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At least 829 records · Page 46Linked to original sources
PubMed1979

Biosynthesis of proline in Pseudomonas aeruginosa. Properties of gamma-glutamyl phosphate reductase and 1-pyrroline-5-carboxylate reductase.

gamma-Glutamyl phosphate reductase, the second enzyme of proline biosynthesis, catalyses the formation of l-glutamic acid 5-semialdehyde from gamma-glutamyl phosphate with NAD(P)H as cofactor. It was purified 150-fold from crude extracts of Pseudomonas aeruginosa PAO 1 by DEAE-cellulose chromatography and hydroxyapatite adsorption chromatography. The partially purified preparation, when assayed in the reverse of the biosynthetic direction, utilized l-1-pyrroline-5-carboxylic acid as substrate and reduced NAD(P)(+). The apparent K(m) values were: NAD(+), 0.36mm; NADP(+), 0.31mm; l-1-pyrroline-5-carboxylic acid, 4mm with NADP(+) and 8mm with NAD(+); P(i), 28mm. 3-(Phosphonoacetylamido)-l-alanine, a structural analogue of gamma-glutamyl phosphate, inhibited this enzyme competitively (K(i)=7mm). 1-Pyrroline-5-carboxylate reductase (EC 1.5.1.2), the third enzyme of proline biosynthesis, was purified 56-fold by (NH(4))(2)SO(4) fractionation, Sephadex G-150 gel filtration and DEAE-cellulose chromatography. It reduced l-1-pyrroline-5-carboxylate with NAD(P)H as a cofactor to l-proline. NADH (K(m)=0.05mm) was a better substrate than NADPH (K(m)=0.02mm). The apparent K(m) values for l-1-pyrroline-5-carboxylate were 0.12mm with NADPH and 0.09mm with NADH. The 3-acetylpyridine analogue of NAD(+) at 2mm caused 95% inhibition of the enzyme, which was also inhibited by thio-NAD(P)(+), heavy-metal ions and thiol-blocking reagents. In cells of strain PAO 1 grown on a proline-medium the activity of gamma-glutamyl kinase and gamma-glutamyl phosphate reductase was about 40% lower than in cells grown on a glutamate medium. No repressive effect of proline on 1-pyrroline-5-carboxylate reductase was observed.

Genes↗
PubMed1981

Chemical and physical characterization of a proline-rich polypeptide from sheep colostrum.

A proline-rich polypeptide isolated from sheep colostrum is described. The molecular weight of the polypeptide determined by gel filtration is 17 200. However, in the presence of guanidinium chloride the molecular weight found is about 6000. The polypeptide contains about 22% of proline, a high proportion of non-polar amino acids, a low percentage of glycine, and no alanine, arginine and cysteine residues. The only N-terminal amino acid found is leucine. C.d. spectra in water and in 50% (v/v) trifluoroethanol suggest the presence of block sequences of proline residues forming helices of polyproline II type. The proline-rich polypeptide is soluble at 4 degrees C but is reversibly precipitated on warming to room temperature. Maximal precipitation is observed at pH 4.6 and at ionic strength above 0.6. The precipitation depends on the concentration of the polypeptide. No effect of other proteins, Ca2+ and Zn2+ ions on the precipitation of the polypeptide was found. The proline-rich polypeptide is not an amphipathic protein. The lack of effect of the polypeptide on proteolytic enzymes ruled out the possibility that it is an inhibitor of proteinases.

Amino Acids↗
PubMed1996

The focal-adhesion vasodilator-stimulated phosphoprotein (VASP) binds to the proline-rich domain in vinculin.

In mammalian cells vasodilator-stimulated phosphoprotein (VASP) is localized to focal adhesions and areas of dynamic membrane activity where it is thought to have a role in actinfilament assembly. The proteins responsible for recruiting VASP to these sites within the cell are not known. The bacterial protein ActA binds VASP via a proline-rich motif that is very similar to a sequence in the proline-rich region of the focal-adhesion protein vinculin. We have examined the ability of VASP, synthesized using an in vitro transcription/translation system, to bind to a series of vinculin peptides expressed as glutathione S-transferase fusion proteins, and have shown that it binds specifically to the proline-rich region in vinculin. Using immobilized peptides corresponding to the two proline-rich motifs within this domain, the VASP-binding site was localized to proline-rich motif-l (residues 839-850). Binding to this motif was not affected by the phosphorylation state of VASP. The C-terminal region of VASP, which is known to be important in targeting VASP to focal adhesions, was shown to be required for binding. These results identify vinculin as a VASP-binding protein likely to be important in recruiting VASP to focal adhesions and the cell membrane.

