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At least 865 records · Page 48Linked to original sources
PubMed2006

Functional effects of intramembranous proline substitutions in the staphylococcal multidrug transporter QacA.

The QacA multidrug transporter is encoded on Staphylococcus aureus multidrug resistance plasmids and confers broad-range antimicrobial resistance to more than 30 monovalent and bivalent lipophilic, cationic compounds from at least 12 different chemical classes. QacA contains 10 proline residues predicted to be within transmembrane regions, several of which are conserved in related export proteins. Proline residues are classically known as helix-breakers and are highly represented within the transmembrane helices of membrane transport proteins, where they can mediate the formation of structures essential for protein stability and transport function. The importance of these 10 intramembranous proline residues for QacA-mediated transport function was determined by examining the functional effect of substituting these residues with glycine, alanine or serine. Several proline-substituted QacA mutants failed to confer high-level resistance to selected QacA substrates. However, no single proline mutation, including those at conserved positions, significantly disrupted QacA protein expression or QacA-mediated resistance to all representative substrates, suggesting that these residues are not essential for the formation of structures requisite to the QacA substrate transport mechanism.

Amino Acid Sequence↗
PubMed1997

Inverse correlation between CD34 expression and proline-4-hydroxylase immunoreactivity on spindle cells noted in hypertrophic scars and keloids.

The CD34 positive (CD34+) spindle cells constitute a special population of spindle cells which shows a unique distribution in the skin. So far, however, the functional role of CD34+ spindle cells and the regulation of CD34 expression on dermal spindle cells are totally unknown. We examined immunohistologically the pattern of the expression of CD34 and proline-4-hydroxylase, a marker for the fibroblasts that participate in active collagen synthesis, on dermal spindle cells at various stages of scar and keloidal tissues. Dermal spindle cells in the lesions of hypertrophic scar and those at inflammatory expanding borders of keloids totally lost CD34 expression, but they strongly expressed proline-4-hydroxylase. On the other hand, they expressed CD34, together with decreased immunoreactivity to anti-proline-4-hydroxylase antibody, in non-inflammatory scars or in a non-inflammatory central portion of keloid. In two cases of scars, in which inflammation began to subside, double immunofluorescence demonstrated that both CD 34 and proline-4-hydroxylase were expressed on the same spindle cells. CD34 expression, once disappeared from the lesions of hypertrophic scar or keloid, seems to return on CD34-proline-4-hydroxylase+ cells, when the initial inflammatory changes begin to regress. There is a reverse correlation between CD34 expression on spindle cells and the synthesis of type I collagen in the skin.

Adolescent↗
PubMed1975

Autoradiographic study of 3-h-proline incorporation by rat periodontal ligament, gingival connective tissue and dental pulp.

The rates of 3-H-proline incorporation by the rat periodontal ligament, the gingival connective tissue and the dental pulp were studied by autoradiography. The rate of 3-H-proline incorporation by the periodontal ligament was 2.8 times higher than by the gingival connective tissue and 5 times higher than by the dental pulp. These differences were significant (p smaller than 0.01). The pattern of 3-H-proline incorporation by the periodontal ligament was significantly different (p smaller than 0.01) from that by the gingival connective tissue and the dental pulp. The latter two tissues exhibited a similar pattern of 3-H-proline incorporation. The ratio of the rates of 3-H-proline incorporation by the three tissues did not correlate with the ratio of the cellular densities in the same three tissues.

Animals↗
PubMed2005

A second novel dye-linked L-proline dehydrogenase complex is present in the hyperthermophilic archaeon Pyrococcus horikoshii OT-3.

Two distinguishable activity bands for dye-linked l-proline dehydrogenase (PDH1 and PDH2) were detected when crude extract of the hyperthermophilic archaeon Pyrococcus horikoshii OT-3 was run on a polyacrylamide gel. After purification, PDH1 was found to be composed of two different subunits with molecular masses of 56 and 43 kDa, whereas PDH2 was composed of four different subunits with molecular masses of 52, 46, 20 and 8 kDa. The native molecular masses of PDH1 and PDH2 were 440 and 101 kDa, respectively, indicating that PDH1 has an alpha4beta4 structure, while PDH2 has an alphabetagammadelta structure. PDH2 was found to be similar to the dye-linked l-proline dehydrogenase complex from Thermococcus profundus, but PDH1 is a different type of enzyme. After production of the enzyme in Escherichia coli, high-performance liquid chromatography showed the PDH1 complex to contain the flavins FMN and FAD as well as ATP. Gene expression and biochemical analyses of each subunit revealed that the beta subunit bound FAD and exhibited proline dehydrogenase activity, while the alpha subunit bound ATP, but unlike the corresponding subunit in the T. profundus enzyme, it exhibited neither proline dehydrogenase nor NADH dehydrogenase activity. FMN was not bound to either subunit, suggesting it is situated at the interface between the alpha and beta subunits. A comparison of the amino-acid sequences showed that the ADP-binding motif in the alpha subunit of PDH1 clearly differs from that in the alpha subunit of PDH2. It thus appears that a second novel dye-linked l-proline dehydrogenase complex is produced in P. horikoshii.

