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PubMed1988

Regulation of proline-rich protein and alpha-amylase genes in parotid-hepatoma hybrid cells.

Salivary proline-rich proteins are encoded by tissue-specific multigene families, and are dramatically induced in mice, rats, and hamsters by treatment with the beta agonist isoproterenol. Salivary gland cells, however, are difficult to maintain in a differentiated state in culture and can be induced to synthesize proline-rich protein mRNAs for only a few days. In an attempt to establish a cell line in which it would be possible to regulate proline-rich protein gene transcription, rat parotid gland cells were fused with the rat hepatoma cell line, FTO-2B. Fused cells were obtained that had a frequency of 7.5 x 10(-6), which is about 125-fold greater than the reversion rate of FTO-2B. The hybrid cells exhibited induced proline-rich protein mRNA synthesis when incubated with either dibutyryl cyclic AMP or forskolin. The ability to induce these genes was maintained for at least 20 passages. Most of the fused cell populations also synthesized elevated levels of alpha-amylase mRNA, another tissue-specific gene.

Animals↗
PubMed1992

Analysis of the mutant proBA operon from a proline-producing strain of Serratia marcescens.

The nucleotide sequence of the proBA operon from a proline-hyperproducing mutant of Serratia marcescens was determined. Two base substitutions were found: one in the proB structural gene, coding for gamma-glutamyl kinase (GK), and a second one in the promoter region of the operon. The former base substitution led to a change of the predicted amino acid at position 117 from an alanine to a valine in GK. This mutation rendered GK 700-fold less sensitive to proline-mediated feedback inhibition than the wild-type enzyme. The other base substitution, a transversion from a G-C to an A-T, was located in the spacer region between the '-35' and '-10' sequences of the promoter, and it increased the transcriptional activity of this operon fourfold. Both these two base substitutions, which were acquired at the step of selecting mutants resistant to a toxic proline analogue, 3,4-dehydroproline, confer upon cells a high proline productivity and an increased osmotolerance.

Amino Acid Sequence↗
PubMed1996

Proline biosynthesis in Streptococcus thermophilus: characterization of the proBA operon and its products.

The presence of proline in the medium was not essential for growth of Streptococcus thermophilus, indicating that there is a proline biosynthetic pathway in this organism. Genetic and biochemical analysis identified and characterized this pathway. Two genes, designated proB and proA, were cloned, sequenced and characterized. Biochemical analysis of the proB- and proA-encoded enzymes showed that the proline biosynthetic pathway of S. thermophilus is similar to the one previously described in Escherichia coli. The deduced amino acid sequence of a 2-408 kb DNA region containing the genes revealed the similarity of the S. thermophilus gene products to ProB and ProA of E. coli and Serratia marcescens, and to the corresponding N- and C-terminal domains of the bifunctional plant enzyme delta 1-pyrroline-5-carboxylate synthetase of Vigna aconitifolia. Northern blot analysis showed that the two genes in S. thermophilus are organized in a single operon with proB proximal and proA distal to the promoter; primer extension analysis indicated that proBA transcription is not under repressive control by exogenously supplied proline.

Aldehyde Oxidoreductases↗
PubMed1989

Characterization of two soybean repetitive proline-rich proteins and a cognate cDNA from germinated axes.

We have resolved and analyzed two proline-rich proteins isolated from the walls of soybean cells in culture. The proteins are similar in amino acid content, containing 20% proline, 20% hydroxyproline, 20% lysine, 16% valine, 10% tyrosine, and 10% glutamate. The proteins undergo a rearrangement or a limited cleavage in dilute NaOH, but are otherwise remarkably stable to a high concentration of alkali. We have cloned and sequenced a cDNA from soybean axes germinated for 31 hours (1A10-2) coding for a protein that closely corresponds in its amino acid content to that of the proline-rich proteins. The cDNA sequence predicts a decameric repeat of Pro-Pro-Val-Tyr-Lys-Pro-Pro-Val-Glu-Lys. Consequently, this class of proteins is referred to as repetitive proline-rich proteins, i.e., RPRP2 and RPRP3. We have also analyzed RNA gel blots with probes that discriminate between the new cDNA clone and a related cDNA previously reported [SbPRP1; Hong, Nagao, and Key (1987). J. Biol. Chem. 262, 8367-8376]. Messenger RNAs from young seedlings and from soybean suspension cultures correspond primarily to the new RPRP clone (1A10-2), whereas the predominant mRNA accumulating later in the roots corresponds to SbPRP1.

Amino Acid Sequence↗
PubMed1998

Pollen profilin function depends on interaction with proline-rich motifs.

