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PubMed1996

Ontogenetic development of proline intestinal transport in the domestic fowl.

1. The proline disappearance from the jejunal and ileal lumen of chickens aged from 1 day to 15 weeks were studied using a perfusion method "in vivo". 2. A decrease in proline transport was observed from younger to older animals. The 2 intestinal segments showed different behaviours, the 1-day and 1-week-old animals showed the same value in the jejunum, whereas in the ileum a progressive decrease in proline transport was observed from the first week of life. 3. The differences observed in the 2 segments could be attributed to the different rates of growth of the jejunum and ileum with age; the jejunum showed a peak of growth in the second week of life whereas the ileum showed a peak of growth in the first week. Proline transport in the jejunum decreased until the fifth week and remained constant thereafter.

Aging↗

PubMed1991

Endogenous nitrosation of L-proline by dietary-derived nitrate.

Two studies were conducted to assess the availability of normal dietary sources of nitrate for endogenous nitrosation of an amino acid substrate, L-proline, and to investigate the potential for dietary ascorbic acid to inhibit such nitrosation. In the first study, 16 subjects consumed a salad meal (containing about 172 mg nitrate) with and without a loading dose of proline. A significant increase (10.8 compared with 2.7 micrograms/24 hrs, p less than 0.0001) in mean urinary N-nitrosoproline (NPRO) excretion following meal plus substrate ingestion was indicative of intragastric proline nitrosation by meal-derived nitrate. In the second study (19 different subjects), the mean urinary NPRO level was significantly decreased (15.8 micrograms compared with 28.4, p = 0.022) by inclusion of ascorbic acid sources in the meal. This demonstrated inhibition of proline nitrosation by dietary sources of the vitamin. Large interindividual variation in nitrosating ability was apparent that was associated with variation in meal nitrate to salivary nitrite conversion. Although meals containing fresh vegetables, as tested here, could provide sufficient nitrate to result in endogenous formation of N-nitroso compounds, there will be considerable inter- and intraindividual variation in the extent of this process.

Administration, Oral↗

PubMed1998

Effects on larvicidal activity of single proline substitutions in alpha3 or alpha4 of the Bacillus thuringiensis Cry4B toxin.

The possible role of alpha-helices 3 and 4 in toxicity of the dipteran-active Bacillus thuringiensis Cry4B delta-endotoxin was investigated by employing proline substitutions via site-directed mutagenesis. Similar to the wild-type Cry4B, the mutant toxins were over-expressed in Escherichia coli as cytoplasmic inclusions and were structurally stable upon solubilization and trypsin activation. The substitution of glutamine 149 by proline in the center of helix 4 (Q149P) resulted in a nearly complete loss of toxicity against Aedes aegypti mosquito-larvae. However, single proline replacements near the center of helix 3 (V119P) and at the N-terminus of helix 4 (Q140P) did not decrease larvicidal activity. The toxicity of E. coli cells expressing the wild-type toxin was significantly reduced by two-hour preincubation with the non-toxic mutant (Q149P), thus indicating that the primary binding step was not affected by the proline substitution in helix 4. The results therefore reveal a crucial role for helix 4 of the Cry4B toxin in toxicity, possibly in membrane insertion and pore formation rather than in receptor recognition.

Aedes↗

PubMed2005

Modification of porcine pancreatic lipase with Z-proline.

Porcine pancreatic lipase was modified with Z-proline via the constitution of amide bonds between the free amino groups of lipase and the carboxyl groups of Z-proline, which were activated by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC). Different amounts of Z-proline were bound to lipase. Modification degree was determined by 2,4,6-trinitrobenzene sulphonic acid (TNBS), by means of the decrease in free amino groups on lipase. The reason for choosing Z-proline was its unique structural characteristics, protected amino groups, and its effect on protein conformation by reducing the flexibility of the lipase molecule, thus achieving stabilization against changes in pH and temperature. After the modification, porcine pancreatic lipase was found to have new physicochemical characteristics, such as optimum alkaline pH stability and thermal stability at elevated temperatures.

Animals↗

PubMed1992

Do the ends justify the mean? Proline mutations at the ends of the keratin coiled-coil rod segment are more disruptive than internal mutations.

