Influence of wine pectic polysaccharides on the interactions between condensed tannins and salivary proteins.

PubMed

Carvalho, Elisabete; Mateus, Nuno; Plet, Benoit; Pianet, Isabelle; Dufourc, Erick; De Freitas, Victor

2006-11-15

Alpha-amylase, a major human salivary protein, and IB8c, a representative of the proline-rich proteins, were obtained by isolation from saliva and by solid-phase synthesis, respectively. The interactions between these proteins and condensed tannins isolated from grape seeds were studied at different protein and tannin concentrations by measuring their aggregation. Pectic polysaccharides were isolated from wine, and their effect on protein tannin aggregation was assessed. The results presented in this study showed that the most acidic fractions of arabinogalactan proteins have the ability to inhibit the formation of aggregates between the grape seed tannins and the two different salivary proteins. Rhamnogalacturonan II has the same ability toward alpha-amylase but not IB8c under the conditions of the present study. Polysaccharides show effects at concentrations at which they are present in wine, which could mean an influence in wine astringency. The interaction between condensed tannins and alpha-amylase is differently affected by ionic strength when compared with IB8c.

Changes in mouse whole saliva soluble proteome induced by tannin-enriched diet

PubMed Central

2010-01-01

Background Previous studies suggested that dietary tannin ingestion may induce changes in mouse salivary proteins in addition to the primarily studied proline-rich proteins (PRPs). The aim of the present study was to determine the protein expression changes induced by condensed tannin intake on the fraction of mouse whole salivary proteins that are unable to form insoluble tannin-protein complexes. Two-dimensional polyacrylamide gel electrophoresis protein separation was used, followed by protein identification by mass spectrometry. Results Fifty-seven protein spots were excised from control group gels, and 21 different proteins were identified. With tannin consumption, the expression levels of one α-amylase isoform and one unidentified protein increased, whereas acidic mammalian chitinase and Muc10 decreased. Additionally, two basic spots that stained pink with Coomassie Brilliant Blue R-250 were newly observed, suggesting that some induced PRPs may remain uncomplexed or form soluble complexes with tannins. Conclusion This proteomic analysis provides evidence that other salivary proteins, in addition to tannin-precipitating proteins, are affected by tannin ingestion. Changes in the expression levels of the acidic mammalian chitinase precursor and in one of the 14 salivary α-amylase isoforms underscores the need to further investigate their role in tannin ingestion. PMID:21159160

Susceptibility to Dental Caries and the Salivary Proline-Rich Proteins

PubMed Central

Levine, Martin

2011-01-01

Early childhood caries affects 28% of children aged 2–6 in the US and is not decreasing. There is a well-recognized need to identify susceptible children at birth. Caries-free adults neutralize bacterial acids in dental biofilms better than adults with severe caries. Saliva contains acidic and basic proline-rich proteins (PRPs) which attach to oral streptococci. The PRPs are encoded within a small region of chromosome 12. An acidic PRP allele (Db) protects Caucasian children from caries but is more common in African Americans. Some basic PRP allelic phenotypes have a three-fold greater frequency in caries-free adults than in those with severe caries. Early childhood caries may associate with an absence of certain basic PRP alleles which bind oral streptococci, neutralize biofilm acids, and are in linkage disequilibrium with Db in Caucasians. The encoding of basic PRP alleles is updated and a new technology for genotyping them is described. PMID:22190937

Turbidity as a measure of salivary protein reactions with astringent substances.

PubMed

Horne, John; Hayes, John; Lawless, Harry T

2002-09-01

Binding of tannins to proline-rich proteins has been proposed as an initial step in the development of astringent sensations. In beer and fruit juices, formation of tannin-protein complexes leads to the well-known effect of haze development or turbidity. Two experiments examined the development of turbidity in human saliva when mixed with tannins as a potential in vitro correlate of astringent sensations. In the first study, haze was measured in filtered human saliva mixed with a range of tannic acid concentrations known to produce supra-threshold psychophysical responses. The second study examined relationships among individual differences in haze development and the magnitude of astringency ratings. Mostly negative correlations were found, consistent with the notion that high levels of salivary proteins protect oral tissues from the drying effects of tannic acid.

Does flavor impact function? Potential consequences of polyphenol-protein interactions in delivery and bioactivity of flavan-3-ols from foods.

PubMed

Ferruzzi, Mario G; Bordenave, Nicolas; Hamaker, Bruce R

2012-11-05

Astringency is a component of the overall flavor experienced when consuming polyphenol rich foods and beverages such as tea, wine, cocoa and select fruits. Following consumption, the astringent sensation results from the well documented ability of polyphenols to bind to salivary proline rich proteins (PRP) and facilitate their precipitation in the oral cavity. In a similar fashion, polyphenols are also known to non-specifically bind food and other biological proteins. While much is known regarding the polyphenol-protein interactions leading to astringency, significantly less information is available regarding the impact of these polyphenol-protein interactions with food or other biological proteins on relevant physiological outcomes. This paper focuses on the interactions between flavan-3-ols, one of the most abundant dietary polyphenol forms, with proteins in food, salivary PRP and other physiological proteins. The physiological implications of these interactions in food and through the gut will be discussed in relation to manipulation of flavan-3-ol bioavailability, metabolism and biological activities including inhibition of digestive enzymes in the gut. Copyright © 2012 Elsevier Inc. All rights reserved.

Salivary amylase induction by tannin-enriched diets as a possible countermeasure against tannins.

PubMed

da Costa, G; Lamy, E; Capela e Silva, F; Andersen, J; Sales Baptista, E; Coelho, A V

2008-03-01

Tannins are characterized by protein-binding affinity. They have astringent/bitter properties that act as deterrents, affecting diet selection. Two groups of salivary proteins, proline-rich proteins and histatins, are effective precipitators of tannin, decreasing levels of available tannins. The possibility of other salivary proteins having a co-adjuvant role on host defense mechanisms against tannins is unknown. In this work, we characterized and compared the protein profile of mice whole saliva from animals fed on three experimental diets: tannin-free diet, diet with the incorporation of 5% hydrolyzable tannins (tannic acid), or diet with 5% condensed tannins (quebracho). Protein analysis was performed by one-dimensional gel electrophoresis combined with Matrix-Assisted Laser Desorption Ionization-Time of Flight mass spectrometry to allow the dynamic study of interactions between diet and saliva. Since abundant salivary proteins obscure the purification and identification of medium and low expressed salivary proteins, we used centrifugation to obtain saliva samples free from proteins that precipitate after tannin binding. Data from Peptide Mass Fingerprinting allowed us to identify ten different proteins, some of them showing more than one isoform. Tannin-enriched diets were observed to change the salivary protein profile. One isoform of alpha-amylase was overexpressed with both types of tannins. Aldehyde reductase was only identified in saliva of the quebracho group. Additionally, a hypertrophy of parotid salivary gland acini was observed by histology, along with a decrease in body mass in the first 4 days of the experimental period.

Characterization, stoichiometry, and stability of salivary protein-tannin complexes by ESI-MS and ESI-MS/MS.

PubMed

Canon, Francis; Paté, Franck; Meudec, Emmanuelle; Marlin, Thérèse; Cheynier, Véronique; Giuliani, Alexandre; Sarni-Manchado, Pascale

2009-12-01

Numerous protein-polyphenol interactions occur in biological and food domains particularly involving proline-rich proteins, which are representative of the intrinsically unstructured protein group (IUP). Noncovalent protein-ligand complexes are readily detected by electrospray ionization mass spectrometry (ESI-MS), which also gives access to ligand binding stoichiometry. Surprisingly, the study of interactions between polyphenolic molecules and proteins is still an area where ESI-MS has poorly benefited, whereas it has been extensively applied to the detection of noncovalent complexes. Electrospray ionization mass spectrometry has been applied to the detection and the characterization of the complexes formed between tannins and a human salivary proline-rich protein (PRP), namely IB5. The study of the complex stability was achieved by low-energy collision-induced dissociation (CID) measurements, which are commonly implemented using triple quadrupole, hybrid quadrupole time-of-flight, or ion trap instruments. Complexes composed of IB5 bound to a model polyphenol EgCG have been detected by ESI-MS and further analyzed by MS/MS. Mild ESI interface conditions allowed us to observe intact noncovalent PRP-tannin complexes with stoichiometries ranging from 1:1 to 1:5. Thus, ESI-MS shows its efficiency for (1) the study of PRP-tannin interactions, (2) the determination of stoichiometry, and (3) the study of complex stability. We were able to establish unambiguously both their stoichiometries and their overall subunit architecture via tandem mass spectrometry and solution disruption experiments. Our results prove that IB5.EgCG complexes are maintained intact in the gas phase.

Toward the optical tongue: flow-through sensing of tannin-protein interactions based on FTIR spectroscopy.

PubMed

Edelmann, Andrea; Lendl, Bernhard

2002-12-11

The interaction of polyphenols (tannins) with proline-rich proteins (gelatin) has been studied using an automated flow injection system with Fourier transform infrared spectroscopic detection to gain insight into chemical aspects related to astringency. In the perception of astringency, a major taste property in red wines and other beverages such as beer, tea, or fruit juices, an interaction between proline-rich salivary proteins and tannins present in the sample takes place. To study this interaction, agarose beads carrying gelatin (a proline-rich protein) were placed in the IR flow cell in such a way that the beads were probed by the IR beam. Using an automated flow system, we injected samples in a carrier stream and flushed over the proteins in a highly reproducible manner. Simultaneously, any retardation due to tannin-protein interactions taking place inside the flow cell was monitored by infrared spectroscopy. Tannins of different sources (grapes, wooden barrels, formulations used in wine making) were investigated, and their flow-through behavior was characterized. Significant differences in their affinity toward gelatin could be observed. Furthermore, because of small but characteristic differences in the IR spectrum, it is possible to distinguish condensed from hydrolyzable tannins. Nonastringent substances such as alcohols, sugars, and acids did not show retention on gelatin. The selectivity of the flow-through sensor was also demonstrated on the example of red and white wine. In contrast to white wine, where no interaction could be observed, in red wine a major interaction of the red wine tannins was found.

Proline-rich proteins moderate the inhibitory effect of tea on iron absorption in rats.

PubMed

Kim, Hee-Seon; Miller, Dennis D

2005-03-01

Tea inhibits iron absorption in studies in which tea is given with radiolabeled iron to humans as a single dose. Our objective was to test the hypothesis that proline-rich proteins (PRPs) may act as a defense against this effect by forming complexes with tannins, thereby preventing them from inhibiting iron absorption. Two studies were conducted. In study 1, rats were given test solutions containing (59)FeCl(3) in water, tea, or tea + gelatin (T/G). In study 2, the rats were divided into 3 groups and assigned to one of 3 nutritionally complete diets: control, tea (5 g tea tannin/kg diet), or T/G (5 g tea tannin + 60 g gelatin/kg diet). Rats were fed the respective diets for 5 d and then given a single (59)Fe-labeled meal of the diet. Iron absorption was measured by whole-body retention of the (59)Fe over a 2-wk period. Iron absorption in study 1 was lower in the tea group (24 +/- 9.6%, P < 0.05) than in the T/G (42 +/- 19.4%) or water groups (50 +/- 7.5%). In study 2, iron absorption did not differ among the groups. Rats fed the tea diet had dramatic hypertrophy of the parotid salivary glands. Adding gelatin as a proxy for salivary PRPs to the tea eliminated the inhibitory effect of tea on iron absorption. The results suggest that PRPs, whether from salivary glands or diet, can protect against the inhibition of iron absorption by tea.

Structural studies and SH3 domain binding properties of a human antiviral salivary proline-rich peptide.

PubMed

Righino, Benedetta; Pirolli, Davide; Radicioni, Giorgia; Marzano, Valeria; Longhi, Renato; Arcovito, Alessandro; Sanna, Maria Teresa; De Rosa, Maria Cristina; Paoluzi, Serena; Cesareni, Gianni; Messana, Irene; Castagnola, Massimo; Vitali, Alberto

2016-09-01

Human saliva contains hundreds of small proline-rich peptides originated by the proteolytic cleavage of the salivary basic Proline-Rich Proteins. Nevertheless only for few of them a specific biological activity has been assigned to date. Among them, the 1932 Da peptide (p1932) has been patented as an anti-HIV agent. In order to shed light on the possible mechanism of action of this peptide, we assessed in this study, by means of molecular dynamics calculations, circular dichroism and FTIR spectroscopic techniques, that p1932 has an intrinsic propensity to adopt a polyproline-II helix arrangement. This structural feature combined with the presence of PxxP motifs in its primary structure, represents an essential property for the exploitation of several biological activities. Next to these findings, we recently demonstrated the ability of this peptide to be internalized within cells of the oral mucosa, thus we focused onto a possible intracellular target, represented by the SH3 domains family. Its ability to interact with selected SH3 domains was finally assayed by Surface Plasmon Resonance spectroscopy. As a result, only Fyn, Hck, and c-Src SH3 domains gave positive results in terms of interaction, showing dissociation constants ranging from nanomolar to micromolar values having the best performer a KD of 148 nM. It is noteworthy that all the interacting domains belong to the Src kinases family, suggesting a role for p1932 as a modulator of the signal transduction pathways mediated by these kinases. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 714-725, 2016. © 2016 Wiley Periodicals, Inc.

Coarse-grained modeling of proline rich protein 1 (PRP-1) in bulk solution and adsorbed to a negatively charged surface.

PubMed

Skepö, Marie; Linse, Per; Arnebrant, Thomas

2006-06-22

Structural properties of the acidic proline rich protein PRP-1 of salivary origin in bulk solution and adsorbed onto a negatively charged surface have been studied by Monte Carlo simulations. A simple model system with focus on electrostatic interactions and short-ranged attractions among the uncharged amino acids has been used. In addition to PRP-1, some mutants were considered to assess the role of the interactions in the systems. Contrary to polyelectrolytes, the protein has a compact structure in salt-free bulk solutions, whereas at high salt concentration the protein becomes more extended. The protein adsorbs to a negatively charged surface, although its net charge is negative. The adsorbed protein displays an extended structure, which becomes more compact upon addition of salt. Hence, the conformational response upon salt addition in the adsorbed state is the opposite as compared to that in bulk solution. The conformational behavior of PRP-1 in bulk solution and at charged surfaces as well as its propensity to adsorb to surfaces with the same net charge are rationalized by the block polyampholytic character of the protein. The presence of a triad of positively charged amino acids in the C-terminal was found to be important for the adsorption of the protein.

A physiological model of tea-induced astringency.

PubMed

Nayak, A; Carpenter, G H

2008-10-20

The mechanism by which solutions containing polyphenols are perceived as astringent is not clearly understood. Salivary proline-rich proteins and histatins are products of salivary glands and rapidly bind polyphenols - thought to be the main astringent compound in such as tea and wine. However it is unclear how this interaction leads to the altered oral mouthfeel known as astringency which is characterised by a dry, puckered feeling all around the mouth. To determine the role of saliva in the perception of astringency a protocol was used to decrease the volume of saliva from the mouth (by washing with water) and then by chewing to increase the volume of saliva above resting levels. Following each of these conditions subjects tasted the same solution of black tea and were asked to rate the relative astringency. Compared to the astringency rating of black tea at rest the majority of subjects (10 out of 15) perceived an increase in astringency following washing the mouth with water. Most subjects then perceived a decrease in astringency following chewing compared to the previous state. In all subjects a reduction in salivary proteins was detected following water washout and an increase above resting levels detected following chewing although there was no change in oral mucosal wetness. A separate experiment revealed several of the proteins interacting following the water washout were salivary in origin. We conclude that salivary proteins in solution inhibit the mouthfeeling of astringency which is mediated, at least in part, by salivary proteins adhered to buccal mucosal cells.

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.

What interactions drive the salivary mucosal pellicle formation?

PubMed Central

Gibbins, Hannah L.; Yakubov, Gleb E.; Proctor, Gordon B.; Wilson, Stephen; Carpenter, Guy H.

2014-01-01

The bound salivary pellicle is essential for protection of both the enamel and mucosa in the oral cavity. The enamel pellicle formation is well characterised, however the mucosal pellicle proteins have only recently been clarified and what drives their formation is still unclear. The aim of this study was to examine the salivary pellicle on particles with different surface properties (hydrophobic or hydrophilic with a positive or negative charge), to determine a suitable model to mimic the mucosal pellicle. A secondary aim was to use the model to test how transglutaminase may alter pellicle formation. Particles were incubated with resting whole mouth saliva, parotid saliva and submandibular/sublingual saliva. Following incubation and two PBS and water washes bound salivary proteins were eluted with two concentrations of SDS, which were later analysed using SDS-PAGE and Western blotting. Experiments were repeated with purified transglutaminase to determine how this epithelial-derived enzyme may alter the bound pellicle. Protein pellicles varied according to the starting salivary composition and the particle chemistry. Amylase, the single most abundant protein in saliva, did not bind to any particle indicating specific protein binding. Most proteins bound through hydrophobic interactions and a few according to their charges. The hydrophobic surface most closely matched the known salivary mucosal pellicle by containing mucins, cystatin and statherin but an absence of amylase and proline-rich proteins. This surface was further used to examine the effect of added transglutaminase. At the concentrations used only statherin showed any evidence of crosslinking with itself or another saliva protein. In conclusion, the formation of the salivary mucosal pellicle is probably mediated, at least in part, by hydrophobic interactions to the epithelial cell surface. PMID:24921197

Saliva tannin interactions.

PubMed

Prinz, J F; Lucas, P W

2000-11-01

Many plant foods contain tannins, compounds that bind proteins, such as mammalian enzymes. Although described as tasteless, tannins can be detected orally by their astringency. However, the actual mechanism of oral detection and the effect of tannins on mastication and swallowing have been little investigated. Here, we show from in vitro tests that tannic acid, a common standard in tests used to detect tannins, significantly reduces the lubricating qualities of human saliva both by decreasing its viscosity and increasing friction, both factors lending support to the notion that astringency is a tactile phenomenon. From the literature, it is clear that this effect depends on the presence of salivary proline-rich proteins (PRP). In a mammalian context, ingestion of tannin-rich foods in a species with salivary PRP will be signalled by interference with bolus formation during mastication while the increase in friction may also be detectable and lead to increased tooth wear if the signal is ignored. In a human context, cross-cultural preferences for tannin-rich beverages such as tea, coffee and red wine at the end of meals may be explained by reduction in adhesion of food particles to the oral mucosa allowing their rapid oral clearance.

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.

The colloidal state of tannins impacts the nature of their interaction with proteins: the case of salivary proline-rich protein/procyanidins binding.

PubMed

Cala, Olivier; Dufourc, Erick J; Fouquet, Eric; Manigand, Claude; Laguerre, Michel; Pianet, Isabelle

2012-12-18

While the definition of tannins has been historically associated with its propensity to bind proteins in a nonspecific way, it is now admitted that specific interaction also occurs. The case of the astringency perception is a good example to illustrate this phenomenon: astringency is commonly described as a tactile sensation induced by the precipitation of a complex composed of proline-rich proteins present in the human saliva and tannins present in beverages such as tea or red wines. In the present work, the interactions between a human saliva protein segment and three different procyanidins (B1, B3, and C2) were investigated at the atomic level by NMR and molecular dynamics. The data provided evidence for (i) an increase in affinity compared to shortest human saliva peptides, which is accounted for by protein "wraping around" the tannin, (ii) a specificity in the interaction below tannin critical micelle concentration (CMC) of ca. 10 mM, with an affinity scale such that C2 > B1 > B3, and (iii) a nonspecific binding above tannin CMC that conducts irremediably to the precipitation of the tannins/protein complex. Such physicochemical findings describe in accurate terms saliva protein-tannin interactions and provide support for a more subtle description by oenologists of wine astringency perception in the mouth.

Comparative salivary proteomics analysis of children with and without dental caries using the iTRAQ/MRM approach.

PubMed

Wang, Kun; Wang, Yufei; Wang, Xiuqing; Ren, Qian; Han, Sili; Ding, Longjiang; Li, Zhongcheng; Zhou, Xuedong; Li, Wei; Zhang, Linglin

2018-01-19

Dental caries is a major worldwide oral disease afflicting a large proportion of children. As an important host factor of caries susceptibility, saliva plays a significant role in the occurrence and development of caries. The aim of the present study was to characterize the healthy and cariogenic salivary proteome and determine the changes in salivary protein expression of children with varying degrees of active caries, also to establish salivary proteome profiles with a potential therapeutic use against dental caries. In this study, unstimulated saliva samples were collected from 30 children (age 10-12 years) with no dental caries (NDC, n = 10), low dental caries (LDC, n = 10), and high dental caries (HDC, n = 10). Salivary proteins were extracted, reduced, alkylated, trypsin digested and labeled with isobaric tags for relative and absolute quantitation, and then they were analyzed with GO annotation, biological pathway analysis, hierarchical clustering analysis, and protein-protein interaction analysis. Targeted verifications were then performed using multiple reaction monitoring mass spectrometry. A total of 244 differentially expressed proteins annotated with GO annotation in biological processes, cellular component and molecular function were identified in comparisons among children with varying degrees of active caries. A number of caries-related proteins as well as pathways were identified in this study. As compared with caries-free children, the most significantly enriched pathways involved by the up-regulated proteins in LDC and HDC were the ubiquitin mediated proteolysis pathway and African trypanosomiasis pathway, respectively. Subsequently, we selected 53 target proteins with differential expression in different comparisons, including mucin 7, mucin 5B, histatin 1, cystatin S and cystatin SN, basic salivary proline rich protein 2, for further verification using MRM assays. Protein-protein interaction analysis of these proteins revealed complex protein interaction networks, indicating synergistic action of salivary proteins in caries resistance or cariogenicity. Overall, our results afford new insight into the salivary proteome of children with dental caries. These findings might have bright prospect in future in developing novel biomimetic peptides with preventive and therapeutic benefits for childhood caries.

Elongated fibrillar structure of a streptococcal adhesin assembled by the high-affinity association of [alpha]- and PPII-helices

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

Larson, Matthew R.; Rajashankar, Kanagalaghatta R.; Patel, Manisha H.

2010-08-18

Streptococcus mutans antigen I/II (AgI/II) is a cell surface-localized protein adhesin that interacts with salivary components within the salivary pellicle. AgI/II contributes to virulence and has been studied as an immunological and structural target, but a fundamental understanding of its underlying architecture has been lacking. Here we report a high-resolution (1.8 {angstrom}) crystal structure of the A{sub 3}VP{sub 1} fragment of S. mutans AgI/II that demonstrates a unique fibrillar form (155 {angstrom}) through the interaction of two noncontiguous regions in the primary sequence. The A{sub 3} repeat of the alanine-rich domain adopts an extended {alpha}-helix that intertwines with the P{submore » 1} repeat polyproline type II (PPII) helix to form a highly extended stalk-like structure heretofore unseen in prokaryotic or eukaryotic protein structures. Velocity sedimentation studies indicate that full-length AgI/II that contains three A/P repeats extends over 50 nanometers in length. Isothermal titration calorimetry revealed that the high-affinity association between the A{sub 3} and P{sub 1} helices is enthalpically driven. Two distinct binding sites on AgI/II to the host receptor salivary agglutinin (SAG) were identified by surface plasmon resonance (SPR). The current crystal structure reveals that AgI/II family proteins are extended fibrillar structures with the number of alanine- and proline-rich repeats determining their length.« less

Salivary proteins and early childhood caries: A gel electrophoretic analysis

PubMed Central

Bhalla, Sumati; Tandon, Shobha; Satyamoorthy, K.

2010-01-01

Background: Early childhood caries (ECC) is a common disease process that afflicts a large proportion of the child population worldwide. Extensive research in past indicates that it is the result of bacterial infection, also influenced by host and dietary factors. Current caries research seeks to identify risk factors as well as natural oral defenses that may protect against or prevent caries development. Saliva, in spite of being the strongest defense system, still has a wide array of properties and proteins whose role is yet not clearly known. Aim: To compare the resting human whole salivary characteristics in children with ECC and those who are caries free. Settings and Design: The study was conducted over a period of 9 months in 4- to 6-year-old 100 children comprising two groups – 50 with ECC and 50 caries free. Materials and Methods: The whole salivary flow rate, pH, mean protein concentration, and the electrophoretic profile of salivary proteins by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) were compared among both groups. Statistical Analysis: The SPSS (version 11.0) software package was used to conduct the chi-square, Fisher's exact and Pearson's chi-square tests to compare the data. Results: On gel electrophoresis, there was a significant difference among both groups with caries-free subjects having a higher number of proline-rich protein bands, substantiating the protective role of this protein. A significantly higher number of glycoprotein bands were observed in the whole saliva of subjects with ECC. A significant inverse correlation between the mean protein concentration and the whole salivary flow rate was observed in both groups. PMID:22114372

Codominant expression of genes coding for different sets of inducible salivary polypeptides associated with parotid hypertrophy in two inbred mouse strains.

PubMed

López-Solís, Remigio O; Kemmerling, Ulrike

2005-05-01

Experimental mouse parotid hypertrophy has been associated with the expression of a number of isoproterenol-induced salivary proline-rich polypeptides (IISPs). Mouse salivary proline-rich proteins (PRPs) have been mapped both to chromosomes 6 and 8. Recently, mice of two inbred strains (A/Snell and A. Swiss) have been found to differ drastically in the IISPs. In this study, mice of both strains were used for cross-breeding experiments addressed to define the pattern of inheritance of the IISP phenotype and to establish whether the IISPs are coded on a single or on several chromosomes. The IISP phenotype of individual mice was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of whole saliva collected after three daily stimulations by isoproterenol. Parental A/Snell and A. Swiss mice were homogeneous for distinctive strain-associated IISP-patterns. First filial generation (F1) mice obtained from the cross of A/Snell with A. Swiss mice expressed with no exception both the A/Snell and A. Swiss IISPs (coexpression). In the second filial generation (F2) both parental IISP phenotypes reappeared together with a majority of mice expressing the F1-hybrid phenotype (1:2:1 ratio). Backcrosses of F1 x A/Snell and F1 x A. Swiss produced offsprings displaying the F1 and the corresponding parental phenotypes with a 1:1 ratio. No recombinants were observed among F2 mice or among mice resulting from backcrosses. Thus, genes coding for the IISPs that are expressed differentially in both mouse strains are located on the same chromosome, probably at the same locus (alleles) or at quite closely linked loci (nonalleles). 2005 Wiley-Liss, Inc

Saliva and Serum Protein Exchange at the Tooth Enamel Surface

PubMed Central

Heller, D.; Helmerhorst, E.J.; Oppenheim, F.G.

2016-01-01

The acquired enamel pellicle is an oral, fluid-derived protein layer that forms on the tooth surface. It is a biologically and clinically important integument that protects teeth against enamel demineralization, and abrasion. Tooth surfaces are exposed to different proteinaceous microenvironments depending on the enamel location. For instance, tooth surfaces close to the gingival sulcus contact serum proteins that emanate via this sulcus, which may impact pellicle composition locally. The aims of this study were to define the major salivary and serum components that adsorb to hydroxyapatite, to study competition among them, and to obtain preliminary evidence in an in vivo saliva/serum pellicle model. Hydroxyapatite powder was incubated with saliva and serum, and the proteins that adsorbed were identified by mass spectrometry. To study competition, saliva and serum proteins were labeled with CyDyes, mixed in various proportions, and incubated with hydroxyapatite. In vivo competition was assessed using a split-mouth design, with half the buccal tooth surfaces coated with serum and the other half with saliva. After exposure to the oral environment for 0 min, 30 min and 2 h, the pellicles were analyzed by SDS-PAGE. In pure saliva- or serum-derived pellicles, 82 and 84 proteins were identified, respectively. When present concomitantly, salivary protein adsorbers effectively competed with serum protein adsorbers for the hydroxyapatite surface. Specifically, acidic proline-rich protein, cystatin, statherin and protein S100-A9 proteins competed off apolipoproteins, complement C4-A, haptoglobin, transthyretin and serotransferrin. In vivo evidence further supported the replacement of serum proteins by salivary proteins. In conclusion, although significant numbers of serum proteins emanate from the gingival sulcus, their ability to participate in dental pellicle formation is likely reduced in the presence of strong salivary protein adsorbers. The functional properties of the acquired enamel pellicle will therefore be mostly dictated by the salivary component. PMID:27879420

Changes in rat parotid saliva protein composition following chronic reserpine treatment and their relation to inanition.

PubMed

Johnson, D A

1988-01-01

Chronic administration of the catecholamine-depleting agent, reserpine (0.5 mg/kg), resulted in a reduction in food intake after 3 days. To differentiate effects of the drug from those of reduced food intake a pair-fed group, whose daily caloric intake was restricted to the amount consumed by the reserpine-treated rats, was included. After 7 days, both the reserpine-treated and pair-fed control exhibited a marked reduction in the volume of saliva collected in a 30 min interval following a secretory stimulus compared to untreated ad libitum-fed controls, and the proportion of salivary proteins attributable to acidic and basic proline-rich proteins and to minor 1b protein were decreased whereas deoxyribonuclease was increased. For two of the salivary proteins (fractions I and V) changes for the reserpine-treated and pair-fed groups were different. Fraction I was reduced in both groups, but exhibited a greater decrease in the pair-fed than in the reserpine-treated, whereas fraction V was significantly increased only in the pair-fed group. Thus many of the salivary changes associated with reserpine treatment may have resulted from the change in feeding habits and not from reserpine treatment per se. The study demonstrates the importance of controlling for food intake under experimental circumstances which may lead to a marked change in daily feeding habits.

Salivary proline-rich protein may reduce tannin-iron chelation: a systematic narrative review.

PubMed

Delimont, Nicole M; Rosenkranz, Sara K; Haub, Mark D; Lindshield, Brian L

2017-01-01

Tannins are often cited for antinutritional effects, including chelation of non-heme iron. Despite this, studies exploring non-heme iron bioavailability inhibition with long-term consumption have reported mixed results. Salivary proline-rich proteins (PRPs) may mediate tannin-antinutritional effects on non-heme iron bioavailability. To review evidence regarding biochemical binding mechanisms and affinity states between PRPs and tannins, as well as effects of PRPs on non-heme iron bioavailability with tannin consumption in vivo. Narrative systematic review and meta-analysis. Common themes in biochemical modeling and affinity studies were collated for summary and synthesis; data were extracted from in vivo experiments for meta-analysis. Thirty-two studies were included in analysis. Common themes that positively influenced tannin-PRP binding included specificity of tannin-PRP binding, PRP and tannin stereochemistry. Hydrolyzable tannins have different affinities than condensed tannins when binding to PRPs. In vivo, hepatic iron stores and non-heme iron absorption are not significantly affected by tannin consumption ( d  = -0.64-1.84; -2.7-0.13 respectively), and PRP expression may increase non-heme iron bioavailability with tannin consumption. In vitro modeling suggests that tannins favor PRP binding over iron chelation throughout digestion. Hydrolyzable tannins are not representative of tannin impact on non-heme iron bioavailability in food tannins because of their unique structural properties and PRP affinities. With tannin consumption, PRP production is increased, and may be an initial line of defense against tannin-non-heme iron chelation in vivo . More research is needed to compare competitive binding of tannin-PRP to tannin-non-heme iron complexes, and elucidate PRPs' role in adaption to non-heme iron bioavailability in vivo.

Proline: The Distribution, Frequency, Positioning, and Common Functional Roles of Proline and Polyproline Sequences in the Human Proteome

PubMed Central

Morgan, Alexander A.; Rubenstein, Edward

2013-01-01

Proline is an anomalous amino acid. Its nitrogen atom is covalently locked within a ring, thus it is the only proteinogenic amino acid with a constrained phi angle. Sequences of three consecutive prolines can fold into polyproline helices, structures that join alpha helices and beta pleats as architectural motifs in protein configuration. Triproline helices are participants in protein-protein signaling interactions. Longer spans of repeat prolines also occur, containing as many as 27 consecutive proline residues. Little is known about the frequency, positioning, and functional significance of these proline sequences. Therefore we have undertaken a systematic bioinformatics study of proline residues in proteins. We analyzed the distribution and frequency of 687,434 proline residues among 18,666 human proteins, identifying single residues, dimers, trimers, and longer repeats. Proline accounts for 6.3% of the 10,882,808 protein amino acids. Of all proline residues, 4.4% are in trimers or longer spans. We detected patterns that influence function based on proline location, spacing, and concentration. We propose a classification based on proline-rich, polyproline-rich, and proline-poor status. Whereas singlet proline residues are often found in proteins that display recurring architectural patterns, trimers or longer proline sequences tend be associated with the absence of repetitive structural motifs. Spans of 6 or more are associated with DNA/RNA processing, actin, and developmental processes. We also suggest a role for proline in Kruppel-type zinc finger protein control of DNA expression, and in the nucleation and translocation of actin by the formin complex. PMID:23372670

Effect of condensed tannins addition on the astringency of red wines.

PubMed

Soares, Susana; Sousa, André; Mateus, Nuno; de Freitas, Victor

2012-02-01

Astringency has been defined as a group of sensations involving dryness, tightening, and shrinking of the oral surface. It has been accepted that astringency is due to the tannin-induced interaction and/or precipitation of the salivary proline-rich proteins (PRPs) in the oral cavity, as a result of the ingestion of food products rich in tannins, for example, red wine. The sensory evaluation of astringency is difficult, and the existence of fast and reliable methods to its study in vitro is scarce. So, in this work, the astringency of red wine supplemented with oligomeric procyanidins (condensed tannins), and the salivary proteins (SP) involved in its development were evaluated by high-performance liquid chromatography analysis of human saliva after its interaction with red wine and by sensorial evaluation. The results show that for low concentration of tannins, the decrease of acidic PRPs and statherin is correlated with astringency intensity, with these families having a high relative complexation and precipitation toward condensed tannins comparatively to the other SP. However, for higher concentrations of tannins, the relative astringency between wines seems to correlate's to the glycosylated PRPs changes. This work shows for the first time that the several families of SP could be involved in different stages of the astringency development.

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

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.

No major role for binding by salivary proteins as a defense against dietary tannins in Mediterranean goats.

PubMed

Hanovice-Ziony, Michal; Gollop, Nathan; Landau, Serge Yan; Ungar, Eugene David; Muklada, Hussein; Glasser, Tzach Aharon; Perevolotsky, Avi; Walker, John Withers

2010-07-01

We investigated whether Mediterranean goats use salivary tannin-binding proteins to cope with tannin-rich forages by determining the affinity of salivary or parotid gland proteins for tannic acid or quebracho tannin. Mixed saliva, sampled from the oral cavity, or parotid gland contents were compared to the intermediate affinity protein bovine serum albumin with a competitive binding assay. Goats that consume tannin-rich browse (Damascus) and goats that tend to avoid tannins (Mamber) were sequentially fed high (Pistacia lentiscus L.), low (vetch hay), or zero (wheat hay) tannin forages. Affinity of salivary proteins for tannins did not differ between goat breeds and did not respond to presence or absence of tannins in the diet. Proteins in mixed saliva had slightly higher affinity for tannins than those in parotid saliva, but neither source contained proteins with higher affinity for tannins than bovine serum albumin. Similarly, 3 months of browsing in a tannin-rich environment had little effect on the affinity of salivary proteins for tannin in adult goats of either breed. We sampled mixed saliva from young kids before they consumed forage and after 3 months of foraging in a tannin-rich environment. Before foraging, the saliva of Mamber kids had higher affinity for tannic acid (but not quebracho tannin) than the saliva of Damascus kids, but there was no difference after 3 months of exposure to tannin-rich browse, and the affinity of the proteins was always similar to the affinity of bovine serum albumin. Our results suggest there is not a major role for salivary tannin-binding proteins in goats. Different tendencies of goat breeds to consume tannin-rich browse does not appear be related to differences in salivary tannin-binding proteins.

Ethanol Concentration Influences the Mechanisms of Wine Tannin Interactions with Poly(L-proline) in Model Wine.

PubMed

McRae, Jacqui M; Ziora, Zyta M; Kassara, Stella; Cooper, Matthew A; Smith, Paul A

2015-05-06

Changes in ethanol concentration influence red wine astringency, and yet the effect of ethanol on wine tannin-salivary protein interactions is not well understood. Isothermal titration calorimetry (ITC) was used to measure the binding strength between the model salivary protein, poly(L-proline) (PLP) and a range of wine tannins (tannin fractions from a 3- and a 7-year old Cabernet Sauvignon wine) across different ethanol concentrations (5, 10, 15, and 40% v/v). Tannin-PLP interactions were stronger at 5% ethanol than at 40% ethanol. The mechanism of interaction changed for most tannin samples across the wine-like ethanol range (10-15%) from a combination of hydrophobic and hydrogen binding at 10% ethanol to only hydrogen binding at 15% ethanol. These results indicate that ethanol concentration can influence the mechanisms of wine tannin-protein interactions and that the previously reported decrease in wine astringency with increasing alcohol may, in part, relate to a decrease tannin-protein interaction strength.

Discovery of putative salivary biomarkers for Sjögren's syndrome using high resolution mass spectrometry and bioinformatics.

PubMed

Zoukhri, Driss; Rawe, Ian; Singh, Mabi; Brown, Ashley; Kublin, Claire L; Dawson, Kevin; Haddon, William F; White, Earl L; Hanley, Kathleen M; Tusé, Daniel; Malyj, Wasyl; Papas, Athena

2012-03-01

The purpose of the current study was to determine if saliva contains biomarkers that can be used as diagnostic tools for Sjögren's syndrome (SjS). Twenty seven SjS patients and 27 age-matched healthy controls were recruited for these studies. Unstimulated glandular saliva was collected from the Wharton's duct using a suction device. Two µl of salvia were processed for mass spectrometry analyses on a prOTOF 2000 matrix-assisted laser desorption/ionization orthogonal time of flight (MALDI O-TOF) mass spectrometer. Raw data were analyzed using bioinformatic tools to identify biomarkers. MALDI O-TOF MS analyses of saliva samples were highly reproducible and the mass spectra generated were very rich in peptides and peptide fragments in the 750-7,500 Da range. Data analysis using bioinformatic tools resulted in several classification models being built and several biomarkers identified. One model based on 7 putative biomarkers yielded a sensitivity of 97.5%, specificity of 97.8% and an accuracy of 97.6%. One biomarker was present only in SjS samples and was identified as a proteolytic peptide originating from human basic salivary proline-rich protein 3 precursor. We conclude that salivary biomarkers detected by high-resolution mass spectrometry coupled with powerful bioinformatic tools offer the potential to serve as diagnostic/prognostic tools for SjS.

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...

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.

Salivary α-amylase, serum albumin, and myoglobin protect against DNA-damaging activities of ingested dietary agents in vitro

PubMed Central

Hossain, M. Zulfiquer; Patel, Kalpesh; Kern, Scott E.

2014-01-01

Potent DNA-damaging activities were seen in vitro from dietary chemicals found in coffee, tea, and liquid smoke. A survey of tea varieties confirmed genotoxic activity to be widespread. Constituent pyrogallol-like polyphenols (PLPs) such as epigallocatechin-3-gallate (EGCG), pyrogallol, and gallic acid were proposed as a major source of DNA-damaging activities, inducing DNA double-strand breaks in the p53R assay, a well characterized assay sensitive to DNA strand breaks, and comet assay. Paradoxically, their consumption does not lead to the kind of widespread cellular toxicity and acute disease that might be expected from genotoxic exposure. Existing physiological mechanisms could limit DNA damage from dietary injurants. Serum albumin and salivary α-amylase are known to bind EGCG. Salivary α-amylase, serum albumin, and myoglobin, but not salivary proline-rich proteins, reduced damage from tea, coffee, and PLPs, but did not inhibit damage from the chemotherapeutics etoposide and camptothecin. This represents a novel function for saliva in addition to its known functions including protection against tannins. Cell populations administered repeated pyrogallol exposures had abatement of measured DNA damage by two weeks, indicating an innate cellular adaptation. We suggest that layers of physiological protections may exist toward natural dietary products to which animals have had high-level exposure over evolution. PMID:24842839

Salivary α-amylase, serum albumin, and myoglobin protect against DNA-damaging activities of ingested dietary agents in vitro.

PubMed

Hossain, M Zulfiquer; Patel, Kalpesh; Kern, Scott E

2014-08-01

Potent DNA-damaging activities were seen in vitro from dietary chemicals found in coffee, tea, and liquid smoke. A survey of tea varieties confirmed genotoxic activity to be widespread. Constituent pyrogallol-like polyphenols (PLPs) such as epigallocatechin-3-gallate (EGCG), pyrogallol, and gallic acid were proposed as a major source of DNA-damaging activities, inducing DNA double-strand breaks in the p53R assay, a well characterized assay sensitive to DNA strand breaks, and comet assay. Paradoxically, their consumption does not lead to the kind of widespread cellular toxicity and acute disease that might be expected from genotoxic exposure. Existing physiological mechanisms could limit DNA damage from dietary injurants. Serum albumin and salivary α-amylase are known to bind EGCG. Salivary α-amylase, serum albumin, and myoglobin, but not salivary proline-rich proteins, reduced damage from tea, coffee, and PLPs, but did not inhibit damage from the chemotherapeutics etoposide and camptothecin. This represents a novel function for saliva in addition to its known functions including protection against tannins. Cell populations administered repeated pyrogallol exposures had abatement of measured DNA damage by two weeks, indicating an innate cellular adaptation. We suggest that layers of physiological protections may exist toward natural dietary products to which animals have had high-level exposure over evolution. Copyright © 2014 Elsevier Ltd. All rights reserved.

Modifications of the acidic soluble salivary proteome in human children from birth to the age of 48months investigated by a top-down HPLC-ESI-MS platform.

PubMed

Manconi, B; Cabras, T; Pisano, E; Sanna, M T; Olianas, A; Fanos, V; Faa, G; Nemolato, S; Iavarone, F; Castagnola, M; Messana, I

2013-10-08

During the first year of life the infant oral environment undergoes dramatic changes. To investigate how the salivary proteome of human children evolves during infant development we have analyzed whole saliva of 88 children aged between 0 and 48months by a top-down platform based on RP-HPLC-ESI-MS. Children were divided according to their age into five groups (A, 0-6months, N=17; B, 7-12months, N=14; C, 13-24months, N=32; D, 25-36months, N=16; E, 37-48months, N=9). The proteins and peptides analyzed were histatins (histatin-1, histatin-3 1/24), acidic proline-rich proteins, statherin, P-B peptide, and salivary cystatins. Protein and peptide quantification based on the area of the RP-HPLC-ESI-MS extracted ion current peak evidenced that: (i) concentrations of the major salivary proteins/peptides showed a minimum in the 0-6-month-old group and increased with age; (ii) the level of histatin-1 reached a maximum in the 7-12-month-old group, a minimum in the 13-24-month-aged babies and it increased again in the 25-36-month-old group; (iii) S-type cystatins were almost undetectable in the 0-6-month-old group; (iv) P-B peptide concentration greatly increased with age; (v) histatin-3 1/24 and statherin concentrations did not show any age-related variation. The top-down proteomic approach undertaken in this work reveals that the salivary proteome of human children from birth to 48months of age shows important quantitative modifications. The concentrations of the major salivary proteins, with the exception of statherin and histatin-3 1/24, showed a minimum in the 0-6-month-old group when the expression in salivary glands is probably not fully activated. Concentrations of the salivary proteins slowly increased with age, with different trends. Only histatin-1 showed the highest concentration in the 7-12-month-old group, followed by a decrease in the 13-24-month-aged children. This particular trend could be related to the phenomenon of eruption of primary dentition. This study gives a contribution to the knowledge on the physiological variability occurring in human saliva during the early childhood. It could represent a strong and reliable basis for further investigation of saliva to develop diagnostic and prognostic biomarkers. © 2013.

Different Type 1 Fimbrial Genes and Tropisms of Commensal and Potentially Pathogenic Actinomyces spp. with Different Salivary Acidic Proline-Rich Protein and Statherin Ligand Specificities

PubMed Central

Li, Tong; Khah, Massoud Kheir; Slavnic, Snjezana; Johansson, Ingegerd; Strömberg, Nicklas

2001-01-01

Actinomyces spp. exhibit type 1 fimbria-mediated adhesion to salivary acidic proline-rich proteins (PRPs) and statherin ligands. Actinomyces spp. with different animal and tissue origins belong to three major adhesion types as relates to ligand specificity and type 1 fimbria genes. (i) In preferential acidic-PRP binding, strains of Actinomyces naeslundii genospecies 1 and 2 from human and monkey mouths displayed at least three ligand specificities characterized by preferential acidic-PRP binding. Slot blot DNA hybridization showed seven highly conserved type 1 fimbria genes (orf1- to -6 and fimP) in genospecies 1 and 2 strains, except that orf5 and orf3 were divergent in genospecies 1. (ii) In preferential statherin binding, oral Actinomyces viscosus strains of rat and hamster origin (and strain 19246 from a human case of actinomycosis) bound statherin preferentially. DNA hybridization and characterization of the type 1 fimbria genes from strain 19246 revealed a homologous gene cluster of four open reading frames (orfA to -C and fimP). Bioinformatics suggested sortase (orfB, orf4, and part of orf5), prepilin peptidase (orfC and orf6), fimbria subunit (fimP), and usher- and autotransporter-like (orfA and orf1 to -3) functions. Those gene regions corresponding to orf3 and orf5 were divergent, those corresponding to orf2, orf1, and fimP were moderately conserved, and those corresponding to orf4 and orf6 were highly conserved. Restriction fragment length polymorphism analyses using a fimP probe separated human and monkey and rat and hamster strains into phylogenetically different groups. (iii) In statherin-specific binding, strains of A. naeslundii genospecies 1 from septic and other human infections displayed a low-avidity binding to statherin. Only the orf4 and orf6 gene regions were highly conserved. Finally, rat saliva devoid of statherin bound bacterial strains avidly irrespective of ligand specificity, and specific antisera detected either type 1, type 2, or both types of fimbria on the investigated Actinomyces strains. PMID:11705891

mRNAs for PRPs, statherin, and histatins in von Ebner's gland tissues.

PubMed

Azen, E A; Hellekant, G; Sabatini, L M; Warner, T F

1990-11-01

A search was made for expression of genes for proline-rich proteins (PRPs) and other salivary-type proteins, including statherin and histatins, in taste-bud tissues of mice and primates because of previous genetic findings in mice (Azen et al., 1986) that Prp and taste genes for certain bitter substances are either the same or closely linked. Taste-bud tissues and other tissues were tested for specific mRNAs with labeled DNA probes by Northern blotting and in situ hybridization. It was found that PRP mRNAs were present in von Ebner's glands of mice and macaques, and that there was a much greater degree of PRP mRNA induction in mouse parotid (16-fold) than in von Ebner's gland (two-fold) after in vivo isoproterenol stimulation. This difference may be due, in part, to differences in autonomic nerve innervation. Statherin and histatin mRNAs were found in macaque taste-bud tissues containing von Ebner's gland, and statherin protein was found in human von Ebner's gland by immunohistochemistry. The finding of PRP gene expression in von Ebner's gland, whose secretions have been suggested to play a role in taste stimulation, adds further support to a possible function of PRPs in bitter tasting. The possible functions of statherin and histatins in von Ebner's gland secretions may be related to statherin's regulation of salivary calcium and histatins' antibacterial and antifungal properties.

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.

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.

Inhibition of hydroxyapatite growth by casein, a potential salivary phosphoprotein homologue.

PubMed

Romero, Maria J R H; Nakashima, Syozi; Nikaido, Toru; Ichinose, Shizuko; Sadr, Alireza; Tagami, Junji

2015-08-01

Salivary phosphoproteins are essential in tooth mineral regulation but are often overlooked in vitro. This study aimed to evaluate the effect of casein, as a salivary phosphoprotein homologue, on the deposition and growth of hydroxyapatite (HA) on tooth surfaces. Hydroxyapatite growth was quantified using seeded crystal systems. Artificial saliva (AS) containing HA powder and 0, 10, 20, 50, or 100 μg ml(-1) of casein, or 100 μg ml(-1) of dephosphorylated casein (Dcasein), was incubated for 0-8 h at 37°C, pH 7.2. Calcium concentrations were measured using atomic absorption spectroscopy (AAS). Surface precipitation of HA on bovine enamel and dentine blocks, incubated in similar conditions for 7 d, was examined using field emission scanning electron microscopy (FE-SEM) and transmission electron microscopy (TEM) with selected area electron diffraction (SAED). Casein adsorption was assessed using modified Lowry assays and zeta-potential measurements. The AAS results revealed a concentration-dependent inhibition of calcium consumption. Hydroxyapatite precipitation occurred when no casein was present, whereas precipitation of HA was apparently completely inhibited in casein-containing groups. Adsorption data demonstrated increasingly negative zeta-potential with increased casein concentration and an affinity constant similar to proline-rich proteins with Langmuir modelling. Casein inhibited the deposition and growth of HA primarily through the binding of esterized phosphate to HA active sites, indicating its potential as a mineral-regulating salivary phosphoprotein homologue in vitro. © 2015 Eur J Oral Sci.

Effect of a protein-rich meal on urinary and salivary free amino acid concentrations in human subjects.

PubMed

Brand, H S; Jörning, G G; Chamuleau, R A; Abraham-Inpijn, L

1997-08-08

The aim of the present study was to investigate whether in healthy volunteers acute changes in plasma free amino acid composition after a protein-rich test meal are reflected in the urinary and salivary concentrations of the corresponding amino acids. The ingestion of a protein-rich meal elicited a significant increase of plasma and urine amino acid concentrations. The postprandial salivary amino acid excretion showed only minor changes. For several amino acids (alanine, arginine, asparagine, glycine, threonine and valine) significant relations were observed between the increase in concentration of these amino acids in venous plasma and urine. In whole saliva, only threonine and valine showed a significant relationship with the corresponding plasma concentration. Our data suggest that the urinary amino acid excretion of several amino acids has the potential for estimating short-term changes in plasma concentrations. Determination of salivary amino acid concentrations seems less appropriate for this purpose.

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.

Dose-Dependent Effects of L-Arginine on PROP Bitterness Intensity and Latency and Characteristics of the Chemical Interaction between PROP and L-Arginine.

PubMed

Melis, Melania; Arca, Massimiliano; Aragoni, Maria Carla; Cabras, Tiziana; Caltagirone, Claudia; Castagnola, Massimo; Crnjar, Roberto; Messana, Irene; Tepper, Beverly J; Tomassini Barbarossa, Iole

2015-01-01

Genetic variation in the ability to taste the bitterness of 6-n-propylthiouracil (PROP) is a complex trait that has been used to predict food preferences and eating habits. PROP tasting is primarily controlled by polymorphisms in the TAS2R38 gene. However, a variety of factors are known to modify the phenotype. Principle among them is the salivary protein Ps-1 belonging to the basic proline-rich protein family (bPRP). Recently, we showed that oral supplementation with Ps-1 as well as its related free amino acids (L-Arg and L-Lys) enhances PROP bitterness perception, especially for PROP non-tasters who have low salivary levels of Ps-1. Here, we show that salivary L-Arg levels are higher in PROP super-tasters compared to medium tasters and non-tasters, and that oral supplementation with free L-Arg enhances PROP bitterness intensity as well as reduces bitterness latency in a dose-dependent manner, particularly in individuals with low salivary levels of both free L-Arg and Ps-1 protein. Supplementation with L-Arg also enhanced the bitterness of caffeine. We also used 1H-NMR spectroscopy and quantum-mechanical calculations carried out by Density Functional Theory (DFT) to characterize the chemical interaction between free L-Arg and the PROP molecule. Results showed that the -NH2 terminal group of the L-ArgH+ side chain interacts with the carbonyl or thiocarbonyl groups of PROP by forming two hydrogen bonds with the resulting charged adduct. The formation of this PROP•ArgH+ hydrogen-bonded adduct could enhance bitterness intensity by increasing the solubility of PROP in saliva and its availability to receptor sites. Our data suggest that L-Arg could act as a 'carrier' of various bitter molecules in saliva.

Dose-Dependent Effects of L-Arginine on PROP Bitterness Intensity and Latency and Characteristics of the Chemical Interaction between PROP and L-Arginine

PubMed Central

Melis, Melania; Arca, Massimiliano; Aragoni, Maria Carla; Cabras, Tiziana; Caltagirone, Claudia; Castagnola, Massimo; Crnjar, Roberto; Messana, Irene; Tepper, Beverly J.; Tomassini Barbarossa, Iole

2015-01-01

Genetic variation in the ability to taste the bitterness of 6-n-propylthiouracil (PROP) is a complex trait that has been used to predict food preferences and eating habits. PROP tasting is primarily controlled by polymorphisms in the TAS2R38 gene. However, a variety of factors are known to modify the phenotype. Principle among them is the salivary protein Ps-1 belonging to the basic proline-rich protein family (bPRP). Recently, we showed that oral supplementation with Ps-1 as well as its related free amino acids (L-Arg and L-Lys) enhances PROP bitterness perception, especially for PROP non-tasters who have low salivary levels of Ps-1. Here, we show that salivary L-Arg levels are higher in PROP super-tasters compared to medium tasters and non-tasters, and that oral supplementation with free L-Arg enhances PROP bitterness intensity as well as reduces bitterness latency in a dose-dependent manner, particularly in individuals with low salivary levels of both free L-Arg and Ps-1 protein. Supplementation with L-Arg also enhanced the bitterness of caffeine. We also used 1H-NMR spectroscopy and quantum-mechanical calculations carried out by Density Functional Theory (DFT) to characterize the chemical interaction between free L-Arg and the PROP molecule. Results showed that the –NH2 terminal group of the L-ArgH+ side chain interacts with the carbonyl or thiocarbonyl groups of PROP by forming two hydrogen bonds with the resulting charged adduct. The formation of this PROP•ArgH+ hydrogen-bonded adduct could enhance bitterness intensity by increasing the solubility of PROP in saliva and its availability to receptor sites. Our data suggest that L-Arg could act as a ‘carrier’ of various bitter molecules in saliva. PMID:26103639

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

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

Differential expression of isoproterenol-induced salivary polypeptides in two mouse strains that are congenic for the H-2 histocompatibility gene complex.

PubMed

López Solís, Remigio O; Weis, Ulrike Kemmerling; Ceballos, Alicia Ramos; Salas, Gustavo Hoecker

2003-12-01

Two inbred mouse strains, A/Snell and A.Swiss, which were produced as congenic with regard to the H-2 histocompatibility gene complex, are homozygous for two different groups of isoproterenol-induced salivary polypeptides (IISP). These polypeptides, which have been considered as markers of the hypertrophic growth of the parotid acinar cells, are members of the complex family of salivary proline-rich proteins (PRP) on the basis of both their massive accumulation in the parotid acinar cells in response to chronic isoproterenol, secretory character, high solubility in trichloroacetic acid and metachromatic staining by Coomassie blue. IISP expressed in both mouse strains were identified by unidimensional SDS-polyacrylamide electrophoresis and Coomassie blue staining both in parotid gland homogenates and in whole salivas obtained from mice repeatedly stimulated at 24-h intervals with isoproterenol. Parotid glands from 40 mice (20 A/Snell and 20 A.Swiss) and salivas from 270 mice (200 A/Snell and 70 A.Swiss) were analyzed. One of the congenic strains (A/Snell) expressed five IISP (Mr 65, 61, 51.5, 38, and 37 kDa) and the other strain (A.Swiss) expressed six IISP (Mr 59, 57, 54.5, 46, 36, and 34 kDa). No inter-individual intra-strain variations were observed, thus defining strain-associated patterns of IISP (PRP). Copyright 2003 Wiley-Liss, Inc.

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

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.

Strains of Actinomyces naeslundii and Actinomyces viscosus Exhibit Structurally Variant Fimbrial Subunit Proteins and Bind to Different Peptide Motifs in Salivary Proteins

PubMed Central

Li, Tong; Johansson, Ingegerd; Hay, Donald I.; Strömberg, Nicklas

1999-01-01

Oral strains of Actinomyces spp. express type 1 fimbriae, which are composed of major FimP subunits, and bind preferentially to salivary acidic proline-rich proteins (APRPs) or to statherin. We have mapped genetic differences in the fimP subunit genes and the peptide recognition motifs within the host proteins associated with these differential binding specificities. The fimP genes were amplified by PCR from Actinomyces viscosus ATCC 19246, with preferential binding to statherin, and from Actinomyces naeslundii LY7, P-1-K, and B-1-K, with preferential binding to APRPs. The fimP gene from the statherin-binding strain 19246 is novel and has about 80% nucleotide and amino acid sequence identity to the highly conserved fimP genes of the APRP-binding strains (about 98 to 99% sequence identity). The novel FimP protein contains an amino-terminal signal peptide, randomly distributed single-amino-acid substitutions, and structurally different segments and ends with a cell wall-anchoring and a membrane-spanning region. When agarose beads with CNBr-linked host determinant-specific decapeptides were used, A. viscosus 19246 bound to the Thr42Phe43 terminus of statherin and A. naeslundii LY7 bound to the Pro149Gln150 termini of APRPs. Furthermore, while the APRP-binding A. naeslundii strains originate from the human mouth, A. viscosus strains isolated from the oral cavity of rat and hamster hosts showed preferential binding to statherin and contained the novel fimP gene. Thus, A. viscosus and A. naeslundii display structurally variant fimP genes whose protein products are likely to interact with different peptide motifs and to determine animal host tropism. PMID:10225854

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.

The Surprising Composition of the Salivary Proteome of Preterm Human Newborn

PubMed Central

Castagnola, Massimo; Inzitari, Rosanna; Fanali, Chiara; Iavarone, Federica; Vitali, Alberto; Desiderio, Claudia; Vento, Giovanni; Tirone, Chiara; Romagnoli, Costantino; Cabras, Tiziana; Manconi, Barbara; Teresa Sanna, Maria; Boi, Roberto; Pisano, Elisabetta; Olianas, Alessandra; Pellegrini, Mariagiuseppina; Nemolato, Sonia; Wilhelm Heizmann, Claus; Faa, Gavino; Messana, Irene

2011-01-01

Saliva is a body fluid of a unique composition devoted to protect the mouth cavity and the digestive tract. Our high performance liquid chromatography (HPLC)-electrospray ionization-MS analysis of the acidic soluble fraction of saliva from preterm human newborn surprisingly revealed more than 40 protein masses often undetected in adult saliva. We were able to identify the following proteins: stefin A and stefin B, S100A7 (two isoforms), S100A8, S100A9 (four isoforms), S100A11, S100A12, small proline-rich protein 3 (two isoforms), lysozyme C, thymosins β4 and β10, antileukoproteinase, histone H1c, and α and γ globins. The average mass value reported in international data banks was often incongruent with our experimental results mostly because of post-translational modifications of the proteins, e.g. acetylation of the N-terminal residue. A quantitative label-free MS analysis showed protein levels altered in relation to the postconceptional age and suggested coordinate and hierarchical functions for these proteins during development. In summary, this study shows for the first time that analysis of these proteins in saliva of preterm newborns might represent a noninvasive way to obtain precious information of the molecular mechanisms of development of human fetal oral structures. PMID:20943598

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.

Evidence for functional heterogeneity both between and within four sources of condensed tannin

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

Asquith, T.N.

1985-01-01

Condensed tannins are polymers of flavan-3-ols that are produced by many plants in a wide variety of tissues. The ability of these compounds to actively precipitate proteins has been linked to nutritional deficiencies in many animals. Four purified tannins (quebracho, wattle, pinto bean and sorghum) were compared to chemical assays and astringency towards (/sup 14/C)-BSA. Quebracho and wattle tannins were much less astringent and had longer chain lengths that sorghum or pinto bean tannins. Quebracho tannin had a very high affinity for salivary proline-rich glycoproteins (PRPs) and pinto bean tannin alone had a measurable affinity for soybean trypsin inhibitor. Thismore » suggests that tannin/protein interactions in vivo may be very specific. Protein bound carbohydrate enhanced the binding of PRPs to tanning and conferred specificity on the interactions. Carbohydrate also increases the solubility of protein/tanning complexes, which may aid the animal in eliminating the complexes. (/sup 125/I)-labeled condensed tannin was shown to retain the ability to discriminate between high and low affinity proteins. (/sup 125/I)-labeled phenols were isolated from livers and kidneys of rats fed (/sup 125/I)-labeled tannin. The techniques described in this thesis should be widely applicable to studying in vivo functions of condensed tannins.« less

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

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.

Salivary agglutinin, which binds Streptococcus mutans and Helicobacter pylori, is the lung scavenger receptor cysteine-rich protein gp-340.

PubMed

Prakobphol, A; Xu, F; Hoang, V M; Larsson, T; Bergstrom, J; Johansson, I; Frängsmyr, L; Holmskov, U; Leffler, H; Nilsson, C; Borén, T; Wright, J R; Strömberg, N; Fisher, S J

2000-12-22

Salivary agglutinin is a high molecular mass component of human saliva that binds Streptococcus mutans, an oral bacterium implicated in dental caries. To study its protein sequence, we isolated the agglutinin from human parotid saliva. After trypsin digestion, a portion was analyzed by matrix-assisted laser/desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), which gave the molecular mass of 14 unique peptides. The remainder of the digest was subjected to high performance liquid chromatography, and the separated peptides were analyzed by MALDI-TOF/post-source decay; the spectra gave the sequences of five peptides. The molecular mass and peptide sequence information showed that salivary agglutinin peptides were identical to sequences in lung (lavage) gp-340, a member of the scavenger receptor cysteine-rich protein family. Immunoblotting with antibodies that specifically recognized either lung gp-340 or the agglutinin confirmed that the salivary agglutinin was gp-340. Immunoblotting with an antibody specific to the sialyl Le(x) carbohydrate epitope detected expression on the salivary but not the lung glycoprotein, possible evidence of different glycoforms. The salivary agglutinin also interacted with Helicobacter pylori, implicated in gastritis and peptic ulcer disease, Streptococcus agalactiae, implicated in neonatal meningitis, and several oral commensal streptococci. These results identify the salivary agglutinin as gp-340 and suggest it binds bacteria that are important determinants of either the oral ecology or systemic diseases.

Identification of a glycine-rich protein from the tick Rhipicephalus haemaphysaloides and evaluation of its vaccine potential against tick feeding.

PubMed

Zhou, Jinlin; Gong, Haiyan; Zhou, Yongzhi; Xuan, Xuenan; Fujisaki, Kozo

2006-12-01

A cDNA coding a glycine-rich protein was identified from the Rhipicephalus haemaphysaloides tick. The cDNA named here as RH50 was 1,823 bp, including a single open reading frame (ORF) of 1,518 nucleotides. The ORF encodes a polypeptide of 506 amino acid residues with a size of 50 kDa, as calculated by a computer. The predicted amino acid sequence of RH50 showed a low homology to sequences of some known extracellular matrix-like proteins. The native protein was identified in both the fed tick salivary gland lysates and extracts of cement material using the serum against the recombinant protein. Reverse transcription polymerase chain reaction results showed that RH50 mRNA was only transcribed in partially fed tick salivary glands, not in unfed tick salivary glands or partially fed tick midgut, fat body, or ovary. The differential expression of RH50 protein in fed tick salivary glands was confirmed by immunofluorescence. The low attachment rate both in the adult and nymphal tick, and the high mortality of immature ticks (nymph) feeding on recombinant RH50-immunized rabbits were found. These results show that the RH50 protein could be a useful candidate for anti-tick vaccine development.

Mass Spectrometric Identification of the Arginine and Lysine deficient Proline Rich Glutamine Rich Wheat Storage Proteins

USDA-ARS?s Scientific Manuscript database

Tandem mass spectrometry (MS/MS) of enzymatic digest has made possible identification of a wide variety of proteins and complex samples prepared by such techniques as RP-HPLC or 2-D gel electrophoresis. Success requires peptide fragmentation to be indicative of the peptide amino acid sequence. The f...

PRELP (proline/arginine-rich end leucine-rich repeat protein) promotes osteoblastic differentiation of preosteoblastic MC3T3-E1 cells by regulating the β-catenin pathway

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

Li, Haiying; Cui, Yazhou; Luan, Jing

Proline/arginine-rich end leucine-rich repeat protein (PRELP) is a collagen-binding proteoglycan highly expressed in the developing bones. Recent studies indicated that PRELP could inhibit osteoclastogenesis as a NF-κB inhibitor. However, its role during osteoblast differentiation is still unclear. In this study, we confirmed that the expression of PRELP increased with the osteogenesis induction of preosteoblastic MC3T3-E1 cells. Down-regulation of PRELP expression by shRNA reduced ALP activity, mineralization and expression of osteogenic marker gene Runx2. Our microarray analysis data suggested that β-catenin may act as a hub gene in the PRELP-mediated gene network. We validated furtherly that PRELP knockdown could inhibit themore » level of connexin43, a key regulator of osteoblast differentiation by affecting β-catenin protein expression, and its nuclear translocation in MC3T3-E1 preosteoblasts. Therefore, this study established a new role of PRELP in modulating β-catenin/connexin43 pathway and osteoblast differentiation.« less

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

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.

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.

IRBIT plays an important role in NHE3-mediated pHi regulation in HSG cells.

PubMed

Tran, Tien Manh; Park, Moon-Yong; Lee, Jiyeon; Bae, Jun-Seok; Hwang, Sung-Min; Choi, Se-Young; Mikoshiba, Katsuhiko; Park, Kyungpyo

2013-07-19

Expression of inositol-1,4,5-trisphosphate (IP3) receptor-binding protein (IRBIT) has been reported in epithelial cells. However, its role in pHi regulation is not well understood. In this study, we investigated the role of IRBIT in pHi regulation, mediated by Na(+)/H(+) exchangers (NHEs), in salivary glands. We measured pHi recovery from cell acidification in BCECF-loaded salivary HSG cells. Western blot and co-immunoprecipitation (CO-IP) assays were also performed, showing that NHE1, 2 and 3 are expressed, and IRBIT binds to NHE3. HOE642, a specific NHE1 blocker, inhibited pHi recovery, but 40% pH(i) recovery was still observed even at the highest concentration of HOE642. Furthermore, pretreatment of the cells with siIRBIT significantly inhibited pHi recovery, indicating that NHE3 potentially plays a role in pHi recovery as well. The amount of membrane-localized NHE3 and its interaction with IRBIT are also significantly increased by cell acidification. In addition, we found that Ste20p-related proline alanine-rich kinase (SPAK) reverses the effect of IRBIT on membrane NHE3 translocation. Taken together, we conclude that IRBIT plays an important role in pHi regulation, mediated by NHE3, and further regulated by SPAK. Copyright © 2013 Elsevier Inc. All rights reserved.

A Novel Topology of Proline-rich Transmembrane Protein 2 (PRRT2)

PubMed Central

Rossi, Pia; Sterlini, Bruno; Castroflorio, Enrico; Marte, Antonella; Onofri, Franco; Valtorta, Flavia; Maragliano, Luca; Corradi, Anna; Benfenati, Fabio

2016-01-01

Proline-rich transmembrane protein 2 (PRRT2) has been identified as the single causative gene for a group of paroxysmal syndromes of infancy, including epilepsy, paroxysmal movement disorders, and migraine. On the basis of topology predictions, PRRT2 has been assigned to the recently characterized family of Dispanins, whose members share the two-transmembrane domain topology with a large N terminus and short C terminus oriented toward the outside of the cell. Because PRRT2 plays a role at the synapse, it is important to confirm the exact orientation of its N and C termini with respect to the plasma membrane to get clues regarding its possible function. Using a combination of different experimental approaches, including live immunolabeling, immunogold electron microscopy, surface biotinylation and computational modeling, we demonstrate a novel topology for this protein. PRRT2 is a type II transmembrane protein in which only the second hydrophobic segment spans the plasma membrane, whereas the first one is associated with the internal surface of the membrane and forms a helix-loop-helix structure without crossing it. Most importantly, the large proline-rich N-terminal domain is not exposed to the extracellular space but is localized intracellularly, and only the short C terminus is extracellular (Ncyt/Cexo topology). Accordingly, we show that PRRT2 interacts with the Src homology 3 domain-bearing protein Intersectin 1, an intracellular protein involved in synaptic vesicle cycling. These findings will contribute to the clarification of the role of PRRT2 at the synapse and the understanding of pathogenic mechanisms on the basis of PRRT2-related neurological disorders. PMID:26797119

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.

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

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.

Abscisic Acid- and Stress-Induced Highly Proline-Rich Glycoproteins Regulate Root Growth in Rice1[W][OPEN

PubMed Central

Tseng, I-Chieh; Hong, Chwan-Yang; Yu, Su-May; Ho, Tuan-Hua David

2013-01-01

In the root of rice (Oryza sativa), abscisic acid (ABA) treatment, salinity, or water deficit stress induces the expression of a family of four genes, REPETITIVE PROLINE-RICH PROTEIN (RePRP). These genes encode two subclasses of novel proline-rich glycoproteins with highly repetitive PX1PX2 motifs, RePRP1 and RePRP2. RePRP orthologs exist only in monocotyledonous plants, and their functions are virtually unknown. Rice RePRPs are heavily glycosylated with arabinose and glucose on multiple hydroxyproline residues. They are significantly different from arabinogalactan proteins that have glycan chains composed of arabinose and galactose. Transient and stable expressions of RePRP-green fluorescent protein reveal that a fraction of this protein is localized to the plasma membrane. In rice roots, ABA treatment increases RePRP expression preferentially in the elongation zone. Overexpression of RePRP in transgenic rice reduces root cell elongation in the absence of ABA, similar to the effect of ABA on wild-type roots. Conversely, simultaneous knockdown of the expression of RePRP1 and RePRP2 reduces the root sensitivity to ABA, indicating that RePRP proteins play an essential role in ABA/stress regulation of root growth and development. Moreover, rice RePRPs specifically interact with a polysaccharide, arabinogalactan, in a dosage-dependent manner. It is suggested that RePRP1 and RePRP2 are functionally redundant suppressors of root cell expansion and probably act through interactions with cell wall components near the plasma membrane. PMID:23886623

Tetramer-organizing polyproline-rich peptides differ in CHO cell-expressed and plasma-derived human butyrylcholinesterase tetramers.

