembled from public databases and used for phylogeny reconstruction by means of the Bayesian method. Since resolution in some clades of the obtained tree was too low, syntenic analysis was carried out to define the evolutionary history of GAPD more precisely. The performed selection tests showed that selective pressure varies across lineages and isoenzymes, as well as across different regions of the same sequences. Conclusions The obtained results suggest that GAPD-1 and GAPD-2 emerged after duplication during the early evolution of chordates. GAPD-2 was subsequently lost by most lineages except lizards, mammals, as well as cartilaginous and bony fishes. In reptilians and mammals, GAPD-2 specialized to a testis-specific protein and acquired the novel N-terminal proline-rich domain anchoring the protein in the sperm tail cytoskeleton. This domain is likely to have originated by exonization of a microsatellite genomic region. Recognition of the proline-rich domain by cytoskeletal proteins seems to be unspecific. Besides testis, GAPD-2 of lizards was also found in some regenerating tissues, but it lacks the proline-rich domain due to tissue-specific alternative splicing. PMID:21663662

Evolution of hard proteins in the sauropsid integument in relation to the cornification of skin derivatives in amniotes.

PubMed

Alibardi, Lorenzo; Dalla Valle, Luisa; Nardi, Alessia; Toni, Mattia

2009-04-01

Hard skin appendages in amniotes comprise scales, feathers and hairs. The cell organization of these appendages probably derived from the localization of specialized areas of dermal-epidermal interaction in the integument. The horny scales and the other derivatives were formed from large areas of dermal-epidermal interaction. The evolution of these skin appendages was characterized by the production of specific coiled-coil keratins and associated proteins in the inter-filament matrix. Unlike mammalian keratin-associated proteins, those of sauropsids contain a double beta-folded sequence of about 20 amino acids, known as the core-box. The core-box shows 60%-95% sequence identity with known reptilian and avian proteins. The core-box determines the polymerization of these proteins into filaments indicated as beta-keratin filaments. The nucleotide and derived amino acid sequences for these sauropsid keratin-associated proteins are presented in conjunction with a hypothesis about their evolution in reptiles-birds compared to mammalian keratin-associated proteins. It is suggested that genes coding for ancestral glycine-serine-rich sequences of alpha-keratins produced a new class of small matrix proteins. In sauropsids, matrix proteins may have originated after mutation and enrichment in proline, probably in a central region of the ancestral protein. This mutation gave rise to the core-box, and other regions of the original protein evolved differently in the various reptilians orders. In lepidosaurians, two main groups, the high glycine proline and the high cysteine proline proteins, were formed. In archosaurians and chelonians two main groups later diversified into the high glycine proline tyrosine, non-feather proteins, and into the glycine-tyrosine-poor group of feather proteins, which evolved in birds. The latter proteins were particularly suited for making the elongated barb/barbule cells of feathers. In therapsids-mammals, mutations of the ancestral proteins formed the high glycine-tyrosine or the high cysteine proteins but no core-box was produced in the matrix proteins of the hard corneous material of mammalian derivatives.

Testis-specific glyceraldehyde-3-phosphate dehydrogenase: origin and evolution.

PubMed

Kuravsky, Mikhail L; Aleshin, Vladimir V; Frishman, Dmitrij; Muronetz, Vladimir I

2011-06-10

Glyceraldehyde-3-phosphate dehydrogenase (GAPD) catalyses one of the glycolytic reactions and is also involved in a number of non-glycolytic processes, such as endocytosis, DNA excision repair, and induction of apoptosis. Mammals are known to possess two homologous GAPD isoenzymes: GAPD-1, a well-studied protein found in all somatic cells, and GAPD-2, which is expressed solely in testis. GAPD-2 supplies energy required for the movement of spermatozoa and is tightly bound to the sperm tail cytoskeleton by the additional N-terminal proline-rich domain absent in GAPD-1. In this study we investigate the evolutionary history of GAPD and gain some insights into specialization of GAPD-2 as a testis-specific protein. A dataset of GAPD sequences was assembled from public databases and used for phylogeny reconstruction by means of the Bayesian method. Since resolution in some clades of the obtained tree was too low, syntenic analysis was carried out to define the evolutionary history of GAPD more precisely. The performed selection tests showed that selective pressure varies across lineages and isoenzymes, as well as across different regions of the same sequences. The obtained results suggest that GAPD-1 and GAPD-2 emerged after duplication during the early evolution of chordates. GAPD-2 was subsequently lost by most lineages except lizards, mammals, as well as cartilaginous and bony fishes. In reptilians and mammals, GAPD-2 specialized to a testis-specific protein and acquired the novel N-terminal proline-rich domain anchoring the protein in the sperm tail cytoskeleton. This domain is likely to have originated by exonization of a microsatellite genomic region. Recognition of the proline-rich domain by cytoskeletal proteins seems to be unspecific. Besides testis, GAPD-2 of lizards was also found in some regenerating tissues, but it lacks the proline-rich domain due to tissue-specific alternative splicing.