Intermediate filament (IF) assembly is remarkable, in that it appears to be self-driven by the primary sequence of IF proteins, a family (40-220 kd) with diverse sequences, but similar secondary structures. Each IF polypeptide has a central 310 amino acid residue alpha-helical rod domain, involved in coiled-coil dinner formation. Two short (approximately 10 amino acid residue) stretches at the ends of this rod are more highly conserved than the rest, although the molecular basis for this is unknown. In addition, the rod is segmented by three short nonhelical linkers of conserved location, but not sequence. To examine the degree to which different conserved helical and nonhelical rod sequences contribute to dimer, tetramer, and higher ordered interactions, we introduced proline mutations in residues throughout the rod of a type I keratin, and we removed existing proline residues from the linker regions. To further probe the role of the rod ends, we introduced more subtle mutations near the COOH-terminus. We examined the consequences of these mutations on (a) IF network formation in vivo, and (b) 10-nm filament assembly in vitro. Surprisingly, all proline mutations located deep in the coiled-coil rod segment showed rather modest effects on filament network formation and 10-nm filament assembly. In addition, removing the existing proline residues was without apparent effect in vivo, and in vitro, these mutants assembled into 10-nm filaments with a tendency to aggregate, but with otherwise normal appearance. The most striking effects on filament network formation and IF assembly were observed with mutations at the very ends of the rod. These data indicate that sequences throughout the rod are not equal with respect to their role in filament network formation and in 10-nm filament assembly. Specifically, while the internal rod segments seem able to tolerate considerable changes in alpha-helical conformation, the conserved ends seem to be essential for creating a very specific structure, in which even small perturbations can lead to loss of IF stability and disruption of normal cellular interactions. These findings have important implications for the disease Epidermolysis Bullosa Simplex, arising from point mutations in keratins K5 or K14.

Amino Acid Sequence↗

PubMed1974

The labeling of cultured cells of Acer with (14C)proline and its significance.

The distribution of the radioactivity from [(14)C]proline that is bound in cultured cells of Acer has been determined by electron microscope autoradiography. In this way proline may be related to the cell wall as a morphological entity rather than as a fraction in a biochemical separation of a heterogeneous crop of cells. The cells in culture may vary greatly. Some are active growing, turgid cells, with thin protoplasts tightly pressed against their walls; in others the protoplasts may spontaneously withdraw from the wall; in still others the protoplasts disorganize, and walls thicken and become sculptured as the cells differentiate and even senesce. Different culturing practices may affect the status of the cells, and this, in turn, affects the distribution of radioactivity from proline in the cells. Cells which are actively growing, turgid, and nucleated have the highest grain density in their protoplasts and nuclei; as the protoplasts of such cells withdraw from their walls, they retain the bulk of the radioactivity. On the other hand, in cells which have thickened walls and sparse protoplast contents, the radioactivity is accumulated in their walls. A high content of proline and hydroxyproline-rich protein is, therefore, not a necessary or invariable feature of the cell walls of cultured Acer cells but depends on the state of development of these cells.

Carbon Radioisotopes↗

PubMed2000

A proline-rich region and nearby cysteine residues target XLalphas to the Golgi complex region.

XLalphas is a splice variant of the heterotrimeric G protein, Galpha(s), found on Golgi membranes in cells with regulated and constitutive secretion. We examined the role of the alternatively spliced amino terminus of XLalphas for Golgi targeting with the use of subcellular fractionation and fluorescence microscopy. XLalphas incorporated [(3)H]palmitate, and mutation of cysteines in a cysteine-rich region inhibited this incorporation and lessened membrane attachment. Deletion of a proline-rich region abolished Golgi localization of XLalphas without changing its membrane attachment. The proline-rich and cysteine-rich regions together were sufficient to target the green fluorescent protein, a cytosolic protein, to Golgi membranes. The membrane attachment and Golgi targeting of the fusion protein required the putative palmitoylation sites, the cysteine residues in the cysteine-rich region. Several peripheral membrane proteins found at the Golgi have proline-rich regions, including a Galpha(i2) splice variant, dynamin II, betaIII spectrin, comitin, and a Golgi SNARE, GS32. Our results suggest that proline-rich regions can be a Golgi-targeting signal for G protein alpha subunits and possibly for other peripheral membrane proteins as well.

Alternative Splicing↗

PubMed1985

The role of oral nitrate in the nitrosation of [14C]proline by conventional microflora and germ-free rats.