Actins↗
PubMed2001

Conformational changes play a role in regulating the activity of the proline utilization pathway-specific regulator in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the ability to use proline as a nitrogen source requires the Put3p transcriptional regulator, which turns on the expression of the proline utilization genes, PUT1 and PUT2, in the presence of the inducer proline and in the absence of preferred nitrogen sources. Changes in target gene expression occur through an alteration in activity of the DNA-bound Put3p, a member of the Zn(II)2Cys6 binuclear cluster family of proteins. Here, we report that the 'on' conformation can be mimicked in the absence of proline by the insertion of an epitope tag in several different places in the protein, as well as by specific amino acid changes that suppress a put3 mutation leading to non-inducibility of the pathway. In addition, the presence of proline causes a conformational change in the Put3 protein detected by increased sensitivity to thrombin or V8 protease. These findings suggest that Put3p shifts from an inactive to an activate state via conformational changes.

Base Sequence↗
PubMed1991

Negative and positive regulation of a novel proline-rich protein mRNA by fungal elicitor and wounding.

The structure and expression of a cDNA clone (PvPRP1) isolated from a cDNA library prepared from bean (Phaseolus vulgaris) cells treated with fungal elicitor have been characterized. Sequence analysis of the 1.1 kb insert revealed a complete open reading frame which encodes a 32 kDa protein. The protein resembles other proline-rich proteins in plants but possesses several unique features: (i) the N-terminal half of the protein is proline rich and contains three identical repeats of Pro-Val-His-Pro-Pro-Val-Lys-Pro-Pro-Val and six related repeats; (ii) the proline-rich region contains two tracts of six histidine residues; and (iii) the C-terminal half is low in proline and lacks repeats. Genomic blotting experiments suggest the presence of a single PvPRP1 gene as well as more distantly related genes within the bean genome. A dramatic decrease in PvPRP1 mRNA levels occurs within 2 h of elicitor treatment of cell cultures. The PvPRP1 mRNA is present at a moderate level in hypocotyls. Upon wounding, the mRNA level initially decreases over 5 h and then accumulates over 25 h to levels which are higher than the basal level in unwounded hypocotyls. Based on the similarity to other proline-rich proteins with repeated motifs, including the presence of a putative signal peptide, it is likely that the PvPRP1 protein is targetted to the cell wall. The expression of the PvPRP1 gene appears to be integrated with the remodeling of the plant cell wall during the defense response.

Amino Acid Sequence↗
PubMed2000

Evidence supporting the requirement for two proline residues for expression of c.

BACKGROUND: In humans, c antigen expression is associated with a proline residue at amino acid position 103 in the second extracellular loop of the CE protein. Comparison of nonhuman primate Rh proteins suggested that c reactivity might actually involve two proline residues. It has been shown that the RBCs of New World capuchin monkeys (Cebus apella) react with anti-c. To further define the amino acid residues involved in c expression, Rh cDNA from the capuchin was analyzed. STUDY DESIGN AND METHODS: Rh transcripts were amplified by reverse transcription PCR from RNA isolated from the reticulocytes of a capuchin monkey and were cloned and sequenced. RESULTS: Rh transcripts from the capuchin monkey, whose RBCs react with anti-c, were found to encode adjacent proline residues at 102 and 103. CONCLUSION: Sequencing of Rh transcripts from the capuchin monkey supports the hypothesis that the expression of c requires two adjacent proline residues. Proline causes bends or loops in proteins, which, in this case, might form a unique, stable structure resistant to perturbations induced by changes in upstream or downstream residues. This would explain the scarcity in humans of c variants as compared to the other major Rh antigen variants, and the preservation of c reactivity despite 24-percent divergence between the human and capuchin Rh proteins.