Water-Deficit Tolerance in Sweet Potato [Ipomoea batatas (L.) Lam.] by Foliar Application of Paclobutrazol: Role of Soluble Sugar and Free Proline

PubMed Central

Yooyongwech, Suravoot; Samphumphuang, Thapanee; Tisarum, Rujira; Theerawitaya, Cattarin; Cha-um, Suriyan

2017-01-01

The objective of this study was to elevate water deficit tolerance by improving soluble sugar and free proline accumulation, photosynthetic pigment stabilization, photosynthetic abilities, growth performance and storage root yield in sweet potato cv. ‘Tainung 57’ using a foliar application of paclobutrazol (PBZ). The experiment followed a Completely Randomized Block Design with four concentrations of PBZ: 0 (control), 17, 34, and 51 μM before exposure to 47.5% (well irrigation), 32.3% (mild water deficit) or 17.5% (severe water deficit) soil water content. A sweet potato cultivar, ‘Japanese Yellow’, with water deficit tolerance attributes was the positive check in this study. Total soluble sugar content (sucrose, glucose, and fructose) increased by 3.96-folds in ‘Tainung 57’ plants treated with 34 μM PBZ grown under 32.3% soil water content (SWC) compared to the untreated plants, adjusting osmotic potential in the leaves and controlling stomatal closure (represented by stomatal conductance and transpiration rate). In addition, under the same treatment, free proline content (2.15 μmol g-1 FW) increased by 3.84-folds when exposed to 17.5% SWC. PBZ had an improved effect on leaf size, vine length, photosynthetic pigment stability, chlorophyll fluorescence, and net photosynthetic rate; hence, delaying wilting symptoms and maintaining storage root yield (26.93 g plant-1) at the harvesting stage. A positive relationship between photon yield of PSII (ΦPSII) and net photosynthetic rate was demonstrated (r2 = 0.73). The study concludes that soluble sugar and free proline enrichment in PBZ-pretreated plants may play a critical role as major osmoprotectant to control leaf osmotic potential and stomatal closure when plants were subjected to low soil water content, therefore, maintaining the physiological and morphological characters as well as storage root yield. PMID:28848596

Impaired helix 12 dynamics due to proline 892 substitutions in the androgen receptor are associated with complete androgen insensitivity.

PubMed

Elhaji, Youssef A; Stoica, Ileana; Dennis, Sheldon; Purisima, Enrico O; Lumbroso, Rose; Beitel, Lenore K; Trifiro, Mark A

2006-03-15

Structural studies of the ligand-binding domain (LBD) of several steroid receptors have revealed that the dynamic properties of the C-terminal helix 12 (H12) are the major determinant of the activation mode of these receptors. H12 exhibits high mobility and different conformations in the absence of ligand. Upon ligand binding, H12 is stabilized in a precise position to seal the ligand-binding pocket and finalize the assembly of the activation function (AF-2) domain. In this study, we investigated the role of the conserved proline 892 of the androgen receptor (AR) in directing the dynamic location and orientation of the AR-H12. We used a combined approach including kinetic and biochemical assays with molecular dynamic simulations to analyze two substitutions (P892A and P892L) identified in individuals with complete androgen insensitivity syndrome. Our analyses revealed distinct mechanisms by which these substitutions impair H12 function resulting in severely defective receptors. The AR-P892A receptor exhibited reduced ligand binding and transactivational potential because of an increased flexibility in H12. The AR-P892L substitution renders the receptor inactive due to a distorted, unstructured and misplaced H12. To confirm the mutants' inability to stabilize H12 in an active position, we have developed a novel in vivo assay to evaluate the accessibility of the H12-docking site on the AR-LBD surface. An extrinsic AR-H12 peptide was able to interact with wild-type and mutant LBDs in the absence of ligand. Ligand-induced proper positioning of the intrinsic H12 of wild-type AR prevented these interactions, whereas the misplacement of the mutants' H12 did not. Proline at this position may be critical for H12 dynamics not only in the AR, but also in other nuclear receptors where this proline is conserved.