The urinary excretion of N-nitroso-L-[U-14C]proline by conventional microflora and germ free rats was used to assess the role of the gut bacteria and oral nitrate in the endogenous formation of N-nitroso compounds. The formation of nitrosoproline was qualitatively similar in conventional and germfree rats (equivalent to nitrosation of approximately 0.01-0.05% of the initial dose of [U-14C]proline) suggesting no involvement of the intestinal flora in this reaction. Furthermore, nitrosamino acid production was similar following the administration of nitrate and [U-14C]proline or [U-14C]proline alone, demonstrating no involvement of exogenous nitrate under the conditions of the experiment. Dietary contamination with nitrate/nitrite was negligible. The results are consistent with the suggestion that nitrate/nitrite reserves in the body are important in the formation of nitrosoproline in vivo.

Administration, Oral↗

PubMed1997

FBP WW domains and the Abl SH3 domain bind to a specific class of proline-rich ligands.

WW domains are conserved protein motifs of 38-40 amino acids found in a broad spectrum of proteins. They mediate protein-protein interactions by binding proline-rich modules in ligands. A 10 amino acid proline-rich portion of the morphogenic protein, formin, is bound in vitro by both the WW domain of the formin-binding protein 11 (FBP11) and the SH3 domain of Abl. To explore whether the FBP11 WW domain and Abl SH3 domain bind to similar ligands, we screened a mouse limb bud expression library for putative ligands of the FBP11 WW domain. In so doing, we identified eight ligands (WBP3 through WBP10), each of which contains a proline-rich region or regions. Peptide sequence comparisons of the ligands revealed a conserved motif of 10 amino acids that acts as a modular sequence binding the FBP11 WW domain, but not the WW domain of the putative signal transducing factor, hYAP65. Interestingly, the consensus ligand for the FBP11 WW domain contains residues that are also required for binding by the Abl SH3 domain. These findings support the notion that the FBP11 WW domain and the Abl SH3 domain can compete for the same proline-rich ligands and suggest that at least two subclasses of WW domains exist, namely those that bind a PPLP motif, and those that bind a PPXY motif.

Adaptor Proteins, Signal Transducing↗

PubMed2000

SH3 domain recognition of a proline-independent tyrosine-based RKxxYxxY motif in immune cell adaptor SKAP55.

Src-homology 3 (SH3) domains recognize PXXP core motif preceded or followed by positively charged residue(s). Whether SH3 domains recognize motifs other than proline-based sequences is unclear. In this study, we report SH3 domain binding to a novel proline-independent motif in immune cell adaptor SKAP55, which is comprised of two N-terminal lysine and arginine residues followed by two tyrosines (i.e. RKxxYxxY). Domains capable of binding to class I proline motifs bound to the motif, while the class II domains failed to bind. Peptide precipitation, alanine scanning and in vivo co-expression studies demonstrated a requirement for the arginine, lysine and tandem tyrosines of the motif. Two-dimensional NMR analysis of the peptide bound FYN-SH3 domain showed overlap with the binding site of a proline-rich peptide on the charged surface of the SH3 domain, while resonance signals for other residues (W119, W120, Y137) were not perturbed by the RKGDYASY based peptide. Expression of the RKGDYASY peptide potently inhibited TcRzeta/CD3-mediated NF-AT transcription in T cells. Our findings extend the repertoire of SH3 domain binding motifs to include a tyrosine-based motif and demonstrate a regulatory role for this motif in receptor signaling.

Amino Acids, Diamino↗

PubMed1987

Evidence for positive regulation of the proline utilization pathway in Saccharomyces cerevisiae.

A mutation has been identified that prevents Saccharomyces cerevisiae cells from growing on proline as the sole source of nitrogen, causes noninducible expression of the PUT1 and PUT2 genes, and is completely recessive. In the put3-75 mutant, the basal level of expression (ammonia as nitrogen source) of PUT1-lacZ and PUT2-lacZ gene fusions as measured by beta-galactosidase activity is reduced 4- and 7-fold, respectively, compared with the wild-type strain. Normal regulation is not restored when the cells are grown on arginine as the sole nitrogen source and put3-75 cells remain sensitive to the proline analog, L-azetidine-2-carboxylic acid, indicating that the block is not at the level of transport of the inducer, proline. In a cross between the put3-75 strain and the semidominant, constitutive mutation PUT3c-68, only parental ditype tetrads were found, indicating allelism of the two mutations. Further support for allelism derives from the comparison of enzyme levels in heteroallelic and heterozygous diploid strains. The constitutive allele appears to be fully dominant to the noninducible allele but only partially dominant to the wild type, suggesting an interaction between the wild-type and PUT3c-68 gene products. The PUT3 gene maps on chromosome XI, about 5.7 cM from the centromere. The phenotypes of alleles of the PUT3 gene, either recessive and noninducible (the put3-75 phenotype) or semidominant and constitutive (the PUT3c-68 phenotype), and their pleiotropy suggest that the PUT3 gene product is a positive activator of the proline utilization pathway.