PubMed

Schopfer, Lawrence M; Lockridge, Oksana

2016-06-01

Tetrameric butyrylcholinesterase (BChE) in human plasma is the product of multiple genes, namely one BCHE gene on chromosome 3q26.1 and multiple genes that encode polyproline-rich peptides. The function of the polyproline-rich peptides is to assemble BChE into tetramers. CHO cells transfected with human BChE cDNA express BChE monomers and dimers, but only low quantities of tetramers. Our goal was to identify the polyproline-rich peptides in CHO-cell derived human BChE tetramers. CHO cell-produced human BChE tetramers were purified from serum-free culture medium. Peptides embedded in the tetramerization domain were released from BChE tetramers by boiling and identified by liquid chromatography-tandem mass spectrometry. A total of 270 proline-rich peptides were sequenced, ranging in size from 6-41 residues. The peptides originated from 60 different proteins that reside in multiple cell compartments including the nucleus, cytoplasm, and endoplasmic reticulum. No single protein was the source of the polyproline-rich peptides in CHO cell-expressed human BChE tetramers. In contrast, 70% of the tetramer-organizing peptides in plasma-derived BChE tetramers originate from lamellipodin. No protein source was identified for polyproline peptides containing up to 41 consecutive proline residues. In conclusion, the use of polyproline-rich peptides as a tetramerization motif is documented only for the cholinesterases, but is expected to serve other tetrameric proteins as well. The CHO cell data suggest that the BChE tetramer-organizing peptide can arise from a variety of proteins. Copyright © 2016 Elsevier B.V. All rights reserved.

Induction of salivary polypeptides associated with parotid hypertrophy by gallotannins administered topically into the mouse mouth.

PubMed

Gho, Francesca; Peña-Neira, Alvaro; López-Solís, Remigio O

2007-02-01

Isoproterenol-induced salivary polypeptides (IISP), a group of proline-rich proteins synthesized by mouse parotids, have been considered as markers for isoproterenol-induced parotid hypertrophy. Rodents fed diets containing high-tannin cereals (sorghum), also develop parotid hypertrophy. To test whether tannins are directly involved in provoking sialotrophic growth, we studied the effect of intraperitoneal and topical oral administrations of tannic acid (TA) on the induction of IISP polypeptides in endogamic mice (A/Snell). TA was characterized by HPLC chromatography and spectral analysis and shown to be composed solely of gallotannins, a complex family of glucose and gallic acid esters. IISP polypeptides were monitored in saliva by SDS-polyacrylamide gel electrophoresis during 36 h after ending TA stimulation. Single daily intraperitoneal administrations of TA for 3 consecutive days (0.033 mg/g bw/day), at variance of parallel administrations of isoproterenol (0.042 mg/g bw/day) failed to induce IISP polypeptides. However, repeated topical applications of TA into the mouse mouths (1.21 mg/g bw divided into three equal doses given at 4-h intervals within a single day) resulted in unequivocal induction of IISP polypeptides. That response was clearly intensified by increasing the stimulation frequency to eight equivalent doses given at 1.5-h intervals within a single day (corresponding to 3.23 mg/g bw) and even further by repeating this protocol for 3 days. Under these productive schemes of stimulations by TA, electrophoretic fractionation of parotid homogenates showed new polypeptide bands migrating in parallel to salivary IISP. These results suggest that topically administered gallotannins are effective inducers of trophic growth in mouse parotids.

A Novel Topology of Proline-rich Transmembrane Protein 2 (PRRT2): HINTS FOR AN INTRACELLULAR FUNCTION AT THE SYNAPSE.

PubMed

Rossi, Pia; Sterlini, Bruno; Castroflorio, Enrico; Marte, Antonella; Onofri, Franco; Valtorta, Flavia; Maragliano, Luca; Corradi, Anna; Benfenati, Fabio

2016-03-18

Proline-rich transmembrane protein 2 (PRRT2) has been identified as the single causative gene for a group of paroxysmal syndromes of infancy, including epilepsy, paroxysmal movement disorders, and migraine. On the basis of topology predictions, PRRT2 has been assigned to the recently characterized family of Dispanins, whose members share the two-transmembrane domain topology with a large N terminus and short C terminus oriented toward the outside of the cell. Because PRRT2 plays a role at the synapse, it is important to confirm the exact orientation of its N and C termini with respect to the plasma membrane to get clues regarding its possible function. Using a combination of different experimental approaches, including live immunolabeling, immunogold electron microscopy, surface biotinylation and computational modeling, we demonstrate a novel topology for this protein. PRRT2 is a type II transmembrane protein in which only the second hydrophobic segment spans the plasma membrane, whereas the first one is associated with the internal surface of the membrane and forms a helix-loop-helix structure without crossing it. Most importantly, the large proline-rich N-terminal domain is not exposed to the extracellular space but is localized intracellularly, and only the short C terminus is extracellular (N cyt/C exo topology). Accordingly, we show that PRRT2 interacts with the Src homology 3 domain-bearing protein Intersectin 1, an intracellular protein involved in synaptic vesicle cycling. These findings will contribute to the clarification of the role of PRRT2 at the synapse and the understanding of pathogenic mechanisms on the basis of PRRT2-related neurological disorders. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

The Effects of Threonine Phosphorylation on the Stability and Dynamics of the Central Molecular Switch Region of 18.5-kDa Myelin Basic Protein

PubMed Central

De Avila, Miguel; Polverini, Eugenia; Harauz, George

2013-01-01

The classic isoforms of myelin basic protein (MBP) are essential for the formation and maintenance of myelin in the central nervous system of higher vertebrates. The protein is involved in all facets of the development, compaction, and stabilization of the multilamellar myelin sheath, and also interacts with cytoskeletal and signaling proteins. The predominant 18.5-kDa isoform of MBP is an intrinsically-disordered protein that is a candidate auto-antigen in the human demyelinating disease multiple sclerosis. A highly-conserved central segment within classic MBP consists of a proline-rich region (murine 18.5-kDa sequence –T92-P93-R94-T95-P96-P97-P98-S99–) containing a putative SH3-ligand, adjacent to a region that forms an amphipathic α-helix (P82-I90) upon interaction with membranes, or under membrane-mimetic conditions. The T92 and T95 residues within the proline-rich region can be post-translationally modified through phosphorylation by mitogen-activated protein (MAP) kinases. Here, we have investigated the structure of the α-helical and proline-rich regions in dilute aqueous buffer, and have evaluated the effects of phosphorylation at T92 and T95 on the stability and dynamics of the α-helical region, by utilizing four 36-residue peptides (S72–S107) with differing phosphorylation status. Nuclear magnetic resonance spectroscopy reveals that both the α-helical as well as the proline-rich regions are disordered in aqueous buffer, whereas they are both structured in a lipid environment (cf., Ahmed et al., Biochemistry 51, 7475-9487, 2012). Thermodynamic analysis of trifluoroethanol-titration curves monitored by circular dichroism spectroscopy reveals that phosphorylation, especially at residue T92, impedes formation of the amphipathic α-helix. This conclusion is supported by molecular dynamics simulations, which further illustrate that phosphorylation reduces the folding reversibility of the α-helix upon temperature perturbation and affect the global structure of the peptides through altered electrostatic interactions. The results support the hypothesis that the central conserved segment of MBP constitutes a molecular switch in which the conformation and/or intermolecular interactions are mediated by phosphorylation/dephosphorylation at T92 and T95. PMID:23861868

The effects of threonine phosphorylation on the stability and dynamics of the central molecular switch region of 18.5-kDa myelin basic protein.

PubMed

Vassall, Kenrick A; Bessonov, Kyrylo; De Avila, Miguel; Polverini, Eugenia; Harauz, George

2013-01-01

The classic isoforms of myelin basic protein (MBP) are essential for the formation and maintenance of myelin in the central nervous system of higher vertebrates. The protein is involved in all facets of the development, compaction, and stabilization of the multilamellar myelin sheath, and also interacts with cytoskeletal and signaling proteins. The predominant 18.5-kDa isoform of MBP is an intrinsically-disordered protein that is a candidate auto-antigen in the human demyelinating disease multiple sclerosis. A highly-conserved central segment within classic MBP consists of a proline-rich region (murine 18.5-kDa sequence -T92-P93-R94-T95-P96-P97-P98-S99-) containing a putative SH3-ligand, adjacent to a region that forms an amphipathic α-helix (P82-I90) upon interaction with membranes, or under membrane-mimetic conditions. The T92 and T95 residues within the proline-rich region can be post-translationally modified through phosphorylation by mitogen-activated protein (MAP) kinases. Here, we have investigated the structure of the α-helical and proline-rich regions in dilute aqueous buffer, and have evaluated the effects of phosphorylation at T92 and T95 on the stability and dynamics of the α-helical region, by utilizing four 36-residue peptides (S72-S107) with differing phosphorylation status. Nuclear magnetic resonance spectroscopy reveals that both the α-helical as well as the proline-rich regions are disordered in aqueous buffer, whereas they are both structured in a lipid environment (cf., Ahmed et al., Biochemistry 51, 7475-9487, 2012). Thermodynamic analysis of trifluoroethanol-titration curves monitored by circular dichroism spectroscopy reveals that phosphorylation, especially at residue T92, impedes formation of the amphipathic α-helix. This conclusion is supported by molecular dynamics simulations, which further illustrate that phosphorylation reduces the folding reversibility of the α-helix upon temperature perturbation and affect the global structure of the peptides through altered electrostatic interactions. The results support the hypothesis that the central conserved segment of MBP constitutes a molecular switch in which the conformation and/or intermolecular interactions are mediated by phosphorylation/dephosphorylation at T92 and T95.

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

Motility Determinants in WASP Family ProteinsD⃞

PubMed Central

Yarar, Defne; D'Alessio, Joseph A.; Jeng, Robert L.; Welch, Matthew D.

2002-01-01

In response to upstream signals, proteins in the Wiskott-Aldrich Syndrome protein (WASP) family regulate actin nucleation via the Arp2/3 complex. Despite intensive study of the function of WASP family proteins in nucleation, it is not yet understood how their distinct structural organization contributes to actin-based motility. Herein, we analyzed the activities of WASP and Scar1 truncation derivatives by using a bead-based motility assay. The minimal region of WASP sufficient to direct movement was the C-terminal WCA fragment, whereas the corresponding region of Scar1 was insufficient. In addition, the proline-rich regions of WASP and Scar1 and the Ena/VASP homology 1 (EVH1) domain of WASP independently enhanced motility rates. The contributions of these regions to motility could not be accounted for by their direct effects on actin nucleation with the Arp2/3 complex, suggesting that they stimulate motility by recruiting additional factors. We have identified profilin as one such factor. WASP- and Scar1-coated bead motility rates were significantly reduced by depletion of profilin and VASP and could be more efficiently rescued by a combination of VASP and wild-type profilin than by VASP and a mutant profilin that cannot bind proline-rich sequences. Moreover, motility of WASP WCA beads was not affected by the depletion or addback of VASP and profilin. Our results suggest that recruitment of factors, including profilin, by the proline-rich regions of WASP and Scar1 and the EVH1 domain of WASP stimulates cellular actin-based motility. PMID:12429845

Characterization of salivary alpha-amylase binding to Streptococcus sanguis

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

Scannapieco, F.A.; Bergey, E.J.; Reddy, M.S.

1989-09-01

The purpose of this study was to identify the major salivary components which interact with oral bacteria and to determine the mechanism(s) responsible for their binding to the bacterial surface. Strains of Streptococcus sanguis, Streptococcus mitis, Streptococcus mutans, and Actinomyces viscosus were incubated for 2 h in freshly collected human submandibular-sublingual saliva (HSMSL) or parotid saliva (HPS), and bound salivary components were eluted with 2% sodium dodecyl sulfate. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western transfer, alpha-amylase was the prominent salivary component eluted from S. sanguis. Studies with {sup 125}I-labeled HSMSL or {sup 125}I-labeled HPS also demonstrated a componentmore » with an electrophoretic mobility identical to that of alpha-amylase which bound to S. sanguis. Purified alpha-amylase from human parotid saliva was radiolabeled and found to bind to strains of S. sanguis genotypes 1 and 3 and S. mitis genotype 2, but not to strains of other species of oral bacteria. Binding of ({sup 125}I)alpha-amylase to streptococci was saturable, calcium independent, and inhibitable by excess unlabeled alpha-amylases from a variety of sources, but not by secretory immunoglobulin A and the proline-rich glycoprotein from HPS. Reduced and alkylated alpha-amylase lost enzymatic and bacterial binding activities. Binding was inhibited by incubation with maltotriose, maltooligosaccharides, limit dextrins, and starch.« less

Carbachol-induced in vitro secretion of certain human submandibular proteins investigated by mass-spectrometry.

PubMed

Cabras, Tiziana; Castagnola, Massimo; Inzitari, Rosanna; Ekström, Jörgen; Isola, Michela; Riva, Alessandro; Messana, Irene

2008-11-01

To investigate protein content of saliva produced in vitro by samples of human submandibular gland following stimulation with the muscarinic agent carbachol. Tissue samples, obtained at surgery from seven patients and showing normal morphological appearance, were tested for 30 min: in absence of carbachol and atropine; in presence of carbachol (10 microM); in presence of carbachol (10 microM) and atropine (20 microM); or in presence of just atropine (20 microM). Medium was analysed by high-performance liquid chromatography-mass-spectrometry. Neither before nor during surgery were the patients exposed to drug treatments that were likely to influence the in vitro secretion. Proline-rich proteins (PRP)-1 and -3, peptide PC and PB, statherin, cystatins SN, S1 and S2 were invariably found in control gland tissue medium. Mean concentrations of these proteins/peptides in the medium were non-proportionally elevated following carbachol exposure to the gland tissues. Difference between basal release and carbachol-induced secretion achieved statistical significance as to all the proteins/peptides under study but for statherin. Atropine alone or atropine plus carbachol caused no significant changes compared to the basal release of proteins/peptides. In vitro studies on salivary glands make it possible to study protein secretion from individual glands and thus, to reveal the contribution of the various types of gland to protein/peptide content of whole saliva. The disproportional responses to carbachol may imply that the proteins/peptides are not confined to the same cells or to the same intracellular locations and are therefore not secreted as packages at parasympathetic cholinergic activity.

Effect of WAVE2 phosphorylation on activation of the Arp2/3 complex.

PubMed

Nakanishi, Osamu; Suetsugu, Shiro; Yamazaki, Daisuke; Takenawa, Tadaomi

2007-03-01

Members of the family of WASP-family Verprolin homologous proteins (WAVEs) activate the Arp2/3 complex to induce actin polymerization. The WAVE family comprises three proteins, namely, WAVE1, WAVE2 and WAVE3. Among them, WAVE2 is crucial for activation of the Arp2/3 complex for the formation of branched actin filaments in lamellipodia. Activation of mitogen-activated protein (MAP) kinase signalling results in the phosphorylation of the WAVE family proteins; however, which of the three WAVE proteins is phosphorylated is unclear. We found that in vitro WAVE2 is directly phosphorylated by a MAP kinase, i.e. extracellular signal-regulated kinase (ERK) 2. The proline-rich region and the verprolin, cofilin and acidic (VCA) region of WAVE2 were phosphorylated. Interestingly, the phosphorylated VCA region had a higher affinity for the Arp2/3 complex. However, the phosphorylation of the VCA region resulted in reduced induction of Arp2/3-mediated actin polymerization in vitro. The role of the phosphorylation of the proline-rich region was not determined.

Salivary proteomics of healthy dogs: An in depth catalog

PubMed Central

Furrow, Eva; Souza, Clarissa P.; Granick, Jennifer L.; de Jong, Ebbing P.; Griffin, Timothy J.; Wang, Xiong

2018-01-01

Objective To provide an in-depth catalog of the salivary proteome and endogenous peptidome of healthy dogs, evaluate proteins and peptides with antimicrobial properties, and compare the most common salivary proteins and peptides between different breed phylogeny groups. Methods 36 healthy dogs without evidence of periodontal disease representing four breed phylogeny groups, based upon single nucleotide polymorphism haplotypes (ancient, herding/sighthound, and two miscellaneous groups). Saliva collected from dogs was pooled by phylogeny group and analyzed using nanoscale liquid chromatography-tandem mass spectrometry. Resulting tandem mass spectra were compared to databases for identification of endogenous peptides and inferred proteins. Results 2,491 proteins and endogenous peptides were found in the saliva of healthy dogs with no periodontal disease. All dog phylogeny groups’ saliva was rich in proteins and peptides with antimicrobial functions. The ancient breeds group was distinct in that it contained unique proteins and was missing many proteins and peptides present in the other groups. Conclusions and clinical relevance Using a sophisticated nanoscale liquid chromatography-tandem mass spectrometry, we were able to identify 10-fold more salivary proteins than previously reported in dogs. Seven of the top 10 most abundant proteins or peptides serve immune functions and many more with various antimicrobial mechanisms were found. This is the most comprehensive analysis of healthy canine saliva to date, and will provide the groundwork for future studies analyzing salivary proteins and endogenous peptides in disease states. PMID:29329347

Salivary proteomics of healthy dogs: An in depth catalog.

PubMed

Torres, Sheila M F; Furrow, Eva; Souza, Clarissa P; Granick, Jennifer L; de Jong, Ebbing P; Griffin, Timothy J; Wang, Xiong

2018-01-01

To provide an in-depth catalog of the salivary proteome and endogenous peptidome of healthy dogs, evaluate proteins and peptides with antimicrobial properties, and compare the most common salivary proteins and peptides between different breed phylogeny groups. 36 healthy dogs without evidence of periodontal disease representing four breed phylogeny groups, based upon single nucleotide polymorphism haplotypes (ancient, herding/sighthound, and two miscellaneous groups). Saliva collected from dogs was pooled by phylogeny group and analyzed using nanoscale liquid chromatography-tandem mass spectrometry. Resulting tandem mass spectra were compared to databases for identification of endogenous peptides and inferred proteins. 2,491 proteins and endogenous peptides were found in the saliva of healthy dogs with no periodontal disease. All dog phylogeny groups' saliva was rich in proteins and peptides with antimicrobial functions. The ancient breeds group was distinct in that it contained unique proteins and was missing many proteins and peptides present in the other groups. Using a sophisticated nanoscale liquid chromatography-tandem mass spectrometry, we were able to identify 10-fold more salivary proteins than previously reported in dogs. Seven of the top 10 most abundant proteins or peptides serve immune functions and many more with various antimicrobial mechanisms were found. This is the most comprehensive analysis of healthy canine saliva to date, and will provide the groundwork for future studies analyzing salivary proteins and endogenous peptides in disease states.

A novel zinc-finger protein with a proline-rich domain mediates ABA-regulated seed dormancy in Arabidopsis.

PubMed

He, Yuehui; Gan, Susheng

2004-01-01

Seed dormancy is an important developmental process that prevents pre-harvest sprouting in many grains and other seeds. Abscisic acid (ABA), a plant hormone, plays a crucial role in regulating dormancy but the underlying molecular regulatory mechanisms are not fully understood. An Arabidopsis zinc-finger gene, MEDIATOR OF ABA-REGULATED DORMANCY 1 ( MARD1 ) was identified and functionally analyzed. MARD1 expression is up-regulated by ABA. A T-DNA insertion in the promoter region downstream of two ABA-responsive elements (ABREs) renders MARD1 unable to respond to ABA. The mard1 seeds are less dormant and germinate in total darkness; their germination is resistant to external ABA at the stage of radicle protrusion. These results suggest that this novel zinc-finger protein with a proline-rich N-terminus is an important downstream component of the ABA signaling pathway that mediates ABA-regulated seed dormancy in Arabidopsis.

Antimicrobial proline-rich peptides from the hemolymph of marine snail Rapana venosa.

PubMed

Dolashka, Pavlina; Moshtanska, Vesela; Borisova, Valika; Dolashki, Aleksander; Stevanovic, Stefan; Dimanov, Tzvetan; Voelter, Wolfgang

2011-07-01

Hemolymph of Rapana venosa snails is a complex mixture of biochemically and pharmacologically active components such as peptides and proteins. Antimicrobial peptides are gaining attention as antimicrobial alternatives to chemical food preservatives and commonly used antibiotics. Therefore, for the first time we have explored the isolation, identification and characterisation of 11 novel antimicrobial peptides produced by the hemolymph of molluscs. The isolated peptides from the hemolymph applying ultrafiltration and reverse-phase high-performance liquid chromatography (RP-HPLC) have molecular weights between 3000 and 9500 Da, determined by mass spectrometric analysis. The N-terminal sequences of the peptides identified by Edman degradation matched no peptides in the MASCOT search database, indicating novel proline-rich peptides. UV spectra revealed that these substances possessed the characteristics of protein peptides with acidic isoelectric points. However, no Cotton effects were observed between 190 and 280 nm by circular dichroism spectroscopy. Four of the pro-rich peptides also showed strong antimicrobial activities against tested microorganisms including Gram-positive and Gram-negative bacteria. Copyright © 2011 Elsevier Inc. All rights reserved.

Thermodynamic contribution of backbone conformational entropy in the binding between SH3 domain and proline-rich motif.

PubMed

Zeng, Danyun; Shen, Qingliang; Cho, Jae-Hyun

2017-02-26

Biological functions of intrinsically disordered proteins (IDPs), and proteins containing intrinsically disordered regions (IDRs) are often mediated by short linear motifs, like proline-rich motifs (PRMs). Upon binding to their target proteins, IDPs undergo a disorder-to-order transition which is accompanied by a large conformational entropy penalty. Hence, the molecular mechanisms underlying control of conformational entropy are critical for understanding the binding affinity and selectivity of IDPs-mediated protein-protein interactions (PPIs). Here, we investigated the backbone conformational entropy change accompanied by binding of the N-terminal SH3 domain (nSH3) of CrkII and PRM derived from guanine nucleotide exchange factor 1 (C3G). In particular, we focused on the estimation of conformational entropy change of disordered PRM upon binding to the nSH3 domain. Quantitative characterization of conformational dynamics of disordered peptides like PRMs is limited. Hence, we combined various methods, including NMR model-free analysis, δ2D, DynaMine, and structure-based calculation of entropy loss. This study demonstrates that the contribution of backbone conformational entropy change is significant in the PPIs mediated by IDPs/IDRs. Copyright © 2017 Elsevier Inc. All rights reserved.

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.

Prolyl hydroxylation regulates protein degradation, synthesis, and splicing in human induced pluripotent stem cell-derived cardiomyocytes

PubMed Central

Stoehr, Andrea; Yang, Yanqin; Patel, Sajni; Evangelista, Alicia M.; Aponte, Angel; Wang, Guanghui; Liu, Poching; Boylston, Jennifer; Kloner, Philip H.; Lin, Yongshun; Gucek, Marjan; Zhu, Jun; Murphy, Elizabeth

2016-01-01

Aims Protein hydroxylases are oxygen- and α-ketoglutarate-dependent enzymes that catalyse hydroxylation of amino acids such as proline, thus linking oxygen and metabolism to enzymatic activity. Prolyl hydroxylation is a dynamic post-translational modification that regulates protein stability and protein–protein interactions; however, the extent of this modification is largely uncharacterized. The goals of this study are to investigate the biological consequences of prolyl hydroxylation and to identify new targets that undergo prolyl hydroxylation in human cardiomyocytes. Methods and results We used human induced pluripotent stem cell-derived cardiomyocytes in combination with pulse-chase amino acid labelling and proteomics to analyse the effects of prolyl hydroxylation on protein degradation and synthesis. We identified 167 proteins that exhibit differences in degradation with inhibition of prolyl hydroxylation by dimethyloxalylglycine (DMOG); 164 were stabilized. Proteins involved in RNA splicing such as serine/arginine-rich splicing factor 2 (SRSF2) and splicing factor and proline- and glutamine-rich (SFPQ) were stabilized with DMOG. DMOG also decreased protein translation of cytoskeletal and sarcomeric proteins such as α-cardiac actin. We searched the mass spectrometry data for proline hydroxylation and identified 134 high confidence peptides mapping to 78 unique proteins. We identified SRSF2, SFPQ, α-cardiac actin, and cardiac titin as prolyl hydroxylated. We identified 29 prolyl hydroxylated proteins that showed a significant difference in either protein degradation or synthesis. Additionally, we performed next-generation RNA sequencing and showed that the observed decrease in protein synthesis was not due to changes in mRNA levels. Because RNA splicing factors were prolyl hydroxylated, we investigated splicing ± inhibition of prolyl hydroxylation and detected 369 alternative splicing events, with a preponderance of exon skipping. Conclusions This study provides the first extensive characterization of the cardiac prolyl hydroxylome and demonstrates that inhibition of α-ketoglutarate hydroxylases alters protein stability, translation, and splicing. PMID:27095734

Salivary flow rate and biochemical composition analysis in stimulated whole saliva of children with cystic fibrosis.

PubMed

da Silva Modesto, Karine Barros; de Godói Simões, Jéssica Bueno; de Souza, Amanda Ferreira; Damaceno, Neiva; Duarte, Danilo Antonio; Leite, Mariana Ferreira; de Almeida, Eliete Rodrigues

2015-11-01

It is recognized that cystic fibrosis (CF) patients present a risk for oral diseases, since it affects exocrine glands, and the treatment consists of a carbohydrate-rich diet. Recognizing the protective function of saliva on maintaining oral health, the aim of the study was to evaluate salivary parameters in stimulated whole saliva from children with CF. A case-control study was conducted comparing stimulated whole saliva of healthy (n=28; control group) and CF children (n=21; experimental group). Salivary flow rate, initial pH, buffer capacity (total and in each range of pH), total protein and sialic acid (total, free, and conjugated) concentration, α-amylase and salivary peroxidase activities were evaluated. Data were compared by two-tailed Student t test (95% CI; p ≤ 0.05). CF patients presented a significant reduction in salivary parameters compared with the control group (p ≤ 0.05): salivary flow rate (36%), buffer capacity (pH range from 6.9 to 6.0), sialic acid concentration (total 75%, free 61%, and conjugated 83%); α-amylase and salivary peroxidase activities (55%). Additionally, a significant increase in total protein concentration (180%) of stimulated whole saliva from CF patients was verified compared with the control group (p ≤ 0.05). Children with CF presented significant changes in salivary composition, including salivary flow rate, buffering capacity and protective proteins of the oral cavity, compared with children without CF. Copyright © 2015 Elsevier Ltd. All rights reserved.

Is there adaptation of the exocrine pancreas in wild animal? The case of the Roe deer.

PubMed

Guilloteau, Paul; Vitari, Francesca; Metzinger-Le Meuth, Valérie; Le Normand, Laurence; Romé, Véronique; Savary, Gérard; Delaby, Luc; Domeneghini, Cinzia; Morisset, Jean

2012-05-28

Physiology of the exocrine pancreas has been well studied in domestic and in laboratory animals as well as in humans. However, it remains quite unknown in wildlife mammals. Roe deer and cattle (including calf) belong to different families but have a common ancestor. This work aimed to evaluate in the Roe deer, the adaptation to diet of the exocrine pancreatic functions and regulations related to animal evolution and domestication. Forty bovine were distributed into 2 groups of animals either fed exclusively with a milk formula (monogastric) or fed a dry feed which allowed for rumen function to develop, they were slaughtered at 150 days of age. The 35 Roe deer were wild animals living in the temperate broadleaf and mixed forests, shot during the hunting season and classified in two groups adult and young. Immediately after death, the pancreas was removed for tissue sample collection and then analyzed. When expressed in relation to body weight, pancreas, pancreatic protein weights and enzyme activities measured were higher in Roe deer than in calf. The 1st original feature is that in Roe deer, the very high content in pancreatic enzymes seems to be related to specific digestive products observed (proline-rich proteins largely secreted in saliva) which bind tannins, reducing their deleterious effects on protein digestion. The high chymotrypsin and elastase II quantities could allow recycling of proline-rich proteins. In contrast, domestication and rearing cattle resulted in simplified diet with well digestible components. The 2nd feature is that in wild animal, both receptor subtypes of the CCK/gastrin family peptides were present in the pancreas as in calf, although CCK-2 receptor subtype was previously identified in higher mammals. Bovine species could have lost some digestive capabilities (no ingestion of great amounts of tannin-rich plants, capabilities to secrete high amounts of proline-rich proteins) compared with Roe deer species. CCK and gastrin could play an important role in the regulation of pancreatic secretion in Roe deer as in calf. This work, to the best of our knowledge is the first study which compared the Roe deer adaptation to diet with a domesticated animal largely studied.

Tyrosine phosphorylation of WW proteins

PubMed Central

Reuven, Nina; Shanzer, Matan

2015-01-01

A number of key regulatory proteins contain one or two copies of the WW domain known to mediate protein–protein interaction via proline-rich motifs, such as PPxY. The Hippo pathway components take advantage of this module to transduce tumor suppressor signaling. It is becoming evident that tyrosine phosphorylation is a critical regulator of the WW proteins. Here, we review the current knowledge on the involved tyrosine kinases and their roles in regulating the WW proteins. PMID:25627656

Inhibition of PKR Activation by the Proline-Rich RNA Binding Domain of the Herpes Simplex Virus Type 1 Us11 Protein

PubMed Central

Poppers, Jeremy; Mulvey, Matthew; Khoo, David; Mohr, Ian

2000-01-01

Upon activation by double-stranded RNA in virus-infected cells, the cellular PKR kinase phosphorylates the translation initiation factor eukaryotic initiation factor 2 (eIF2) and thereby inhibits protein synthesis. The γ34.5 and Us11 gene products encoded by herpes simplex virus type 1 (HSV-1) are dedicated to preventing the accumulation of phosphorylated eIF2. While the γ34.5 gene specifies a regulatory subunit for protein phosphatase 1α, the Us11 gene encodes an RNA binding protein that also prevents PKR activation. γ34.5 mutants fail to grow on a variety of human cells as phosphorylated eIF2 accumulates and protein synthesis ceases prior to the completion of the viral life cycle. We demonstrate that expression of a 68-amino-acid fragment of Us11 containing a novel proline-rich basic RNA binding domain allows for sustained protein synthesis and enhanced growth of γ34.5 mutants. Furthermore, this fragment is sufficient to inhibit activation of the cellular PKR kinase in a cell-free system, suggesting that the intrinsic activities of this small fragment, notably RNA binding and ribosome association, may be required to prevent PKR activation. PMID:11070019

Inhibition of PKR activation by the proline-rich RNA binding domain of the herpes simplex virus type 1 Us11 protein.

PubMed

Poppers, J; Mulvey, M; Khoo, D; Mohr, I

2000-12-01

Upon activation by double-stranded RNA in virus-infected cells, the cellular PKR kinase phosphorylates the translation initiation factor eukaryotic initiation factor 2 (eIF2) and thereby inhibits protein synthesis. The gamma 34.5 and Us11 gene products encoded by herpes simplex virus type 1 (HSV-1) are dedicated to preventing the accumulation of phosphorylated eIF2. While the gamma 34.5 gene specifies a regulatory subunit for protein phosphatase 1 alpha, the Us11 gene encodes an RNA binding protein that also prevents PKR activation. gamma 34.5 mutants fail to grow on a variety of human cells as phosphorylated eIF2 accumulates and protein synthesis ceases prior to the completion of the viral life cycle. We demonstrate that expression of a 68-amino-acid fragment of Us11 containing a novel proline-rich basic RNA binding domain allows for sustained protein synthesis and enhanced growth of gamma 34.5 mutants. Furthermore, this fragment is sufficient to inhibit activation of the cellular PKR kinase in a cell-free system, suggesting that the intrinsic activities of this small fragment, notably RNA binding and ribosome association, may be required to prevent PKR activation.

Deciphering the complexities of the wheat flour proteome using quantitative two-dimensional electrophoresis, three proteases and tandem mass spectrometry

USDA-ARS?s Scientific Manuscript database

Wheat flour is one of the world's major food ingredients, but it is difficult to distinguish and identify the many proteins in a flour sample. The abundant glutamine and proline rich gluten proteins are responsible for many of the unique end-use qualities of wheat flour but it is challenging to dis...

Regulation of Steroid Hormone Receptor Function By the 52-kDa FK506-Binding Protein (FKBP52)

PubMed Central

Sivils, Jeffrey C.; Storer, Cheryl L.; Galigniana, Mario D.; Cox, Marc B.

2011-01-01

The large FK506-binding protein FKBP52 has been characterized as an important positive regulator of androgen, glucocorticoid and progesterone receptor signaling pathways. FKBP52 associates with receptor-Hsp90 complexes and is proposed to have roles in both receptor hormone binding and receptor subcellular localization. Data from biochemical and cellular studies has been corroborated in whole animal models as fkbp52-deficient male and female mice display characteristics of androgen, glucocorticoid and/or progesterone insensitivity. FKBP52 receptor specificity and the specific phenotypes displayed by the fkbp52-deficient mice have firmly established FKBP52 as a promising target for the treatment of a variety of hormone-dependent diseases. Recent studies demonstrated that the FKBP52 FK1 domain and the proline-rich loop within this domain are functionally important for FKBP52 regulation of receptor function. Based on these data, efforts are currently underway to target the FKBP52 FK1 domain and the proline-rich loop with small molecule inhibitors. PMID:21511531

Occurrence, Functions and Biological Significance of Arginine-Rich Proteins.

PubMed

Chandana, Thimmegowda; Venkatesh, Yeldur P

2016-01-01

Arginine, the most basic among the 20 amino acids, occurs less frequently than lysine in proteins despite being coded by six codons. Only a few important proteins of biological significance have been found to be abundant in arginine. It has been established that these arginine-rich proteins have been assigned important roles in the biological systems. Arginine-rich cationic proteins are known to stabilize macromolecular structures by establishing appropriate interactions (salt bridges, hydrogen bonds and cation-π interactions). These proteins are also known to be the key members of many regulatory pathways such as gene expression, chromatin stability, expurgation of introns from naïve mRNA, mRNA splicing, membrane-penetrating activity and pathogenesis-related defense, to name a few. Further, arginine occurs in various combinations with other amino acids (serine, lysine, proline, tryptophan, valine, glycine and glutamic acid) which diversify the potential functions of arginine-rich proteins. Arginine-rich proteins known till date from dietary sources have been described in terms of their structure and functional properties. A variety of activities such as bactericidal, membrane-penetrating, antimicrobial, anti-hypertensive, pro-angiogenic and others have been reported for arginine-rich proteins. This review attempts to collate the occurrence, functions and the biological significance of this unique class of proteins rich in arginine.

Specific serine-proline phosphorylation and glycogen synthase kinase 3β-directed subcellular targeting of stathmin 3/Sclip in neurons.

PubMed

Devaux, Sara; Poulain, Fabienne E; Devignot, Véronique; Lachkar, Sylvie; Irinopoulou, Theano; Sobel, André

2012-06-22

During nervous system development, neuronal growth, migration, and functional morphogenesis rely on the appropriate control of the subcellular cytoskeleton including microtubule dynamics. Stathmin family proteins play major roles during the various stages of neuronal differentiation, including axonal growth and branching, or dendritic development. We have shown previously that stathmins 2 (SCG10) and 3 (SCLIP) fulfill distinct, independent and complementary regulatory roles in axonal morphogenesis. Although the two proteins have been proposed to display the four conserved phosphorylation sites originally identified in stathmin 1, we show here that they possess distinct phosphorylation sites within their specific proline-rich domains (PRDs) that are differentially regulated by phosphorylation by proline-directed kinases involved in the control of neuronal differentiation. ERK2 or CDK5 phosphorylate the two proteins but with different site specificities. We also show for the first time that, unlike stathmin 2, stathmin 3 is a substrate for glycogen synthase kinase (GSK) 3β both in vitro and in vivo. Interestingly, stathmin 3 phosphorylated at its GSK-3β target site displays a specific subcellular localization at neuritic tips and within the actin-rich peripheral zone of the growth cone of differentiating hippocampal neurons in culture. Finally, pharmacological inhibition of GSK-3β induces a redistribution of stathmin 3, but not stathmin 2, from the periphery toward the Golgi region of neurons. Stathmin proteins can thus be either regulated locally or locally targeted by specific phosphorylation, each phosphoprotein of the stathmin family fulfilling distinct and specific roles in the control of neuronal differentiation.

Resolution of Site-Specific Conformational Heterogeneity in Proline-Rich Molecular Recognition by Src Homology 3 Domains.

PubMed

Horness, Rachel E; Basom, Edward J; Mayer, John P; Thielges, Megan C

2016-02-03

Conformational heterogeneity and dynamics are increasingly evoked in models of protein molecular recognition but are challenging to experimentally characterize. Here we combine the inherent temporal resolution of infrared (IR) spectroscopy with the spatial resolution afforded by selective incorporation of carbon-deuterium (C-D) bonds, which provide frequency-resolved absorptions within a protein IR spectrum, to characterize the molecular recognition of the Src homology 3 (SH3) domain of the yeast protein Sho1 with its cognate proline-rich (PR) sequence of Pbs2. The IR absorptions of C-D bonds introduced at residues along a peptide of the Pbs2 PR sequence report on the changes in the local environments upon binding to the SH3 domain. Interestingly, upon forming the complex the IR spectra of the peptides labeled with C-D bonds at either of the two conserved prolines of the PXXP consensus recognition sequence show more absorptions than there are C-D bonds, providing evidence for the population of multiple states. In contrast, the NMR spectra of the peptides labeled with (13)C at the same residues show only single resonances, indicating rapid interconversion on the NMR time scale. Thus, the data suggest that the SH3 domain recognizes its cognate peptide with a component of induced fit molecular recognition involving the adoption of multiples states, which have previously gone undetected due to interconversion between the populated states that is too fast to resolve using conventional methods.

GSK3β phosphorylates newly identified site in the proline-alanine-rich region of cardiac myosin-binding protein C and alters cross-bridge cycling kinetics in human: short communication.

PubMed

Kuster, Diederik W D; Sequeira, Vasco; Najafi, Aref; Boontje, Nicky M; Wijnker, Paul J M; Witjas-Paalberends, E Rosalie; Marston, Steven B; Dos Remedios, Cristobal G; Carrier, Lucie; Demmers, Jeroen A A; Redwood, Charles; Sadayappan, Sakthivel; van der Velden, Jolanda

2013-02-15

Cardiac myosin-binding protein C (cMyBP-C) regulates cross-bridge cycling kinetics and, thereby, fine-tunes the rate of cardiac muscle contraction and relaxation. Its effects on cardiac kinetics are modified by phosphorylation. Three phosphorylation sites (Ser275, Ser284, and Ser304) have been identified in vivo, all located in the cardiac-specific M-domain of cMyBP-C. However, recent work has shown that up to 4 phosphate groups are present in human cMyBP-C. To identify and characterize additional phosphorylation sites in human cMyBP-C. Cardiac MyBP-C was semipurified from human heart tissue. Tandem mass spectrometry analysis identified a novel phosphorylation site on serine 133 in the proline-alanine-rich linker sequence between the C0 and C1 domains of cMyBP-C. Unlike the known sites, Ser133 was not a target of protein kinase A. In silico kinase prediction revealed glycogen synthase kinase 3β (GSK3β) as the most likely kinase to phosphorylate Ser133. In vitro incubation of the C0C2 fragment of cMyBP-C with GSK3β showed phosphorylation on Ser133. In addition, GSK3β phosphorylated Ser304, although the degree of phosphorylation was less compared with protein kinase A-induced phosphorylation at Ser304. GSK3β treatment of single membrane-permeabilized human cardiomyocytes significantly enhanced the maximal rate of tension redevelopment. GSK3β phosphorylates cMyBP-C on a novel site, which is positioned in the proline-alanine-rich region and increases kinetics of force development, suggesting a noncanonical role for GSK3β at the sarcomere level. Phosphorylation of Ser133 in the linker domain of cMyBP-C may be a novel mechanism to regulate sarcomere kinetics.

Elevated immunoglobulin G antibodies to the proline-rich amino-terminal region of Epstein-Barr virus nuclear antigen-2 in sera from patients with systemic connective tissue diseases and from a subgroup of Sjögren's syndrome patients with pulmonary involvements.

PubMed

Yamazaki, M; Kitamura, R; Kusano, S; Eda, H; Sato, S; Okawa-Takatsuji, M; Aotsuka, S; Yanagi, K

2005-03-01

Associations of Epstein-Barr virus (EBV) and autoimmune diseases have been hypothesized. We have analysed IgG antibodies to EBV nuclear antigen (EBNA)-2 in sera from Japanese patients with autoimmune systemic connective tissue diseases (CTD), exemplified by systemic lupus erythematosus (SLE), primary Sjogren's syndrome (SS), rheumatoid arthritis (RA), systemic sclerosis (SSc) and secondary SS (classical CTDs complicated with SS). An enzyme-linked immunosorbent assay (ELISA) which uses glutathione-S-transferase polypeptides fused to EBV nuclear antigen (EBNA)-2 and EBNA-1 was developed. Ratios of IgG antibody reactivity to whole IgG concentrations of sera were calculated to normalize EBNA-2 and EBNA-1 antibody levels to the hypergammaglobulinaemia that occurs in CTD. The ELISA optical density OD(450) readings of IgG antibodies to both the amino-terminal aa 1-116 of EBNA-2 and carboxyl-terminal aa 451-641 of EBNA-1 were elevated significantly in patients with SLE, primary SS, RA, SSc and secondary SS when compared to EBNA-1. The OD readings were divided by serum IgG concentrations to normalize for the hypergammaglobulinaemia. The specific levels of IgG antibodies to the amino-terminal region of EBNA-2 were elevated in patients with SLE, primary SS or RA, as well as those with secondary SS complicated with SLE or RA. The EBNA-2 amino-terminal region contains a polyproline tract and a proline-rich sequence and has considerable amino acid sequence homology with many cellular proline-rich proteins. High ratios of EBNA-2 aa 1-116 to EBNA-1 aa 451-641 IgG antibody levels which probably suggest reactivation of EBV latent infection were associated significantly with pulmonary involvement in SS patients. These results are consistent with the hypothesis that the sequence similarity between the amino-terminal region of EBNA-2 and proline-rich cellular proteins is associated with pathogenesis in a subpopulation of CTD patients, possibly by the molecular mimicry-epitope shift mechanism.

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.

Prolyl hydroxylation regulates protein degradation, synthesis, and splicing in human induced pluripotent stem cell-derived cardiomyocytes.

PubMed

Stoehr, Andrea; Yang, Yanqin; Patel, Sajni; Evangelista, Alicia M; Aponte, Angel; Wang, Guanghui; Liu, Poching; Boylston, Jennifer; Kloner, Philip H; Lin, Yongshun; Gucek, Marjan; Zhu, Jun; Murphy, Elizabeth

2016-06-01

Protein hydroxylases are oxygen- and α-ketoglutarate-dependent enzymes that catalyse hydroxylation of amino acids such as proline, thus linking oxygen and metabolism to enzymatic activity. Prolyl hydroxylation is a dynamic post-translational modification that regulates protein stability and protein-protein interactions; however, the extent of this modification is largely uncharacterized. The goals of this study are to investigate the biological consequences of prolyl hydroxylation and to identify new targets that undergo prolyl hydroxylation in human cardiomyocytes. We used human induced pluripotent stem cell-derived cardiomyocytes in combination with pulse-chase amino acid labelling and proteomics to analyse the effects of prolyl hydroxylation on protein degradation and synthesis. We identified 167 proteins that exhibit differences in degradation with inhibition of prolyl hydroxylation by dimethyloxalylglycine (DMOG); 164 were stabilized. Proteins involved in RNA splicing such as serine/arginine-rich splicing factor 2 (SRSF2) and splicing factor and proline- and glutamine-rich (SFPQ) were stabilized with DMOG. DMOG also decreased protein translation of cytoskeletal and sarcomeric proteins such as α-cardiac actin. We searched the mass spectrometry data for proline hydroxylation and identified 134 high confidence peptides mapping to 78 unique proteins. We identified SRSF2, SFPQ, α-cardiac actin, and cardiac titin as prolyl hydroxylated. We identified 29 prolyl hydroxylated proteins that showed a significant difference in either protein degradation or synthesis. Additionally, we performed next-generation RNA sequencing and showed that the observed decrease in protein synthesis was not due to changes in mRNA levels. Because RNA splicing factors were prolyl hydroxylated, we investigated splicing ± inhibition of prolyl hydroxylation and detected 369 alternative splicing events, with a preponderance of exon skipping. This study provides the first extensive characterization of the cardiac prolyl hydroxylome and demonstrates that inhibition of α-ketoglutarate hydroxylases alters protein stability, translation, and splicing. Published by Oxford University Press on behalf of the European Society of Cardiology 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.

A WASp–VASP complex regulates actin polymerization at the plasma membrane

PubMed Central

Castellano, Flavia; Le Clainche, Christophe; Patin, Delphine; Carlier, Marie-France; Chavrier, Philippe

2001-01-01

Proteins of the Wiskott–Aldrich syndrome and Ena/VASP families both play essential functions in the regulation of actin dynamics at the cell leading edge. However, possibilities of functional interplay between members of these two families have not been addressed. Here we show that, in hemopoietic cells, recruitment of the C-terminal VCA (Verprolin homology, Cofilin homology, Acidic) domain of WASp at the plasma membrane by a ligand technique using rapamycin as an intermediate is not sufficient to elicit efficient Arp2/3 complex-mediated actin polymerization. Other domains of WASp, in particular the proline-rich domain, are required for the formation of actin-rich structures. An in vitro analysis demonstrates that the proline-rich domain of WASp binds VASP with an affinity of ∼106 M–1. In addition, WASp and VASP both accumulate in actin-rich phagocytic cups. Finally, in a reconstituted motility medium, VASP enhances actin-based propulsion of WASp-coated beads in a fashion reminiscent of its effect on Listeria movement. We propose that VASP and WASp cooperation is essential in stimulating actin assembly and membrane protrusion at the leading edge. PMID:11598004

The N-terminal domain of the thermo-regulated surface protein PrpA of Enterococcus faecium binds to fibrinogen, fibronectin and platelets

PubMed Central

Guzmán Prieto, Ana M.; Urbanus, Rolf T.; Zhang, Xinglin; Bierschenk, Damien; Koekman, C. Arnold; van Luit-Asbroek, Miranda; Ouwerkerk, Janneke P.; Pape, Marieke; Paganelli, Fernanda L.; Wobser, Dominique; Huebner, Johannes; Hendrickx, Antoni P. A.; Bonten, Marc J. M.; Willems, Rob J. L.; van Schaik, Willem

2015-01-01

Enterococcus faecium is a commensal of the mammalian gastrointestinal tract, but is also found in non-enteric environments where it can grow between 10 °C and 45 °C. E. faecium has recently emerged as a multi-drug resistant nosocomial pathogen. We hypothesized that genes involved in the colonization and infection of mammals exhibit temperature-regulated expression control and we therefore performed a transcriptome analysis of the clinical isolate E. faecium E1162, during mid-exponential growth at 25 °C and 37 °C. One of the genes that exhibited differential expression between 25 °C and 37 °C, was predicted to encode a peptidoglycan-anchored surface protein. The N-terminal domain of this protein is unique to E. faecium and closely related enterococci, while the C-terminal domain is homologous to the Streptococcus agalactiae surface protein BibA. This region of the protein contains proline-rich repeats, leading us to name the protein PrpA for proline-rich protein A. We found that PrpA is a surface-exposed protein which is most abundant during exponential growth at 37 °C in E. faecium E1162. The heterologously expressed and purified N-terminal domain of PrpA was able to bind to the extracellular matrix proteins fibrinogen and fibronectin. In addition, the N-terminal domain of PrpA interacted with both non-activated and activated platelets. PMID:26675410

The N-terminal domain of the thermo-regulated surface protein PrpA of Enterococcus faecium binds to fibrinogen, fibronectin and platelets.

PubMed

Guzmán Prieto, Ana M; Urbanus, Rolf T; Zhang, Xinglin; Bierschenk, Damien; Koekman, C Arnold; van Luit-Asbroek, Miranda; Ouwerkerk, Janneke P; Pape, Marieke; Paganelli, Fernanda L; Wobser, Dominique; Huebner, Johannes; Hendrickx, Antoni P A; Bonten, Marc J M; Willems, Rob J L; van Schaik, Willem

2015-12-17

Enterococcus faecium is a commensal of the mammalian gastrointestinal tract, but is also found in non-enteric environments where it can grow between 10 °C and 45 °C. E. faecium has recently emerged as a multi-drug resistant nosocomial pathogen. We hypothesized that genes involved in the colonization and infection of mammals exhibit temperature-regulated expression control and we therefore performed a transcriptome analysis of the clinical isolate E. faecium E1162, during mid-exponential growth at 25 °C and 37 °C. One of the genes that exhibited differential expression between 25 °C and 37 °C, was predicted to encode a peptidoglycan-anchored surface protein. The N-terminal domain of this protein is unique to E. faecium and closely related enterococci, while the C-terminal domain is homologous to the Streptococcus agalactiae surface protein BibA. This region of the protein contains proline-rich repeats, leading us to name the protein PrpA for proline-rich protein A. We found that PrpA is a surface-exposed protein which is most abundant during exponential growth at 37 °C in E. faecium E1162. The heterologously expressed and purified N-terminal domain of PrpA was able to bind to the extracellular matrix proteins fibrinogen and fibronectin. In addition, the N-terminal domain of PrpA interacted with both non-activated and activated platelets.

The Smooth Muscle of the Artery

DTIC Science & Technology

1975-01-01

membrpne structure (343). Certainly, the transported proteins may serve is A source of amino acids (lysin-rich and/or proline rich proceins?) for bio...into Long-Chain Fatty Acids by Cellular Fractions from Normal Rabbit Aorts Suprrnatant fraction 1(# 4 hTABOLI CHIAACTERIStICS OF SMOOH MUSCLE 3 I shall...acetate into fatty acids in a homogenate of rat aorta. VT It took many more years and much refine- Lipid Synthesis ment of techniques to analyze more by

Studies of the structure-activity relationships of peptides and proteins involved in growth and development based on their three-dimensional structures.

PubMed

Nagata, Koji

2010-01-01

Peptides and proteins with similar amino acid sequences can have different biological functions. Knowledge of their three-dimensional molecular structures is critically important in identifying their functional determinants. In this review, I describe the results of our and other groups' structure-based functional characterization of insect insulin-like peptides, a crustacean hyperglycemic hormone-family peptide, a mammalian epidermal growth factor-family protein, and an intracellular signaling domain that recognizes proline-rich sequence.

Hybrid proline-rich proteins: novel players in plant cell elongation?

PubMed Central

Dvořáková, Lenka; Srba, Miroslav; Opatrny, Zdenek; Fischer, Lukas

2012-01-01

Background and Aims Hybrid proline-rich proteins (HyPRPs) represent a large family of putative cell-wall proteins characterized by the presence of a variable N-terminal domain and a conserved C-terminal domain that is related to non-specific lipid transfer proteins. The function of HyPRPs remains unclear, but their widespread occurrence and abundant expression patterns indicate that they may be involved in a basic cellular process. Methods To elucidate the cellular function of HyPRPs, we modulated the expression of three HyPRP genes in tobacco (Nicotiana tabacum) BY-2 cell lines and in potato (Solanum tuberosum) plants. Key Results In BY-2 lines, over-expression of the three HyPRP genes with different types of N-terminal domains resulted in similar phenotypic changes, namely increased cell elongation, both in suspension culture and on solid media where the over-expression resulted in enhanced calli size. The over-expressing cells showed increased plasmolysis in a hypertonic mannitol solution and accelerated rate of protoplast release, suggesting loosening of the cell walls. In contrast to BY-2 lines, no phenotypic changes were observed in potato plants over-expressing the same or analogous HyPRP genes, presumably due to more complex compensatory mechanisms in planta. Conclusions Based on the results from BY-2 lines, we propose that HyPRPs, more specifically their C-terminal domains, represent a novel group of proteins involved in cell expansion. PMID:22028464

Beta-keratins of differentiating epidermis of snake comprise glycine-proline-serine-rich proteins with an avian-like gene organization.

PubMed

Dalla Valle, Luisa; Nardi, Alessia; Belvedere, Paola; Toni, Mattia; Alibardi, Lorenzo

2007-07-01

Beta-keratins of reptilian scales have been recently cloned and characterized in some lizards. Here we report for the first time the sequence of some beta-keratins from the snake Elaphe guttata. Five different cDNAs were obtained using 5'- and 3'-RACE analyses. Four sequences differ by only few nucleotides in the coding region, whereas the last cDNA shows, in this region, only 84% of identity. The gene corresponding to one of the cDNA sequences has a single intron present in the 5'-untranslated region. This genomic organization is similar to that of birds' beta-keratins. Cloning and Southern blotting analysis suggest that snake beta-keratins belong to a family of high-related genes as for geckos. PCR analysis suggests a head-to-tail orientation of genes in the same chromosome. In situ hybridization detected beta-keratin transcripts almost exclusively in differentiating oberhautchen and beta-cells of the snake epidermis in renewal phase. This is confirmed by Northern blotting that showed, in this phase, a high expression of two different transcripts whereas only the longer transcript is expressed at a much lower level in resting skin. The cDNA coding sequences encoded putative glycine-proline-serine rich proteins containing 137-139 amino acids, with apparent isoelectric point at 7.5 and 8.2. A central region, rich in proline, shows over 50% homology with avian scale, claw, and feather keratins. The prediction of secondary structure shows mainly a random coil conformation and few beta-strand regions in the central region, likely involved in the formation of a fibrous framework of beta-keratins. This region was possibly present in basic reptiles that originated reptiles and birds. Copyright 2007 Wiley-Liss, Inc.

Absence of Capsule Reveals Glycan-Mediated Binding and Recognition of Salivary Mucin MUC7 by Streptococcus pneumoniae

PubMed Central

Thamadilok, Supaporn; Roche-Håkansson, Hazeline; Håkansson, Anders P.; Ruhl, Stefan

2015-01-01

SUMMARY Salivary proteins modulate bacterial colonization in the oral cavity and interact with systemic pathogens that pass through the oropharynx. An interesting example is the opportunistic respiratory pathogen Streptococcus pneumoniae that normally resides in the nasopharynx, but belongs to the greater Mitis group of streptococci, most of which colonize the oral cavity. S. pneumoniae also expresses a serine-rich repeat (SRR) adhesin, PsrP, that is a homologue to oral Mitis group SRR adhesins, such as Hsa of S. gordonii and SrpA of S. sanguinis. Since the latter bind to salivary glycoproteins through recognition of terminal sialic acids, we wanted to determine whether S. pneumoniae also binds to salivary proteins through possibly the same mechanism. We found that only a capsule-free mutant of S. pneumoniae TIGR4 binds to salivary proteins, most prominently to mucin MUC7, but that this binding was not mediated through PsrP or recognition of sialic acid. We also found, however, that PsrP is involved in agglutination of human red blood cells (RBCs). After removal of PsrP, an additional previously masked lectin-like adhesin activity mediating agglutination of sialidase-treated RBCs becomes revealed. Using a custom-spotted glycoprotein and neoglycoprotein dot blot array, we identify candidate glycan motifs recognized by PsrP and by the putative S. pneumoniae adhesin that could perhaps be responsible for pneumococcal binding to salivary MUC7 and glycoproteins on RBCs. PMID:26172471

NO-Rich Diet for Lifestyle-Related Diseases

PubMed Central

Kobayashi, Jun; Ohtake, Kazuo; Uchida, Hiroyuki

2015-01-01

Decreased nitric oxide (NO) availability due to obesity and endothelial dysfunction might be causally related to the development of lifestyle-related diseases such as insulin resistance, ischemic heart disease, and hypertension. In such situations, instead of impaired NO synthase (NOS)-dependent NO generation, the entero-salivary nitrate-nitrite-NO pathway might serve as a backup system for NO generation by transmitting NO activities in the various molecular forms including NO and protein S-nitrosothiols. Recently accumulated evidence has demonstrated that dietary intake of fruits and vegetables rich in nitrate/nitrite is an inexpensive and easily-practicable way to prevent insulin resistance and vascular endothelial dysfunction by increasing the NO availability; a NO-rich diet may also prevent other lifestyle-related diseases, including osteoporosis, chronic obstructive pulmonary disease (COPD), and cancer. This review provides an overview of our current knowledge of NO generation through the entero-salivary pathway and discusses its safety and preventive effects on lifestyle-related diseases. PMID:26091235

The PBII gene of the human salivary proline-rich protein P-B produces another protein, Q504X8, with an opiorphin homolog, QRGPR.

PubMed

Saitoh, Eiichi; Sega, Takuya; Imai, Akane; Isemura, Satoko; Kato, Tetsuo; Ochiai, Akihito; Taniguchi, Masayuki

2018-04-01

The NCBI gene database and human-transcriptome database for alternative splicing were used to determine the expression of mRNAs for P-B (SMR3B) and variant form of P-B. The translational product from the former mRNA was identified as the protein named P-B, whereas that from the latter has not yet been elucidated. In the present study, we investigated the expression of P-B and its variant form at the protein level. To identify the variant protein of P-B, (1) cationic proteins with a higher isoelectric point in human pooled whole saliva were purified by a two dimensional liquid chromatography; (2) the peptide fragments generated from the in-solution of all proteins digested with trypsin separated and analyzed by MALDI-TOF-MS; and (3) the presence or absence of P-B in individual saliva was examined by 15% SDS-PAGE. The peptide sequences (I 37 PPPYSCTPNMNNCSR 52 , C 53 HHHHKRHHYPCNYCFCYPK 72 , R 59 HHYPCNYCFCYPK 72 and H 60 HYPCNYCFCYPK 72 ) present in the variant protein of P-B were identified. The peptide sequence (G 6 PYPPGPLAPPQPFGPGFVPPPPPPPYGPGR 36 ) in P-B (or the variant) and sequence (I 37 PPPPPAPYGPGIFPPPPPQP 57 ) in P-B were identified. The sum of the sequences identified indicated a 91.23% sequence identity for P-B and 79.76% for the variant. There were cases in which P-B existed in individual saliva, but there were cases in which it did not exist in individual saliva. The variant protein is produced by excising a non-canonical intron (CC-AC pair) from the 3'-noncoding sequence of the PBII gene. Both P-B and the variant are subject to proteolysis in the oral cavity. Copyright © 2018 Elsevier Ltd. All rights reserved.

Evolution of hard proteins in the sauropsid integument in relation to the cornification of skin derivatives in amniotes

PubMed Central

Alibardi, Lorenzo; Valle, Luisa Dalla; Nardi, Alessia; Toni, Mattia

2009-01-01

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. PMID:19422429

Proline: Mother Nature’s cryoprotectant applied to protein crystallography

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

Pemberton, Travis A.; Still, Brady R.; Christensen, Emily M.

The amino acid l-proline is shown to be a good cryoprotectant for protein crystals. Four examples are provided; the range of proline used for cryoprotection is 2.0–3.0 M. l-Proline is one of Mother Nature’s cryoprotectants. Plants and yeast accumulate proline under freeze-induced stress and the use of proline in the cryopreservation of biological samples is well established. Here, it is shown that l-proline is also a useful cryoprotectant for protein crystallography. Proline was used to prepare crystals of lysozyme, xylose isomerase, histidine acid phosphatase and 1-pyrroline-5-carboxylate dehydrogenase for low-temperature data collection. The crystallization solutions in these test cases included themore » commonly used precipitants ammonium sulfate, sodium chloride and polyethylene glycol and spanned the pH range 4.6–8.5. Thus, proline is compatible with typical protein-crystallization formulations. The proline concentration needed for cryoprotection of these crystals is in the range 2.0–3.0 M. Complete data sets were collected from the proline-protected crystals. Proline performed as well as traditional cryoprotectants based on the diffraction resolution and data-quality statistics. The structures were refined to assess the binding of proline to these proteins. As observed with traditional cryoprotectants such as glycerol and ethylene glycol, the electron-density maps clearly showed the presence of proline molecules bound to the protein. In two cases, histidine acid phosphatase and 1-pyrroline-5-carboxylate dehydrogenase, proline binds in the active site. It is concluded that l-proline is an effective cryoprotectant for protein crystallography.« less

A Deep Insight into the Sialome of Male and Female Aedes aegypti Mosquitoes

PubMed Central

Ribeiro, José M. C.; Martin-Martin, Ines; Arcà, Bruno; Calvo, Eric

2016-01-01

Only adult female mosquitoes feed on blood, while both genders take sugar meals. Accordingly, several compounds associated with blood feeding (i.e. vasodilators, anti-clotting, anti-platelets) are found only in female glands, while enzymes associated with sugar feeding or antimicrobials (such as lysozyme) are found in the glands of both sexes. We performed de novo assembly of reads from adult Aedes aegypti female and male salivary gland libraries (285 and 90 million reads, respectively). By mapping back the reads to the assembled contigs, plus mapping the reads from a publicly available Ae. aegypti library from adult whole bodies, we identified 360 transcripts (including splice variants and alleles) overexpressed tenfold or more in the glands when compared to whole bodies. Moreover, among these, 207 were overexpressed fivefold or more in female vs. male salivary glands, 85 were near equally expressed and 68 were overexpressed in male glands. We call in particular the attention to C-type lectins, angiopoietins, female-specific Antigen 5, the 9.7 kDa, 12–14 kDa, 23.5 kDa, 62/34 kDa, 4.2 kDa, proline-rich peptide, SG8, 8.7 kDa family and SGS fragments: these polypeptides are all of unknown function, but due to their overexpression in female salivary glands and putative secretory nature they are expected to affect host physiology. We have also found many transposons (some of which novel) and several endogenous viral transcripts (probably acquired by horizontal transfer) which are overexpressed in the salivary glands and may play some role in tissue-specific gene regulation or represent a mechanism of virus interference. This work contributes to a near definitive catalog of male and female salivary gland transcripts from Ae. aegypti, which will help to direct further studies aiming at the functional characterization of the many transcripts with unknown function and the understanding of their role in vector-host interaction and pathogen transmission. PMID:26999592

Combined Transcriptomic and Proteomic Analysis of the Posterior Salivary Gland from the Southern Blue-Ringed Octopus and the Southern Sand Octopus.

PubMed

Whitelaw, Brooke L; Strugnell, Jan M; Faou, Pierre; da Fonseca, Rute R; Hall, Nathan E; Norman, Mark; Finn, Julian; Cooke, Ira R

2016-09-02

This study provides comprehensive proteomic profiles from the venom producing posterior salivary glands of octopus (superorder Octopodiformes) species. A combined transcriptomic and proteomic approach was used to identify 1703 proteins from the posterior salivary gland of the southern blue-ringed octopus, Hapalochlaena maculosa and 1300 proteins from the posterior salivary gland of the southern sand octopus, Octopus kaurna. The two proteomes were broadly similar; clustering of proteins into orthogroups revealed 937 that were shared between species. Serine proteases were particularly diverse and abundant in both species. Other abundant proteins included a large number of secreted proteins, many of which had no known conserved domains, or homology to proteins with known function. On the basis of homology to known venom proteins, 23 putative toxins were identified in H. maculosa and 24 in O. kaurna. These toxins span nine protein families: CAP (cysteine rich secretory proteins, antigen 5, parthenogenesis related), chitinase, carboxylesterase, DNase, hyaluronidase, metalloprotease, phospholipase, serine protease and tachykinin. Serine proteases were responsible for 70.9% and 86.3% of putative toxin expression in H. maculosa and O. kaurna, respectively, as determined using intensity based absolute quantification (iBAQ) measurements. Phylogenetic analysis of the putative toxin serine proteases revealed a similar suite of diverse proteins present in both species. Posterior salivary gland composition of H. maculosa and O. kaurna differ in several key aspects. While O. kaurna expressed the proteinaceous neurotoxin, tachykinin, this was absent from H. maculosa, perhaps reflecting the acquisition of a potent nonproteinaceous neurotoxin, tetrodotoxin (TTX) produced by bacteria in the salivary glands of that species. The dispersal factor, hyaluronidase was particularly abundant in H. maculosa. Chitinase was abundant in both species and is believed to facilitate envenomation in chitinous prey such as crustaceans. Cephalopods represent a largely unexplored source of novel proteins distinct from all other venomous taxa and are of interest for further inquiry, as novel proteinaceous toxins derived from venoms may contribute to pharmaceutical design.

Precipitation of salivary proteins after the interaction with wine: the effect of ethanol, pH, fructose, and mannoproteins.

PubMed

Rinaldi, Alessandra; Gambuti, Angelita; Moio, Luigi

2012-04-01

Astringency is a complex sensation mainly caused by the precipitation of salivary proteins with polyphenols. In wine it can be enhanced or reduced depending on the composition of the medium. In order to investigate the effect of ethanol, tartaric acid, fructose, and commercial mannoproteins (MPs) addition on the precipitation of salivary proteins, the saliva precipitation index (SPI) was determined by means of the sodium dodecyl sulphate polyacrylamide gel electrophoresis of human saliva after the reaction with Merlot wines and model solutions. Gelatin index, ethanol index, and Folin-Ciocalteu index were also determined. As resulted by Pearson's correlation, data on SPI were well correlated with the sensory analysis performed on the same samples. In a second experiment, increasing the ethanol (11%-13%-17%), MPs (0-2-8 g/L), fructose (0-2-6 g/L) level, and pH values (2.9-3.0-3.6), a decrease in the precipitation of salivary proteins was observed. A difference in the SPI between model solution and red wine stated that an influence of wine matrix on the precipitation of salivary proteins occurred. Results provide interesting suggestions for enologists, which could modulate the astringency of red wine by: (i) leaving some residual reducing sugars (such as fructose) in red wine during winemaking of grapes rich in tannins; (ii) avoiding the lowering of pH; (iii) adding commercial mannoproteins or promoting a "sur lie" aging; and (iv) harvesting grapes at high technological maturity in order to obtain wines with a satisfactory alcoholic content when possible. © 2012 Institute of Food Technologists®

Proline: Mother Nature;s cryoprotectant applied to protein crystallography

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

Pemberton, Travis A.; Still, Brady R.; Christensen, Emily M.

L-Proline is one of Mother Nature's cryoprotectants. Plants and yeast accumulate proline under freeze-induced stress and the use of proline in the cryopreservation of biological samples is well established. Here, it is shown that L-proline is also a useful cryoprotectant for protein crystallography. Proline was used to prepare crystals of lysozyme, xylose isomerase, histidine acid phosphatase and 1-pyrroline-5-carboxylate dehydrogenase for low-temperature data collection. The crystallization solutions in these test cases included the commonly used precipitants ammonium sulfate, sodium chloride and polyethylene glycol and spanned the pH range 4.6-8.5. Thus, proline is compatible with typical protein-crystallization formulations. The proline concentration neededmore » for cryoprotection of these crystals is in the range 2.0-3.0 M. Complete data sets were collected from the proline-protected crystals. Proline performed as well as traditional cryoprotectants based on the diffraction resolution and data-quality statistics. The structures were refined to assess the binding of proline to these proteins. As observed with traditional cryoprotectants such as glycerol and ethylene glycol, the electron-density maps clearly showed the presence of proline molecules bound to the protein. In two cases, histidine acid phosphatase and 1-pyrroline-5-carboxylate dehydrogenase, proline binds in the active site. It is concluded that L-proline is an effective cryoprotectant for protein crystallography.« less

Proline: Mother Nature’s cryoprotectant applied to protein crystallography

PubMed Central

Pemberton, Travis A.; Still, Brady R.; Christensen, Emily M.; Singh, Harkewal; Srivastava, Dhiraj; Tanner, John J.

2012-01-01

l-Proline is one of Mother Nature’s cryoprotectants. Plants and yeast accumulate proline under freeze-induced stress and the use of proline in the cryopreservation of biological samples is well established. Here, it is shown that l-proline is also a useful cryoprotectant for protein crystallography. Proline was used to prepare crystals of lysozyme, xylose isomerase, histidine acid phosphatase and 1-pyrroline-5-carboxylate dehydrogenase for low-temperature data collection. The crystallization solutions in these test cases included the commonly used precipitants ammonium sulfate, sodium chloride and polyethylene glycol and spanned the pH range 4.6–8.5. Thus, proline is compatible with typical protein-crystallization formulations. The proline concentration needed for cryoprotection of these crystals is in the range 2.0–3.0 M. Complete data sets were collected from the proline-protected crystals. Proline performed as well as traditional cryoprotectants based on the diffraction resolution and data-quality statistics. The structures were refined to assess the binding of proline to these proteins. As observed with traditional cryoprotectants such as glycerol and ethylene glycol, the electron-density maps clearly showed the presence of proline molecules bound to the protein. In two cases, histidine acid phosphatase and 1-pyrroline-5-carboxylate dehydrogenase, proline binds in the active site. It is concluded that l-proline is an effective cryoprotectant for protein crystallography. PMID:22868767

A Solution NMR Investigation into the Murine Amelogenin Splice-Variant LRAP (Leucine-Rich Amelogenin Protein).

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

Buchko, Garry W.; Tarasevich, Barbara J.; Roberts, Jacky

2010-09-01

Amelogenins are the dominant proteins present in ameloblasts during the early stages of enamel biomineralization, making up >90% of the matrix protein. Along with the full-length protein there are several splice-variant isoforms of amelogenin present including LRAP (Leucine-Rich Amelogenin Protein), a protein that consists of the first 33 and the last 26 residues of full-length amelogenin. Using solution-state NMR spectroscopy we have assigned the 1H-15N HSQC spectrum of murine LRAP (rp(H)LRAP) in 2% acetic acid at pH 3.0 by making extensive use of previous chemical shift assignments for full-length murine amelogenin (rp(H)M180). This correlation was possible because LRAP, like themore » full-length protein, is intrinsically disordered under these solution conditions. The major difference between the 1H-15N HSQC spectra of rp(H)M180 and rp(H)LRAP was an additional set of amide resonances for each of the seven non-proline residues between S12* and Y12 at the N-terminus of rp(H)LRAP indicating that the N-terminal region of LRAP exists in two different conformations. Analysis of the proline carbon chemical shifts suggest that the molecular basis for the two states is not a cis-trans isomerization of one or more of the proline residues in the N-terminal region and is likely due to a slow exchange process. As observed with rp(H)M180, residue specific changes in molecular dynamics, manifested by the reduction in intensity and disappearance of 1H-15N HSQC cross peaks, were observed with the addition of NaCl to rp(H)LRAP. These perturbations may signal early events governing supramolecular self-assembly of rp(H)LRAP into nanospheres. However, the different pattern of 1H-15N HSQC cross peak perturbation between rp(H)LRAP and rp(H)M180 in high salt suggest that the termini may behave differently in their respective nanospheres, and perhaps, these differences account for the cell signaling properties attributable to LRAP but not the full-length protein.« less

Study of intermolecular contacts in the proline-rich homeodomain (PRH)-DNA complex using molecular dynamics simulations.

PubMed

Jalili, Seifollah; Karami, Leila

2012-03-01

The proline-rich homeodomain (PRH)-DNA complex consists of a protein with 60 residues and a 13-base-pair DNA. The PRH protein is a transcription factor that plays a key role in the regulation of gene expression. PRH is a significant member of the Q50 class of homeodomain proteins. The homeodomain section of PRH is essential for binding to DNA and mediates sequence-specific DNA binding. Three 20-ns molecular dynamics (MD) simulations (free protein, free DNA and protein-DNA complex) in explicit solvent water were performed to elucidate the intermolecular contacts in the PRH-DNA complex and the role of dynamics of water molecules forming water-mediated contacts. The simulation provides a detailed explanation of the trajectory of hydration water molecules. The simulations show that some water molecules in the protein-DNA interface exchange with bulk waters. The simulation identifies that most of the contacts consisted of direct interactions between the protein and DNA including specific and non-specific contacts, but several water-mediated polar contacts were also observed. The specific interaction between Gln50 and C18 and water-mediated hydrogen bond between Gln50 and T7 were found to be present during almost the entire time of the simulation. These results show good consistency with experimental and previous computational studies. Structural properties such as root-mean-square deviations (RMSD), root-mean-square fluctuations (RMSF) and secondary structure were also analyzed as a function of time. Analyses of the trajectories showed that the dynamic fluctuations of both the protein and the DNA were lowered by the complex formation.

