Comparative analysis of human milk and infant formula derived peptides following in vitro digestion.

It has long been recognised that there are differences between human milk and infant formulas which lead to differences in health and nutrition for the neonate. In this study we examine and compare the peptide profile of human milk and an exemplar infant formula. The study identifies both similarities and differences in the endogenous and postdigestion peptide profiles of human milk and infant formula. This includes differences in the protein source of these peptides but also with the region within the protein producing the dominant proteins. Clustering of similar peptides around regions of high sequence identity and known bioactivity was also observed. Together the data may explain some of the functional differences between human milk and infant formula, while identifying some aspects of conserved function between bovine and human milks which contribute to the effectiveness of modern infant formula as a substitute for human milk.

Redox proteomic evaluation of oxidative modification and recovery in a 3D reconstituted human skin tissue model exposed to UVB.

OBJECTIVE: Exposure to UV in humans resulting in sunburn triggers a complex series of events that are a mix of immediate and delayed damage mediation and healing. While studies on the effects of UV exposure on DNA damage and repair have been reported, changes in the oxidative modification of skin proteins are poorly understood at the molecular level, despite the important role played by structural proteins in skin tissue, and the effect of the integrity of these proteins on skin appearance and health. Proteomic molecular mapping of oxidation was here applied to try to enhance understanding of skin damage and recovery from oxidative damage and UVB exposure. METHODS: A redox proteomic-based approach was applied to evaluating skin protein modification when exposed to varying doses of UVB after initial oxidative stress, via tracking changes in protein oxidation during the healing process in vitro using a full-thickness reconstituted human skin tissue model. Bioassays and structural evaluation confirmed that our cultured skin tissues underwent a normal physiological response to UVB exposure. RESULTS: A set of potential skin marker peptides was generated, for use in tracking skin protein oxidative modification. Exposure to UVB after thermal oxidative stress was found to result in higher levels of skin protein oxidation than a non-irradiated control for up to seven days after exposure. Recovery of the skin proteins from oxidative stress, as assessed by the overall protein oxidation levels, was found to be impaired by UVB exposure. Oxidative modification was largely observed in skin structural proteins. CONCLUSION: Exposure of skin proteins to UVB exacerbates oxidative damage to structural skin proteins, with higher exposure levels leading to increasingly impaired recovery from this damage. This has potential implications for the functional performance of the proteins and inter-related skin health and cosmetic appearance.

Resistivity coefficients for body composition analysis using bioimpedance spectroscopy: effects of body dominance and mixture theory algorithm.

Body composition is commonly predicted from bioelectrical impedance spectroscopy using mixture theory algorithms. Mixture theory algorithms require the input of values for the resistivities of intra-and extracellular water of body tissues. Various derivations of these algorithms have been published, individually requiring resistivity values specific for each algorithm. This study determined apparent resistivity values in 85 healthy males and 66 healthy females for each of the four published mixture theory algorithms. The resistivity coefficients determined here are compared to published values and the inter-individual (biological) variation discussed with particular reference to consequential error in prediction of body fluid volumes. In addition, the relationships between the four algorithmic approaches are derived and methods for the inter-conversion of coefficients between algorithms presented.

Redox proteomic evaluation of bleaching and alkali damage in human hair.

OBJECTIVE: Protein modification and damage in human hair, resulting from environmental, cosmetic and grooming stresses, create changes to visual and tactile characteristics and correlates with consumer perception of quality. This study outlines molecular-level evaluation of modification resulting from peroxide (bleaching) and alkaline straightening (relaxing) treatments. METHODS: Redox proteomic profiling of virgin, bleached and relaxed hair tresses was performed, with comprehensive qualitative characterization of modification and semi-quantitative evaluation of damage through adaptation of a new damage scoring system. Modifications were mapped to specific locations in the hair proteome and a range of potential damage marker peptides identified. RESULTS: Virgin hair contained a baseline level of modification, consistent with environmental oxidative insult during hair growth. Hydrogen peroxide bleaching resulted in significantly increased levels of oxidative damage observable at the molecular level. This treatment also resulted in enhanced levels of dehydroalanine and dehydration products; modifications typically associated with alkali or thermal treatment and not previously been reported as a product of hair bleaching. Relaxation treatment with sodium hydroxide increased the formation of dehydroalanine and dehydration products and moderately enhanced the levels of oxidation. Cysteine was the predominant modification site for both bleaching and alkali damage. CONCLUSION: This study validates the utility and power of redox proteomic-based approaches to characterizing hair modification. This offers potential application to a wide range of damage types, as well as evaluation of new damage mitigation and repair technologies.