Alleles↗

PubMed1983

Complete amino acid sequence of a basic proline-rich peptide, P-F, from human parotid saliva.

The complete amino acid sequence of a basic proline-rich peptide, P-F, isolated from human parotid saliva was determined to be Ser-Pro-Pro-Gly-Lys-Pro-Gln-Gly-Pro-Pro-Pro-Gln-Gly-Gly-Asn-Gln-Pro-Gln-Gly-Pro-Pro-Pro-Pro-Pro-Gly-Lys-Pro-Gln-Gly-Pro-Pro-Pro-Gln-Gly-Asn-Lys-Pro-Gln-Gly-Pro-Pro-Pro-Pro-Gly-Lys-Pro-Gln-Gly-Pro-Pro-Pro-Gln-Gly-Ser-Lys-Ser-Arg-Ser-Ala by conventional methods. P-F contains a number of repeating sequences and oligo-proline structures identical with those in other proline-rich peptides such as P-C, P-D, P-E, and Protein C. P-F has the highest degree of homology with P-E among these proline-rich peptides.

Amino Acid Sequence↗

PubMed1992

Incorporation of [3H]proline into collagen and other proteins in rats fed diets with various zinc concentrations.

Incorporation of labeled proline into liver and skin proteins of weanling rats was studied during zinc restriction. Reduced incorporation of labeled proline into skin proteins including collagen hydroxyproline and an increase in its incorporation into liver and plasma proteins was observed with 0 or 0.046 mumol zinc/g diet, compared with 0.092, 0.153 or 0.306 mumol/g diet. Food (energy) restriction was then compared with zinc restriction, during recovery from protein restriction. A shift in proline incorporation from skin protein and skin collagen hydroxyproline, towards liver and plasma proteins, was closely correlated with body weight gain, whether it was limited by zinc restriction or by energy restriction. This diversion of label from skin to liver and plasma is probably mediated through mechanisms that limit growth during food restriction. Proline-incorporation patterns during zinc or energy restriction are clearly different from those of vitamin-C deficiency in guinea pigs.

Animals↗

PubMed1998

Adjacent proline residues in the inhibitory domain of the Oct-2 transcription factor play distinct functional roles.

A 40 amino acid region of Oct-2 from amino acids 142 to 181 functions as an active repressor domain capable of inhibiting both basal activity and activation of promoters containing a TATA box, but not of those that contain an initiator element. Based on our observation that the equivalent region of the closely related Oct-1 factor does not act as an inhibitory domain, we have mutated specific residues in the Oct-2 domain in an attempt to probe their importance in repressor domain function. Although mutations of several residues have no or minimal effect, mutation of proline 175 to arginine abolishes the ability to inhibit both basal and activated transcription. In contrast, mutation of proline 174 to arginine confers upon the domain the ability to repress activation of an initiator-containing promoter by acidic activation domains, and also suppresses the effect of the proline 175 mutation. Hence, adjacent proline residues play key roles in the functioning of the inhibitory domain and in limiting its specificity to TATA-box-containing promoters.

Amino Acid Sequence↗

PubMed2001

Importance of a proline-rich sequence in the amino-terminal region for correct folding of mitochondrial and soluble microbial p450s.

All microsomal P450s have a proline-rich sequence (PR) in the amino-terminal region that is needed for proper folding [Kusano, K., Sakaguchi, M., Kagawa, N., Waterman, M.R. and Omura, T. (2001) J. Biochem., 129, 259-269]. There are also multiple proline residues near the amino-termini of the mature forms of all mitochondrial P450s and the amino-termini of soluble microbial P450s. To examine the functional significance of the PR in mitochondrial P450s, we expressed human P450c27 (CYP27) and bovine P450scc (CYP11A1) in an Escherichia coli heterologous expression system, and found that in each one specific proline residue is important for correct folding. Deletions from the amino-terminus further indicated the importance of the PR for the expression of a spectrally normal P450c27. Essentially the same results were obtained with two soluble microbial P450s, P450cam (CYP101) and P450nor, in each of which a PR is important for proper folding. We conclude that in all P450s (mitochondrial, microbial and microsomal P450s), a proline-rich sequence located in the amino-terminal region is important for proper folding. Furthermore, we predict that the importance of the PR in P450 folding is to reduce the tendency of the polypeptide to misfold prior to heme binding.