HOXB7: An Oncogenic Gene in Breast Cancer

DTIC Science & Technology

2006-05-01

novel roles for homeodomain-containing proteins include the role of human proline-rich homeodomain protein, PRH (known as Hex in studies on...immunofluorescence but not by IHC. We have now generated antibodies in chicken since the antigenicity of the human peptide may not be high in rabbits or mice since...Pandita, T. K. (2004). The role of the DNA double-strand break response network in meiosis . DNA Repair (Amst) 3, 1149-1164. Rubin, E., Mittnacht, S

Proteomic Analysis of Cattle Tick Rhipicephalus (Boophilus) microplus Saliva: A Comparison between Partially and Fully Engorged Females

PubMed Central

Terra, Renata Maria Soares; Martins, João Ricardo; Mulenga, Albert; Sherman, Nicholas E.; Fox, Jay W.; Yates, John R.; Termignoni, Carlos; Pinto, Antônio F. M.; da Silva Vaz, Itabajara

2014-01-01

The cattle tick Rhipicephalus (Boophilus) microplus is one of the most harmful parasites affecting bovines. Similarly to other hematophagous ectoparasites, R. microplus saliva contains a collection of bioactive compounds that inhibit host defenses against tick feeding activity. Thus, the study of tick salivary components offers opportunities for the development of immunological based tick control methods and medicinal applications. So far, only a few proteins have been identified in cattle tick saliva. The aim of this work was to identify proteins present in R. microplus female tick saliva at different feeding stages. Proteomic analysis of R. microplus saliva allowed identifying peptides corresponding to 187 and 68 tick and bovine proteins, respectively. Our data confirm that (i) R. microplus saliva is complex, and (ii) that there are remarkable differences in saliva composition between partially engorged and fully engorged female ticks. R. microplus saliva is rich mainly in (i) hemelipoproteins and other transporter proteins, (ii) secreted cross-tick species conserved proteins, (iii) lipocalins, (iv) peptidase inhibitors, (v) antimicrobial peptides, (vii) glycine-rich proteins, (viii) housekeeping proteins and (ix) host proteins. This investigation represents the first proteomic study about R. microplus saliva, and reports the most comprehensive Ixodidae tick saliva proteome published to date. Our results improve the understanding of tick salivary modulators of host defense to tick feeding, and provide novel information on the tick-host relationship. PMID:24762651

SH2/SH3 adaptor proteins can link tyrosine kinases to a Ste20-related protein kinase, HPK1.

PubMed

Anafi, M; Kiefer, F; Gish, G D; Mbamalu, G; Iscove, N N; Pawson, T

1997-10-31

Ste20-related protein kinases have been implicated as regulating a range of cellular responses, including stress-activated protein kinase pathways and the control of cytoskeletal architecture. An important issue involves the identities of the upstream signals and regulators that might control the biological functions of mammalian Ste20-related protein kinases. HPK1 is a protein-serine/threonine kinase that possesses a Ste20-like kinase domain, and in transfected cells activates a protein kinase pathway leading to the stress-activated protein kinase SAPK/JNK. Here we have investigated candidate upstream regulators that might interact with HPK1. HPK1 possesses an N-terminal catalytic domain and an extended C-terminal tail with four proline-rich motifs. The SH3 domains of Grb2 bound in vitro to specific proline-rich motifs in the HPK1 tail and functioned synergistically to direct the stable binding of Grb2 to HPK1 in transfected Cos1 cells. Epidermal growth factor (EGF) stimulation did not affect the binding of Grb2 to HPK1 but induced recruitment of the Grb2.HPK1 complex to the autophosphorylated EGF receptor and to the Shc docking protein. Several activated receptor and cytoplasmic tyrosine kinases, including the EGF receptor, stimulated the tyrosine phosphorylation of the HPK1 serine/threonine kinase. These results suggest that HPK1, a mammalian Ste20-related protein-serine/threonine kinase, can potentially associate with protein-tyrosine kinases through interactions mediated by SH2/SH3 adaptors such as Grb2. Such interaction may provide a possible mechanism for cross-talk between distinct biochemical pathways following the activation of tyrosine kinases.

Binding preference of carbon nanotube over proline-rich motif ligand on SH3-domain: a comparison with different force fields.

PubMed

Shi, Biyun; Zuo, Guanghong; Xiu, Peng; Zhou, Ruhong

2013-04-04

With the widespread applications of nanomaterials such as carbon nanotubes, there is a growing concern on the biosafety of these engineered nanoparticles, in particular their interactions with proteins. In molecular simulations of nanoparticle-protein interactions, the choice of empirical parameters (force fields) plays a decisive role, and thus is of great importance and should be examined carefully before wider applications. Here we compare three commonly used force fields, CHARMM, OPLSAA, and AMBER in study of the competitive binding of a single wall carbon nanotube (SWCNT) with a native proline-rich motif (PRM) ligand on its target protein SH3 domain, a ubiquitous protein-protein interaction mediator involved in signaling and regulatory pathways. We find that the SWCNT displays a general preference over the PRM in binding with SH3 domain in all the three force fields examined, although the degree of preference can be somewhat different, with the AMBER force field showing the highest preference. The SWCNT prevents the ligand from reaching its native binding pocket by (i) occupying the binding pocket directly, and (ii) binding with the ligand itself and then being trapped together onto some off-sites. The π-π stacking interactions between the SWCNT and aromatic residues are found to play a significant role in its binding to the SH3 domain in all the three force fields. Further analyses show that even the SWCNT-ligand binding can also be relatively more stable than the native ligand-protein binding, indicating a serious potential disruption to the protein SH3 function.

Novel double-stranded RNA viruses of plant-feeding insects encode a serine-alanine-proline rich protein and a polymerase distantly related to fungal viruses

USDA-ARS?s Scientific Manuscript database

Novel double stranded RNAs (~8 kbp) were isolated from the three cornered alfalfa hopper (Spissistilus festinus) and beet leafhopper (Circulifer tenellus), two plant-feeding hemipteran insect pests. Genome organization of the two new viruses, designated as Spissistilus festinus virus 1 (SpFV1) and ...

Plant-feeding insects harbor double-stranded RNA viruses encoding a novel proline-alanine rich protein and a polymerase distantly related to that of fungal viruses

USDA-ARS?s Scientific Manuscript database

Novel double-stranded RNAs (~8 kbp) were isolated from three cornered alfalfa hopper (Spissistilus festinus) and beet leafhopper (Circulifer tenellus), two plant-feeding hemipteran insect pests. Genomes of the two new viruses, designated as Spissistilus festinus virus 1 (SpFV1) and Circulifer tenell...

Structures of proline-rich peptides bound to the ribosome reveal a common mechanism of protein synthesis inhibition

DOE PAGES

Gagnon, Matthieu G.; Roy, Raktim N.; Lomakin, Ivan B.; ...

2016-01-24

Here, with bacterial resistance becoming a serious threat to global public health, antimicrobial peptides (AMPs) have become a promising area of focus in antibiotic research. AMPs are derived from a diverse range of species, from prokaryotes to humans, with a mechanism of action that often involves disruption of the bacterial cell membrane. Proline-rich antimicrobial peptides (PrAMPs) are instead actively transported inside the bacterial cell where they bind and inactivate specific targets. Recently, it was reported that some PrAMPs, such as Bac7 1–35, oncocins and apidaecins, bind and inactivate the bacterial ribosome. Here we report the crystal structures of Bac7 1–35,more » Pyrrhocoricin, Metalnikowin and two oncocin derivatives, bound to the Thermus thermophilus 70S ribosome. Each of the PrAMPs blocks the peptide exit tunnel of the ribosome by simultaneously occupying three well characterized antibioticbinding sites and interferes with the initiation step of translation, thereby revealing a common mechanism of action used by these PrAMPs to inactivate protein synthesis. Our study expands the repertoire of PrAMPs and provides a framework for designing new-generation therapeutics.« less

Structures of proline-rich peptides bound to the ribosome reveal a common mechanism of protein synthesis inhibition

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

Gagnon, Matthieu G.; Roy, Raktim N.; Lomakin, Ivan B.

Here, with bacterial resistance becoming a serious threat to global public health, antimicrobial peptides (AMPs) have become a promising area of focus in antibiotic research. AMPs are derived from a diverse range of species, from prokaryotes to humans, with a mechanism of action that often involves disruption of the bacterial cell membrane. Proline-rich antimicrobial peptides (PrAMPs) are instead actively transported inside the bacterial cell where they bind and inactivate specific targets. Recently, it was reported that some PrAMPs, such as Bac7 1–35, oncocins and apidaecins, bind and inactivate the bacterial ribosome. Here we report the crystal structures of Bac7 1–35,more » Pyrrhocoricin, Metalnikowin and two oncocin derivatives, bound to the Thermus thermophilus 70S ribosome. Each of the PrAMPs blocks the peptide exit tunnel of the ribosome by simultaneously occupying three well characterized antibioticbinding sites and interferes with the initiation step of translation, thereby revealing a common mechanism of action used by these PrAMPs to inactivate protein synthesis. Our study expands the repertoire of PrAMPs and provides a framework for designing new-generation therapeutics.« less

Essential motions and energetic contributions of individual residues in a peptide bound to an SH3 domain.

PubMed Central

Kolafa, J; Perram, J W; Bywater, R P

2000-01-01

We have studied protein-ligand interactions by molecular dynamics simulations using software designed to exploit parallel computing architectures. The trajectories were analyzed to extract the essential motions and to estimate the individual contributions of fragments of the ligand to overall binding enthalpy. Two forms of the bound ligand are compared, one with the termini blocked by covalent derivatization, and one in the underivatized, zwitterionic form. The ends of the peptide tend to bind more loosely in the capped form. We can observe significant motions in the bound ligand and distinguish between motions of the peptide backbone and of the side chains. This could be useful in designing ligands, which fit optimally to the binding protein. We show that it is possible to determine the different contributions of each residue in a peptide to the enthalpy of binding. Proline is a major net contributor to binding enthalpy, in keeping with the known propensity for this family of proteins to bind proline-rich peptides. PMID:10919999

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.

Transgenic Mice Expressing Yeast CUP1 Exhibit Increased Copper Utilization from Feeds

PubMed Central

Chen, Zhenliang; Liao, Rongrong; Zhang, Xiangzhe; Wang, Qishan; Pan, Yuchun

2014-01-01

Copper is required for structural and catalytic properties of a variety of enzymes participating in many vital biological processes for growth and development. Feeds provide most of the copper as an essential micronutrient consumed by animals, but inorganic copper could not be utilized effectively. In the present study, we aimed to develop transgenic mouse models to test if copper utilization will be increased by providing the animals with an exogenous gene for generation of copper chelatin in saliva. Considering that the S. cerevisiae CUP1 gene encodes a Cys-rich protein that can bind copper as specifically as copper chelatin in yeast, we therefore constructed a transgene plasmid containing the CUP1 gene regulated for specific expression in the salivary glands by a promoter of gene coding pig parotid secretory protein. Transgenic CUP1 was highly expressed in the parotid and submandibular salivary glands and secreted in saliva as a 9-kDa copper-chelating protein. Expression of salivary copper-chelating proteins reduced fecal copper contents by 21.61% and increased body-weight by 12.97%, suggesting that chelating proteins improve the utilization and absorbed efficacy of copper. No negative effects on the health of the transgenic mice were found by blood biochemistry and histology analysis. These results demonstrate that the introduction of the salivary CUP1 transgene into animals offers a possible approach to increase the utilization efficiency of copper and decrease the fecal copper contents. PMID:25265503

The sialotranscriptome of Antricola delacruzi female ticks is compatible with non-hematophagous behavior and an alternative source of food

PubMed Central

Ribeiro, José Marcos C.; Labruna, Marcelo B.; Mans, Ben J.; Maruyama, Sandra Regina; Francischetti, Ivo M. B.; Barizon, Gustavo Canavaci; de Miranda Santos, Isabel K. F.

2012-01-01

The hosts for Antricola delacruzi ticks are insectivorous, cave-dwelling bats on which only larvae are found. The mouthparts of nymphal and adult A. delacruzi are compatible with scavenging feeding because the hypostome is small and toothless. How a single blood meal of a larva provides energy for several molts as well as for oviposition by females is not known. Adults of A. delacruzi possibly feed upon an unknown food source in bat guano, a substrate on which nymphal and adult stages are always found. Guano produced by insectivorous bats contains twice the amount of protein and 60 times the amount of iron as beef. In addition, bacteria and chitin-rich fungi proliferate on guano. Comparative data on the transcriptome of the salivary glands of A. delacruzi is nonexistent and would help to understand the physiological adaptations of salivary glands that accompany different sources of food as well as the steps taken by the Acari towards haematophagy, believed to have evolved from scavenging dead animals. Annotation of the transcriptome of salivary glands from female instars of A. delacruzi collected on guano categorized 5.7% of the clusters of expressed genes as putative secreted proteins. They included abundantly expressed TIL domain-containing proteins (possible anti-microbials), an abundantly expressed protein similar to a serum amyloid found in the sialotranscriptomes of Ornithodoros spp., a savignygrin, a family of mucin/peritrophin/cuticle-like proteins, antimicrobials and an HIV envelope-like glycoprotein also found in soft ticks. When comparing the transcriptome of A. delacruzi with those of blood-feeding female soft and hard ticks some notable differences were observed; they consisted of the following transcripts over- or under-represented or absent in the sialotranscriptome of A. delacuzi that may reflect its source of food: ferritin, mucins with chitin-binding domains and TIL domain-containing proteins versus lipocalins, basic tail proteins, metalloproteases, glycine-rich proteins and Kunitz protease inhibitors, respectively. PMID:22306723

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.

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

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.

A mental retardation gene, motopsin/prss12, modulates cell morphology by interaction with seizure-related gene 6.

PubMed

Mitsui, Shinichi; Hidaka, Chiharu; Furihata, Mutsuo; Osako, Yoji; Yuri, Kazunari

2013-07-12

A serine protease, motopsin (prss12), plays a significant role in cognitive function and the development of the brain, since the loss of motopsin function causes severe mental retardation in humans and enhances social behavior in mice. Motopsin is activity-dependently secreted from neuronal cells, is captured around the synaptic cleft, and cleaves a proteoglycan, agrin. The multi-domain structure of motopsin, consisting of a signal peptide, a proline-rich domain, a kringle domain, three scavenger receptor cysteine-rich domains, and a protease domain at the C-terminal, suggests the interaction with other molecules through these domains. To identify a protein interacting with motopsin, we performed yeast two-hybrid screening and found that seizure-related gene 6 (sez-6), a transmembrane protein on the plasma membrane of neuronal cells, bound to the proline-rich/kringle domain of motopsin. Pull-down and immunoprecipitation analyses indicated the interaction between these proteins. Immunocytochemical and immunohistochemical analyses suggested the co-localization of motopsin and sez-6 at neuronal cells in the developmental mouse brain and at motor neurons in the anterior horn of human spinal cords. Transient expression of motopsin in neuro2a cells increased the number and length of neurites as well as the level of neurite branching. Interestingly, co-expression of sez-6 with motopsin restored the effect of motopsin at the basal level, while sez-6 expression alone showed no effects on cell morphology. Our results suggest that the interaction of motopsin and sez-6 modulates the neuronal cell morphology. Copyright © 2013 Elsevier Inc. All rights reserved.

Structural Landscape of the Proline-Rich Domain of Sos1 Nucleotide Exchange Factor

PubMed Central

McDonald, Caleb B.; Bhat, Vikas; Kurouski, Dmitry; Mikles, David C.; Deegan, Brian J.; Seldeen, Kenneth L.; Lednev, Igor K.; Farooq, Amjad

2013-01-01

Despite its key role in mediating a plethora of cellular signaling cascades pertinent to health and disease, little is known about the structural landscape of the proline-rich (PR) domain of Sos1 guanine nucleotide exchange factor. Herein, using a battery of biophysical tools, we provide evidence that the PR domain of Sos1 is structurally disordered and adopts an extended random coil-like conformation in solution. Of particular interest is the observation that while chemical denaturation of PR domain results in the formation of a significant amount of polyproline II (PPII) helices, it has little or negligible effect on its overall size as measured by its hydrodynamic radius. Our data also show that the PR domain displays a highly dynamic conformational basin in agreement with the knowledge that the intrinsically unstructured proteins rapidly interconvert between an ensemble of conformations. Collectively, our study provides new insights into the conformational equilibrium of a key signaling molecule with important consequences on its physiological function. PMID:23528987

Plasmodium cysteine repeat modular proteins 1-4: complex proteins with roles throughout the malaria parasite life cycle.

PubMed

Thompson, Joanne; Fernandez-Reyes, Delmiro; Sharling, Lisa; Moore, Sally G; Eling, Wijnand M; Kyes, Sue A; Newbold, Christopher I; Kafatos, Fotis C; Janse, Chris J; Waters, Andrew P

2007-06-01

The Cysteine Repeat Modular Proteins (PCRMP1-4) of Plasmodium, are encoded by a small gene family that is conserved in malaria and other Apicomplexan parasites. They are very large, predicted surface proteins with multipass transmembrane domains containing motifs that are conserved within families of cysteine-rich, predicted surface proteins in a range of unicellular eukaryotes, and a unique combination of protein-binding motifs, including a >100 kDa cysteine-rich modular region, an epidermal growth factor-like domain and a Kringle domain. PCRMP1 and 2 are expressed in life cycle stages in both the mosquito and vertebrate. They colocalize with PfEMP1 (P. falciparum Erythrocyte Membrane Antigen-1) during its export from P. falciparum blood-stage parasites and are exposed on the surface of haemolymph- and salivary gland-sporozoites in the mosquito, consistent with a role in host tissue targeting and invasion. Gene disruption of pcrmp1 and 2 in the rodent malaria model, P. berghei, demonstrated that both are essential for transmission of the parasite from the mosquito to the mouse and has established their discrete and important roles in sporozoite targeting to the mosquito salivary gland. The unprecedented expression pattern and structural features of the PCRMPs thus suggest a variety of roles mediating host-parasite interactions throughout the parasite life cycle.

SNPing at the Epidermal Barrier.

PubMed

Kelsell, David P; Byrne, Carolyn

2011-08-01

Filaggrin variants are well-established risk factors for atopic eczema (AE). Recent studies suggest additional epidermal differentiation complex (EDC) gene associations with AE. In this issue, Marenholz and colleagues confirm this prediction and show that a small proline-rich protein 3 (SPRR3) variant confers susceptibility to AE. This finding suggests that further genetic and functional characterization of SPRR3 should be performed in patients with AE.

Pigeonpea Hybrid-Proline-Rich Protein (CcHyPRP) Confers Biotic and Abiotic Stress Tolerance in Transgenic Rice

PubMed Central

Mellacheruvu, Sunitha; Tamirisa, Srinath; Vudem, Dashavantha Reddy; Khareedu, Venkateswara Rao

2016-01-01

In this study, we report the overexpression of Cajanus cajan hybrid-proline-rich protein encoding gene (CcHyPRP) in rice which resulted in increased tolerance to both abiotic and biotic stresses. Compared to the control plants, the transgenic rice lines, expressing CcHyPRP, exhibited high-level tolerance against major abiotic stresses, viz., drought, salinity, and heat, as evidenced by increased biomass, chlorophyll content, survival rate, root, and shoot growth. Further, transgenic rice lines showed increased panicle size and grain number compared to the control plants under different stress conditions. The CcHyPRP transgenics, as compared to the control, revealed enhanced activities of catalase and superoxide dismutase (SOD) enzymes and reduced malondialdehyde (MDA) levels. Expression pattern of CcHyPRP::GFP fusion-protein confirmed its predominant localization in cell walls. Moreover, the CcHyPRP transgenics, as compared to the control, exhibited increased resistance to the fungal pathogen Magnaporthe grisea which causes blast disease in rice. Higher levels of bZIP and endochitinase transcripts as well as endochitinase activity were observed in transgenic rice compared to the control plants. The overall results demonstrate the intrinsic role of CcHyPRP in conferring multiple stress tolerance at the whole-plant level. The multipotent CcHyPRP seems promising as a prime candidate gene to fortify crop plants for enhanced tolerance/resistance to different stress factors. PMID:26834756

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.

β-Sheet Containment by Flanking Prolines: Molecular Dynamic Simulations of the Inhibition of β-Sheet Elongation by Proline Residues in Human Prion Protein

PubMed Central

Shamsir, Mohd S.; Dalby, Andrew R.

2007-01-01

Previous molecular dynamic simulations have reported elongation of the existing β-sheet in prion proteins. Detailed examination has shown that these elongations do not extend beyond the proline residues flanking these β-sheets. In addition, proline has also been suggested to possess a possible structural role in preserving protein interaction sites by preventing invasion of neighboring secondary structures. In this work, we have studied the possible structural role of the flanking proline residues by simulating mutant structures with alternate substitution of the proline residues with valine. Simulations showed a directional inhibition of elongation, with the elongation progressing in the direction of valine including evident inhibition of elongation by existing proline residues. This suggests that the flanking proline residues in prion proteins may have a containment role and would confine the β-sheet within a specific length. PMID:17172295

Proline puckering parameters for collagen structure simulations

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

Wu, Di, E-mail: diwu@fudan.edu.cn

Collagen is made of triple helices rich in proline residues, and hence is influenced by the conformational motions of prolines. Because the backbone motions of prolines are restricted by the helical structures, the only side chain motion—proline puckering—becomes an influential factor that may affect the stability of collagen structures. In molecular simulations, a proper proline puckering population is desired so to yield valid results of the collagen properties. Here we design the proline puckering parameters in order to yield suitable proline puckering populations as demonstrated in the experimental results. We test these parameters in collagen and the proline dipeptide simulations.more » Compared with the results of the PDB and the quantum calculations, we propose the proline puckering parameters for the selected collagen model simulations.« less

Structure of the dimeric exonuclease TREX1 in complex with DNA displays a proline-rich binding site for WW Domains.

PubMed

Brucet, Marina; Querol-Audí, Jordi; Serra, Maria; Ramirez-Espain, Ximena; Bertlik, Kamila; Ruiz, Lidia; Lloberas, Jorge; Macias, Maria J; Fita, Ignacio; Celada, Antonio

2007-05-11

TREX1 is the most abundant mammalian 3' --> 5' DNA exonuclease. It has been described to form part of the SET complex and is responsible for the Aicardi-Goutières syndrome in humans. Here we show that the exonuclease activity is correlated to the binding preferences toward certain DNA sequences. In particular, we have found three motifs that are selected, GAG, ACA, and CTGC. To elucidate how the discrimination occurs, we determined the crystal structures of two murine TREX1 complexes, with a nucleotide product of the exonuclease reaction, and with a single-stranded DNA substrate. Using confocal microscopy, we observed TREX1 both in nuclear and cytoplasmic subcellular compartments. Remarkably, the presence of TREX1 in the nucleus requires the loss of a C-terminal segment, which we named leucine-rich repeat 3. Furthermore, we detected the presence of a conserved proline-rich region on the surface of TREX1. This observation points to interactions with proline-binding domains. The potential interacting motif "PPPVPRPP" does not contain aromatic residues and thus resembles other sequences that select SH3 and/or Group 2 WW domains. By means of nuclear magnetic resonance titration experiments, we show that, indeed, a polyproline peptide derived from the murine TREX1 sequence interacted with the WW2 domain of the elongation transcription factor CA150. Co-immunoprecipitation studies confirmed this interaction with the full-length TREX1 protein, thereby suggesting that TREX1 participates in more functional complexes than previously thought.

Study of base pair mutations in proline-rich homeodomain (PRH)-DNA complexes using molecular dynamics.

PubMed

Jalili, Seifollah; Karami, Leila; Schofield, Jeremy

2013-06-01

Proline-rich homeodomain (PRH) is a regulatory protein controlling transcription and gene expression processes by binding to the specific sequence of DNA, especially to the sequence 5'-TAATNN-3'. The impact of base pair mutations on the binding between the PRH protein and DNA is investigated using molecular dynamics and free energy simulations to identify DNA sequences that form stable complexes with PRH. Three 20-ns molecular dynamics simulations (PRH-TAATTG, PRH-TAATTA and PRH-TAATGG complexes) in explicit solvent water were performed to investigate three complexes structurally. Structural analysis shows that the native TAATTG sequence forms a complex that is more stable than complexes with base pair mutations. It is also observed that upon mutation, the number and occupancy of the direct and water-mediated hydrogen bonds decrease. Free energy calculations performed with the thermodynamic integration method predict relative binding free energies of 0.64 and 2 kcal/mol for GC to AT and TA to GC mutations, respectively, suggesting that among the three DNA sequences, the PRH-TAATTG complex is more stable than the two mutated complexes. In addition, it is demonstrated that the stability of the PRH-TAATTA complex is greater than that of the PRH-TAATGG complex.

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.

Quantitative Analysis of Human Salivary Gland-Derived Intact Proteome Using Top-Down Mass Spectrometry

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

Wu, Si; Brown, Joseph N.; Tolic, Nikola

There are several notable challenges inherent to fully characterizing the entirety of the human saliva proteome using bottom-up approaches, including polymorphic isoforms, post-translational modifications, unique splice variants, deletions, and truncations. To address these challenges, we have developed a top-down based liquid chromatography-mass spectrometry (LC-MS) approach, which cataloged 20 major human salivary proteins with a total of 83 proteoforms, containing a broad range of post-translational modifications. Among these proteins, several previously reported disease biomarker proteins were identified at the intact protein level, such as beta-2 microglobulin (B2M). In addition, intact glycosylated proteoforms of several saliva proteins were also characterized, including intactmore » N-glycosylated protein prolactin inducible protein (PIP) and O-glycosylated acidic protein rich protein (aPRP). These characterized proteoforms constitute an intact saliva proteoform database, which was used for quantitative comparison of intact salivary proteoforms among six healthy individuals. Human parotid (PS) and submandibular/sublingual gland (SMSL) secretion samples (2 μg of protein each) from six healthy individuals were compared using RPLC coupled with the 12T FTICR mass spectrometer. Significantly different protein and PTM patterns were resolved with high reproducibility between PS and SMSL glands. The results from this study provide further insight into the potential mechanisms of PTM pathways in oral glandular secretion, expanding our knowledge of this complex yet easily accessible fluid. Intact protein LC-MS approach presented herein can potentially be applied for rapid and accurate identification of biomarkers from only a few microliters of human glandular saliva.« less

Cellular Localization and Characterization of Cytosolic Binding Partners for Gla Domain-containing Proteins PRRG4 and PRRG2*

PubMed Central

Yazicioglu, Mustafa N.; Monaldini, Luca; Chu, Kirk; Khazi, Fayaz R.; Murphy, Samuel L.; Huang, Heshu; Margaritis, Paris; High, Katherine A.

2013-01-01

The genes encoding a family of proteins termed proline-rich γ-carboxyglutamic acid (PRRG) proteins were identified and characterized more than a decade ago, but their functions remain unknown. These novel membrane proteins have an extracellular γ-carboxyglutamic acid (Gla) protein domain and cytosolic WW binding motifs. We screened WW domain arrays for cytosolic binding partners for PRRG4 and identified novel protein-protein interactions for the protein. We also uncovered a new WW binding motif in PRRG4 that is essential for these newly found protein-protein interactions. Several of the PRRG-interacting proteins we identified are essential for a variety of physiologic processes. Our findings indicate possible novel and previously unidentified functions for PRRG proteins. PMID:23873930

Enhancement of both salivary protein-enological tannin interactions and astringency perception by ethanol.

PubMed

Obreque-Slíer, Elías; Peña-Neira, Alvaro; López-Solís, Remigio

2010-03-24

Red wine astringency has been associated with interactions of tannins with salivary proteins. Tannins are active protein precipitants. Not much evidence exists demonstrating contribution of other wine components to astringency. We aimed to investigate an eventual role of ethanol both in astringency and salivary protein-enological tannin interactions. A trained sensory panel scored perceived astringency. Salivary protein-tannin interactions were assessed by observing both tannin-dependent changes in salivary protein diffusion on cellulose membranes and tannin-induced salivary protein precipitation. Proanthocyanidins and gallotannins in aqueous and hydroalcoholic solutions were assayed. A biphasic mode of diffusion on cellulose membranes displayed by salivary proteins was unaffected after dilution with water or enological concentrations of ethanol. At those concentrations ethanol was not astringent. In aqueous solution, tannins provoked both restriction of salivary protein diffusion, protein precipitation, and astringency. Those effects were exacerbated by 13% ethanol. In summary, enological concentrations of ethanol exacerbate astringency and salivary protein-tannin interactions.

The sialotranscriptome of Antricola delacruzi female ticks is compatible with non-hematophagous behavior and an alternative source of food.

PubMed

Ribeiro, José Marcos C; Labruna, Marcelo B; Mans, Ben J; Maruyama, Sandra Regina; Francischetti, Ivo M B; Barizon, Gustavo Canavaci; de Miranda Santos, Isabel K F

2012-05-01

The hosts for Antricola delacruzi ticks are insectivorous, cave-dwelling bats on which only larvae are found. The mouthparts of nymphal and adult A. delacruzi are compatible with scavenging feeding because the hypostome is small and toothless. How a single blood meal of a larva provides energy for several molts as well as for oviposition by females is not known. Adults of A. delacruzi possibly feed upon an unknown food source in bat guano, a substrate on which nymphal and adult stages are always found. Guano produced by insectivorous bats contains twice the amount of protein and 60 times the amount of iron as beef. In addition, bacteria and chitin-rich fungi proliferate on guano. Comparative data on the transcriptome of the salivary glands of A. delacruzi is nonexistent and would help to understand the physiological adaptations of salivary glands that accompany different sources of food as well as the steps taken by the Acari toward haematophagy, believed to have evolved from scavenging dead animals. Annotation of the transcriptome of salivary glands from female instars of A. delacruzi collected on guano categorized 5.7% of the clusters of expressed genes as putative secreted proteins. They included abundantly expressed TIL-domain-containing proteins (possible anti-microbials), an abundantly expressed protein similar to a serum amyloid found in the sialotranscriptomes of Ornithodoros spp., a savignygrin, a family of mucin/peritrophin/cuticle-like proteins, anti-microbials and an HIV envelope-like glycoprotein also found in soft ticks. When comparing the transcriptome of A. delacruzi with those of blood-feeding female soft and hard ticks some notable differences were observed; they consisted of the following transcripts over- or under-represented or absent in the sialotranscriptome of A. delacruzi that may reflect its source of food: ferritin, mucins with chitin-binding domains and TIL-domain-containing proteins versus lipocalins, basic tail proteins, metalloproteases, glycine-rich proteins and Kunitz protease inhibitors, respectively. Copyright © 2012 Elsevier Ltd. All rights reserved.

Conformation of a Group 2 Late Embryogenesis Abundant Protein from Soybean. Evidence of Poly (l-Proline)-type II Structure1

PubMed Central

Soulages, Jose L.; Kim, Kangmin; Arrese, Estela L.; Walters, Christina; Cushman, John C.

2003-01-01

Late embryogenesis abundant (LEA) proteins are members of a large group of hydrophilic, glycine-rich proteins found in plants, algae, fungi, and bacteria known collectively as hydrophilins that are preferentially expressed in response to dehydration or hyperosmotic stress. Group 2 LEA (dehydrins or responsive to abscisic acid) proteins are postulated to stabilize macromolecules against damage by freezing, dehydration, ionic, or osmotic stress. However, the structural and physicochemical properties of group 2 LEA proteins that account for such functions remain unknown. We have analyzed the structural properties of a recombinant form of a soybean (Glycine max) group 2 LEA (rGmDHN1). Differential scanning calorimetry of purified rGmDHN1 demonstrated that the protein does not display a cooperative unfolding transition upon heating. Ultraviolet absorption and circular dichroism spectroscopy revealed that the protein is in a largely hydrated and unstructured conformation in solution. However, ultraviolet absorption and circular dichroism measurements collected at different temperatures showed that the protein exists in equilibrium between two extended conformational states: unordered and left-handed extended helical or poly (l-proline)-type II structures. It is estimated that 27% of the residues of rGmDHN1 adopt or poly (l-proline)-type II-like helical conformation at 12°C. The content of extended helix gradually decreases to 15% as the temperature is increased to 80°C. Studies of the conformation of the protein in solution in the presence of liposomes, trifluoroethanol, and sodium dodecyl sulfate indicated that rGmDHN1 has a very low intrinsic ability to adopt α-helical structure and to interact with phospholipid bilayers through amphipathic α-helices. The ability of the protein to remain in a highly extended conformation at low temperatures could constitute the basis of the functional role of GmDHN1 in the prevention of freezing, desiccation, ionic, or osmotic stress-related damage to macromolecular structures. PMID:12644649

RNA sequencing and pathway analysis identify tumor necrosis factor alpha driven small proline-rich protein dysregulation in chronic rhinosinusitis.

PubMed

Ramakrishnan, Vijay R; Gonzalez, Joseph R; Cooper, Sarah E; Barham, Henry P; Anderson, Catherine B; Larson, Eric D; Cool, Carlyne D; Diller, John D; Jones, Kenneth; Kinnamon, Sue C

2017-09-01

Chronic rhinosinusitis (CRS) is a heterogeneous inflammatory disorder in which many pathways contribute to end-organ disease. Small proline-rich proteins (SPRR) are polypeptides that have recently been shown to contribute to epithelial biomechanical properties relevant in T-helper type 2 inflammation. There is evidence that genetic polymorphism in SPRR genes may predict the development of asthma in children with atopy and, correlatively, that expression of SPRRs is increased under allergic conditions, which leads to epithelial barrier dysfunction in atopic disease. RNAs from uncinate tissue specimens from patients with CRS and control subjects were compared by RNA sequencing by using Ingenuity Pathway Analysis (n = 4 each), and quantitative polymerase chain reaction (PCR) (n = 15). A separate cohort of archived sinus tissue was examined by immunohistochemistry (n = 19). A statistically significant increase of SPRR expression in CRS sinus tissue was identified that was not a result of atopic presence. SPRR1 and SPRR2A expressions were markedly increased in patients with CRS (p < 0.01) on RNA sequencing, with confirmation by using real-time PCR. Immunohistochemistry of archived surgical samples demonstrated staining of SPRR proteins within squamous epithelium of both groups. Pathway analysis indicated tumor necrosis factor (TNF) alpha as a master regulator of the SPRR gene products. Expression of SPRR1 and of SPRR2A is increased in mucosal samples from patients with CRS and appeared as a downstream result of TNF alpha modulation, which possibly resulted in epithelial barrier dysfunction.

Fertility and polarized cell growth depends on eIF5A for translation of polyproline-rich formins in Saccharomyces cerevisiae.

PubMed

Li, Tianlu; Belda-Palazón, Borja; Ferrando, Alejandro; Alepuz, Paula

2014-08-01

eIF5A is an essential and evolutionary conserved translation elongation factor, which has recently been proposed to be required for the translation of proteins with consecutive prolines. The binding of eIF5A to ribosomes occurs upon its activation by hypusination, a modification that requires spermidine, an essential factor for mammalian fertility that also promotes yeast mating. We show that in response to pheromone, hypusinated eIF5A is required for shmoo formation, localization of polarisome components, induction of cell fusion proteins, and actin assembly in yeast. We also show that eIF5A is required for the translation of Bni1, a proline-rich formin involved in polarized growth during shmoo formation. Our data indicate that translation of the polyproline motifs in Bni1 is eIF5A dependent and this translation dependency is lost upon deletion of the polyprolines. Moreover, an exogenous increase in Bni1 protein levels partially restores the defect in shmoo formation seen in eIF5A mutants. Overall, our results identify eIF5A as a novel and essential regulator of yeast mating through formin translation. Since eIF5A and polyproline formins are conserved across species, our results also suggest that eIF5A-dependent translation of formins could regulate polarized growth in such processes as fertility and cancer in higher eukaryotes. Copyright © 2014 by the Genetics Society of America.

Structural landscape of the proline-rich domain of Sos1 nucleotide exchange factor.

PubMed

McDonald, Caleb B; Bhat, Vikas; Kurouski, Dmitry; Mikles, David C; Deegan, Brian J; Seldeen, Kenneth L; Lednev, Igor K; Farooq, Amjad

2013-01-01

Despite its key role in mediating a plethora of cellular signaling cascades pertinent to health and disease, little is known about the structural landscape of the proline-rich (PR) domain of Sos1 guanine nucleotide exchange factor. Herein, using a battery of biophysical tools, we provide evidence that the PR domain of Sos1 is structurally disordered and adopts an extended random coil-like conformation in solution. Of particular interest is the observation that while chemical denaturation of PR domain results in the formation of a significant amount of polyproline II (PPII) helices, it has little or negligible effect on its overall size as measured by its hydrodynamic radius. Our data also show that the PR domain displays a highly dynamic conformational basin in agreement with the knowledge that the intrinsically unstructured proteins rapidly interconvert between an ensemble of conformations. Collectively, our study provides new insights into the conformational equilibrium of a key signaling molecule with important consequences on its physiological function. Copyright © 2013 Elsevier B.V. All rights reserved.

Structural insights into the specific binding of huntingtin proline-rich region with the SH3 and WW domains.

PubMed

Gao, Yong-Guang; Yan, Xian-Zhong; Song, Ai-Xin; Chang, Yong-Gang; Gao, Xue-Chao; Jiang, Nan; Zhang, Qi; Hu, Hong-Yu

2006-12-01

The interactions of huntingtin (Htt) with the SH3 domain- or WW domain-containing proteins have been implicated in the pathogenesis of Huntington's disease (HD). We report the specific interactions of Htt proline-rich region (PRR) with the SH3GL3-SH3 domain and HYPA-WW1-2 domain pair by NMR. The results show that Htt PRR binds with the SH3 domain through nearly its entire chain, and that the binding region on the domain includes the canonical PxxP-binding site and the specificity pocket. The C terminus of PRR orients to the specificity pocket, whereas the N terminus orients to the PxxP-binding site. Htt PRR can also specifically bind to WW1-2; the N-terminal portion preferentially binds to WW1, while the C-terminal portion binds to WW2. This study provides structural insights into the specific interactions between Htt PRR and its binding partners as well as the alteration of these interactions that involve PRR, which may have implications for the understanding of HD.

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.

Anopheles salivary gland proteomes from major malaria vectors

PubMed Central

2012-01-01

Background Antibody responses against Anopheles salivary proteins can indicate individual exposure to bites of malaria vectors. The extent to which these salivary proteins are species-specific is not entirely resolved. Thus, a better knowledge of the diversity among salivary protein repertoires from various malaria vector species is necessary to select relevant genus-, subgenus- and/or species-specific salivary antigens. Such antigens could be used for quantitative (mosquito density) and qualitative (mosquito species) immunological evaluation of malaria vectors/host contact. In this study, salivary gland protein repertoires (sialomes) from several Anopheles species were compared using in silico analysis and proteomics. The antigenic diversity of salivary gland proteins among different Anopheles species was also examined. Results In silico analysis of secreted salivary gland protein sequences retrieved from an NCBInr database of six Anopheles species belonging to the Cellia subgenus (An. gambiae, An. arabiensis, An. stephensi and An. funestus) and Nyssorhynchus subgenus (An. albimanus and An. darlingi) displayed a higher degree of similarity compared to salivary proteins from closely related Anopheles species. Additionally, computational hierarchical clustering allowed identification of genus-, subgenus- and species-specific salivary proteins. Proteomic and immunoblot analyses performed on salivary gland extracts from four Anopheles species (An. gambiae, An. arabiensis, An. stephensi and An. albimanus) indicated that heterogeneity of the salivary proteome and antigenic proteins was lower among closely related anopheline species and increased with phylogenetic distance. Conclusion This is the first report on the diversity of the salivary protein repertoire among species from the Anopheles genus at the protein level. This work demonstrates that a molecular diversity is exhibited among salivary proteins from closely related species despite their common pharmacological activities. The involvement of these proteins as antigenic candidates for genus-, subgenus- or species-specific immunological evaluation of individual exposure to Anopheles bites is discussed. PMID:23148599

A proline-rich sequence unique to MEK1 and MEK2 is required for raf binding and regulates MEK function.

PubMed

Catling, A D; Schaeffer, H J; Reuter, C W; Reddy, G R; Weber, M J

1995-10-01

Mammalian MEK1 and MEK2 contain a proline-rich (PR) sequence that is absent both from the yeast homologs Ste7 and Byr1 and from a recently cloned activator of the JNK/stress-activated protein kinases, SEK1/MKK4. Since this PR sequence occurs in MEKs that are regulated by Raf family enzymes but is missing from MEKs and SEKs activated independently of Raf, we sought to investigate the role of this sequence in MEK1 and MEK2 regulation and function. Deletion of the PR sequence from MEK1 blocked the ability of MEK1 to associate with members of the Raf family and markedly attenuated activation of the protein in vivo following growth factor stimulation. In addition, this sequence was necessary for efficient activation of MEK1 in vitro by B-Raf but dispensable for activation by a novel MEK1 activator which we have previously detected in fractionated fibroblast extracts. Furthermore, we found that a phosphorylation site within the PR sequence of MEK1 was required for sustained MEK1 activity in response to serum stimulation of quiescent fibroblasts. Consistent with this observation, we observed that MEK2, which lacks a phosphorylation site at the corresponding position, was activated only transiently following serum stimulation. Finally, we found that deletion of the PR sequence from a constitutively activated MEK1 mutant rendered the protein nontransforming in Rat1 fibroblasts. These observations indicate a critical role for the PR sequence in directing specific protein-protein interactions important for the activation, inactivation, and downstream functioning of the MEKs.

A proline-rich sequence unique to MEK1 and MEK2 is required for raf binding and regulates MEK function.

PubMed Central

Catling, A D; Schaeffer, H J; Reuter, C W; Reddy, G R; Weber, M J

1995-01-01

Mammalian MEK1 and MEK2 contain a proline-rich (PR) sequence that is absent both from the yeast homologs Ste7 and Byr1 and from a recently cloned activator of the JNK/stress-activated protein kinases, SEK1/MKK4. Since this PR sequence occurs in MEKs that are regulated by Raf family enzymes but is missing from MEKs and SEKs activated independently of Raf, we sought to investigate the role of this sequence in MEK1 and MEK2 regulation and function. Deletion of the PR sequence from MEK1 blocked the ability of MEK1 to associate with members of the Raf family and markedly attenuated activation of the protein in vivo following growth factor stimulation. In addition, this sequence was necessary for efficient activation of MEK1 in vitro by B-Raf but dispensable for activation by a novel MEK1 activator which we have previously detected in fractionated fibroblast extracts. Furthermore, we found that a phosphorylation site within the PR sequence of MEK1 was required for sustained MEK1 activity in response to serum stimulation of quiescent fibroblasts. Consistent with this observation, we observed that MEK2, which lacks a phosphorylation site at the corresponding position, was activated only transiently following serum stimulation. Finally, we found that deletion of the PR sequence from a constitutively activated MEK1 mutant rendered the protein nontransforming in Rat1 fibroblasts. These observations indicate a critical role for the PR sequence in directing specific protein-protein interactions important for the activation, inactivation, and downstream functioning of the MEKs. PMID:7565670

Pyrrhocoricin, a proline-rich antimicrobial peptide derived from insect, inhibits the translation process in the cell-free Escherichia coli protein synthesis system.

PubMed

Taniguchi, Masayuki; Ochiai, Akihito; Kondo, Hiroshi; Fukuda, Shun; Ishiyama, Yohei; Saitoh, Eiichi; Kato, Tetsuo; Tanaka, Takaaki

2016-05-01

Previous studies have shown that pyrrhocoricin, a proline-rich antimicrobial peptide (PrAMP), killed sensitive species in a dose-dependent manner by specifically binding to DnaK. Here, on the basis of the finding that DnaK-deficient Escherichia coli strains are susceptible to PrAMPs, we used pyrrhocoricin to investigate internal targets other than DnaK. Using conventional antibiotics (bleomycin, streptomycin, and fosfomycin) that have known modes of action, first, we validated the availability of an assay using a cell-free rapid translation system (RTS), which is an in vitro protein synthesis system based on E. coli lysate, for evaluating inhibition of protein synthesis. We found that, similarly to bleomycin and streptomycin, pyrrhocoricin inhibited GFP synthesis in RTS in a concentration-dependent manner. In addition, blockage of transcription and translation steps in RTS was individually estimated using RT-PCR after gene expression to determine mRNA products and using sodium dodecyl sulfate-polyacrylamide gel electrophoresis to determine the amounts of GFP expressed from purified mRNA, respectively. The results demonstrated that this inhibition of GFP synthesis by pyrrhocoricin did not occur at the transcription step but rather at the translation step, in a manner similar to that of GFP synthesis by streptomycin, an inhibitor of the translation step by causing misreading of tRNA. These results suggest that RTS is a powerful assay system for determining if antimicrobial peptides inhibit protein synthesis and its transcription and/or translation steps. This is the first study to have shown that pyrrhocoricin inhibited protein synthesis by specifically repressing the translation step. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

The ASPP interaction network: electrostatic differentiation between pro- and anti-apoptotic proteins.

PubMed

Benyamini, Hadar; Friedler, Assaf

2011-01-01

The ASPP proteins are apoptosis regulators: ASPP1 and ASPP2 promote, while iASPP inhibits, apoptosis. The mechanism by which these different outcomes are achieved is still unknown. The C-terminal ankyrin repeats and SH3 domain (ANK-SH3) mediate the interactions of the ASPP proteins with major apoptosis regulators such as p53, Bcl-2, and NFκB. The structure of the complex between ASPP2(ANK-SH3) and the core domain of p53 (p53CD) was previously determined. We have recently characterized the individual interactions of ASPP2(ANK-SH3) with Bcl-2 and NFκB, as well as a regulatory intramolecular interaction with the proline rich domain of ASPP2. Here we compared the ASPP interactions at two levels: ASPP2(ANK-SH3) with different proteins, and different ASPP family members with each protein partner. We found that the binding sites of ASPP2 to p53CD, Bcl-2, and NFκB are different, yet lie on the same face of ASPP2(ANK-SH3) . The intramolecular binding site to the proline rich domain overlaps the three intermolecular binding sites. To reveal the basis of functional diversity in the ASPP family, we compared their protein-binding domains. A subset of surface-exposed residues differentiates ASPP1 and ASPP2 from iASPP: ASPP1/2 are more negatively charged in specific residues that contact positively charged residues of p53CD, Bcl-2, and NFκB. We also found a gain of positive charge at the non-protein binding face of ASPP1/2, suggesting a role in electrostatic direction towards the negatively charged protein binding face. The electrostatic differences in binding interfaces between the ASPP proteins may be one of the causes for their different function. Copyright © 2010 John Wiley & Sons, Ltd.

Characterization of the human submandibular/sublingual saliva glycoproteome using lectin affinity chromatography coupled to Multidimensional Protein Identification Technology

PubMed Central

Gonzalez-Begne, Mireya; Lu, Bingwen; Liao, Lujian; Xu, Tao; Bedi, Gurrinder; Melvin, James E.; Yates, John R.

2011-01-01

In-depth analysis of the salivary proteome is fundamental to understanding the functions of salivary proteins in the oral cavity and to reveal disease biomarkers involved in different pathophysiological conditions, with the ultimate goal of improving patient diagnosis and prognosis. Submandibular and sublingual glands contribute saliva rich in glycoproteins to the total saliva output, making them valuable sources for glycoproteomic analysis. Lectin-affinity chromatography coupled to mass spectrometry-based shotgun proteomics was used to explore the submandibular/sublingual (SM/SL) saliva glycoproteome. A total of 262 N- and O-linked glycoproteins were identified by multidimensional protein identification technology (MudPIT). Only 38 were previously described in SM and SL salivas from the human salivary N-linked glycoproteome, while 224 were unique. Further comparison analysis with SM/SL saliva of the human saliva proteome, revealed 125 glycoproteins not formerly reported in this secretion. KEGG pathway analyses demonstrated that many of these glycoproteins are involved in processes such as complement and coagulation cascades, cell communication, glycosphingolipid biosynthesis neo-lactoseries, O-glycan biosynthesis, glycan structures-biosynthesis 2, starch and sucrose metabolism, peptidoglycan biosynthesis or others pathways. In summary, lectin-affinity chromatography coupled to MudPIT mass spectrometry identified many novel glycoproteins in SM/SL saliva. These new additions to the salivary proteome may prove to be a critical step for providing reliable biomarkers in the diagnosis of a myriad of oral and systemic diseases. PMID:21936497

Associations of Salivary BPIFA1 Protein in Chronic Periodontitis Patients with Type 2 Diabetes Mellitus

PubMed Central

Tang, Chen-Yi

2017-01-01

Aims To explore the differences in salivary BPI fold containing family A, member 1 (BPIFA1) concentration among type 2 diabetes mellitus (T2DM) subjects with various severities of chronic periodontitis and to determine whether BPIFA1 in saliva can be used as a potential biomarker of T2DM. Methods Unstimulated saliva samples were collected from 44 subjects with T2DM and 44 without T2DM (NDM). Additionally, demographic data and general health parameters, including fasting blood glucose (FBG) and body mass index (BMI), were collected. We also detected full-mouth clinical periodontal parameters including probing pocket depth (PPD), clinical attachment level (CAL), bleeding index (BI), and plaque index (PLI). Salivary BPIFA1, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) concentrations were also detected. Results BPIFA1 in saliva was detected at relatively high levels. T2DM subjects had decreased salivary BPIFA1 concentrations (P = 0.031). In T2DM subjects with nonperiodontitis or severe periodontitis, the level of BPIFA1 was significantly lower compared with that of NDM. Salivary TNF-α concentration displayed a similar trend to BPIFA1 in the NDM group. Conclusions BPIFA1 protein is rich in saliva and might be used as a potential predictive biomarker of T2DM, especially in patients with severe periodontitis and nonperiodontitis. This trial is registered with ChiCTR-ROC-17010310. PMID:29109737

Molecular Diversity between Salivary Proteins from New World and Old World Sand Flies with Emphasis on Bichromomyia olmeca, the Sand Fly Vector of Leishmania mexicana in Mesoamerica

PubMed Central

Townsend, Shannon; Pasos-Pinto, Silvia; Sanchez, Laura; Rasouli, Manoochehr; B. Guimaraes-Costa, Anderson; Aslan, Hamide; Francischetti, Ivo M. B.; Oliveira, Fabiano; Becker, Ingeborg; Kamhawi, Shaden; Ribeiro, Jose M. C.; Jochim, Ryan C.; Valenzuela, Jesus G.

2016-01-01

Background Sand fly saliva has been shown to have proteins with potent biological activities, salivary proteins that can be used as biomarkers of vector exposure, and salivary proteins that are candidate vaccines against different forms of leishmaniasis. Sand fly salivary gland transcriptomic approach has contributed significantly to the identification and characterization of many of these salivary proteins from important Leishmania vectors; however, sand fly vectors in some regions of the world are still neglected, as Bichromomyia olmeca (formerly known as Lutzomyia olmeca olmeca), a proven vector of Leishmania mexicana in Mexico and Central America. Despite the importance of this vector in transmitting Leishmania parasite in Mesoamerica there is no information on the repertoire of B. olmeca salivary proteins and their relationship to salivary proteins from other sand fly species. Methods and Findings A cDNA library of the salivary glands of wild-caught B. olmeca was constructed, sequenced, and analyzed. We identified transcripts encoding for novel salivary proteins from this sand fly species and performed a comparative analysis between B. olmeca salivary proteins and those from other sand fly species. With this new information we present an updated catalog of the salivary proteins specific to New World sand flies and salivary proteins common to all sand fly species. We also report in this work the anti-Factor Xa activity of Lofaxin, a salivary anticoagulant protein present in this sand fly species. Conclusions This study provides information on the first transcriptome of a sand fly from Mesoamerica and adds information to the limited repertoire of salivary transcriptomes from the Americas. This comparative analysis also shows a fast degree of evolution in salivary proteins from New World sand flies as compared with Old World sand flies. PMID:27409591

Three-dimensional structure and dynamics of wine tannin-saliva protein complexes. A multitechnique approach.

PubMed

Simon, Cécile; Barathieu, Karine; Laguerre, Michel; Schmitter, Jean-Marie; Fouquet, Eric; Pianet, Isabelle; Dufourc, Erick J

2003-09-09

The interactions between the B3 (catechin-4alpha,8-catechin) red wine tannin and the human salivary protein fragment IB7(14) (SPPGKPQGPPPQGG) were monitored by (1)H magic angle spinning NMR, circular dichroism, electrospray ionization mass spectrometry, and molecular modeling. It is found that the secondary structure of IB7(14) is made of a type II helix (collagen helix) and random coil. The central glycine 8 appears to act as a flexible rotula separating two helix II regions. Three tannin molecules tightly complex the peptide, without modifying its secondary structure, but seem to reduce its conformational dynamics. The binding dissociation constant is in the millimolar range. B3 tannins with a "tweezers" conformation bind to the hydrophilic side of the saliva peptide, suggesting that the principal driving forces toward association are governed by hydrogen bonding between the carbonyl functions of proline residues and both the phenol and catechol OH groups. These findings are further discussed in the frame of an astringency phenomenon.

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

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.

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

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

Comparative Protein Profiling of Intraphagosomal Expressed Proteins of Mycobacterium bovis BCG.

PubMed

Singhal, Neelja; Kumar, Manish; Sharma, Divakar; Bisht, Deepa

2016-01-01

BCG, the only available vaccine against tuberculosis affords a variable protection which wanes with time. In this study we have analyzed and compared the proteins which are expressed differentially during broth-culture and intraphagosomal growth of M.bovis BCG. Eight proteins which showed increased expression during the intraphagosomal growth were identified by MALDI-TOF/MS. These were - a precursor of alanine and proline-rich secreted protein apa, isoforms of malate dehydrogenase, large subunit alpha (Alpha-ETF) of electron transfer flavoprotein, immunogenic protein MPB64 precursor, UPF0036 protein, and two proteins with unknown function. Based on these findings we speculate that higher expression of these proteins has a probable role in intracellular survival, adaptation and/or immunoprotective effect of BCG. Further, these proteins might also be used as gene expression markers for endosome trafficking events of BCG.

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.

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

Effect of salivary pellicle on antibacterial activity of novel antibacterial dental adhesives using a dental plaque microcosm biofilm model

PubMed Central

Li, Fang; Weir, Michael D.; Fouad, Ashraf F.; Xu, Hockin H.K.

2014-01-01

Objectives Antibacterial primer and adhesive are promising to inhibit biofilms and caries. Since restorations in vivo are exposed to saliva, one concern is the attenuation of antibacterial activity due to salivary pellicles. The objective of this study was to investigate the effects of salivary pellicles on bonding agents containing a new monomer dimethylaminododecyl methacrylate (DMADDM) or nanoparticles of silver (NAg) against biofilms for the first time. Methods DMADDM and NAg were synthesized and incorporated into Scotchbond Multi-Purpose adhesive and primer. Specimens were either coated or not coated with salivary pellicles. A microcosm biofilm model was used with mixed saliva from ten donors. Two types of culture medium were used: an artificial saliva medium (McBain), and Brain Heart Infusion (BHI) medium without salivary proteins. Metabolic activity, colony-forming units (CFU), and lactic acid production of plaque microcosm biofilms were measured (n = 6). Results Bonding agents containing DMADDM and NAg greatly inhibited biofilm activities, even with salivary pellicles. When using BHI, the pre-coating of salivary pellicles on resin surfaces significantly decreased the antibacterial effect (p < 0.05). When using artificial saliva medium, pre-coating of salivary pellicles on resin did not decrease the antibacterial effect. These results suggest that artificial saliva yielded medium-derived pellicles on resin surfaces, which provided attenuating effects on biofilms similar to salivary pellicles. Compared with the commercial control, the DMADDM-containing bonding agent reduced biofilm CFU by about two orders of magnitude. Significance Novel DMADDM- and NAg-containing bonding agents substantially reduced biofilm growth even with salivary pellicle coating on surfaces, indicating a promising usage in saliva-rich environment. DMADDM and NAg may be useful in a wide range of primers, adhesives and other restoratives to achieve antibacterial and anti-caries capabilities. PMID:24332270

Effect of salivary pellicle on antibacterial activity of novel antibacterial dental adhesives using a dental plaque microcosm biofilm model.

PubMed

Li, Fang; Weir, Michael D; Fouad, Ashraf F; Xu, Hockin H K

2014-02-01

Antibacterial primer and adhesive are promising to inhibit biofilms and caries. Since restorations in vivo are exposed to saliva, one concern is the attenuation of antibacterial activity due to salivary pellicles. The objective of this study was to investigate the effects of salivary pellicles on bonding agents containing a new monomer dimethylaminododecyl methacrylate (DMADDM) or nanoparticles of silver (NAg) against biofilms for the first time. DMADDM and NAg were synthesized and incorporated into Scotchbond Multi-Purpose adhesive and primer. Specimens were either coated or not coated with salivary pellicles. A microcosm biofilm model was used with mixed saliva from ten donors. Two types of culture medium were used: an artificial saliva medium (McBain), and Brain Heart Infusion (BHI) medium without salivary proteins. Metabolic activity, colony-forming units (CFU), and lactic acid production of plaque microcosm biofilms were measured (n=6). Bonding agents containing DMADDM and NAg greatly inhibited biofilm activities, even with salivary pellicles. When using BHI, the pre-coating of salivary pellicles on resin surfaces significantly decreased the antibacterial effect (p<0.05). When using artificial saliva medium, pre-coating of salivary pellicles on resin did not decrease the antibacterial effect. These results suggest that artificial saliva yielded medium-derived pellicles on resin surfaces, which provided attenuating effects on biofilms similar to salivary pellicles. Compared with the commercial control, the DMADDM-containing bonding agent reduced biofilm CFU by about two orders of magnitude. Novel DMADDM- and NAg-containing bonding agents substantially reduced biofilm growth even with salivary pellicle coating on surfaces, indicating a promising usage in saliva-rich environment. DMADDM and NAg may be useful in a wide range of primers, adhesives and other restoratives to achieve antibacterial and anti-caries capabilities. Published by Elsevier Ltd.

Bridge over troubled proline: assignment of intrinsically disordered proteins using (HCA)CON(CAN)H and (HCA)N(CA)CO(N)H experiments concomitantly with HNCO and i(HCA)CO(CA)NH.

PubMed

Hellman, Maarit; Piirainen, Henni; Jaakola, Veli-Pekka; Permi, Perttu

2014-01-01

NMR spectroscopy is by far the most versatile and information rich technique to study intrinsically disordered proteins (IDPs). While NMR is able to offer residue level information on structure and dynamics, assignment of chemical shift resonances in IDPs is not a straightforward process. Consequently, numerous pulse sequences and assignment protocols have been developed during past several years, targeted especially for the assignment of IDPs, including experiments that employ H(N), H(α) or (13)C detection combined with two to six indirectly detected dimensions. Here we propose two new HN-detection based pulse sequences, (HCA)CON(CAN)H and (HCA)N(CA)CO(N)H, that provide correlations with (1)H(N)(i - 1), (13)C'(i - 1) and (15)N(i), and (1)H(N)(i + 1), (13)C'(i) and (15)N(i) frequencies, respectively. Most importantly, they offer sequential links across the proline bridges and enable filling the single proline gaps during the assignment. We show that the novel experiments can efficiently complement the information available from existing HNCO and intraresidual i(HCA)CO(CA)NH pulse sequences and their concomitant usage enabled >95 % assignment of backbone resonances in cytoplasmic tail of adenosine receptor A2A in comparison to 73 % complete assignment using the HNCO/i(HCA)CO(CA)NH data alone.

Effect of mobile phone use on salivary concentrations of protein, amylase, lipase, immunoglobulin A, lysozyme, lactoferrin, peroxidase and C-reactive protein of the parotid gland.

PubMed

Hashemipour, M S; Yarbakht, M; Gholamhosseinian, A; Famori, H

2014-05-01

The possibility of side effects associated with the electromagnetic waves emitted from mobile phones is a controversial issue. The present study aimed to evaluate the effect of mobile phone use on parotid gland salivary concentrations of protein, amylase, lipase, immunoglobulin A, lysozyme, lactoferrin, peroxidase and C-reactive protein. Stimulated salivary samples were collected simultaneously from both parotid glands of 86 healthy volunteers. Salivary flow rate and salivary concentrations of proteins, amylase, lipase, lysozyme, lactoferrin, peroxidase, C-reactive protein and immunoglobulin A, were measured. Data were analysed using t-tests and one-way analyses of variance. Salivary flow rate and parotid gland salivary concentrations of protein were significantly higher on the right side compared to the left in those that predominantly held mobile phones on the right side. In addition, there was a decrease in concentrations of amylase, lipase, lysozyme, lactoferrin and peroxidase. The side of dominant mobile phone use was associated with differences in salivary flow rate and parotid gland salivary concentrations, in right-dominant users. Although mobile phone use influenced salivary composition, the relationship was not significant.

Tick salivary secretion as a source of antihemostatics

PubMed Central

Chmelar, Jindrich; Calvo, Eric; Pedra, Joao H.F.; Francischetti, Ivo M. B.; Kotsyfakis, Michail

2012-01-01

Ticks are mostly obligatory blood feeding ectoparasites that have an impact on human and animal health. In addition to direct damage due to feeding, some tick species serve as the vectors for the causative agents of several diseases, such as the spirochetes of the genus Borrelia causing Lyme disease, the virus of tick-borne encephalitis, various Rickettsial pathogens or even protozoan parasites like Babesia spp. Hard ticks are unique among bloodfeeders because of their prolonged feeding period that may last up to two weeks. During such a long period of blood uptake, the host develops a wide range of mechanisms to prevent blood loss. The arthropod ectoparasite, in turn, secretes saliva in the sites of bite that assists blood feeding. Indeed, tick saliva represents a rich source of proteins with potent pharmacologic action that target different mechanisms of coagulation, platelet aggregation and vasoconstriction. Tick adaptation to their vertebrate hosts led to the inclusion of a powerful protein armamentarium in their salivary secretion that has been investigated by high throughput methods. The resulting knowledge can be exploited for the isolation of novel antihemostatic agents. Here we review the tick salivary antihemostatics and their characterized functions at the molecular and cellular levels. PMID:22564820

Expression, Refolding and Crystallizations of the Grb2-like (GADS) C-Terminal SH3 Domain Complexed with a SLP-76 Motif Peptide

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

Faravelli,A.; Dimasi, N.

The Grb2-like adaptor protein GADS is composed of an N-terminal SH3 domain, an SH2 domain, a proline-rich region and a C-terminal SH3 domain. GADS interacts through its C-terminal SH3 domain with the adaptor protein SLP-76, thus recruiting this protein and other associated molecules to the linker for activation of T-cell (LAT) protein. The DNA encoding the C-terminal SH3 domain of GADS (GADS-cSH3) was assembled synthetically using a recursive PCR technique and the protein was overexpressed in Escherichia coli, refolded and purified. Several crystals of this domain in complex with the SLP-76 peptide were obtained and characterized.

Comparative Transcriptome Analysis of Salivary Glands of Two Populations of Rice Brown Planthopper, Nilaparvata lugens, That Differ in Virulence

PubMed Central

Chen, Hongdan; Ye, Wenfeng; Li, Shaohui; Lou, Yonggen

2013-01-01

Background The brown planthopper (BPH), Nilaparvata lugens (Stål), a destructive rice pest in Asia, can quickly overcome rice resistance by evolving new virulent populations. Herbivore saliva plays an important role in plant–herbivore interactions, including in plant defense and herbivore virulence. However, thus far little is known about BPH saliva at the molecular level, especially its role in virulence and BPH–rice interaction. Methodology/Principal Findings Using cDNA amplification in combination with Illumina short-read sequencing technology, we sequenced the salivary-gland transcriptomes of two BPH populations with different virulence; the populations were derived from rice variety TN1 (TN1 population) and Mudgo (M population). In total, 37,666 and 38,451 unigenes were generated from the salivary glands of these populations, respectively. When combined, a total of 43,312 unigenes were obtained, about 18 times more than the number of expressed sequence tags previously identified from these glands. Gene ontology annotations and KEGG orthology classifications indicated that genes related to metabolism, binding and transport were significantly active in the salivary glands. A total of 352 genes were predicted to encode secretory proteins, and some might play important roles in BPH feeding and BPH–rice interactions. Comparative analysis of the transcriptomes of the two populations revealed that the genes related to ‘metabolism,’ ‘digestion and absorption,’ and ‘salivary secretion’ might be associated with virulence. Moreover, 67 genes encoding putative secreted proteins were differentially expressed between the two populations, suggesting these genes may contribute to the change in virulence. Conclusions/Significance This study was the first to compare the salivary-gland transcriptomes of two BPH populations having different virulence traits and to find genes that may be related to this difference. Our data provide a rich molecular resource for future functional studies on salivary glands and will be useful for elucidating the molecular mechanisms underlying BPH feeding and virulence differences. PMID:24244529

Comparative transcriptome analysis of salivary glands of two populations of rice brown planthopper, Nilaparvata lugens, that differ in virulence.

PubMed

Ji, Rui; Yu, Haixin; Fu, Qiang; Chen, Hongdan; Ye, Wenfeng; Li, Shaohui; Lou, Yonggen

2013-01-01

The brown planthopper (BPH), Nilaparvata lugens (Stål), a destructive rice pest in Asia, can quickly overcome rice resistance by evolving new virulent populations. Herbivore saliva plays an important role in plant-herbivore interactions, including in plant defense and herbivore virulence. However, thus far little is known about BPH saliva at the molecular level, especially its role in virulence and BPH-rice interaction. Using cDNA amplification in combination with Illumina short-read sequencing technology, we sequenced the salivary-gland transcriptomes of two BPH populations with different virulence; the populations were derived from rice variety TN1 (TN1 population) and Mudgo (M population). In total, 37,666 and 38,451 unigenes were generated from the salivary glands of these populations, respectively. When combined, a total of 43,312 unigenes were obtained, about 18 times more than the number of expressed sequence tags previously identified from these glands. Gene ontology annotations and KEGG orthology classifications indicated that genes related to metabolism, binding and transport were significantly active in the salivary glands. A total of 352 genes were predicted to encode secretory proteins, and some might play important roles in BPH feeding and BPH-rice interactions. Comparative analysis of the transcriptomes of the two populations revealed that the genes related to 'metabolism,' 'digestion and absorption,' and 'salivary secretion' might be associated with virulence. Moreover, 67 genes encoding putative secreted proteins were differentially expressed between the two populations, suggesting these genes may contribute to the change in virulence. This study was the first to compare the salivary-gland transcriptomes of two BPH populations having different virulence traits and to find genes that may be related to this difference. Our data provide a rich molecular resource for future functional studies on salivary glands and will be useful for elucidating the molecular mechanisms underlying BPH feeding and virulence differences.

1H and 15N NMR resonance assignments and secondary structure of titin type I domains.

PubMed

Muhle-Goll, C; Nilges, M; Pastore, A

1997-01-01

Titin/connectin is a giant muscle protein with a highly modular architecture consisting of multiple repeats of two sequence motifs, named type I and type II. Type I modules have been suggested to be intracellular members of the fibronectin type III (Fn3) domain family. Along the titin sequence they are exclusively present in the region of the molecule located in the sarcomere A-band. This region has been shown to interact with myosin and C-protein. One of the most noticeable features of type I modules is that they are particularly rich in semiconserved prolines, since these residues account for about 8% of their sequence. We have determined the secondary structure of a representative type I domain (A71) by 15N and 1H NMR. We show that the type I domains of titin have the Fn3 fold as proposed, consisting of a three- and a four-stranded beta-sheet. When the two sheets are placed on top of each other to form the beta-sandwich characteristic of the Fn3 fold, 8 out of 10 prolines are found on the same side of the molecule and form an exposed hydrophobic patch. This suggests that the semiconserved prolines might be relevant for the function of type I modules, providing a surface for binding to other A-band proteins. The secondary structure of A71 was structurally aligned to other extracellular Fn3 modules of known 3D structure. The alignment shows that titin type I modules have closest similarity to the first Fn3 domain of Drosophila neuroglian.

Antisense Proline-Arginine RAN dipeptides linked to C9ORF72-ALS/FTD form toxic nuclear aggregates that initiate in vitro and in vivo neuronal death

PubMed Central

Wen, Xinmei; Tan, Wenzhi; Westergard, Thomas; Krishnamurthy, Karthik; ShamamandriMarkandaiah, Shashirekha; Shi, Yingxiao; Lin, Shaoyu; Shneider, Neil A.; Monaghan, John; Pandey, Udai B.; Pasinelli, Piera; Ichida, Justin K.; Trotti, Davide

2015-01-01

SUMMARY Expanded GGGGCC nucleotide repeats within the C9ORF72 gene are the most common genetic mutation associated with both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Sense and antisense transcripts of these expansions are translated to form five dipeptide repeat proteins (DRPs). We employed primary cortical and motor neuron cultures, live-cell imaging, and transgenic fly models and found that the arginine-rich dipeptides, in particular Proline-Arginine (PR), are potently neurotoxic. Factors that anticipated their neurotoxicity included aggregation in nucleoli, decreased number of processing bodies, and stress granules formation, implying global translational dysregulation as path accountable for toxicity. Nuclear PR aggregates were also found in human-induced motor neurons and postmortem spinal cord tissues from C9ORF72 ALS and ALS/FTD patients. Intronic G4C2 transcripts, but not loss of C9ORF72 protein, are also toxic to motor and cortical neurons. Interestingly, G4C2 transcript-mediated neurotoxicity synergizes with that of PR aggregates, suggesting convergence of mechanisms. PMID:25521377

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

Structural Characterization of Proline-rich Tyrosine Kinase 2 (PYK2) Reveals a Unique (DFG-out) Conformation and Enables Inhibitor Design

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

Han, Seungil; Mistry, Anil; Chang, Jeanne S.