Characterisation of photo-oxidation products within photoyellowed wool proteins: tryptophan and tyrosine derived chromophores.

Understanding the photodegradation of complex protein systems represents a significant goal in protein science. The photo-oxidation and resultant photoyellowing of wool in sunlight is a severe impediment to its marketability. However, although some photomodifications have been found in irradiated model amino acid systems, direct identification of the chromophoric photoproducts responsible for photoyellowing in irradiated wool itself has proved elusive. We here describe the direct characterisation and location of yellow chromophores and related photomodifications within the proteins of photoyellowed wool fabric, utilising a quasi-proteomic approach. In total, eight distinct photoproducts were characterised. Of these, five were derived from tryptophan; namely hydroxytryptophan, N-formylkynurenine, kynurenine, residues consistent with the dehydration of kynurenine, and hydroxykynurenine, while three were derived from tyrosine; namely dihydroxyphenylalanine, dityrosine, and a cross-linked residue consistent with a hydroxylated dityrosine residue. Fourteen modified peptide sequences were identified and the positions of modification for thirteen of these were located within the primary structure of known wool proteins. The nature of the photoproducts characterised offer valuable insight into the reaction pathways followed in the UV-induced photoyellowing of wool proteins.

Metabolic engineering of Saccharomyces cerevisiae for production of novel lipid compounds.

The yeast Saccharomyces cerevisiae has been modified successfully for production of numerous metabolites and therapeutic proteins through metabolic engineering, but has not been utilized to date for the production of lipid-derived compounds. We developed a lipid metabolic engineering strategy in S. cerevisiae based upon culturing techniques that are typically employed for studies of peroxisomal biogenesis; cells were grown in media containing fatty acids as a sole carbon source, which promotes peroxisomal proliferation and induction of enzymes associated with fatty acid beta-oxidation. Our results indicate that growth of yeast on fatty acids such as oleate results in extensive uptake of these fatty acids from the media and a subsequent increase in total cellular lipid content from 2% to 15% dry cell weight. We also show that co-expression of plant fatty acid desaturases 2 and 3 ( FAD2 and FAD3), using a fatty acid-inducible peroxisomal gene promoter, coupled the processes of fatty acid uptake with the induction of a new metabolic pathway leading from oleic acid (18:1) to linolenic acid (18:3). Finally, we show that cultivation of yeast cells in the presence of triacylglycerols and exogenously supplied lipase promotes extensive incorporation of triglyceride fatty acids into yeast cells. Collectively, these results provide a framework for bioconversion of low-cost oils into value-added lipid products.

Disruption of a gene encoding a putative gamma-butyrolactone-binding protein in Streptomyces tendae affects nikkomycin production.

A 2.6-kb BamHI fragment from the genome of the wild-type, nikkomycin-producing strain of Streptomyces tendae ATCC 31160 was cloned and sequenced. This 2.6-kb BamHI fragment corresponds to the DNA site where transposon Tn4560 had inserted to create a nikkomycin-nonproducing mutant. A possible ORF of 660 nucleotides was found in this 2.6-kb BamHI fragment, in which the third base of each codon was either G or C in 92% of the codons. The deduced amino acid sequence coded by this ORF (TarA, tendae autoregulator receptor) shows strong homology with several Gamma-butyrolactone-binding proteins that negatively regulate antibiotic production in other streptomycetes and have a helix-turn-helix DNA-binding motif. A portion (179 nucleotides) of tarA that encodes the helix-turn-helix motif was replaced with ermE, and wild-type S. tendae was transformed with this construct borne in pDH5, a gene-disruption vector. Southern hybridization indicated that ermE had inserted in the 2.6-kb BamHI region in one isolate that is erythromycin resistant. Northern hybridization indicated that tarA disruption significantly increased the amount of disrupted-tarA mRNA. This suggests that TarA negatively regulates its own synthesis. Nikkomycin production by the tarA disruptant was delayed but reached the wild-type level after longer incubation in production medium.