Field controlled experiments on the physiological responses of maize (Zea mays L.) leaves to low-level air and soil mercury exposures.

PubMed

Niu, Zhenchuan; Zhang, Xiaoshan; Wang, Sen; Zeng, Ming; Wang, Zhangwei; Zhang, Yi; Ci, Zhijia

2014-01-01

Thousands of tons of mercury (Hg) are released from anthropogenic and natural sources to the atmosphere in a gaseous elemental form per year, yet little is known regarding the influence of airborne Hg on the physiological activities of plant leaves. In the present study, the effects of low-level air and soil Hg exposures on the gas exchange parameters of maize (Zea mays L.) leaves and their accumulation of Hg, proline, and malondialdehyde (MDA) were examined via field open-top chamber and Hg-enriched soil experiments, respectively. Low-level air Hg exposures (<50 ng m(-3)) had little effects on the gas exchange parameters of maize leaves during most of the daytime (p > 0.05). However, both the net photosynthesis rate and carboxylation efficiency of maize leaves exposed to 50 ng m(-3) air Hg were significantly lower than those exposed to 2 ng m(-3) air Hg in late morning (p < 0.05). Additionally, the Hg, proline, and MDA concentrations in maize leaves exposed to 20 and 50 ng m(-3) air Hg were significantly higher than those exposed to 2 ng m(-3) air Hg (p < 0.05). These results indicated that the increase in airborne Hg potentially damaged functional photosynthetic apparatus in plant leaves, inducing free proline accumulation and membrane lipid peroxidation. Due to minor translocation of soil Hg to the leaves, low-level soil Hg exposures (<1,000 ng g(-1)) had no significant influences on the gas exchange parameters, or the Hg, proline, and MDA concentrations in maize leaves (p > 0.05). Compared to soil Hg, airborne Hg easily caused physiological stress to plant leaves. The effects of increasing atmospheric Hg concentration on plant physiology should be of concern.

Bacillus anthracis Prolyl 4-Hydroxylase Interacts with and Modifies Elongation Factor Tu

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schnicker, Nicholas J.; Razzaghi, Mortezaali; Guha Thakurta, Sanjukta

Prolyl hydroxylation is a very common post-translational modification and plays many roles in eukaryotes such as collagen stabilization, hypoxia sensing, and controlling protein transcription and translation. There is a growing body of evidence that suggests that prokaryotes contain prolyl 4-hydroxylases (P4Hs) homologous to the hypoxia-inducible factor (HIF) prolyl hydroxylase domain (PHD) enzymes that act on elongation factor Tu (EFTu) and are likely involved in the regulation of bacterial translation. Recent biochemical and structural studies with a PHD from Pseudomonas putida (PPHD) determined that it forms a complex with EFTu and hydroxylates a prolyl residue of EFTu. Moreover, while animal, plant,more » and viral P4Hs act on peptidyl proline, most prokaryotic P4Hs have been known to target free l-proline; the exceptions include PPHD and a P4H from Bacillus anthracis (BaP4H) that modifies collagen-like proline-rich peptides. Here we use biophysical and mass spectrometric methods to demonstrate that BaP4H recognizes full-length BaEFTu and a BaEFTu 9-mer peptide for site-specific proline hydroxylation. Using size-exclusion chromatography coupled small-angle X-ray scattering (SEC–SAXS) and binding studies, we determined that BaP4H forms a 1:1 heterodimeric complex with BaEFTu. The SEC–SAXS studies reveal dissociation of BaP4H dimeric subunits upon interaction with BaEFTu. While BaP4H is unusual within bacteria in that it is structurally and functionally similar to the animal PHDs and collagen P4Hs, respectively, this work provides further evidence of its promiscuous substrate recognition. It is possible that the enzyme might have evolved to hydroxylate a universally conserved protein in prokaryotes, similar to the PHDs, and implies a functional role in B. anthracis.« less

Inhibition of Arabidopsis growth by the allelopathic compound azetidine-2-carboxylate is due to the low amino acid specificity of cytosolic prolyl-tRNA synthetase.