Proline-rich tyrosine kinase 2 (PYK2) is a cytoplasmic, non-receptor tyrosine kinase implicated in multiple signaling pathways. It is a negative regulator of osteogenesis and considered a viable drug target for osteoporosis treatment. The high-resolution structures of the human PYK2 kinase domain with different inhibitor complexes establish the conventional bilobal kinase architecture and show the conformational variability of the DFG loop. The basis for the lack of selectivity for the classical kinase inhibitor, PF-431396, within the FAK family is explained by our structural analyses. Importantly, the novel DFG-out conformation with two diarylurea inhibitors (BIRB796, PF-4618433) reveals a distinct subclass of non-receptormore » tyrosine kinases identifiable by the gatekeeper Met-502 and the unique hinge loop conformation of Leu-504. This is the first example of a leucine residue in the hinge loop that blocks the ATP binding site in the DFG-out conformation. Our structural, biophysical, and pharmacological studies suggest that the unique features of the DFG motif, including Leu-504 hinge-loop variability, can be exploited for the development of selective protein kinase inhibitors.« less

Mass Spectrometry Based Proteomic Analysis of Salivary Glands of Urban Malaria Vector Anopheles stephensi

PubMed Central

Vijay, Sonam

2014-01-01

Salivary gland proteins of Anopheles mosquitoes offer attractive targets to understand interactions with sporozoites, blood feeding behavior, homeostasis, and immunological evaluation of malaria vectors and parasite interactions. To date limited studies have been carried out to elucidate salivary proteins of An. stephensi salivary glands. The aim of the present study was to provide detailed analytical attributives of functional salivary gland proteins of urban malaria vector An. stephensi. A proteomic approach combining one-dimensional electrophoresis (1DE), ion trap liquid chromatography mass spectrometry (LC/MS/MS), and computational bioinformatic analysis was adopted to provide the first direct insight into identification and functional characterization of known salivary proteins and novel salivary proteins of An. stephensi. Computational studies by online servers, namely, MASCOT and OMSSA algorithms, identified a total of 36 known salivary proteins and 123 novel proteins analysed by LC/MS/MS. This first report describes a baseline proteomic catalogue of 159 salivary proteins belonging to various categories of signal transduction, regulation of blood coagulation cascade, and various immune and energy pathways of An. stephensi sialotranscriptome by mass spectrometry. Our results may serve as basis to provide a putative functional role of proteins in concept of blood feeding, biting behavior, and other aspects of vector-parasite host interactions for parasite development in anopheline mosquitoes. PMID:25126571

Mass spectrometry based proteomic analysis of salivary glands of urban malaria vector Anopheles stephensi.

PubMed

Vijay, Sonam; Rawat, Manmeet; Sharma, Arun

2014-01-01

Salivary gland proteins of Anopheles mosquitoes offer attractive targets to understand interactions with sporozoites, blood feeding behavior, homeostasis, and immunological evaluation of malaria vectors and parasite interactions. To date limited studies have been carried out to elucidate salivary proteins of An. stephensi salivary glands. The aim of the present study was to provide detailed analytical attributives of functional salivary gland proteins of urban malaria vector An. stephensi. A proteomic approach combining one-dimensional electrophoresis (1DE), ion trap liquid chromatography mass spectrometry (LC/MS/MS), and computational bioinformatic analysis was adopted to provide the first direct insight into identification and functional characterization of known salivary proteins and novel salivary proteins of An. stephensi. Computational studies by online servers, namely, MASCOT and OMSSA algorithms, identified a total of 36 known salivary proteins and 123 novel proteins analysed by LC/MS/MS. This first report describes a baseline proteomic catalogue of 159 salivary proteins belonging to various categories of signal transduction, regulation of blood coagulation cascade, and various immune and energy pathways of An. stephensi sialotranscriptome by mass spectrometry. Our results may serve as basis to provide a putative functional role of proteins in concept of blood feeding, biting behavior, and other aspects of vector-parasite host interactions for parasite development in anopheline mosquitoes.

Nitrogen reserves, spring regrowth and winter survival of field-grown alfalfa (Medicago sativa) defoliated in the autumn.

PubMed

Dhont, Catherine; Castonguay, Yves; Nadeau, Paul; Bélanger, Gilles; Drapeau, Raynald; Laberge, Serge; Avice, Jean-Christophe; Chalifour, François-P

2006-01-01

The objective of the study was to characterize variations in proline, arginine, histidine, vegetative storage proteins, and cold-inducible gene expression in overwintering roots of field-grown alfalfa, in response to autumn defoliation, and in relation to spring regrowth and winter survival. Field trials, established in 1996 in eastern Canada, consisted of two alfalfa cultivars ('AC Caribou' and 'WL 225') defoliated in 1997 and 1998 either only twice during the summer or three times with the third defoliation taken 400, 500 or 600 growing degree days (basis 5 degrees C) after the second summer defoliation. The root accumulation of proline, arginine, histidine and soluble proteins of 32, 19 and 15 kDa, characterized as alfalfa vegetative storage proteins, was reduced the following spring by an early autumn defoliation at 400 or 500 growing degree days in both cultivars; the 600-growing-degree-days defoliation treatment had less or no effect. Transcript levels of the cold-inducible gene msaCIA, encoding a glycine-rich protein, were markedly reduced by autumn defoliation in 'WL 225', but remained unaffected in the more winter-hardy cultivar 'AC Caribou'. The expression of another cold-inducible gene, the dehydrin homologue msaCIG, was not consistently affected by autumn defoliation. Principal component analyses, including components of root organic reserves at the onset of winter, along with yield and plant density in the following spring, revealed that (a) amino acids and soluble proteins are positively related to the vigour of spring regrowth but poorly related to winter survival and (b) winter survival, as indicated by plant density in the spring, is associated with higher concentrations of cryoprotective sugars in alfalfa roots the previous autumn. An untimely autumn defoliation of alfalfa reduces root accumulation of specific N reserves such as proline, arginine, histidine and vegetative storage proteins that are positively related to the vigour of spring regrowth but poorly related to winter survival.

Salivary protein concentration, flow rate, buffer capacity and pH estimation: A comparative study among young and elderly subjects, both normal and with gingivitis and periodontitis.

PubMed

Shaila, Mulki; Pai, G Prakash; Shetty, Pushparaj

2013-01-01

To evaluate the salivary protein concentration in gingivitis and periodontitis patients and compare the parameters like salivary total protein, salivary albumin, salivary flow rate, pH, buffer capacity and flow rate in both young and elderly patients with simple methods. One hundred and twenty subjects were grouped based on their age as young and elderly. Each group was subgrouped (20 subjects) as controls, gingivitis and periodontitis. Unstimulated whole saliva was collected from patients and flow rate was noted down during collection of the sample. Salivary protein estimation was done using the Biuret method and salivary albumin was assessed using the Bromocresol green method. pH was estimated with a pHmeter and buffering capacity was analyzed with the titration method. Student's t-test, Fisher's test (ANOVA) and Tukey HSD (ANOVA) tests were used for statistical analysis. A very highly significant rise in the salivary total protein and albumin concentration was noted in gingivitis and periodontitis subjects of both young and elderly. An overall decrease in salivary flow rate was observed among the elderly, and also the salivary flow rate of women was significantly lower than that of men. Significant associations between salivary total protein and albumin in gingivitis and periodontitis were found with simple biochemical tests. A decrease in salivary flow rate among elderly and among women was noted.

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

Immune recognition of salivary proteins from the cattle tick Rhipicephalus microplus differs according to the genotype of the bovine host.

PubMed

Garcia, Gustavo Rocha; Maruyama, Sandra Regina; Nelson, Kristina T; Ribeiro, José Marcos Chaves; Gardinassi, Luiz Gustavo; Maia, Antonio Augusto Mendes; Ferreira, Beatriz Rossetti; Kooyman, Frans N J; de Miranda Santos, Isabel K F

2017-03-14

Males of the cattle tick Rhipicephalus microplus produce salivary immunoglobulin-binding proteins and allotypic variations in IgG are associated with tick loads in bovines. These findings indicate that antibody responses may be essential to control tick infestations. Infestation loads with cattle ticks are heritable: some breeds carry high loads of reproductively successful ticks, in others, few ticks feed and they reproduce inefficiently. Different patterns of humoral immunity against tick salivary proteins may explain these phenotypes. We describe the profiles of humoral responses against tick salivary proteins elicited during repeated artificial infestations of bovines of a tick-resistant (Nelore) and a tick-susceptible (Holstein) breed. We measured serum levels of total IgG1, IgG2 and IgE immunoglobulins and of IgG1 and IgG2 antibodies specific for tick salivary proteins. With liquid chromatography followed by mass spectrometry we identified tick salivary proteins that were differentially recognized by serum antibodies from tick-resistant and tick-susceptible bovines in immunoblots of tick salivary proteins separated by two-dimensional electrophoresis. Baseline levels of total IgG1 and IgG2 were significantly higher in tick-susceptible Holsteins compared with resistant Nelores. Significant increases in levels of total IgG1, but not of IgG2 accompanied successive infestations in both breeds. Resistant Nelores presented with significantly higher levels of salivary-specific antibodies before and at the first challenge with tick larvae; however, by the third challenge, tick-susceptible Holsteins presented with significantly higher levels of IgG1 and IgG2 tick salivary protein-specific antibodies. Importantly, sera from tick-resistant Nelores reacted with 39 tick salivary proteins in immunoblots of salivary proteins separated in two dimensions by electrophoresis versus only 21 spots reacting with sera from tick-susceptible Holsteins. Levels of tick saliva-specific antibodies were not directly correlated with infestation phenotypes. However, in spite of receiving apparently lower amounts of tick saliva, tick-resistant bovines recognized more tick salivary proteins. These reactive salivary proteins are putatively involved in several functions of parasitism and blood-feeding. Our results indicate that neutralization by host antibodies of tick salivary proteins involved in parasitism is essential to control tick infestations.

In Vitro Identification of Histatin 5 Salivary Complexes

PubMed Central

Moffa, Eduardo B.; Machado, Maria A. A. M.; Mussi, Maria C. M.; Xiao, Yizhi; Garrido, Saulo S.; Giampaolo, Eunice T.; Siqueira, Walter L.

2015-01-01

With recent progress in the analysis of the salivary proteome, the number of salivary proteins identified has increased dramatically. However, the physiological functions of many of the newly discovered proteins remain unclear. Closely related to the study of a protein’s function is the identification of its interaction partners. Although in saliva some proteins may act primarily as single monomeric units, a significant percentage of all salivary proteins, if not the majority, appear to act in complexes with partners to execute their diverse functions. Coimmunoprecipitation (Co-IP) and pull-down assays were used to identify the heterotypic complexes between histatin 5, a potent natural antifungal protein, and other salivary proteins in saliva. Classical protein–protein interaction methods in combination with high-throughput mass spectrometric techniques were carried out. Co-IP using protein G magnetic Sepharose TM beads suspension was able to capture salivary complexes formed between histatin 5 and its salivary protein partners. Pull-down assay was used to confirm histatin 5 protein partners. A total of 52 different proteins were identified to interact with histatin 5. The present study used proteomic approaches in conjunction with classical biochemical methods to investigate protein–protein interaction in human saliva. Our study demonstrated that when histatin 5 is complexed with salivary amylase, one of the 52 proteins identified as a histatin 5 partner, the antifungal activity of histatin 5 is reduced. We expected that our proteomic approach could serve as a basis for future studies on the mechanism and structural-characterization of those salivary protein interactions to understand their clinical significance. PMID:26544073

Chemistry of gluten proteins.

PubMed

Wieser, Herbert

2007-04-01

Gluten proteins play a key role in determining the unique baking quality of wheat by conferring water absorption capacity, cohesivity, viscosity and elasticity on dough. Gluten proteins can be divided into two main fractions according to their solubility in aqueous alcohols: the soluble gliadins and the insoluble glutenins. Both fractions consist of numerous, partially closely related protein components characterized by high glutamine and proline contents. Gliadins are mainly monomeric proteins with molecular weights (MWs) around 28,000-55,000 and can be classified according to their different primary structures into the alpha/beta-, gamma- and omega-type. Disulphide bonds are either absent or present as intrachain crosslinks. The glutenin fraction comprises aggregated proteins linked by interchain disulphide bonds; they have a varying size ranging from about 500,000 to more than 10 million. After reduction of disulphide bonds, the resulting glutenin subunits show a solubility in aqueous alcohols similar to gliadins. Based on primary structure, glutenin subunits have been divided into the high-molecular-weight (HMW) subunits (MW=67,000-88,000) and low-molecular-weight (LMW) subunits (MW=32,000-35,000). Each gluten protein type consists or two or three different structural domains; one of them contains unique repetitive sequences rich in glutamine and proline. Native glutenins are composed of a backbone formed by HMW subunit polymers and of LMW subunit polymers branched off from HMW subunits. Non-covalent bonds such as hydrogen bonds, ionic bonds and hydrophobic bonds are important for the aggregation of gliadins and glutenins and implicate structure and physical properties of dough.

Exploring the Roles of Proline in Three-Dimensional Domain Swapping from Structure Analysis and Molecular Dynamics Simulations.

PubMed

Huang, Yongqi; Gao, Meng; Su, Zhengding

2018-02-01

Three-dimensional (3D) domain swapping is a mechanism to form protein oligomers. It has been proposed that several factors, including proline residues in the hinge region, may affect the occurrence of 3D domain swapping. Although introducing prolines into the hinge region has been found to promote domain swapping for some proteins, the opposite effect has also been observed in several studies. So far, how proline affects 3D domain swapping remains elusive. In this work, based on a large set of 3D domain-swapped structures, we performed a systematic analysis to explore the correlation between the presence of proline in the hinge region and the occurrence of 3D domain swapping. We further analyzed the conformations of proline and pre-proline residues to investigate the roles of proline in 3D domain swapping. We found that more than 40% of the domain-swapped structures contained proline residues in the hinge region. Unexpectedly, conformational transitions of proline residues were rarely observed upon domain swapping. Our analyses showed that hinge regions containing proline residues preferred more extended conformations, which may be beneficial for the occurrence of domain swapping by facilitating opening of the exchanged segments.

Metallofullerenol Gd@C82(OH)22 distracts the proline-rich-motif from putative binding on the SH3 domain

NASA Astrophysics Data System (ADS)

Kang, Seung-Gu; Huynh, Tien; Zhou, Ruhong

2013-03-01

Biocompatibility is often regarded as one important aspect of de novo designed nanomaterials for biosafety. However, the toxicological effect, appearing along with its latency, is much more difficult to address by linearly mapping physicochemical properties of related nanomaterials with biological effects such as immune or cellular regulatory responses due to the complicated protein-protein interactions. Here, we investigate a potential interference of a metallofullerenol, Gd@C82(OH)22, on the function of SH3 domain, a highly promiscuous protein-protein interaction mediator involved in signaling and regulatory pathways through its binding with the proline-rich motif (PRM) peptides, using the atomistic molecular dynamics simulation. Our study shows that when only Gd@C82(OH)22 and the SH3 domain are present (without the PRM ligand), Gd@C82(OH)22 can interact with the SH3 domain by either directly blocking the hydrophobic active site or binding with a hydrophilic off-site with almost equal probability, which can be understood from its intrinsic amphiphilic nature. In a binding competition with the PRM onto the SH3 domain, however, the on-site binding mode is depleted while Gd@C82(OH)22 effectively intercepts the PRM from the putative binding site of the SH3 domain, implying that Gd@C82(OH)22 can disturb protein-protein interactions mediated by the SH3 domain. Despite a successful surface modification in an aqueous biological medium and a more recent demonstration as potential de novo cancer therapeutics, our study indicates that greater attention is needed in assessing the potential cytotoxicity of these nanomaterials.Biocompatibility is often regarded as one important aspect of de novo designed nanomaterials for biosafety. However, the toxicological effect, appearing along with its latency, is much more difficult to address by linearly mapping physicochemical properties of related nanomaterials with biological effects such as immune or cellular regulatory responses due to the complicated protein-protein interactions. Here, we investigate a potential interference of a metallofullerenol, Gd@C82(OH)22, on the function of SH3 domain, a highly promiscuous protein-protein interaction mediator involved in signaling and regulatory pathways through its binding with the proline-rich motif (PRM) peptides, using the atomistic molecular dynamics simulation. Our study shows that when only Gd@C82(OH)22 and the SH3 domain are present (without the PRM ligand), Gd@C82(OH)22 can interact with the SH3 domain by either directly blocking the hydrophobic active site or binding with a hydrophilic off-site with almost equal probability, which can be understood from its intrinsic amphiphilic nature. In a binding competition with the PRM onto the SH3 domain, however, the on-site binding mode is depleted while Gd@C82(OH)22 effectively intercepts the PRM from the putative binding site of the SH3 domain, implying that Gd@C82(OH)22 can disturb protein-protein interactions mediated by the SH3 domain. Despite a successful surface modification in an aqueous biological medium and a more recent demonstration as potential de novo cancer therapeutics, our study indicates that greater attention is needed in assessing the potential cytotoxicity of these nanomaterials. Electronic supplementary information (ESI) available. See DOI: 10.1039/c3nr33756a

Chemical shift assignments of the partially deuterated Fyn SH2-SH3 domain.

PubMed

Kieken, Fabien; Loth, Karine; van Nuland, Nico; Tompa, Peter; Lenaerts, Tom

2018-04-01

Src Homology 2 and 3 (SH2 and SH3) are two key protein interaction modules involved in regulating the activity of many proteins such as tyrosine kinases and phosphatases by respective recognition of phosphotyrosine and proline-rich regions. In the Src family kinases, the inactive state of the protein is the direct result of the interaction of the SH2 and the SH3 domain with intra-molecular regions, leading to a closed structure incompetent with substrate modification. Here, we report the 1 H, 15 N and 13 C backbone- and side-chain chemical shift assignments of the partially deuterated Fyn SH3-SH2 domain and structural differences between tandem and single domains. The BMRB accession number is 27165.

Updating the Salivary Gland Transcriptome of Phlebotomus papatasi (Tunisian Strain): The Search for Sand Fly-Secreted Immunogenic Proteins for Humans

PubMed Central

Ben Ahmed, Melika; Zhioua, Elyes; Chelbi, Ifhem; Cherni, Saifedine; Louzir, Hechmi; Ribeiro, José M. C.; Valenzuela, Jesus G.

2012-01-01

Introduction Sand fly saliva plays an important role in both blood feeding and outcome of Leishmania infection. A cellular immune response against a Phlebotomus papatasi salivary protein was shown to protect rodents against Leishmania major infection. In humans, P. papatasi salivary proteins induce a systemic cellular immune response as well as a specific antisaliva humoral immune response, making these salivary proteins attractive targets as markers of exposure for this Leishmania vector. Surprisingly, the repertoire of salivary proteins reported for P. papatasi–a model sand fly for Leishmania-vector-host molecular interactions–is very limited compared with other sand fly species. We hypothesize that a more comprehensive study of the transcripts present in the salivary glands of P. papatasi will provide better knowledge of the repertoire of proteins of this important vector and will aid in selection of potential immunogenic proteins for humans and of those proteins that are highly conserved between different sand fly strains. Methods and Findings A cDNA library from P. papatasi (Tunisian strain) salivary glands was constructed, and randomly selected transcripts were sequenced and analyzed. The most abundant transcripts encoding secreted proteins were identified and compared with previously reported sequences. Importantly, we identified salivary proteins not described before in this sand fly species. Conclusions Comparative analysis between the salivary proteins of P. papatasi from Tunisia and Israel strains shows a high level of identity, suggesting these proteins as potential common targets for markers of vector exposure or inducers of cellular immune responses in humans for different geographic areas. PMID:23139741

Updating the salivary gland transcriptome of Phlebotomus papatasi (Tunisian strain): the search for sand fly-secreted immunogenic proteins for humans.

PubMed

Abdeladhim, Maha; Jochim, Ryan C; Ben Ahmed, Melika; Zhioua, Elyes; Chelbi, Ifhem; Cherni, Saifedine; Louzir, Hechmi; Ribeiro, José M C; Valenzuela, Jesus G

2012-01-01

Sand fly saliva plays an important role in both blood feeding and outcome of Leishmania infection. A cellular immune response against a Phlebotomus papatasi salivary protein was shown to protect rodents against Leishmania major infection. In humans, P. papatasi salivary proteins induce a systemic cellular immune response as well as a specific antisaliva humoral immune response, making these salivary proteins attractive targets as markers of exposure for this Leishmania vector. Surprisingly, the repertoire of salivary proteins reported for P. papatasi-a model sand fly for Leishmania-vector-host molecular interactions-is very limited compared with other sand fly species. We hypothesize that a more comprehensive study of the transcripts present in the salivary glands of P. papatasi will provide better knowledge of the repertoire of proteins of this important vector and will aid in selection of potential immunogenic proteins for humans and of those proteins that are highly conserved between different sand fly strains. A cDNA library from P. papatasi (Tunisian strain) salivary glands was constructed, and randomly selected transcripts were sequenced and analyzed. The most abundant transcripts encoding secreted proteins were identified and compared with previously reported sequences. Importantly, we identified salivary proteins not described before in this sand fly species. Comparative analysis between the salivary proteins of P. papatasi from Tunisia and Israel strains shows a high level of identity, suggesting these proteins as potential common targets for markers of vector exposure or inducers of cellular immune responses in humans for different geographic areas.

New procyanidin B3-human salivary protein complexes by mass spectrometry. Effect of salivary protein profile, tannin concentration, and time stability.

PubMed

Perez-Gregorio, Maria Rosa; Mateus, Nuno; De Freitas, Victor

2014-10-15

Several factors could influence the tannin-protein interaction such as the human salivary protein profile, the tannin tested, and the tannin/protein ratio. The goal of this study aims to study the effect of different salivas (A, B, and C) and different tannin concentrations (0.5 and 1 mg/mL) on the interaction process as well as the complex's stability over time. This study is focused on the identification of new procyanidin B3-human salivary protein complexes. Thus, 48 major B3-human salivary protein aggregates were identified regardless of the saliva and tannin concentration tested. A higher number of aggregates was found at lower tannin concentration. Moreover, the number of protein moieties involved in the aggregation process was higher when the tannin concentration was also higher. The selectivity of the different groups of proteins to bind tannin was also confirmed. It was also verified that the B3-human salivary protein complexes formed evolved over time.

Tropical tannin-rich fodder intake modifies saliva-binding capacity in growing sheep.

PubMed

Vargas-Magaña, J J; Aguilar-Caballero, A J; Torres-Acosta, J F J; Sandoval-Castro, C A; Hoste, H; Capetillo-Leal, C M

2013-12-01

We evaluated the effect of feeding dietary tannins from Lysiloma latisiliquum fresh forage on the saliva tannin-binding capacity of hair sheep lambs without previous exposure to tannin-rich (TR) fodder. Twenty-four hair sheep lambs (13.6±3.04 kg LW) were fed a tannin-free diet at the beginning of the experimental period (from day 10 to 13). On day 14, lambs were distributed into three groups (n=8): control group (CG), fed with the tannin-free diet (from D10 to D112); tannin short-term group (TST), fed the basal diet and 650 g of L. latisiliquum forage (from D14 to D55); tannin long-term group (TLT), fed the basal diet and 650 g of L. latisiliquum forage (from D14 to D112). Saliva samples were collected from the mouth of each lamb in the morning before feeding time on D10 and D14 (baseline period), on D49 and D56 (period 1) and on D97 and D112 (period 2). The tannin binding response of salivary protein (∆% turbidity) was determined with the haze development test (HDT) using either tannic acid or L. latisiliquum forage acetone extract. A turbidity protein index (TPI) was calculated as (∆% turbidity/[salivary protein (mg)]). Differences in HDT and TPI in the different groups were compared by repeated measures ANOVA using Proc Mixed. All groups had similar ∆% turbidity throughout the experiment (P>0.05). At baseline and period 1, the TPI of the different groups was similar (P>0.05). On period 2 the TLT group showed higher TPI compared with CG (P<0.05). Meanwhile, CG and TST showed similar salivary TPI. The saliva of hair sheep lambs consuming TR L. latisiliquum fresh fodder (TLT group) increased their TPI compared with control lambs not exposed to tannins.

The salivary glands of two sand fly vectors of Leishmania: Lutzomyia migonei (França) and Lutzomyia ovallesi (Ortiz)(Diptera: Psychodidae).

PubMed

Nieves, Elsa; Buelvas, Neudo; Rondón, Maritza; González, Néstor

2010-01-01

Leishmaniasis is a vector-borne disease transmitted by the intradermal inoculation of Leishmania (Kinetoplastida: Trypanosomatidae) promastigotes together with saliva during the bite of an infected sand fly. The salivary glands were compared from two vector species, Lutzomyia ovallesi (Ortiz,1952) and Lutzomyia migonei (França,1920) (Diptera: Psychodidae). Protein profiles by SDS PAGE of salivary glands were compared among species as well as their development at several times post feeding. First, mice were immunized to salivary proteins by exposure to biting by L. ovallesi and of L. migonei. Antibodies in these mice against salivary gland-specific proteins were evaluated by immunoblotting. No apparent change was revealed in the kinetic expression of salivary proteins induced by the different physiological states post feeding. Qualitative and quantitative variations were detected in16-18 polypeptides with molecular weights ranging from 6 to 180 kDa. Species-specific proteins were demonstrated for L. migonei and L. ovallesi. In addition, antibodies against salivary gland specific proteins were found in mice immunized by the saliva of both species. Basic information was obtained concerning the nature of salivary gland proteins of L. migonei and L. ovallesi. This information helps to elucidate the role of salivary proteins and their potential as effective tools in screening risk factors in human and other vertebrate hosts.

Numb endocytic adapter proteins regulate the transport and processing of the amyloid precursor protein in an isoform-dependent manner: implications for Alzheimer disease pathogenesis.

PubMed

Kyriazis, George A; Wei, Zelan; Vandermey, Miriam; Jo, Dong-Gyu; Xin, Ouyang; Mattson, Mark P; Chan, Sic L

2008-09-12

Central to the pathogenesis of Alzheimer disease is the aberrant processing of the amyloid precursor protein (APP) to generate amyloid beta-peptide (Abeta), the principle component of amyloid plaques. The cell fate determinant Numb is a phosphotyrosine binding domain (PTB)-containing endocytic adapter protein that interacts with the carboxyl-terminal domain of APP. The physiological relevance of this interaction is unknown. Mammals produce four alternatively spliced variants of Numb that differ in the length of their PTB and proline-rich region. In the current study, we determined the influence of the four human Numb isoforms on the intracellular trafficking and processing of APP. Stable expression of Numb isoforms that differ in the PTB but not in the proline-rich region results in marked differences in the sorting of APP to the recycling and degradative pathways. Neural cells expressing Numb isoforms that lack the insert in the PTB (short PTB (SPTB)) exhibited marked accumulation of APP in Rab5A-labeled early endosomal and recycling compartments, whereas those expressing isoforms with the insertion in the PTB (long PTB (LPTB)) exhibited reduced amounts of cellular APP and its proteolytic derivatives relative to parental control cells. Neither the activities of the beta- and gamma-secretases nor the expression of APP mRNA were significantly different in the stably transfected cells, suggesting that the differential effects of the Numb proteins on APP metabolism is likely to be secondary to altered APP trafficking. In addition, the expression of SPTB-Numb increases at the expense of LPTB-Numb in neuronal cultures subjected to stress, suggesting a role for Numb in stress-induced Abeta production. Taken together, these results suggest distinct roles for the human Numb isoforms in APP metabolism and may provide a novel potential link between altered Numb isoform expression and increased Abeta generation.

Salivary agglutinin/glycoprotein-340/DMBT1: a single molecule with variable composition and with different functions in infection, inflammation and cancer.

PubMed

Ligtenberg, Antoon J M; Veerman, Enno C I; Nieuw Amerongen, Arie V; Mollenhauer, Jan

2007-12-01

Salivary agglutinin (SAG), lung glycoprotein-340 (gp-340) and Deleted in Malignant Brain Tumours 1 (DMBT1) are three names for identical proteins encoded by the dmbt1 gene. DMBT1/SAG/gp-340 belongs to the scavenger receptor cysteine-rich (SRCR) superfamily of proteins, a superfamily of secreted or membrane-bound proteins with SRCR domains that are highly conserved down to sponges, the most ancient metazoa. On the one hand, DMBT1 may represent an innate defence factor acting as a pattern recognition molecule. It interacts with a broad range of pathogens, including cariogenic streptococci and Helicobacter pylori, influenza viruses and HIV, but also with mucosal defence proteins, such as IgA, surfactant proteins and MUC5B. Stimulation of alveolar macrophage migration, suppression of neutrophil oxidative burst and activation of the complement cascade point further to an important role in the regulation of inflammatory responses. On the other hand, DMBT1 has been demonstrated to play a role in epithelial and stem cell differentiation. Inactivation of the gene coding for this protein may lead to disturbed differentiation, possibly resulting in tumour formation. These data strongly point to a role for DMBT1 as a molecule linking innate immune processes with regenerative processes.

Second-site changes affect viability of amphotropic/ecotropic chimeric enveloped murine leukemia viruses.

PubMed

O'Reilly, L; Roth, M J

2000-01-01

Chimeras were previously generated between the ecotropic (Moloney-MuLV) and amphotropic (4070A) SU and TM proteins of murine leukemia virus (MuLV). After passage in D17 cells, three chimeras with junctions in the C terminus of SU (AE5, AE6, and AE7), showed improved kinetics of viral spreading, suggesting that they had adapted. Sequencing of the viruses derived from the D17 cell lines revealed second-site changes within the env gene. Changes were detected in the receptor binding domain, the proline-rich region, the C terminus of SU, and the ectodomain of TM. Second-site changes were subcloned into the parental DNA, singly and in combination, and tested for viability. All viruses had maintained their original cloned mutations and junctions. Reconstruction and passage of AE7 or AE6 virus with single point mutations recovered the additional second-site changes identified in the parental population. The AE5 isolate required changes in the VRA, the VRC, the VRB-hinge region, and the C terminus of SU for efficient infection. Passage of virus, including the parental 4070A, in D17 cells resulted in a predominant G100R mutation within the receptor binding domain. Viruses were subjected to titer determination in three cell types, NIH 3T3, canine D17, and 293T. AE6 viruses with changes in the proline-rich region initially adapted for growth on D17 cells could infect all cell types tested. AE6-based chimeras with additional mutations in the C terminus of SU could infect D17 and 293T cells. Infection of NIH 3T3 cells was dependent on the proline-rich mutation. AE7-based chimeras encoding L538Q and G100R were impaired in infecting NIH 3T3 and 293T cells.

Second-Site Changes Affect Viability of Amphotropic/Ecotropic Chimeric Enveloped Murine Leukemia Viruses

PubMed Central

O'Reilly, Lucille; Roth, Monica J.

2000-01-01

Chimeras were previously generated between the ecotropic (Moloney-MuLV) and amphotropic (4070A) SU and TM proteins of murine leukemia virus (MuLV). After passage in D17 cells, three chimeras with junctions in the C terminus of SU (AE5, AE6, and AE7), showed improved kinetics of viral spreading, suggesting that they had adapted. Sequencing of the viruses derived from the D17 cell lines revealed second-site changes within the env gene. Changes were detected in the receptor binding domain, the proline-rich region, the C terminus of SU, and the ectodomain of TM. Second-site changes were subcloned into the parental DNA, singly and in combination, and tested for viability. All viruses had maintained their original cloned mutations and junctions. Reconstruction and passage of AE7 or AE6 virus with single point mutations recovered the additional second-site changes identified in the parental population. The AE5 isolate required changes in the VRA, the VRC, the VRB-hinge region, and the C terminus of SU for efficient infection. Passage of virus, including the parental 4070A, in D17 cells resulted in a predominant G100R mutation within the receptor binding domain. Viruses were subjected to titer determination in three cell types, NIH 3T3, canine D17, and 293T. AE6 viruses with changes in the proline-rich region initially adapted for growth on D17 cells could infect all cell types tested. AE6-based chimeras with additional mutations in the C terminus of SU could infect D17 and 293T cells. Infection of NIH 3T3 cells was dependent on the proline-rich mutation. AE7-based chimeras encoding L538Q and G100R were impaired in infecting NIH 3T3 and 293T cells. PMID:10623753

The hrp23 Protein in the Balbiani Ring Pre-mRNP Particles Is Released Just before or at the Binding of the Particles to the Nuclear Pore Complex

PubMed Central

Sun, Xin; Alzhanova-Ericsson, Alla T.; Visa, Neus; Aissouni, Youssef; Zhao, Jian; Daneholt, Bertil

1998-01-01

Balbiani ring (BR) pre-mRNP particles reside in the nuclei of salivary glands of the dipteran Chironomus tentans and carry the message for giant-sized salivary proteins. In the present study, we identify and characterize a new protein component in the BR ribonucleoprotein (RNP) particles, designated hrp23. The protein with a molecular mass of 20 kD has a single RNA-binding domain and a glycine-arginine-serine–rich auxiliary domain. As shown by immunoelectron microscopy, the hrp23 protein is added to the BR transcript concomitant with transcription, is still present in the BR particles in the nucleoplasm, but is absent from the BR particles that are bound to the nuclear pore complex or are translocating through the central channel of the complex. Thus, hrp23 is released just before or at the binding of the particles to the nuclear pore complex. It is noted that hrp23 behaves differently from two other BR RNP proteins earlier studied: hrp36 and hrp45. These proteins both reach the nuclear pore complex, and hrp36 even accompanies the RNA into the cytoplasm. It is concluded that each BR RNA-binding protein seems to have a specific flow pattern, probably related to the particular role of the protein in gene expression. PMID:9732280

Genetic variation in small proline rich protein 2B as a predictor for asthma among children with eczema

PubMed Central

Epstein, Tolly G.; LeMasters, Grace K.; Bernstein, David I.; Ericksen, Mark B.; Martin, Lisa J.; Ryan, Patrick H.; Biagini Myers, Jocelyn M.; Butsch Kovacic, Melinda S.; Lindsey, Mark A.; He, Hua; Reponen, Tiina; Villareal, Manuel S.; Lockey, James E.; Bernstein, Cheryl K.; Khurana Hershey, Gurjit K.

2013-01-01

Background Small proline rich protein 2B (SPRR2B) is a skin and lung epithelial protein associated with allergic inflammation in mice that has not been evaluated in human atopic diseases. Objective To determine whether single-nucleotide polymorphisms (SNPs) in SPRR2B are associated with childhood eczema and with the phenotype of childhood eczema combined with asthma. Methods Genotyping for SPRR2B and filaggrin (FLG) was performed in 2 independent populations: the Cincinnati Childhood Allergy & Air Pollution Study (CCAAPS; N = 762; birth-age, 4 years) and the Greater Cincinnati Pediatric Clinical Repository (GCPCR;N = 1152; ages 5–10 years). Eczema and eczema plus asthma were clinical outcomes based on parental report and clinician’s diagnosis. Genetic analyses were restricted to whites and adjusted for sex in both cohorts and adjusted for environmental covariates in CCAAPS. Results Variants in SPRR2B were not significantly associated with eczema in either cohort after Bonferroni adjustment. Children from both cohorts with the CC genotype of the SPRR2B rs6693927 SNP were at 4 times the risk for eczema plus asthma (adjusted odds ratio, 4.1; 95% confidence interval, 1.5– 10.9; P = .005 in CCAAPS; and adjusted odds ratio, 4.0; 95% confidence interval, 1.8 –9.1; P <.001 in the GCPCR), however. SNPs in SPRR2B were not in strong linkage disequilibrium with the R501X and del2282 FLG mutations, and these findings were independent of FLG. Conclusions An SNP in SPRR2B was predictive of asthma among white children with eczema from 2 independent populations. SPRR2B polymorphisms may serve as important predictive markers for the combined eczema plus asthma phenotype. PMID:22374195

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.

Analysis of salivary transcripts and antigens of the sand fly Phlebotomus arabicus

PubMed Central

Hostomská, Jitka; Volfová, Věra; Mu, Jianbing; Garfield, Mark; Rohoušová, Iva; Volf, Petr; Valenzuela, Jesus G; Jochim, Ryan C

2009-01-01

Background Sand fly saliva plays an important role in blood feeding and Leishmania transmission as it was shown to increase parasite virulence. On the other hand, immunity to salivary components impedes the establishment of infection. Therefore, it is most desirable to gain a deeper insight into the composition of saliva in sand fly species which serve as vectors of various forms of leishmaniases. In the present work, we focused on Phlebotomus (Adlerius) arabicus, which was recently shown to transmit Leishmania tropica, the causative agent of cutaneous leishmaniasis in Israel. Results A cDNA library from salivary glands of P. arabicus females was constructed and transcripts were sequenced and analyzed. The most abundant protein families identified were SP15-like proteins, ParSP25-like proteins, D7-related proteins, yellow-related proteins, PpSP32-like proteins, antigen 5-related proteins, and 34 kDa-like proteins. Sequences coding for apyrases, hyaluronidase and other putative secreted enzymes were also represented, including endonuclease, phospholipase, pyrophosphatase, amylase and trehalase. Mass spectrometry analysis confirmed the presence of 20 proteins predicted to be secreted in the salivary proteome. Humoral response of mice bitten by P. arabicus to salivary antigens was assessed and many salivary proteins were determined to be antigenic. Conclusion This transcriptomic analysis of P. arabicus salivary glands is the first description of salivary proteins of a sand fly in the subgenus Adlerius. Proteomic analysis of P. arabicus salivary glands produced the most comprehensive account in a single sand fly species to date. Detailed information and phylogenetic relationships of the salivary proteins are provided, expanding the knowledge base of molecules that are likely important factors of sand fly-host and sand fly-Leishmania interactions. Enzymatic and immunological investigations further demonstrate the value of functional transcriptomics in advancing biological and epidemiological research that can impact leishmaniasis. PMID:19555500

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.

Expression of small leucine-rich proteoglycans in rat anterior pituitary gland.

PubMed

Horiguchi, Kotaro; Syaidah, Rahimi; Fujiwara, Ken; Tsukada, Takehiro; Ramadhani, Dini; Jindatip, Depicha; Kikuchi, Motoshi; Yashiro, Takashi

2013-01-01

Proteoglycans are components of the extracellular matrix and comprise a specific core protein substituted with covalently linked glycosaminoglycan chains. Small leucine-rich proteoglycans (SLRPs) are a major family of proteoglycans and have key roles as potent effectors in cellular signaling pathways. Research during the last two decades has shown that SLRPs regulate biological functions in many tissues such as skin, tendon, kidney, liver, and heart. However, little is known of the expression of SLRPs, or the characteristics of the cells that produce them, in the anterior pituitary gland. Therefore, we have determined whether SLRPs are present in rat anterior pituitary gland. We have used real-time reverse transcription with the polymerase chain reaction to analyze the expression of SLRP genes and have identified the cells that produce SLRPs by using in situ hybridization with a digoxigenin-labeled cRNA probe. We have clearly detected the mRNA expression of SLRP genes, and cells expressing decorin, biglycan, fibromodulin, lumican, proline/arginine-rich end leucine-rich repeat protein (PRELP), and osteoglycin are located in the anterior pituitary gland. We have also investigated the possible double-staining of SLRP mRNA and pituitary hormones, S100 protein (a marker of folliculostellate cells), desmin (a marker of capillary pericytes), and isolectin B4 (a marker of endothelial cells). Decorin, biglycan, fibromodulin, lumican, PRELP, and osteoglycin mRNA have been identified in S100-protein-positive and desmin-positive cells. Thus, we conclude that folliculostellate cells and pericytes produce SLRPs in rat anterior pituitary gland.

Photosynthetic and antioxidant responses of Liquidambar formosana and Schima superba seedlings to sulfuric-rich and nitric-rich simulated acid rain.

PubMed

Chen, Juan; Wang, Wen-Hua; Liu, Ting-Wu; Wu, Fei-Hua; Zheng, Hai-Lei

2013-03-01

To study whether differential responses occur in photosynthesis and antioxidant system for seedlings of Liquidambar formosana, an acid rain (AR)-sensitive tree species and Schima superba, an AR-tolerant tree species treated with three types of pH 3.0 simulated AR (SiAR) including sulfuric-rich (S-SiAR), nitric-rich (N-SiAR), sulfate and nitrate mixed (SN-SiAR), we investigated the changes of leaf necrosis, chlorophyll content, soluble protein and proline content, photosynthesis and chlorophyll fluorescence characteristics, reactive oxygen species production, membrane lipid peroxidation, small molecular antioxidant content, antioxidant enzyme activities and related protein expressions. Our results showed that SiAR significantly caused leaf necrosis, inhibited photosynthesis, induced superoxide radical and hydrogen peroxide generation, aggravated membrane lipid peroxidation, changed antioxidant enzyme activities, modified related protein expressions such as Cu/Zn superoxide dismutase (SOD), l-ascorbate peroxidase (APX, EC 1. 11. 1. 11), glutathione S transferase (GST, EC 2. 5. 1. 18) and Rubisco large subunit (RuBISCO LSU), altered non-protein thiols (NPT) and glutathione (GSH) content in leaves of L. formosana and S. superba. Taken together, we concluded that the damages caused by SiAR in L. formosana were more severe and suffered from more negative impacts than in S. superba. S-SiAR induced more serious damages for the plants than did SN-SiAR and N-SiAR. Crown Copyright © 2013. Published by Elsevier Masson SAS. All rights reserved.

BPI-fold (BPIF) containing/plunc protein expression in human fetal major and minor salivary glands.

PubMed

Alves, Daniel Berretta Moreira; Bingle, Lynne; Bingle, Colin David; Lourenço, Silvia Vanessa; Silva, Andréia Aparecida; Pereira, Débora Lima; Vargas, Pablo Agustin

2017-01-16

The aim of this study was to determine expression, not previously described, of PLUNC (palate, lung, and nasal epithelium clone) (BPI-fold containing) proteins in major and minor salivary glands from very early fetal tissue to the end of the second trimester and thus gain further insight into the function of these proteins. Early fetal heads, and major and minor salivary glands were collected retrospectively and glands were classified according to morphodifferentiation stage. Expression of BPI-fold containing proteins was localized through immunohistochemistry. BPIFA2, the major BPI-fold containing protein in adult salivary glands, was detected only in the laryngeal pharynx; the lack of staining in salivary glands suggested salivary expression is either very late in development or is only in adult tissues. Early expression of BPIFA1 was seen in the trachea and nasal cavity with salivary gland expression only seen in late morphodifferentiation stages. BPIFB1 was seen in early neural tissue and at later stages in submandibular and sublingual glands. BPIFA1 is significantly expressed in early fetal oral tissue but BPIFB1 has extremely limited expression and the major salivary BPIF protein (BPIFA2) is not produced in fetal development. Further studies, with more sensitive techniques, will confirm the expression pattern and enable a better understanding of embryonic BPIF protein function.

The Salivary Protein Repertoire of the Polyphagous Spider Mite Tetranychus urticae: A Quest for Effectors.

PubMed

Jonckheere, Wim; Dermauw, Wannes; Zhurov, Vladimir; Wybouw, Nicky; Van den Bulcke, Jan; Villarroel, Carlos A; Greenhalgh, Robert; Grbić, Mike; Schuurink, Rob C; Tirry, Luc; Baggerman, Geert; Clark, Richard M; Kant, Merijn R; Vanholme, Bartel; Menschaert, Gerben; Van Leeuwen, Thomas

2016-12-01

The two-spotted spider mite Tetranychus urticae is an extremely polyphagous crop pest. Alongside an unparalleled detoxification potential for plant secondary metabolites, it has recently been shown that spider mites can attenuate or even suppress plant defenses. Salivary constituents, notably effectors, have been proposed to play an important role in manipulating plant defenses and might determine the outcome of plant-mite interactions. Here, the proteomic composition of saliva from T. urticae lines adapted to various host plants-bean, maize, soy, and tomato-was analyzed using a custom-developed feeding assay coupled with nano-LC tandem mass spectrometry. About 90 putative T. urticae salivary proteins were identified. Many are of unknown function, and in numerous cases belonging to multimembered gene families. RNAseq expression analysis revealed that many genes coding for these salivary proteins were highly expressed in the proterosoma, the mite body region that includes the salivary glands. A subset of genes encoding putative salivary proteins was selected for whole-mount in situ hybridization, and were found to be expressed in the anterior and dorsal podocephalic glands. Strikingly, host plant dependent expression was evident for putative salivary proteins, and was further studied in detail by micro-array based genome-wide expression profiling. This meta-analysis revealed for the first time the salivary protein repertoire of a phytophagous chelicerate. The availability of this salivary proteome will assist in unraveling the molecular interface between phytophagous mites and their host plants, and may ultimately facilitate the development of mite-resistant crops. Furthermore, the technique used in this study is a time- and resource-efficient method to examine the salivary protein composition of other small arthropods for which saliva or salivary glands cannot be isolated easily. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

The Salivary Protein Repertoire of the Polyphagous Spider Mite Tetranychus urticae: A Quest for Effectors*

PubMed Central

Jonckheere, Wim; Zhurov, Vladimir; Villarroel, Carlos A.; Greenhalgh, Robert; Grbić, Mike; Schuurink, Rob C.; Tirry, Luc; Kant, Merijn R.; Vanholme, Bartel

2016-01-01

The two-spotted spider mite Tetranychus urticae is an extremely polyphagous crop pest. Alongside an unparalleled detoxification potential for plant secondary metabolites, it has recently been shown that spider mites can attenuate or even suppress plant defenses. Salivary constituents, notably effectors, have been proposed to play an important role in manipulating plant defenses and might determine the outcome of plant-mite interactions. Here, the proteomic composition of saliva from T. urticae lines adapted to various host plants—bean, maize, soy, and tomato—was analyzed using a custom-developed feeding assay coupled with nano-LC tandem mass spectrometry. About 90 putative T. urticae salivary proteins were identified. Many are of unknown function, and in numerous cases belonging to multimembered gene families. RNAseq expression analysis revealed that many genes coding for these salivary proteins were highly expressed in the proterosoma, the mite body region that includes the salivary glands. A subset of genes encoding putative salivary proteins was selected for whole-mount in situ hybridization, and were found to be expressed in the anterior and dorsal podocephalic glands. Strikingly, host plant dependent expression was evident for putative salivary proteins, and was further studied in detail by micro-array based genome-wide expression profiling. This meta-analysis revealed for the first time the salivary protein repertoire of a phytophagous chelicerate. The availability of this salivary proteome will assist in unraveling the molecular interface between phytophagous mites and their host plants, and may ultimately facilitate the development of mite-resistant crops. Furthermore, the technique used in this study is a time- and resource-efficient method to examine the salivary protein composition of other small arthropods for which saliva or salivary glands cannot be isolated easily. PMID:27703040

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.

Isolation and characterization of cDNA clones for carrot extensin and a proline-rich 33-kDa protein

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

Chen, J.; Varner, J.E.

1985-07-01

Extensins are hydroxyproline-rich glycoproteins associated with most dicotyledonous plant cell walls. To isolate cDNA clones encoding extensin, the authors started by isolating poly(A) RNA from carrot root tissue, and then translating the RNA in vitro, in the presence of tritiated leucine or proline. A 33-kDa peptide was identified in the translation products as a putative extensin precursor. From a cDNA library constructed with poly(A) RNA from wounded carrots, one cDNA clone (pDC5) was identified that specifically hybridized to poly(A) RNA encoding this 33-kDa peptide. They isolated three cDNA clones (pDC11, pDC12, and pDC16) from another cDNA library using pCD5 asmore » a probe. DNA sequence data, RNA hybridization analysis, and hybrid released in vitro translation indicate that the cDNA clones pDC11 encodes extensin and that cDNA clones pDC12 and pDC16 encode the 33-kDa peptide, which as yet has an unknown identity and function. The assumption that the 33-kDa peptide was an extensin precursor was invalid. RNA hybridization analysis showed that RNA encoded by both clone types is accumulated upon wounding.« less

PaASK1, a mitogen-activated protein kinase kinase kinase that controls cell degeneration and cell differentiation in Podospora anserina.

PubMed Central

Kicka, Sébastien; Silar, Philippe

2004-01-01

MAPKKK are kinases involved in cell signaling. In fungi, these kinases are known to regulate development, pathogenicity, and the sensing of external conditions. We show here that Podospora anserina strains mutated in PaASK1, a MAPKKK of the MEK family, are impaired in the development of crippled growth, a cell degeneration process caused by C, a nonconventional infectious element. They also display defects in mycelium pigmentation, differentiation of aerial hyphae, and making of fruiting bodies, three hallmarks of cell differentiation during stationary phase in P. anserina. Overexpression of PaASK1 results in exacerbation of crippled growth. PaASK1 is a large protein of 1832 amino acids with several domains, including a region rich in proline and a 60-amino-acid-long polyglutamine stretch. Deletion analysis reveals that the polyglutamine stretch is dispensable for PaASK1 activity, whereas the region that contains the prolines is essential but insufficient to promote full activity. We discuss a model based on the hysteresis of a signal transduction cascade to account for the role of PaASK1 in both cell degeneration and stationary-phase cell differentiation. PMID:15082544

PaASK1, a mitogen-activated protein kinase kinase kinase that controls cell degeneration and cell differentiation in Podospora anserina.

PubMed

Kicka, Sébastien; Silar, Philippe

2004-03-01

MAPKKK are kinases involved in cell signaling. In fungi, these kinases are known to regulate development, pathogenicity, and the sensing of external conditions. We show here that Podospora anserina strains mutated in PaASK1, a MAPKKK of the MEK family, are impaired in the development of crippled growth, a cell degeneration process caused by C, a nonconventional infectious element. They also display defects in mycelium pigmentation, differentiation of aerial hyphae, and making of fruiting bodies, three hallmarks of cell differentiation during stationary phase in P. anserina. Overexpression of PaASK1 results in exacerbation of crippled growth. PaASK1 is a large protein of 1832 amino acids with several domains, including a region rich in proline and a 60-amino-acid-long polyglutamine stretch. Deletion analysis reveals that the polyglutamine stretch is dispensable for PaASK1 activity, whereas the region that contains the prolines is essential but insufficient to promote full activity. We discuss a model based on the hysteresis of a signal transduction cascade to account for the role of PaASK1 in both cell degeneration and stationary-phase cell differentiation.

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

The Nck family of adapter proteins: regulators of actin cytoskeleton.

PubMed

Buday, László; Wunderlich, Livius; Tamás, Peter

2002-09-01

SH2/SH3 domain-containing adapter proteins, such as the Nck family, play a major role in regulating tyrosine kinase signalling. They serve to recruit proline-rich effector molecules to tyrosine-phosphorylated kinases or their substrates. Initially, it was not clear why cells from nematodes to vertebrates contain redundant and closely related SH2/SH3 adapters, such as Grb2, Crk and Nck. Recent evidence suggests that their biological roles are clearly different, whereas, for example, Grb2 connects activated receptor tyrosine kinases to Sos and Ras, leading to cell proliferation. The proteins of Nck family are implicated in organisation of actin cytoskeleton, cell movement or axon guidance in flies. In this review, the author attempts to summarise signalling pathways in which Nck plays a critical role.

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.

An ELISA for SGP28/CRISP-3, a cysteine-rich secretory protein in human neutrophils, plasma, and exocrine secretions.

PubMed

Udby, Lene; Cowland, Jack B; Johnsen, Anders H; Sørensen, Ole E; Borregaard, Niels; Kjeldsen, Lars

2002-05-01

Specific granule protein of 28 kDa (SGP28), also termed cysteine-rich secretory protein 3 (CRISP-3), is a glycoprotein that belongs to a family of cysteine-rich secretory proteins (CRISPs). SGP28 was originally discovered in human neutrophils, but transcripts are widely distributed in exocrine glands (salivary glands, pancreas, and prostate) and also found at lower levels in epididymis, ovary, thymus, and colon. The function of SGP28/CRISP-3 is not yet known. Similarities to pathogenesis-related proteins in plants and the expression in neutrophils and exocrine glands suggest that SGP28/CRISP-3 may play a role in innate host defense. We describe here the production of a recombinant, C-terminally truncated form of CRISP-3 (rCRISP-3Delta) and the generation of polyclonal antibodies against rCRISP-3Delta that are useful in immunoblotting and immunocytochemistry. We present a specific, accurate, and reproducible enzyme-linked immunosorbant assay (ELISA) for the measurement of CRISP-3 with a detection limit of 2 ng/ml. We further demonstrate the presence of CRISP-3 protein in human plasma (6.3 microg/ml), saliva (21.8 microg/ml), seminal plasma (11.2 microg/ml), and sweat (0.15 microg/ml), and describe the coexistence of two different molecular weight forms of CRISP-3, representing an N-glycosylated and a non-glycosylated form of the mature protein.

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

The heptad repeats region is essential for AcMNPV P10 filament formation and not the proline-rich or the C-terminus basic regions.

PubMed

Dong, Chunsheng; Deng, Fei; Li, Dan; Wang, Hualin; Hu, Zhihong

2007-09-01

Baculovirus P10 protein is a small conserved protein and is expressed as bundles of filaments in the host cell during the late phase of virus infection. So far the published results on the domain responsible for filament structural formation have been contradictory. Electron microscopy revealed that the C-terminus basic region was involved in filament structural formation in the Autographa californica multiple nucleocapsid nucleopolyhedrovirus (AcMNPV) [van Oers, M.M., Flipsen, J.T., Reusken, C.B., Sliwinsky, E.L., Vlak, J.M., 1993. Functional domains of the p10 protein of Autographa californica nuclear polyhedorsis virus. J. Gen. Virol. 74, 563-574.]. While in the Helicoverpa armigera nucleopolyhedrovirus (HearNPV), the heptad repeats region but not the C-terminus domain was proven to be responsible for filament formation [Dong, C., Li, D., Long, G., Deng, F., Wang, H., Hu, Z., 2005. Identification of functional domains required for HearNPV P10 filament formation. Virology 338, 112-120.]. In this manuscript, fluorescence confocal microscopy was applied to study AcMNPV P10 filament formation. A set of plasmids containing different P10 structural domains fused with a fluorescent protein were constructed and transfected into Sf-9 cells. The data indicated that the heptad repeats region, but not the proline-rich region or the C-terminus basic region, is essential for AcMNPV P10 filament formation. Co-transfection of P10s tagged with different fluorescent revealed that P10s with defective heptad repeats region could not interact with intact heptad repeats region or even full-length P10s to form filament structure. Within the heptad repeats region, deletion of the three amino acids spacing of AcMNPV P10 appeared to have no significant impact on the formation of filament structures, but the content of the heptad repeats region appeared to play a role in the morphology of the filaments.

Tyrosine Nitration within the Proline-Rich Region of Tau in Alzheimer's Disease

PubMed Central

Reyes, Juan F.; Fu, Yifan; Vana, Laurel; Kanaan, Nicholas M.; Binder, Lester I.

2011-01-01

A substantial body of evidence suggests that nitrative injury contributes to neurodegeneration in Alzheimer's disease (AD) and other neurodegenerative disorders. Previously, we showed in vitro that within the tau protein the N-terminal tyrosine residues (Y18 and Y29) are more susceptible to nitrative modifications than other tyrosine sites (Y197 and Y394). Using site-specific antibodies to nitrated tau at Y18 and Y29, we identified tau nitrated in both glial (Y18) and neuronal (Y29) tau pathologies. In this study, we report the characterization of two novel monoclonal antibodies, Tau-nY197 and Tau-nY394, recognizing tau nitrated at Y197 and Y394, respectively. By Western blot analysis, Tau-nY197 labeled soluble tau and insoluble paired helical filament proteins (PHF-tau) nitrated at Y197 from control and AD brain samples. Tau-nY394 failed to label soluble tau isolated from control or severe AD samples, but labeled insoluble PHF-tau to a limited extent. Immunohistochemical analysis using Tau-nY197 revealed the hallmark tau pathology associated with AD; Tau-nY394 did not detect any pathological lesions characteristic of the disorder. These data suggest that a subset of the hallmark pathological inclusions of AD contain tau nitrated at Y197. However, nitration at Y197 was also identified in soluble tau from all control samples, including those at Braak stage 0, suggesting that nitration at this site in the proline-rich region of tau may have normal biological functions in the human brain. PMID:21514440

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

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.

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.

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

Evolution of sfbI Encoding Streptococcal Fibronectin-Binding Protein I: Horizontal Genetic Transfer and Gene Mosaic Structure

PubMed Central

Towers, Rebecca J.; Fagan, Peter K.; Talay, Susanne R.; Currie, Bart J.; Sriprakash, Kadaba S.; Walker, Mark J.; Chhatwal, Gursharan S.

2003-01-01

Streptococcal fibronectin-binding protein is an important virulence factor involved in colonization and invasion of epithelial cells and tissues by Streptococcus pyogenes. In order to investigate the mechanisms involved in the evolution of sfbI, the sfbI genes from 54 strains were sequenced. Thirty-four distinct alleles were identified. Three principal mechanisms appear to have been involved in the evolution of sfbI. The amino-terminal aromatic amino acid-rich domain is the most variable region and is apparently generated by intergenic recombination of horizontally acquired DNA cassettes, resulting in a genetic mosaic in this region. Two distinct and divergent sequence types that shared only 61 to 70% identity were identified in the central proline-rich region, while variation at the 3′ end of the gene is due to deletion or duplication of defined repeat units. Potential antigenic and functional variabilities in SfbI imply significant selective pressure in vivo with direct implications for the microbial pathogenesis of S. pyogenes. PMID:14662917

A novel source of biofertilizer from feather biomass for banana cultivation.

PubMed

Gurav, Ranjit G; Jadhav, Jyoti P

2013-07-01

Feather waste is a promising protein biomass available as by-product from poultry processing was found to be rich in peptides, amino acids, and minerals like nitrogen, phosphorus, potassium, calcium, magnesium, iron, manganese, zinc, and copper. Soil and foliar application of these products, besides representing a sustainable solution to the problem of feather disposal, may also represent an effective strategy to tackle the environmental effluence. As a consequence, they were also found to be very attractive in elevating the protein, amino acids, reducing sugar, total chlorophyll, and proline content of plants. On the other side, fertilizing effect enhanced the antioxidant potential of banana fruit which was assessed using 2, 2-diphenyl-1-picrylhydrazyl, ferric reducing/antioxidant power, and N, N-dimethyl-p-phenylendiamine. This was associated with considerably higher antioxidant contents like total phenolics and flavonoids. Therefore, the application of this organic amendment could promote and improve the agro-ecosystem, human health; soil biological activities, and at the same time enhance the production of plant or products rich in bioactive substances.

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

Roles of larval sea urchin spicule SM50 domains in organic matrix self-assembly and calcium carbonate mineralization.

PubMed

Rao, Ashit; Seto, Jong; Berg, John K; Kreft, Stefan G; Scheffner, Martin; Cölfen, Helmut

2013-08-01

The larval spicule matrix protein SM50 is the most abundant occluded matrix protein present in the mineralized larval sea urchin spicule. Recent evidence implicates SM50 in the stabilization of amorphous calcium carbonate (ACC). Here, we investigate the molecular interactions of SM50 and CaCO3 by investigating the function of three major domains of SM50 as small ubiquitin-like modifier (SUMO) fusion proteins - a C-type lectin domain (CTL), a glycine rich region (GRR) and a proline rich region (PRR). Under various mineralization conditions, we find that SUMO-CTL is monomeric and influences CaCO3 mineralization, SUMO-GRR aggregates into large protein superstructures and SUMO-PRR modifies the early CaCO3 mineralization stages as well as growth. The combination of these mineralization and self-assembly properties of the major domains synergistically enable the full-length SM50 to fulfill functions of constructing the organic spicule matrix as well as performing necessary mineralization activities such as Ca(2+) ion recruitment and organization to allow for proper growth and development of the mineralized larval sea urchin spicule. Copyright © 2013 Elsevier Inc. All rights reserved.

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

An N-terminal glycine-rich sequence contributes to retrovirus trimer of hairpins stability

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

Wilson, Kirilee A.; Maerz, Anne L.; Baer, Severine

2007-08-10

Retroviral transmembrane proteins (TMs) contain a glycine-rich segment linking the N-terminal fusion peptide and coiled coil core. Previously, we reported that the glycine-rich segment (Met-326-Ser-337) of the human T-cell leukemia virus type 1 (HTLV-1) TM, gp21, is a determinant of membrane fusion function [K.A. Wilson, S. Baer, A.L. Maerz, M. Alizon, P. Poumbourios, The conserved glycine-rich segment linking the N-terminal fusion peptide to the coiled coil of human T-cell leukemia virus type 1 transmembrane glycoprotein gp21 is a determinant of membrane fusion function, J. Virol. 79 (2005) 4533-4539]. Here we show that the reduced fusion activity of an I334A mutantmore » correlated with a decrease in stability of the gp21 trimer of hairpins conformation, in the context of a maltose-binding protein-gp21 chimera. The stabilizing influence of Ile-334 required the C-terminal membrane-proximal sequence Trp-431-Ser-436. Proline substitution of four of five Gly residues altered gp21 trimer of hairpins stability. Our data indicate that flexibility within and hydrophobic interactions mediated by this region are determinants of gp21 stability and membrane fusion function.« less

Sialome of a Generalist Lepidopteran Herbivore: Identification of Transcripts and Proteins from Helicoverpa armigera Labial Salivary Glands

PubMed Central

Celorio-Mancera, Maria de la Paz; Courtiade, Juliette; Muck, Alexander; Heckel, David G.; Musser, Richard O.; Vogel, Heiko

2011-01-01

Although the importance of insect saliva in insect-host plant interactions has been acknowledged, there is very limited information on the nature and complexity of the salivary proteome in lepidopteran herbivores. We inspected the labial salivary transcriptome and proteome of Helicoverpa armigera, an important polyphagous pest species. To identify the majority of the salivary proteins we have randomly sequenced 19,389 expressed sequence tags (ESTs) from a normalized cDNA library of salivary glands. In parallel, a non-cytosolic enriched protein fraction was obtained from labial salivary glands and subjected to two-dimensional gel electrophoresis (2-DE) and de novo peptide sequencing. This procedure allowed comparison of peptides and EST sequences and enabled us to identify 65 protein spots from the secreted labial saliva 2DE proteome. The mass spectrometry analysis revealed ecdysone, glucose oxidase, fructosidase, carboxyl/cholinesterase and an uncharacterized protein previously detected in H. armigera midgut proteome. Consistently, their corresponding transcripts are among the most abundant in our cDNA library. We did find redundancy of sequence identification of saliva-secreted proteins suggesting multiple isoforms. As expected, we found several enzymes responsible for digestion and plant offense. In addition, we identified non-digestive proteins such as an arginine kinase and abundant proteins of unknown function. This identification of secreted salivary gland proteins allows a more comprehensive understanding of insect feeding and poses new challenges for the elucidation of protein function. PMID:22046331

Anopheles gambiae Circumsporozoite Protein–Binding Protein Facilitates Plasmodium Infection of Mosquito Salivary Glands

PubMed Central

Wang, Jiuling; Zhang, Yue; Zhao, Yang O.; Li, Michelle W. M.; Zhang, Lili; Dragovic, Srdjan; Abraham, Nabil M.; Fikrig, Erol

2013-01-01

Malaria, a mosquito-borne disease caused by Plasmodium species, causes substantial morbidity and mortality throughout the world. Plasmodium sporozoites mature in oocysts formed in the mosquito gut wall and then invade the salivary glands, where they remain until transmitted to the vertebrate host during a mosquito bite. The Plasmodium circumsporozoite protein (CSP) binds to salivary glands and plays a role in the invasion of this organ by sporozoites. We identified an Anopheles salivary gland protein, named CSP-binding protein (CSPBP), that interacts with CSP. Downregulation of CSPBP in mosquito salivary glands inhibited invasion by Plasmodium organisms. In vivo bioassays showed that mosquitoes that were fed blood with CSPBP antibody displayed a 25% and 90% reduction in the parasite load in infected salivary glands 14 and 18 days after the blood meal, respectively. These results suggest that CSPBP is important for the infection of the mosquito salivary gland by Plasmodium organisms and that blocking CSPBP can interfere with the Plasmodium life cycle. PMID:23801601

Functional genomics of gam56: characterisation of the role of a 56 kilodalton sexual stage antigen in oocyst wall formation in Eimeria maxima.

PubMed

Belli, Sabina I; Witcombe, David; Wallach, Michael G; Smith, Nicholas C

2002-12-19

Gam56 (M(r) 56,000) is an antigen found in the sexual (macrogametocyte) stage of the intestinal parasite Eimeria maxima that is implicated in protective immunity. The gene (gam56) encoding this protein was cloned and sequenced. It is a single-copy, intronless gene, that localises to a 1,754 bp transcript, and is first detected at 120 h p.i. The gene predicts two distinct protein domains; a tyrosine-serine rich region, composed of amino acids implicated in oocyst wall formation in Eimeria spp., and a proline-methionine rich region often detected in extensins, protein components of plant cell walls. The tyrosine-serine rich region predicts a secondary structure commonly seen in the structural protein fibroin, a component of the cocoon of the caterpillar Bombyx mori. The inference that gam56 is a structural component of the oocyst wall was confirmed when a specific antibody to gam56 recognised the wall forming bodies in macrogametocytes, and the walls of oocysts and sporocysts. Together, these data identify a developmentally regulated, sexual stage gene in E. maxima that shares primary and secondary structure features in common with intrinsic structural proteins in other parasites such as Schistosoma mansoni and Fasciola hepatica, and other organisms across different phyla, including the caterpillar Bombyx mori. In addition, these findings provide evidence for the molecular mechanisms underlying oocyst wall formation in Eimeria and the role of gametocyte antigens in this process.

Latherin: A Surfactant Protein of Horse Sweat and Saliva

PubMed Central

Beeley, John G.; Bovell, Douglas L.; Lu, Jian R.; Zhao, Xiubo; Cooper, Alan; Kennedy, Malcolm W.

2009-01-01

Horses are unusual in producing protein-rich sweat for thermoregulation, a major component of which is latherin, a highly surface-active, non-glycosylated protein. The amino acid sequence of latherin, determined from cDNA analysis, is highly conserved across four geographically dispersed equid species (horse, zebra, onager, ass), and is similar to a family of proteins only found previously in the oral cavity and associated tissues of mammals. Latherin produces a significant reduction in water surface tension at low concentrations (≤1 mg ml−1), and therefore probably acts as a wetting agent to facilitate evaporative cooling through a waterproofed pelt. Neutron reflection experiments indicate that this detergent-like activity is associated with the formation of a dense protein layer, about 10 Å thick, at the air-water interface. However, biophysical characterization (circular dichroism, differential scanning calorimetry) in solution shows that latherin behaves like a typical globular protein, although with unusual intrinsic fluorescence characteristics, suggesting that significant conformational change or unfolding of the protein is required for assembly of the air-water interfacial layer. RT-PCR screening revealed latherin transcripts in horse skin and salivary gland but in no other tissues. Recombinant latherin produced in bacteria was also found to be the target of IgE antibody from horse-allergic subjects. Equids therefore may have adapted an oral/salivary mucosal protein for two purposes peculiar to their lifestyle, namely their need for rapid and efficient heat dissipation and their specialisation for masticating and processing large quantities of dry food material. PMID:19478940

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

Jia, Xiaofei; Singh, Rajendra; Homann, Stefanie

The HIV-1 protein Nef inhibits antigen presentation by class I major histocompatibility complex (MHC-I). We determined the mechanism of this activity by solving the crystal structure of a protein complex comprising Nef, the MHC-I cytoplasmic domain (MHC-I CD) and the {mu}1 subunit of the clathrin adaptor protein complex 1. A ternary, cooperative interaction clamps the MHC-I CD into a narrow binding groove at the Nef-{mu}1 interface, which encompasses the cargo-recognition site of {mu}1 and the proline-rich strand of Nef. The Nef C terminus induces a previously unobserved conformational change in {mu}1, whereas the N terminus binds the Nef core tomore » position it optimally for complex formation. Positively charged patches on {mu}1 recognize acidic clusters in Nef and MHC-I. The structure shows how Nef functions as a clathrin-associated sorting protein to alter the specificity of host membrane trafficking and enable viral evasion of adaptive immunity.« less

First large scale chemical synthesis of the 72 amino acid HIV-1 nucleocapsid protein NCp7 in an active form.

PubMed

de Rocquigny, H; Ficheux, D; Gabus, C; Fournié-Zaluski, M C; Darlix, J L; Roques, B P

1991-10-31

The nucleocapsid protein (NC) of the human immunodeficiency virus type 1 plays a crucial role in the formation of infectious viral particles and therefore should be a major target for the development of antiviral agents. This requires an investigation of NC protein structure and of its interactions with both primer tRNA(Lys,3) and genomic RNA. Nucleocapsid protein NCp7, which results from the maturation of NCp15, contains two zinc fingers flanked by sequences rich in basic and proline residues. Here we report the first synthesis of large quantities of NCp7 able to activate HIV-1 RNA dimerization and replication primer tRNA(Lys,3) annealing to the initiation site of reverse transcription. In addition UV spectroscopic analyses performed to characterize the Co2+ binding properties of each zinc finger suggest that the two fingers probably interact in NCp7.

Solution Model of the Intrinsically Disordered Polyglutamine Tract-Binding Protein-1

PubMed Central

Rees, Martin; Gorba, Christian; de Chiara, Cesira; Bui, Tam T.T.; Garcia-Maya, Mitla; Drake, Alex F.; Okazawa, Hitoshi; Pastore, Annalisa; Svergun, Dmitri; Chen, Yu Wai

2012-01-01

Polyglutamine tract-binding protein-1 (PQBP-1) is a 265-residue nuclear protein that is involved in transcriptional regulation. In addition to its role in the molecular pathology of the polyglutamine expansion diseases, mutations of the protein are associated with X-linked mental retardation. PQBP-1 binds specifically to glutamine repeat sequences and proline-rich regions, and interacts with RNA polymerase II and the spliceosomal protein U5-15kD. In this work, we obtained a biophysical characterization of this protein by employing complementary structural methods. PQBP-1 is shown to be a moderately compact but largely disordered molecule with an elongated shape, having a Stokes radius of 3.7 nm and a maximum molecular dimension of 13 nm. The protein is monomeric in solution, has residual β-structure, and is in a premolten globule state that is unaffected by natural osmolytes. Using small-angle x-ray scattering data, we were able to generate a low-resolution, three-dimensional model of PQBP-1. PMID:22500761

Human mRNA polyadenylate binding protein: evolutionary conservation of a nucleic acid binding motif.

PubMed Central

Grange, T; de Sa, C M; Oddos, J; Pictet, R

1987-01-01

We have isolated a full length cDNA (cDNA) coding for the human poly(A) binding protein. The cDNA derived 73 kd basic translation product has the same Mr, isoelectric point and peptidic map as the poly(A) binding protein. DNA sequence analysis reveals a 70,244 dalton protein. The N terminal part, highly homologous to the yeast poly(A) binding protein, is sufficient for poly(A) binding activity. This domain consists of a four-fold repeated unit of approximately 80 amino acids present in other nucleic acid binding proteins. In the C terminal part there is, as in the yeast protein, a sequence of approximately 150 amino acids, rich in proline, alanine and glutamine which together account for 48% of the residues. A 2,9 kb mRNA corresponding to this cDNA has been detected in several vertebrate cell types and in Drosophila melanogaster at every developmental stage including oogenesis. Images PMID:2885805

Host association influences variation at salivary protein genes in the bat ectoparasite Cimex adjunctus.

