Dietary Effects of Copper and Iron Deficiency on Rat Intestine: A Differential Display Proteome Analysis Alessandra Tosco,†,‡ Rosa Anna Siciliano,†,§ Giuseppina Cacace,§ Maria Fiorella Mazzeo,§ Roberta Capone,‡ Antonio Malorni,§ Arturo Leone,*,‡,§ and Liberato Marzullo‡ Dipartimento di Scienze Farmaceutiche Universita` degli Studi di Salernos Via Ponte Don Melillo 84084 Fisciano (SA) Italy, Istituto di Scienze dell’Alimentazione, Consiglio Nazionale delle RicerchesVia Roma 52 a/c, 83100 Avellino Italy Received April 13, 2005
Copper and iron are cofactors of many metallo-proteins that accomplish vital functions, such as oxygen and electron transport. Specific metabolic pathways have been selected through evolution, although still not fully elucidated, to confine the dangerous reactivity of their free ionic forms. Inadequate supply of both metals can severly affect basic physiological funtions. A differential analysis of the rat intestinal proteome evidenced the following dietary copper- and iron-deficiencies, i.e., significant changes in the levels of proteins belonging to different functional classes (glucose and fatty acid metabolism, molecular chaperones, cytoskeleton plasticity, vitamin transporters). The presented results bring new perspectives to understand the role of copper and iron in the metabolic pathways and provide novel diagnostic tools to characterize the effects of subclinical deficiencies of both metals in unbalanced nutritional disorders. Keywords: copper • iron; intestine • proteomics • mass spectrometry • FABP • molecular chaperone • enolase • VDBP • filamin
Introduction Copper and/or iron deficiencies are associated, in their most serious forms, with different pathological symptoms. It is wellknown that severe copper deficiency can lead to anemia, neuronal degeneration, cardiac hypertrophy, and impaired elastin cross-linking. Many of the deleterious effects of copper deficiency have been ascribed to the loss of cuproenzyme activity, copper being an essential cofactor for enzyme functions such as Cu, Zn-superoxide dismutase, cytochrome c oxidase, dopamine-monooxygenase, lysyl oxidase, and peptidylglycine-amidating monooxygenase.1 Iron deficiency is associated with reduced work and intellectual capacity, diminished growth, and impaired immune response. Iron deficiency anemia (IDA) is estimated to affect 30% of the world’s population.2 An adequate iron supply is essential for the function of many biochemical processes, including electron-transfer reactions, gene regulation, binding and transport of oxygen, and regulation of cell growth and differentiation. Close correlations exist between the two micronutrient’s metabolisms, and a deficient supply of one metal may alter the uptake and utilization of the other.3 In a recent study carried out on the intestine of rats fed on diets deficient in the two metals, we analyzed, by DDRT-PCR technique, the differential expression induced at transcriptional * To whom correspondence should be addressed. Tel: +39-089-962812. Fax: +39-089-962828. E-mail:
[email protected]. † These authors equally contributed to the work described in this article. ‡ Dipartimento di Scienze Farmaceutiche, Universita` degli Studi di Salerno. § Istituto di Scienze dell’Alimentazione, Consiglio Nazionale delle Ricerche. 10.1021/pr0501012 CCC: $30.25
2005 American Chemical Society
level by such dietary restrictions.4 These results prompted us to extend such analyses at a proteomic level on the same tissues, to gain a global view of the effects of the metal-deprived diets on the expression of genes in the intestine, the primary site of entry of dietary nutrients. In detail, the 2-DE separation step and the further image analysis were complemented with mass spectrometric methodologies for the identification of proteins contained in the spots of interest through the wellknown Peptide Mass Fingerprinting (PMF) strategy.5,6 Significant quantitative alterations of proteins belonging to different functional classes were found, in particular in the levels of fatty acid binding proteins and molecular chaperons.