Chilling-sensitive, post-transcriptional regulation of a plant fatty acid desaturase expressed in yeast.

Plants respond to chilling exposure by increasing the relative proportion of polyunsaturated fatty acids in their lipids. However, unlike the response in many other organisms, plant fatty acid desaturase genes are typically not upregulated during this process. We expressed the Brassica napus FAD3 gene, which encodes an enzyme for synthesis of linolenic acid, in Saccharomyces cerevisiae and observed a temperature-dependent increase in linolenic acid production at cooler growth temperatures. Untransformed yeast cells, however, responded to cooler temperatures primarily by shortening fatty acid chains, even when polyunsaturated fatty acids were supplied in the growth media. Measurement of the steady-state levels of Fad3 protein in transformed yeast revealed an 8.5-fold increase in steady-state amount of desaturase enzyme when cells were cultivated at cooler temperatures. The increase was not due to changes in transcriptional activity, since Northern hybridization revealed no appreciable changes in abundance of FAD3 transcripts at cooler temperatures. Taken together, the results suggest that the increase in linolenic acid content in cells containing Fad3 was not due to enhanced physiological demand for polyunsaturated fatty acids by yeast, but rather a cold-inducible, post-transcriptional increase in steady-state amount of plant desaturase enzyme. Implications for plant adaptation to chilling are discussed.

Immunocytological localization of two plant fatty acid desaturases in the endoplasmic reticulum.

The subcellular location of two integral membrane-bound fatty acid desaturases (Fads), Fad2 and Fad3, was elucidated by immunofluorescence microscopic analyses of tobacco suspension cells transiently transformed with different epitope-tagged versions of the enzymes. Both myc- or hemagglutinin-tagged Fad2 and Fad3 localized to the same region of the endoplasmic reticulum (ER), as evidenced by their co-localization with the ER lumenal protein calreticulin. Results from differential permeabilization experiments revealed that the N-termini of both epitope-tagged Fad2 and Fad3 were exposed on the cytosolic side of ER membranes. These data define the subcellular location and topological orientation of plant desaturases in ER membranes.

Characterization of the critical amino acids of an Aspergillus parasiticus cytochrome P-450 monooxygenase encoded by ordA that is involved in the biosynthesis of aflatoxins B1, G1, B2, and G2.

The conversion of O-methylsterigmatocystin (OMST) and dihydro-O-methylsterigmatocystin to aflatoxins B1, G1, B2, and G2 requires a cytochrome P-450 type of oxidoreductase activity. ordA, a gene adjacent to the omtA gene, was identified in the aflatoxin-biosynthetic pathway gene cluster by chromosomal walking in Aspergillus parasiticus. The ordA gene was a homolog of the Aspergillus flavus ord1 gene, which is involved in the conversion of OMST to aflatoxin B1. Complementation of A. parasiticus SRRC 2043, an OMST-accumulating strain, with the ordA gene restored the ability to produce aflatoxins B1, G1, B2, and G2. The ordA gene placed under the control of the GAL1 promoter converted exogenously supplied OMST to aflatoxin B1 in Saccharomyces cerevisiae. In contrast, the ordA gene homolog in A. parasiticus SRRC 2043, ordA1, was not able to carry out the same conversion in the yeast system. Sequence analysis revealed that the ordA1 gene had three point mutations which resulted in three amino acid changes (His-400-->Leu-400, Ala-143-->Ser-143, and Ile-528-->Tyr-528). Site-directed mutagenesis studies showed that the change of His-400 to Leu-400 resulted in a loss of the monooxygenase activity and that Ala-143 played a significant role in the catalytic conversion. In contrast, Ile-528 was not associated with the enzymatic activity. The involvement of the ordA gene in the synthesis of aflatoxins G1, and G2 in A. parasiticus suggests that enzymes required for the formation of aflatoxins G1 and G2 are not present in A. flavus. The results showed that in addition to the conserved heme-binding and redox reaction domains encoded by ordA, other seemingly domain-unrelated amino acid residues are critical for cytochrome P-450 catalytic activity. The ordA gene has been assigned to a new cytochrome P-450 gene family named CYP64 by The Cytochrome P450 Nomenclature Committee.