PubMed

Talbot, Benoit; Vonhof, Maarten J; Broders, Hugh G; Fenton, Brock; Keyghobadi, Nusha

2018-05-01

Parasite-host relationships create strong selection pressures that can lead to adaptation and increasing specialization of parasites to their hosts. Even in relatively loose host-parasite relationships, such as between generalist ectoparasites and their hosts, we may observe some degree of specialization of parasite populations to one of the multiple potential hosts. Salivary proteins are used by blood-feeding ectoparasites to prevent hemostasis in the host and maximize energy intake. We investigated the influence of association with specific host species on allele frequencies of salivary protein genes in Cimex adjunctus, a generalist blood-feeding ectoparasite of bats in North America. We analysed two salivary protein genes: an apyrase, which hydrolyses ATP at the feeding site and thus inhibits platelet aggregation, and a nitrophorin, which brings nitrous oxide to the feeding site, inhibiting platelet aggregation and vasoconstriction. We observed more variation at both salivary protein genes among parasite populations associated with different host species than among populations from different spatial locations associated with the same host species. The variation in salivary protein genes among populations on different host species was also greater than expected under a neutral scenario of genetic drift and gene flow. Finally, host species was an important predictor of allelic divergence in genotypes of individual C. adjunctus at both salivary protein genes. Our results suggest differing selection pressures on these two salivary protein genes in C. adjunctus depending on the host species. © 2018 European Society For Evolutionary Biology. Journal of Evolutionary Biology © 2018 European Society For Evolutionary Biology.

Mining a differential sialotranscriptome of Rhipicephalus microplus guides antigen discovery to formulate a vaccine that reduces tick infestations.

PubMed

Maruyama, Sandra R; Garcia, Gustavo R; Teixeira, Felipe R; Brandão, Lucinda G; Anderson, Jennifer M; Ribeiro, José M C; Valenzuela, Jesus G; Horackova, Jana; Veríssimo, Cecília J; Katiki, Luciana M; Banin, Tamy M; Zangirolamo, Amanda F; Gardinassi, Luiz G; Ferreira, Beatriz R; de Miranda-Santos, Isabel K F

2017-04-26

Ticks cause massive damage to livestock and vaccines are one sustainable substitute for the acaricides currently heavily used to control infestations. To guide antigen discovery for a vaccine that targets the gamut of parasitic strategies mediated by tick saliva and enables immunological memory, we exploited a transcriptome constructed from salivary glands from all stages of Rhipicephalus microplus ticks feeding on genetically tick-resistant and susceptible bovines. Different levels of host anti-tick immunity affected gene expression in tick salivary glands; we thus selected four proteins encoded by genes weakly expressed in ticks attempting to feed on resistant hosts or otherwise abundantly expressed in ticks fed on susceptible hosts; these sialoproteins mediate four functions of parasitism deployed by male ticks and that do not induce antibodies in naturally infected, susceptible bovines. We then evaluated in tick-susceptible heifers an alum-adjuvanted vaccine formulated with recombinant proteins. Parasite performance (i.e. weight and numbers of females finishing their parasitic cycle) and titres of antigen-specific antibodies were significantly reduced or increased, respectively, in vaccinated versus control heifers, conferring an efficacy of 73.2%; two of the antigens were strong immunogens, rich in predicted T-cell epitopes and challenge infestations boosted antibody responses against them. Mining sialotranscriptomes guided by the immunity of tick-resistant hosts selected important targets and infestations boosted immune memory against salivary antigens.

Na+-glucose cotransporter SGLT1 protein in salivary glands: potential involvement in the diabetes-induced decrease in salivary flow.

PubMed

Sabino-Silva, R; Freitas, H S; Lamers, M L; Okamoto, M M; Santos, M F; Machado, U F

2009-03-01

Oral health complications in diabetes include decreased salivary secretion. The SLC5A1 gene encodes the Na(+)-glucose cotransporter SGLT1 protein, which not only transports glucose, but also acts as a water channel. Since SLC5A1 expression is altered in kidneys of diabetic subjects, we hypothesize that it could also be altered in salivary glands, contributing to diabetic dysfunction. The present study shows a diabetes-induced decrease (p < 0.001) in salivary secretion, which was accompanied by enhanced (p < 0.05) SGLT1 mRNA expression in parotid (50%) and submandibular (30%) glands. Immunohistochemical analysis of parotid gland of diabetic rats revealed that SGLT1 protein expression increased in the luminal membrane of ductal cells, which can stimulate water reabsorption from primary saliva. Furthermore, SGLT1 protein was reduced in myoepithelial cells of the parotid from diabetic animals, and that, by reducing cellular contractile activity, might also be related to reduced salivary flux. Six-day insulin-treated diabetic rats reversed all alterations. In conclusion, diabetes increases SLC5A1 gene expression in salivary glands, increasing the SGLT1 protein content in the luminal membrane of ductal cells, which, by increasing water reabsorption, might explain the diabetes-induced decrease in salivary secretion.

Proline-Rich Peptide Mimics Effects of Enamel Matrix Derivative on Rat Oral Mucosa Incisional Wound Healing.

PubMed

Villa, Oscar; Wohlfahrt, Johan C; Mdla, Ibrahimu; Petzold, Christiane; Reseland, Janne E; Snead, Malcolm L; Lyngstadaas, Staale P

2015-12-01

Proline-rich peptides have been shown to promote periodontal regeneration. However, their effect on soft tissue wound healing has not yet been investigated. The aim of this study is to evaluate the effect of enamel matrix derivative (EMD), tyrosine-rich amelogenin peptide (TRAP), and a synthetic proline-rich peptide (P2) on acute wound healing after a full-thickness flap procedure in an incisional rat model. This experimental study has a split-mouth, randomized, placebo-controlled design. Test and control wounds were created on the palatal mucosa of 54 Sprague-Dawley rats. Wounds were histologically processed, and reepithelialization, leukocyte infiltration, and angiogenesis were assessed at days 1, 3, and 7 post-surgery. EMD and P2 significantly promoted early wound closure at day 1 (P <0.001 and P = 0.004, respectively). EMD maintained a significant acceleration of reepithelialization at day 3 (P = 0.004). Wounds treated by EMD and P2 showed increased angiogenesis during the first 3 days of healing (P = 0.03 and 0.001, respectively). Leukocyte infiltration was decreased in EMD-treated wounds at day 1 (P = 0.03), and P2 and TRAP induced a similar effect at days 3 (P = 0.002 and P <0.0001, respectively) and 7 (P = 0.005 and P <0.001). EMD and P2 promoted reepithelialization and neovascularization in full-thickness surgical wounds on rat oral mucosa.

Next-Generation Sequencing of Protein-Coding and Long Non-protein-Coding RNAs in Two Types of Exosomes Derived from Human Whole Saliva.

PubMed

Ogawa, Yuko; Tsujimoto, Masafumi; Yanoshita, Ryohei

2016-01-01

Exosomes are small extracellular vesicles containing microRNAs and mRNAs that are produced by various types of cells. We previously used ultrafiltration and size-exclusion chromatography to isolate two types of human salivary exosomes (exosomes I, II) that are different in size and proteomes. We showed that salivary exosomes contain large repertoires of small RNAs. However, precise information regarding long RNAs in salivary exosomes has not been fully determined. In this study, we investigated the compositions of protein-coding RNAs (pcRNAs) and long non-protein-coding RNAs (lncRNAs) of exosome I, exosome II and whole saliva (WS) by next-generation sequencing technology. Although 11% of all RNAs were commonly detected among the three samples, the compositions of reads mapping to known RNAs were similar. The most abundant pcRNA is ribosomal RNA protein, and pcRNAs of some salivary proteins such as S100 calcium-binding protein A8 (protein S100-A8) were present in salivary exosomes. Interestingly, lncRNAs of pseudogenes (presumably, processed pseudogenes) were abundant in exosome I, exosome II and WS. Translationally controlled tumor protein gene, which plays an important role in cell proliferation, cell death and immune responses, was highly expressed as pcRNA and pseudogenes in salivary exosomes. Our results show that salivary exosomes contain various types of RNAs such as pseudogenes and small RNAs, and may mediate intercellular communication by transferring these RNAs to target cells as gene expression regulators.

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.

Dual Function of a Tip Fimbrillin of Actinomyces in Fimbrial Assembly and Receptor Binding▿

PubMed Central

Wu, Chenggang; Mishra, Arunima; Yang, Jinghua; Cisar, John O.; Das, Asis; Ton-That, Hung

2011-01-01

Interaction of Actinomyces oris with salivary proline-rich proteins (PRPs), which serve as fimbrial receptors, involves type 1 fimbriae. Encoded by the gene locus fimQ-fimP-srtC1, the type 1 fimbria is comprised of the fimbrial shaft FimP and the tip fimbrillin FimQ. Fimbrial polymerization requires the fimbria-specific sortase SrtC1, which catalyzes covalent linkage of fimbrial subunits. Using genetics, biochemical methods, and electron microscopy, we provide evidence that the tip fimbrillin, FimQ, is involved in fimbrial assembly and interaction with PRPs. Specifically, while deletion of fimP completely abolished the type 1 fimbrial structures, surface display of monomeric FimQ was not affected by this mutation. Surprisingly, deletion of fimQ significantly reduced surface assembly of the type 1 fimbriae. This defect was rescued by recombinant FimQ ectopically expressed from a plasmid. In agreement with the role of type 1 fimbriae in binding to PRPs, aggregation of A. oris with PRP-coated beads was abrogated in cells lacking srtC1 or fimP. This aggregation defect of the ΔfimP mutant was mainly due to significant reduction of FimQ on the bacterial surface, as the aggregation was not observed in a strain lacking fimQ. Increasing expression of FimQ in the ΔfimP mutant enhanced aggregation, while overexpression of FimP in the ΔfimQ mutant did not. Furthermore, recombinant FimQ, not FimP, bound surface-associated PRPs in a dose-dependent manner. Thus, not only does FimQ function as the major adhesin of the type 1 fimbriae, it also plays an important role in fimbrial assembly. PMID:21531799

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.

Sustaining elevated levels of nitrite in the oral cavity through consumption of nitrate-rich beetroot juice in young healthy adults reduces salivary pH.

PubMed

Hohensinn, Barbara; Haselgrübler, Renate; Müller, Ulrike; Stadlbauer, Verena; Lanzerstorfer, Peter; Lirk, Gerald; Höglinger, Otmar; Weghuber, Julian

2016-11-30

Dietary inorganic nitrate (NO 3 - ) and its reduced forms nitrite (NO 2 - ) and nitric oxide (NO), respectively, are of critical importance for host defense in the oral cavity. High concentrations of salivary nitrate are linked to a lower prevalence of caries due to growth inhibition of cariogenic bacteria. In-vitro studies suggest that the formation of antimicrobial NO results in an increase of the pH preventing erosion of tooth enamel. The purpose of this study was to prove this effect in-vivo. In a randomized clinical study with 46 subjects we investigated whether NO 3 - rich beetroot juice exhibits a protective effect against caries by an increase of salivary pH. Our results show that, in comparison to a placebo group, consumption of beetroot juice that contains 4000 mg/L NO 3 - results in elevated levels of salivary NO 2 - , nitrite NO 3 - , and NO. Furthermore, we determined an increase of the mean pH of saliva from 7.0 to 7.5, confirming the anti-cariogenic effect of the used NO 3 - -rich beetroot juice. Taken together, we have found that NO 3 - -rich beetroot juice holds potential effects against dental caries by preventing acidification of human saliva. C-87-15 (Ethics Commissions of Upper Austria). Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

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.

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

Change of salivary stress marker concentrations during pregnancy: maternal depressive status suppress changes of those levels.

PubMed

Tsubouchi, Hiroaki; Nakai, Yuichiro; Toda, Masahiro; Morimoto, Kanehisa; Chang, Yang Sil; Ushioda, Norichika; Kaku, Shoji; Nakamura, Takafumi; Kimura, Tadashi; Shimoya, Koichiro

2011-08-01

The aim of the present study was to show changes in salivary cortisol and chromogranin A/protein concentrations as stress markers during pregnancy and to clarify the effect of chronic stress on stress markers. Salivary samples were collected from 69 pregnant women during pregnancy. Salivary cortisol levels and chromogranin A/protein titers were determined. We surveyed the women's chronic stress using the Zung self-rating depression scale and General Health Questionnaire-28. Cortisol levels in the saliva of pregnant women showed biphasic change during pregnancy. Chromogranin A/protein levels in the saliva of pregnant women increased in the second and the early third trimesters and decreased to the puerperal period. Salivary cortisol concentrations of the chronic high stress group were significantly lower compared with those of the normal group. Salivary chromogranin A/protein concentrations of the chronic high stress group were also significantly lower than those of the normal group. The titration of salivary cortisol concentrations and chromogranin A/protein levels is a useful tool to determine maternal stress levels. The elevation of cortisol and chromogranin A/protein in the saliva was suppressed in the chronic high stress group during pregnancy. © 2011 The Authors. Journal of Obstetrics and Gynaecology Research © 2011 Japan Society of Obstetrics and Gynecology.

Human Common Salivary Protein 1 (CSP-1) Promotes Binding of Streptococcus mutans to Experimental Salivary Pellicle and Glucans Formed on Hydroxyapatite Surface

PubMed Central

Ambatipudi, Kiran S.; Hagen, Fred K.; Delahunty, Claire M.; Han, Xuemei; Shafi, Rubina; Hryhorenko, Jennifer; Gregoire, Stacy; Marquis, Robert E.; Melvin, James E.; Koo, Hyun; Yates, John R.

2010-01-01

Summary The saliva proteome includes host defense factors and specific bacterial-binding proteins that modulate microbial growth and colonization of tooth surface in the oral cavity. A multidimensional mass spectrometry approach identified the major host-derived salivary proteins which interacted with Streptococcus mutans (strain UA159), the primary microorganism associated with the pathogenesis of dental caries. Two abundant host proteins were found to tightly bind to S. mutans cells, common salivary protein-1 (CSP-1) and deleted in malignant brain tumor 1 (DMBT1, also known as salivary agglutinin or gp340). In contrast to gp340, limited functional information is available on CSP-1. The sequence of CSP-1 shares 38.1% similarity with rat CSP-1. Recombinant CSP-1 (rCSP-1) protein did not cause aggregation of S. mutans cells and was devoid of any significant biocidal activity (2.5 to 10 μg/ml). However, S. mutans cells exposed to rCSP-1 (10 μg/ml) in saliva displayed enhanced adherence to experimental salivary pellicle and to glucans in the pellicle formed on hydroxyapatite surfaces. Thus, our data demonstrate that the host salivary protein CSP-1 binds to S. mutans cells and may influence the initial colonization of this pathogenic bacterium onto tooth surface. PMID:20858015

Distribution of serotonergic and dopaminergic nerve fibers in the salivary gland complex of the cockroach Periplaneta americana

PubMed Central

Baumann, Otto; Dames, Petra; Kühnel, Dana; Walz, Bernd

2002-01-01

Background The cockroach salivary gland consists of secretory acini with peripheral ion-transporting cells and central protein-producing cells, an extensive duct system, and a pair of reservoirs. Salivation is controled by serotonergic and dopaminergic innervation. Serotonin stimulates the secretion of a protein-rich saliva, dopamine causes the production of a saliva without proteins. These findings suggest a model in which serotonin acts on the central cells and possibly other cell types, and dopamine acts selectively on the ion-transporting cells. To examine this model, we have analyzed the spatial relationship of dopaminergic and serotonergic nerve fibers to the various cell types. Results The acinar tissue is entangled in a meshwork of serotonergic and dopaminergic varicose fibers. Dopaminergic fibers reside only at the surface of the acini next to the peripheral cells. Serotonergic fibers invade the acini and form a dense network between central cells. Salivary duct segments close to the acini are locally associated with dopaminergic and serotonergic fibers, whereas duct segments further downstream have only dopaminergic fibers on their surface and within the epithelium. In addition, the reservoirs have both a dopaminergic and a serotonergic innervation. Conclusion Our results suggest that dopamine is released on the acinar surface, close to peripheral cells, and along the entire duct system. Serotonin is probably released close to peripheral and central cells, and at initial segments of the duct system. Moreover, the presence of serotonergic and dopaminergic fiber terminals on the reservoir indicates that the functions of this structure are also regulated by dopamine and serotonin. PMID:12095424

Distribution of serotonergic and dopaminergic nerve fibers in the salivary gland complex of the cockroach Periplaneta americana.

PubMed

Baumann, Otto; Dames, Petra; Kühnel, Dana; Walz, Bernd

2002-06-24

The cockroach salivary gland consists of secretory acini with peripheral ion-transporting cells and central protein-producing cells, an extensive duct system, and a pair of reservoirs. Salivation is controlled by serotonergic and dopaminergic innervation. Serotonin stimulates the secretion of a protein-rich saliva, dopamine causes the production of a saliva without proteins. These findings suggest a model in which serotonin acts on the central cells and possibly other cell types, and dopamine acts selectively on the ion-transporting cells. To examine this model, we have analyzed the spatial relationship of dopaminergic and serotonergic nerve fibers to the various cell types. The acinar tissue is entangled in a meshwork of serotonergic and dopaminergic varicose fibers. Dopaminergic fibers reside only at the surface of the acini next to the peripheral cells. Serotonergic fibers invade the acini and form a dense network between central cells. Salivary duct segments close to the acini are locally associated with dopaminergic and serotonergic fibers, whereas duct segments further downstream have only dopaminergic fibers on their surface and within the epithelium. In addition, the reservoirs have both a dopaminergic and a serotonergic innervation. Our results suggest that dopamine is released on the acinar surface, close to peripheral cells, and along the entire duct system. Serotonin is probably released close to peripheral and central cells, and at initial segments of the duct system. Moreover, the presence of serotonergic and dopaminergic fiber terminals on the reservoir indicates that the functions of this structure are also regulated by dopamine and serotonin.

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

Expression, refolding and crystallizations of the Grb2-like (GADS) C-terminal SH3 domain complexed with a SLP-76 motif peptide

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

Faravelli, Alessandro; Dimasi, Nazzareno, E-mail: ndimasi@gmail.com

Several crystals of the Grb2-like C-terminal SH3 domain in complex with a motif peptide from the SLP-76 protein were obtained and characterized. The Grb2-like adaptor protein GADS is composed of an N-terminal SH3 domain, an SH2 domain, a proline-rich region and a C-terminal SH3 domain. GADS interacts through its C-terminal SH3 domain with the adaptor protein SLP-76, thus recruiting this protein and other associated molecules to the linker for activation of T-cell (LAT) protein. The DNA encoding the C-terminal SH3 domain of GADS (GADS-cSH3) was assembled synthetically using a recursive PCR technique and the protein was overexpressed in Escherichia coli,more » refolded and purified. Several crystals of this domain in complex with the SLP-76 peptide were obtained and characterized.« less

A high affinity RIM-binding protein/Aplip1 interaction prevents the formation of ectopic axonal active zones.

PubMed

Siebert, Matthias; Böhme, Mathias A; Driller, Jan H; Babikir, Husam; Mampell, Malou M; Rey, Ulises; Ramesh, Niraja; Matkovic, Tanja; Holton, Nicole; Reddy-Alla, Suneel; Göttfert, Fabian; Kamin, Dirk; Quentin, Christine; Klinedinst, Susan; Andlauer, Till Fm; Hell, Stefan W; Collins, Catherine A; Wahl, Markus C; Loll, Bernhard; Sigrist, Stephan J

2015-08-14

Synaptic vesicles (SVs) fuse at active zones (AZs) covered by a protein scaffold, at Drosophila synapses comprised of ELKS family member Bruchpilot (BRP) and RIM-binding protein (RBP). We here demonstrate axonal co-transport of BRP and RBP using intravital live imaging, with both proteins co-accumulating in axonal aggregates of several transport mutants. RBP, via its C-terminal Src-homology 3 (SH3) domains, binds Aplip1/JIP1, a transport adaptor involved in kinesin-dependent SV transport. We show in atomic detail that RBP C-terminal SH3 domains bind a proline-rich (PxxP) motif of Aplip1/JIP1 with submicromolar affinity. Pointmutating this PxxP motif provoked formation of ectopic AZ-like structures at axonal membranes. Direct interactions between AZ proteins and transport adaptors seem to provide complex avidity and shield synaptic interaction surfaces of pre-assembled scaffold protein transport complexes, thus, favouring physiological synaptic AZ assembly over premature assembly at axonal membranes.

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

Hoekstra, M.F.; Ou, A.C.; DeMaggio, A.J.

In simple eukaryotes, protein kinases regulate mitotic and meiotic cell cycles, the response to polypeptide pheromones, and the initiation of nuclear DNA synthesis. The protein HRR25 from the budding yeast Saccharomyces cerevisiae was defined by the mutation hrr25-1. This mutation resulted in sensitivity to continuous expression of the HO double-strand endonuclease, to methyl methanesulfonate, and to x-irradiation. Homozygotes of hrr25-1 were unable to sporulate and disruption and deletion of HRR25 interfered with mitotic and meiotic cell division. Sequence analysis revealed two distinctive regions in the protein. The NH{sub 2}-terminus of HRR25 contains the hallmark features of protein kinases, whereas themore » COOH-terminus is rich in proline and glutamine. Mutations in HRR25 at conserved residues found in all protein kinases inactivated the gene, and these mutants exhibited the hrr25 null phenotypes. Taken together, the hrr25 mutant phenotypes and the features of the gene product indicate that HRR25 is a distinctive member of the protein kinase superfamily.« less

A deep insight into the sialotranscriptome of the mosquito, Psorophora albipes

PubMed Central

2013-01-01

Background Psorophora mosquitoes are exclusively found in the Americas and have been associated with transmission of encephalitis and West Nile fever viruses, among other arboviruses. Mosquito salivary glands represent the final route of differentiation and transmission of many parasites. They also secrete molecules with powerful pharmacologic actions that modulate host hemostasis, inflammation, and immune response. Here, we employed next generation sequencing and proteome approaches to investigate for the first time the salivary composition of a mosquito member of the Psorophora genus. We additionally discuss the evolutionary position of this mosquito genus into the Culicidae family by comparing the identity of its secreted salivary compounds to other mosquito salivary proteins identified so far. Results Illumina sequencing resulted in 13,535,229 sequence reads, which were assembled into 3,247 contigs. All families were classified according to their in silico-predicted function/ activity. Annotation of these sequences allowed classification of their products into 83 salivary protein families, twenty (24.39%) of which were confirmed by our subsequent proteome analysis. Two protein families were deorphanized from Aedes and one from Ochlerotatus, while four protein families were described as novel to Psorophora genus because they had no match with any other known mosquito salivary sequence. Several protein families described as exclusive to Culicines were present in Psorophora mosquitoes, while we did not identify any member of the protein families already known as unique to Anophelines. Also, the Psorophora salivary proteins had better identity to homologs in Aedes (69.23%), followed by Ochlerotatus (8.15%), Culex (6.52%), and Anopheles (4.66%), respectively. Conclusions This is the first sialome (from the Greek sialo = saliva) catalog of salivary proteins from a Psorophora mosquito, which may be useful for better understanding the lifecycle of this mosquito and the role of its salivary secretion in arboviral transmission. PMID:24330624

The CDM Superfamily Protein MBC Directs Myoblast Fusion through a Mechanism That Requires Phosphatidylinositol 3,4,5-Triphosphate Binding but Is Independent of Direct Interaction with DCrk▿§

PubMed Central

Balagopalan, Lakshmi; Chen, Mei-Hui; Geisbrecht, Erika R.; Abmayr, Susan M.

2006-01-01

myoblast city (mbc), a member of the CDM superfamily, is essential in the Drosophila melanogaster embryo for fusion of myoblasts into multinucleate fibers. Using germ line clones in which both maternal and zygotic contributions were eliminated and rescue of the zygotic loss-of-function phenotype, we established that mbc is required in the fusion-competent subset of myoblasts. Along with its close orthologs Dock180 and CED-5, MBC has an SH3 domain at its N terminus, conserved internal domains termed DHR1 and DHR2 (or “Docker”), and C-terminal proline-rich domains that associate with the adapter protein DCrk. The importance of these domains has been evaluated by the ability of MBC mutations and deletions to rescue the mbc loss-of-function muscle phenotype. We demonstrate that the SH3 and Docker domains are essential. Moreover, ethyl methanesulfonate-induced mutations that change amino acids within the MBC Docker domain to residues that are conserved in other CDM family members nevertheless eliminate MBC function in the embryo, which suggests that these sites may mediate interactions specific to Drosophila MBC. A functional requirement for the conserved DHR1 domain, which binds to phosphatidylinositol 3,4,5-triphosphate, implicates phosphoinositide signaling in myoblast fusion. Finally, the proline-rich C-terminal sites mediate strong interactions with DCrk, as expected. These sites are not required for MBC to rescue the muscle loss-of-function phenotype, however, which suggests that MBC's role in myoblast fusion can be carried out independently of direct DCrk binding. PMID:17030600

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

The scientific exploration of saliva in the post-proteomic era: from database back to basic function

PubMed Central

Ruhl, Stefan

2012-01-01

The proteome of human saliva can be considered as being essentially completed. Diagnostic markers for a number of diseases have been identified among salivary proteins and peptides, taking advantage of saliva as an easy-to-obtain biological fluid. Yet, the majority of disease markers identified so far are serum components and not intrinsic proteins produced by the salivary glands. Furthermore, despite the fact that saliva is essential for protecting the oral integuments and dentition, little progress has been made in finding risk predictors in the salivary proteome for dental caries or periodontal disease. Since salivary proteins, and in particular the attached glycans, play an important role in interactions with the microbial world, the salivary glycoproteome and other post-translational modifications of salivary proteins need to be studied. Risk markers for microbial diseases, including dental caries, are likely to be discovered among the highly glycosylated major protein species in saliva. This review will attempt to raise new ideas and also point to under-researched areas that may hold promise for future applicability in oral diagnostics and prediction of oral disease. PMID:22292826

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

Systematic Analysis of Intracellular-targeting Antimicrobial Peptides, Bactenecin 7, Hybrid of Pleurocidin and Dermaseptin, Proline-Arginine-rich Peptide, and Lactoferricin B, by Using Escherichia coli Proteome Microarrays.

PubMed

Ho, Yu-Hsuan; Shah, Pramod; Chen, Yi-Wen; Chen, Chien-Sheng

2016-06-01

Antimicrobial peptides (AMPs) act either through membrane lysis or by attacking intracellular targets. Intracellular targeting AMPs are a resource for antimicrobial agent development. Several AMPs have been identified as intracellular targeting peptides; however, the intracellular targets of many of these peptides remain unknown. In the present study, we used an Escherichia coli proteome microarray to systematically identify the protein targets of three intracellular targeting AMPs: bactenecin 7 (Bac7), a hybrid of pleurocidin and dermaseptin (P-Der), and proline-arginine-rich peptide (PR-39). In addition, we also included the data of lactoferricin B (LfcinB) from our previous study for a more comprehensive analysis. We analyzed the unique protein hits of each AMP in the Kyoto Encyclopedia of Genes and Genomes. The results indicated that Bac7 targets purine metabolism and histidine kinase, LfcinB attacks the transcription-related activities and several cellular carbohydrate biosynthetic processes, P-Der affects several catabolic processes of small molecules, and PR-39 preferentially recognizes proteins involved in RNA- and folate-metabolism-related cellular processes. Moreover, both Bac7 and LfcinB target purine metabolism, whereas LfcinB and PR-39 target lipopolysaccharide biosynthesis. This suggested that LfcinB and Bac7 as well as LfcinB and PR-39 have a synergistic effect on antimicrobial activity, which was validated through antimicrobial assays. Furthermore, common hits of all four AMPs indicated that all of them target arginine decarboxylase, which is a crucial enzyme for Escherichia coli survival in extremely acidic environments. Thus, these AMPs may display greater inhibition to bacterial growth in extremely acidic environments. We have also confirmed this finding in bacterial growth inhibition assays. In conclusion, this comprehensive identification and systematic analysis of intracellular targeting AMPs reveals crucial insights into the intracellular mechanisms of the action of AMPs. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Apoptotic actions of p53 require transcriptional activation of PUMA and do not involve a direct mitochondrial/cytoplasmic site of action in postnatal cortical neurons.

PubMed

Uo, Takuma; Kinoshita, Yoshito; Morrison, Richard S

2007-11-07

Recent studies in non-neuronal cells have shown that the tumor suppressor p53 can promote cell death through a transcription-independent mechanism involving its direct action with a subset of Bcl-2 family member proteins in the cytosol and at the mitochondria. In cultured cortical neurons, however, we could not find evidence supporting a significant contribution of the cytosolic/mitochondrial p53 pathway, and available evidence instead corroborated the requirement for the transcriptional activity of p53. When directly targeted to the cytosol/mitochondria, wild-type p53 lost its apoptosis-inducing activity in neurons but not in non-neuronal cells. The N-terminal p53 fragment (transactivation and proline-rich domains), which induces apoptosis in non-neuronal cells via the cytosolic/mitochondrial pathway, displayed no apoptogenic activity in neurons. In neuronal apoptosis induced by camptothecin or an MDM2 (murine double minute 2) inhibitor, nutlin-3, endogenous p53 protein did not accumulate in the cytosol/mitochondria, and transcriptional inhibition after p53 induction effectively blocked cell death. In addition, overexpression of a dominant-negative form of p53 (R273H) completely suppressed induction of proapoptotic p53 target genes and cell death. PUMA (p53-upregulated modulator of apoptosis) was one such gene induced by camptothecin, and its overexpression was sufficient to induce Bax (Bcl-2-associated X protein)-dependent neuronal death, whereas Noxa was not apoptogenic. These results collectively demonstrate that, in contrast to non-neuronal cells, the apoptotic activity of p53 in postnatal cortical neurons does not rely on its direct action at the cytosol/mitochondria but is exclusively mediated through its transcription-dependent functions. The uniqueness of p53-mediated apoptotic signaling in postnatal cortical neurons was further illustrated by the dispensable function of the proline-rich domain of p53.

Profiling of Human Acquired Immunity Against the Salivary Proteins of Phlebotomus papatasi Reveals Clusters of Differential Immunoreactivity

PubMed Central

Geraci, Nicholas S.; Mukbel, Rami M.; Kemp, Michael T.; Wadsworth, Mariha N.; Lesho, Emil; Stayback, Gwen M.; Champion, Matthew M.; Bernard, Megan A.; Abo-Shehada, Mahmoud; Coutinho-Abreu, Iliano V.; Ramalho-Ortigão, Marcelo; Hanafi, Hanafi A.; Fawaz, Emadeldin Y.; El-Hossary, Shabaan S.; Wortmann, Glenn; Hoel, David F.; McDowell, Mary Ann

2014-01-01

Phlebotomus papatasi sand flies are among the primary vectors of Leishmania major parasites from Morocco to the Indian subcontinent and from southern Europe to central and eastern Africa. Antibody-based immunity to sand fly salivary gland proteins in human populations remains a complex contextual problem that is not yet fully understood. We profiled the immunoreactivities of plasma antibodies to sand fly salivary gland sonicates (SGSs) from 229 human blood donors residing in different regions of sand fly endemicity throughout Jordan and Egypt as well as 69 US military personnel, who were differentially exposed to P. papatasi bites and L. major infections in Iraq. Compared with plasma from control region donors, antibodies were significantly immunoreactive to five salivary proteins (12, 26, 30, 38, and 44 kDa) among Jordanian and Egyptian donors, with immunoglobulin G4 being the dominant anti-SGS isotype. US personnel were significantly immunoreactive to only two salivary proteins (38 and 14 kDa). Using k-means clustering, donors were segregated into four clusters distinguished by unique immunoreactivity profiles to varying combinations of the significantly immunogenic salivary proteins. SGS-induced cellular proliferation was diminished among donors residing in sand fly-endemic regions. These data provide a clearer picture of human immune responses to sand fly vector salivary constituents. PMID:24615125

The Role of Circulating Amino Acids in the Hypothalamic Regulation of Liver Glucose Metabolism123

PubMed Central

Arrieta-Cruz, Isabel; Gutiérrez-Juárez, Roger

2016-01-01

A pandemic of diabetes and obesity has been developing worldwide in close association with excessive nutrient intake and a sedentary lifestyle. Variations in the protein content of the diet have a direct impact on glucose homeostasis because amino acids (AAs) are powerful modulators of insulin action. In this work we review our recent findings on how elevations in the concentration of the circulating AAs leucine and proline activate a metabolic mechanism located in the mediobasal hypothalamus of the brain that sends a signal to the liver via the vagus nerve, which curtails glucose output. This neurogenic signal is strictly dependent on the metabolism of leucine and proline to acetyl-coenzyme A (CoA) and the subsequent production of malonyl-CoA; the signal also requires functional neuronal ATP-sensitive potassium channels. The liver then responds by lowering the rate of gluconeogenesis and glycogenolysis, ultimately leading to a net decrease in glucose production and in concentrations of circulating glucose. Furthermore, we review here how our work with proline suggests a new role of astrocytes in the central regulation of glycemia. Last, we outline how factors such as the consumption of fat-rich diets can interfere with glucoregulatory mechanisms and, in the long term, may contribute to the development of hyperglycemia, a hallmark of type 2 diabetes. PMID:27422516

The Role of Circulating Amino Acids in the Hypothalamic Regulation of Liver Glucose Metabolism.

PubMed

Arrieta-Cruz, Isabel; Gutiérrez-Juárez, Roger

2016-07-01

A pandemic of diabetes and obesity has been developing worldwide in close association with excessive nutrient intake and a sedentary lifestyle. Variations in the protein content of the diet have a direct impact on glucose homeostasis because amino acids (AAs) are powerful modulators of insulin action. In this work we review our recent findings on how elevations in the concentration of the circulating AAs leucine and proline activate a metabolic mechanism located in the mediobasal hypothalamus of the brain that sends a signal to the liver via the vagus nerve, which curtails glucose output. This neurogenic signal is strictly dependent on the metabolism of leucine and proline to acetyl-coenzyme A (CoA) and the subsequent production of malonyl-CoA; the signal also requires functional neuronal ATP-sensitive potassium channels. The liver then responds by lowering the rate of gluconeogenesis and glycogenolysis, ultimately leading to a net decrease in glucose production and in concentrations of circulating glucose. Furthermore, we review here how our work with proline suggests a new role of astrocytes in the central regulation of glycemia. Last, we outline how factors such as the consumption of fat-rich diets can interfere with glucoregulatory mechanisms and, in the long term, may contribute to the development of hyperglycemia, a hallmark of type 2 diabetes. © 2016 American Society for Nutrition.

Transcriptomic Analysis of the Salivary Glands of an Invasive Whitefly

PubMed Central

Su, Yun-Lin; Li, Jun-Min; Li, Meng; Luan, Jun-Bo; Ye, Xiao-Dong; Wang, Xiao-Wei; Liu, Shu-Sheng

2012-01-01

Background Some species of the whitefly Bemisia tabaci complex cause tremendous losses to crops worldwide through feeding directly and virus transmission indirectly. The primary salivary glands of whiteflies are critical for their feeding and virus transmission. However, partly due to their tiny size, research on whitefly salivary glands is limited and our knowledge on these glands is scarce. Methodology/Principal Findings We sequenced the transcriptome of the primary salivary glands of the Mediterranean species of B. tabaci complex using an effective cDNA amplification method in combination with short read sequencing (Illumina). In a single run, we obtained 13,615 unigenes. The quantity of the unigenes obtained from the salivary glands of the whitefly is at least four folds of the salivary gland genes from other plant-sucking insects. To reveal the functions of the primary glands, sequence similarity search and comparisons with the whole transcriptome of the whitefly were performed. The results demonstrated that the genes related to metabolism and transport were significantly enriched in the primary salivary glands. Furthermore, we found that a number of highly expressed genes in the salivary glands might be involved in secretory protein processing, secretion and virus transmission. To identify potential proteins of whitefly saliva, the translated unigenes were put into secretory protein prediction. Finally, 295 genes were predicted to encode secretory proteins and some of them might play important roles in whitefly feeding. Conclusions/Significance: The combined method of cDNA amplification, Illumina sequencing and de novo assembly is suitable for transcriptomic analysis of tiny organs in insects. Through analysis of the transcriptome, genomic features of the primary salivary glands were dissected and biologically important proteins, especially secreted proteins, were predicted. Our findings provide substantial sequence information for the primary salivary glands of whiteflies and will be the basis for future studies on whitefly-plant interactions and virus transmission. PMID:22745728

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.

MDC9, a widely expressed cellular disintegrin containing cytoplasmic SH3 ligand domains

PubMed Central

1996-01-01

Cellular disintegrins are a family of proteins that are related to snake venom integrin ligands and metalloproteases. We have cloned and sequenced the mouse and human homologue of a widely expressed cellular disintegrin, which we have termed MDC9 (for metalloprotease/disintegrin/cysteine-rich protein 9). The deduced mouse and human protein sequences are 82% identical. MDC9 contains several distinct protein domains: a signal sequence is followed by a prodomain and a domain with sequence similarity to snake venom metalloproteases, a disintegrin domain, a cysteine-rich region, an EGF repeat, a membrane anchor, and a cytoplasmic tail. The cytoplasmic tail of MDC9 has two proline-rich sequences which can bind the SH3 domain of Src, and may therefore function as SH3 ligand domains. Western blot analysis shows that MDC9 is an approximately 84-kD glycoprotein in all mouse tissues examined, and in NIH 3T3 fibroblast and C2C12 myoblast mouse cell lines. MDC9 can be both cell surface biotinylated and 125I-labeled in NIH 3T3 mouse fibroblasts, indicating that the protein is present on the plasma membrane. Expression of MDC9 in COS-7 cells yields an 84-kD protein, and immunofluorescence analysis of COS-7 cells expressing MDC9 shows a staining pattern that is consistent with a plasma membrane localization. The apparent molecular mass of 84 kD suggests that MDC9 contains a membrane-anchored metalloprotease and disintegrin domain. We propose that MDC9 might function as a membrane-anchored integrin ligand or metalloprotease, or that MDC9 may combine both activities in one protein. PMID:8647900

Centromeric binding and activity of Protein Phosphatase 4

PubMed Central

Lipinszki, Zoltan; Lefevre, Stephane; Savoian, Matthew S.; Singleton, Martin R.; Glover, David M.; Przewloka, Marcin R.

2015-01-01

The cell division cycle requires tight coupling between protein phosphorylation and dephosphorylation. However, understanding the cell cycle roles of multimeric protein phosphatases has been limited by the lack of knowledge of how their diverse regulatory subunits target highly conserved catalytic subunits to their sites of action. Phosphoprotein phosphatase 4 (PP4) has been recently shown to participate in the regulation of cell cycle progression. We now find that the EVH1 domain of the regulatory subunit 3 of Drosophila PP4, Falafel (Flfl), directly interacts with the centromeric protein C (CENP-C). Unlike other EVH1 domains that interact with proline-rich ligands, the crystal structure of the Flfl amino-terminal EVH1 domain bound to a CENP-C peptide reveals a new target-recognition mode for the phosphatase subunit. We also show that binding of Flfl to CENP-C is required to bring PP4 activity to centromeres to maintain CENP-C and attached core kinetochore proteins at chromosomes during mitosis. PMID:25562660

The U24 Protein from Human Herpesvirus 6 and 7 Affects Endocytic Recycling▿

PubMed Central

Sullivan, Brian M.; Coscoy, Laurent

2010-01-01

Modulation of T-cell receptor expression and signaling is essential to the survival of many viruses. The U24 protein expressed by human herpesvirus 6A, a ubiquitous human pathogen, has been previously shown to downregulate the T-cell receptor. Here, we show that U24 also mediates cell surface downregulation of a canonical early endosomal recycling receptor, the transferrin receptor, indicating that this viral protein acts by blocking early endosomal recycling. We present evidence that U24 is a C-tail-anchored protein that is dependent for its function on TRC40/Asna-1, a component of a posttranslational membrane insertion pathway. Finally, we find that U24 proteins from other roseoloviruses have a similar genetic organization and a conserved function that is dependent on a proline-rich motif. Inhibition of a basic cellular process by U24 has interesting implications not only for the pathogenicity of roseoloviruses but also for our understanding of the biology of endosomal transport. PMID:19923186

Affinity chromatography for purification of the modular protein growth factor receptor-bound protein 2 and development of a screening test for growth factor receptor-bound protein 2 Src homology 3 domain inhibitor using peroxidase-linked ligand.

PubMed

Gril, B; Liu, W Q; Lenoir, C; Garbay, C; Vidal, M

2006-04-01

Growth factor receptor-bound protein 2 (Grb2) is an adapter protein involved in the Ras-dependent signaling pathway that plays an important role in human cancers initiated by oncogenic receptors. Grb2 is constituted by one Src homology 2 domain surrounded by two SH3 domains, and the inhibition of the interactions produced by these domains could provide an antitumor approach. In evaluating chemical libraries, to search for potential Grb2 inhibitors, it was necessary to elaborate a rapid test for their screening. We have developed, first, a batch method based on the use of an affinity column bearing a Grb2-SH3 peptide ligand to isolate highly purified Grb2. We subsequently describe a very rapid 96-well screening of inhibitors based on a simple competition between purified Grb2 and a peroxidase-coupled proline-rich peptide.

A further study on the dietary-regulated biosynthesis of high-sulphur wool proteins

PubMed Central

Gillespie, J. M.; Broad, Andrea; Reis, P. J.

1969-01-01

When the diet of sheep is supplemented by the infusion of sulphur-containing amino acids or casein into the abomasum, the newly synthesized wool shows characteristic changes in its amino acid composition, with significant increases in cystine, proline and serine and decreases in aspartic acid and phenylalanine. This modification seems to be due entirely to an alteration in the overall composition of the high-sulphur proteins and to an increase in their proportion in the fibre. These variations are not the result of a change in the composition of individual proteins, but are due to alterations in their relative proportions and to the initiation of the synthesis of `new' proteins, many of which are extremely rich in cystine. It is suggested that the heterogeneity of the high-sulphur proteins may be due, in part, to similar changes in composition caused by natural variations in the nutrition of sheep. ImagesFig. 3.Fig. 4. PMID:5774505

Plasmodium vivax: a monoclonal antibody recognizes a circumsporozoite protein precursor on the sporozoite surface.

PubMed

Gonzalez-Ceron, L; Rodriguez, M H; Wirtz, R A; Sina, B J; Palomeque, O L; Nettel, J A; Tsutsumi, V

1998-11-01

The major surface circumsporozoite (CS) proteins are known to play a role in malaria sporozoite development and invasion of invertebrate and vertebrate host cells. Plasmodium vivax CS protein processing during mosquito midgut oocyst and salivary gland sporozoite development was studied using monoclonal antibodies which recognize different CS protein epitopes. Monoclonal antibodies which react with the CS amino acid repeat sequences by ELISA recognized a 50-kDa precursor protein in immature oocyst and additional 47- and 42-kDa proteins in older oocysts. A 42-kDa CS protein was detected after initial sporozoite invasion of mosquito salivary glands and an additional 50-kDa precursor CS protein observed later in infected salivary glands. These data confirm previous results with other Plasmodium species, in which more CS protein precursors were detected in oocysts than in salivary gland sporozoites. A monoclonal antibody (PvPCS) was characterized which reacts with an epitope found only in the 50-kDa precursor CS protein. PvPCS reacted with all P. vivax sporozoite strains tested by indirect immunofluorescent assay, homogeneously staining the sporozoite periphery with much lower intensity than that produced by anti-CS repeat antibodies. Immunoelectron microscopy using PvPCS showed that the CS protein precursor was associated with peripheral cytoplasmic vacuoles and membranes of sporoblast and budding sporozoites in development oocysts. In salivary gland sporozoites, the CS protein precursor was primarily associated with micronemes and sporozoite membranes. Our results suggest that the 50-kDa CS protein precursor is synthesized intracellularly and secreted on the membrane surface, where it is proteolytically processed to form the 42-kDa mature CS protein. These data indicate that differences in CS protein processing in oocyst and salivary gland sporozoites development may occur. Copyright 1998 Academic Press.

Salivary protein adsorption and Streptococccus gordonii adhesion to dental material surfaces.

PubMed

Schweikl, Helmut; Hiller, Karl-Anton; Carl, Ulrich; Schweiger, Rainer; Eidt, Andreas; Ruhl, Stefan; Müller, Rainer; Schmalz, Gottfried

2013-10-01

The initial adhesion of microorganisms to clinically used dental biomaterials is influenced by physico-chemical parameters like hydrophobicity and pre-adsorption of salivary proteins. Here, polymethyl methacrylate (PMMA), polyethylene (PE), polytetrafluoroethylene (PTFE), silicone (Mucopren soft), silorane-based (Filtek Silorane) and methacrylate-based (Tetric EvoCeram) dental composites, a conventional glassionomer cement as well as cobalt-chromium-molybdenum (Co28Cr6Mo) and titanium (Ti6Al4V) were tested for adsorption of salivary proteins and adhesion of Streptococcus gordonii DL1. Wettability of material surfaces precoated with salivary proteins or left in phosphate-buffered saline was determined by the measurement of water contact angles. Amounts of adsorbed proteins were determined directly on material surfaces after biotinylation of amino groups and detection by horseradish peroxidase-conjugated avidin-D. The same technique was used to analyze for the binding of biotinylated bacteria to material surfaces. The highest amount of proteins (0.18μg/cm(2)) adsorbed to hydrophobic PTFE samples, and the lowest amount (0.025μg/cm(2)) was detected on silicone. The highest number of S. gordonii (3.2×10(4)CFU/mm(2)) adhered to the hydrophilic glassionomer cement surface coated with salivary proteins, and the lowest number (4×10(3)CFU/mm(2)) was found on the hydrophobic silorane-based composite. Hydrophobicity of pure material surfaces and the number of attached microorganisms were weakly negatively correlated. No such correlation between hydrophobicity and the number of bacteria was detected when surfaces were coated with salivary proteins. Functional groups added by the adsorption of specific salivary proteins to material surfaces are more relevant for initial bacterial adhesion than hydrophobicity as a physical property. Copyright © 2013 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Role of BAG3 in cancer progression: A therapeutic opportunity.

PubMed

De Marco, Margot; Basile, Anna; Iorio, Vittoria; Festa, Michelina; Falco, Antonia; Ranieri, Bianca; Pascale, Maria; Sala, Gianluca; Remondelli, Paolo; Capunzo, Mario; Firpo, Matthew A; Pezzilli, Raffaele; Marzullo, Liberato; Cavallo, Pierpaolo; De Laurenzi, Vincenzo; Turco, Maria Caterina; Rosati, Alessandra

2018-06-01

BAG3 is a multifunctional protein that can bind to heat shock proteins (Hsp) 70 through its BAG domain and to other partners through its WW domain, proline-rich (PXXP) repeat and IPV (Ile-Pro-Val) motifs. Its intracellular expression can be induced by stressful stimuli, while is constitutive in skeletal muscle, cardiac myocytes and several tumour types. BAG3 can modulate the levels, localisation or activity of its partner proteins, thereby regulating major cell pathways and functions, including apoptosis, autophagy, mechanotransduction, cytoskeleton organisation, motility. A secreted form of BAG3 has been identified in studies on pancreatic ductal adenocarcinoma (PDAC). Secreted BAG3 can bind to a specific receptor, IFITM2, expressed on macrophages, and induce the release of factors that sustain tumour growth and the metastatic process. BAG3 neutralisation therefore appears to constitute a novel potential strategy in the therapy of PDAC and, possibly, other tumours. Copyright © 2017 Elsevier Ltd. All rights reserved.

Molecular organization of excitatory chemical synapses in the mammalian brain

NASA Astrophysics Data System (ADS)

Gundelfinger, E. D.; tom Dieck, S.

Chemical synapses are highly specialized cell-cell junctions designed for efficient signaling between nerve cells. Distinct cytoskeletal matrices are assembled at either side of the synaptic junction. The presynaptic cytomatrix at the active zone (CAZ) defines and organizes the site of neurotransmitter release from presynaptic nerve terminals. The postsynaptic density (PSD) tethers neurotransmitter receptors and the postsynaptic signal transduction machinery. Recent progress in the identification and characterization of novel CAZ and PSD components has revealed new insights into the molecular organization and assembly mechanisms of the synaptic neurotransmission apparatus. On the presynaptic side, Bassoon and Piccolo, two related giant proteins, are crucially involved in scaffolding the CAZ. On the postsynaptic side, two families of multi-domain adaptor proteins, the MAGuKs (membrane-associated guanylate kinase homologs) and the ProSAP (proline-rich synapse-associated protein, also termed Shank) family members are thought to be major organizing molecules of the PSD.

Molecular Physiology of SPAK and OSR1: Two Ste20-Related Protein Kinases Regulating Ion Transport

PubMed Central

Gagnon, Kenneth B.; Delpire, Eric

2015-01-01

SPAK (Ste20-related proline alanine rich kinase) and OSR1 (oxidative stress responsive kinase) are members of the germinal center kinase VI sub-family of the mammalian Ste20 (Sterile20)-related protein kinase family. Although there are 30 enzymes in this protein kinase family, their conservation across the fungi, plant and animal kingdom confirms their evolutionary importance. Already, a large volume of work has accumulated on the tissue distribution, binding partners, signaling cascades, and physiological roles of mammalian SPAK and OSR1 in multiple organ systems. After reviewing this basic information, we will examine newer studies that demonstrate the pathophysiological consequences to SPAK and/or OSR1 disruption, discuss the development and analysis of genetically-engineered mouse models, and address the possible role these serine/threonine kinases might have in cancer proliferation and migration. PMID:23073627

SOME PROPERTIES OF HYALIN

PubMed Central

Stephens, R. E.; Kane, R. E.

1970-01-01

The principal protein component of the hyaline layer of sea urchin eggs is the calcium-insoluble protein first described by Kane and Hersh. The protein hyalin is abnormally high in acidic amino acids, almost devoid of basic amino acids, and characteristically rich in valine and proline. Essentially all of the cysteine present is found in the disulfide form; no evidence points to intermolecular disulfide linkages. Hyalin from several species has a minimal subunit weight of about 100,000, though evidence exists for a particle three times this weight in urea or guanidine hydrochloride from one species. Optical rotatory dispersion measurements indicate no α-helix content, though the dispersion has unique characteristic features. Addition of small quantities of calcium causes hyalin to gel to a birefringent fibrous form. The fibrous, birefringent form of hyalin is rendered isotropic upon addition of EDTA, but the birefringence is restored with re-addition of divalent cation. PMID:4190067

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.

Molecular characterization of Trypanosoma cruzi SAP proteins with host-cell lysosome exocytosis-inducing activity required for parasite invasion.

PubMed

Zanforlin, Tamiris; Bayer-Santos, Ethel; Cortez, Cristian; Almeida, Igor C; Yoshida, Nobuko; da Silveira, José Franco

2013-01-01

To invade target cells, Trypanosoma cruzi metacyclic forms engage distinct sets of surface and secreted molecules that interact with host components. Serine-, alanine-, and proline-rich proteins (SAP) comprise a multigene family constituted of molecules with a high serine, alanine and proline residue content. SAP proteins have a central domain (SAP-CD) responsible for interaction with and invasion of mammalian cells by metacyclic forms. Using a 513 bp sequence from SAP-CD in blastn analysis, we identified 39 full-length SAP genes in the genome of T. cruzi. Although most of these genes were mapped in the T. cruzi in silico chromosome TcChr41, several SAP sequences were spread out across the genome. The level of SAP transcripts was twice as high in metacyclic forms as in epimastigotes. Monoclonal (MAb-SAP) and polyclonal (anti-SAP) antibodies produced against the recombinant protein SAP-CD were used to investigate the expression and localization of SAP proteins. MAb-SAP reacted with a 55 kDa SAP protein released by epimastigotes and metacyclic forms and with distinct sets of SAP variants expressed in amastigotes and tissue culture-derived trypomastigotes (TCTs). Anti-SAP antibodies reacted with components located in the anterior region of epimastigotes and between the nucleus and the kinetoplast in metacyclic trypomastigotes. In contrast, anti-SAP recognized surface components of amastigotes and TCTs, suggesting that SAP proteins are directed to different cellular compartments. Ten SAP peptides were identified by mass spectrometry in vesicle and soluble-protein fractions obtained from parasite conditioned medium. Using overlapping sequences from SAP-CD, we identified a 54-aa peptide (SAP-CE) that was able to induce host-cell lysosome exocytosis and inhibit parasite internalization by 52%. This study provides novel information about the genomic organization, expression and cellular localization of SAP proteins and proposes a triggering role for extracellular SAP proteins in host-cell lysosome exocytosis during metacyclic internalization.

Molecular Characterization of Trypanosoma cruzi SAP Proteins with Host-Cell Lysosome Exocytosis-Inducing Activity Required for Parasite Invasion

PubMed Central

Zanforlin, Tamiris; Bayer-Santos, Ethel; Cortez, Cristian; Almeida, Igor C.; Yoshida, Nobuko; da Silveira, José Franco

2013-01-01

Background To invade target cells, Trypanosoma cruzi metacyclic forms engage distinct sets of surface and secreted molecules that interact with host components. Serine-, alanine-, and proline-rich proteins (SAP) comprise a multigene family constituted of molecules with a high serine, alanine and proline residue content. SAP proteins have a central domain (SAP-CD) responsible for interaction with and invasion of mammalian cells by metacyclic forms. Methods and Findings Using a 513 bp sequence from SAP-CD in blastn analysis, we identified 39 full-length SAP genes in the genome of T. cruzi. Although most of these genes were mapped in the T. cruzi in silico chromosome TcChr41, several SAP sequences were spread out across the genome. The level of SAP transcripts was twice as high in metacyclic forms as in epimastigotes. Monoclonal (MAb-SAP) and polyclonal (anti-SAP) antibodies produced against the recombinant protein SAP-CD were used to investigate the expression and localization of SAP proteins. MAb-SAP reacted with a 55 kDa SAP protein released by epimastigotes and metacyclic forms and with distinct sets of SAP variants expressed in amastigotes and tissue culture-derived trypomastigotes (TCTs). Anti-SAP antibodies reacted with components located in the anterior region of epimastigotes and between the nucleus and the kinetoplast in metacyclic trypomastigotes. In contrast, anti-SAP recognized surface components of amastigotes and TCTs, suggesting that SAP proteins are directed to different cellular compartments. Ten SAP peptides were identified by mass spectrometry in vesicle and soluble-protein fractions obtained from parasite conditioned medium. Using overlapping sequences from SAP-CD, we identified a 54-aa peptide (SAP-CE) that was able to induce host-cell lysosome exocytosis and inhibit parasite internalization by 52%. Conclusions This study provides novel information about the genomic organization, expression and cellular localization of SAP proteins and proposes a triggering role for extracellular SAP proteins in host-cell lysosome exocytosis during metacyclic internalization. PMID:24391838

Polygalacturonase isozymes in Lygus hesperus Salivary Glands

USDA-ARS?s Scientific Manuscript database

The feeding strategy of mirids has been referred to as “lacerate or macerate and flush feeding” which supports high rates of food intake. In other words, plant bugs digest the plant tissue extra-orally, producing a liquefied brew rich in simple nutrient molecules. The insect's salivary polygalacturo...

Recombinant Salivary Proteins of Phlebotomus orientalis are Suitable Antigens to Measure Exposure of Domestic Animals to Sand Fly Bites

PubMed Central

Sima, Michal; Ferencova, Blanka; Warburg, Alon; Rohousova, Iva; Volf, Petr

2016-01-01

Background Certain salivary proteins of phlebotomine sand flies injected into the host skin during blood-feeding are highly antigenic and elicit strong antibody-mediated immune responses in repeatedly-exposed hosts. These antibodies can be measured by enzyme-linked immuno sorbent assays (ELISAs) using salivary gland homogenates (SGHs) as the source of antigens and serve as a markers for exposure to biting sand flies. Large-scale screening for anti-sand fly saliva antibodies requires replacement of SGH with recombinant salivary proteins. In East Africa, Phlebotomus orientalis is the main vector of Leishmania donovani, a trypanosomatid parasite causing visceral leishmaniasis. We tested recombinant salivary proteins derived from Ph. orientalis saliva to study exposure of domestic animals to this sand fly species. Methodology/Principal Findings Antigenic salivary proteins from Ph. orientalis were identified by immunoblot and mass spectrometry. Recombinant apyrase rPorSP15, yellow-related protein rPorSP24, ParSP25-like protein rPorSP65, D7-related protein rPorSP67, and antigen 5-related protein rPorSP76 were tested using ELISA with sera of domestic animals from L. donovani foci in Ethiopia where Ph. orientalis is present. Our results highlighted recombinant yellow-related protein rPorSP24 as the most promising antigen, displaying a high positive correlation coefficient as well as good sensitivity and specificity when compared to SGH. This recombinant protein was the most suitable one for testing sera of dogs, sheep, and goats. In addition, a different antigen, rPorSP65 was found efficacious for testing canine sera. Conclusions/Significance Recombinant salivary proteins of Ph. orientalis, specifically rPorSP24, were shown to successfully substitute SGH in serological experiments to measure exposure of domestic animals to Ph. orientalis, the vector of L. donovani. The results suggest that rPorSP24 might be a suitable antigen for detecting anti-Ph. orientalis antibody-mediated reactions also in other host species. PMID:26986566

Recombinant Salivary Proteins of Phlebotomus orientalis are Suitable Antigens to Measure Exposure of Domestic Animals to Sand Fly Bites.

PubMed

Sima, Michal; Ferencova, Blanka; Warburg, Alon; Rohousova, Iva; Volf, Petr

2016-03-01

Certain salivary proteins of phlebotomine sand flies injected into the host skin during blood-feeding are highly antigenic and elicit strong antibody-mediated immune responses in repeatedly-exposed hosts. These antibodies can be measured by enzyme-linked immuno sorbent assays (ELISAs) using salivary gland homogenates (SGHs) as the source of antigens and serve as a markers for exposure to biting sand flies. Large-scale screening for anti-sand fly saliva antibodies requires replacement of SGH with recombinant salivary proteins. In East Africa, Phlebotomus orientalis is the main vector of Leishmania donovani, a trypanosomatid parasite causing visceral leishmaniasis. We tested recombinant salivary proteins derived from Ph. orientalis saliva to study exposure of domestic animals to this sand fly species. Antigenic salivary proteins from Ph. orientalis were identified by immunoblot and mass spectrometry. Recombinant apyrase rPorSP15, yellow-related protein rPorSP24, ParSP25-like protein rPorSP65, D7-related protein rPorSP67, and antigen 5-related protein rPorSP76 were tested using ELISA with sera of domestic animals from L. donovani foci in Ethiopia where Ph. orientalis is present. Our results highlighted recombinant yellow-related protein rPorSP24 as the most promising antigen, displaying a high positive correlation coefficient as well as good sensitivity and specificity when compared to SGH. This recombinant protein was the most suitable one for testing sera of dogs, sheep, and goats. In addition, a different antigen, rPorSP65 was found efficacious for testing canine sera. Recombinant salivary proteins of Ph. orientalis, specifically rPorSP24, were shown to successfully substitute SGH in serological experiments to measure exposure of domestic animals to Ph. orientalis, the vector of L. donovani. The results suggest that rPorSP24 might be a suitable antigen for detecting anti-Ph. orientalis antibody-mediated reactions also in other host species.

Flexible docking of a ligand peptide to a receptor protein by multicanonical molecular dynamics simulation

NASA Astrophysics Data System (ADS)

Nakajima, Nobuyuki; Higo, Junichi; Kidera, Akinori; Nakamura, Haruki

1997-10-01

A new method for flexible docking by multicanonical molecular dynamics simulation is presented. The method was applied to the binding of a short proline-rich peptide to a Src homology 3 (SH3) domain. The peptide and the side-chains at the ligand binding cleft of SH3 were completely flexible and the large number of possible conformations and dispositions of the peptide were sampled. The reweighted canonical resemble at 300 K resulted in only a few predominant binding modes, one of which was similar to the complex crystal structure. The inverted peptide orientation was also observed in the other binding modes.

Characterization of a cDNA encoding a protein involved in formation of the skeleton during development of the sea urchin Lytechinus pictus.

PubMed

Livingston, B T; Shaw, R; Bailey, A; Wilt, F

1991-12-01

In order to investigate the role of proteins in the formation of mineralized tissues during development, we have isolated a cDNA that encodes a protein that is a component of the organic matrix of the skeletal spicule of the sea urchin, Lytechinus pictus. The expression of the RNA encoding this protein is regulated over development and is localized to the descendents of the micromere lineage. Comparison of the sequence of this cDNA to homologous cDNAs from other species of urchin reveal that the protein is basic and contains three conserved structural motifs: a signal peptide, a proline-rich region, and an unusual region composed of a series of direct repeats. Studies on the protein encoded by this cDNA confirm the predicted reading frame deduced from the nucleotide sequence and show that the protein is secreted and not glycosylated. Comparison of the amino acid sequence to databases reveal that the repeat domain is similar to proteins that form a unique beta-spiral supersecondary structure.

Gliadins from wheat grain: an overview, from primary structure to nanostructures of aggregates.

PubMed

Urade, Reiko; Sato, Nobuhiro; Sugiyama, Masaaki

2018-04-01

Gliadins are well-known wheat grain proteins, particularly important in food science. They were studied as early as the 1700s. Despite their long history, it has been difficult to identify their higher-order structure as they aggregate in aqueous solution. Consequently, most studies have been performed by extracting the proteins in 70% ethanol or dilute acidic solutions. The carboxy-terminal half of α- and γ-gliadins have α-helix-rich secondary structures stabilized with intramolecular disulfide bonds, which are present in either aqueous ethanol or pure water. The amino-terminal-repeat region of α- and γ-gliadins has poly-L-proline II and β-reverse-turn structures. ω-Gliadins also have poly-L-proline II and β-reverse-turn structures, but no α-helix structure. The size and shape of gliadin molecules have been determined by assessing a variety of parameters: their sedimentation velocity in the analytical ultracentrifuge, intrinsic viscosity, small-angle X-ray scattering profile, and images of the proteins from scanning probe microscopes such as a tunneling electron microscope and atomic force microscope. Models for gliadins are either rods or prolate ellipsoids whether in aqueous ethanol, dilute acid, or pure water. Recently, gliadins have been shown to be soluble in pure water, and a novel extraction method into pure water has been established. This has made it possible to analyze gliadins in pure water at neutral pH, and permitted the characterization of hydrated gliadins. They formed hierarchical nanoscale structures with internal density fluctuations at high protein concentrations.

Proteomic Profiling of Cereal Aphid Saliva Reveals Both Ubiquitous and Adaptive Secreted Proteins

PubMed Central

Wilkinson, Tom L.

2013-01-01

The secreted salivary proteins from two cereal aphid species, Sitobion avenae and Metopolophium dirhodum, were collected from artificial diets and analysed by tandem mass spectrometry. Protein identification was performed by searching MS data against the official protein set from the current pea aphid (Acyrthosiphon pisum) genome assembly and revealed 12 and 7 proteins in the saliva of S. avenae and M. dirhodum, respectively. When combined with a comparable dataset from A. pisum, only three individual proteins were common to all the aphid species; two paralogues of the GMC oxidoreductase family (glucose dehydrogenase; GLD) and ACYPI009881, an aphid specific protein previously identified as a putative component of the salivary sheath. Antibodies were designed from translated protein sequences obtained from partial cDNA sequences for ACYPI009881 and both saliva associated GLDs. The antibodies detected all parent proteins in secreted saliva from the three aphid species, but could only detect ACYPI009881, and not saliva associated GLDs, in protein extractions from the salivary glands. This result was confirmed by immunohistochemistry using whole and sectioned salivary glands, and in addition, localised ACYPI009881 to specific cell types within the principal salivary gland. The implications of these findings for the origin of salivary components and the putative role of the proteins identified are discussed in the context of our limited understanding of the functional relationship between aphid saliva and the plants they feed on. The mass spectrometry data have been deposited to the ProteomeXchange and can be accessed under the identifier PXD000113. PMID:23460852

Proteomic profiling of cereal aphid saliva reveals both ubiquitous and adaptive secreted proteins.

PubMed

Rao, Sohail A K; Carolan, James C; Wilkinson, Tom L

2013-01-01

The secreted salivary proteins from two cereal aphid species, Sitobion avenae and Metopolophium dirhodum, were collected from artificial diets and analysed by tandem mass spectrometry. Protein identification was performed by searching MS data against the official protein set from the current pea aphid (Acyrthosiphon pisum) genome assembly and revealed 12 and 7 proteins in the saliva of S. avenae and M. dirhodum, respectively. When combined with a comparable dataset from A. pisum, only three individual proteins were common to all the aphid species; two paralogues of the GMC oxidoreductase family (glucose dehydrogenase; GLD) and ACYPI009881, an aphid specific protein previously identified as a putative component of the salivary sheath. Antibodies were designed from translated protein sequences obtained from partial cDNA sequences for ACYPI009881 and both saliva associated GLDs. The antibodies detected all parent proteins in secreted saliva from the three aphid species, but could only detect ACYPI009881, and not saliva associated GLDs, in protein extractions from the salivary glands. This result was confirmed by immunohistochemistry using whole and sectioned salivary glands, and in addition, localised ACYPI009881 to specific cell types within the principal salivary gland. The implications of these findings for the origin of salivary components and the putative role of the proteins identified are discussed in the context of our limited understanding of the functional relationship between aphid saliva and the plants they feed on. The mass spectrometry data have been deposited to the ProteomeXchange and can be accessed under the identifier PXD000113.

A Sand Fly Salivary Protein Vaccine Shows Efficacy Against Vector-Transmitted Cutaneous Leishmaniasis in Nonhuman Primates

DTIC Science & Technology

2015-06-03

demonstrating its immunogenicity in humans. PdSP15 sequence and structure show no homol- ogy to mammalian proteins, further demonstrating its potential...sequence or structure homology to known human proteins The protective salivary antigen PdSP15 shares sequence homology only to the small odorant binding...salivary proteins PpSP15 and PsSP15, respectively (Fig. 4B). To exclude any structural similarities to human pro teins, the crystal structure of PdPS15

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

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

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.

Functional transcriptomics of wild-caught Lutzomyia intermedia salivary glands: identification of a protective salivary protein against Leishmania braziliensis infection.

PubMed

de Moura, Tatiana R; Oliveira, Fabiano; Carneiro, Marcia W; Miranda, José Carlos; Clarêncio, Jorge; Barral-Netto, Manoel; Brodskyn, Cláudia; Barral, Aldina; Ribeiro, José M C; Valenzuela, Jesus G; de Oliveira, Camila I

2013-01-01

Leishmania parasites are transmitted in the presence of sand fly saliva. Together with the parasite, the sand fly injects salivary components that change the environment at the feeding site. Mice immunized with Phlebotomus papatasi salivary gland (SG) homogenate are protected against Leishmania major infection, while immunity to Lutzomyia intermedia SG homogenate exacerbated experimental Leishmania braziliensis infection. In humans, antibodies to Lu. intermedia saliva are associated with risk of acquiring L. braziliensis infection. Despite these important findings, there is no information regarding the repertoire of Lu. intermedia salivary proteins. A cDNA library from the Salivary Glands (SGs) of wild-caught Lu. intermedia was constructed, sequenced, and complemented by a proteomic approach based on 1D SDS PAGE and mass/mass spectrometry to validate the transcripts present in this cDNA library. We identified the most abundant transcripts and proteins reported in other sand fly species as well as novel proteins such as neurotoxin-like proteins, peptides with ML domain, and three small peptides found so far only in this sand fly species. DNA plasmids coding for ten selected transcripts were constructed and used to immunize BALB/c mice to study their immunogenicity. Plasmid Linb-11--coding for a 4.5-kDa protein--induced a cellular immune response and conferred protection against L. braziliensis infection. This protection correlated with a decreased parasite load and an increased frequency of IFN-γ-producing cells. We identified the most abundant and novel proteins present in the SGs of Lu. intermedia, a vector of cutaneous leishmaniasis in the Americas. We also show for the first time that immunity to a single salivary protein from Lu. intermedia can protect against cutaneous leishmaniasis caused by L. braziliensis.

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.

TAT-Mediated Delivery of Tousled Protein to Salivary Glands Protects Against Radiation-Induced Hypofunction

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

Sunavala-Dossabhoy, Gulshan, E-mail: gsunav@lsuhsc.edu; Palaniyandi, Senthilnathan; Richardson, Charles

2012-09-01

Purpose: Patients treated with radiotherapy for head-and-neck cancer invariably suffer its deleterious side effect, xerostomia. Salivary hypofunction ensuing from the irreversible destruction of glands is the most common and debilitating oral complication affecting patients undergoing regional radiotherapy. Given that the current management of xerostomia is palliative and ineffective, efforts are now directed toward preventive measures to preserve gland function. The human homolog of Tousled protein, TLK1B, facilitates chromatin remodeling at DNA repair sites and improves cell survival against ionizing radiation (IR). Therefore, we wanted to determine whether a direct transfer of TLK1B protein to rat salivary glands could protect againstmore » IR-induced salivary hypofunction. Methods: The cell-permeable TAT-TLK1B fusion protein was generated. Rat acinar cell line and rat salivary glands were pretreated with TAT peptide or TAT-TLK1B before IR. The acinar cell survival in vitro and salivary function in vivo were assessed after radiation. Results: We demonstrated that rat acinar cells transduced with TAT-TLK1B were more resistant to radiation (D{sub 0} = 4.13 {+-} 1.0 Gy; {alpha}/{beta} = 0 Gy) compared with cells transduced with the TAT peptide (D{sub 0} = 4.91 {+-} 1.0 Gy; {alpha}/{beta} = 20.2 Gy). Correspondingly, retroductal instillation of TAT-TLK1B in rat submandibular glands better preserved salivary flow after IR (89%) compared with animals pretreated with Opti-MEM or TAT peptide (31% and 39%, respectively; p < 0.01). Conclusions: The results demonstrate that a direct transfer of TLK1B protein to the salivary glands effectively attenuates radiation-mediated gland dysfunction. Prophylactic TLK1B-protein therapy could benefit patients undergoing radiotherapy for head-and-neck cancer.« less

Effects of Repeated Administration of Pilocarpine and Isoproterenol on Aquaporin-5 Expression in Rat Salivary Glands

PubMed Central

Susa, Taketo; Sawai, Nobuhiko; Aoki, Takeo; Iizuka-Kogo, Akiko; Kogo, Hiroshi; Negishi, Akihide; Yokoo, Satoshi; Takata, Kuniaki; Matsuzaki, Toshiyuki

2013-01-01

Aquaporins are water channel proteins which enable rapid water movement across the plasma membrane. Aquaporin-5 (AQP5) is the major aquaporin and is expressed on the apical membrane of salivary gland acinar cells. We examined the effects of repeated administration of pilocarpine, a clinically useful stimulant for salivary fluid secretion, and isoproterenol (IPR), a stimulant for salivary protein secretion, on the abundance of AQP5 protein in rat salivary glands by immunofluorescence microscopy and semi-quantitative immunoblotting. Unexpectedly AQP5 was decreased in pilocarpine-administered salivary glands, in which fluid secretion must be highly stimulated, implying that AQP5 might not be required for fluid secretion at least in pilocarpine-administered state. The abundance of AQP5, on the other hand, was found to be significantly increased in IPR-administered submandibular and parotid glands. To address the possible mechanism of the elevation of AQP5 abundance in IPR-administered animals, changes of AQP5 level in fasting animals, in which the exocytotic events are reduced, were examined. AQP5 was found to be decreased in fasting animals as expected. These results suggested that the elevation of cAMP and/or frequent exocytotic events could increase AQP5 protein. AQP5 expression seems to be easily changed by salivary stimulants, although these changes do not always reflect the ability in salivary fluid secretion. PMID:24610966

Smoking influences salivary histamine levels in periodontal disease.