PubMed

Lee, Jiyeon; Joshi, Naveen; Pasini, Rita; Dobson, Renwick C J; Allison, Jane; Leustek, Thomas

2016-10-01

The toxicity of azetidine-2-carboxylic acid (A2C), a structural analogue of L-proline, results from its incorporation into proteins due to misrecognition by prolyl-tRNA synthetase (ProRS). The growth of Arabidopsis thaliana seedling roots is more sensitive to inhibition by A2C than is cotyledon growth. Arabidopsis contains two ProRS isozymes. AtProRS-Org (At5g52520) is localized in chloroplasts/mitochondria, and AtProRS-Cyt (At3g62120) is cytosolic. AtProRS-Cyt mRNA is more highly expressed in roots than in cotyledons. Arabidopsis ProRS isoforms were expressed as His-tagged recombinant proteins in Escherichia coli. Both enzymes were functionally active in ATP-PPi exchange and aminoacylation assays, and showed similar K m for L-proline. A major difference was observed in the substrate specificity of the two enzymes. AtProRS-Cyt showed nearly identical substrate specificity for L-proline and A2C, but for AtProRS-Org the specificity constant was 77.6 times higher for L-proline than A2C, suggesting that A2C-sensitivity may result from lower amino acid specificity of AtProRS-Cyt. Molecular modelling and simulation results indicate that this specificity difference between the AtProRS isoforms may result from altered modes of substrate binding. Similar kinetic results were obtained with the ProRSs from Zea mays, suggesting that the difference in substrate specificity is a conserved feature of ProRS isoforms from plants that do not accumulate A2C and are sensitive to A2C toxicity. The discovery of the mode of action of A2C toxicity could lead to development of biorational weed management strategies. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

Properties of a proline-containing glass ionomer dental cement.

PubMed

Ansari, Sahar; Moshaverinia, Maryam; Roohpour, Nima; Chee, Winston W L; Schricker, Scott R; Moshaverinia, Alireza

2013-11-01

Proline-containing glass ionomers are promising fast-set dental restorative materials with superior mechanical properties; however, little information is available on other physical properties of this type of glass ionomer. The objectives of this study were to synthesize and characterize a polyacrylic acid terpolymer containing proline derivative (PD) and to investigate the physical properties of this glass ionomer cement (GIC) and its cytotoxicity in vitro. A terpolymer of AA (acrylic acid), IA (itaconic acid), and proline derivative (MP) with an 8:1:1 molar ratio was synthesized and characterized. Experimental GIC specimens were made from the synthetized terpolymer with Fuji IX (GC America, Alsip, Ill) commercial glass ionomer powder as recommended by the manufacturer. Specimens were mixed and fabricated at room temperature and were conditioned in distilled water at 37°C for 1 day and 1 week. Vickers hardness was determined with a microhardness tester. The water sorption characteristics and fluoride releasing properties of the specimens were investigated. The in vitro cytotoxicity of the experimental glass ionomer was assessed by evaluating the C2C12 cell metabolism with methyltetrazolium (MTT) assay. Commercial Fuji IX was used as a control for comparison. The data obtained for the experimental GIC (PD) were compared with the control group by using 1- and 2-way ANOVA and the Tukey multiple range test at α=.05. Proline-modified GIC (PD) exhibited significantly higher surface hardness values (Vickers hardness number [VHN] 58 ±6.1) in comparison to Fuji IX GIC (VHN 47 ±5.3) after 1 week of maturation. Statistical analysis of data showed that the water sorption properties of the experimental cement (PD) were significantly greater than those of the control group (P<.05). The experimental GIC showed a significant increase in the amounts of initial fluoride release (P<.05) with continued fluoride release from the bulk of the material. The experimental group showed slightly reduced cell metabolism and cell number in comparison to the control group. However, the results were not statistically different (P>.05). An amino acid-containing GIC had better surface hardness properties than commercial Fuji IX GIC. This formulation of fast-set glass ionomer showed increased water sorption without adversely affecting the amount of fluoride release. Considering its biocompatibility, this material shows promise not only as a dental restorative material but also as a bone cement with low cytotoxicity. Copyright © 2013 Editorial Council for the Journal of Prosthetic Dentistry. Published by Mosby, Inc. All rights reserved.