Amino Acid Sequence↗
PubMed2006

Na+ and pH dependence of proline and beta-alanine absorption in rat small intestine.

AIMS: Early characterization of intestinal absorption of imino acids in mammals has demonstrated the existence of a Na+-dependent, Cl- -independent transport system in rat small intestine, which is the only carrier for beta-alanine. Based on the substrate selectivity, it was proposed that the Proton Amino Acid Transporter 1 (PAT1) could be the same as this imino acid carrier. The present study characterizes the pH and Na+ dependence of proline and beta-alanine uptake in rat small intestine. METHODS: Intestinal uptake of radiolabelled l-proline or beta-alanine was measured in brush border membrane vesicles and everted intestinal rings, in the presence and absence of Na+ and at different pH values. RESULTS: The existence of an inwardly directed H+ gradient in the absence of Na+ enhanced the initial entry of proline and beta-alanine in brush border membrane vesicles, that reached a transient overshoot with maximal value around 30 s. In the absence of pH gradient, no overshoot was shown. In entire tissue, there was an increase of proline and beta-alanine uptake at acidic pH that was higher in the presence of Na+ than in its absence. This ion dependence and pH effect of the amino acids uptake also increased with the incubation period. Substrate inhibition studies confirmed that intestinal proline absorption in rat occurs mainly by system B and PAT1-like transporter. CONCLUSIONS: There is a Na+ -independent, H+ -dependent transporter of amino acids at the apical membrane of the rat enterocytes.

Amino Acid Transport Systems↗
PubMed2002

The critical role of transmembrane prolines in human prostacyclin receptor activation.

The human prostacyclin receptor (hIP), a G protein-coupled receptor (GPCR), plays important roles in vascular smooth muscle relaxation as well as the prevention of platelet aggregation. It has been postulated that GPCR transmembrane (TM) prolines serve as molecular hinges or swivels and are necessary for proper binding and activation. By individually (as well as collectively) mutating these hIP prolines to alanine, the ability to form key structural and functional configurations was removed. Significant effects on both binding and activation were observed. Two highly conserved prolines across GPCRs, Pro-154, and Pro-254 (TMVI), showed the greatest effect on decreasing both binding and activation when changed to alanine. Along the extracellular boundary of the highly conserved transmembrane III domain, a proline-to-alanine mutation at position 89 (P89A) revealed normal binding affinity in comparison with the 1D4-epitope-tagged hIP (hIP1D4) wild-type control (K(i), iloprost = 3 +/- 2 versus 7 +/- 3 nM, respectively). In contrast, activation was markedly affected, with an EC(50) of 12.0 +/- 2.5 nM compared with that of 1.2 +/- 0.3 nM (10-fold difference) for the hIP1D4. Movement within TMIII has been shown to be necessary for effective GPCR activation. Both the extracellular location (above the putative binding pocket) along with an exclusive effect upon activation suggest that this movement is facilitated by the presence of Pro-89 and independent from the actions of ligand binding. This finding strongly supports a model in which proline residues serve as molecular hinges or swivels, essential for coupling receptor binding to activation.

Amino Acid Sequence↗
PubMed1997

Sequence-specific and phosphorylation-dependent proline isomerization: a potential mitotic regulatory mechanism.

Pin1 is an essential and conserved mitotic peptidyl-prolyl isomerase (PPIase) that is distinct from members of two other families of conventional PPIases, cyclophilins and FKBPs (FK-506 binding proteins). In response to their phosphorylation during mitosis, Pin1 binds and regulates members of a highly conserved set of proteins that overlaps with antigens recognized by the mitosis-specific monoclonal antibody MPM-2. Pin1 is here shown to be a phosphorylation-dependent PPIase that specifically recognizes the phosphoserine-proline or phosphothreonine-proline bonds present in mitotic phosphoproteins. Both Pin1 and MPM-2 selected similar phosphorylated serine-proline-containing peptides, providing the basis for the specific interaction between Pin1 and MPM-2 antigens. Pin1 preferentially isomerized proline residues preceded by phosphorylated serine or threonine with up to 1300-fold selectivity compared with unphosphorylated peptides. Pin1 may thus regulate mitotic progression by catalyzing sequence-specific and phosphorylation-dependent proline isomerization.