Redox-sensitive homodimerization of Pex11p: a proposed mechanism to regulate peroxisomal division.

Pex11p (formerly Pmp27) has been implicated in peroxisomal proliferation (Erdmann, R., and G. Blobel. 1995. J. Cell Biol. 128; 509-523; Marshall, P.A., Y.I. Krimkevich, R.H. Lark, J.M. Dyer, M. Veenhuis, and J.M. Goodman, 1995. J. Cell Biol. 129; 345-355). In its absence, peroxisomes in Saccharomyces cerevisiae fail to proliferate in response to oleic acid; instead, one or two large peroxisomes are formed. Conversely, overproduction of Pex11p causes an increase in peroxisomal number. In this report, we confirm the function of Pex11p in organelle proliferation by demonstrating that this protein can cause fragmentation in vivo of large peroxisomes into smaller organelles. Pex11p is on the inner surface of the peroxisomal membrane. It can form homodimers, and this species is more abundant in mature peroxisomes than in proliferating organelles. Removing one of the three cysteines in the protein inhibits homodimerization. This cysteine 3-->alanine mutation leads to an increase in number and a decrease in peroxisomal density, compared with the wild-type protein, in response to oleic acid. We propose that the active species is the "monomeric" form, and that the increasing oxidative metabolism within maturing peroxisomes causes dimer formation and inhibition of further organelle division.

The sorting sequence of the peroxisomal integral membrane protein PMP47 is contained within a short hydrophilic loop.

No targeting sequence for peroxisomal integral membrane proteins has yet been identified. We have previously shown that a region of 67 amino acids is necessary to target Pmp47, a protein that spans the membrane six times, to peroxisomes. This region comprises two membrane spans and the intervening loop. We now demonstrate that the 20 amino acid loop, which is predicted to face the matrix, is both necessary and sufficient for peroxisomal targeting. Sufficiency was demonstrated with both chloramphenicol acetyltransferase and green fluorescent protein as carriers. There is a cluster of basic amino acids in the middle of the loop that we predict protrudes from the membrane surface into the matrix by a flanking stem structure. We show that the targeting signal is composed of this basic cluster and a block of amino acids immediately down-stream from it.

Extensive modifications for methionine enhancement in the beta-barrels do not alter the structural stability of the bean seed storage protein phaseolin.

Common beans are widely utilized as a food source, yet are low in the essential amino acid methionine. As an initial step to overcome this defect the methionine content of the primary bean seed storage protein phaseolin was increased by replacing 20 evolutionarily variant hydrophobic residues with methionine and inserting short, methionine-rich sequences into turn and loop regions of the protein structure. Methionine enhancement ranged from 5 to 30 residues. An Escherichia coli expression system was developed to characterize the structural stability of the mutant proteins. Proteins of expected sizes were obtained for all constructs except for negative controls, which were rapidly degraded in E. coli. Thermal denaturation of the purified proteins demonstrated that both wild-type and mutant phaseolin proteins denatured reversibly at approximately 61 degrees C. In addition, urea denaturation experiments of the wild-type and a mutant protein (with 30 additional methionines) confirmed that the structural stability of the proteins was very similar. Remarkably, these results indicate that the phaseolin protein tolerates extensive modifications, including 20 substitutions and two loop inserts for methionine enhancement in the beta-barrel and loop structures, with extremely small effects on protein stability.

Pmp27 promotes peroxisomal proliferation.