Experimental Section Reagents. Immobiline DryStrip, IPG buffer, DryStrip cover fluid, protein molecular weight markers for SDS-PAGE analyses, and Agarose for IEF were purchased from Amersham Biosciences. 30% Acrylamide/bis solution (37.5:1, 2.6%C), Comassie Brilliant Blue G-250 from Bio-Rad. Urea, Chaps, dithiothreitol (DTT), trypsin, R-cyano-4-hydroxycinnamic acid, angiotensin III, adrenocorticotropic hormone (clip 18-39) (ACTH) were from Sigma. Tris, SDS, glycine, iodoacetamide, bromophenol blue, ammonium persulfate, TEMED were purchased from ICN. Glycerol, methanol, acetonitrile (ACN), and glacial acetic acid were from Carlo Erba. Animals and Diets. Animal housing, diet composition, and determination of copper and iron tissue concentrations by atomic absorption spectrometry have already been described elsewhere.4 Briefly, 24 male weanling, 15-day old, SpragueDawley rats (Charles River Italia, Italy) weighing 25-50 g, were individually housed in stainless steel cages with a 12 h light/ dark cycle and had free access to food and to deionized and Journal of Proteome Research 2005, 4, 1781-1788
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research articles distilled water. With the aim of establishing a mild metal deficiency at the end of the treatments, the animals were randomly divided after 21 days into three dietary groups (eight subjects per group): control (C), copper- deficient (CuD) and iron- deficient group (FeD). Rats were sacrificed by intraperitoneal injection of pentobarbital, and the organs of interest were then explanted, quickly washed with phosphate-buffered saline (PBS), and immediately frozen in liquid nitrogen. The care and use of rats were approved by the Animal Care and Ethic Committee of INRAN (Roma, Italy). Sample Preparation. A 200-µg portion of small intestine was suspended in 500 µL buffer (20% (v/v) glycerol, 1% (v/v) Tween 20, 20 mM Hepes pH 7.5, 1 mM EDTA, 1 mM DTT), supplemented with protease inhibitor cocktail (Sigma, cat. No. P2714). Intestinal tissues were homogenized on ice with 20 strokes in a tight fitting Dounce homogenizer (Wheaton). The homogenates were centrifuged at 20 000 × g for 20 min at 4 °C and sample protein concentration was determined using the Bradford method7 (Bio-Rad). Animals from each dietary group (C, CuD, and FeD) were randomly divided into two sub-groups (four subjects each): A and B. Equal amounts of individual tissue lysates were then pooled accordingly to the sub-group division (CA, CB, CuDA, CuDB, FeDA, FeDB). Each pool sample was finally analyzed in triplicate. 2-D Electrophoresis. 2-D Electrophoresis was performed as described by O’Farrell.8 The equipment was purchased from Amersham Biosciences. The first-dimensional isoelectric focusing (IEF) was performed using the Ettan IPGphor, while the second dimensional SDS-PAGE was carried out using the Ettan DALT twelve System. Gels were poured using the Gel Caster system. Twelve samples were run simultaneously in each experiment. Seven-hundred microgram portions of each protein sample were diluted in rehydration solution (8 M Urea, 2% Chaps, 100 mM DTT, 0.5% v/v IPG Buffer pH 3-10, 0.002% bromophenol blue) to a final volume of 350 µL and applied by in-gel rehydration (according to the manufacturer’s instructions) in IPGStrip 18 cm, pH 3-10 L for 12 h; then proteins were focused up to 32 000 Vh at a maximum voltage of 8000 V. After IEF, proteins were reduced and alkylated by soaking the IPGstrips in the equilibration solution (6 M Urea, 2% SDS, 30% glycerol, 50 mM Tris-HCl, pH 8.8) containing 130 mM DTT for 10 min at RT and then in equilibration solution containing 135 mM iodoacetamide for 5 min. The equilibrated IPGstrips were sealed on top of the SDS-PAGE gel (21 × 25 cm, 12% polyacrylamide), using 0.5% agarose. The second dimensional SDS-PAGE was carried out using the Tris-glycine-SDS buffer system (25 mM Tris, 192 mM glycine and 0.1% SDS) at 5 W/gel for 1 h and then at 15 W/gel until the dye front reached the bottom edge of the gel. Protein spots were visualized by staining with Comassie Brilliant Blue G-250. Image Analysis. The 2-DE protein patterns were recorded as digitalized images using a high-resolution scanner (GS-710 Calibrated Imaging Densitometer, Bio-Rad). Spot detection, quantization and analysis were performed using the PDQuest 2-D Analysis Software, Version 6.2 by Bio-Rad. Spots whose intensity was lower than 1400 were not considered in the analysis. To correct for differences in sample loading or staining intensity among gels, the “normalized spot volume” was utilized. Spots whose intensity showed a 3-fold or higher change were selected for further analysis. For each 1782