Amino Acid Isomerases↗
PubMed1987

Nuclear magnetic resonance spectroscopy reveals the metabolic origins of proline excreted by an Escherichia coli derivative during growth on [13C]acetate.

We have used 13C nuclear magnetic resonance to monitor acetate metabolism in a proline-overproducing strain of Escherichia coli growing on 13C-labeled acetate. The conversion of 13C-labeled acetate to proline by actively dividing cells was followed in vivo, and the site specificity of the incorporation of the acetate carbons in the proline was determined from spectra of butanol extracts of the growth media. The degree of incorporation of deuterium from partially deuterated water into various sites on the proline was monitored from the beta-deuterium-shifted signals in the 13C spectra. The spectra provide information on the origin of the carbons and the protons during proline biosynthesis.

Acetates↗
PubMed2000

Control of expression of divergent Pseudomonas putida put promoters for proline catabolism.

Pseudomonas putida KT2440 uses proline as the sole C and N source. Utilization of this amino acid involves its uptake, which is mediated by the PutP protein, and its conversion into glutamate, mediated by the PutA protein. Sequence analysis revealed that the putA and putP genes are transcribed divergently. Expression from the putP and putA genes was analyzed at the mRNA level in different host backgrounds in the absence and presence of proline. Expression from the put promoters was induced by proline. The transcription initiation points of the putP and putA genes were precisely mapped via primer extension, and sequence analysis of the upstream DNA region showed well-separated promoters for these two genes. The PutA protein acts as a repressor of put gene expression in P. putida because expression from the put promoters is constitutive in a host background with a knockout putA gene. This regulatory activity is independent of the catabolic activity of PutA, because we show that a point mutation (Glu896-->Lys) that prevents catalytic activity allowed the protein to retain its regulatory activity. Expression from the put promoters in the presence of proline in a putA-proficient background requires a positive regulatory protein, still unidentified, whose expression seems to be sigma(54) dependent because the put genes were not expressed in a sigma(54)-deficient background. Expression of the putA and putP genes was equally high in the presence of proline in sigma(38)- and ihf-deficient P. putida backgrounds.

Amino Acid Transport Systems, Neutral↗
PubMed2006

Transcriptional activation of the Staphylococcus aureus putP gene by low-proline-high osmotic conditions and during infection of murine and human tissues.

Staphylococcus aureus can grow virtually anywhere in the human body but needs to import proline through low- and high-affinity proline transporters to survive. This study examined the regulation of the S. aureus putP gene, which encodes a high-affinity proline permease. putP::lacZ and putP::lux transcriptional fusions were constructed and integrated into the genomes of several S. aureus strains. Enzyme activity was measured after growth in media with various osmolyte concentrations. As osmolarity rose, putP expression increased, with a plateau at 2 M for NaCl in strain LL3-1. Proline concentrations as low as 17.4 muM activated expression of the putP gene. The putP::lux fusion was also integrated into the genomes of S. aureus strains that were either SigB inactive (LL3-1, 8325-4, and SH1003) or SigB active (Newman and SH1000). SigB inactive strains showed increased putP gene expression as NaCl concentrations rose, whereas SigB active strains displayed a dramatic decrease in putP expression, suggesting that the alternative sigma factor B plays a negative role in putP regulation. Mice inoculated with S. aureus strains containing the putP::lux fusion exhibited up to a 715-fold increase in putP expression, although levels in the various murine organs differed. Moreover, urine from human patients infected with S. aureus showed elevated putP levels by use of a PCR procedure, whereas blood and some abscess material had no significant increase. Thus, putP is transcriptionally activated by a low-proline and high osmotic environment both in growth media and in murine or human clinical specimens.

Amino Acid Transport Systems, Neutral↗
PubMed2006

Analysis of proline reduction in the nosocomial pathogen Clostridium difficile.