The actin binding protein profilin has dramatic effects on actin polymerization in vitro and in living cells. Plants have large multigene families encoding profilins, and many cells or tissues can express multiple profilin isoforms. Recently, we characterized several profilin isoforms from maize pollen for their ability to alter cytoarchitecture when microinjected into living plant cells and for their association with poly-L-proline and monomeric actin from maize pollen. In this study, we characterize a new profilin isoform from maize, which has been designated ZmPRO4, that is expressed predominantly in endosperm but is also found at low levels in all tissues examined, including mature and germinated pollen. The affinity of ZmPRO4 for monomeric actin, which was measured by two independent methods, is similar to that of the three profilin isoforms previously identified in pollen. In contrast, the affinity of ZmPRO4 for poly-L-proline is nearly twofold higher than that of native pollen profilin and the other recombinant profilin isoforms. When ZmPRO4 was microinjected into plant cells, the effect on actin-dependent nuclear position was significantly more rapid than that of another pollen profilin isoform, ZmPRO1. A gain-of-function mutant (ZmPRO1-Y6F) was created and found to enhance poly-L-proline binding activity and to disrupt cytoarchitecture as effectively as ZmPRO4. In this study, we demonstrate that profilin isoforms expressed in a single cell can have different effects on actin in living cells and that the poly-L-proline binding function of profilin may have important consequences for the regulation of actin cytoskeletal dynamics in plant cells.

Actins↗
PubMed2001

Crystallization and preliminary crystallographic analysis of the proline dehydrogenase domain of the multifunctional PutA flavoprotein from Escherichia coli.

The PutA flavoprotein from Escherichia coli is a multifunctional protein that plays pivotal roles in proline catabolism by functioning as both a membrane-associated bifunctional enzyme and a transcriptional repressor. Peripherally membrane-bound PutA catalyzes the two-step oxidation of proline to glutamate, while cytoplasmic PutA represses the transcription of its own gene and the gene for a proline-transporter protein. X-ray crystallographic studies on PutA have been initiated to determine how the PutA structural scaffold enables it to be both an enzyme and a repressor, and to understand the mechanism by which PutA switches between its enzymatic and DNA-binding functions. To facilitate crystallization, a recombinant protein (PutA669) corresponding to the N-terminal 669 amino-acid residues of the 1320 residues of PutA was engineered. Activity assays demonstrated that PutA669 catalyzes the first step of chemistry performed by PutA, the conversion of proline to Delta1-pyrroline-5-carboxylate. Crystals of PutA669 have been obtained from PEG 3000 buffered at pH 6-7. The crystals occupy an I-centered orthorhombic lattice with unit-cell parameters a = 72.5, b = 140.2, c = 146.8 A; a 2.15 A data set was collected using a rotating-anode source. Assuming one molecule per asymmetric unit, the Matthews coefficient V(M) is 2.5 A(3) Da(-1), with a solvent content of 50%. The structure of PutA669 will be solved by multiple isomorphous replacement.

Bacterial Proteins↗
PubMed2005

The flexibility in the proline ring couples to the protein backbone.

In proteins, the proline ring exists predominantly in two discrete states. However, there is also a small but significant amount of flexibility in the proline ring of high-resolution protein structures. We have found that this side-chain flexibility is coupled to the backbone conformation. To study this coupling, we have developed a model that is simply based on geometric and steric factors and not on energetics. We show that the coupling between phi and chi1 torsions in the proline ring can be described by an analytic equation that was developed by Bricard in 1897, and we describe a computer algorithm that implements the equation. The model predicts the observed coupling very well. The strain in the C(gamma)-C(delta)-N angle appears to be the principal barrier between the UP and DOWN pucker. This strain is relaxed to allow the proline ring to flatten in the rare PLANAR conformation.

Algorithms↗
PubMed2006

The SH3 domain of a M7 interacts with its C-terminal proline-rich region.