PubMed

Bertl, K; Haririan, H; Laky, M; Matejka, M; Andrukhov, O; Rausch-Fan, X

2012-05-01

Histamine, a potent vasoactive amine, is increased in saliva of periodontitis patients. The present study aimed to further investigate the diagnostic potential of histamine for periodontal disease and assessed smoking, a major risk factor of periodontitis, as a possible influencing factor. Salivary and serum samples of 106 participants (60 periodontitis patients, 46 controls) were collected. Salivary histamine was determined by a commercially available ELISA kit, and serum C-reactive protein was measured by a routine laboratory test. Cigarettes per day and packyears were assessed as smoking exposure parameters. Statistically significantly increased levels of salivary histamine and serum C-reactive protein were detected between the patient and control group (P = 0.022 and P = 0.001). Salivary histamine levels were significantly higher in smoking compared with non-smoking patients (P < 0.001), and salivary histamine as well as serum C-reactive protein correlated significantly positively with smoking exposure parameters (P < 0.05). Smoking, an established and common risk factor of periodontitis, was assessed as a possible influencing factor for salivary histamine. Most interestingly, salivary histamine differed highly significantly between smoking and non-smoking periodontitis patients. Our results suggest a possible involvement of histamine in tobacco-exacerbated periodontal disease, but do not suggest salivary histamine as a reliable diagnostic marker for periodontitis. © 2011 John Wiley & Sons A/S.

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

Induction of Salivary Proteins Modifies Measures of Both Orosensory and Postingestive Feedback during Exposure to a Tannic Acid Diet

PubMed Central

Torregrossa, Ann-Marie; Nikonova, Larissa; Bales, Michelle B.; Villalobos Leal, Maria; Smith, James C.; Contreras, Robert J.; Eckel, Lisa A.

2014-01-01

There are hundreds of proteins in saliva. Although it has long been hypothesized that these proteins modulate taste by interacting with taste receptors or taste stimuli, the functional impact of these proteins on feeding remains relatively unexplored. We have developed a new technique for saliva collection that does not interfere with daily behavioral testing and allows us to explore the relationship between feeding behavior and salivary protein expression. First, we monitored the alterations in salivary protein expression while simultaneously monitoring the animals' feeding behavior and meal patterns on a custom control diet or on the same diet mixed with 3% tannic acid. We demonstrated that six protein bands increased in density with dietary tannic acid exposure. Several of these bands were significantly correlated with behaviors thought to represent both orosensory and postingestive signaling. In a follow-up experiment, unconditioned licking to 0.01–3% tannic acid solutions was measured during a brief-access taste test before and after exposure to the tannic acid diet. In this experiment, rats with salivary proteins upregulated found the tannin solution less aversive (i.e., licked more) than those in the control condition. These data suggest a role for salivary proteins in mediating changes in both orosensory and postingestive feedback. PMID:25162297

An insight into the sialome of the blood-sucking bug Triatoma infestans, a vector of Chagas' disease

PubMed Central

Assumpção, Teresa C. F.; Francischetti, Ivo M. B.; Andersen, John F.; Schwarz, Alexandra; Santana, Jaime M.; Ribeiro, José M. C.

2008-01-01

Triatoma infestans is a hemiptera, vector of Chagas’ disease, that feeds exclusively on vertebrate blood in all life stages. Hematophagous insects’ salivary glands (SG) produce potent pharmacological compounds that counteract host hemostasis, including anti-clotting, anti-platelet, and vasodilatory molecules. To obtain a further insight into the salivary biochemical and pharmacological complexity of this insect, a cDNA library from its salivary glands was randomly sequenced. Also, salivary proteins were submitted to two dimentional gel (2D-gel) electrophoresis followed by MS analysis. We present the analysis of a set of 1,534 (SG) cDNA sequences, 645 of which coded for proteins of a putative secretory nature. Most salivary proteins described as lipocalins matched peptide sequences obtained from proteomic results. PMID:18207082

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.

Mapping and mutagenesis of the amino-terminal transcriptional repression domain of the Drosophila Krüppel protein.

PubMed Central

Licht, J D; Hanna-Rose, W; Reddy, J C; English, M A; Ro, M; Grossel, M; Shaknovich, R; Hansen, U

1994-01-01

We previously demonstrated that the Drosophila Krüppel protein is a transcriptional repressor with separable DNA-binding and transcriptional repression activities. In this study, the minimal amino (N)-terminal repression region of the Krüppel protein was defined by transferring regions of the Krüppel protein to a heterologous DNA-binding protein, the lacI protein. Fusion of a predicted alpha-helical region from amino acids 62 to 92 in the N terminus of the Krüppel protein was sufficient to transfer repression activity. This putative alpha-helix has several hydrophobic surfaces, as well as a glutamine-rich surface. Mutants containing multiple amino acid substitutions of the glutamine residues demonstrated that this putative alpha-helical region is essential for repression activity of a Krüppel protein containing the entire N-terminal and DNA-binding regions. Furthermore, one point mutant with only a single glutamine on this surface altered to lysine abolished the ability of the Krüppel protein to repress, indicating the importance of the amino acid at residue 86 for repression. The N terminus also contained an adjacent activation region localized between amino acids 86 and 117. Finally, in accordance with predictions from primary amino acid sequence similarity, a repression region from the Drosophila even-skipped protein, which was six times more potent than that of the Krüppel protein in the mammalian cells, was characterized. This segment included a hydrophobic stretch of 11 consecutive alanine residues and a proline-rich region. Images PMID:8196644

Proteome analysis of gut and salivary gland proteins of fifth-instar nymph and adults of the sunn pest, Eurygaster integriceps.

PubMed

Bezdi, Mohammad Saadati; Toorchi, Mahmoud; Pourabad, Reza Farshbaf; Zarghami, Nosratollah; Nouri, Mohammad-Zaman; Komatsu, Setsuko

2012-10-01

In the digestive system of the sunn pest, Eurygaster integriceps Puton (Hemiptera: Scutelleridae), the salivary gland has a key role in extra oral digestion and the gut is the main site for digestion of food. In this study, proteomics was used to study the role of proteins involved in digestion. The amount of feeding on wheat grain by adult insects increased by comparison to fifth-instar nymphs. Proteins of the gut and salivary gland in adults and fifth-instar nymphs were analyzed 1 day after feeding. The proteins related to digestion, metabolism, and defense against toxins were accumulated in the gut of adult insects. Three plant proteins including serpin, dehydroascorbate reductase, and β-amylase were accumulated in guts of adults. In the salivary gland, phospholipase A2 and arginine kinase were increased in adults. Heat shock protein 70 increased in the gut of fifth-instar nymphs. Proteomic analysis revealed that most of changed proteins in digestive system of sunn pest were increased in adults. This study provided more targets derived from gut and salivary gland for pest management. © 2012 Wiley Periodicals, Inc.

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

Towards a Proteomic Catalogue and Differential Annotation of Salivary Gland Proteins in Blood Fed Malaria Vector Anopheles culicifacies by Mass Spectrometry.

PubMed

Rawal, Ritu; Vijay, Sonam; Kadian, Kavita; Singh, Jagbir; Pande, Veena; Sharma, Arun

2016-01-01

In order to understand the importance of functional proteins in mosquito behavior, following blood meal, a baseline proteomic dataset is essential for providing insights into the physiology of blood feeding. Therefore, in this study as first step, in solution and 1-D electrophoresis digestion approach combined with tandem mass spectrometry (nano LC-MS/MS) and computational bioinformatics for data mining was used to prepare a baseline proteomic catalogue of salivary gland proteins of sugar fed An. culicifacies mosquitoes. A total of 106 proteins were identified and analyzed by SEQUEST algorithm against mosquito protein database from Uniprot/NCBI. Importantly, D7r1, D7r2, D7r4, salivary apyrase, anti-platelet protein, calreticulin, antigen 5 family proteins were identified and grouped on the basis of biological and functional roles. Secondly, differential protein expression and annotations between salivary glands of sugar fed vs blood fed mosquitoes was analyzed using 2-Delectrophoresis combined with MALDI-TOF mass spectrometry. The alterations in the differential expression of total 38 proteins was observed out of which 29 proteins like beclin-1, phosphorylating proteins, heme oxygenase 1, ferritin, apoptotic proteins, coagulation and immunity like, serine proteases, serpins, c-type lectin and protein in regulation of blood feeding behavior were found to be up regulated while 9 proteins related to blood feeding, juvenile hormone epoxide hydrolase ii, odorant binding proteins and energy metabolic enzymes were found to be down regulated. To our knowledge, this study provides a first time baseline proteomic dataset and functional annotations of An. culicifacies salivary gland proteins that may be involved during the blood feeding. Identification of differential salivary proteins between sugar fed and blood fed mosquitoes and their plausible role may provide insights into the physiological processes associated with feeding behavior and sporozoite transmission during the process of blood feeding.

Towards a Proteomic Catalogue and Differential Annotation of Salivary Gland Proteins in Blood Fed Malaria Vector Anopheles culicifacies by Mass Spectrometry

PubMed Central

Rawal, Ritu; Vijay, Sonam; Kadian, Kavita; Singh, Jagbir; Pande, Veena; Sharma, Arun

2016-01-01

In order to understand the importance of functional proteins in mosquito behavior, following blood meal, a baseline proteomic dataset is essential for providing insights into the physiology of blood feeding. Therefore, in this study as first step, in solution and 1-D electrophoresis digestion approach combined with tandem mass spectrometry (nano LC-MS/MS) and computational bioinformatics for data mining was used to prepare a baseline proteomic catalogue of salivary gland proteins of sugar fed An. culicifacies mosquitoes. A total of 106 proteins were identified and analyzed by SEQUEST algorithm against mosquito protein database from Uniprot/NCBI. Importantly, D7r1, D7r2, D7r4, salivary apyrase, anti-platelet protein, calreticulin, antigen 5 family proteins were identified and grouped on the basis of biological and functional roles. Secondly, differential protein expression and annotations between salivary glands of sugar fed vs blood fed mosquitoes was analyzed using 2-Delectrophoresis combined with MALDI-TOF mass spectrometry. The alterations in the differential expression of total 38 proteins was observed out of which 29 proteins like beclin-1, phosphorylating proteins, heme oxygenase 1, ferritin, apoptotic proteins, coagulation and immunity like, serine proteases, serpins, c-type lectin and protein in regulation of blood feeding behavior were found to be up regulated while 9 proteins related to blood feeding, juvenile hormone epoxide hydrolase ii, odorant binding proteins and energy metabolic enzymes were found to be down regulated. To our knowledge, this study provides a first time baseline proteomic dataset and functional annotations of An. culicifacies salivary gland proteins that may be involved during the blood feeding. Identification of differential salivary proteins between sugar fed and blood fed mosquitoes and their plausible role may provide insights into the physiological processes associated with feeding behavior and sporozoite transmission during the process of blood feeding. PMID:27602567

Dynamic changes in saliva after acute mental stress

PubMed Central

Naumova, Ella A.; Sandulescu, Tudor; Bochnig, Clemens; Khatib, Philipp Al; Lee, Wing-Kee; Zimmer, Stefan; Arnold, Wolfgang H.

2014-01-01

Stress-related variations of fluoride concentration in supernatant saliva and salivary sediment, salivary cortisol, total protein and pH after acute mental stress were assessed. The hypothesis was that stress reactions have no influence on these parameters. Thirty-four male students were distributed into two groups: first received the stress exposure followed by the same protocol two weeks later but without stress exposure, second underwent the protocol without stress exposure followed by the stress exposure two weeks later. The stressor was a public speech followed by tooth brushing. Saliva was collected before, immediately after stress induction and immediately, at 10, 30 and 120 min. after tooth brushing. Cortisol concentrations, total protein, intraoral pH, and fluoride content in saliva were measured. The data were analyzed statistically. Salivary sediment was ca 4.33% by weight of whole unstimulated saliva. Fluoride bioavailability was higher in salivary sediment than in supernatant saliva. The weight and fluoride concentration was not altered during 2 hours after stress exposure. After a public speech, the salivary cortisol concentration significantly increased after 20 minutes compared to the baseline. The salivary protein concentration and pH also increased. Public speaking influences protein concentration and salivary pH but does not alter the fluoride concentration of saliva. PMID:24811301

A Potential Role for Drosophila Mucins in Development and Physiology

PubMed Central

Syed, Zulfeqhar A.; Härd, Torleif; Uv, Anne; van Dijk-Härd, Iris F.

2008-01-01

Vital vertebrate organs are protected from the external environment by a barrier that to a large extent consists of mucins. These proteins are characterized by poorly conserved repeated sequences that are rich in prolines and potentially glycosylated threonines and serines (PTS). We have now used the characteristics of the PTS repeat domain to identify Drosophila mucins in a simple bioinformatics approach. Searching the predicted protein database for proteins with at least 4 repeats and a high ST content, more than 30 mucin-like proteins were identified, ranging from 300–23000 amino acids in length. We find that Drosophila mucins are present at all stages of the fly life cycle, and that their transcripts localize to selective organs analogous to sites of vertebrate mucin expression. The results could allow for addressing basic questions about human mucin-related diseases in this model system. Additionally, many of the mucins are expressed in selective tissues during embryogenesis, thus revealing new potential functions for mucins as apical matrix components during organ morphogenesis. PMID:18725942

CD6 and Linker of Activated T Cells are Potential Interaction Partners for T Cell-Specific Adaptor Protein.

PubMed

Hem, C D; Ekornhol, M; Granum, S; Sundvold-Gjerstad, V; Spurkland, A

2017-02-01

The T cell-specific adaptor protein (TSAd) contains several protein interaction domains, and is merging as a modulator of T cell activation. Several interaction partners for the TSAd proline-rich region and phosphotyrosines have been identified, including the Src and Tec family kinases lymphocyte-specific protein tyrosine kinase and interleukin 2-inducible T cell kinase. Via its Src homology 2 (SH2) domain, TSAd may thus function as a link between these enzymes and other signalling molecules. However, few binding partners to the TSAd SH2 domain in T cells are hitherto known. Through the use of in silico ligand prediction, peptide spot arrays, pull-down and immunoprecipitation experiments, we here report novel interactions between the TSAd SH2 domain and CD6 phosphotyrosine (pTyr) 629 and linker of activated T cells (LAT) pTyr 171 , pTyr 191 and pTyr 226 . © 2016 The Foundation for the Scandinavian Journal of Immunology.

Armadillo Repeat Containing 8α Binds to HRS and Promotes HRS Interaction with Ubiquitinated Proteins

PubMed Central

Tomaru, Koji; Ueda, Atsuhisa; Suzuki, Takeyuki; Kobayashi, Nobuaki; Yang, Jun; Yamamoto, Masaki; Takeno, Mitsuhiro; Kaneko, Takeshi; Ishigatsubo, Yoshiaki

2010-01-01

Recently, we reported that a complex with an essential role in the degradation of Fructose-1,6-bisphosphatase in yeast is well conserved in mammalian cells; we named this mammalian complex C-terminal to the Lissencephaly type-1-like homology (CTLH) complex. Although the function of the CTLH complex remains unclear, here we used yeast two-hybrid screening to isolate Hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) as a protein binding to a key component of CTLH complex, Armadillo repeat containing 8 (ARMc8) α. The association was confirmed by a yeast two-hybrid assay and a co-immunoprecipitation assay. The proline-rich domain of HRS was essential for the association. As demonstrated through immunofluorescence microscopy, ARMc8α co-localized with HRS. ARMc8α promoted the interaction of HRS with various ubiquitinated proteins through the ubiquitin-interacting motif. These findings suggest that HRS mediates protein endosomal trafficking partly through its interaction with ARMc8α. PMID:20224683

Changes in the folding landscape of the WW domain provide a molecular mechanism for an inherited genetic syndrome.

PubMed

Pucheta-Martinez, Encarna; D'Amelio, Nicola; Lelli, Moreno; Martinez-Torrecuadrada, Jorge L; Sudol, Marius; Saladino, Giorgio; Gervasio, Francesco Luigi

2016-07-26

WW domains are small domains present in many human proteins with a wide array of functions and acting through the recognition of proline-rich sequences. The WW domain belonging to polyglutamine tract-binding protein 1 (PQBP1) is of particular interest due to its direct involvement in several X chromosome-linked intellectual disabilities, including Golabi-Ito-Hall (GIH) syndrome, where a single point mutation (Y65C) correlates with the development of the disease. The mutant cannot bind to its natural ligand WBP11, which regulates mRNA processing. In this work we use high-field high-resolution NMR and enhanced sampling molecular dynamics simulations to gain insight into the molecular causes the disease. We find that the wild type protein is partially unfolded exchanging among multiple beta-strand-like conformations in solution. The Y65C mutation further destabilizes the residual fold and primes the protein for the formation of a disulphide bridge, which could be at the origin of the loss of function.

Changes in the folding landscape of the WW domain provide a molecular mechanism for an inherited genetic syndrome

NASA Astrophysics Data System (ADS)

Pucheta-Martinez, Encarna; D'Amelio, Nicola; Lelli, Moreno; Martinez-Torrecuadrada, Jorge L.; Sudol, Marius; Saladino, Giorgio; Gervasio, Francesco Luigi

2016-07-01

WW domains are small domains present in many human proteins with a wide array of functions and acting through the recognition of proline-rich sequences. The WW domain belonging to polyglutamine tract-binding protein 1 (PQBP1) is of particular interest due to its direct involvement in several X chromosome-linked intellectual disabilities, including Golabi-Ito-Hall (GIH) syndrome, where a single point mutation (Y65C) correlates with the development of the disease. The mutant cannot bind to its natural ligand WBP11, which regulates mRNA processing. In this work we use high-field high-resolution NMR and enhanced sampling molecular dynamics simulations to gain insight into the molecular causes the disease. We find that the wild type protein is partially unfolded exchanging among multiple beta-strand-like conformations in solution. The Y65C mutation further destabilizes the residual fold and primes the protein for the formation of a disulphide bridge, which could be at the origin of the loss of function.

Purification and molecular cloning of SH2- and SH3-containing inositol polyphosphate-5-phosphatase, which is involved in the signaling pathway of granulocyte-macrophage colony-stimulating factor, erythropoietin, and Bcr-Abl.

PubMed

Odai, H; Sasaki, K; Iwamatsu, A; Nakamoto, T; Ueno, H; Yamagata, T; Mitani, K; Yazaki, Y; Hirai, H

1997-04-15

Grb2/Ash and Shc are the adapter proteins that link tyrosine-kinase receptors to Ras and make tyrosine-kinase functionally associated with receptors and Ras in fibroblasts and hematopoietic cells. Grb2/Ash and Shc have the SH3, SH2, or phosphotyrosine binding domains. These domains bind to proteins containing proline-rich regions or tyrosine-phosphorylated proteins and contribute to the association of Grb2/Ash and Shc with other signaling molecules. However, there could remain unidentified signaling molecules that physically and functionally interact with these adapter proteins and have biologically important roles in the signaling pathways. By using the GST fusion protein including the full length of Grb2/Ash, we have found that c-Cbl and an unidentified 135-kD protein (pp135) are associated with Grb2/Ash. We have also found that they become tyrosine-phosphorylated by treatment of a human leukemia cell line, UT-7, with granulocyte-macrophage colony-stimulating factor (GM-CSF). We have purified the pp135 by using GST-Grb2/Ash affinity column and have isolated the full-length complementary DNA (cDNA) encoding the pp135 using a cDNA probe, which was obtained by the degenerate polymerase chain reaction based on a peptide sequence of the purified pp135. The cloned cDNA has 3,958 nucleotides that contain a single long open reading frame of 3,567 nucleotides, encoding a 1,189 amino acid protein with a predicted molecular weight of approximately 133 kD. The deduced amino acid sequence reveals that pp135 is a protein that has one SH2, one SH3, and one proline-rich domain. The pp135, which contains two motifs conserved among the inositol polyphosphate-5-phosphatase proteins, was shown to have the inositol polyphosphate-5-phosphatase activity. The pp135 was revealed to associate constitutively with Grb2/Ash and inducibly with Shc using UT-7 cells stimulated with GM-CSF. In the cell lines derived from human chronic myelogenous leukemia, pp135 was constitutively tyrosine-phosphorylated and associated with Shc and Bcr-Abl. These facts suggest that pp135 is a signaling molecule that has a unique enzymatic activity and should play an important role in the signaling pathway triggered by GM-CSF and in the transformation of hematopoietic cells caused by Bcr-Abl.

Estimation of salivary glucose, salivary amylase, salivary total protein and salivary flow rate in diabetics in India.

PubMed

Panchbhai, Arati S; Degwekar, Shirish S; Bhowte, Rahul R

2010-09-01

Diabetes is known to influence salivary composition and function, eventually affecting the oral cavity. We thus evaluated saliva samples for levels of glucose, amylase and total protein, and assessed salivary flow rate in diabetics and healthy non-diabetics. We also analyzed these parameters with regard to duration and type of diabetes mellitus and gender, and aimed to assess the interrelationships among the variables included in the study. A total of 120 age- and sex-matched participants were divided into 3 groups of 40 each; the uncontrolled diabetic group, the controlled diabetic group and the healthy non-diabetic group. Salivary investigations were performed using unstimulated whole saliva. Mean salivary glucose levels were found to be significantly elevated in both uncontrolled and controlled diabetics, as compared to healthy non-diabetics. There were significant decreases in mean salivary amylase levels in controlled diabetics when compared to healthy non-diabetics. Other than salivary glucose, no other parameters were found to be markedly affected in diabetes mellitus. Further research is needed to explore the clinical implications of these study results.

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.

Evidence that a laminin-like insect protein mediates early events in the interaction of a Phytoparasite with its vector's salivary gland.

PubMed

de Almeida Dias, Felipe; Souza dos Santos, Andre Luis; Santos Lery, Letícia Miranda; Alves e Silva, Thiago Luiz; Oliveira, Mauricio Martins; Bisch, Paulo Mascarello; Saraiva, Elvira Maria; Souto-Padrón, Thaïs Cristina; Lopes, Angela Hampshire

2012-01-01

Phytomonas species are plant parasites of the family Trypanosomatidae, which are transmitted by phytophagous insects. Some Phytomonas species cause major agricultural damages. The hemipteran Oncopeltus fasciatus is natural and experimental host for several species of trypanosomatids, including Phytomonas spp. The invasion of the insect vectors' salivary glands is one of the most important events for the life cycle of Phytomonas species. In the present study, we show the binding of Phytomonas serpens at the external face of O. fasciatus salivary glands by means of scanning electron microscopy and the in vitro interaction of living parasites with total proteins from the salivary glands in ligand blotting assays. This binding occurs primarily through an interaction with a 130 kDa salivary gland protein. The mass spectrometry of the trypsin-digest of this protein matched 23% of human laminin-5 β3 chain precursor sequence by 16 digested peptides. A protein sequence search through the transcriptome of O. fasciatus embryo showed a partial sequence with 51% similarity to human laminin β3 subunit. Anti-human laminin-5 β3 chain polyclonal antibodies recognized the 130 kDa protein by immunoblotting. The association of parasites with the salivary glands was strongly inhibited by human laminin-5, by the purified 130 kDa insect protein, and by polyclonal antibodies raised against the human laminin-5 β3 chain. This is the first report demonstrating that a laminin-like molecule from the salivary gland of O. fasciatus acts as a receptor for Phytomonas binding. The results presented in this investigation are important findings that will support further studies that aim at developing new approaches to prevent the transmission of Phytomonas species from insects to plants and vice-versa.

Evidence That a Laminin-Like Insect Protein Mediates Early Events in the Interaction of a Phytoparasite with Its Vector's Salivary Gland

PubMed Central

Dias, Felipe de Almeida; dos Santos, Andre Luis Souza; Lery, Letícia Miranda Santos; Alves e Silva, Thiago Luiz; Oliveira, Mauricio Martins; Bisch, Paulo Mascarello; Saraiva, Elvira Maria; Souto-Padrón, Thaïs Cristina; Lopes, Angela Hampshire

2012-01-01

Phytomonas species are plant parasites of the family Trypanosomatidae, which are transmitted by phytophagous insects. Some Phytomonas species cause major agricultural damages. The hemipteran Oncopeltus fasciatus is natural and experimental host for several species of trypanosomatids, including Phytomonas spp. The invasion of the insect vectors' salivary glands is one of the most important events for the life cycle of Phytomonas species. In the present study, we show the binding of Phytomonas serpens at the external face of O. fasciatus salivary glands by means of scanning electron microscopy and the in vitro interaction of living parasites with total proteins from the salivary glands in ligand blotting assays. This binding occurs primarily through an interaction with a 130 kDa salivary gland protein. The mass spectrometry of the trypsin-digest of this protein matched 23% of human laminin-5 β3 chain precursor sequence by 16 digested peptides. A protein sequence search through the transcriptome of O. fasciatus embryo showed a partial sequence with 51% similarity to human laminin β3 subunit. Anti-human laminin-5 β3 chain polyclonal antibodies recognized the 130 kDa protein by immunoblotting. The association of parasites with the salivary glands was strongly inhibited by human laminin-5, by the purified 130 kDa insect protein, and by polyclonal antibodies raised against the human laminin-5 β3 chain. This is the first report demonstrating that a laminin-like molecule from the salivary gland of O. fasciatus acts as a receptor for Phytomonas binding. The results presented in this investigation are important findings that will support further studies that aim at developing new approaches to prevent the transmission of Phytomonas species from insects to plants and vice-versa. PMID:23118944

Functional Transcriptomics of Wild-Caught Lutzomyia intermedia Salivary Glands: Identification of a Protective Salivary Protein against Leishmania braziliensis Infection

PubMed Central

Carneiro, Marcia W.; Miranda, José Carlos; Clarêncio, Jorge; Barral-Netto, Manoel; Brodskyn, Cláudia; Barral, Aldina; Ribeiro, José M. C.; Valenzuela, Jesus G.; de Oliveira, Camila I.

2013-01-01

Background Leishmania parasites are transmitted in the presence of sand fly saliva. Together with the parasite, the sand fly injects salivary components that change the environment at the feeding site. Mice immunized with Phlebotomus papatasi salivary gland (SG) homogenate are protected against Leishmania major infection, while immunity to Lutzomyia intermedia SG homogenate exacerbated experimental Leishmania braziliensis infection. In humans, antibodies to Lu. intermedia saliva are associated with risk of acquiring L. braziliensis infection. Despite these important findings, there is no information regarding the repertoire of Lu. intermedia salivary proteins. Methods and Findings A cDNA library from the Salivary Glands (SGs) of wild-caught Lu. intermedia was constructed, sequenced, and complemented by a proteomic approach based on 1D SDS PAGE and mass/mass spectrometry to validate the transcripts present in this cDNA library. We identified the most abundant transcripts and proteins reported in other sand fly species as well as novel proteins such as neurotoxin-like proteins, peptides with ML domain, and three small peptides found so far only in this sand fly species. DNA plasmids coding for ten selected transcripts were constructed and used to immunize BALB/c mice to study their immunogenicity. Plasmid Linb-11—coding for a 4.5-kDa protein—induced a cellular immune response and conferred protection against L. braziliensis infection. This protection correlated with a decreased parasite load and an increased frequency of IFN-γ-producing cells. Conclusions We identified the most abundant and novel proteins present in the SGs of Lu. intermedia, a vector of cutaneous leishmaniasis in the Americas. We also show for the first time that immunity to a single salivary protein from Lu. intermedia can protect against cutaneous leishmaniasis caused by L. braziliensis. PMID:23717705

Self-perceived oral health and salivary proteins in children with type 1 diabetes.

PubMed

Javed, F; Sundin, U; Altamash, M; Klinge, B; Engström, P-E

2009-01-01

The aim was to validate self-perceived oral health with salivary IgG as an inflammatory parameter in children with type 1 diabetes. Unstimulated whole saliva samples were collected from 36 children with well controlled and 12 with poorly controlled type 1 diabetes and 40 non-diabetic children (Controls). Salivary flow rate, random blood glucose level, salivary protein concentration and immunoglobulin A and G levels were recorded using standard techniques. Data concerning oral health and diabetes status were collected. Self-perceived gingival bleeding (bleeding gums), bad breath and dry mouth were higher in diabetic children when compared with those in controls (P < 0.05). Gingival bleeding was frequently perceived by children with poorly controlled compared to well-controlled type 1 diabetes (P < 0.05) and controls (P < 0.001). Bad breath was common perceived by children with poorly controlled compared to well-controlled type 1 diabetes (P < 0.05) and controls (P < 0.0001). Salivary flow rate was lower in the diabetic children compared to controls (P < 0.01) with no difference between children with poorly controlled and well-controlled type 1 diabetes. Salivary IgG per mg protein concentration was higher in the diabetics when compared with the control group (P < 0.0001). IgG per mg protein levels were also higher in children with poorly controlled when compared with well-controlled type 1 diabetes (P < 0.05). There was no difference in IgA per mg protein and total protein concentrations between children with poorly controlled and well-controlled type 1 diabetes. Self-perceived gingival bleeding and salivary IgG per mg protein concentration were increased in children with type 1 diabetes compared with controls. These variables were also increased in children with poorly controlled compared with well-controlled type 1 diabetes.

Functional characterization of a competitive peptide antagonist of p65 in human macrophage-like cells suggests therapeutic potential for chronic inflammation

PubMed Central

Srinivasan, Mythily; Blackburn, Corinne; Lahiri, Debomoy K

2014-01-01

Glucocorticoid-induced leucine zipper (GILZ) is a glucocorticoid responsive protein that links the nuclear factor-kappa B (NFκB) and the glucocorticoid signaling pathways. Functional and binding studies suggest that the proline-rich region at the carboxy terminus of GILZ binds the p65 subunit of NFκB and suppresses the immunoinflammatory response. A widely-used strategy in the discovery of peptide drugs involves exploitation of the complementary surfaces of naturally occurring binding partners. Previously, we observed that a synthetic peptide (GILZ-P) derived from the proline-rich region of GILZ bound activated p65 and ameliorated experimental encephalomyelitis. Here we characterize the secondary structure of GILZ-P by circular dichroic analysis. GILZ-P adopts an extended polyproline type II helical conformation consistent with the structural conformation commonly observed in interfaces of transient intermolecular interactions. To determine the potential application of GILZ-P in humans, we evaluated the toxicity and efficacy of the peptide drug in mature human macrophage-like THP-1 cells. Treatment with GILZ-P at a wide range of concentrations commonly used for peptide drugs was nontoxic as determined by cell viability and apoptosis assays. Functionally, GILZ-P suppressed proliferation and glutamate secretion by activated macrophages by inhibiting nuclear translocation of p65. Collectively, our data suggest that the GILZ-P has therapeutic potential in chronic CNS diseases where persistent inflammation leads to neurodegeneration such as multiple sclerosis and Alzheimer’s disease. PMID:25584020

Biophysical properties of intrinsically disordered p130Cas substrate domain--implication in mechanosensing.

PubMed

Hotta, Kinya; Ranganathan, Soumya; Liu, Ruchuan; Wu, Fei; Machiyama, Hiroaki; Gao, Rong; Hirata, Hiroaki; Soni, Neelesh; Ohe, Takashi; Hogue, Christopher W V; Madhusudhan, M S; Sawada, Yasuhiro

2014-04-01

Mechanical stretch-induced tyrosine phosphorylation in the proline-rich 306-residue substrate domain (CasSD) of p130Cas (or BCAR1) has eluded an experimentally validated structural understanding. Cellular p130Cas tyrosine phosphorylation is shown to function in areas without internal actomyosin contractility, sensing force at the leading edge of cell migration. Circular dichroism shows CasSD is intrinsically disordered with dominant polyproline type II conformations. Strongly conserved in placental mammals, the proline-rich sequence exhibits a pseudo-repeat unit with variation hotspots 2-9 residues before substrate tyrosine residues. Atomic-force microscopy pulling experiments show CasSD requires minimal extension force and exhibits infrequent, random regions of weak stability. Proteolysis, light scattering and ultracentrifugation results show that a monomeric intrinsically disordered form persists for CasSD in solution with an expanded hydrodynamic radius. All-atom 3D conformer sampling with the TraDES package yields ensembles in agreement with experiment when coil-biased sampling is used, matching the experimental radius of gyration. Increasing β-sampling propensities increases the number of prolate conformers. Combining the results, we conclude that CasSD has no stable compact structure and is unlikely to efficiently autoinhibit phosphorylation. Taking into consideration the structural propensity of CasSD and the fact that it is known to bind to LIM domains, we propose a model of how CasSD and LIM domain family of transcription factor proteins may function together to regulate phosphorylation of CasSD and effect machanosensing.

Functional Evaluation of Proteins in Watery and Gel Saliva of Aphids

PubMed Central

van Bel, Aart J. E.; Will, Torsten

2016-01-01

Gel and watery saliva are regarded as key players in aphid–pIant interactions. The salivary composition seems to be influenced by the variable environment encountered by the stylet tip. Milieu sensing has been postulated to provide information needed for proper stylet navigation and for the required switches between gel and watery saliva secretion during stylet progress. Both the chemical and physical factors involved in sensing of the stylet’s environment are discussed. To investigate the salivary proteome, proteins were collected from dissected gland extracts or artificial diets in a range of studies. We discuss the advantages and disadvantages of either collection method. Several proteins were identified by functional assays or by use of proteomic tools, while most of their functions still remain unknown. These studies disclosed the presence of at least two proteins carrying numerous sulfhydryl groups that may act as the structural backbone of the salivary sheath. Furthermore, cell-wall degrading proteins such a pectinases, pectin methylesterases, polygalacturonases, and cellulases as well as diverse Ca2+-binding proteins (e.g., regucalcin, ARMET proteins) were detected. Suppression of the plant defense may be a common goal of salivary proteins. Salivary proteases are likely involved in the breakdown of sieve-element proteins to invalidate plant defense or to increase the availability of organic N compounds. Salivary polyphenoloxidases, peroxidases and oxidoreductases were suggested to detoxify, e.g., plant phenols. During the last years, an increasing number of salivary proteins have been categorized under the term ‘effector’. Effectors may act in the suppression (C002 or MIF cytokine) or the induction (e.g., Mp10 or Mp 42) of plant defense, respectively. A remarkable component of watery saliva seems the protein GroEL that originates from Buchnera aphidicola, the obligate symbiont of aphids and probably reflects an excretory product that induces plant defense responses. Furthermore, chitin fragments in the saliva may trigger defense reactions (e.g., callose deposition). The functions of identified proteins and protein classes are discussed with regard to physical and chemical characteristics of apoplasmic and symplasmic plant compartments. PMID:28018380

Two Proline-Rich Nuclear Localization Signals in the Amino- and Carboxyl-Terminal Regions of the Borna Disease Virus Phosphoprotein

PubMed Central

Shoya, Yuko; Kobayashi, Takeshi; Koda, Toshiaki; Ikuta, Kazuyoshi; Kakinuma, Mitsuaki; Kishi, Masahiko

1998-01-01

Borna disease virus (BDV) uses a unique strategy of replication and transcription which takes place in the nucleus, unlike other known, nonsegmented, negative-stranded RNA viruses of animal origin. In this process, viral constituents necessary for replication must be transported to the nucleus from the cytoplasm. We report here the evidence that BDV P protein, which may play an important role in viral replication and transcription, is transported into the nucleus in the absence of other viral constituents. This transportation is accomplished by its own nuclear localization signals (NLSs), which are present in both N-terminal (29PRPRKIPR36) and C-terminal (181PPRIYPQLPSAPT193) regions of the protein. These two NLSs can function independently and both have several Pro residues as key amino acids. PMID:9811710

Jasmonic acid/methyl jasmonate accumulate in wounded soybean hypocotyls and modulate wound gene expression.

PubMed

Creelman, R A; Tierney, M L; Mullet, J E

1992-06-01

Jasmonic acid (JA) and its methyl ester, methyl jasmonate (MeJA), are plant lipid derivatives that resemble mammalian eicosanoids in structure and biosynthesis. These compounds are proposed to play a role in plant wound and pathogen responses. Here we report the quantitative determination of JA/MeJA in planta by a procedure based on the use of [13C,2H3]MeJA as an internal standard. Wounded soybean (Glycine max [L] Merr. cv. Williams) stems rapidly accumulated MeJA and JA. Addition of MeJA to soybean suspension cultures also increased mRNA levels for three wound-responsive genes (chalcone synthase, vegetative storage protein, and proline-rich cell wall protein) suggesting a role for MeJA/JA in the mediation of several changes in gene expression associated with the plants' response to wounding.

Protein Binding and Astringent Taste of a Polymeric Procyanidin, 1,2,3,4,6-Penta-O-galloyl-β-D-glucopyranose, Castalagin and Grandinin

PubMed Central

Hofmann, Thomas; Glabasnia, Arne; Schwarz, Bernd; Wisman, Kimberly N.; Gangwer, Kelly A.; Hagerman, Ann E.

2008-01-01

The objective of the present investigation was to examine oral astringency and protein binding activity of four structurally well-defined tannins, namely procyanidin (epicatechin16(4→8)catechin), pentagalloyl glucose (1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose), castalagin, and grandinin, representing the three main structural categories of tannins, the proanthocyanidins, the gallotannins, and the ellagitannins. Astringency threshold and dose response were determined by the half-tongue test using a trained human panel. Protein binding stoichiometry and relative affinity were determined using radioiodinated bovine serum albumin in precipitation or competitive binding assays. Procyanidin and pentagalloyl glucose were perceived as highly astringent compounds and had relatively steep dose response curves but castalagin and grandinin had a lower mass threshold for detection. In vitro, procyanidin was the most effective protein precipitating agent, and grandinin the least. Increasing the temperature increased protein precipitation by the hydrolysable tannins, especially grandinin. All four polyphenols had higher relative affinity for proline-rich proteins than for bovine serum albumin. PMID:17147439

Interactions between whey proteins and salivary proteins as related to astringency of whey protein beverages at low pH.

PubMed

Ye, A; Streicher, C; Singh, H

2011-12-01

Whey protein beverages have been shown to be astringent at low pH. In the present study, the interactions between model whey proteins (β-lactoglobulin and lactoferrin) and human saliva in the pH range from 7 to 2 were investigated using particle size, turbidity, and ζ-potential measurements and sodium dodecyl sulfate-PAGE. The correlation between the sensory results of astringency and the physicochemical data was discussed. Strong interactions between β-lactoglobulin and salivary proteins led to an increase in the particle size and turbidity of mixtures of both unheated and heated β-lactoglobulin and human saliva at pH ∼3.4. However, the large particle size and high turbidity that occurred at pH 2.0 were the result of aggregation of human salivary proteins. The intense astringency in whey protein beverages may result from these increases in particle size and turbidity at these pH values and from the aggregation and precipitation of human salivary proteins alone at pH <3.0. The involvement of salivary proteins in the interaction is a key factor in the perception of astringency in whey protein beverages. At any pH, the increases in particle size and turbidity were much smaller in mixtures of lactoferrin and saliva, which suggests that aggregation and precipitation may not be the only mechanism linked to the perception of astringency in whey protein. Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Probiotic bacteria affect the composition of salivary pellicle and streptococcal adhesion in vitro.

PubMed

Haukioja, A; Loimaranta, V; Tenovuo, J

2008-08-01

The use of probiotic bacteria is increasing worldwide and at least some of them can transiently colonize the oral cavity. Several studies have shown that probiotic bacteria, which are often thought of in relation only to intestinal health, can also affect the oral ecology, but the mechanisms for this are largely unknown. The aim of this study was to investigate in vitro if the probiotic bacteria used in commercial products affect the protein composition of the salivary pellicle and the adherence of other oral bacteria. Salivary pellicle on hydroxyapatite and the adhesion of two oral streptococci, Streptococcus mutans and Streptococcus gordonii, were used as a model. Probiotic bacteria that bound to saliva-coated hydroxyapatite reduced the adhesion of S. mutans but the inhibitory effect on the adherence of S. gordonii was weaker. Salivary pellicle protein composition was modified by all the strains tested. The modifications in the pellicle affected the adherence of S. mutans but not of S. gordonii. Two of the proteins missing from the pellicles made of saliva-treated with the probiotic bacteria were identified as salivary agglutinin gp340 and salivary peroxidase. All bacterial strains bound salivary agglutinin gp340. The ability of the probiotic bacteria to degrade peroxidase was demonstrated with purified bovine lactoperoxidase and two of the probiotic strains. This in vitro study showed that probiotic strains used in commercial products may affect the oral ecology by specifically preventing the adherence of other bacteria and by modifying the protein composition of the salivary pellicle.

Green Tea Consumption after Intense Taekwondo Training Enhances Salivary Defense Factors and Antibacterial Capacity

PubMed Central

Lin, Shiuan-Pey; Li, Chia-Yang; Suzuki, Katsuhiko; Chang, Chen-Kang; Chou, Kuei-Ming; Fang, Shih-Hua

2014-01-01

The aim of this study was to investigate the short-term effects of green tea consumption on selected salivary defense proteins, antibacterial capacity and anti-oxidation activity in taekwondo (TKD) athletes, following intensive training. Twenty-two TKD athletes performed a 2-hr TKD training session. After training, participants ingested green tea (T, caffeine 6 mg/kg and catechins 22 mg/kg) or an equal volume of water (W). Saliva samples were collected at three time points: before training (BT-T; BT-W), immediately after training (AT-T; AT-W), and 30 min after drinking green tea or water (Rec-T; Rec-W). Salivary total protein, immunoglobulin A (SIgA), lactoferrin, α-amylase activity, free radical scavenger activity (FRSA) and antibacterial capacity were measured. Salivary total protein, lactoferrin, SIgA concentrations and α-amylase activity increased significantly immediately after intensive TKD training. After tea drinking and 30 min rest, α-amylase activity and the ratio of α-amylase to total protein were significantly higher than before and after training. In addition, salivary antibacterial capacity was not affected by intense training, but green tea consumption after training enhanced salivary antibacterial capacity. Additionally, we observed that salivary FRSA was markedly suppressed immediately after training and quickly returned to pre-exercise values, regardless of which fluid was consumed. Our results show that green tea consumption significantly enhances the activity of α-amylase and salivary antibacterial capacity. PMID:24498143

Molecular analysis of two cDNA clones encoding acidic class I chitinase in maize.

PubMed Central

Wu, S; Kriz, A L; Widholm, J M

1994-01-01

The cloning and analysis of two different cDNA clones encoding putative maize (Zea mays L.) chitinases obtained by polymerase chain reaction (PCR) and cDNA library screening is described. The cDNA library was made from poly(A)+ RNA from leaves challenged with mercuric chloride for 2 d. The two clones, pCh2 and pCh11, appear to encode class I chitinase isoforms with cysteine-rich domains (not found in pCh11 due to the incomplete sequence) and proline-/glycine-rich or proline-rich hinge domains, respectively. The pCh11 clone resembles a previously reported maize seed chitinase; however, the deduced proteins were found to have acidic isoelectric points. Analysis of all monocot chitinase sequences available to date shows that not all class I chitinases possess the basic isoelectric points usually found in dicotyledonous plants and that monocot class II chitinases do not necessarily exhibit acidic isoelectric points. Based on sequence analysis, the pCh2 protein is apparently synthesized as a precursor polypeptide with a signal peptide. Although these two clones belong to class I chitinases, they share only about 70% amino acid homology in the catalytic domain region. Southern blot analysis showed that pCh2 may be encoded by a small gene family, whereas pCh11 was single copy. Northern blot analysis demonstrated that these genes are differentially regulated by mercuric chloride treatment. Mercuric chloride treatment caused rapid induction of pCh2 from 6 to 48 h, whereas pCh11 responded only slightly to the same treatment. During seed germination, embryos constitutively expressed both chitinase genes and the phytohormone abscisic acid had no effect on the expression. The fungus Aspergillus flavus was able to induce both genes to comparable levels in aleurone layers and embryos but not in endosperm tissue. Maize callus growth on the same plate with A. flavus for 1 week showed induction of the transcripts corresponding to pCh2 but not to pCh11. These studies indicate that the different chitinase isoforms in maize might have different functions in the plant, since they show differential expression patterns under different conditions. PMID:7972490

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

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.

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

Structural and energetic basis of ALS-causing mutations in the atypical proline-tyrosine nuclear localization signal of the Fused in Sarcoma protein (FUS)

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

Zhang, Zi Chao; Chook, Yuh Min

Mutations in the proline/tyrosine–nuclear localization signal (PY-NLS) of the Fused in Sarcoma protein (FUS) cause amyotrophic lateral sclerosis (ALS). Here we report the crystal structure of the FUS PY-NLS bound to its nuclear import receptor Karyopherinβ2 (Kapβ2; also known as Transportin). The FUS PY-NLS occupies the structurally invariant C-terminal arch of Kapβ2, tracing a path similar to that of other characterized PY-NLSs. Unlike other PY-NLSs, which generally bind Kapβ2 in fully extended conformations, the FUS peptide is atypical as its central portion forms a 2.5-turn α-helix. The Kapβ2-binding epitopes of the FUS PY-NLS consist of an N-terminal PGKM hydrophobic motif,more » a central arginine-rich α-helix, and a C-terminal PY motif. ALS mutations are found almost exclusively within these epitopes. Each ALS mutation site makes multiple contacts with Kapβ2 and mutations of these residues decrease binding affinities for Kapβ2 (K D for wild-type FUS PY-NLS is 9.5 nM) up to ninefold. Thermodynamic analyses of ALS mutations in the FUS PY-NLS show that the weakening of FUS-Kapβ2 binding affinity, the degree of cytoplasmic mislocalization, and ALS disease severity are correlated.« less

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

Salivary gland dysfunction markers in type 2 diabetes mellitus patients.

PubMed

Aitken-Saavedra, Juan; Rojas-Alcayaga, Gonzalo; Maturana-Ramírez, Andrea; Escobar-Álvarez, Alejandro; Cortes-Coloma, Andrea; Reyes-Rojas, Montserrat; Viera-Sapiain, Valentina; Villablanca-Martínez, Claudia; Morales-Bozo, Irene

2015-10-01

Diabetes mellitus (DM) is a chronic disease of the carbohydrate metabolism that, when not rigorously controlled, compromises systemic and organ integrity, thereby causing renal diseases, blindness, neuropathy, arteriosclerosis, infections, and glandular dysfunction, including the salivary glands. The aim of this study was to determine the relationship between the qualitative and quantitative parameters of salivary alteration, which are indicators of salivary gland dysfunction, and the level of metabolic control of type 2 diabetes patients. A convenience sample of 74 voluntary patients with type 2 DM was selected, each of whom donated a sample of unstimulated saliva. Salivary parameters such as salivary flow rate, protein concentration, pH, and xerostomia were studied. There is a positive relationship between the level of metabolic control measured with HbA1 and the protein concentration in saliva (Spearman rho = 0.329 and p = 0.004). The same assay showed an inverse correlation between HbA1 and pH (Spearman rho = -0.225 and p = 0.05). The protein concentration in saliva and, to a lesser extent, the pH may be useful as glandular dysfunction indicators in DM2 patients. Saliva, type 2 diabetes mellitus, pH, protein concentration, xerostomia.

Identification of Regulatory Factors for Mesenchymal Stem Cell-Derived Salivary Epithelial Cells in a Co-Culture System

PubMed Central

Park, Yun-Jong; Koh, Jin; Gauna, Adrienne E.; Chen, Sixue; Cha, Seunghee

2014-01-01

Patients with Sjögren’s syndrome or head and neck cancer patients who have undergone radiation therapy suffer from severe dry mouth (xerostomia) due to salivary exocrine cell death. Regeneration of the salivary glands requires a better understanding of regulatory mechanisms by which stem cells differentiate into exocrine cells. In our study, bone marrow-derived mesenchymal stem cells were co-cultured with primary salivary epithelial cells from C57BL/6 mice. Co-cultured bone marrow-derived mesenchymal stem cells clearly resembled salivary epithelial cells, as confirmed by strong expression of salivary gland epithelial cell-specific markers, such as alpha-amylase, muscarinic type 3 receptor, aquaporin-5, and cytokeratin 19. To identify regulatory factors involved in this differentiation, transdifferentiated mesenchymal stem cells were analyzed temporarily by two-dimensional-gel-electrophoresis, which detected 58 protein spots (>1.5 fold change, p<0.05) that were further categorized into 12 temporal expression patterns. Of those proteins only induced in differentiated mesenchymal stem cells, ankryin-repeat-domain-containing-protein 56, high-mobility-group-protein 20B, and transcription factor E2a were selected as putative regulatory factors for mesenchymal stem cell transdifferentiation based on putative roles in salivary gland development. Induction of these molecules was confirmed by RT-PCR and western blotting on separate sets of co-cultured mesenchymal stem cells. In conclusion, our study is the first to identify differentially expressed proteins that are implicated in mesenchymal stem cell differentiation into salivary gland epithelial cells. Further investigation to elucidate regulatory roles of these three transcription factors in mesenchymal stem cell reprogramming will provide a critical foundation for a novel cell-based regenerative therapy for patients with xerostomia. PMID:25402494

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 Same Microbiota and a Potentially Discriminant Metabolome in the Saliva of Omnivore, Ovo-Lacto-Vegetarian and Vegan Individuals

PubMed Central

De Filippis, Francesca; Vannini, Lucia; La Storia, Antonietta; Laghi, Luca; Piombino, Paola; Stellato, Giuseppina; Serrazanetti, Diana I.; Gozzi, Giorgia; Turroni, Silvia; Ferrocino, Ilario; Lazzi, Camilla; Di Cagno, Raffaella; Gobbetti, Marco; Ercolini, Danilo

2014-01-01

The salivary microbiota has been linked to both oral and non-oral diseases. Scant knowledge is available on the effect of environmental factors such as long-term dietary choices on the salivary microbiota and metabolome. This study analyzed the microbial diversity and metabolomic profiles of the saliva of 161 healthy individuals who followed an omnivore or ovo-lacto-vegetarian or vegan diet. A large core microbiota was identified, including 12 bacterial genera, found in >98% of the individuals. The subjects could be stratified into three “salivary types” that differed on the basis of the relative abundance of the core genera Prevotella, Streptococcus/Gemella and Fusobacterium/Neisseria. Statistical analysis indicated no effect of dietary habit on the salivary microbiota. Phylogenetic beta-diversity analysis consistently showed no differences between omnivore, ovo-lacto-vegetarian and vegan individuals. Metabolomic profiling of saliva using 1H-NMR and GC-MS/SPME identified diet-related biomarkers that enabled a significant discrimination between the 3 groups of individuals on the basis of their diet. Formate, urea, uridine and 5-methyl-3-hexanone could discriminate samples from omnivores, whereas 1-propanol, hexanoic acid and proline were characteristic of non-omnivore diets. Although the salivary metabolome can be discriminating for diet, the microbiota has a remarkable inter-individual stability and did not vary with dietary habits. Microbial homeostasis might be perturbed with sub-standard oral hygiene or other environmental factors, but there is no current indication that a choice of an omnivore, ovo-lacto-vegetarian or vegan diet can lead to a specific composition of the oral microbiota with consequences on the oral homeostasis. PMID:25372853

The same microbiota and a potentially discriminant metabolome in the saliva of omnivore, ovo-lacto-vegetarian and Vegan individuals.

PubMed

De Filippis, Francesca; Vannini, Lucia; La Storia, Antonietta; Laghi, Luca; Piombino, Paola; Stellato, Giuseppina; Serrazanetti, Diana I; Gozzi, Giorgia; Turroni, Silvia; Ferrocino, Ilario; Lazzi, Camilla; Di Cagno, Raffaella; Gobbetti, Marco; Ercolini, Danilo

2014-01-01

The salivary microbiota has been linked to both oral and non-oral diseases. Scant knowledge is available on the effect of environmental factors such as long-term dietary choices on the salivary microbiota and metabolome. This study analyzed the microbial diversity and metabolomic profiles of the saliva of 161 healthy individuals who followed an omnivore or ovo-lacto-vegetarian or vegan diet. A large core microbiota was identified, including 12 bacterial genera, found in >98% of the individuals. The subjects could be stratified into three "salivary types" that differed on the basis of the relative abundance of the core genera Prevotella, Streptococcus/Gemella and Fusobacterium/Neisseria. Statistical analysis indicated no effect of dietary habit on the salivary microbiota. Phylogenetic beta-diversity analysis consistently showed no differences between omnivore, ovo-lacto-vegetarian and vegan individuals. Metabolomic profiling of saliva using (1)H-NMR and GC-MS/SPME identified diet-related biomarkers that enabled a significant discrimination between the 3 groups of individuals on the basis of their diet. Formate, urea, uridine and 5-methyl-3-hexanone could discriminate samples from omnivores, whereas 1-propanol, hexanoic acid and proline were characteristic of non-omnivore diets. Although the salivary metabolome can be discriminating for diet, the microbiota has a remarkable inter-individual stability and did not vary with dietary habits. Microbial homeostasis might be perturbed with sub-standard oral hygiene or other environmental factors, but there is no current indication that a choice of an omnivore, ovo-lacto-vegetarian or vegan diet can lead to a specific composition of the oral microbiota with consequences on the oral homeostasis.

Two novel mutations in the glycine-rich region of human PAX6 gene: Implications for an association of cataracts and anosmia with aniridia

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

Martha, A.; Ferrel, R.E.; Hittner, H.M.

1994-09-01

Aniridia (iris hyplasia) is a autosomal dominant congenital disorder of the eye. Mutations in the human aniridia (PAX6) gene have now been identified in many patients from various ethnic groups. In the present study we describe new mutations in this gene. Out of four mutations found, three were novel mutations; the fourth one is identical to the previously reported mutations (C{yields}T transition at nt 240). The three novel mutations analyzed were in the glycine-rich region (two) and in the proline/serine/threonine-rich (PST) region (one). Previously no mutations were reported for the glycine-rich region in humans. One of the mutations found inmore » this region is associated with cataracts in an aniridia family. The other splice mutation found in the PST domain is associated with anosmia (lack of sensation to smell) in a sporadic aniridia case. Two of the mutations presented here, one in the glycine-rich region and the other in the PST domain, were not detected by SSCR. These mutations could be detected by using MDE gel and heteroduplex information. All mutations found in the present study are similar in that 32 of 33 PAX6 mutations result in protein truncation and haploinsufficiency.« less

The puckering free-energy surface of proline

NASA Astrophysics Data System (ADS)

Wu, Di

2013-03-01

Proline has two preferred puckering states, which are often characterized by the pseudorotation phase angle and amplitude. Although proline's five endocyclic torsion angles can be utilized to calculate the phase angle and amplitude, it is not clear if there is any direct correlation between each torsion angle and the proline-puckering pathway. Here we have designed five proline puckering pathways utilizing each torsion angle χj (j = 1˜5) as the reaction coordinate. By examining the free-energy surfaces of the five puckering pathways, we find they can be categorized into two groups. The χ2 pathway (χ2 is about the Cβ—Cγ bond) is especially meaningful in describing proline puckering: it changes linearly with the puckering amplitude and symmetrically with the phase angle. Our results show that this conclusion applies to both trans and cis proline conformations. We have also analyzed the correlations of proline puckering and its backbone torsion angles ϕ and ψ. We show proline has preferred puckering states at the specific regions of ϕ, ψ angles. Interestingly, the shapes of ψ-χ2 free-energy surfaces are similar among the trans proline in water, cis proline in water and cis proline in the gas phase, but they differ substantially from that of the trans proline in the gas phase. Our calculations are conducted using molecular simulations; we also verify our results using the proline conformations selected from the Protein Data Bank. In addition, we have compared our results with those calculated by the quantum mechanical methods.

The saliva proteome of the blood-feeding insect Triatoma infestans is rich in platelet-aggregation inhibitors

NASA Astrophysics Data System (ADS)

Charneau, Sébastien; Junqueira, Magno; Costa, Camila M.; Pires, Daniele L.; Fernandes, Ellen S.; Bussacos, Ana C.; Sousa, Marcelo V.; Ricart, Carlos André O.; Shevchenko, Andrej; Teixeira, Antonio R. L.

2007-12-01

The saliva of the bloodsucking bug Triatoma infestans vector of Chagas disease contains an anti-hemostatic molecular cocktail that prevents coagulation, vasoconstriction and platelet aggregation in a vertebrate prey. In order to characterize T. infestans saliva proteome, we separated the secreted saliva by two-dimensional gel electrophoresis (2-DE). More than 200 salivary proteins were detected on the 2-DE map, mainly in the alkaline region. By nanoLC-MS/MS analysis using a LTQ-Orbitrap equipment followed by a combination of conventional and sequence-similarity searches, we identified 58 main protein spots. Most of such proteins possess potential blood-feeding associated functions, particularly anti-platelet aggregation proteins belonging to lipocalin and apyrase families. The saliva protein composition indicates a highly specific molecular mechanism of early response to platelet aggregation. This first proteome analysis of the T. infestans secreted saliva provides a basis for a better understanding of this fluid protein composition highly directed to counterpart hemostasis of the prey, thus promoting the bug's blood-feeding.

The effect of physical exercise on salivary secretion of MUC5B, amylase and lysozyme.

PubMed

Ligtenberg, Antoon J M; Brand, Henk S; van den Keijbus, Petra A M; Veerman, Enno C I

2015-11-01

Saliva secretion is regulated by the autonomic nervous system. Parasympathic stimuli increase the secretion of water and mucin MUC5B, whereas sympathetic stimuli such as physical exercise increase the secretion of amylase and other proteins. In the present study we investigated the effect of physical exercise, as a sympathetic stimulus, on salivary flow rate and output of MUC5B, amylase, lysozyme and total protein. Unstimulated whole saliva was collected before exercise (1), after 10 min exercise with moderate intensity by running with a heart rate around 130 beats per minute (2), followed by 10 min exercise with high intensity by running to exhaustion (3) and after 30 min recovery (4). Salivary flow rate, protein and MUC5B concentration, and amylase and lysozyme activity were determined. Saliva protein composition was analysed using SDS-PAGE and immunoblotting. Salivary flow rate, protein and lysozyme secretion increased after exercise with moderate intensity and increased further after exercise with high intensity (p<0.01). Amylase and MUC5B increased after exercise with moderate intensity (p<0.0001), but did not differ significantly between moderate and high exercise intensity. SDS-PAGE analysis and immunoblotting showed that, especially after exercise with high intensity, the concentrations of several other salivary proteins, including MUC7, albumin, and extra-parotid glycoprotein, also increased. Exercise may not only lead to the anticipated increase in amylase and protein secretion, but also to an increase in salivary flow rate and MUC5B secretion. Copyright © 2015 Elsevier Ltd. All rights reserved.

Antioxidant Defence, Oxidative Stress and Oxidative Damage in Saliva, Plasma and Erythrocytes of Dementia Patients. Can Salivary AGE be a Marker of Dementia?

PubMed Central

Choromańska, Magdalena; Klimiuk, Anna; Kostecka-Sochoń, Paula; Wilczyńska, Karolina; Kwiatkowski, Mikołaj; Okuniewska, Natalia; Waszkiewicz, Napoleon; Zalewska, Anna

2017-01-01

Oxidative stress plays a crucial role in dementia pathogenesis; however, its impact on salivary secretion and salivary qualities is still unknown. This study included 80 patients with moderate dementia and 80 healthy age- and sex-matched individuals. Salivary flow, antioxidants (salivary peroxidase, catalase, superoxide dismutase, uric acid and total antioxidant capacity), and oxidative damage products (advanced oxidation protein products, advanced glycation end products (AGE), 8-isoprostanes, 8-hydroxy-2’-deoxyguanosine and total oxidant status) were estimated in non-stimulated and stimulated saliva, as well as in plasma and erythrocytes. We show that in dementia patients the concentration/activity of major salivary antioxidants changes, and the level of oxidative damage to DNA, proteins and lipids is increased compared to healthy controls. Non-stimulated and stimulated salivary secretions were significantly reduced in dementia patients. The deterioration in mini mental state examination (MMSE) score correlated with salivary AGE levels, which when considered with receiver operating characteristic (ROC) analysis, suggests their potential role in the non-invasive diagnosis of dementia. In conclusion, dementia is associated with disturbed salivary redox homeostasis and impaired secretory function of the salivary glands. Salivary AGE may be useful in the diagnosis of dementia. PMID:29053628

The host antimicrobial peptide Bac71-35 binds to bacterial ribosomal proteins and inhibits protein synthesis.

PubMed

Mardirossian, Mario; Grzela, Renata; Giglione, Carmela; Meinnel, Thierry; Gennaro, Renato; Mergaert, Peter; Scocchi, Marco

2014-12-18

Antimicrobial peptides (AMPs) are molecules from innate immunity with high potential as novel anti-infective agents. Most of them inactivate bacteria through pore formation or membrane barrier disruption, but others cross the membrane without damages and act inside the cells, affecting vital processes. However, little is known about their intracellular bacterial targets. Here we report that Bac71-35, a proline-rich AMP belonging to the cathelicidin family, can reach high concentrations (up to 340 μM) inside the E. coli cytoplasm. The peptide specifically and completely inhibits in vitro translation in the micromolar concentration range. Experiments of incorporation of radioactive precursors in macromolecules with E. coli cells confirmed that Bac71-35 affects specifically protein synthesis. Ribosome coprecipitation and crosslinking assays showed that the peptide interacts with ribosomes, binding to a limited subset of ribosomal proteins. Overall, these results indicate that the killing mechanism of Bac71-35 is based on a specific block of protein synthesis. Copyright © 2014 Elsevier Ltd. All rights reserved.

Consumption of apple-boysenberry beverage decreases salivary Actinomyces naeslundii and their adhesion in a multi-species biofilm model.

PubMed

Parkar, S G; Eady, S; Cabecinha, M; Skinner, M A

2017-04-26

We hypothesised that consumption of beverage rich in both fibre and polyphenols, rather than each bioactive alone, will modulate populations of selected salivary bacteria, and their adhesion characteristics and that some of these effects may be due to the anti-microbial activity of the beverage bioactives. We investigated the effect of 4 weeks' consumption of beverages, rich in apple fibre, boysenberry polyphenols, or both on salivary bacteria in healthy subjects. In this placebo-controlled crossover study, saliva samples were collected at the beginning and end of each treatment period, and used for qPCR quantitation of Lactobacillus spp., Actinomyces naeslundii and Streptococcus mutans. The counts of salivary A. naeslundii decreased after the consumption of the apple-boysenberry beverage (P<0.05, Student's t-test). We also examined the effect of the subjects' saliva on bacterial adhesion using a mixed species biofilm model. The salivary pellicles prepared before and after each treatment were inoculated with laboratory strains of A. naeslundii, Lactobacillus rhamnosus and S. mutans and tested for biofilm formation. The post appleboysenberry beverage salivary pellicle significantly decreased the adhesion of A. naeslundii at the end of both 3 and 24 h, in the in vitro biofilm. A 1/16 dilution of the apple-boysenberry beverage itself decreased the proliferation of test strains of A. naeslundii and S. mutans by 51 and 55%, respectively (P<0.005), indicating the antimicrobial activity of its bioactives. This study demonstrated that consumption of apple-boysenberry beverage, rather than apple or the boysenberry beverage alone or the placebo, decreased salivary A. naeslundii and their adhesion under laboratory conditions. These changes are factors that influence oral microecology and potentially oral health.

Distinct signatures of dental plaque metabolic byproducts dictated by periodontal inflammatory status

PubMed Central

Sakanaka, Akito; Kuboniwa, Masae; Hashino, Ei; Bamba, Takeshi; Fukusaki, Eiichiro; Amano, Atsuo

2017-01-01

Onset of chronic periodontitis is associated with an aberrant polymicrobial community, termed dysbiosis. Findings regarding its etiology obtained using high-throughput sequencing technique suggested that dysbiosis holds a conserved metabolic signature as an emergent property. The purpose of this study was to identify robust biomarkers for periodontal inflammation severity. Furthermore, we investigated disease-associated metabolic signatures of periodontal microbiota using a salivary metabolomics approach. Whole saliva samples were obtained from adult subjects before and after removal of supragingival plaque (debridement). Periodontal inflamed surface area (PISA) was employed as an indicator of periodontal inflammatory status. Based on multivariate analyses using pre-debridement salivary metabolomics data, we found that metabolites associated with higher PISA included cadaverine and hydrocinnamate, while uric acid and ethanolamine were associated with lower PISA. Next, we focused on dental plaque metabolic byproducts by selecting salivary metabolites significantly decreased following debridement. Metabolite set enrichment analysis revealed that polyamine metabolism, arginine and proline metabolism, butyric acid metabolism, and lysine degradation were distinctive metabolic signatures of dental plaque in the high PISA group, which may be related to the metabolic signatures of disease-associated communities. Collectively, our findings identified potential biomarkers of periodontal inflammatory status and also provide insight into metabolic signatures of dysbiotic communities. PMID:28220901

Titanium Surface Roughing Treatments contribute to Higher Interaction with Salivary Proteins MG2 and Lactoferrin.

PubMed

Cavalcanti, Yuri Wanderley; Soare, Rodrigo Villamarim; Leite Assis, Marina Araújo; Zenóbio, Elton Gonçalves; Girundi, Francisco Mauro da Silva

2015-02-01

Some surface treatments performed on titanium can alter the composition of salivary pellicle formed on this abiotic surface. Such treatments modify the titanium's surface properties and can promote higher adsorption of proteins, which allow better integration of titanium to the biotic system. This study aimed to evaluate the interactions between salivary proteins and titanium disks with different surface treatments. Machined titanium disks (n = 48) were divided into four experimental groups (n = 12), according to their surface treatments: surface polishing (SP); acid etching (A); spot-blasting plus acid etching (SB-A); spot-blasting followed by acid etching and nano-functionalization (SB-A-NF). Titanium surfaces were characterized by surface roughness and scanning electron microscopy (SEM). Specimens were incubated with human saliva extracted from submandibular and sublingual glands. Total salivary protein adsorbed to titanium was quantified and samples were submitted to western blotting for mucin glycoprotein 2 (MG2) and lactoferrin identification. Surface roughness was statistically higher for SB-A and SB-A-NF groups. Scanning electron microscopy images confirmed that titanium surface treatments increased surface roughness with higher number of porous and scratches for SB-A and SB-A-NF groups. Total protein adsorption was significantly higher for SB-A and SB-A-NF groups (p < 0.05), which also presented higher interactions with MG2 and lactoferrin proteins. The roughing of titanium surface by spot-blasting plus acid etching treatments contribute to higher interaction with salivary proteins, such as MG2 and lactoferrin. Titanium surface roughing increases the interactions of the substratum with salivary proteins, which can influence the integration of dental implants and their components to the oral environment. However, those treatments should be used carefully intraorally, avoiding increase biofilm formation.

Bioprospection of immature salivary glands of Chrysomya megacephala (Fabricius, 1794) (Diptera: Calliphoridae).

PubMed

Caleffe, Ronaldo Roberto Tait; de Oliveira, Stefany Rodrigues; Gigliolli, Adriana Aparecida Sinópolis; Ruvolo-Takasusuki, Maria Claudia Colla; Conte, Helio

2018-06-08

Larval therapy (LT) comprises the application of sterile Calliphoridae larvae for wound debridement, disinfection, and healing in humans and animals. Larval digestion plays a key role in LT, where the salivary glands and gut produce and secrete proteolytic and antimicrobial substances. The objective of this work was to bioprospect the salivary glands of Chrysomya megacephala (Fabricius, 1794) larvae, using ultrastructural, morphological, and histological observations, and the total protein electrophoretic profile. The salivary glands present a deferent duct, originating from the buccal cavity, which bifurcates into efferent ducts that insert through a slight dilatation to a pair of tubular-shaped tissues, united in the region of fat cells. Histologically, the secretion had protein characteristics. Cell cytoplasm presented numerous free ribosomes, autophagic vacuoles, spherical and elongated mitochondria, atypical Golgi complexes, and dilated rough endoplasmic reticulum. In the apical cytoplasm, secretory granules and microvilli secretions demonstrated intense protein synthesis, basal cytoplasm with trachea insertions, and numerous mitochondria. The present work described the ultrastructure and morphology of C. megacephala third instar salivary glands, confirming intense protein synthesis and the molecular weight of soluble proteins. Copyright © 2018 Elsevier Ltd. All rights reserved.

Interplay of biopharmaceutics, biopharmaceutics drug disposition and salivary excretion classification systems

PubMed Central

Idkaidek, Nasir M.