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spot, the mean spot intensity was calculated by the software, averaging its intensity measured in the six gels run for each dietary group. Spots whose mean intensity showed a 1.5-fold or higher change were grouped in a set and, among those spots showing a significant change in intensity, detected reproducibly in all the acquired gels were chosen for further mass spectrometric analyses. Protein Identification. In-gel digestion was carried out according to Shevchenko and co-workers.9 Briefly, Comassiestained protein spots were manually excised from 2-D gels and destained with 50% ACN in 50 mM ammonium bicarbonate, dehydrated in ACN and vacuum-dried in a Speed-Vac centrifuge (Savant). Each gel piece was then re-swollen in 10 µL of 25 mM ammonium bicarbonate pH 8.4 containing 10 ng/µL of trypsin at 4 °C for 15 min. The excess of enzymatic solution was removed and 20 µL of buffer solution was added to the gel pieces. Digestion proceeded overnight at 37 °C. A 0.5-µL portion of the obtained peptide mixture was mixed with 0.5 µL of a saturated solution of R-cyano-4-hydroxycinnaminic acid (10 mg/mL in 50% ACN containing 25 fmol/µL angiotensin and 125 fmol/µL ACTH as internal standards), spotted directly on a MALDI target plate and dried under ambient condition. All mass spectra were generated on a MALDI-TOF mass spectrometer Voyager DE PRO (Applied Biosystems), operating in positive-ion reflectron mode. The laser intensity (N2, 337 ns) was set just above the ion generation threshold and pulsed every 10 ns. Mass spectra were acquired from each sample by accumulating 100 laser shots and were calibrated using as internal standards the monoisotopic peak of angiotensin (m/z 931.5154) and that of ACTH (m/z 2465.1989). All mass values are reported as monoisotopic masses. Protein identification was achieved by using the MALDI mass spectral data for database search against the NCBInr database using the MASCOT search algorithm (http://www.matrixscience.com/).10 Parameters for all searches were as follows: mammalian as taxonomic category, trypsin as enzyme, carbamidomethyl as fixed modification for cysteine residues, and methionine oxidation as variable modification, two missing cleavages, and 30 ppm as mass tolerance for the monoisotopic peptide masses.
Results and Discussion Twenty-four rats were randomly divided into three dietary groups, including a control (C), a copper- (CuD) and an iron(FeD) deficient treatment. The dietary administration of low contents of these oligoelements did not affect the mean body weights of rats, which did not even show any evident physiological or behavioral alteration during the period of treatment and before the sacrifice. Analyses by atomic absorption spectroscopy of liver and intestine samples ascertained the copper or iron deficient status of the animals.4 Moreover, to confirm the homeostatic response to low dietary iron, we analyzed the expression of the Divalent Metal Transporter 1 (DMT1). This protein is able to transport dietary non-heme iron, and other divalent metal cations and is localized on the apical surface of absorptive enterocytes. Its expression is positively regulated in the proximal duodenum of mice fed on iron-deficient diet.11 Real-Time-PCR analysis showed a positive regulation of DMT1 in the intestines of both FeD and CuD rats.4 Gut samples were analyzed in order to detect changes in protein levels under these different dietary treatments. 2-DE Separation and MS Identification of Differentially Expressed Proteins. To minimize differences in protein pro-
Dietary Effects of Copper and Iron Deficiency
Figure 1. 2-DE map of proteins extracted from gut tissue of Control group. Proteins were separated in the first dimension on IPG strip pH 3-10 L followed by SDS-PAGE on 12% polyacrilamide gel. 20 spots whose intensity differed by a 1,5-fold or more between the C samples and the CuD and FeD samples are indicated on the 2-DE map. Identification of proteins present in those spots is reported in Table 1.