Myosins play essential roles in migration, cytokinesis, endocytosis, and adhesion. They are composed of a large N-terminal motor domain with ATPase and actin binding sites and C-terminal neck and tail regions, whose functional roles and structural context in the protein are less well characterized. The tail regions of myosins I, IV, VII, XII, and XV each contain a putative SH3 domain that may be involved in protein-protein interactions. SH3 domains are reported to bind proline-rich motifs, especially "PxxP" sequences, and such interactions serve regulatory functions. The activity of Src, PI3, and Itk kinases, for example, is regulated by intramolecular interactions between their SH3 domain and internal proline-rich sequences. Here, we use NMR spectroscopy to reveal the structure of a protein construct from Dictyostelium myosin VII (DdM7) spanning A1620-T1706, which contains its SH3 domain and adjacent proline-rich region. The SH3 domain forms the signature beta-barrel architecture found in other SH3 domains, with conserved tryptophan and tyrosine residues forming a hydrophobic pocket known to bind "PxxP" motifs. In addition, acidic residues in the RT or n-Src loops are available to interact with the basic anchoring residues that are typically found in ligands or proteins that bind SH3 domains. The DdM7 SH3 differs in the hydrophobicity of the second pocket formed by the 3(10) helix and following beta-strand, which contains polar rather than hydrophobic side chains. Most unusual, however, is that this domain binds its adjacent proline-rich region at a surface remote from the region previously identified to bind "PxxP" motifs. The interaction may affect the orientation of the tail without sacrificing the availability of the canonical "PxxP"-binding surface.

Amino Acid Motifs↗
PubMed1976

Cell-mediated cytotoxicity for melanoma tumor cells: detection by a (3H) proline release assay.

An in vitro lymphocyte-mediated cytotoxicity assay using [3H]proline-labeled target cells is described. The assay, modified from an original procedure of Bean et al., assesses the release of [3-H]proline by filtering the total culture fluid containing both trypsinized tumor cells and effector cells. Filtration is performed with a semiautomatic harvesting device using low suction pressure and large-diameter glass filters. Pretreatment of filters with whole serum diminishes adsorption of cell-free radioactive material considerably and thus increases the sensitivity of the assay. Nearly 100% of the radioactivity could be recovered with this harvesting device. The technique allowed the detection of cytolytic activities of lymphocytes after 6 h of incubation. Lymphocytes from patients with primary malignant melanoma showed a significantly higher cytolytic reactivity (P less than 0.001) than normal donors' lymphocytes against three different melanoma cell lines. In a series of parallel experiments on 36 patients and 18 normal donors, this modification of the [3H]proline test was compared with three different assays: the conventional microcytotoxicity test of Takasugi and Klein, the original [3H]proline microcytotoxicity test of Bean et al., and the viability count of tumor cells.

Cell Line↗
PubMed1988

N.m.r. and computer-simulated conformational analyses of a nonapeptide found in a human salivary proline-rich glycoprotein.

The amino acid sequence G(1)-P(2)-P(3)-P(4)-H(5)-P(6)-G(7)-K(8)-P(9) occurs twice in the proline-rich glycoprotein (PRG) found in human parotid saliva. As part of our efforts to elucidate the structure-function relationships of PRG, this nonapeptide sequence (PRG9) was synthesized for the purpose of conformational analyses by high-resolution proton n.m.r. spectroscopy and computer-modeling. The empirical n.m.r. spectrum differed from the simulated spectrum in that the overall chemical shift locations were displaced from their random coil positions and the five proline residues had non-degenerate C alpha H alpha protons. Other n.m.r. data indicated that no intramolecular hydrogen-bonding was present in the PRG. In conjunction with X-ray crystallographic data on a triproline-containing model compound (Kartha, g., Ashida, T. & Kakudo, M. (1974) Acta Cryst. B30, 1861-1866), four energy-minimized PRG9 structures were obtained. Two of the structures were energetically unfavorable, while the other two conformations were reasonable. The two most likely structures gave all prolines an S-type ring pucker, the P(2)-P(3)-P(4) sequence as a poly-L-proline II helix, the H(5) phi = -90.3 degrees, P(6) and P(9) with trans peptide bond orientation, G(7) in an extended state, and the K(8) phi = -93.2 degrees or -146.8 degrees for structures #1 and #2, respectively.

Computer Simulation↗
PubMed1987

Nitrogen catabolite regulation of proline permease in Saccharomyces cerevisiae. Cloning of the PUT4 gene and study of PUT4 RNA levels in wild-type and mutant strains.

The proline permease gene PUT4 has been cloned. Nitrogen-source regulation ('ammonia sensitivity') of this and at least two other amino-acid permeases is believed to occur at two distinct levels, i.e. permease synthesis and permease activity. Therefore, PUT4 transcription/messenger stability was examined in the ammonia- and proline-grown wild type as well as in mutant strains supposedly affected at only one or at both of these levels. We report transcript-level repression of proline permease synthesis in ammonia-grown cells. Repression is lifted at this level in gdhCR, gln1ts and gdhA mutants which exhibit pleiotropically derepressed permease and catabolic enzyme activities. On the other hand, the npi1 and npi2 mutations, formerly called mut2 and mut4, relieve an inactivation process which seems only to affect permeases. These mutations do not affect the detected PUT4 RNA level. The only known positive factor in proline permease regulation, the nitrogen permease reactivator protein Npr1, is believed to counteract the inactivation process on derepressing media. This protein appears to have an additional, indirect effect on PUT4 transcription/messenger stability: it would actually mediate repression via its activating effect on ammonia uptake.