2013-01-01

The aim of this commentary is to investigate the interplay of Biopharmaceutics Classification System (BCS), Biopharmaceutics Drug Disposition Classification System (BDDCS) and Salivary Excretion Classification System (SECS). BCS first classified drugs based on permeability and solubility for the purpose of predicting oral drug absorption. Then BDDCS linked permeability with hepatic metabolism and classified drugs based on metabolism and solubility for the purpose of predicting oral drug disposition. On the other hand, SECS classified drugs based on permeability and protein binding for the purpose of predicting the salivary excretion of drugs. The role of metabolism, rather than permeability, on salivary excretion is investigated and the results are not in agreement with BDDCS. Conclusion The proposed Salivary Excretion Classification System (SECS) can be used as a guide for drug salivary excretion based on permeability (not metabolism) and protein binding. PMID:24493977

An insight into the sialome of Glossina morsitans morsitans

PubMed Central

2010-01-01

Background Blood feeding evolved independently in worms, arthropods and mammals. Among the adaptations to this peculiar diet, these animals developed an armament of salivary molecules that disarm their host's anti-bleeding defenses (hemostasis), inflammatory and immune reactions. Recent sialotranscriptome analyses (from the Greek sialo = saliva) of blood feeding insects and ticks have revealed that the saliva contains hundreds of polypeptides, many unique to their genus or family. Adult tsetse flies feed exclusively on vertebrate blood and are important vectors of human and animal diseases. Thus far, only limited information exists regarding the Glossina sialome, or any other fly belonging to the Hippoboscidae. Results As part of the effort to sequence the genome of Glossina morsitans morsitans, several organ specific, high quality normalized cDNA libraries have been constructed, from which over 20,000 ESTs from an adult salivary gland library were sequenced. These ESTs have been assembled using previously described ESTs from the fat body and midgut libraries of the same fly, thus totaling 62,251 ESTs, which have been assembled into 16,743 clusters (8,506 of which had one or more EST from the salivary gland library). Coding sequences were obtained for 2,509 novel proteins, 1,792 of which had at least one EST expressed in the salivary glands. Despite library normalization, 59 transcripts were overrepresented in the salivary library indicating high levels of expression. This work presents a detailed analysis of the salivary protein families identified. Protein expression was confirmed by 2D gel electrophoresis, enzymatic digestion and mass spectrometry. Concurrently, an initial attempt to determine the immunogenic properties of selected salivary proteins was undertaken. Conclusions The sialome of G. m. morsitans contains over 250 proteins that are possibly associated with blood feeding. This set includes alleles of previously described gene products, reveals new evidence that several salivary proteins are multigenic and identifies at least seven new polypeptide families unique to Glossina. Most of these proteins have no known function and thus, provide a discovery platform for the identification of novel pharmacologically active compounds, innovative vector-based vaccine targets, and immunological markers of vector exposure. PMID:20353571

Suppression of white light generation (supercontinuum) in biological media: a pilot study using human salivary proteins

NASA Astrophysics Data System (ADS)

Santhosh, C.; Dharmadhikari, A. K.; Alti, K.; Dharmadhikari, J. A.; Mathur, D.

2007-02-01

Propagation of ultrashort pulses of intense, infrared light through transparent medium gives rise to a visually spectacular phenomenon known as supercontinuum (white light) generation wherein the spectrum of transmitted light is very considerably broader than that of the incident light. We have studied the propagation of ultrafast (<45 fs) pulses of intense infrared light through biological media (water, and water doped with salivary proteins) which reveal that white light generation is severely suppressed in the presence of a major salivary protein, α-amylase.

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.

Mithramycin inhibits epithelial-to-mesenchymal transition and invasion by downregulating SP1 and SNAI1 in salivary adenoid cystic carcinoma.

PubMed

Li, Jiasu; Gao, Hongmei; Meng, Lingxu; Yin, Lin

2017-06-01

Mithramycin exhibits certain anticancer effects in glioma, metastatic cerebral carcinoma, malignant lymphoma, chorionic carcinoma and breast cancer. However, its effects on salivary adenoid cystic carcinoma remain unclear. Here, we report that mithramycin significantly inhibited epithelial-to-mesenchymal transition and invasion in human salivary adenoid cystic carcinoma cell lines. The underlying mechanism for this activity was further demonstrated to involve decreasing the expression of the transcription factors specificity protein 1 and SNAI1. Specificity protein 1 is a pro-tumourigenic transcription factor that is overexpressed in SACC-LM and SACC-83 cells, and its expression is inhibited by mithramycin. Moreover, chromatin immunoprecipitation assays showed that specificity protein 1 induced SNAI1 transcription through direct binding to the SNAI1 promoter. In summary, this study uncovered the mechanism through which mithramycin inhibits epithelial-to-mesenchymal transition and invasion in salivary adenoid cystic carcinoma cell lines, namely, via downregulating specificity protein 1 and SNAI1 expression, which suggests mithramycin may be a promising therapeutic option for salivary adenoid cystic carcinoma.

Insights into the sand fly saliva: Blood-feeding and immune interactions between sand flies, hosts, and Leishmania.

PubMed

Lestinova, Tereza; Rohousova, Iva; Sima, Michal; de Oliveira, Camila I; Volf, Petr

2017-07-01

Leishmaniases are parasitic diseases present worldwide that are transmitted to the vertebrate host by the bite of an infected sand fly during a blood feeding. Phlebotomine sand flies inoculate into the mammalian host Leishmania parasites embedded in promastigote secretory gel (PSG) with saliva, which is composed of a diverse group of molecules with pharmacological and immunomodulatory properties. In this review, we focus on 3 main aspects of sand fly salivary molecules: (1) structure and composition of salivary glands, including the properties of salivary molecules related to hemostasis and blood feeding, (2) immunomodulatory properties of salivary molecules and the diverse impacts of these molecules on leishmaniasis, ranging from disease exacerbation to vaccine development, and (3) use of salivary molecules for field applications, including monitoring host exposure to sand flies and the risk of Leishmania transmission. Studies showed interesting differences between salivary proteins of Phlebotomus and Lutzomyia species, however, no data were ever published on salivary proteins of Sergentomyia species. In the last 15 years, numerous studies have characterized sand fly salivary proteins and, in parallel, have addressed the impact of such molecules on the biology of the host-sand fly-parasite interaction. The results obtained shall pave the way for the development of field-application tools that could contribute to the management of leishmaniasis in endemic areas.

Salivary gland dysfunction markers in type 2 diabetes mellitus patients

PubMed Central

Aitken-Saavedra, Juan; Rojas-Alcayaga, Gonzalo; Maturana-Ramírez, Andrea; Escobar-Álvarez, Alejandro; Cortes-Coloma, Andrea; Reyes-Rojas, Montserrat; Viera -Sapiain, Valentina; Villablanca-Martínez, Claudia

2015-01-01

Background Diabetes mellitus (DM) is a chronic disease of the carbohydrate metabolism that, when not rigorously controlled, compromises systemic and organ integrity, thereby causing renal diseases, blindness, neuropathy, arteriosclerosis, infections, and glandular dysfunction, including the salivary glands. The aim of this study was to determine the relationship between the qualitative and quantitative parameters of salivary alteration, which are indicators of salivary gland dysfunction, and the level of metabolic control of type 2 diabetes patients. Material and Methods A convenience sample of 74 voluntary patients with type 2 DM was selected, each of whom donated a sample of unstimulated saliva. Salivary parameters such as salivary flow rate, protein concentration, pH, and xerostomia were studied. Results There is a positive relationship between the level of metabolic control measured with HbA1 and the protein concentration in saliva (Spearman rho = 0.329 and p = 0.004). The same assay showed an inverse correlation between HbA1 and pH (Spearman rho = -0.225 and p = 0.05). Conclusions The protein concentration in saliva and, to a lesser extent, the pH may be useful as glandular dysfunction indicators in DM2 patients. Key words:Saliva, type 2 diabetes mellitus, pH, protein concentration, xerostomia. PMID:26535097

Salivary Sialic Acid Levels in Smokeless Tobacco Users

PubMed Central

Farhad Mollashahi, Leila; Honarmand, Marieh; Nakhaee, Alireza; Mollashahi, Ghasem

2016-01-01

Background Smokeless tobacco chewing is one of the known risk factors for oral cancer. It is consumed widely by residents of southeastern Iran. Objectives In this study, salivary free and total sialic acid, and total protein were compared in paan consumers and non-consumers. Patients and Methods In this cross-sectional study, unstimulated saliva of 94 subjects (44 paan consumers and 50 non-consumers) who were referred to the oral medicine department of the dentistry school of Zahedan were collected. Salivary free and total sialic acid, and total protein concentration were measured by standard biochemical methods, and the obtained data were analyzed using SPSS 20 through the non-parametric Mann-Whitney test. Results The concentration of salivary free sialic acid (23.21 ± 18.98 mg/L) was significantly increased in paan consumers. The concentration of salivary Total sialic acid (TSA) (39.57 ± 26.58 mg/L) and total protein (0.77 ± 0.81 mg/mL) showed increases in paan consumers, however, the results were not statistically significant. Conclusions Salivary free and total sialic acid, and total protein were higher in the paan consumers compared to non-consumers. Due to the carcinogenic effect of smokeless tobacco, measurement of these parameters in saliva may be useful in early detection of oral cancer. PMID:27622172

Thermodynamic dissection of the binding energetics of proline-rich peptides to the Abl-SH3 domain: implications for rational ligand design.

PubMed

Palencia, Andrés; Cobos, Eva S; Mateo, Pedro L; Martínez, Jose C; Luque, Irene

2004-02-13

The inhibition of the interactions between SH3 domains and their targets is emerging as a promising therapeutic strategy. To date, rational design of potent ligands for these domains has been hindered by the lack of understanding of the origins of the binding energy. We present here a complete thermodynamic analysis of the binding energetics of the p41 proline-rich decapeptide (APSYSPPPPP) to the SH3 domain of the c-Abl oncogene. Isothermal titration calorimetry experiments have revealed a thermodynamic signature for this interaction (very favourable enthalpic contributions opposed by an unfavourable binding entropy) inconsistent with the highly hydrophobic nature of the p41 ligand and the Abl-SH3 binding site. Our structural and thermodynamic analyses have led us to the conclusion, having once ruled out any possible ionization events or conformational changes coupled to the association, that the establishment of a complex hydrogen-bond network mediated by water molecules buried at the binding interface is responsible for the observed thermodynamic behaviour. The origin of the binding energetics for proline-rich ligands to the Abl-SH3 domain is further investigated by a comparative calorimetric analysis of a set of p41-related ligands. The striking effects upon the enthalpic and entropic contributions provoked by conservative substitutions at solvent-exposed positions in the ligand confirm the complexity of the interaction. The implications of these results for rational ligand design are discussed.

Immediate erosive potential of cola drinks and orange juices.

PubMed

Jensdottir, T; Holbrook, P; Nauntofte, B; Buchwald, C; Bardow, A

2006-03-01

Little is known about the erosive potential of soft drinks within the first minutes of exposure to teeth, and about the potentially protective role of salivary proteins. We hypothesized that the erosive potential is determined primarily by pH and decreases in the presence of salivary proteins. To investigate this, we first added uncoated hydroxyapatite crystals and, second, salivary-protein-coated hydroxyapatite crystals to 20 commercially available cola drinks and orange juices simultaneously, with pH recordings every 15 sec for 3 min. The amount of apatite lost per liter of soft drink per sec was calculated from titratable acidity values to each pH obtained by crystal addition. The erosive potential within the first minutes of exposure was determined solely by the pH of the drink, and the erosive potential was ten-fold higher in cola drinks compared with juices. However, salivary proteins reduced the erosive potential of cola drinks by up to 50%.

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.

An abundant nucleolar phosphoprotein is associated with ribosomal DNA in Tetrahymena macronuclei.

PubMed Central

McGrath, K E; Smothers, J F; Dadd, C A; Madireddi, M T; Gorovsky, M A; Allis, C D

1997-01-01

An abundant 52-kDa phosphoprotein was identified and characterized from macronuclei of the ciliated protozoan Tetrahymena thermophila. Immunoblot analyses combined with light and electron microscopic immunocytochemistry demonstrate that this polypeptide, termed Nopp52, is enriched in the nucleoli of transcriptionally active macronuclei and missing altogether from transcriptionally inert micronuclei. The cDNA sequence encoding Nopp52 predicts a polypeptide whose amino-terminal half consists of multiple acidic/serine-rich regions alternating with basic/proline-rich regions. Multiple serines located in these acidic stretches lie within casein kinase II consensus motifs, and Nopp52 is an excellent substrate for casein kinase II in vitro. The carboxyl-terminal half of Nopp52 contains two RNA recognition motifs and an extreme carboxyl-terminal domain rich in glycine, arginine, and phenylalanine, motifs common in many RNA processing proteins. A similar combination and order of motifs is found in vertebrate nucleolin and yeast NSR1, suggesting that Nopp52 is a member of a family of related nucleolar proteins. NSR1 and nucleolin have been implicated in transcriptional regulation of rDNA and rRNA processing. Consistent with a role in ribosomal gene metabolism, rDNA and Nopp52 colocalize in situ, as well as by cross-linking and immunoprecipitation experiments, demonstrating an association between Nopp52 and rDNA in vivo. Images PMID:9017598

Mechanism of SOS PR-domain autoinhibition revealed by single-molecule assays on native protein from lysate

PubMed Central

Lee, Young Kwang; Low-Nam, Shalini T.; Chung, Jean K.; Hansen, Scott D.; Lam, Hiu Yue Monatrice; Alvarez, Steven; Groves, Jay T.

2017-01-01

The guanine nucleotide exchange factor (GEF) Son of Sevenless (SOS) plays a critical role in signal transduction by activating Ras. Here we introduce a single-molecule assay in which individual SOS molecules are captured from raw cell lysate using Ras-functionalized supported membrane microarrays. This enables characterization of the full-length SOS protein, which has not previously been studied in reconstitution due to difficulties in purification. Our measurements on the full-length protein reveal a distinct role of the C-terminal proline-rich (PR) domain to obstruct the engagement of allosteric Ras independently of the well-known N-terminal domain autoinhibition. This inhibitory role of the PR domain limits Grb2-independent recruitment of SOS to the membrane through binding of Ras·GTP in the SOS allosteric binding site. More generally, this assay strategy enables characterization of the functional behaviour of GEFs with single-molecule precision but without the need for purification. PMID:28452363

Mechanism of SOS PR-domain autoinhibition revealed by single-molecule assays on native protein from lysate.

PubMed

Lee, Young Kwang; Low-Nam, Shalini T; Chung, Jean K; Hansen, Scott D; Lam, Hiu Yue Monatrice; Alvarez, Steven; Groves, Jay T

2017-04-28

The guanine nucleotide exchange factor (GEF) Son of Sevenless (SOS) plays a critical role in signal transduction by activating Ras. Here we introduce a single-molecule assay in which individual SOS molecules are captured from raw cell lysate using Ras-functionalized supported membrane microarrays. This enables characterization of the full-length SOS protein, which has not previously been studied in reconstitution due to difficulties in purification. Our measurements on the full-length protein reveal a distinct role of the C-terminal proline-rich (PR) domain to obstruct the engagement of allosteric Ras independently of the well-known N-terminal domain autoinhibition. This inhibitory role of the PR domain limits Grb2-independent recruitment of SOS to the membrane through binding of Ras·GTP in the SOS allosteric binding site. More generally, this assay strategy enables characterization of the functional behaviour of GEFs with single-molecule precision but without the need for purification.

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

Apyrase activity and adenosine diphosphate induced platelet aggregation inhibition by the salivary gland proteins of Culicoides variipennis, the North American vector of bluetongue viruses.

PubMed

Pérez de León, A A; Tabachnick, W J

1996-02-01

Salivary gland homogenates of Culicoides variipennis, the primary vector of bluetongue (BLU) viruses in North America, were analyzed for apyrase activity. Apyrase (ATP diphosphohydrolase, EC 3.6.1.5) is an anti-hemostatic and anti-inflammatory salivary enzyme of most hematophagous arthropods. The enzyme activity was measured by the release of orthophosphate using ATP, ADP, and AMP as substrates with Ca2+ as the divalent cation. ATPase (11.5 +/- 1 mU/pair of glands), ADPase (7.3 +/- 0.7 mU/pair of glands), and insignificant (P < 0.05) AMPase (0.07 mU/pair of glands) activities were detected in female salivary glands. Male salivary glands contained lower amounts of ATPase and ADPase activity (P < 0.05). The ATPase and ADPase activities were greatest at pH 8.5, and were similarly activated by Mg2+. Molecular sieving HPLC of salivary gland homogenates generated a single peak which coincided with ATPase and ADPase, but no AMPase, activity; the protein has an estimated molecular mass of 35,000 Da. ATPase and ADPase activity, and total protein concentration, were reduced (P < 0.05) in the salivary glands of females after taking a blood meal from a sheep. Salivary gland homogenates also inhibited ADP-induced platelet aggregation in vitro. It is concluded that the salivary ATPase and ADPase activities of C. variipennis reside in one enzyme, and that this enzyme is likely an apyrase. The apyrase activity is thought to be responsible for the inhibition of ADP-induced platelet aggregation, as indicated by the apparent discharge of apyrase from salivary glands into the host during blood feeding. This suggests that apyrase is one of the salivary proteins present in C. variipennis acting as antigens in the development of Culicoides hypersensitivity in ruminants and horses. Apyrase may inhibit an inflammatory response at the feeding site through the subsequent degradation of its end-product, AMP, to adenosine, a potent anti-inflammatory substance, by the ecto-5' nucleotidase activity of neutrophils.

Physiopathology and Management of Gluten-Induced Celiac Disease.

PubMed

Kumar, Jitendra; Kumar, Manoj; Pandey, Rajesh; Chauhan, Nar Singh

2017-02-01

Proline- and glutamine-rich gluten proteins are one of the major constituents of cereal dietary proteins, which are largely resistant to complete cleavage by the human gastrointestinal (GI) digestive enzymes. Partial digestion of gluten generates approximately 35 amino acids (aa) immunomodulatory peptides which activate T-cell-mediated immune system, followed by immunological inflammation of mucosa leading to the onset of celiac disease (CD). CD is an autoimmune disease associated with HLA-DQ2/DQ8 polymorphism and dysbiosis of gut microbiota. CD is either diagnosed using duodenal mucosal biopsis or serological testing for transglutaminase 2 (TG2) specific antibodies (IgA and IgG). Current therapy for CD management is gluten-free diet, while other therapies like glutenase, probiotics, immunomodulation, jamming of HLA-DQ2, inhibition of TG2, and gluten tolerance aided by gluten tolerizing vaccines are being developed. © 2017 Institute of Food Technologists®.

The seahorse genome and the evolution of its specialized morphology.

PubMed

Lin, Qiang; Fan, Shaohua; Zhang, Yanhong; Xu, Meng; Zhang, Huixian; Yang, Yulan; Lee, Alison P; Woltering, Joost M; Ravi, Vydianathan; Gunter, Helen M; Luo, Wei; Gao, Zexia; Lim, Zhi Wei; Qin, Geng; Schneider, Ralf F; Wang, Xin; Xiong, Peiwen; Li, Gang; Wang, Kai; Min, Jiumeng; Zhang, Chi; Qiu, Ying; Bai, Jie; He, Weiming; Bian, Chao; Zhang, Xinhui; Shan, Dai; Qu, Hongyue; Sun, Ying; Gao, Qiang; Huang, Liangmin; Shi, Qiong; Meyer, Axel; Venkatesh, Byrappa

2016-12-14

Seahorses have a specialized morphology that includes a toothless tubular mouth, a body covered with bony plates, a male brood pouch, and the absence of caudal and pelvic fins. Here we report the sequencing and de novo assembly of the genome of the tiger tail seahorse, Hippocampus comes. Comparative genomic analysis identifies higher protein and nucleotide evolutionary rates in H. comes compared with other teleost fish genomes. We identified an astacin metalloprotease gene family that has undergone expansion and is highly expressed in the male brood pouch. We also find that the H. comes genome lacks enamel matrix protein-coding proline/glutamine-rich secretory calcium-binding phosphoprotein genes, which might have led to the loss of mineralized teeth. tbx4, a regulator of hindlimb development, is also not found in H. comes genome. Knockout of tbx4 in zebrafish showed a 'pelvic fin-loss' phenotype similar to that of seahorses.

Profilin as a regulator of the membrane-actin cytoskeleton interface in plant cells

PubMed Central

Sun, Tiantian; Li, Shanwei; Ren, Haiyun

2013-01-01

Membrane structures and cytoskeleton dynamics are intimately inter-connected in the eukaryotic cell. Recently, the molecular mechanisms operating at this interface have been progressively addressed. Many experiments have revealed that the actin cytoskeleton can interact with membranes through various discrete membrane domains. The actin-binding protein, profilin has been proven to inhibit actin polymerization and to promote F-actin elongation. This is dependent on many factors, such as the profilin/G-actin ratio and the ionic environment of the cell. Additionally, profilin has specific domains that interact with phosphoinositides and poly-L-proline rich proteins; theoretically, this gives profilin the opportunity to interact with membranes, and a large number of experiments have confirmed this possibility. In this article, we summarize recent findings in plant cells, and discuss the evidence of the connections among actin cytoskeleton, profilin and biomembranes through direct or indirect relationships. PMID:24391654

PRRT2 Is a Key Component of the Ca2+-Dependent Neurotransmitter Release Machinery

PubMed Central

Valente, Pierluigi; Castroflorio, Enrico; Rossi, Pia; Fadda, Manuela; Sterlini, Bruno; Cervigni, Romina Ines; Prestigio, Cosimo; Giovedì, Silvia; Onofri, Franco; Mura, Elisa; Guarnieri, Fabrizia C.; Marte, Antonella; Orlando, Marta; Zara, Federico; Fassio, Anna; Valtorta, Flavia; Baldelli, Pietro; Corradi, Anna; Benfenati, Fabio

2016-01-01

Summary Heterozygous mutations in proline-rich transmembrane protein 2 (PRRT2) underlie a group of paroxysmal disorders, including epilepsy, kinesigenic dyskinesia, and migraine. Most of the mutations lead to impaired PRRT2 expression, suggesting that loss of PRRT2 function may contribute to pathogenesis. We show that PRRT2 is enriched in presynaptic terminals and that its silencing decreases the number of synapses and increases the number of docked synaptic vesicles at rest. PRRT2-silenced neurons exhibit a severe impairment of synchronous release, attributable to a sharp decrease in release probability and Ca2+ sensitivity and associated with a marked increase of the asynchronous/synchronous release ratio. PRRT2 interacts with the synaptic proteins SNAP-25 and synaptotagmin 1/2. The results indicate that PRRT2 is intimately connected with the Ca2+-sensing machinery and that it plays an important role in the final steps of neurotransmitter release. PMID:27052163

Jasmonic acid/methyl jasmonate accumulate in wounded soybean hypocotyls and modulate wound gene expression.

PubMed Central

Creelman, R A; Tierney, M L; Mullet, J E

1992-01-01

Jasmonic acid (JA) and its methyl ester, methyl jasmonate (MeJA), are plant lipid derivatives that resemble mammalian eicosanoids in structure and biosynthesis. These compounds are proposed to play a role in plant wound and pathogen responses. Here we report the quantitative determination of JA/MeJA in planta by a procedure based on the use of [13C,2H3]MeJA as an internal standard. Wounded soybean (Glycine max [L] Merr. cv. Williams) stems rapidly accumulated MeJA and JA. Addition of MeJA to soybean suspension cultures also increased mRNA levels for three wound-responsive genes (chalcone synthase, vegetative storage protein, and proline-rich cell wall protein) suggesting a role for MeJA/JA in the mediation of several changes in gene expression associated with the plants' response to wounding. Images PMID:1594598

Actinomyces naeslundii Displays Variant fimP and fimA Fimbrial Subunit Genes Corresponding to Different Types of Acidic Proline-Rich Protein and β-Linked Galactosamine Binding Specificity

PubMed Central

Hallberg, K.; Holm, C.; Öhman, U.; Strömberg, N.

1998-01-01

Actinomyces naeslundii genospecies 1 and 2 bind to acidic proline-rich proteins (APRPs) and statherin via type 1 fimbriae and to β-linked galactosamine (GalNAcβ) structures via type 2 fimbriae. In addition, A. naeslundii displays two types of binding specificity for both APRPs-statherin and GalNAcβ, while Actinomyces odontolyticus binds to unknown structures. To study the molecular basis for these binding specificities, DNA fragments spanning the entire or central portions of fimP (type 1) and fimA (type 2) fimbrial subunit genes were amplified by PCR from strains of genospecies 1 and 2 and hybridized with DNA from two independent collections of oral Actinomyces isolates. Isolates of genospecies 1 and 2 and A. odontolyticus, but no other Actinomyces species, were positive for hybridization with fimP and fimA full-length probes irrespective of binding to APRPs and statherin, GalNAcβ, or unknown structures. Isolates of genospecies 1 and 2, with deviating patterns of GalNAcβ1-3Galα-O-ethyl-inhibitable coaggregation with Streptococcus oralis Ss34 and MPB1, were distinguished by a fimA central probe from genospecies 1 and 2, respectively. Furthermore, isolates of genospecies 1 and 2 displaying preferential binding to APRPs over statherin were positive with a fimP central probe, while a genospecies 2 strain with the opposite binding preference was not. The sequences of fimP and fimA central gene segments were highly conserved among isolates with the same, but diversified between those with a variant, binding specificity. In conclusion, A. naeslundii exhibits variant fimP and fimA genes corresponding to diverse APRP and GalNAcβ specificities, respectively, while A. odontolyticus has a genetically related but distinct adhesin binding specificity. PMID:9712794

Free-Energy Landscape of the Amino-Terminal Fragment of Huntingtin in Aqueous Solution

PubMed Central

Binette, Vincent; Côté, Sébastien; Mousseau, Normand

2016-01-01

The first exon of Huntingtin—a protein with multiple biological functions whose misfolding is related to Huntington’s disease—modulates its localization, aggregation, and function within the cell. It is composed of a 17-amino-acid amphipathic segment (Htt17), an amyloidogenic segment of consecutive glutamines (QN), and a proline-rich segment. Htt17 is of fundamental importance: it serves as a membrane anchor to control the localization of huntingtin, it modulates huntingtin’s function through posttranslational modifications, and it controls the self-assembly of the amyloidogenic QN segment into oligomers and fibrils. Experimentally, the conformational ensemble of the Htt17 monomer, as well as the impact of the polyglutamine and proline-rich segments, remains, however, mostly uncharacterized at the atomic level due to its intrinsic flexibility. Here, we unveil the free-energy landscape of Htt17, Htt17Q17, and Htt17Q17P11 using Hamiltonian replica exchange combined with well-tempered metadynamics. We characterize the free-energy landscape of these three fragments in terms of a few selected collective variables. Extensive simulations reveal that the free energy of Htt17 is dominated by a broad ensemble of configurations that agree with solution NMR chemical shifts. Addition of Q17 at its carboxy-terminus reduces the extent of the main basin to more extended configurations of Htt17 with lower helix propensity. Also, the aliphatic carbons of Q17 partially sequester the nonpolar amino acids of Htt17. For its part, addition of Q17P11 shifts the overall landscape to a more extended and helical Htt17 stabilized by interactions with Q17 and P11, which almost exclusively form a PPII-helix, as well as by intramolecular H-bonds and salt bridges. Our characterization of Huntingtin’s amino-terminus provides insights into the structural origin of its ability to oligomerize and interact with phospholipid bilayers, processes closely linked to the biological functions of this protein. PMID:26958885

Activation of the Lbc Rho Exchange Factor Proto-Oncogene by Truncation of an Extended C Terminus That Regulates Transformation and Targeting

PubMed Central

Sterpetti, Paola; Hack, Andrew A.; Bashar, Mariam P.; Park, Brian; Cheng, Sou-De; Knoll, Joan H. M.; Urano, Takeshi; Feig, Larry A.; Toksoz, Deniz

1999-01-01

The human lbc oncogene product is a guanine nucleotide exchange factor that specifically activates the Rho small GTP binding protein, thus resulting in biologically active, GTP-bound Rho, which in turn mediates actin cytoskeletal reorganization, gene transcription, and entry into the mitotic S phase. In order to elucidate the mechanism of onco-Lbc transformation, here we report that while proto- and onco-lbc cDNAs encode identical N-terminal dbl oncogene homology (DH) and pleckstrin homology (PH) domains, proto-Lbc encodes a novel C terminus absent in the oncoprotein that includes a predicted α-helical region homologous to cyto-matrix proteins, followed by a proline-rich region. The lbc proto-oncogene maps to chromosome 15, and onco-lbc represents a fusion of the lbc proto-oncogene N terminus with a short, unrelated C-terminal sequence from chromosome 7. Both onco- and proto-Lbc can promote formation of GTP-bound Rho in vivo. Proto-Lbc transforming activity is much reduced compared to that of onco-Lbc, and a significant increase in transforming activity requires truncation of both the α-helical and proline-rich regions in the proto-Lbc C terminus. Deletion of the chromosome 7-derived C terminus of onco-Lbc does not destroy transforming activity, demonstrating that it is loss of the proto-Lbc C terminus, rather than gain of an unrelated C-terminus by onco-Lbc, that confers transforming activity. Mutations of onco-Lbc DH and PH domains demonstrate that both domains are necessary for full transforming activity. The proto-Lbc product localizes to the particulate (membrane) fraction, while the majority of the onco-Lbc product is cytosolic, and mutations of the PH domain do not affect this localization. The proto-Lbc C-terminus alone localizes predominantly to the particulate fraction, indicating that the C terminus may play a major role in the correct subcellular localization of proto-Lbc, thus providing a mechanism for regulating Lbc oncogenic potential. PMID:9891067

Role of proline under changing environments

PubMed Central

Hayat, Shamsul; Hayat, Qaiser; Alyemeni, Mohammed Nasser; Wani, Arif Shafi; Pichtel, John; Ahmad, Aqil

2012-01-01

When exposed to stressful conditions, plants accumulate an array of metabolites, particularly amino acids. Amino acids have traditionally been considered as precursors to and constituents of proteins, and play an important role in plant metabolism and development. A large body of data suggests a positive correlation between proline accumulation and plant stress. Proline, an amino acid, plays a highly beneficial role in plants exposed to various stress conditions. Besides acting as an excellent osmolyte, proline plays three major roles during stress, i.e., as a metal chelator, an antioxidative defense molecule and a signaling molecule. Review of the literature indicates that a stressful environment results in an overproduction of proline in plants which in turn imparts stress tolerance by maintaining cell turgor or osmotic balance; stabilizing membranes thereby preventing electrolyte leakage; and bringing concentrations of reactive oxygen species (ROS) within normal ranges, thus preventing oxidative burst in plants. Reports indicate enhanced stress tolerance when proline is supplied exogenously at low concentrations. However, some reports indicate toxic effects of proline when supplied exogenously at higher concentrations. In this article, we review and discuss the effects of exogenous proline on plants exposed to various abiotic stresses. Numerous examples of successful application of exogenous proline to improve stress tolerance are presented. The roles played by exogenous proline under varying environments have been critically examined and reviewed. PMID:22951402

Identification of factors that function in Drosophila salivary gland cell death during development using proteomics

PubMed Central

McPhee, C K; Balgley, B M; Nelson, C; Hill, J H; Batlevi, Y; Fang, X; Lee, C S; Baehrecke, E H

2013-01-01

Proteasome inhibitors induce cell death and are used in cancer therapy, but little is known about the relationship between proteasome impairment and cell death under normal physiological conditions. Here, we investigate the relationship between proteasome function and larval salivary gland cell death during development in Drosophila. Drosophila larval salivary gland cells undergo synchronized programmed cell death requiring both caspases and autophagy (Atg) genes during development. Here, we show that ubiquitin proteasome system (UPS) function is reduced during normal salivary gland cell death, and that ectopic proteasome impairment in salivary gland cells leads to early DNA fragmentation and salivary gland condensation in vivo. Shotgun proteomic analyses of purified dying salivary glands identified the UPS as the top category of proteins enriched, suggesting a possible compensatory induction of these factors to maintain proteolysis during cell death. We compared the proteome following ectopic proteasome impairment to the proteome during developmental cell death in salivary gland cells. Proteins that were enriched in both populations of cells were screened for their function in salivary gland degradation using RNAi knockdown. We identified several factors, including trol, a novel gene CG11880, and the cop9 signalsome component cop9 signalsome 6, as required for Drosophila larval salivary gland degradation. PMID:22935612

A salivary sheath protein essential for the interaction of the brown planthopper with rice plants.

PubMed

Huang, Hai-Jian; Liu, Cheng-Wen; Cai, Ye-Fang; Zhang, Min-Zhu; Bao, Yan-Yuan; Zhang, Chuan-Xi

2015-11-01

Salivary secretions, including gel saliva and watery saliva, play crucial roles in the interaction between the insect and plant during feeding. In this study, we identified a salivary gland-specific gene encoding a salivary sheath protein (NlShp) in Nilaparvata lugens. NlShp has two alternative splicing variants; both are expressed at high levels during the nymph and adult stages. Immunohistochemical staining showed that the NlShp were synthesized in the principal gland cells of the salivary gland. LC-MS/MS and western blot analysis confirmed that NlShp was one of the components of the salivary sheath. Simultaneously knocking down the two NlShp variants by RNA interference inhibited both salivary flange and salivary sheath formation and resulted in a lethal phenotype within four days for the brown planthopper (BPH) feeding on rice plants, indicating that the salivary sheath and salivary flanges were essential for plant-associated feeding. Despite the salivary sheath deficiency, no obvious phenotype was observed in the NlShp-knockdown BPHs fed on artificial diet. The electrical penetration graph (EPG) results showed that salivary sheath-deficient BPHs exhibited a prolonged nonpenetration period, scarce sap period, and increased stylet movement on rice plants and eventually starved to death. Our results provided evidence that the interaction between the salivary sheath and host plant might be a critical step in successful BPH feeding. According to present research, we propose a salivary sheath required feeding model for piercing-sucking insects and provide a potential target for rice planthopper management. Copyright © 2015 Elsevier Ltd. All rights reserved.

Humoral response of captive zebra sharks Stegostoma fasciatum to salivary gland proteins of the leech Branchellion torpedinis.

PubMed

Marancik, David P; Leary, John H; Fast, Mark M; Flajnik, Martin F; Camus, Alvin C

2012-10-01

Parasitism by the marine leech Branchellion torpedinis is known to cause disease and mortality in captive elasmobranchs and is difficult to control when inadvertently introduced into public aquaria. Preliminary characterization of the salivary gland transcriptome of B. torpedinis has identified anticoagulants, proteases, and immunomodulators that may be secreted into host tissues to aid leech feeding. This retrospective study examined antigen-specific serum IgM responses in captive zebra sharks Stegostoma fasciatum to leech salivary gland extract. Antibody response was examined by ELISA and Western blot assays in 20 serum samples from six zebra sharks, with a 5 year history of leech infection, and 18 serum samples from 8 captive bred zebra sharks, with no history of leech exposure. ELISA demonstrated significantly higher serum IgM titers to salivary gland extract in exposed zebra sharks compared to the non-exposed population. No obvious trends in antibody titers were appreciated in exposed zebra sharks over a four-year period. One-dimensional and two-dimensional Western blot assays revealed IgM targeted specific salivary gland proteins within the 40, 55, 70 and 90 kD range. Antigenic proteins identified by liquid chromatography-tandem mass spectrometry and de novo peptide sequencing include a secreted disintegrin, metalloproteinase and thrombospondin motif containing protein (ADAMTS), tubulin, aldehyde dehydrogenase and two unknown proteins. Humoral immune responses to leech salivary gland proteins warrants further investigation as there may be options to exploit immune mechanisms to reduce parasite burdens in aquaria. Copyright © 2012 Elsevier Ltd. All rights reserved.

Salivary flow and composition in diabetic and non-diabetic subjects.

PubMed

Lasisi, T J; Fasanmade, A A

2012-06-07

The study investigated the effects of type 2 diabetes mellitus on salivary flow and composition in humans compared to healthy sex and age matched controls. Forty adult human subjects divided into 20 diabetic and 20 non-diabetic healthy subjects were included. Saliva samples were collected and analysed for glucose, total protein, calcium, sodium, potassium, chloride and bicarbonate. Salivary flow rate was also determined. The results showed that salivary glucose and potassium levels were significantly higher (p = 0.01 and 0.002 respectively) in diabetic patients compared with non-diabetic participants. It was also found that the diabetic patients had significant reduction in salivary flow rate when compared with non-diabetic individuals. In contrast, there was no significant difference in levels of total protein, Na+, Ca++, Cl- and HCO3- between the two groups. These results suggest that some oral diseases associated with diabetes mellitus may be due to altered levels of salivary glucose, potassium and flow.

Proline as a stress protectant in yeast: physiological functions, metabolic regulations, and biotechnological applications.

PubMed

Takagi, Hiroshi

2008-11-01

Proline is an important amino acid in terms of its biological functions and biotechnological applications. In response to osmotic stress, proline is accumulated in many bacterial and plant cells as an osmoprotectant. However, it has been shown that proline levels are not increased under various stress conditions in the yeast Saccharomyces cerevisiae cells. Proline is believed to serve multiple functions in vitro such as protein and membrane stabilization, lowering the T (m) of DNA, and scavenging of reactive oxygen species, but the mechanisms of these functions in vivo are poorly understood. Yeast cells biosynthesize proline from glutamate in the cytoplasm via the same pathway found in bacteria and plants and also convert excess proline to glutamate in the mitochondria. Based on the fact that proline has stress-protective activity, S. cerevisiae cells that accumulate proline were constructed by disrupting the PUT1 gene involved in the degradation pathway and by expressing the mutant PRO1 gene encoding the feedback inhibition-less sensitive gamma-glutamate kinase to enhance the biosynthetic activity. The engineered yeast strains successfully showed enhanced tolerance to many stresses, including freezing, desiccation, oxidation, and ethanol. However, the appropriate cellular level and localization of proline play pivotal roles in the stress-protective effect. These results indicate that the increased stress protection is observed in yeast cells under the artificial condition of proline accumulation. Proline is expected to contribute to yeast-based industries by improving the production of frozen dough and alcoholic beverages or breakthroughs in bioethanol production.

A Catalogue of Altered Salivary Proteins Secondary to Invasive Ductal Carcinoma: A Novel In Vivo Paradigm to Assess Breast Cancer Progression

PubMed Central

Streckfus, Charles F.; Bigler, Lenora

2016-01-01

The objective of this manuscript is to introduce a catalogue of salivary proteins that are altered secondary to carcinoma of the breast. The catalogue of salivary proteins is a compilation of twenty years of research by the authors and consists of 233 high and low abundant proteins which have been identified by LC-MS/MS mass spectrometry, 2D-gel analysis and by enzyme-linked immunosorbent assay. The body of research suggests that saliva is a fluid suffused with solubilized by-products of oncogenic expression and that these proteins may be useful in the study of breast cancer progress, treatment efficacy and the tailoring of individualized patient care. PMID:27477923

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.

Salivary histatins in human deep posterior lingual glands (of von Ebner).

PubMed

Piludu, Marco; Lantini, Maria Serenella; Cossu, Margherita; Piras, Monica; Oppenheim, Frank G; Helmerhorst, Eva J; Siqueira, Walter; Hand, Arthur R

2006-11-01

Human saliva contains a family of low molecular weight histidine-rich proteins, named histatins, characterised by bactericidal and fungicidal activities in vitro against several microbial pathogens, such as Streptococcus mutans and Candida albicans. They represent a major component of an innate host non-immune defense system. In an earlier study we described the distribution of histatins in the glandular parenchyma of human major salivary glands, confirming that all human major salivary glands are involved in the secretion of histatins into saliva. In the present study we determined the expression and localisation of histatins in human posterior deep lingual glands (von Ebner's glands) by means of immunoelectron microscopy. Thin sections of normal human salivary glands, embedded in Epon resin, were incubated with rabbit polyclonal antibodies specific for human histatins and successively with a gold conjugated goat anti-rabbit IgG used as secondary antibody. Sections incubated with medium devoid of primary antibody or containing non-immune serum were used as controls. The serous secreting cells represented the main source of histatins in the glandular parenchyma of von Ebner's glands. At the electron microscopic level, labeling was associated with rough endoplasmic reticulum, Golgi complex and secretory granules that represented the main cytoplasmic site of histatin localisation. However, variability in the intensity of labeling was observed among adjacent cells. The present results show for the first time that human von Ebner's glands produce and represent a significant source of histatins, supporting the hypothesis of their important role in preventing microbial assaults on the tissues in the posterior region of the tongue and in the circumvallate papillae.

Development of a Multiplexed Liquid Chromatography Multiple-Reaction-Monitoring Mass Spectrometry (LC-MRM/MS) Method for Evaluation of Salivary Proteins as Oral Cancer Biomarkers*

PubMed Central

Chen, Hsiao-Wei; Wu, Chun-Feng; Chu, Lichieh Julie; Chiang, Wei-Fang; Wu, Chih-Ching; Yu, Jau-Song; Tsai, Cheng-Han; Liang, Kung-Hao; Chang, Yu-Sun; Wu, Maureen; Ou Yang, Wei-Ting

2017-01-01

Multiple (selected) reaction monitoring (MRM/SRM) of peptides is a growing technology for target protein quantification because it is more robust, precise, accurate, high-throughput, and multiplex-capable than antibody-based techniques. The technique has been applied clinically to the large-scale quantification of multiple target proteins in different types of fluids. However, previous MRM-based studies have placed less focus on sample-preparation workflow and analytical performance in the precise quantification of proteins in saliva, a noninvasively sampled body fluid. In this study, we evaluated the analytical performance of a simple and robust multiple reaction monitoring (MRM)-based targeted proteomics approach incorporating liquid chromatography with mass spectrometry detection (LC-MRM/MS). This platform was used to quantitatively assess the biomarker potential of a group of 56 salivary proteins that have previously been associated with human cancers. To further enhance the development of this technology for assay of salivary samples, we optimized the workflow for salivary protein digestion and evaluated quantification performance, robustness and technical limitations in analyzing clinical samples. Using a clinically well-characterized cohort of two independent clinical sample sets (total n = 119), we quantitatively characterized these protein biomarker candidates in saliva specimens from controls and oral squamous cell carcinoma (OSCC) patients. The results clearly showed a significant elevation of most targeted proteins in saliva samples from OSCC patients compared with controls. Overall, this platform was capable of assaying the most highly multiplexed panel of salivary protein biomarkers, highlighting the clinical utility of MRM in oral cancer biomarker research. PMID:28235782

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

Molecular and Immunogenic Properties of Apyrase SP01B and D7-Related SP04 Recombinant Salivary Proteins of Phlebotomus perniciosus from Madrid, Spain

PubMed Central

Martín-Martín, Inés

2013-01-01

Sand fly salivary proteins are on the spotlight to become vaccine candidates against leishmaniasis and to markers of exposure to sand fly bites due to the host immune responses they elicit. Working with the whole salivary homogenate entails serious drawbacks such as the need for maintaining sand fly colonies and the laborious task of glands dissection. In order to overcome these difficulties, producing recombinant proteins of different vectors has become a major task. In this study, a cDNA library was constructed with the salivary glands of Phlebotomus perniciosus from Madrid, Spain, the most widespread vector of Leishmania infantum in the Mediterranean basin. Analysis of the cDNA sequences showed several polymorphisms among the previously described salivary transcripts. The apyrase SP01B and the D7-related protein SP04 were successfully cloned, expressed in Escherichia coli, and purified. Besides, recombinant proteins were recognized by sera of hamsters and mice previously immunized with saliva through the exposure to uninfected sand fly bites. These results suggest that these two recombinant proteins conserved their immunogenic properties after expression in a prokaryote system. Therefore, this work contributes to expand the knowledge of P. perniciosus saliva that would be eventually used for the development of tools for vector control programs. PMID:24171166

Characterization of Guinea Pig Antibody Responses to Salivary Proteins of Triatoma infestans for the Development of a Triatomine Exposure Marker

PubMed Central

Dorňáková, Veronika; Salazar-Sanchez, Renzo; Borrini-Mayori, Katty; Carrion-Navarro, Oscar; Levy, Michael Z.; Schaub, Günter A.; Schwarz, Alexandra

2014-01-01

Background Salivary proteins of Triatoma infestans elicit humoral immune responses in their vertebrate hosts. These immune responses indicate exposure to triatomines and thus can be a useful epidemiological tool to estimate triatomine infestation. In the present study, we analyzed antibody responses of guinea pigs to salivary antigens of different developmental stages of four T. infestans strains originating from domestic and/or peridomestic habitats in Argentina, Bolivia, Chile and Peru. We aimed to identify developmental stage- and strain-specific salivary antigens as potential markers of T. infestans exposure. Methodology and Principal Findings In SDS-PAGE analysis of salivary proteins of T. infestans the banding pattern differed between developmental stages and strains of triatomines. Phenograms constructed from the salivary profiles separated nymphal instars, especially the 5th instar, from adults. To analyze the influence of stage- and strain-specific differences in T. infestans saliva on the antibody response of guinea pigs, twenty-one guinea pigs were exposed to 5th instar nymphs and/or adults of different T. infestans strains. Western blot analyses using sera of exposed guinea pigs revealed stage- and strain-specific variations in the humoral response of animals. In total, 27 and 17 different salivary proteins reacted with guinea pig sera using IgG and IgM antibodies, respectively. Despite all variations of recognized salivary antigens, an antigen of 35 kDa reacted with sera of almost all challenged guinea pigs. Conclusion Salivary antigens are increasingly considered as an epidemiological tool to measure exposure to hematophagous arthropods, but developmental stage- and strain-specific variations in the saliva composition and the respective differences of immunogenicity are often neglected. Thus, the development of a triatomine exposure marker for surveillance studies after triatomine control campaigns requires detailed investigations. Our study resulted in the identification of a potential antigen as useful marker of T. infestans exposure. PMID:24699441

Exploring monovalent and multivalent peptides for the inhibition of FBP21-tWW.

PubMed

Henning, Lisa Maria; Bhatia, Sumati; Bertazzon, Miriam; Marczynke, Michaela; Seitz, Oliver; Volkmer, Rudolf; Haag, Rainer; Freund, Christian

2015-01-01

The coupling of peptides to polyglycerol carriers represents an important route towards the multivalent display of protein ligands. In particular, the inhibition of low affinity intracellular protein-protein interactions can be addressed by this design. We have applied this strategy to develop binding partners for FBP21, a protein which is important for the splicing of pre-mRNA in the nucleus of eukaryotic cells. Firstly, by using phage display the optimized sequence WPPPPRVPR was derived which binds with K Ds of 80 μM and 150 µM to the individual WW domains and with a K D of 150 μM to the tandem-WW1-WW2 construct. Secondly, this sequence was coupled to a hyperbranched polyglycerol (hPG) that allowed for the multivalent display on the surface of the dendritic polymer. This novel multifunctional hPG-peptide conjugate displayed a K D of 17.6 µM which demonstrates that the new carrier provides a venue for the future inhibition of proline-rich sequence recognition by FBP21 during assembly of the spliceosome.

Interaction with Polyglutamine-expanded Huntingtin Alters Cellular Distribution and RNA Processing of Huntingtin Yeast Two-hybrid Protein A (HYPA)*

PubMed Central

Jiang, Ya-Jun; Che, Mei-Xia; Yuan, Jin-Qiao; Xie, Yuan-Yuan; Yan, Xian-Zhong; Hu, Hong-Yu

2011-01-01

Huntington disease (HD) is an autosomal inherited disorder that causes the deterioration of brain cells. The polyglutamine (polyQ) expansion of huntingtin (Htt) is implicated in the pathogenesis of HD via interaction with an RNA splicing factor, Htt yeast two-hybrid protein A/forming-binding protein 11 (HYPA/FBP11). Besides the pathogenic polyQ expansion, Htt also contains a proline-rich region (PRR) located exactly in the C terminus to the polyQ tract. However, how the polyQ expansion influences the PRR-mediated protein interaction and how this abnormal interaction leads to the biological consequence remain elusive. Our NMR structural analysis indicates that the PRR motif of Htt cooperatively interacts with the tandem WW domains of HYPA through domain chaperoning effect of WW1 on WW2. The polyQ-expanded Htt sequesters HYPA to the cytosolic location and then significantly reduces the efficiency of pre-mRNA splicing. We propose that the toxic gain-of-function of the polyQ-expanded Htt that causes dysfunction of cellular RNA processing contributes to the pathogenesis of HD. PMID:21566141

Interaction with polyglutamine-expanded huntingtin alters cellular distribution and RNA processing of huntingtin yeast two-hybrid protein A (HYPA).

PubMed

Jiang, Ya-Jun; Che, Mei-Xia; Yuan, Jin-Qiao; Xie, Yuan-Yuan; Yan, Xian-Zhong; Hu, Hong-Yu

2011-07-15

Huntington disease (HD) is an autosomal inherited disorder that causes the deterioration of brain cells. The polyglutamine (polyQ) expansion of huntingtin (Htt) is implicated in the pathogenesis of HD via interaction with an RNA splicing factor, Htt yeast two-hybrid protein A/forming-binding protein 11 (HYPA/FBP11). Besides the pathogenic polyQ expansion, Htt also contains a proline-rich region (PRR) located exactly in the C terminus to the polyQ tract. However, how the polyQ expansion influences the PRR-mediated protein interaction and how this abnormal interaction leads to the biological consequence remain elusive. Our NMR structural analysis indicates that the PRR motif of Htt cooperatively interacts with the tandem WW domains of HYPA through domain chaperoning effect of WW1 on WW2. The polyQ-expanded Htt sequesters HYPA to the cytosolic location and then significantly reduces the efficiency of pre-mRNA splicing. We propose that the toxic gain-of-function of the polyQ-expanded Htt that causes dysfunction of cellular RNA processing contributes to the pathogenesis of HD.

Changes in the folding landscape of the WW domain provide a molecular mechanism for an inherited genetic syndrome

PubMed Central

Pucheta-Martinez, Encarna; D’Amelio, Nicola; Lelli, Moreno; Martinez-Torrecuadrada, Jorge L.; Sudol, Marius; Saladino, Giorgio; Gervasio, Francesco Luigi

2016-01-01

WW domains are small domains present in many human proteins with a wide array of functions and acting through the recognition of proline-rich sequences. The WW domain belonging to polyglutamine tract-binding protein 1 (PQBP1) is of particular interest due to its direct involvement in several X chromosome-linked intellectual disabilities, including Golabi-Ito-Hall (GIH) syndrome, where a single point mutation (Y65C) correlates with the development of the disease. The mutant cannot bind to its natural ligand WBP11, which regulates mRNA processing. In this work we use high-field high-resolution NMR and enhanced sampling molecular dynamics simulations to gain insight into the molecular causes the disease. We find that the wild type protein is partially unfolded exchanging among multiple beta-strand-like conformations in solution. The Y65C mutation further destabilizes the residual fold and primes the protein for the formation of a disulphide bridge, which could be at the origin of the loss of function. PMID:27456546

The secreted salivary proteome of the pea aphid Acyrthosiphon pisum characterised by mass spectrometry.

PubMed

Carolan, James C; Fitzroy, Carol I J; Ashton, Peter D; Douglas, Angela E; Wilkinson, Thomas L

2009-05-01

Nine proteins secreted in the saliva of the pea aphid Acyrthosiphon pisum were identified by a proteomics approach using GE-LC-MS/MS and LC-MS/MS, with reference to EST and genomic sequence data for A. pisum. Four proteins were identified by their sequences: a homolog of angiotensin-converting enzyme (an M2 metalloprotease), an M1 zinc-dependant metalloprotease, a glucose-methanol-choline (GMC)-oxidoreductase and a homolog to regucalcin (also known as senescence marker protein 30). The other five proteins are not homologous to any previously described sequence and included an abundant salivary protein (represented by ACYPI009881), with a predicted length of 1161 amino acids and high serine, tyrosine and cysteine content. A. pisum feeds on plant phloem sap and the metalloproteases and regucalcin (a putative calcium-binding protein) are predicted determinants of sustained feeding, by inactivation of plant protein defences and inhibition of calcium-mediated occlusion of phloem sieve elements, respectively. The amino acid composition of ACYPI009881 suggests a role in the aphid salivary sheath that protects the aphid mouthparts from plant defences, and the oxidoreductase may promote gelling of the sheath protein or mediate oxidative detoxification of plant allelochemicals. Further salivary proteins are expected to be identified as more sensitive MS technologies are developed.

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

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.

Salt Stress in Arabidopsis: Lipid Transfer Protein AZI1 and Its Control by Mitogen-Activated Protein Kinase MPK3

PubMed Central

Pitzschke, Andrea

2014-01-01

A plant’s capability to cope with environmental challenges largely relies on signal transmission through mitogen-activated protein kinase (MAPK) cascades. In Arabidopsis thaliana, MPK3 is particularly strongly associated with numerous abiotic and biotic stress responses. Identification of MPK3 substrates is a milestone towards improving stress resistance in plants. Here, we characterize AZI1, a lipid transfer protein (LTP)-related hybrid proline-rich protein (HyPRP), as a novel target of MPK3. AZI1 is phosphorylated by MPK3 in vitro. As documented by co-immunoprecipitation and bimolecular fluorescence complementation experiments, AZI1 interacts with MPK3 to form protein complexes in planta. Furthermore, null mutants of azi1 are hypersensitive to salt stress, while AZI1-overexpressing lines are markedly more tolerant. AZI1 overexpression in the mpk3 genetic background partially alleviates the salt-hypersensitive phenotype of this mutant, but functional MPK3 appears to be required for the full extent of AZI1-conferred robustness. Notably, this robustness does not come at the expense of normal development. Immunoblot and RT–PCR data point to a role of MPK3 as positive regulator of AZI1 abundance. PMID:24214892

Collection of salivary proteins of psyllids (Hemiptera: Psylloidea)

USDA-ARS?s Scientific Manuscript database

Phloem-feeding insects discharge into the phloem of host plants copious amounts of enzymatically active saliva which prevents phloem occlusion and suppresses plant defenses. Although previous reports have documented the composition and roles of salivary proteins from aphids, there are no published ...

Protein kinase A-dependent increase in WAVE2 expression induced by the focal adhesion protein vinexin.

PubMed

Mitsushima, Masaru; Sezaki, Takuhito; Akahane, Rie; Ueda, Kazumitsu; Suetsugu, Shiro; Takenawa, Tadaomi; Kioka, Noriyuki

2006-03-01

The focal adhesion protein vinexin is a member of a family of adaptor proteins that are thought to participate in the regulation of cell adhesion, cytoskeletal reorganization, and growth factor signaling. Here, we show that vinexin beta increases the amount of and reduces the mobility on SDS-PAGE of Wiskott-Aldrich syndrome protein family verprolin-homologous protein (WAVE) 2 protein, which is a key factor modulating actin polymerization in migrating cells. This mobility retardation disappeared after in vitro phosphatase treatment. Co-immunoprecipitation assays revealed the interaction of vinexin beta with WAVE2 as well as WAVE1 and N-WASP. Vinexin beta interacts with the proline-rich region of WAVE2 through the first and second SH3 domains of vinexin beta. Mutations disrupting the interaction impaired the ability of vinexin beta to increase the amount of WAVE2 protein. Treatments with proteasome inhibitors increased the amount of WAVE2, but did not have an additive effect with vinexin beta. Inhibition of protein kinase A (PKA) activity suppressed the vinexin-induced increase in WAVE2 protein, while activation of PKA increased WAVE2 expression without vinexin beta. These results suggest that vinexin beta regulates the proteasome-dependent degradation of WAVE2 in a PKA-dependent manner.

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

Dietary Vitamin C, E and β-Carotene Intake Does Not Significantly Affect Plasma or Salivary Antioxidant Indices and Salivary C-Reactive Protein in Older Subjects.

PubMed

Gawron-Skarbek, Anna; Guligowska, Agnieszka; Prymont-Przymińska, Anna; Godala, Małgorzata; Kolmaga, Agnieszka; Nowak, Dariusz; Szatko, Franciszek; Kostka, Tomasz

2017-07-09

It is not clear whether habitual dietary intake influences the antioxidant or inflammatory status. The aim of the present study was to assess the impact of antioxidative vitamins C, E, and β-carotene obtained from daily food rations on plasma and salivary Total Antioxidant Capacity (TAC), uric acid and salivary C-reactive protein (CRP). The study involved 80 older subjects (66.9 ± 4.3 years), divided into two groups: group 1 ( n = 43) with lower and group 2 ( n = 37) with higher combined vitamins C, E and β-carotene intake. A 24-h dietary recall was obtained from each individual. TAC was assessed simultaneously with two methods in plasma (Ferric Reducing Ability of Plasma-FRAP, 2.2-diphenyl-1-picryl-hydrazyl-DPPH) and in saliva (FRAS and DPPHS test). Lower vitamin C intake corresponded to higher FRAS. There were no other correlations between vitamins C, E or β-carotene intake and antioxidant indices. Salivary CRP was not related to any antioxidant indices. FRAS was decreased in group 2 ( p < 0.01) but no other group differences for salivary or for plasma antioxidant parameters and salivary CRP were found. Habitual, not extra supplemented dietary intake does not significantly affect plasma or salivary TAC and salivary CRP.

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.

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

[Effect of citric acid stimulation on salivary alpha-amylase, total protein, salivary flow rate and pH value in Pi deficiency children].

PubMed

Yang, Ze-min; Chen, Long-hui; Lin, Jing; Zhang, Min; Yang, Xiao-rong; Chen, Wei-wen

2015-02-01

To compare the effect of citric acid stimulation on salivary alpha-amylase (sAA), total protein (TP), salivary flow rate, and pH value between Pi deficiency (PD) children and healthy children, thereby providing evidence for Pi controlling saliva theory. Twenty PD children were recruited, and 29 healthy children were also recruited at the same time. Saliva samples from all subjects were collected before and after citric acid stimulation. The sAA activity and amount, TP contents, salivary flow rate, and pH value were determined and compared. (1) Citric acid stimulation was able to significantly increase salivary flow rate, pH value, sAA activities, sAA specific activity and sAA amount (including glycosylated and non-glycosylated sAA amount) in healthy children (P<0.05), while it could markedly increase salivary flow rate, pH value, and glycosylated sAA levels in PD children (P<0.05); (2) Although there was no statistical difference in determined salivary indices between the two groups (P>0.05), salivary indices except salivary flow rate and glycosylated sAA levels decreased more in PD children. There was statistical difference in sAA activity ratio, sAA specific activity ratio, and the ratio of glycosylated sAA levels between PD children and healthy children (P<0.05). PD children had decreased response to citric acid stimulation.

Estimation of salivary flow rate, pH, buffer capacity, calcium, total protein content and total antioxidant capacity in relation to dental caries severity, age and gender.

PubMed

Pandey, Pallavi; Reddy, N Venugopal; Rao, V Arun Prasad; Saxena, Aditya; Chaudhary, C P

2015-03-01

The aim of the study was to evaluate salivary flow rate, pH, buffering capacity, calcium, total protein content and total antioxidant capacity in relation to dental caries, age and gender. The study population consisted of 120 healthy children aged 7-15 years that was further divided into two groups: 7-10 years and 11-15 years. In this 60 children with DMFS/dfs = 0 and 60 children with DMFS/dfs ≥5 were included. The subjects were divided into two groups; Group A: Children with DMFS/dfs = 0 (caries-free) Group B: Children with DMFS/dfs ≥5 (caries active). Unstimulated saliva samples were collected from all groups. Flow rates were determined, and samples analyzed for pH, buffer capacity, calcium, total protein and total antioxidant status. Salivary antioxidant activity is measured with spectrophotometer by an adaptation of 2,2'-azino-di-(3-ethylbenzthiazoline-6-sulphonate) assays. The mean difference of the two groups; caries-free and caries active were proved to be statistically significant (P < 0.05) for salivary calcium, total protein and total antioxidant level for both the sexes in the age group 7-10 years and for the age 11-15 years the mean difference of the two groups were proved to be statistically significant (P < 0.05) for salivary calcium level for both the sexes. Salivary total protein and total antioxidant level were proved to be statistically significant for male children only. In general, total protein and total antioxidants in saliva were increased with caries activity. Calcium content of saliva was found to be more in caries-free group and increased with age.

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.

Glycoinositolphospholipids from Trypanosomatids Subvert Nitric Oxide Production in Rhodnius prolixus Salivary Glands

PubMed Central

Gazos-Lopes, Felipe; Mesquita, Rafael Dias; Silva-Cardoso, Lívia; Senna, Raquel; Silveira, Alan Barbosa; Jablonka, Willy; Cudischevitch, Cecília Oliveira; Carneiro, Alan Brito; Machado, Ednildo Alcantara; Lima, Luize G.; Monteiro, Robson Queiroz; Nussenzveig, Roberto Henrique; Folly, Evelize; Romeiro, Alexandre; Vanbeselaere, Jorick; Mendonça-Previato, Lucia; Previato, José Osvaldo; Valenzuela, Jesus G.; Ribeiro, José Marcos Chaves; Atella, Georgia Correa; Silva-Neto, Mário Alberto Cardoso

2012-01-01

Background Rhodnius prolixus is a blood-sucking bug vector of Trypanosoma cruzi and T. rangeli. T. cruzi is transmitted by vector feces deposited close to the wound produced by insect mouthparts, whereas T. rangeli invades salivary glands and is inoculated into the host skin. Bug saliva contains a set of nitric oxide-binding proteins, called nitrophorins, which deliver NO to host vessels and ensure vasodilation and blood feeding. NO is generated by nitric oxide synthases (NOS) present in the epithelium of bug salivary glands. Thus, T. rangeli is in close contact with NO while in the salivary glands. Methodology/Principal Findings Here we show by immunohistochemical, biochemical and molecular techniques that inositolphosphate-containing glycolipids from trypanosomatids downregulate NO synthesis in the salivary glands of R. prolixus. Injecting insects with T. rangeli-derived glycoinositolphospholipids (Tr GIPL) or T. cruzi-derived glycoinositolphospholipids (Tc GIPL) specifically decreased NO production. Salivary gland treatment with Tc GIPL blocks NO production without greatly affecting NOS mRNA levels. NOS protein is virtually absent from either Tr GIPL- or Tc GIPL-treated salivary glands. Evaluation of NO synthesis by using a fluorescent NO probe showed that T. rangeli-infected or Tc GIPL-treated glands do not show extensive labeling. The same effect is readily obtained by treatment of salivary glands with the classical protein tyrosine phosphatase (PTP) inhibitor, sodium orthovanadate (SO). This suggests that parasite GIPLs induce the inhibition of a salivary gland PTP. GIPLs specifically suppressed NO production and did not affect other anti-hemostatic properties of saliva, such as the anti-clotting and anti-platelet activities. Conclusions/Significance Taken together, these data suggest that trypanosomatids have overcome NO generation using their surface GIPLs. Therefore, these molecules ensure parasite survival and may ultimately enhance parasite transmission. PMID:23077586

Saliva Proteomics Analysis Offers Insights on Type 1 Diabetes Pathology in a Pediatric Population

PubMed Central

Pappa, Eftychia; Vastardis, Heleni; Mermelekas, George; Gerasimidi-Vazeou, Andriani; Zoidakis, Jerome; Vougas, Konstantinos

2018-01-01

The composition of the salivary proteome is affected by pathological conditions. We analyzed by high resolution mass spectrometry approaches saliva samples collected from children and adolescents with type 1 diabetes and healthy controls. The list of more than 2000 high confidence protein identifications constitutes a comprehensive characterization of the salivary proteome. Patients with good glycemic regulation and healthy individuals have comparable proteomic profiles. In contrast, a significant number of differentially expressed proteins were identified in the saliva of patients with poor glycemic regulation compared to patients with good glycemic control and healthy children. These proteins are involved in biological processes relevant to diabetic pathology such as endothelial damage and inflammation. Moreover, a putative preventive therapeutic approach was identified based on bioinformatic analysis of the deregulated salivary proteins. Thus, thorough characterization of saliva proteins in diabetic pediatric patients established a connection between molecular changes and disease pathology. This proteomic and bioinformatic approach highlights the potential of salivary diagnostics in diabetes pathology and opens the way for preventive treatment of the disease. PMID:29755368

Advances in basic and clinical immunology in 2016.

PubMed

Chinen, Javier; Badran, Yousef R; Geha, Raif S; Chou, Janet S; Fried, Ari J

2017-10-01

Advances in basic immunology in 2016 included studies that further characterized the role of different proteins in the differentiation of effector T and B cells, including cytokines and proteins involved in the actin cytoskeleton. Regulation of granule formation and secretion in cytotoxic cells was also further described by examining patients with familial hemophagocytic lymphohistiocytosis. The role of prenylation in patients with mevalonate kinase deficiency leading to inflammation has been established. We reviewed advances in clinical immunology, as well as new approaches of whole-genome sequencing and genes newly reported to be associated with immunodeficiency, such as linker of activation of T cells (LAT); B-cell CLL/lymphoma 11B (BCL11B); RGD, leucine-rich repeat, tropomodulin domain, and proline-rich domain-containing protein (RLTPR); moesin; and Janus kinase 1 (JAK1). Trials of hematopoietic stem cell transplantation and gene therapy for primary immunodeficiency have had relative success; the use of autologous virus-specific cytotoxic T cells has proved effective as well. New medications are being explored, such as pioglitazone, which is under study for its role in enhancing the oxidative burst in patients with chronic granulomatous disease. Development of vaccines for HIV infection continues to provide insight into the immune response against a virus with an extraordinary mutation rate. Copyright © 2017 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

The aberrant cell walls of boron-deficient bean root nodules have no covalently bound hydroxyproline-/proline-rich proteins.

PubMed Central

Bonilla, I; Mergold-Villaseñor, C; Campos, M E; Sánchez, N; Pérez, H; López, L; Castrejón, L; Sánchez, F; Cassab, G I

1997-01-01

B-deficient bean (Phaseolus vulgaris L.) nodules examined by light microscopy showed dramatic anatomical changes, mainly in the parenchyma region. Western analysis of total nodule extracts examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that one 116-kD polypeptide was recognized by antibodies raised against hydroxyproline-rich glycoproteins (HRGPs) from the soybean (Glycine max) seed coat. A protein with a comparable molecular mass of 116 kD was purified from the cell walls of soybean root nodules. The amino acid composition of this protein is similar to the early nodulin (ENOD2) gene. Immunoprecipitation of the soybean ENOD2 in vitro translation product showed that the soybean seed coat anti-HRGP antibodies recognized this early nodulin. Furthermore, we used these antibodies to localize the ENOD2 homolog in bean nodules. Immunocytochemistry revealed that in B-deficient nodules ENOD2 was absent in the walls of the nodule parenchyma. The absence of ENOD2 in B-deficient nodules was corroborated by performing hydroxyproline assays. Northern analysis showed that ENOD2 mRNA is present in B-deficient nodules; therefore, the accumulation of ENOD2 is not affected by B deficiency, but its assembly into the cell wall is. B-deficient nodules fix much less N2 than control nodules, probably because the nodule parenchyma is no longer an effective O2 barrier. PMID:9414547

Isolation and N-terminal sequencing of a novel cadmium-binding protein from Boletus edulis

NASA Astrophysics Data System (ADS)

Collin-Hansen, C.; Andersen, R. A.; Steinnes, E.

2003-05-01

A Cd-binding protein was isolated from the popular edible mushroom Boletus edulis, which is a hyperaccumulator of both Cd and Hg. Wild-growing samples of B. edulis were collected from soils rich in Cd. Cd radiotracer was added to the crude protein preparation obtained from ethanol precipitation of heat-treated cytosol. Proteins were then further separated in two consecutive steps; gel filtration and anion exchange chromatography. In both steps the Cd radiotracer profile showed only one distinct peak, which corresponded well with the profiles of endogenous Cd obtained by atomic absorption spectrophotometry (AAS). Concentrations of the essential elements Cu and Zn were low in the protein fractions high in Cd. N-terminal sequencing performed on the Cd-binding protein fractions revealed a protein with a novel amino acid sequence, which contained aromatic amino acids as well as proline. Both the N-terminal sequencing and spectrofluorimetric analysis with EDTA and ABD-F (4-aminosulfonyl-7-fluoro-2, 1, 3-benzoxadiazole) failed to detect cysteine in the Cd-binding fractions. These findings conclude that the novel protein does not belong to the metallothionein family. The results suggest a role for the protein in Cd transport and storage, and they are of importance in view of toxicology and food chemistry, but also for environmental protection.

Histone and RNA-binding protein interaction creates crosstalk network for regulation of alternative splicing.

PubMed

Kim, Yong-Eun; Park, Chungoo; Kim, Kyoon Eon; Kim, Kee K

2018-04-30

Alternative splicing is an essential process in eukaryotes, as it increases the complexity of gene expression by generating multiple proteins from a single pre-mRNA. However, information on the regulatory mechanisms for alternative splicing is lacking, because splicing occurs over a short period via the transient interactions of proteins within functional complexes of the spliceosome. Here, we investigated in detail the molecular mechanisms connecting alternative splicing with epigenetic mechanisms. We identified interactions between histone proteins and splicing factors such as Rbfox2, Rbfox3, and splicing factor proline and glutamine rich protein (SFPQ) by in vivo crosslinking and immunoprecipitation. Furthermore, we confirmed that splicing factors were bound to specific modified residues of histone proteins. Additionally, changes in histone methylation due to histone methyltransferase inhibitor treatment notably affected alternative splicing in selected genes. Therefore, we suggested that there may be crosstalk mechanisms connecting histone modifications and RNA-binding proteins that increase the local concentration of RNA-binding proteins in alternative exon loci of nucleosomes by binding specific modified histone proteins, leading to alternative splicing. This crosstalk mechanism may play a major role in epigenetic processes such as histone modification and the regulation of alternative splicing. Copyright © 2018 Elsevier Inc. All rights reserved.

Determination of amino acid contents of manketti seeds (Schinziophyton rautanenii) by pre-column derivatisation with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate and RP-HPLC.

PubMed

Gwatidzo, Luke; Botha, Ben M; McCrindle, Rob I

2013-12-01

Defatted kernel flour from manketti seed kernels (Schinziophyton rautanenii) is an underutilised natural product. The plant grows in the wild, on sandy soils little used for agriculture in Southern Africa. The kernels are rich in protein and have a great potential for improving nutrition. The protein content and amino acid profile of manketti seed kernel were studied, using a new analytical method, in order to evaluate the nutritional value. The crude protein content of the press cake and defatted kernel flour was 29.0% and 67.5%, respectively. Leucine and arginine were found to be the most abundant essential and non-essential amino acids, respectively. The seed kernel contained 4.77 g leucine and 12.34 g arginine/100 g of defatted seed kernel flour. Methionine and proline were the least abundant essential and non-essential amino acids to with 0.23 g methionine and 0.36 g proline/100 g of defatted seed kernel flour, respectively. Validation of the pre-column derivatisation procedure with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) for the determination of amino acids was carried out. The analytical parameters were determined: linearity (0.0025-0.20 mM), accuracy of the derivatisation procedure: 86.7-109.8%, precision (method: 0.72-5.04%, instrumental: 0.14-1.88% and derivatisation: 0.15-2.94% and 0.41-4.32% for intraday and interday, respectively). Limits of detection and quantification were 6.80-157 mg/100 g and 22.7-523 mg/100 g kernel flour, respectively. Copyright © 2013 Elsevier Ltd. All rights reserved.

Proline substitutions and threonine pseudophosphorylation of the SH3 ligand of 18.5-kDa myelin basic protein decrease its affinity for the Fyn-SH3 domain and alter process development and protein localization in oligodendrocytes.