Clostridium difficile, a proteolytic strict anaerobe, has emerged as a clinically significant nosocomial pathogen in recent years. Pathogenesis is due to the production of lethal toxins, A and B, members of the large clostridial cytotoxin family. Although it has been established that alterations in the amino acid content of the growth medium affect toxin production, the molecular mechanism for this observed effect is not yet known. Since there is a paucity of information on the amino acid fermentation pathways used by this pathogen, we investigated whether Stickland reactions might be at the heart of its bioenergetic pathways. Growth of C. difficile on Stickland pairs yielded large increases in cell density in a limiting basal medium, demonstrating that these reactions are tied to ATP production. Selenium supplementation was required for this increase in cell yield. Analysis of genome sequence data reveals genes encoding the protein components of two key selenoenzyme reductases, glycine reductase and d-proline reductase (PR). These selenoenzymes were expressed upon the addition of the corresponding Stickland acceptor (glycine, proline, or hydroxyproline). Purification of the selenoenzyme d-proline reductase revealed a mixed complex of PrdA and PrdB (SeCys-containing) proteins. PR utilized only d-proline but not l-hydroxyproline, even in the presence of an expressed and purified proline racemase. PR was found to be independent of divalent cations, and zinc was a potent inhibitor of PR. These results show that Stickland reactions are key to the growth of C. difficile and that the mechanism of PR may differ significantly from that of previously studied PR from nonpathogenic species.

Amino Acid Isomerases↗
PubMed1977

Biosynthesis of prodigiosin by non-proliferating wild-type Serratia marcescens and mutants deficient in catabolism of alanine, histidine, and proline.

Mutants of Serratia marcescens Nima, designated as Aut, Hut, or Put, did not utilize L-alanine, L-histidine, or L-proline, respectively, as a sole carbon source but did utilize other amino acids or glycerol as carbon sources. The bacteria were permeable to alanine, histidine, and proline but lacked the enzymes responsible for degradation of these amino acids. The Aut mutant contained no L-alanine dehydrogenase activity, whereas the Hut and Put mutants contained only 7 and 4% of the histidase and proline oxidase activities, respectively, found in the wild-type strain. Rates of oxygen uptake and protein synthesis were significantly lower when the mutants were incubated in the presence of amino acids they could not degrade. Studies of L-[14C]alanine, L-[14C]histidine, and L-[14C]proline incorporation into prodigiosin synthesized by these mutants and the wild-type strain revealed that proline was incorporated intact, whereas all of alanine except the carboxyl group was incorporated into the pigment molecule. Histidine did not enter prodigiosin directly. These data suggested that the presence of unique biosynthetic pathways, independent of primary metabolism, leads to formation of prodigiosin from specific amino acids.

Alanine↗
PubMed1978

Cluster of genes controlling proline degradation in Salmonella typhimurium.

A cluster of genes essential for degradation of proline to glutamate (put) is located between the pyrC and pyrD loci at min 22 of the Salmonella chromosome. A series of 25 deletion mutants of this region have been isolated and used to construct a fine-structure map of the put genes. The map includes mutations affecting the proline degradative activities, proline oxidase and pyrroline-5-carboxylic dehydrogenase. Also included are mutations affecting the major proline permease and a regulatory mutation that affects both enzyme and permease production. The two enzymatic activities appear to be encoded by a single gene (putA). The regulatory mutation maps between the putA gene and the proline permease gene (putP).

Amino Acid Oxidoreductases↗
PubMed1979

Isolation and preliminary characterization of Saccharomyces cerevisiae proline auxotrophs.

Proline-requiring mutants of Saccharomyces cerevisiae were isolated. Each mutation is recessive and is inherited as expected for a single nuclear gene. Three complementation groups cold be defined which are believed to correspond to mutations in the three genes (pro1, pro2, and pro3) coding for the three enzymes of the pathway. Mutants defective in the pro1 and pro2 genes can be satisfied by arginine or ornithine as well as proline. This suggests that the blocks are in steps leading to glutamate semialdehyde, either in glutamyl kinase or glutamyl phosphate reductase. A pro3 mutant has been shown by enzyme assay to be deficient in delta 1-pyrroline-5-carboxylate reductase which converts pyrroline-5-carboxylate to proline. A unique feature of yeast proline auxotrophs is their failure to grown on the rich medium, yeast extract-peptone-glucose. This failure is not understood at present, although it accounts for the absence of proline auxotrophs in previous screening for amino acid auxotrophy.

Cell-Free System↗
PubMed1980

Identification and mapping of a second proline permease Salmonella typhimurium.