Amino Acid Transport Systems, Neutral↗
PubMed2006

Mouse tryptophan hydroxylase isoform 2 and the role of proline 447 in enzyme function.

Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in the synthesis of the neurotransmitter serotonin (5-HT). Once thought to be a single gene product, TPH is now known to exist in two isoforms. Isoform 1 (TPH1) is found in the pineal gland and gut, and isoform 2 (TPH2) is selectively expressed in brain. A single-nucleotide polymorphism in TPH2 results in a proline-to-arginine mutation at residue 447 and substantially lowers catalytic activity. In view of the importance of TPH in determining brain 5-HT function, we cloned TPH2 and produced the P447R mutant to assess the importance of this proline in enzyme function. Catalytically active TPH2 and the P447R mutant were expressed at the predicted subunit molecular mass of 56 kDa. The P447R mutant expressed less than 50% of the activity of TPH2. Mutation of this conserved proline in TPH1 (P403R) also resulted in an enzyme with significantly lower activity than the wild-type enzyme. The P447R mutant had a V(max) 50% lower than that of TPH2. The P447R mutation did not alter the oligomeric assembly of the protein, nor change its responsiveness to cysteine modification. The P447R mutation did not alter enzyme substrate specificity or stability, but conferred slightly enhanced sensitivity to inhibition by dopamine and diminished sensitivity to iron in catalysis. The conserved proline in TPH (residue 447 in TPH2 and 403 in TPH1) plays an important role in enzyme function by regulating V(max) of the catalytic reaction.

Animals↗
PubMed2006

Susceptibility of Escherichia coli to L-selenaproline and other L-proline analogues in laboratory culture media and normal human urine.

AIMS: The aims of this study were to identify analogues of L-proline which inhibit the growth of Escherichia coli in both laboratory culture media and normal human urine and to study their mechanisms of uptake. METHODS AND RESULTS: The susceptibility of E. coli to L-proline analogues was studied by radial streak assays on agar plates and by minimal inhibitory concentration determinations in liquid media. Only L-selenaproline (SCA) inhibited growth in Mueller-Hinton medium and human urine as well as in glucose minimal medium. L-Proline did not prevent the inhibition of growth by SCA and strains defective in L-proline transport were as susceptible to SCA as wild-type strains. However, E. coli was resistant to SCA in the presence of L-cysteine and L-cystine. Spontaneous mutants selected for resistance to SCA or L-selenocystine were resistant to the other compound and had reduced growth in minimal medium containing L-cysteine or L-cystine as the sole sulfur source. CONCLUSIONS: L-selenaproline inhibited the growth of E. coli under conditions that may occur in the urinary tract and appeared to be taken up by the L-cystine transport system. SIGNIFICANCE AND IMPACT OF THE STUDY: Although urinary tract infections caused by E. coli can be treated with sulfamethoxazole/trimethoprim and quinolones, resistance to these antibiotics has been increasing. These results suggest that L-selenaproline may represent a new class of compounds that could be used to treat these infections.

Bacteriological Techniques↗
PubMed1995

Cloning, sequencing and analysis of a gene encoding Escherichia coli proline dehydrogenase.

Using a genomic subtraction technique, we cloned a DNA sequence that is present in wild-type Escherichia coli strain CSH4 but is missing in a presumptive proline dehydrogenase deletion mutant RM2. Experimental evidence indicated that the cloned fragment codes for proline dehydrogenase (EC 1.5.99.8) since RM2 cells transformed with a plasmid containing this sequence was able to survive on minimal medium supplemented with proline as the sole nitrogen and carbon sources. The cloned DNA fragment has an open reading frame of 3942 bp and encodes a protein of 1313 amino acids with a calculated Mr of 143,808. The deduced amino acid sequence of the E. coli proline dehydrogenase has an 84.9% homology to the previously reported Salmonella typhimurium putA gene but it is 111 amino acids longer at the C-terminal than the latter.

Amino Acid Sequence↗
PubMed1995

Post-transcriptional control and kinetic characterization of proline transport in germinating conidiospores of Aspergillus nidulans.