Animals↗
PubMed2004

Reversed voltage-dependent gating of a bacterial sodium channel with proline substitutions in the S6 transmembrane segment.

Members of the voltage-gated-like ion channel superfamily have a conserved pore structure. Transmembrane helices that line the pore (M2 or S6) are thought to gate it at the cytoplasmic end by bending at a hinge glycine residue. Proline residues favor bending of alpha-helices, and substitution of proline for this glycine (G219) dramatically stabilizes the open state of a bacterial Na(+) channel NaChBac. Here we have probed S6 pore-lining residues of NaChBac by proline mutagenesis. Five of 15 proline-substitution mutants yielded depolarization-activated Na(+) channels, but only G219P channels have strongly negatively shifted voltage dependence of activation, demonstrating specificity for bending at G219 for depolarization-activated gating. Remarkably, three proline-substitution mutations on the same face of S6 as G219 yielded channels that activated upon hyperpolarization and inactivated very slowly. Studies of L226P showed that hyperpolarization to -147 mV gives half-maximal activation, 123 mV more negative than WT. Analysis of combination mutations and studies of block by the local anesthetic etidocaine favored the conclusion that hyperpolarization-activated gating results from opening of the cytoplasmic gate formed by S6 helices. Substitution of multiple amino acids for L226 indicated that hyperpolarization-activated gating was correlated with a high propensity for bending, whereas depolarization-activated gating was favored by a low propensity for bending. Our results further define the dominant role of bending of S6 in determining not only the voltage dependence but also the polarity of voltage-dependent gating. Native hyperpolarization-activated gating of hyperpolarization- and cyclic nucleotide-gated (HCN) channels in animals and KAT channels in plants may involve bending at analogous S6 amino acid residues.

Amino Acid Substitution↗
PubMed2000

Structure-activity analysis of buforin II, a histone H2A-derived antimicrobial peptide: the proline hinge is responsible for the cell-penetrating ability of buforin II.

Buforin II is a 21-aa potent antimicrobial peptide that forms, in a hydrophobic medium, an amphipathic structure consisting of an N-terminal random coil region (residues 1-4), an extended helical region (residues 5-10), a hinge (residue 11), and a C-terminal regular alpha-helical region (residues 12-21). To elucidate the structural features of buforin II that are required for its potent antimicrobial activity, we synthesized a series of N- and C-terminally truncated or amino acid-substituted synthetic buforin II analogs and examined their antimicrobial activity and mechanism of action. Deletion of the N-terminal random coil region increased the antibacterial activity approximately 2-fold, but further N-terminal truncation yielded peptide analogs with progressively decreasing activity. Removal of four amino acids from the C-terminal end of buforin II resulted in a complete loss of antimicrobial activity. The substitution of leucine for the proline hinge decreased significantly the antimicrobial activity. Confocal fluorescence microscopic studies showed that buforin II analogs with a proline hinge penetrated the cell membrane without permeabilization and accumulated in the cytoplasm. However, removal of the proline hinge abrogated the ability of the peptide to enter cells, and buforin II analogs without a proline hinge localized on the cell surface, permeabilizing the cell membrane. In addition, the cell-penetrating efficiency of buforin II and its truncated analogs, which depended on the alpha-helical content of the peptides, correlated linearly with their antimicrobial potency. Our results demonstrate clearly that the proline hinge is responsible for the cell-penetrating ability of buforin II, and the cell-penetrating efficiency determines the antimicrobial potency of the peptide.

Amino Acid Sequence↗
PubMed1979

Amplification and characterization of the proline transport carrier of Escherichia coli K-12 by using proT+ hybrid plasmids.