PubMed

Smith, Graham S T; De Avila, Miguel; Paez, Pablo M; Spreuer, Vilma; Wills, Melanie K B; Jones, Nina; Boggs, Joan M; Harauz, George

2012-01-01

The developmentally regulated myelin basic proteins (MBPs), which arise from the golli (gene of oligodendrocyte lineage) complex, are highly positively charged, intrinsically disordered, multifunctional proteins having several alternatively spliced isoforms and posttranslational modifications, and they play key roles in myelin compaction. The classic 18.5-kDa MBP isoform has a proline-rich region comprising amino acids 92-99 (murine sequence -T(92)PRTPPPS(99)-) that contains a minimal SH3 ligand domain. We have previously shown that 18.5-kDa MBP binds to several SH3 domains, including that of Fyn, a member of the Src family of tyrosine kinases involved in a number of signaling pathways during CNS development. To determine the physiological role of this binding as well as the role of phosphorylation of Thr92 and Thr95, in the current study we have produced several MBP variants specifically targeting phosphorylation sites and key structural regions of MBP's SH3 ligand domain. Using isothermal titration calorimetry, we have demonstrated that, compared with the wild-type protein, these variants have lower affinity for the SH3 domain of Fyn. Moreover, overexpression of N-terminal-tagged GFP versions in immortalized oligodendroglial N19 and N20.1 cell cultures results in aberrant elongation of membrane processes and increased branching complexity and inhibits the ability of MBP to decrease Ca(2+) influx. Phosphorylation of Thr92 can also cause MBP to traffic to the nucleus, where it may participate in additional protein-protein interactions. Coexpression of MBP with a constitutively active form of Fyn kinase resulted in membrane process elaboration, a phenomenon that was abolished by point amino acid substitutions in MBP's SH3 ligand domain. These results suggest that MBP's SH3 ligand domain plays a key role in intracellular protein interactions in vivo and may be required for proper membrane elaboration of developing oligodendrocytes and, further, that phosphorylation of Thr92 and Thr95 can regulate this function. Copyright © 2011 Wiley Periodicals, Inc.

Salivary Gland Hypofunction in tyrosylprotein sulfotransferase-2 Knockout Mice Is Due to Primary Hypothyroidism

PubMed Central

Westmuckett, Andrew D.; Siefert, Joseph C.; Tesiram, Yasvir A.; Pinson, David M.; Moore, Kevin L.

2013-01-01

Background Protein-tyrosine sulfation is a post-translational modification of an unknown number of secreted and membrane proteins mediated by two known Golgi tyrosylprotein sulfotransferases (TPST-1 and TPST-2). We reported that Tpst2-/- mice have mild-moderate primary hypothyroidism, whereas Tpst1-/- mice are euthyroid. While using magnetic resonance imaging (MRI) to look at the thyroid gland we noticed that the salivary glands in Tpst2-/- mice appeared smaller than in wild type mice. This prompted a detailed analysis to compare salivary gland structure and function in wild type, Tpst1-/-, and Tpst2 -/- mice. Methodology/Principal Findings Quantitative MRI imaging documented that salivary glands in Tpst2-/- females were ≈ 30% smaller than wild type or Tpst1-/- mice and that the granular convoluted tubules in Tpst2-/- submandibular glands were less prominent and were almost completely devoid of exocrine secretory granules compared to glands from wild type or Tpst1-/- mice. In addition, pilocarpine–induced salivary flow and salivary α-amylase activity in Tpst2-/- mice of both sexes was substantially lower than in wild type and Tpst1-/- mice. Anti-sulfotyrosine Western blots of salivary gland extracts and saliva showed no differences between wild type, Tpst1-/-, and Tpst2-/- mice, suggesting that the salivary gland hypofunction is due to factor(s) extrinsic to the salivary glands. Finally, we found that all indicators of hypothyroidism (serum T4, body weight) and salivary gland hypofunction (salivary flow, salivary α-amylase activity, histological changes) were restored to normal or near normal by thyroid hormone supplementation. Conclusions/Significance Our findings conclusively demonstrate that low body weight and salivary gland hypofunction in Tpst2-/- mice is due solely to primary hypothyroidism. PMID:23951251

Development of a Multiplexed Liquid Chromatography Multiple-Reaction-Monitoring Mass Spectrometry (LC-MRM/MS) Method for Evaluation of Salivary Proteins as Oral Cancer Biomarkers.

PubMed

Chen, Yi-Ting; Chen, Hsiao-Wei; Wu, Chun-Feng; Chu, Lichieh Julie; Chiang, Wei-Fang; Wu, Chih-Ching; Yu, Jau-Song; Tsai, Cheng-Han; Liang, Kung-Hao; Chang, Yu-Sun; Wu, Maureen; Ou Yang, Wei-Ting

2017-05-01

Multiple (selected) reaction monitoring (MRM/SRM) of peptides is a growing technology for target protein quantification because it is more robust, precise, accurate, high-throughput, and multiplex-capable than antibody-based techniques. The technique has been applied clinically to the large-scale quantification of multiple target proteins in different types of fluids. However, previous MRM-based studies have placed less focus on sample-preparation workflow and analytical performance in the precise quantification of proteins in saliva, a noninvasively sampled body fluid. In this study, we evaluated the analytical performance of a simple and robust multiple reaction monitoring (MRM)-based targeted proteomics approach incorporating liquid chromatography with mass spectrometry detection (LC-MRM/MS). This platform was used to quantitatively assess the biomarker potential of a group of 56 salivary proteins that have previously been associated with human cancers. To further enhance the development of this technology for assay of salivary samples, we optimized the workflow for salivary protein digestion and evaluated quantification performance, robustness and technical limitations in analyzing clinical samples. Using a clinically well-characterized cohort of two independent clinical sample sets (total n = 119), we quantitatively characterized these protein biomarker candidates in saliva specimens from controls and oral squamous cell carcinoma (OSCC) patients. The results clearly showed a significant elevation of most targeted proteins in saliva samples from OSCC patients compared with controls. Overall, this platform was capable of assaying the most highly multiplexed panel of salivary protein biomarkers, highlighting the clinical utility of MRM in oral cancer biomarker research. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

New mechanism of oral immunity to mucosal candidiasis in hyper-IgE syndrome

PubMed Central

Conti, HR; Baker, O; Freeman, AF; Jang, WS; Holland, SM; Li, RA; Edgerton, M; Gaffen, SL

2011-01-01

Oropharyngeal candidiasis (OPC, thrush) is an opportunistic infection caused by the commensal fungus Candida albicans. An understanding of immunity to Candida has recently begun to unfold with the identification of fungal pattern-recognition receptors such as C-type lectin receptors, which trigger protective T-helper (Th)17 responses in the mucosa. Hyper-IgE syndrome (HIES/Job’s syndrome) is a rare congenital immunodeficiency characterized by dominant-negative mutations in signal transducer and activator of transcription 3, which is downstream of the Th17-inductive cytokines interleukin (IL)-6 and IL-23, and hence patients with HIES exhibit dramatic Th17 deficits. HIES patients develop oral and mucocutaneous candidiasis, supporting a protective role for Th17 cells in immunity to OPC. However, the Th17-dependent mechanisms of antifungal immunity in OPC are still poorly defined. An often unappreciated aspect of oral immunity is saliva, which is rich in antimicrobial proteins (AMPs) and exerts direct antifungal activity. In this study, we show that HIES patients show significant impairment in salivary AMPs, including β-defensin 2 and Histatins. This tightly correlates with reduced candidacidal activity of saliva and concomitantly elevated colonization with Candida. Moreover, IL-17 induces histatins in cultured salivary gland cells. This is the first demonstration that HIES is associated with defective salivary activity, and provides a mechanism for the severe susceptibility of these patients to OPC. PMID:21346738

New mechanism of oral immunity to mucosal candidiasis in hyper-IgE syndrome.

PubMed

Conti, H R; Baker, O; Freeman, A F; Jang, W S; Holland, S M; Li, R A; Edgerton, M; Gaffen, S L

2011-07-01

Oropharyngeal candidiasis (OPC, thrush) is an opportunistic infection caused by the commensal fungus Candida albicans. An understanding of immunity to Candida has recently begun to unfold with the identification of fungal pattern-recognition receptors such as C-type lectin receptors, which trigger protective T-helper (Th)17 responses in the mucosa. Hyper-IgE syndrome (HIES/Job's syndrome) is a rare congenital immunodeficiency characterized by dominant-negative mutations in signal transducer and activator of transcription 3, which is downstream of the Th17-inductive cytokines interleukin (IL)-6 and IL-23, and hence patients with HIES exhibit dramatic Th17 deficits. HIES patients develop oral and mucocutaneous candidiasis, supporting a protective role for Th17 cells in immunity to OPC. However, the Th17-dependent mechanisms of antifungal immunity in OPC are still poorly defined. An often unappreciated aspect of oral immunity is saliva, which is rich in antimicrobial proteins (AMPs) and exerts direct antifungal activity. In this study, we show that HIES patients show significant impairment in salivary AMPs, including β-defensin 2 and Histatins. This tightly correlates with reduced candidacidal activity of saliva and concomitantly elevated colonization with Candida. Moreover, IL-17 induces histatins in cultured salivary gland cells. This is the first demonstration that HIES is associated with defective salivary activity, and provides a mechanism for the severe susceptibility of these patients to OPC.

Computational prediction of human salivary proteins from blood circulation and application to diagnostic biomarker identification.

PubMed

Wang, Jiaxin; Liang, Yanchun; Wang, Yan; Cui, Juan; Liu, Ming; Du, Wei; Xu, Ying

2013-01-01

Proteins can move from blood circulation into salivary glands through active transportation, passive diffusion or ultrafiltration, some of which are then released into saliva and hence can potentially serve as biomarkers for diseases if accurately identified. We present a novel computational method for predicting salivary proteins that come from circulation. The basis for the prediction is a set of physiochemical and sequence features we found to be discerning between human proteins known to be movable from circulation to saliva and proteins deemed to be not in saliva. A classifier was trained based on these features using a support-vector machine to predict protein secretion into saliva. The classifier achieved 88.56% average recall and 90.76% average precision in 10-fold cross-validation on the training data, indicating that the selected features are informative. Considering the possibility that our negative training data may not be highly reliable (i.e., proteins predicted to be not in saliva), we have also trained a ranking method, aiming to rank the known salivary proteins from circulation as the highest among the proteins in the general background, based on the same features. This prediction capability can be used to predict potential biomarker proteins for specific human diseases when coupled with the information of differentially expressed proteins in diseased versus healthy control tissues and a prediction capability for blood-secretory proteins. Using such integrated information, we predicted 31 candidate biomarker proteins in saliva for breast cancer.

Computational Prediction of Human Salivary Proteins from Blood Circulation and Application to Diagnostic Biomarker Identification

PubMed Central

Wang, Jiaxin; Liang, Yanchun; Wang, Yan; Cui, Juan; Liu, Ming; Du, Wei; Xu, Ying

2013-01-01

Proteins can move from blood circulation into salivary glands through active transportation, passive diffusion or ultrafiltration, some of which are then released into saliva and hence can potentially serve as biomarkers for diseases if accurately identified. We present a novel computational method for predicting salivary proteins that come from circulation. The basis for the prediction is a set of physiochemical and sequence features we found to be discerning between human proteins known to be movable from circulation to saliva and proteins deemed to be not in saliva. A classifier was trained based on these features using a support-vector machine to predict protein secretion into saliva. The classifier achieved 88.56% average recall and 90.76% average precision in 10-fold cross-validation on the training data, indicating that the selected features are informative. Considering the possibility that our negative training data may not be highly reliable (i.e., proteins predicted to be not in saliva), we have also trained a ranking method, aiming to rank the known salivary proteins from circulation as the highest among the proteins in the general background, based on the same features. This prediction capability can be used to predict potential biomarker proteins for specific human diseases when coupled with the information of differentially expressed proteins in diseased versus healthy control tissues and a prediction capability for blood-secretory proteins. Using such integrated information, we predicted 31 candidate biomarker proteins in saliva for breast cancer. PMID:24324552

The green tea polyphenol (-)-epigallocatechin gallate precipitates salivary proteins including alpha-amylase: biochemical implications for oral health.

PubMed

Hara, Kumiko; Ohara, Masaru; Hayashi, Ikue; Hino, Takamune; Nishimura, Rumi; Iwasaki, Yoriko; Ogawa, Tetsuji; Ohyama, Yoshihiko; Sugiyama, Masaru; Amano, Hideaki

2012-04-01

Green tea is a popular drink throughout the world, and it contains various components, including the green tea polyphenol (-)-epigallocatechin gallate (EGCG). Tea interacts with saliva upon entering the mouth, so the interaction between saliva and EGCG interested us, especially with respect to EGCG-protein binding. SDS-PAGE revealed that several salivary proteins were precipitated after adding EGCG to saliva. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) peptide mass fingerprinting indicated that the major proteins precipitated by EGCG were alpha-amylase, S100, and cystatins. Surface plasmon resonance revealed that EGCG bound to alpha-amylase at dissociation constant (K(d)) = 2.74 × 10(-6) M, suggesting that EGCG interacts with salivary proteins with a relatively strong affinity. In addition, EGCG inhibited the activity of alpha-amylase by non-competitive inhibition, indicating that EGCG is effective at inhibiting the formation of fermentable carbohydrates involved in caries formation. Interestingly, alpha-amylase reduced the antimicrobial activity of EGCG against the periodontal bacterium Aggregatibacter actinomycetemcomitans. Therefore, we considered that EGCG-salivary protein interactions might have both protective and detrimental effects with respect to oral health. © 2012 Eur J Oral Sci.

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

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.

Effect of Cell Phone Use on Salivary Total Protein, Enzymes and Oxidative Stress Markers in Young Adults: A Pilot Study

PubMed Central

Joy, Jasmi; Sunitha, Venkatesh; Rai, Manoj P.; Rao, Suresh; Nambranathayil, Shafeeque; Baliga, Manjeshwar Shrinath

2015-01-01

Introduction: The present study aimed to assess the levels of salivary enzymes, protein and oxidant-antioxidant system in young college-going cell phone users. Materials and Methods: The cell users (students) were categorized in to two groups – less mobile users and high mobile users, based on the duration and frequency of cell use. Unstimulated whole saliva samples of the volunteers were analysed for amylase, lactate dehydrogenase (LDH), malondialdehdye (MDA) and glutathione (GSH). Results: High mobile users had significantly higher levels of amylase (p = 0.001), LDH (p = 0.002) and MDA (p = 0.002) in saliva, when compared to less mobile users. The marginal decrease in salivary total proteins, GSH and flow rate were statistically not significant (p >0.05). Conclusion: Significant changes in salivary enzymes and MDA suggest adverse effect of high use of cell phones on cell health. PMID:25859446

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.

Salivary gland proteome analysis reveals modulation of anopheline unique proteins in insensitive acetylcholinesterase resistant Anopheles gambiae mosquitoes.

PubMed

Cornelie, Sylvie; Rossignol, Marie; Seveno, Martial; Demettre, Edith; Mouchet, François; Djègbè, Innocent; Marin, Philippe; Chandre, Fabrice; Corbel, Vincent; Remoué, Franck; Mathieu-Daudé, Françoise

2014-01-01

Insensitive acetylcholinesterase resistance due to a mutation in the acetylcholinesterase (ace) encoding ace-1 gene confers cross-resistance to organophosphate and carbamate insecticides in Anopheles gambiae populations from Central and West Africa. This mutation is associated with a strong genetic cost revealed through alterations of some life history traits but little is known about the physiological and behavioural changes in insects bearing the ace-1(R) allele. Comparative analysis of the salivary gland contents between An. gambiae susceptible and ace-1(R) resistant strains was carried out to charaterize factors that could be involved in modifications of blood meal process, trophic behaviour or pathogen interaction in the insecticide-resistant mosquitoes. Differential analysis of the salivary gland protein profiles revealed differences in abundance for several proteins, two of them showing major differences between the two strains. These two proteins identified as saglin and TRIO are salivary gland-1 related proteins, a family unique to anopheline mosquitoes, one of them playing a crucial role in salivary gland invasion by Plasmodium falciparum sporozoites. Differential expression of two other proteins previously identified in the Anopheles sialome was also observed. The differentially regulated proteins are involved in pathogen invasion, blood feeding process, and protection against oxidation, relevant steps in the outcome of malaria infection. Further functional studies and insect behaviour experiments would confirm the impact of the modification of the sialome composition on blood feeding and pathogen transmission abilities of the resistant mosquitoes. The data supports the hypothesis of alterations linked to insecticide resistance in the biology of the primary vector of human malaria in Africa.

Bidirectional nucleolar dysfunction in C9orf72 frontotemporal lobar degeneration.

PubMed

Mizielinska, Sarah; Ridler, Charlotte E; Balendra, Rubika; Thoeng, Annora; Woodling, Nathan S; Grässer, Friedrich A; Plagnol, Vincent; Lashley, Tammaryn; Partridge, Linda; Isaacs, Adrian M

2017-04-18

An intronic GGGGCC expansion in C9orf72 is the most common known cause of both frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). The repeat expansion leads to the generation of sense and antisense repeat RNA aggregates and dipeptide repeat (DPR) proteins, generated by repeat-associated non-ATG translation. The arginine-rich DPR proteins poly(glycine-arginine or GR) and poly(proline-arginine or PR) are potently neurotoxic and can localise to the nucleolus when expressed in cells, resulting in enlarged nucleoli with disrupted functionality. Furthermore, GGGGCC repeat RNA can bind nucleolar proteins in vitro. However, the relevance of nucleolar stress is unclear, as the arginine-rich DPR proteins do not localise to the nucleolus in C9orf72-associated FTLD/ALS (C9FTLD/ALS) patient brain. We measured nucleolar size in C9FTLD frontal cortex neurons using a three-dimensional, volumetric approach. Intriguingly, we found that C9FTLD brain exhibited bidirectional nucleolar stress. C9FTLD neuronal nucleoli were significantly smaller than control neuronal nucleoli. However, within C9FTLD brains, neurons containing poly(GR) inclusions had significantly larger nucleolar volumes than neurons without poly(GR) inclusions. In addition, expression of poly(GR) in adult Drosophila neurons led to significantly enlarged nucleoli. A small but significant increase in nucleolar volume was also observed in C9FTLD frontal cortex neurons containing GGGGCC repeat-containing RNA foci. These data show that nucleolar abnormalities are a consistent feature of C9FTLD brain, but that diverse pathomechanisms are at play, involving both DPR protein and repeat RNA toxicity.

ADVANCES IN SALIVARY GLAND GENE THERAPY – ORAL AND SYSTEMIC IMPLICATIONS

PubMed Central

Baum, Bruce J.; Alevizos, Ilias; Chiorini, John A.; Cotrim, Ana P.; Zheng, Changyu

2016-01-01

Introduction Much research demonstrates the feasibility and efficacy of gene transfer to salivary glands. Recently, the first clinical trial targeting a salivary gland was completed, yielding positive safety and efficacy results. Areas covered There are two major disorders affecting salivary glands; radiation damage following treatment for head and neck cancers and Sjögren’s syndrome. Salivary gland gene transfer has also been employed in preclinical studies using transgenic secretory proteins for exocrine (upper gastrointestinal tract) and endocrine (systemic) applications. Expert opinion Salivary gland gene transfer is safe and can be beneficial in humans. Applications to treat and prevent radiation damage show considerable promise. A first-in-human clinical trial for the former was recently successfully completed. Studies on Sjögren’s syndrome suffer from an inadequate understanding of its etiology. Proof of concept in animal models has been shown for exocrine and endocrine disorders. Currently, the most promising exocrine application is for the management of obesity. Endocrine applications are limited, as it is currently impossible to predict if systemically required transgenic proteins will be efficiently secreted into the bloodstream. This results from not understanding of how secretory proteins are sorted. Future studies will likely employ ultrasound assisted and pseudotyped adenoassociated viral vector-mediated gene. PMID:26149284

Evaluation of the relationship between passive smoking and salivary electrolytes, protein, secretory IgA, sialic acid and amylase in young children.

PubMed

Avşar, Aysun; Darka, Ozge; Bodrumlu, Ebru Hazar; Bek, Yüksel

2009-05-01

To evaluate the relationship between passive smoking as determined by salivary cotinine levels and salivary electrolytes, protein, secretory IgA, sialic acid and amylase in children. Saliva was collected from 90 passive smoker (PS) subjects (the study group) and 90 healthy age-matched children (the control group). The study group was divided into three subgroups according the number of cigarettes smoked. Socio-economic status, dental and dietary habits were recorded by questionnaire. Stimulated salivary calcium (Ca), phosphate (P), sodium (Na), potassium (P), total protein, amylase activity, sialic acid level, secretory IgA concentration and cotinine level were analysed. All data were analysed using SPSS, version 13.0. Socio-economic status, dental and dietary habits were similar between the two groups. The salivary electrolytes concentrations did not reveal significant difference between the two groups (p>0.05). The mean cotinine levels of PS children were 1.58+/-4.3 ng/mL. The salivary concentrations of protein were similar between the two groups (p>0.05). The salivary secretory IgA concentration was significantly lower in the PS group than controls. The sialic acid level and amylase activity in PS group were found significantly higher compared with the controls (p<0.05). No difference was observed for all these parameters with sex (p>0.05). When saliva samples were analysed for output, the sialic acid level and amylase activity increased significantly in PS subjects (p<0.05). Further, the output of secretory IgA concentration was found significantly lower compared with the controls (p<0.05). In conclusion, we show that passive smoking was associated with a decrease in secretory IgA concentration, whereas with increase in amylase activity and sialic acid level of stimulated whole saliva in young children.

Characterization of the Lassa virus matrix protein Z: electron microscopic study of virus-like particles and interaction with the nucleoprotein (NP).

PubMed

Eichler, Robert; Strecker, Thomas; Kolesnikova, Larissa; ter Meulen, Jan; Weissenhorn, Winfried; Becker, Stephan; Klenk, Hans Dieter; Garten, Wolfgang; Lenz, Oliver

2004-03-15

Lassa virus is the causative agent of a hemorrhagic fever endemic in west Africa. The RNA genome of Lassa virus encodes the glycoprotein precursor GP-C, a nucleoprotein (NP), the viral polymerase L and a small protein Z (11 kDa). Here, we analyze the role of Z protein for virus maturation. We have recently shown that expression of Z protein in the absence of other viral proteins is sufficient for the release of enveloped Z-containing particles. In this study, we examined particles secreted into the supernatant of a stably Z protein-expressing CHO cell line by electron microscopy. The observed Z-induced virus-like particles did not significantly differ in their morphology and size from Lassa virus particles. Mutation of two proline-rich domains within Z which are known to drastically reduce the release of virus-like particles, had no effect on the cellular localization of the protein nor on its membrane-association. Furthermore, we present evidence that Z interacts with the NP. We assume that Z recruits NP to cellular membranes where virus assembly takes place. We conclude from our data that Lassa virus Z protein plays an essential role in Lassa virus maturation.

Salivary proteins and microbiota as biomarkers for early childhood caries risk assessment

PubMed Central

Hemadi, Abdullah S; Huang, Ruijie; Zhou, Yuan; Zou, Jing

2017-01-01

Early childhood caries (ECC) is a term used to describe dental caries in children aged 6 years or younger. Oral streptococci, such as Streptococcus mutans and Streptococcus sorbrinus, are considered to be the main etiological agents of tooth decay in children. Other bacteria, such as Prevotella spp. and Lactobacillus spp., and fungus, that is, Candida albicans, are related to the development and progression of ECC. Biomolecules in saliva, mainly proteins, affect the survival of oral microorganisms by multiple innate defensive mechanisms, thus modulating the oral microflora. Therefore, the protein composition of saliva can be a sensitive indicator for dental health. Resistance or susceptibility to caries may be significantly correlated with alterations in salivary protein components. Some oral microorganisms and saliva proteins may serve as useful biomarkers in predicting the risk and prognosis of caries. Current research has generated abundant information that contributes to a better understanding of the roles of microorganisms and salivary proteins in ECC occurrence and prevention. This review summarizes the microorganisms that cause caries and tooth-protective salivary proteins with their potential as functional biomarkers for ECC risk assessment. The identification of biomarkers for children at high risk of ECC is not only critical for early diagnosis but also important for preventing and treating the disease. PMID:29125139

The nuclear protein PH5P of the inter-alpha-inhibitor superfamily: a missing link between poly(ADP-ribose)polymerase and the inter-alpha-inhibitor family and a novel actor of DNA repair?

PubMed

Jean, L; Risler, J L; Nagase, T; Coulouarn, C; Nomura, N; Salier, J P

1999-03-05

Poly(ADP-ribose)polymerase is a nuclear NAD-dependent enzyme and an essential nick sensor involved in cellular processes where nicking and rejoining of DNA strands are required. The inter-alpha-inhibitor family is comprized of several plasma proteins that all harbor one or more so-called heavy chains designated H1-H4. The latter originate from precursor polypeptides H1P-H4P whose upper two thirds are highly homologous. We now describe a novel protein that includes (i) a so-called BRCT domain found in many proteins involved in DNA repair, (ii) an area that is homologous to the NAD-dependent catalytic domain of poly(ADP-ribose)polymerase, (iii) an area that is homologous to the upper two thirds of precursor polypeptides H1P-H4P and (iv) a proline-rich region with a potential nuclear localization signal. This protein now designated PH5P points to as yet unsuspected links between poly(ADP-ribose)polymerase and the inter-alpha-inhibitor family and is likely to be involved in DNA repair.

Dietary Vitamin C, E and β-Carotene Intake Does Not Significantly Affect Plasma or Salivary Antioxidant Indices and Salivary C-Reactive Protein in Older Subjects

PubMed Central

Gawron-Skarbek, Anna; Prymont-Przymińska, Anna; Godala, Małgorzata; Kolmaga, Agnieszka; Nowak, Dariusz; Szatko, Franciszek; Kostka, Tomasz

2017-01-01

It is not clear whether habitual dietary intake influences the antioxidant or inflammatory status. The aim of the present study was to assess the impact of antioxidative vitamins C, E, and β-carotene obtained from daily food rations on plasma and salivary Total Antioxidant Capacity (TAC), uric acid and salivary C-reactive protein (CRP). The study involved 80 older subjects (66.9 ± 4.3 years), divided into two groups: group 1 (n = 43) with lower and group 2 (n = 37) with higher combined vitamins C, E and β-carotene intake. A 24-h dietary recall was obtained from each individual. TAC was assessed simultaneously with two methods in plasma (Ferric Reducing Ability of Plasma—FRAP, 2.2-diphenyl-1-picryl-hydrazyl—DPPH) and in saliva (FRAS and DPPHS test). Lower vitamin C intake corresponded to higher FRAS. There were no other correlations between vitamins C, E or β-carotene intake and antioxidant indices. Salivary CRP was not related to any antioxidant indices. FRAS was decreased in group 2 (p < 0.01) but no other group differences for salivary or for plasma antioxidant parameters and salivary CRP were found. Habitual, not extra supplemented dietary intake does not significantly affect plasma or salivary TAC and salivary CRP. PMID:28698489

An insight into the sialotranscriptome of the seed-feeding bug, Oncopeltus fasciatus.

PubMed

Francischetti, Ivo M B; Lopes, Angela H; Dias, Felipe A; Pham, Van M; Ribeiro, José M C

2007-09-01

The salivary transcriptome of the seed-feeding hemipteran, Oncopeltus fasciatus (milkweed bug), is described following assembly of 1025 expressed sequence tags (ESTs) into 305 clusters of related sequences. Inspection of these sequences reveals abundance of low complexity, putative secreted products rich in the amino acids (aa) glycine, serine or threonine, which might function as silk or mucins and assist food canal lubrication and sealing of the feeding site around the mouthparts. Several protease inhibitors were found, including abundant expression of cystatin transcripts that may inhibit cysteine proteases common in seeds that might injure the insect or induce plant apoptosis. Serine proteases and lipases are described that might assist digestion and liquefaction of seed proteins and oils. Finally, several novel putative proteins are described with no known function that might affect plant physiology or act as antimicrobials.

Laminin-111-derived peptide conjugated fibrin hydrogel restores salivary gland function.

PubMed

Nam, Kihoon; Maruyama, Christina L; Wang, Ching-Shuen; Trump, Bryan G; Lei, Pedro; Andreadis, Stelios T; Baker, Olga J

2017-01-01

Hyposalivation reduces the patient quality of life, as saliva is important for maintaining oral health. Current treatments for hyposalivation are limited to medications such as the muscarinic receptor agonists, pilocarpine and cevimeline. However, these therapies only provide temporary relief. Therefore, alternative therapies are essential to restore salivary gland function. An option is to use bioengineered scaffolds to promote functional salivary gland regeneration. Previous studies demonstrated that the laminin-111 protein is critical for intact salivary gland cell cluster formation and organization. However, laminin-111 protein as a whole is not suitable for clinical applications as some protein domains may contribute to unwanted side effects such as degradation, tumorigenesis and immune responses. Conversely, the use of synthetic laminin-111 peptides makes it possible to minimize the immune reactivity or pathogen transfer. In addition, it is relatively simple and inexpensive as compared to animal-derived proteins. Therefore, the goal of this study was to demonstrate whether a 20 day treatment with laminin-111-derived peptide conjugated fibrin hydrogel promotes tissue regeneration in submandibular glands of a wound healing mouse model. In this study, laminin-111-derived peptide conjugated fibrin hydrogel significantly accelerated formation of salivary gland tissue. The regenerated gland tissues displayed not only structural but also functional restoration.

Laminin-111-derived peptide conjugated fibrin hydrogel restores salivary gland function

PubMed Central

Nam, Kihoon; Maruyama, Christina L.; Wang, Ching-Shuen; Trump, Bryan G.; Lei, Pedro; Andreadis, Stelios T.

2017-01-01

Hyposalivation reduces the patient quality of life, as saliva is important for maintaining oral health. Current treatments for hyposalivation are limited to medications such as the muscarinic receptor agonists, pilocarpine and cevimeline. However, these therapies only provide temporary relief. Therefore, alternative therapies are essential to restore salivary gland function. An option is to use bioengineered scaffolds to promote functional salivary gland regeneration. Previous studies demonstrated that the laminin-111 protein is critical for intact salivary gland cell cluster formation and organization. However, laminin-111 protein as a whole is not suitable for clinical applications as some protein domains may contribute to unwanted side effects such as degradation, tumorigenesis and immune responses. Conversely, the use of synthetic laminin-111 peptides makes it possible to minimize the immune reactivity or pathogen transfer. In addition, it is relatively simple and inexpensive as compared to animal-derived proteins. Therefore, the goal of this study was to demonstrate whether a 20 day treatment with laminin-111-derived peptide conjugated fibrin hydrogel promotes tissue regeneration in submandibular glands of a wound healing mouse model. In this study, laminin-111-derived peptide conjugated fibrin hydrogel significantly accelerated formation of salivary gland tissue. The regenerated gland tissues displayed not only structural but also functional restoration. PMID:29095857

Identification of the active components in Bone Marrow Soup: a mitigator against irradiation-injury to salivary glands.

PubMed

Fang, Dongdong; Hu, Shen; Liu, Younan; Quan, Vu-Hung; Seuntjens, Jan; Tran, Simon D

2015-11-03

In separate studies, an extract of soluble intracellular contents from whole bone marrow cells, named "Bone Marrow (BM) Soup", was reported to either improve cardiac or salivary functions post-myocardial infarction or irradiation (IR), respectively. However, the active components in BM Soup are unknown. To demonstrate that proteins were the active ingredients, we devised a method using proteinase K followed by heating to deactivate proteins and for safe injections into mice. BM Soup and "deactivated BM Soup" were injected into mice that had their salivary glands injured with 15Gy IR. Control mice received either injections of saline or were not IR. Results at week 8 post-IR showed the 'deactivated BM Soup' was no better than injections of saline, while injections of native BM Soup restored saliva flow, protected salivary cells and blood vessels from IR-damage. Protein arrays detected several angiogenesis-related factors (CD26, FGF, HGF, MMP-8, MMP-9, OPN, PF4, SDF-1) and cytokines (IL-1ra, IL-16) in BM Soup. In conclusion, the native proteins (but not the nucleic acids, lipids or carbohydrates) were the therapeutic ingredients in BM Soup for functional salivary restoration following IR. This molecular therapy approach has clinical potential because it is theoretically less tumorigenic and immunogenic than cell therapies.

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 oral microbiome and human health.

PubMed

Yamashita, Yoshihisa; Takeshita, Toru

2017-01-01

In this brief review, we discuss our previous research on the relationship between the bacterial composition of salivary microbiota and periodontal disease. Analysis using a terminal restriction fragment length polymorphism method and an international comparison suggest that the predominance of the genera Prevotella and Veillonella in the salivary microbiota is attributable to periodontal disease conditions, and that the predominance of the genus Neisseria indicates healthy periodontal conditions. Furthermore, we recently used next-generation sequencing technology to perform a detailed large-scale analysis of the salivary microbiota. An important finding of that study was that high bacterial richness in the salivary microbiota was significantly associated with poor oral health, as indicated by decayed teeth, periodontitis, and poor oral hygiene. Another important result was that relative abundance of predominant bacteria in saliva was significantly associated with oral health-related conditions. Of the two different cohabiting groups of bacteria found in the salivary microbiota, a greater relative abundance of group I bacteria, which include Prevotella and Veillonella species, was associated with poor oral health, high body mass index, and old age. These findings suggest that the salivary microbiota reflects oral and systemic conditions.

Comparative behavioral and protein study of salivary secretions in Homalodisca spp. sharpshooters (Hemiptera: Cicadellidae: Cicadellinae)

USDA-ARS?s Scientific Manuscript database

A novel brush-induced method to physically stimulate salivation was applied to the glassy-winged and smoke tree sharpshooters. This technique enabled the direct observation of salivary secretion processes, solidification of saliva and for collection of salivary secretions. For both species, brush...

Cariogenic potential of foods. II. Relationship of food composition, plaque microbial counts, and salivary parameters to caries in the rat model.

PubMed

Mundorff-Shrestha, S A; Featherstone, J D; Eisenberg, A D; Cowles, E; Curzon, M E; Espeland, M A; Shields, C P

1994-01-01

A series of rat caries experiments was carried out to test the relative cariogenic potential and to identify the major carcinogenic elements of 22 popular snack foods. Parameters that were measured included rat caries, number of cariogenic bacteria in plaque, salivary parameters including flow rate, buffering capacity, total protein, lysozyme and amylase content, and composition of test foods including protein, fat, phosphorus, calcium, fluoride, galactose, glucose, total reducing sugar, sucrose, and starch. Many interesting relationships were observed between food components, numbers of plaque bacteria, salivary components, and specific types of carious lesions. Protein, fat, and phosphorus in foods were all associated with inhibition of both sulcal and buccolingual (smooth-surface) caries. Food fluoride was associated with inhibition of buccolingual caries, whereas calcium was related to inhibition of sulcal caries. Glucose, reducing sugar, and sucrose in foods were all related to promotion of both sulcal and smooth-surface caries. The numbers of Streptococcus sobrinus in plaque were associated with promotion of smooth-surface caries only, whereas lactobacilli, non-mutans bacteria, and total viable flora were related to promotion of both smooth-surface and sulcal caries. The salivary flow rate was associated with inhibition of both buccolingual and sulcal caries. Salivary buffering capacity (at pH 7) and salivary lysozyme delivery were associated with inhibition of number and severity of sulcal caries, while the salivary amylase content was related to the promotion of the number of sulcal lesions.

The Dictyostelium Carmil Protein Links Capping Protein and the Arp2/3 Complex to Type I Myosins through Their Sh3 Domains

PubMed Central

Jung, Goeh; Remmert, Kirsten; Wu, Xufeng; Volosky, Joanne M.; III, John A. Hammer

2001-01-01

Fusion proteins containing the Src homology (SH)3 domains of Dictyostelium myosin IB (myoB) and IC (myoC) bind a 116-kD protein (p116), plus nine other proteins identified as the seven member Arp2/3 complex, and the α and β subunits of capping protein. Immunoprecipitation reactions indicate that myoB and myoC form a complex with p116, Arp2/3, and capping protein in vivo, that the myosins bind to p116 through their SH3 domains, and that capping protein and the Arp2/3 complex in turn bind to p116. Cloning of p116 reveals a protein dominated by leucine-rich repeats and proline-rich sequences, and indicates that it is a homologue of Acan 125. Studies using p116 fusion proteins confirm the location of the myosin I SH3 domain binding site, implicate NH2-terminal sequences in binding capping protein, and show that a region containing a short sequence found in several G-actin binding proteins, as well as an acidic stretch, can activate Arp2/3-dependent actin nucleation. p116 localizes along with the Arp2/3 complex, myoB, and myoC in dynamic actin-rich cellular extensions, including the leading edge of cells undergoing chemotactic migration, and dorsal, cup-like, macropinocytic extensions. Cells lacking p116 exhibit a striking defect in the formation of these macropinocytic structures, a concomitant reduction in the rate of fluid phase pinocytosis, a significant decrease in the efficiency of chemotactic aggregation, and a decrease in cellular F-actin content. These results identify a complex that links key players in the nucleation and termination of actin filament assembly with a ubiquitous barbed end–directed motor, indicate that the protein responsible for the formation of this complex is physiologically important, and suggest that previously reported myosin I mutant phenotypes in Dictyostelium may be due, at least in part, to defects in the assembly state of actin. We propose that p116 and Acan 125, along with homologues identified in Caenorhabditis elegans, Drosophila, mouse, and man, be named CARMIL proteins, for capping protein, Arp2/3, and myosin I linker. PMID:11425877

Proline Substitutions and Threonine Pseudophosphorylation of the SH3 Ligand of 18.5-kDa Myelin Basic Protein Decrease Its Affinity for the Fyn-SH3 Domain and Alter Process Development and Protein Localization in Oligodendrocytes

PubMed Central

Smith, Graham S.T.; De Avila, Miguel; Paez, Pablo M.; Spreuer, Vilma; Wills, Melanie K.B.; Jones, Nina; Boggs, Joan M.; Harauz, George

2012-01-01

The developmentally regulated myelin basic proteins (MBPs), which arise from the golli (gene of oligodendrocyte lineage) complex, are highly positively charged, intrinsically disordered, multifunctional proteins having several alternatively spliced isoforms and posttranslational modifications, and they play key roles in myelin compaction. The classic 18.5-kDa MBP isoform has a proline-rich region comprising amino acids 92–99 (murine sequence –T92PRTPPPS99–) that contains a minimal SH3 ligand domain. We have previously shown that 18.5-kDa MBP binds to several SH3 domains, including that of Fyn, a member of the Src family of tyrosine kinases involved in a number of signaling pathways during CNS development. To determine the physiological role of this binding as well as the role of phosphorylation of Thr92 and Thr95, in the current study we have produced several MBP variants specifically targeting phosphorylation sites and key structural regions of MBP’s SH3 ligand domain. Using isothermal titration calorimetry, we have demonstrated that, compared with the wild-type protein, these variants have lower affinity for the SH3 domain of Fyn. Moreover, overexpression of N-terminal-tagged GFP versions in immortalized oligodendroglial N19 and N20.1 cell cultures results in aberrant elongation of membrane processes and increased branching complexity and inhibits the ability of MBP to decrease Ca2+ influx. Phosphorylation of Thr92 can also cause MBP to traffic to the nucleus, where it may participate in additional protein–protein interactions. Coexpression of MBP with a constitutively active form of Fyn kinase resulted in membrane process elaboration, a phenomenon that was abolished by point amino acid substitutions in MBP’s SH3 ligand domain. These results suggest that MBP’s SH3 ligand domain plays a key role in intracellular protein interactions in vivo and may be required for proper membrane elaboration of developing oligodendrocytes and, further, that phosphorylation of Thr92 and Thr95 can regulate this function. PMID:21887699

Comparison of salivary levels of mucin and amylase and their relation with clinical parameters obtained from patients with aggressive and chronic periodontal disease

PubMed Central

ACQUIER, Andrea Beatriz; PITA, Alejandra Karina De Couto; BUSCH, Lucila; SÁNCHEZ, Gabriel Antonio

2015-01-01

Objective Salivary mucin and amylase levels are increased in patients with chronic periodontitis (CP). Due to the fact that aggressive periodontitis (AgP) not only differs from chronic periodontitis in terms of its clinical manifestation, the aim of this study was to compare salivary mucin and amylase levels and their relation to the clinical parameters of patients with aggressive periodontitis with that of patients with chronic periodontitis. Material and Methods Eighty subjects were divided into two groups: 20 patients with AgP and their 20 matched controls and 20 patients with CP and their 20 matched controls, based on clinical attachment loss (CAL), probing pocket depth (PPD) and bleeding on probing (BOP). Whole unstimulated saliva was obtained and mucin, amylase and protein were determined by colorimetric methods. Pearson’s correlation analysis was used to determine the relationship between salivary mucin, amylase and protein levels and the clinical parameters. Results Salivary mucin, amylase and protein levels were increased in patients with AgP and CP but there were no differences between them or between control groups. Pearson’s correlation analysis, determined in the entire subjects studied, showed a positive and significant correlation of mucin, amylase and proteins with CAL and PPD and a negative correlation with the flow rate. When Pearson’s correlation analysis was carried out in each group separately, Fisher’s z transformation showed no significant difference between both groups. Conclusion Comparison of the salivary levels of mucin, amylase and protein and their relationship with clinical parameters of AgP patients with that of CP patients revealed no differences between both groups. PMID:26221923

Pollen feeding proteomics: Salivary proteins of the passion flower butterfly, Heliconius melpomene.

PubMed

Harpel, Desiree; Cullen, Darron A; Ott, Swidbert R; Jiggins, Chris D; Walters, James R

2015-08-01

While most adult Lepidoptera use flower nectar as their primary food source, butterflies in the genus Heliconius have evolved the novel ability to acquire amino acids from consuming pollen. Heliconius butterflies collect pollen on their proboscis, moisten the pollen with saliva, and use a combination of mechanical disruption and chemical degradation to release free amino acids that are subsequently re-ingested in the saliva. Little is known about the molecular mechanisms of this complex pollen feeding adaptation. Here we report an initial shotgun proteomic analysis of saliva from Heliconius melpomene. Results from liquid-chromatography tandem mass-spectrometry confidently identified 31 salivary proteins, most of which contained predicted signal peptides, consistent with extracellular secretion. Further bioinformatic annotation of these salivary proteins indicated the presence of four distinct functional classes: proteolysis (10 proteins), carbohydrate hydrolysis (5), immunity (6), and "housekeeping" (4). Additionally, six proteins could not be functionally annotated beyond containing a predicted signal sequence. The presence of several salivary proteases is consistent with previous demonstrations that Heliconius saliva has proteolytic capacity. It is likely that these proteins play a key role in generating free amino acids during pollen digestion. The identification of proteins functioning in carbohydrate hydrolysis is consistent with Heliconius butterflies consuming nectar, like other lepidopterans, as well as pollen. Immune-related proteins in saliva are also expected, given that ingestion of pathogens is a likely route to infection. The few "housekeeping" proteins are likely not true salivary proteins and reflect a modest level of contamination that occurred during saliva collection. Among the unannotated proteins were two sets of paralogs, each seemingly the result of a relatively recent tandem duplication. These results offer a first glimpse into the molecular foundation of Heliconius pollen feeding and provide a substantial advance towards comprehensively understanding this striking evolutionary novelty. Copyright © 2015 Elsevier Ltd. All rights reserved.

Quantitative relation between intermolecular and intramolecular binding of pro-rich peptides to SH3 domains.

PubMed

Zhou, Huan-Xiang

2006-11-01

Flexible linkers are often found to tether binding sequence motifs or connect protein domains. Here we analyze three usages of flexible linkers: 1), intramolecular binding of proline-rich peptides (PRPs) to SH3 domains for kinase regulation; 2), intramolecular binding of PRP for increasing the folding stability of SH3 domains; and 3), covalent linking of PRPs and other ligands for high-affinity bivalent binding. The basis of these analyses is a quantitative relation between intermolecular and intramolecular binding constants. This relation has the form K(i) = K(e0)p for intramolecular binding and K(e) = K(e01)K(e02)p for bivalent binding. The effective concentration p depends on the length of the linker and the distance between the linker attachment points in the bound state. Several applications illustrate the usefulness of the quantitative relation. These include intramolecular binding to the Itk SH3 domain by an internal PRP and to a circular permutant of the alpha-spectrin SH3 domain by a designed PRP, and bivalent binding to the two SH3 domains of Grb2 by two linked PRPs. These and other examples suggest that flexible linkers and sequence motifs tethered to them, like folded protein domains, are also subject to tight control during evolution.

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

Toll like receptors TLR1/2, TLR6 and MUC5B as binding interaction partners with cytostatic proline rich polypeptide 1 in human chondrosarcoma.

PubMed

Galoian, Karina; Abrahamyan, Silva; Chailyan, Gor; Qureshi, Amir; Patel, Parthik; Metser, Gil; Moran, Alexandra; Sahakyan, Inesa; Tumasyan, Narine; Lee, Albert; Davtyan, Tigran; Chailyan, Samvel; Galoyan, Armen

2018-01-01

Metastatic chondrosarcoma is a bone malignancy not responsive to conventional therapies; new approaches and therapies are urgently needed. We have previously reported that mTORC1 inhibitor, antitumorigenic cytostatic proline rich polypeptide 1 (PRP-1), galarmin caused a significant upregulation of tumor suppressors including TET1/2 and SOCS3 (known to be involved in inflammatory processes), downregulation of oncoproteins and embryonic stem cell marker miR-302C and its targets Nanog, c-Myc and Bmi-1 in human chondrosarcoma. To understand better the mechanism of PRP-1 action it was very important to identify the receptor it binds to. Nuclear pathway receptor and GPCR assays indicated that PRP-1 receptors are not G protein coupled, neither do they belong to family of nuclear or orphan receptors. In the present study, we have demonstrated that PRP-1 binding interacting partners belong to innate immunity pattern recognition toll like receptors TLR1/2 and TLR6 and gel forming secreted mucin MUC5B. MUC5B was identified as PRP-1 receptor in human chondrosarcoma JJ012 cell line using Ligand-receptor capture technology. Toll like receptors TLR1/2 and TLR6 were identified as binding interaction partners with PRP-1 by western blot analysis in human chondrosarcoma JJ012 cell line lysates. Immunocytochemistry experiments confirmed the finding and indicated the localization of PRP-1 receptors in the tumor nucleus predominantly. TLR1/2, TLR6 and MUC5B were downregulated in human chondrosarcoma and upregulated in dose-response manner upon PRP-1 treatment. Experimental data indicated that in this cellular context the mentioned receptors had tumor suppressive function.

Toll like receptors TLR1/2, TLR6 and MUC5B as binding interaction partners with cytostatic proline rich polypeptide 1 in human chondrosarcoma

PubMed Central

Galoian, Karina; Abrahamyan, Silva; Chailyan, Gor; Qureshi, Amir; Patel, Parthik; Metser, Gil; Moran, Alexandra; Sahakyan, Inesa; Tumasyan, Narine; Lee, Albert; Davtyan, Tigran; Chailyan, Samvel; Galoyan, Armen

2018-01-01

Metastatic chondrosarcoma is a bone malignancy not responsive to conventional therapies; new approaches and therapies are urgently needed. We have previously reported that mTORC1 inhibitor, antitumorigenic cytostatic proline rich polypeptide 1 (PRP-1), galarmin caused a significant upregulation of tumor suppressors including TET1/2 and SOCS3 (known to be involved in inflammatory processes), downregulation of oncoproteins and embryonic stem cell marker miR-302C and its targets Nanog, c-Myc and Bmi-1 in human chondrosarcoma. To understand better the mechanism of PRP-1 action it was very important to identify the receptor it binds to. Nuclear pathway receptor and GPCR assays indicated that PRP-1 receptors are not G protein coupled, neither do they belong to family of nuclear or orphan receptors. In the present study, we have demonstrated that PRP-1 binding interacting partners belong to innate immunity pattern recognition toll like receptors TLR1/2 and TLR6 and gel forming secreted mucin MUC5B. MUC5B was identified as PRP-1 receptor in human chondrosarcoma JJ012 cell line using Ligand-receptor capture technology. Toll like receptors TLR1/2 and TLR6 were identified as binding interaction partners with PRP-1 by western blot analysis in human chondrosarcoma JJ012 cell line lysates. Immunocytochemistry experiments confirmed the finding and indicated the localization of PRP-1 receptors in the tumor nucleus predominantly. TLR1/2, TLR6 and MUC5B were downregulated in human chondrosarcoma and upregulated in dose-response manner upon PRP-1 treatment. Experimental data indicated that in this cellular context the mentioned receptors had tumor suppressive function. PMID:29138803

Stability and function of interdomain linker variants of glucoamylase 1 from Aspergillus niger.

PubMed

Sauer, J; Christensen, T; Frandsen, T P; Mirgorodskaya, E; McGuire, K A; Driguez, H; Roepstorff, P; Sigurskjold, B W; Svensson, B

2001-08-07

Several variants of glucoamylase 1 (GA1) from Aspergillus niger were created in which the highly O-glycosylated peptide (aa 468--508) connecting the (alpha/alpha)(6)-barrel catalytic domain and the starch binding domain was substituted at the gene level by equivalent segments of glucoamylases from Hormoconis resinae, Humicola grisea, and Rhizopus oryzae encoding 5, 19, and 36 amino acid residues. Variants were constructed in which the H. resinae linker was elongated by proline-rich sequences as this linker itself apparently was too short to allow formation of the corresponding protein variant. Size and isoelectric point of GA1 variants reflected differences in linker length, posttranslational modification, and net charge. While calculated polypeptide chain molecular masses for wild-type GA1, a nonnatural proline-rich linker variant, H. grisea, and R. oryzae linker variants were 65,784, 63,777, 63,912, and 65,614 Da, respectively, MALDI-TOF-MS gave values of 82,042, 73,800, 73,413, and 90,793 Da, respectively, where the latter value could partly be explained by an N-glycosylation site introduced near the linker C-terminus. The k(cat) and K(m) for hydrolysis of maltooligodextrins and soluble starch, and the rate of hydrolysis of barley starch granules were essentially the same for the variants as for wild-type GA1. beta-Cyclodextrin, acarbose, and two heterobidentate inhibitors were found by isothermal titration calorimetry to bind to the catalytic and starch binding domains of the linker variants, indicating that the function of the active site and the starch binding site was maintained. The stability of GA1 linker variants toward GdnHCl and heat, however, was reduced compared to wild-type.

In Silico Analysis of Correlations between Protein Disorder and Post-Translational Modifications in Algae

PubMed Central

Kurotani, Atsushi; Sakurai, Tetsuya

2015-01-01

Recent proteome analyses have reported that intrinsically disordered regions (IDRs) of proteins play important roles in biological processes. In higher plants whose genomes have been sequenced, the correlation between IDRs and post-translational modifications (PTMs) has been reported. The genomes of various eukaryotic algae as common ancestors of plants have also been sequenced. However, no analysis of the relationship to protein properties such as structure and PTMs in algae has been reported. Here, we describe correlations between IDR content and the number of PTM sites for phosphorylation, glycosylation, and ubiquitination, and between IDR content and regions rich in proline, glutamic acid, serine, and threonine (PEST) and transmembrane helices in the sequences of 20 algae proteomes. Phosphorylation, O-glycosylation, ubiquitination, and PEST preferentially occurred in disordered regions. In contrast, transmembrane helices were favored in ordered regions. N-glycosylation tended to occur in ordered regions in most of the studied algae; however, it correlated positively with disordered protein content in diatoms. Additionally, we observed that disordered protein content and the number of PTM sites were significantly increased in the species-specific protein clusters compared to common protein clusters among the algae. Moreover, there were specific relationships between IDRs and PTMs among the algae from different groups. PMID:26307970

In Silico Analysis of Correlations between Protein Disorder and Post-Translational Modifications in Algae.

PubMed

Kurotani, Atsushi; Sakurai, Tetsuya

2015-08-20

Recent proteome analyses have reported that intrinsically disordered regions (IDRs) of proteins play important roles in biological processes. In higher plants whose genomes have been sequenced, the correlation between IDRs and post-translational modifications (PTMs) has been reported. The genomes of various eukaryotic algae as common ancestors of plants have also been sequenced. However, no analysis of the relationship to protein properties such as structure and PTMs in algae has been reported. Here, we describe correlations between IDR content and the number of PTM sites for phosphorylation, glycosylation, and ubiquitination, and between IDR content and regions rich in proline, glutamic acid, serine, and threonine (PEST) and transmembrane helices in the sequences of 20 algae proteomes. Phosphorylation, O-glycosylation, ubiquitination, and PEST preferentially occurred in disordered regions. In contrast, transmembrane helices were favored in ordered regions. N-glycosylation tended to occur in ordered regions in most of the studied algae; however, it correlated positively with disordered protein content in diatoms. Additionally, we observed that disordered protein content and the number of PTM sites were significantly increased in the species-specific protein clusters compared to common protein clusters among the algae. Moreover, there were specific relationships between IDRs and PTMs among the algae from different groups.

Correlation between salivary secretion and salivary AQP5 levels in health and disease.

PubMed

Wang, Di; Iwata, Fusako; Muraguchi, Masahiro; Ooga, Keiko; Ohmoto, Yasukazu; Takai, Masaaki; Mori, Toyoki; Ishikawa, Yasuko

2009-01-01

Saliva samples are useful for noninvasive diagnosis of oral and systemic diseases. The water channel protein aquaporin-5 (AQP5) is released into human saliva. Salivary AQP5 levels show a diurnal variation with the secretion of high levels during the waking hours. An age-related decrease in salivary AQP5 levels parallels a decrease in the volume of saliva. Cevimeline, a muscarinic acetylcholine receptor (mAChR) agonist, induces the release of AQP5. Changes in salivary AQP5 levels after cevimeline administration occur simultaneously with changes in saliva flow rate. AQP5 and lipid rafts are released separately from human salivary glands upon M(3) mAChR stimulation. In patients with diabetes mellitus or Sjögren's syndrome, a decrease in salivary secretion occurs concomitantly with low salivary AQP5 levels. Salivary AQP5 levels correlate with salivary secretion in both healthy and disease states, suggesting that changes in salivary AQP5 levels can be used as an indicator of salivary flow rate and the effect of M(3) mAChR agonists on human salivary glands.

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

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.

Tsetse Salivary Gland Proteins 1 and 2 Are High Affinity Nucleic Acid Binding Proteins with Residual Nuclease Activity

PubMed Central

Caljon, Guy; Ridder, Karin De; Stijlemans, Benoît; Coosemans, Marc; Magez, Stefan; De Baetselier, Patrick; Van Den Abbeele, Jan

2012-01-01

Analysis of the tsetse fly salivary gland EST database revealed the presence of a highly enriched cluster of putative endonuclease genes, including tsal1 and tsal2. Tsal proteins are the major components of tsetse fly (G. morsitans morsitans) saliva where they are present as monomers as well as high molecular weight complexes with other saliva proteins. We demonstrate that the recombinant tsetse salivary gland proteins 1&2 (Tsal1&2) display DNA/RNA non-specific, high affinity nucleic acid binding with KD values in the low nanomolar range and a non-exclusive preference for duplex. These Tsal proteins exert only a residual nuclease activity with a preference for dsDNA in a broad pH range. Knockdown of Tsal expression by in vivo RNA interference in the tsetse fly revealed a partially impaired blood digestion phenotype as evidenced by higher gut nucleic acid, hematin and protein contents. PMID:23110062

[Hydroxyproline: Rich glycoproteins of the plant and cell wall]. Annual technical progress report, 1993

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

Varner, J.E.

1993-06-01

Since xylem tissue includes the main cell types which are lignified, we are interested in gene expression of glycine-rich proteins and proline-rich proteins, and other proteins which are involved in secondary cell wall thickening during xylogenesis. Since the main feature of xylogenesis is the deposition of additional wall components, study of the mechanism of xylogenesis will greatly advance our knowledge of the synthesis and assembly of wall macromolecules. We are using the in vitro xylogenesis system from isolated Zinnia mesophyll cells to isolate genes which are specifically expressed during xylogenesis. We have used subtractive hybridization methods to isolate a numbermore » of cDNA clones for differentially regulated genes from the cells after hormonal induction. So far, we have partially characterized 18 different cDNA clones from 239 positive clones. These differentially regulated genes can be divided into three sets according to the characteristics of gene expression in the induction medium and the control medium. The first set is induced in both the induction medium and the control medium without hormones. The second set is induced mainly in the induction medium and in the control medium with the addition of NAA alone. Two of thesegenes are exclusively induced by auxin. The third set of genes is induced mainly in the induction medium. Since these genes are not induced by either auxin or cytokinin alone, they may be directly involved in the process of xylogenesis. Our experiments on the localization of H{sub 2}O{sub 2} production reinforce the earlier ideas of others that H{sub 2}O{sub 2} is involved in normal lignification.« less

[Hydroxyproline: Rich glycoproteins of the plant and cell wall

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

Varner, J.E.

1993-01-01

Since xylem tissue includes the main cell types which are lignified, we are interested in gene expression of glycine-rich proteins and proline-rich proteins, and other proteins which are involved in secondary cell wall thickening during xylogenesis. Since the main feature of xylogenesis is the deposition of additional wall components, study of the mechanism of xylogenesis will greatly advance our knowledge of the synthesis and assembly of wall macromolecules. We are using the in vitro xylogenesis system from isolated Zinnia mesophyll cells to isolate genes which are specifically expressed during xylogenesis. We have used subtractive hybridization methods to isolate a numbermore » of cDNA clones for differentially regulated genes from the cells after hormonal induction. So far, we have partially characterized 18 different cDNA clones from 239 positive clones. These differentially regulated genes can be divided into three sets according to the characteristics of gene expression in the induction medium and the control medium. The first set is induced in both the induction medium and the control medium without hormones. The second set is induced mainly in the induction medium and in the control medium with the addition of NAA alone. Two of thesegenes are exclusively induced by auxin. The third set of genes is induced mainly in the induction medium. Since these genes are not induced by either auxin or cytokinin alone, they may be directly involved in the process of xylogenesis. Our experiments on the localization of H[sub 2]O[sub 2] production reinforce the earlier ideas of others that H[sub 2]O[sub 2] is involved in normal lignification.« less

Switch-like Arp2/3 activation upon WASP and WIP recruitment to an apparent threshold level by multivalent linker proteins in vivo.

PubMed

Sun, Yidi; Leong, Nicole T; Jiang, Tommy; Tangara, Astou; Darzacq, Xavier; Drubin, David G

2017-08-16

Actin-related protein 2/3 (Arp2/3) complex activation by nucleation promoting factors (NPFs) such as WASP, plays an important role in many actin-mediated cellular processes. In yeast, Arp2/3-mediated actin filament assembly drives endocytic membrane invagination and vesicle scission. Here we used genetics and quantitative live-cell imaging to probe the mechanisms that concentrate NPFs at endocytic sites, and to investigate how NPFs regulate actin assembly onset. Our results demonstrate that SH3 (Src homology 3) domain-PRM (proline-rich motif) interactions involving multivalent linker proteins play central roles in concentrating NPFs at endocytic sites. Quantitative imaging suggested that productive actin assembly initiation is tightly coupled to accumulation of threshold levels of WASP and WIP, but not to recruitment kinetics or release of autoinhibition. These studies provide evidence that WASP and WIP play central roles in establishment of a robust multivalent SH3 domain-PRM network in vivo, giving actin assembly onset at endocytic sites a switch-like behavior.

Exploring monovalent and multivalent peptides for the inhibition of FBP21-tWW

PubMed Central

Bertazzon, Miriam; Marczynke, Michaela; Seitz, Oliver; Volkmer, Rudolf; Haag, Rainer

2015-01-01

Summary The coupling of peptides to polyglycerol carriers represents an important route towards the multivalent display of protein ligands. In particular, the inhibition of low affinity intracellular protein–protein interactions can be addressed by this design. We have applied this strategy to develop binding partners for FBP21, a protein which is important for the splicing of pre-mRNA in the nucleus of eukaryotic cells. Firstly, by using phage display the optimized sequence WPPPPRVPR was derived which binds with K Ds of 80 μM and 150 µM to the individual WW domains and with a K D of 150 μM to the tandem-WW1–WW2 construct. Secondly, this sequence was coupled to a hyperbranched polyglycerol (hPG) that allowed for the multivalent display on the surface of the dendritic polymer. This novel multifunctional hPG-peptide conjugate displayed a K D of 17.6 µM which demonstrates that the new carrier provides a venue for the future inhibition of proline-rich sequence recognition by FBP21 during assembly of the spliceosome. PMID:26124874

Impact of O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation and MGMT expression on dacarbazine resistance of Hodgkin's lymphoma cells.

PubMed

Kewitz, Stefanie; Stiefel, Martina; Kramm, Christof M; Staege, Martin S

2014-01-01

We analyzed the methylation status of the O6-methylguanine-DNA methyltransferase (MGMT) promoter and mRNA expression in HL cells and assessed the response of these cells to dacarbazine. Expression of MGMT correlated with the presence of non-methylated promoters and cell lines with non-methylated promoters showed increased resistance against dacarbazine. KM-H2 cells expressed fusion transcripts between MGMT and proline-rich coiled-coil 2B (PRRC2B) but no wild type MGMT transcripts. Dacarbazine sensitivity suggested that fusion transcripts are translated into a protein with reduced functionality. MGMT promoter methylation predicts dacarbazine sensitivity of HL cells and it might be interesting to analyze this factor in HL patients. Copyright © 2013 Elsevier Ltd. All rights reserved.

FKBP51 and FKBP52 in Signaling and Disease

PubMed Central

Storer, Cheryl L.; Dickey, Chad A.; Galigniana, Mario D.; Rein, Theo; Cox, Marc B.

2011-01-01

FKBP51 and FKBP52 are diverse regulators of steroid hormone receptor signaling including regulation of receptor maturation, hormone binding, and nuclear translocation. Although structurally similar, they are functionally divergent, which is largely attributed to differences in the FK1 domain and the proline-rich loop. FKBP51 and FKBP52 have emerged as likely contributors to a variety of hormone-dependent diseases including stress-related diseases, immune function, reproductive functions and a variety of cancers. In addition, recent studies have implicated FKBP51 and FKBP52 in Alzheimer’s disease and other protein aggregation disorders. This review summarizes our current understanding of FKBP51 and FKBP52 interactions within the receptor-chaperone complex, their contributions to health and disease, and their potential as therapeutic targets for the treatment of these diseases. PMID:21889356

Hsp27 (HSPB1) differential expression in normal salivary glands and pleomorphic adenomas and association with an increased Bcl2/Bax ratio.

PubMed

Siqueira, Elisa C de; Souza, Fabrício T A; Diniz, Marina G; Gomez, Ricardo S; Gomes, Carolina C

2015-01-01

Pleomorphic adenoma (PA) is the most common salivary gland neoplasm. The Hsp27 (HSPB1) is an antiapoptotic protein whose synthesis follows cytotoxic stresses and result in a transient increase in tolerance to subsequent cell injury. Although Hsp27 is expressed in a range of normal tissues and neoplasms, a wide variation in its expression exists among different cells and tissues types. In certain tumours of glandular origin (such as oesophageal adenocarcinomas), the level of Hsp27 is decreased. In the present study, Hsp27 protein levels were evaluated by enzyme-linked immunosorbent assay (ELISA) in a set of 18 fresh PA and 12 normal salivary gland samples. In addition, we tested if Hsp27 protein levels correlated with p53 expression and cell proliferation index, as well as with the transcriptional levels of Bcl-2-associated X protein (BAX), B cell lymphoma 2 (BCL2) and Caspase 3 in PA. We further tested the association between Hsp27 expression and PA tumour size. While all normal salivary gland samples expressed Hsp27 protein, only half of the PA samples expressed it, resulting in a reduced expression of Hsp27 in PA when compared with normal salivary glands (P = 0.003). The expression levels of this protein correlated positively with a higher messenger ribonucleic acid (mRNA) ratio of Bcl2/Bax (R = 0.631; P = 0.01). In conclusion, a decreased Hsp27 protein expression level in PA was found. In addition, Hsp27 levels correlated positively with the Bcl2/Bax mRNA ratio, suggesting an antiapoptotic effect.

Immunogenic Salivary Proteins of Triatoma infestans: Development of a Recombinant Antigen for the Detection of Low-Level Infestation of Triatomines

PubMed Central

Schwarz, Alexandra; Helling, Stefan; Collin, Nicolas; Teixeira, Clarissa R.; Medrano-Mercado, Nora; Hume, Jen C. C.; Assumpção, Teresa C.; Marcus, Katrin; Stephan, Christian; Meyer, Helmut E.; Ribeiro, José M. C.; Billingsley, Peter F.; Valenzuela, Jesus G.; Sternberg, Jeremy M.; Schaub, Günter A.

2009-01-01

Background Triatomines are vectors of Trypanosoma cruzi, the etiological agent of Chagas disease in Latin America. The most effective vector, Triatoma infestans, has been controlled successfully in much of Latin America using insecticide spraying. Though rarely undertaken, surveillance programs are necessary in order to identify new infestations and estimate the intensity of triatomine bug infestations in domestic and peridomestic habitats. Since hosts exposed to triatomines develop immune responses to salivary antigens, these responses can be evaluated for their usefulness as epidemiological markers to detect infestations of T. infestans. Methodology/Principal Findings T. infestans salivary proteins were separated by 2D-gel electrophoresis and tested for their immunogenicity by Western blotting using sera from chickens and guinea pigs experimentally exposed to T. infestans. From five highly immunogenic protein spots, eight salivary proteins were identified by nano liquid chromatography-electrospray ionization-tandem mass spectrometry (nanoLC-ESI-MS/MS) and comparison to the protein sequences of the National Center for Biotechnology Information (NCBI) database and expressed sequence tags of a unidirectionally cloned salivary gland cDNA library from T. infestans combined with the NCBI yeast protein sub-database. The 14.6 kDa salivary protein [gi|149689094] was produced as recombinant protein (rTiSP14.6) in a mammalian cell expression system and recognized by all animal sera. The specificity of rTiSP14.6 was confirmed by the lack of reactivity to anti-mosquito and anti-sand fly saliva antibodies. However, rTiSP14.6 was recognized by sera from chickens exposed to four other triatomine species, Triatoma brasiliensis, T. sordida, Rhodnius prolixus, and Panstrongylus megistus and by sera of chickens from an endemic area of T. infestans and Chagas disease in Bolivia. Conclusions/Significance The recombinant rTiSP14.6 is a suitable and promising epidemiological marker for detecting the presence of small numbers of different species of triatomines and could be developed for use as a new tool in surveillance programs, especially to corroborate vector elimination in Chagas disease vector control campaigns. PMID:19841746

Saliva as a tool for monitoring steroid, peptide and immune markers in sport and exercise science.

PubMed

Papacosta, Elena; Nassis, George P

2011-09-01

This paper discusses the use of saliva analysis as a tool for monitoring steroid, peptide, and immune markers of sports training. Salivary gland physiology, regarding the regulation and stimulation of saliva secretion, as well as methodological issues including saliva collection, storage and analysis are addressed in this paper. The effects of exercise on saliva composition are then considered. Exercise elicits changes in salivary levels of steroid hormones, immunoglobulins, antimicrobial proteins and enzymes. Cortisol, testosterone and dehydroepiandrosterone can be assessed in saliva, providing a non-invasive option to assess the catabolic and anabolic effects of exercise. Validation studies using blood and salivary measures of steroid hormones are addressed in this paper. Effects of acute exercise and training on salivary immunoglobulins (SIgA, SIgM, SIgG) and salivary antimicrobial proteins, including α-amylase, lysozyme and lactoferrin, are also discussed. Analysis of cortisol and testosterone in saliva may help detect the onset of non-functional overreaching and subsequently may help to prevent the development of overtraining syndrome. Assessment of salivary immunoglobulins and antimicrobial proteins has been shown to successfully represent the effects of exercise on mucosal immunity. Increases in SIgA and antimicrobial proteins concentration and/or secretion rate are associated with acute exercise whereas conversely, decreases have been reported in athletes over a training season leaving the athlete susceptible for upper respiratory tract infections. The measurement of physiological biomarkers in whole saliva can provide a significant tool for assessing the immunological and endocrinological status associated with exercise and training. Copyright © 2011 Sports Medicine Australia. Published by Elsevier Ltd. All rights reserved.

Assessment of Anopheles salivary antigens as individual exposure biomarkers to species-specific malaria vector bites

PubMed Central

2012-01-01

Background Malaria transmission occurs during the blood feeding of infected anopheline mosquitoes concomitant with a saliva injection into the vertebrate host. In sub-Saharan Africa, most malaria transmission is due to Anopheles funestus s.s and to Anopheles gambiae s.l. (mainly Anopheles gambiae s.s. and Anopheles arabiensis). Several studies have demonstrated that the immune response against salivary antigens could be used to evaluate individual exposure to mosquito bites. The aim of this study was to assess the use of secreted salivary proteins as specific biomarkers of exposure to An. gambiae and/or An. funestus bites. Methods For this purpose, salivary gland proteins 6 (SG6) and 5′nucleotidases (5′nuc) from An. gambiae (gSG6 and g-5′nuc) and An. funestus (fSG6 and f-5′nuc) were selected and produced in recombinant form. The specificity of the IgG response against these salivary proteins was tested using an ELISA with sera from individuals living in three Senegalese villages (NDiop, n = 50; Dielmo, n = 38; and Diama, n = 46) that had been exposed to distinct densities and proportions of the Anopheles species. Individuals who had not been exposed to these tropical mosquitoes were used as controls (Marseille, n = 45). Results The IgG responses against SG6 recombinant proteins from these two Anopheles species and against g-5′nucleotidase from An. gambiae, were significantly higher in Senegalese individuals compared with controls who were not exposed to specific Anopheles species. Conversely, an association was observed between the level of An. funestus exposure and the serological immune response levels against the f-5′nucleotidase protein. Conclusion This study revealed an Anopheles salivary antigenic protein that could be considered to be a promising antigenic marker to distinguish malaria vector exposure at the species level. The epidemiological interest of such species-specific antigenic markers is discussed. PMID:23276246

Postharvest production of ochratoxin A by Aspergillus ochraceus and Penicillium viridicatum in barley with different protein levels.

PubMed Central

Häggblom, P E; Ghosh, J

1985-01-01

The production of ochratoxin A (OA) in barley by Aspergillus ochraceus and Penicillium viridicatum was measured at 12 and 25 degrees C. The grain had been fertilized with various amounts of nitrogen fertilizer (0, 90, or 240 kg/ha) and contained (at crop maturity) 9.1, 10.4, or 12.0% protein, respectively. The production of OA by both fungi increased as the protein concentration increased. Glutamic acid and proline were enriched relative to other amino acids as the protein concentration increased. The differences in OA production could not be explained by a differential effect of protein or amino acids on fungal growth in barley. However, glutamic acid and proline enhanced OA production in liquid cultures of both A. ochraceus and P. viridicatum. PMID:4004212