In the filamentous fungus Aspergillus nidulans, L-proline uptake is mediated by the product of the prnB gene which codes for a member of a family of amino acid transporters found both in pro- and eukaryotes. Regulation of prnB gene expression has previously been studied in great detail at the molecular level. However, no studies have addressed possible post-transcriptional controls or the kinetic characterisation of the PrnB transporter. Here we develop a rapid and efficient method for direct uptake measurements of proline in germinating conidiospores of A. nidulans. We make use of this method and Northern blot analyses in parallel to study the regulation of PrnB expression both at the level of prnB message accumulation and at a post-transcriptional level. These studies show that (i) pathway-specific and wide-domain regulatory systems, previously shown to control prnB gene expression in multicellular mycelia, also operate in unicellular conidia committed to germination; and (ii) PrnB activity is regulated in response to the nitrogen source present in the medium and the level of internally accumulated proline or other amino acids. We also characterise kinetically the PrnB transporter and a secondary proline transport system. Our results open new possibilities for studies using unicellular conidiospores of filamentous fungi and constitute a necessary first step for a subsequent structure-function analysis of the PrnB transporter.

Amino Acids↗
PubMed1979

Dentin formation in formocresol pulpotomized primary monkey teeth studied by tetracycline and 3H-proline incorporation.

The influence of formocresol treatment on the pulp tissue of 24 primary monkey teeth was studied using tetracycline and 3H-proline labeling techniques. Six untreated monkey teeth served as controls. The tetracycline labeled teeth were examined between 352 and 600 d, following treatment. The 3H-proline labeled teeth were observed over a period of 22-607 d, the isotope being administered 15 d before extraction. The labeling was evaluated in the coronal, middle and apical area of the roots. Dentin formation as indicated by tetracycline labeling was observed in both control teeth and successfully treated teeth, as well as in some of the unsuccessfully treated teeth. The average dentin formation rate per day varied from 1 micrometer in the control teeth to 0.14 micrometer in the pulpotomized teeth (P less than 0.01). Success of treatment was of significant importance for the amount of dentin formation (P less than 0.001). Labeling with 3H-proline, indicating collagen synthesis, could be observed in the pulp and predentin of the majority of areas judged to be normal, degenerated or inflamed. Labeling was not observed in fixed or necrotic tissue. In degenerated pulp tissue the proline labeling was clearly reduced. The findings indicate that dentin formation and collagen synthesis may take place in formaldehyde influenced pulp tissue although at a decreased rate.

Animals↗
PubMed1998

Exploiting the basis of proline recognition by SH3 and WW domains: design of N-substituted inhibitors.

Src homology 3 (SH3) and WW protein interaction domains bind specific proline-rich sequences. However, instead of recognizing critical prolines on the basis of side chain shape or rigidity, these domains broadly accepted amide N-substituted residues. Proline is apparently specifically selected in vivo, despite low complementarity, because it is the only endogenous N-substituted amino acid. This discriminatory mechanism explains how these domains achieve specific but low-affinity recognition, a property that is necessary for transient signaling interactions. The mechanism can be exploited: screening a series of ligands in which key prolines were replaced by nonnatural N-substituted residues yielded a ligand that selectively bound the Grb2 SH3 domain with 100 times greater affinity.

Adaptor Proteins, Signal Transducing↗
PubMed1976

Specific action of 4-nitropyridine 1-oxide on Excherichia coli K-12 pro+ strains leading to the isolation of proline-requiring mutants: isolation and characterization of pro-mutants.

A specific action of 4-nitropyridine 1-oxide on Escherichia coli K-12 Pro(+) strains leading to highly efficient, selective isolation of Pro(-) mutants is described. Incubation of Pro(+) cells with a sublethal concentration of 4-nitropyridine 1-oxide in Penassay broth gave Pro(-) mutants, which lacked either the biosynthetic pathway of proline from glutamic acid to glutamyl gamma-phosphate (proB(-)) or the pathway from glutamyl gamma-phosphate to glutamic gamma-semialdehyde (proA(-)) or both. Pro(-) mutants, which have the metabolic block between Delta(1) pyrroline-5-carboxylate (the cyclized dehydration product of glutamic gamma-semialdehyde) and proline (proC(-)) were not found among survivors. Treatment of Pro(+) cells with N-methyl-N'-nitro-N-nitrosoguanidine led to isolation of all three types of Pro(-) mutants, suggesting that the action of 4-nitropyridine 1-oxide on Pro(+) cells is apparently distinct from the action of N-methyl-N'-nitro-N-nitrosoguanidine. F-duction and interrupted mating experiments led to determination of the correlation between proline loci and the biosynthetic pathway of proline from glutamic acid.

Culture Media↗