Understanding the antimicrobial properties/activity of an 11-residue Lys homopeptide by alanine and proline scan.

PubMed

Carvajal-Rondanelli, P; Aróstica, M; Álvarez, C A; Ojeda, C; Albericio, F; Aguilar, L F; Marshall, S H; Guzmán, F

2018-05-01

Previous work demonstrated that lysine homopeptides adopt a polyproline II (PPII) structure. Lysine homopeptides with odd number of residues, especially with 11 residues (K11), were capable of inhibiting the growth of a broader spectrum of bacteria than those with an even number. Confocal studies also determined that K11 was able to localize exclusively in the bacterial membrane, leading to cell death. In this work, the mechanism of action of this peptide was further analyzed focused on examining the structural changes in bacterial membrane induced by K11, and in K11 itself when interacting with bacterial membrane lipids. Moreover, alanine and proline scans were performed for K11 to identify relevant positions in structure conformation and antibacterial activity. To do so, circular dichroism spectroscopy (CD) was conducted in saline phosphate buffer (PBS) and in lipidic vesicles, using large unilamellar vesicles (LUV), composed of 2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) or bacterial membrane lipid. Antimicrobial activity of K11 and their analogs was evaluated in Gram-positive and Gram-negative bacterial strains. The scanning electron microscopy (SEM) micrographs of Staphylococcus aureus ATCC 25923 exposed to the Lys homopeptide at MIC concentration showed blisters and bubbles formed on the bacterial surface, suggesting that K11 exerts its action by destabilizing the bacterial membrane. CD analysis revealed a remarkably enhanced PPII structure of K11 when replacing some of its central residues by proline in PBS. However, when such peptide analogs were confronted with either DMPG-LUV or membrane lipid extract-LUV, the tendency to form PPII structure was severely weakened. On the contrary, K11 peptide showed a remarkably enhanced PPII structure in the presence of DMPG-LUV. Antibacterial tests revealed that K11 was able to inhibit all tested bacteria with an MIC value of 5 µM, while proline and alanine analogs have a reduced activity on Listeria monocytogenes. Besides, the activity against Vibrio parahaemolyticus was affected in most of the alanine-substituted analogs. However, lysine substitutions by alanine or proline at position 7 did not alter the activity against all tested bacterial strains, suggesting that this position can be screened to find a substitute amino acid yielding a peptide with increased antibacterial activity. These results also indicate that the PPII secondary structure of K11 is stabilized by the interaction of the peptide with negatively charged phospholipids in the bacterial membrane, though not being the sole determinant for its antimicrobial activity.

Influence of Hofmeister ions on the structure of proline-based peptide models: A combined experimental and molecular modeling study

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brohl, Andreas; Albrecht, Benjamin; Zhang, Yong

Here, the influence of three sodium salts, covering a wide range of the Hofmeister series, on the conformation of three proline-based peptide models in aqueous solution is examined using a combination of nuclear magnetic resonance spectroscopy and molecular dynamics simulations. The anions preferentially interact with the cis conformers of the peptide models, which is rationalized by the respective electrostatic potential surfaces. These preferred interactions have a strong impact on the thermodynamics of the cis/trans equilibria, leading to a higher population of the cis conformers. In distinct cases, these equilibria are nearly independent of temperature, showing that the salts are alsomore » able to stabilize the conformers over wide temperature ranges.« less

Possible Release of an ArgGlyArgProGln Pentapeptide with Innate Immunity Properties from Acidic Proline-Rich Proteins by Proteolytic Activity in Commensal Streptococcus and Actinomyces Species

PubMed Central

Li, Tong; Bratt, Per; Jonsson, Andreas P.; Ryberg, Mats; Johansson, Ingegerd; Griffiths, William J.; Bergman, Tomas; Strömberg, Nicklas