In this paper we demonstrate the existence of a second proline permease, gene proP, in Salmonella typhimurium. Uptake assays demonstrate that this second proline permease has 5 to 10% the uptake rate of the putP permease, the cell's major proline permease, when assayed at 20 microM proline. Genetic mapping by Hfr and P22-mediated genetic crosses placed the second proline permease gene at 92 min on the S. typhimurium genetic map, near the genes for melibiose utilization. F'-mediated complementation tests indicated that Escherichia coli also has the proP gene.

Chromosome Mapping↗
PubMed1994

Energetics of alanine, lysine, and proline transport in cytoplasmic membranes of the polyphosphate-accumulating Acinetobacter johnsonii strain 210A.

Amino acid transport in right-side-out membrane vesicles of Acinetobacter johnsonii 210A was studied. L-Alanine, L-lysine, and L-proline were actively transported when a proton motive force of -76 mV was generated by the oxidation of glucose via the membrane-bound glucose dehydrogenase. Kinetic analysis of amino acid uptake at concentrations of up to 80 microM revealed the presence of a single transport system for each of these amino acids with a Kt of less than 4 microM. The mode of energy coupling to solute uptake was analyzed by imposition of artificial ion diffusion gradients. The uptake of alanine and lysine was driven by a membrane potential and a transmembrane pH gradient. In contrast, the uptake of proline was driven by a membrane potential and a transmembrane chemical gradient of sodium ions. The mechanistic stoichiometry for the solute and the coupling ion was close to unity for all three amino acids. The Na+ dependence of the proline carrier was studied in greater detail. Membrane potential-driven uptake of proline was stimulated by Na+, with a half-maximal Na+ concentration of 26 microM. At Na+ concentrations above 250 microM, proline uptake was strongly inhibited. Generation of a sodium motive force and maintenance of a low internal Na+ concentration are most likely mediated by a sodium/proton antiporter, the presence of which was suggested by the Na(+)-dependent alkalinization of the intravesicular pH in inside-out membrane vesicles. The results show that both H+ and Na+ can function as coupling ions in amino acid transport in Acinetobacter spp.

Acinetobacter↗
PubMed2002

A proline-rich region with a highly periodic sequence in Streptococcal beta protein adopts the polyproline II structure and is exposed on the bacterial surface.

Proline-rich regions have been identified in many surface proteins of pathogenic streptococci and staphylococci. These regions have been suggested to be located in cell wall-spanning domains and/or to be required for surface expression of the protein. Because little is known about these regions, which are found in extensively studied and biologically important surface proteins, we characterized the proline-rich region in one such protein, the beta protein of group B streptococci. The proline-rich region in beta, designated the XPZ region, has a proline at every third position, and the sequence is highly periodic in other respects. Immunochemical analysis showed that the XPZ region was not associated with the cell wall but was exposed on the bacterial surface. Moreover, characterization of a beta mutant lacking the XPZ region demonstrated that this region was not required for surface expression of the beta protein. Comparison of the XPZ region in different beta proteins showed that it varied in size but always retained the typical sequence periodicity. Circular dichroism spectroscopy indicated that the XPZ region had the structure of a polyproline II helix, an extended and solvent-exposed structure with exactly three residues per turn. Because of the three-residue sequence periodicity in the XPZ region, it is expected to be amphipathic and to have distinct nonpolar and polar surfaces. This study identified a proline-rich structure with unique properties that is exposed on the surface of an important human pathogen.

Amino Acid Sequence↗
PubMed1968

Regulation of arginine and proline catabolism in Bacillus licheniformis.

The enzymes in the arginine breakdown pathway (arginase, ornithine-delta-transaminase, and Delta'-pyrroline-5-carboxylate dehydrogenase) were found to be present in Bacillus licheniformis cells during exponential growth on glutamate. These enzymes could be coincidentally induced by arginine or ornithine to a very high level and their synthesis could be repressed by the addition of glucose, clearly demonstrating catabolite repression control of the arginine degradative pathway. The strongest catabolite repression control of arginase occurred when cells were grown on glucose and this control decreased when cells were grown on glycerol, acetate, pyruvate, or glutamate. The proline catabolite pathway was present in B. licheniformis during exponential growth on glutamate. The proline oxidation and the Delta'-pyrroline-5-carboxylate dehydrogenase in this breakdown pathway were induced by l-proline to a high level. The Delta'-pyrroline-5-carboxylate dehydrogenase was found to be under catabolite repression control. Arginase could be induced by proline and arginine addition induced proline oxidation, suggesting a common in vivo inducer for these convergent pathways.

Acetates↗