Amino Acid Sequence↗

PubMed1998

Zep: A novel zinc finger protein containing a large proline-rich domain.

Zep, a novel 49 kDa zinc finger protein, was found in the brain of day-13 mouse embryos and cloned. Zep contains two C2H2-type zinc finger motifs close to the N-terminal region. The majority of the molecule is composed of a proline-rich domain showing similarity to proline-rich domains in transcription factors and a salivary proline-rich protein. In addition to the proline-rich domain, Zep has an acidic domain and a serine/threonine-rich domain, all of which are frequently found in many transcription factors. The overall organization of Zep shows no similarity to any other proteins. There is a nuclear localization signal in Zep, and the Zep-GFP (green fluorescent protein) fusion protein is located predominantly in the nucleus. In the day-13 mouse embryo, Zep is strongly expressed in the nervous system, i.e. brain, spinal cord, and dorsal root ganglia, with strong to weak expression observed in other regions. Zep continues to be strongly expressed in the neonatal brain; however, its expression is weak in the brain and spinal cord of adult mice. In situ hybridization reveals strong signals for Zep mRNA in the cerebellum and olfactory bulb with moderate signals detected in the hippocampus and cortex. Strong Zep expression is observed in adult thymus, lung, spleen, testis, and ovary. Zep may be involved in the formation and remodeling of various tissues including nervous tissue, probably through transcriptional regulation.

Amino Acid Sequence↗

PubMed1982

Proline-specific dipeptidyl aminopeptidase from Flavobacterium meningosepticum.

A proline-specific dipeptidyl aminopeptidase was highly purified from cell-free extract of Flavobacterium meningosepticum by a series of column chromatographies on DEAE-Sephadex A-50, Sephadex G-150, hydroxyapatite, and a second gel filtration on Sephadex G-150. The enzyme was most active at pH 7.4-7.8 for both Gly-Pro-beta-naphthylamide (Gly-Pro-2-NNap) and Gly-Pro-p-nitroanilide (Gly-Pro-pNA) and was stable between pH 7 and 9.5. The enzyme was markedly inhibited by diisopropylphosphofluoridate (DFP) and mercury ion but not by sulfhydryl-blocking reagents and metal chelators. The molecular weight of the enzyme was about 160,000 as judged by the gel filtration method and the subunit molecular weight was estimated to be 75,000 by sodium dodecyl sulfate (SDS)-gel electrophoresis, suggesting a dimeric form of the native enzyme. The isoelectric point was at pH 9.5. The enzyme hydrolyzed peptides and peptide amides at the carboxyl side of a proline residue penultimate to the amino-terminal amino acid, as did post-proline dipeptidyl aminopeptidases from various mammals. However, antiserum raised against post-proline dipeptidyl aminopeptidase from porcine kidney did not cross-react with the Flavobacterium dipeptidyl aminopeptidase.

Animals↗

PubMed2003

Proline versus charge concept for protein stabilization against proteolytic attack.

The virtue of the so-called 'proline concept' and the 'charge concept' for stabilizing protease-susceptible regions of a protein structure was compared on bovine pancreatic ribonuclease A. Alanine 20 and serine 21, both of which are located in a loop that is susceptible to the unspecific proteases subtilisin Carlsberg, subtilisin BPN', proteinase K and elastase, were replaced with proline or lysine by site-directed mutagenesis. The rate constant of proteolysis was decreased by up to three orders of magnitude for the proline mutants depending on the site of the mutation and the protease used. In contrast, substitution by lysine increased the proteolytic resistance by only one order of magnitude characterizing the 'proline concept' as superior to the 'charge concept'. Although the four applied proteases are considered to be unspecific, the degree of stabilization of the ribonuclease molecule varied considerably, indicating the impact of individual differences in their substrate specificity on the proteolytic resistance and degradation pathway of the target protein.

Amino Acid Substitution↗