2000-01-01

This study suggests degradation of salivary acidic proline-rich proteins (PRPs) into potential innate-immunity-like peptides by oral Streptococcus and Actinomyces species. PRP degradation paralleled cleavage of Pro-containing substrates. PRP degradation by S. gordonii strain SK12 instantly released a Pyr1-Pro104Pro105 and a Gly111-Pro149Gln150 peptide together with a presumed Arg106Gly107Arg108Pro109Gln110 pentapeptide. The synthetic Arg106Gly107Arg108Pro109Gln110 peptide desorbed bound bacteria and counteracted sucrose-induced decrease of dental plaque pH in vitro. PMID:10948176

Abnormal Collagen Metabolism in Cultured Skin Fibroblasts from Patients with Duchenne Muscular Dystrophy

NASA Astrophysics Data System (ADS)

Rodemann, H. Peter; Bayreuther, Klaus

1984-08-01

Total collagen synthesis is decreased by about 29% (P < 0.01) in skin fibroblasts established in vitro from male patients with Duchenne muscular dystrophy (DMD) as compared with that in normal male skin fibroblasts in vitro. The reduction in collagen synthesis is associated with an approximately 2-fold increase in collagen degradation in DMD fibroblasts. Correlated to these alterations in the metabolism of collagen, DMD fibroblasts express a significantly higher hydroxyproline/proline ratio (DMD: 1.36-1.45; P < 0.01) than do normal fibroblasts (controls: 0.86-0.89). The increased hydroxylation of proline residues of collagen (composed of type I and type III) could be the cause for the enhanced degradation of collagen in DMD fibroblasts.

Integrating docking and molecular dynamics approaches for a series of proline-based 2,5-diketopiperazines as novel αβ-tubulin inhibitors.

PubMed

Fani, Najmeh; Bordbar, Abdol-Khalegh; Ghayeb, Yousef; Sepehri, Saghi

2015-01-01

In this work, docking tools were utilized in order to study the binding properties of more than five hundred of proline-based 2,5-diketopiperazine in the binding site of αβ-tubulin. Results revealed that 20 compounds among them showed lower binding energies in comparison with Tryprostatin-A, a well known tubulin inhibitor and therefore could be potential inhibitors of tubulin. However, the precise evaluation of binding poses represents the similar binding modes for all of these compounds and Tryprostatin-A. Finally, the best docked complex was subjected to a 25 ns molecular dynamics simulation to further validate the proposed binding mode of this compound.

Influence of Hofmeister Ions on the Structure of Proline-Based Peptide Models: A Combined Experimental and Molecular Modeling Study.

PubMed

Bröhl, Andreas; Albrecht, Benjamin; Zhang, Yong; Maginn, Edward; Giernoth, Ralf

2017-03-09

The influence of three sodium salts, covering a wide range of the Hofmeister series, on the conformation of three proline-based peptide models in aqueous solution is examined using a combination of nuclear magnetic resonance spectroscopy and molecular dynamics simulations. The anions preferentially interact with the cis conformers of the peptide models, which is rationalized by the respective electrostatic potential surfaces. These preferred interactions have a strong impact on the thermodynamics of the cis/trans equilibria, leading to a higher population of the cis conformers. In distinct cases, these equilibria are nearly independent of temperature, showing that the salts are also able to stabilize the conformers over wide temperature ranges.

Interactions in L-phenylalanine/L-leucine/L-glutamic Acid/L-proline + 2 M aqueous NaCl/2 M NaNO3 systems at different temperatures

NASA Astrophysics Data System (ADS)

Riyazuddeen, Imran Khan; Afrin, Sadaf

2012-12-01

Density (ρ) and speed of sound ( u) in 2 M aqueous NaCl and 2 M NaNO3 solutions of amino acids: L-phenylalanine, L-leucine, L-glutamic acid, and L-proline have been measured for several molal concentrations of amino acids at different temperatures. The ρ and u data have been used to calculate the values of isothermal compressibility and internal pressure at different temperatures. The trends of variations of κ T and P i with an increase in molal concentration of amino acid and temperature have been discussed in terms of solute-solvent and solute-solute interactions in the systems.