A previous report [Motojima, K., Yamato, I. & Anraku, Y. (1978) J. Bacteriol. 136, 5-9) described the characteristics of mutants (proT-) of Escherichia coli K-12 that are defective in proline transport carrier activity. Two hybrid plasmids from the Clarke and Carbon colony bank were found to complement the mutation by conjugation and transformation. Analysis with restriction endonucleases showed that both plasmid DNAs carried the same fragment of the E. coli chromosome. A polypeptide with a molecular weight of 24,000, specifically coded for by the proT+ plasmid, was identified in the cytoplasmic membrane by using a double-isotope labeling method in a minicell system. The strain harboring the proT+ plasmid has 6 times as much proline transport carrier as the wild strain. This amplification of the carrier makes it possible to measure proline binding to the carrier by microequilibrium dialysis. Detailed analysis of binding indicated that the maximal amount of proline bound to the carrier is 0.70 nmol/mg of protein of the cytoplasmic membrane of the amplified strain. From this value and the assumption that the carrier has one binding site per minimal molecular weight of 24,000, the content of the proline transport carrier in the cytoplasmic membrane was estimated to be 1.7%.

Bacterial Proteins↗
PubMed1981

Proline and glucose transport by renal membranes from dogs with spontaneous idiopathic Fanconi syndrome.

The uptake of proline and glucose by renal brush border membrane vesicles isolated from Basenji dogs exhibiting a spontaneous Fanconi syndrome was examined. The magnitude of the 1-min, Na+-gradient uptake of proline (0.02 mM) and glucose (1.0 mM) by vesicles from affected dog kidney was lower than normal. Concentration-dependent 15-sec uptake in vesicles from normal and affected dogs reveals a two-component transport system for proline and glucose. Kinetic analysis shows altered Km and Vmax values for both proline and glucose transport in the affected dogs. The data suggest that, in this model of Fanconi syndrome, the defective renal reabsorption of proline and glucose is associated with an alteration of luminal transport system.

Animals↗
PubMed1986

Developmental aspects of proline transport in rat renal brush border membranes.

Proline uptake by rat renal brush border membrane vesicles from animals 7 days of age and older has been examined to delineate developmental changes in membrane function that may underlie the physiological hyperprolinuria of young animals. Although the two proline transport systems normally present in adult membranes were found in membranes from young animals, the proline "overshoot" resulting from a sodium ion gradient is minimal and increases with age of the animal from which the membranes were isolated. This is associated with a severalfold faster entry of 22Na into vesicles of the 7-day-old animal compared to entry into membranes prepared from adult kidneys. The very rapid dissipation of the sodium gradient thus diminishing the driving force for transmembrane proline movement may explain the changes in proline overshoot observed in membranes from young animals. The altered sodium permeability is consistent with the fact that young animals have a generalized inability to reabsorb other amino acids whose transport is known to be sodium gradient stimulated.

Age Factors↗
PubMed1992

A bifunctional enzyme (delta 1-pyrroline-5-carboxylate synthetase) catalyzes the first two steps in proline biosynthesis in plants.

Many plants synthesize and accumulate proline in response to osmotic stress. Despite the importance of this pathway, however, the exact metabolic route and enzymes involved in the synthesis of proline in plants have not been unequivocally identified. We report here the isolation of a mothbean (Vigna aconitifolia) cDNA clone encoding a bifunctional enzyme, delta 1-pyrroline-5-carboxylate synthetase (P5CS), with both gamma-glutamyl kinase and glutamic-gamma-semialdehyde dehydrogenase activities that catalyzes the first two steps in proline biosynthesis. The two enzymatic domains of P5CS correspond to the ProB and ProA proteins of Escherichia coli and contain a leucine zipper in each domain, which may facilitate inter- or intramolecular interaction of this protein. The Vigna P5CS enzyme activity is feedback regulated by proline but is less sensitive to end-product inhibition than is the E. coli gamma-glutamyl kinase. The P5CS gene is expressed at high levels in Vigna leaves and is inducible in roots subjected to salt stress, suggesting that P5CS plays a key role in proline biosynthesis, leading to osmoregulation in plants.

1-Pyrroline-5-Carboxylate Dehydrogenase↗
PubMed1994

Assembly of the phagocyte NADPH oxidase: binding of Src homology 3 domains to proline-rich targets.