files, due to the variability among individuals, we analyzed two different protein samples for each dietary group, obtained by pooling equal amounts of lysates from three different individuals. Each protein sample was run in triplicate in order to obtain statistically significant results. The 2-DE gel image analysis of the samples showed a similar protein pattern, and, particularly, a similar number of protein spots. Therefore, the 2-DE gel of a control sample showing the best resolution across the whole gel was selected as a reference proteomic map and is reported in Figure 1. Image analyses of 2-DE maps were carried out by means of PDQuest software. 454 different spots were detected in the reference map and 20 of them showed a difference of at least 1.5-fold mean intensity in the 2-DE maps obtained from gut tissue of animals treated with deficient diets. The position of the selected spots in the 2-DE map is reported in Figure 1. As an example, we report the image analysis of spot 3701, whose intensity increased in 2-DE maps obtained from samples extracted from CuD rats, thus indicating that the expression level of the corresponding protein resulted to be up-regulated by CuD diet. Enlargements of the 2-DE maps in the area close to spot 3701 in the gels obtained from C, CuD, and FeD samples are reported in Figure 2A, together with the histograms of the intensities of spot 3701 in all the different gels (Figure 2B) and the mean fold changes in CuD and FeD samples (Figure 2C). Proteins present in all the 20 spots of interest were identified by MALDI-TOF-MS and PMF strategy (Table 1). The database searches were carried out using Mascot as search algorithm and NCBI as data bank. Proteins from Rattus norvegicus usually exhibited the highest score, although in few
research articles cases proteins from Mus musculus were actually the first hits. In all the cases, the reported accession numbers and all the related information in Table 1 refer to proteins from Rattus norvegicus. Some of the analyzed proteins could be identified in more than one spot appearing near in the 2-DE map. This is probably due to the presence of different isoforms or to differential posttranslational processing. The identified proteins belong to different functional classes (Table 2). We have identified enzymes such as enolase, enoylCoa hydratase, ribonuclease, and also chaperons such as grp78, grp75, PDI-ER60, Hsp70, or Hsp60 and fatty acid binding proteins. As described by the short summaries in the following sections, the identified proteins do not seem to share any selfevident correlation with the known metabolic fates of both metals and appear to be part of a more general metabolic response to the nutritional-induced deficiency of the two micronutrients. Enolase. In vertebrates, the enolase enzyme (2-phospho-Dglycerate hydro-lyase) catalyzes the glycolytic step interconverting 2-phosphoglycerate and phosphoenolpyruvate. Enolase is present as homodimers and heterodimers formed from three distinct subunits with the same molecular weight which differ in their biochemical and immunological properties, as well as in tissue distribution: (1) alpha, a nonneuronal enolase (NNE; designated ENO1 in human), is found in many adult cell types; (2) beta enolase (MSE; ENO3 in human) is expressed exclusively in muscle cells; and finally (3) gamma, is a neuron-specific enolase (NSE; ENO2 in human). In our experiments, the levels of alpha enolase are increased in dietary iron restriction. This evidence is supported by other observations that correlate dietary iron deficiency with the regulation of the glycolitic pathway. Previous work has shown that gamma enolase, the neuron-specific isozyme, is upregulated in the brain of iron-deficient rats.12 Furthermore, the rat liver glyceraldehyde 3-phosphate dehydrogenase mRNA showed a 2.3-fold increase in iron deficiency.13 Finally, Oexle14 and co-workers showed that in a human erythroleukemic cell line, the iron chelating agent DFO (Desferioxamine) leads to reduced activities of the citric acid cycle enzymes (citrate synthase, aconitase, isocitrate dehydrogenase, and succinate dehydrogenase) and increased glycolytic utilization of glucose. Iron deprivation results in reduced NADH formation in the citric acid cycle, and a subsequent lower ATP production via oxidative