Influence of Hofmeister ions on the structure of proline-based peptide models: A combined experimental and molecular modeling study

DOE PAGES

Brohl, Andreas; Albrecht, Benjamin; Zhang, Yong; ...

2017-02-13

Here, the influence of three sodium salts, covering a wide range of the Hofmeister series, on the conformation of three proline-based peptide models in aqueous solution is examined using a combination of nuclear magnetic resonance spectroscopy and molecular dynamics simulations. The anions preferentially interact with the cis conformers of the peptide models, which is rationalized by the respective electrostatic potential surfaces. These preferred interactions have a strong impact on the thermodynamics of the cis/trans equilibria, leading to a higher population of the cis conformers. In distinct cases, these equilibria are nearly independent of temperature, showing that the salts are alsomore » able to stabilize the conformers over wide temperature ranges.« less

Study of the interactions between a proline-rich protein and a flavan-3-ol by NMR: residual structures in the natively unfolded protein provides anchorage points for the ligands.

PubMed

Pascal, Christine; Paté, Franck; Cheynier, Véronique; Delsuc, Marc-André

2009-09-01

Astringency is one of the major organoleptic properties of food and beverages that are made from plants, such as tea, chocolate, beer, or red wine. This sensation is thought to be due to interactions between tannins and salivary proline-rich proteins, which are natively unfolded proteins. A human salivary proline-rich protein, namely IB-5, was produced by the recombinant method. Its interactions with a model tannin, epigallocatechin gallate (EGCG), the major flavan-3-ol in green tea, were studied here. Circular dichroism experiments showed that IB-5 presents residual structures (PPII helices) when the ionic strength is close to that in saliva. In the presence of these residual structures, IB-5 undergoes an increase in structural content upon binding to EGCG. NMR data corroborated the presence of preformed structural elements within the protein prior to binding and a partial assignment was proposed, showing partial structuration. TOCSY experiments showed that amino acids that are involved in PPII helices are more likely to interact with EGCG than those in random coil regions, as if they were anchorage points for the ligand. The signal from IB-5 in the DOSY NMR spectrum revealed an increase in polydispersity upon addition of EGCG while the mean hydrodynamic radius remained unchanged. This strongly suggests the formation of IB-5/EGCG aggregates.

Genetic Variability in Energy Balance and Pancreatic Cancer Risk in a Population-Based Case-Control Study in Minnesota

PubMed Central

Zhang, Jianjun; Dhakal, Ishwori B.; Zhang, Xuemei; Prizment, Anna E.; Anderson, Kristin E.

2013-01-01

Objectives Accumulating evidence suggests that energy imbalance plays a role in pancreatic carcinogenesis. However, it remains unclear whether single nucleotide polymorphisms (SNPs) in genes regulating energy homeostasis influence pancreatic cancer risk. We investigated this question in a case-control study conducted from 1994 to 1998. Methods Cases (n=173) were ascertained from hospitals in the Twin Cities and Mayo Clinic, Minnesota. Controls (n=476) were identified from the general population and frequency matched to cases by age and sex. Seven SNPs were evaluated in relation to pancreatic cancer using unconditional logistic regression. Results After adjustment for confounders, the leucine/proline or proline/proline genotype of the neuropeptide Y (NPY) gene rs16139 was associated with a lower risk than the leucine/leucine genotype [odds ratio (OR) (95% confidence interval) (95% CI): 0.40 (0.15, 0.91)]. Conversely, an increased risk was observed for the glycine/arginine or arginine/arginine genotype of the adrenoceptor beta 2, surface (ADRB2) gene rs1042713 as compared with the glycine/glycine genotype [OR (95% CI): 1.52 (1.01, 2.31)]. Conclusions This study first reveals that SNPs in genes modulating energy intake (NPY) and energy expenditure (ADRB2) altered pancreatic cancer risk. If confirmed by other studies, our findings may shed new light on the etiology and prevention of pancreatic cancer. PMID:24201779

Structure-reactivity relationship of Amadori rearrangement products compared to related ketoses.