Peroxisomes perform many essential functions in eukaryotic cells. The weight of evidence indicates that these organelles divide by budding from preexisting peroxisomes. This process is not understood at the molecular level. Peroxisomal proliferation can be induced in Saccharomyces cerevisiae by oleate. This growth substrate is metabolized by peroxisomal enzymes. We have identified a protein, Pmp27, that promotes peroxisomal proliferation. This protein, previously termed Pmp24, was purified from peroxisomal membranes, and the corresponding gene, PMP27, was isolated and sequenced. Pmp27 shares sequence similarity with the Pmp30 family in Candida boidinii. Pmp27 is a hydrophobic peroxisomal membrane protein but it can be extracted by high pH, suggesting that it does not fully span the bilayer. Its expression is regulated by oleate. The function of Pmp27 was probed by observing the phenotype of strains in which the protein was eliminated by gene disruption or overproduced by expression from a multicopy plasmid. The strain containing the disruption (3B) was able to grow on all carbon sources tested, including oleate, although growth on oleate, glycerol, and acetate was slower than wild type. Strain 3B contained peroxisomes with all of the enzymes of beta-oxidation. However, in addition to the presence of a few modestly sized peroxisomes seen in a typical thin section of a cell growing on oleate-containing medium, cells of strain 3B also contained one or two very large peroxisomes. In contrast, cells in a strain in which Pmp27 was overexpressed contained an increased number of normal-sized peroxisomes. We suggest that Pmp27 promotes peroxisomal proliferation by participating in peroxisomal elongation or fission.

Strategies for selecting mutation sites for methionine enhancement in the bean seed storage protein phaseolin.

The complete three-dimensional structure of the bean seed storage protein phaseolin was generated from alpha-carbon coordinates by using molecular mechanic calculations. This structure was used as a template to simulate modifications aimed at increasing the methionine content of phaseolin. A hydrophilic, methionine-rich looping insert sequence was designed. Simulated mutagenesis shows that the insert might be accommodated in turn and loop regions of the protein, but not within an alpha-helix. Methionine content was also increased by the replacement of hydrophobic amino acids with methionine in the central core beta-barrels of the phaseolin protein. Calculations indicated that methionine can effectively replace conserved or variant leucine, isoleucine, and valine residues. However, alanine residues were much more sensitive to substitution, and demonstrated high variability in the effects of methionine replacement. Introduction of multiple substitutions in the barrel interior demonstrated that the replaced residues could interact favorably to relieve local perturbations caused by individual substitutions. Molecular dynamics simulations were also utilized to study the structural organization of phaseolin. The calculations indicate that there are extensive packing interactions between the major domains of phaseolin, which have important implications for protein folding and stability. Since the proposed mutant proteins can be produced and studied, the results presented here provide an ideal test to determine if there is a correlation between the effects obtained by computer simulation and the effects of the mutations on the protein structure expressed in vivo.

Biophysical analysis of phaseolin denaturation induced by urea, guanidinium chloride, pH, and temperature.

The structural stability of phaseolin was determined by using absorbance, circular dichroism (CD), fluorescence emission, and fluorescence polarization anisotropy to monitor denaturation induced by urea, guanidinium chloride (GdmCl), pH changes, increasing temperature, or a combination thereof. Initial results indicated that phaseolin remained folded to a similar extent in the presence or absence of 6.0 M urea or GdmCl at room temperature. In 6.0 M GdmCl, phaseolin denatures at approximately 65 degrees C when probed with absorbance, CD, and fluorescence polarization anisotropy. The transition occurs at lower temperatures by decreasing pH. Kinetic measurements of denaturation using CD indicated that the denaturation is slow below 55 degrees C and is associated with an activation energy of 52 kcal/mol in 6.0 M GdmCl. In addition, kinetic measurement using fluorescence emission indicated that the single tryptophan residue was sensitive to at least two steps of the denaturation process. The fluorescence emission appeared to reflect some other structural perturbation than protein denaturation, as fluorescence inflection occurred approximately 5 degrees C prior to the changes observed in absorbance, CD, and fluorescence polarization anisotropy.

Radical production in liver mitochondria by peroxidic tumor promoters.

Eleven peroxides have been tested to determine if there is a correlation between tumor-promoting activity and the ability to stimulate radical production in mitochondria. When non-respiring rat liver mitochondria are treated with these peroxidic compounds in the presence of DMPO, ESR signals are observed from the spin trapping of carbon- and oxygen-centered radicals in the case of 4 of the 7 peroxides that are known to be tumor promoters. Enhancement of carbon-centered radical production is observed in the presence of respiratory substrate. Thus there does not appear to be a correlation between tumor-promoting activity of peroxidic compounds and radical production in mitochondria. Oxidants can act as promoters either by 1- or 2-electron oxidation pathways; both types of mechanisms may be inhibited by antioxidants, which can scavenge either radicals or electrophiles.