The NADPH oxidase responsible for generation of superoxide anion and related microbicidal oxidants by phagocytes is assembled from at least five distinct proteins. Two are cytosolic components (p47-phox and p67-phox) that contain Src homology 3 (SH3) domains and associate with a transmembrane cytochrome b558 upon activation. We show here that the SH3 domains of p47-phox bind to proline-rich sequences in p47-phox itself and the p22-phox subunit of cytochrome b558. Binding of the p47-phox SH3 domains to p22-phox was abolished by a mutation in one proline-rich sequence (Pro156-->Gln) noted in a distinct form of chronic granulomatous disease and was inhibited by a short proline-rich synthetic peptide corresponding to residues 149-162 of p22-phox. Expression of mutated p22-phox did not restore oxidase activity to p22-phox-deficient B cells and did not enable p22-phox-dependent translocation of p47-phox to membranes in phorbol ester-stimulated cells. We also show that the cytosolic oxidase components associate with one another through the C-terminal SH3 domain of p67-phox and a proline-rich C-terminal sequence in p47-phox. These SH3 target sites conform to consensus features deduced from SH3 binding sites in other systems. We propose a model in which the oxidase complex assembles through a mechanism involving SH3 domains of both cytosolic proteins and cognate proline-rich targets in other oxidase components.

Amino Acid Sequence↗
PubMed1996

Direct binding of the proline-rich region of protein tyrosine phosphatase 1B to the Src homology 3 domain of p130(Cas).

Protein tyrosine phosphatase 1B (PTP1B) is an abundant intracellular enzyme that is thought to act as a negative regulator of certain signaling pathways. The C terminus of PTP1B contains two proline-rich regions which conform to the canonical class II Src homology 3 domain binding motif, Pro-X-X-Pro-X-Arg. In this study, we establish that PTP1B interacts with Crk, Grb2, and p130(Cas) in vitro and with at least one of these, p130(Cas), in intact cells. The interaction of PTP1B and p130(Cas) is independent of tyrosine phosphorylation but can be disrupted by replacing two critical proline residues in the proline-rich domain of PTP1B between amino acids 301 and 315. When wild-type PTP1B is expressed in 3Y1-v-crk cells, p130(Cas) shows substantial dephosphorylation, whereas the PTP1B proline mutant does not have this effect. In 3Y1 and 3Y1 v-crk-transformed fibroblasts, almost all of the total PTP1B and about 40% of total p130(Cas) co-sediment with membranes composed primarily of endoplasmic reticulum. These results suggest that the proline-rich domain between amino acids 301 and 315 in PTP1B binds Src homology 3-containing proteins and that p130(Cas) may be a physiological target of this phosphatase in cells.

Amino Acid Sequence↗
PubMed1997

A ubiquitin-specific protease that efficiently cleaves the ubiquitin-proline bond.

Ubiquitin is a small eukaryotic protein that is synthesized naturally as one of several fusion proteins, which are processed by ubiquitin-specific proteases to release free ubiquitin. The expression of heterologous proteins as fusions to ubiquitin in either prokaryotic or eukaryotic hosts often dramatically enhances their yield, and allows the exposure of any amino acid following cleavage of ubiquitin. The single exception is when proline is the amino acid immediately following ubiquitin; the ubiquitin-proline bond is poorly cleaved by presently studied ubiquitin-specific proteases. We show that the mouse ubiquitin-specific protease Unp, and its human homolog Unph, can efficiently cleave the ubiquitin-proline bond in ubiquitin fusion proteins of different sizes. N-terminal sequencing of the cleavage products reveals that cleavage occurs precisely at the ubiquitin-proline junction. The biological significance of this cleavage activity is unclear, as ubiquitin-proline fusions do not occur naturally. However, it may indicate a different catalytic mechanism for these ubiquitin-specific proteases and/or that they can cleave ubiquitin-like proteins. Unp and Unph thus represent versatile ubiquitin-specific proteases for cleaving ubiquitin-fusion proteins in biotechnology and basic research, regardless of both the amino acid immediately following ubiquitin, and the size of the fusion partner.

Amino Acid Sequence↗
PubMed2001

The first proline of PALP motif at the C terminus of presenilins is obligatory for stabilization, complex formation, and gamma-secretase activities of presenilins.