PubMed

Kaufmann, Martin; Meissner, Philipp M; Pelke, Daniel; Mügge, Clemens; Kroh, Lothar W

2016-06-16

Structure-reactivity relationships of Amadori rearrangement products compared to their related ketoses were derived from multiple NMR spectroscopic techniques. Besides structure elucidation of six Amadori rearrangement products derived from d-glucose and d-galactose with l-alanine, l-phenylalanine and l-proline, especially quantitative (13)C selective saturation transfer NMR spectroscopy was applied to deduce information on isomeric systems. It could be shown exemplarily that the Amadori compound N-(1-deoxy-d-fructos-1-yl)-l-proline exhibits much higher isomerisation rates than d-fructose, which can be explained by C-1 substituent mediated intramolecular catalysis. In combination with a reduced carbonyl activity of Amadori compounds compared to their related ketoses which results in an increased acyclic keto isomer concentration, the results on isomerisation dynamics lead to a highly significant increased reactivity of Amadori compounds. This can be clearly seen, comparing approximated carbohydrate milieu stability time constants (ACuSTiC) which is 1 s for N-(1-deoxy-d-fructos-1-yl)-l-proline and 10 s for d-fructose at pD 4.20 ± 0.05 at 350 K. In addition, first NMR spectroscopic data are provided, which prove that α-pyranose of (amino acid substituted) d-fructose adopts both, (2)C5 and (5)C2 conformation. Copyright © 2016 Elsevier Ltd. All rights reserved.

Identification and specificity studies of small-molecule ligands for SH3 protein domains.

PubMed

Inglis, Steven R; Stojkoski, Cvetan; Branson, Kim M; Cawthray, Jacquie F; Fritz, Daniel; Wiadrowski, Emma; Pyke, Simon M; Booker, Grant W

2004-10-21

The Src Homology 3 (SH3) domains are small protein-protein interaction domains that bind proline-rich sequences and mediate a wide range of cell-signaling and other important biological processes. Since deregulated signaling pathways form the basis of many human diseases, the SH3 domains have been attractive targets for novel therapeutics. High-affinity ligands for SH3 domains have been designed; however, these have all been peptide-based and no examples of entirely nonpeptide SH3 ligands have previously been reported. Using the mouse Tec Kinase SH3 domain as a model system for structure-based ligand design, we have identified several simple heterocyclic compounds that selectively bind to the Tec SH3 domain. Using a combination of nuclear magnetic resonance chemical shift perturbation, structure-activity relationships, and site-directed mutagenesis, the binding of these compounds at the proline-rich peptide-binding site has been characterized. The most potent of these, 2-aminoquinoline, bound with Kd = 125 microM and was able to compete for binding with a proline-rich peptide. Synthesis of 6-substituted-2-aminoquinolines resulted in ligands with up to 6-fold improved affinity over 2-aminoquinoline and enhanced specificity for the Tec SH3 domain. Therefore, 2-aminoquinolines may potentially be useful for the development of high affinity small molecule ligands for SH3 domains.

Analysis of the Anticancer Phytochemicals in Andrographis paniculata Nees. under Salinity Stress

PubMed Central

Valdiani, Alireza; Maziah, Mahmood; Saad, Mohd Said

2013-01-01

Salinity causes the adverse effects in all physiological processes of plants. The present study aimed to investigate the potential of salt stress to enhance the accumulation of the anticancer phytochemicals in Andrographis paniculata accessions. For this purpose, 70-day-old plants were grown in different salinity levels (0.18, 4, 8, 12, and 16 dSm−1) on sand medium. After inducing a period of 30-day salinity stress and before flowering, all plants were harvested and the data on morphological traits, proline content and the three anticancer phytochemicals, including andrographolide (AG), neoandrographolide (NAG), and 14-deoxy-11,12-didehydroandrographolide (DDAG), were measured. The results indicated that salinity had a significant effect on the aforementioned three anticancer phytochemicals. In addition, the salt tolerance index (STI) was significantly decreased, while, except for DDAG, the content of proline, the AG, and NAG was significantly increased (P ≤ 0.01). Furthermore, it was revealed that significant differences among accessions could happen based on the total dry weight, STI, AG, and NAG. Finally, we noticed that the salinity at 12 dSm−1 led to the maximum increase in the quantities of AG, NAG, and DDAG. In other words, under salinity stress, the tolerant accessions were capable of accumulating the higher amounts of proline, AG, and NAG than the sensitive accessions. PMID:24371819