Mutations in presenilin (PS) genes cause early-onset familial Alzheimer's disease by increasing production of the amyloidogenic form of amyloid beta peptides ending at residue 42 (Abeta42). PS is an evolutionarily conserved multipass transmembrane protein, and all known PS proteins contain a proline-alanine-leucine-proline (PALP) motif starting at proline (P) 414 (amino acid numbering based on human PS2) at the C terminus. Furthermore, missense mutations that replace the first proline of PALP with leucine (P414L) lead to a loss-of-function of PS in Drosophila melanogaster and Caenorhabditis elegans. To elucidate the roles of the PALP motif in PS structure and function, we analyzed neuro2a as well as PS1/2 null fibroblast cell lines transfected with human PS harboring mutations at the PALP motif. P414L mutation in PS2 (and its equivalent in PS1) abrogated stabilization, high molecular weight complex formation, and entry to Golgi/trans-Golgi network of PS proteins, resulting in failure of Abeta42 overproduction on familial Alzheimer's disease mutant basis as well as of site-3 cleavage of Notch. These data suggest that the first proline of the PALP motif plays a crucial role in the stabilization and formation of the high molecular weight complex of PS, the latter being the active form with intramembrane proteolytic activities.

Alzheimer Disease↗
PubMed2005

Aspartyl-tRNA synthetase requires a conserved proline in the anticodon-binding loop for tRNA(Asn) recognition in vivo.

Most prokaryotes require Asp-tRNA(Asn) for the synthesis of Asn-tRNA(Asn). This misacylated tRNA species is synthesized by a non-discriminating aspartyl-tRNA synthetase (AspRS) that acylates both tRNA(Asp) and tRNA(Asn) with aspartate. In contrast, a discriminating AspRS forms only Asp-tRNA(Asp). Here we show that a conserved proline (position 77) in the L1 loop of the non-discriminating Deinococcus radiodurans AspRS2 is required for tRNA(Asn) recognition in vivo. Escherichia coli trpA34 was transformed with DNA from a library of D. radiodurans aspS2 genes with a randomized codon 77 and then subjected to in vivo selection for Asp-tRNA(Asn) formation by growth in minimal medium. Only proline codons were found at position 77 in the aspS2 genes isolated from 21 of the resulting viable colonies. However, when the aspS temperature-sensitive E. coli strain CS89 was transformed with the same DNA library and then screened for Asp-tRNA(Asp) formation in vivo by growth at the non-permissive temperature, codons for seven other amino acids besides proline were identified at position 77 in the isolates examined. Thus, replacement of proline 77 by cysteine, isoleucine, leucine, lysine, phenylalanine, serine, or valine resulted in mutant D. radiodurans AspRS2 enzymes still capable of forming Asp-tRNA(Asp) but unable to recognize tRNA(Asn). This strongly suggests that proline 77 is responsible for the non-discriminatory tRNA recognition properties of this enzyme.

Acylation↗
PubMed2005

Main chain hydrogen bond interactions in the binding of proline-rich gluten peptides to the celiac disease-associated HLA-DQ2 molecule.

Binding of peptide epitopes to major histocompatibility complex proteins involves multiple hydrogen bond interactions between the peptide main chain and major histocompatibility complex residues. The crystal structure of HLA-DQ2 complexed with the alphaI-gliadin epitope (LQPFPQPELPY) revealed four hydrogen bonds between DQ2 and peptide main chain amides. This is remarkable, given that four of the nine core residues in this peptide are proline residues that cannot engage in amide hydrogen bonding. Preserving main chain hydrogen bond interactions despite the presence of multiple proline residues in gluten peptides is a key element for the HLA-DQ2 association of celiac disease. We have investigated the relative contribution of each main chain hydrogen bond interaction by preparing a series of N-methylated alphaI epitope analogues and measuring their binding affinity and off-rate constants to DQ2. Additionally, we measured the binding of alphaI-gliadin peptide analogues in which norvaline, which contains a backbone amide hydrogen bond donor, was substituted for each proline. Our results demonstrate that hydrogen bonds at P4 and P2 positions are most important for binding, whereas the hydrogen bonds at P9 and P6 make smaller contributions to the overall binding affinity. There is no evidence for a hydrogen bond between DQ2 and the P1 amide nitrogen in peptides without proline at this position. This is a unique feature of DQ2 and is likely a key parameter for preferential binding of proline-rich gluten peptides and development of celiac disease.

Amino Acid Sequence↗