phosphorylation. To overcome this lack of oxidative ATP formation, cells have to intensify the anaerobic shortcut in glycolysis to cope with NADH and ATP unbalances. In this context, it is not surprising to find an up-regulation of enolase also in the intestine of iron-deficient rats. The enhancement of anaerobic glycolysis increases lactic fermentation with consequent alteration of intracellular acidity. In our experimental model, this effect could alter, at different levels, the intracellular equilibria, thus influencing important physiological functions such as muscular contractility and intestinal peristalsis, as well as the active transport of nutrients and other molecules across the cellular membranes. Vitamin D Binding Protein and Filamin A. Vitamin D binding protein (VDBP), also known as Group specific protein (Gc), is a 52 kDa protein that binds monomeric actin other than Vitamin D. The protein forms three domains, the first of them containing the sterol binding site. One of the putative functions of VDBP is to clear up any actin monomer that accidentally Journal of Proteome Research • Vol. 4, No. 5, 2005 1783
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Figure 2. Differential expression of protein present in spot 3701. (A) Gel regions encompassing spot 3701 are shown for one of the gels obtained from C, CuD, and FeD samples, respectively. (B) Histogram reporting the intensities of spot 3701 in the 18 gels obtained from the six different samples run in triplicate. (C) Histogram of mean spot intensity for Control, CuD, and FeD samples.
enters the blood stream as a result of cell injury.15 The observed increase of VDBP in copper-deficient rats may be related to the ability of this protein to sequester monomeric G-actin that would otherwise be available to polymerize into F-actin. Moreover, in the intestine of copper-deficient rats, we also found a down-regulation in the expression of Filamin A. Filamins are believed to be essential for mammalian cell motility, through the formation of loose microfilaments nets. Filamin A cross-links actin filaments, allowing the formation of a dynamic three-dimensional network. It can link the actin cytoskeleton to the plasma membrane via its association with integral membrane proteins, thereby functioning as an important signaling scaffold.16 Dietary copper is known to have an important role in microvascular control mechanisms, and it was shown that copper deficiency promotes F-actin polymerization in lung microvascular endothelial cells,17 while copper treatment increases the intestinal paracellular permeability of the human intestinal CaCo-2 cells.18 1784
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In our system, dietary copper could influence the reorganization of the cytoskeleton, at least at the intestinal level, thereby altering the levels of VDBP and Filamin A proteins. According to this hypothesis, the luminal and intracellular intestinal copper level could be considered also as modulator of the intestinal permeability, possibly acting through the regulation of motogenic factors and/or the cytoskeleton plasticity. Copper deficiency would then influence the complex series of events encompassing cell proliferation, differentiation, and migration that daily oversee on the integrity and continuity of the intestinal epithelium. Proteins Involved in Fatty Acids Metabolism. An involvement of iron and copper in fatty acid and cholesterol metabolisms was already described, even though the observed effects appear to be a secondary response. None of these metabolic pathways involves metallo-enzymes. Copper deficiency is often associated with hypertrigliceridemia and hypercholesterolemia. It was also reported that these alterations are related to the elevated hepatic iron content that
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Dietary Effects of Copper and Iron Deficiency Table 1. Major Protein Alterations Following Dietary Treatment spot
acc. no.
ID
PM
pI
Mascot score
matched peptides
prot. cov. (%)
regulationa CuD
2002 2803 2905 3701 3702 3703
gi|4033695 gi|25742763 gi|34933178 gi|11560024 gi|476569 gi|38382858
14.592 72.473 124.773 61.098 55.080 57.044
5.52 5.07 5.59 5.91 5.65 5.88
87 210 137 236 77 87
6/16 20/28 11/14 21/29 7/17 8/17
35 35 11 37 13 13
v vv v vvv v vvv
3705
gi|38382858
57.044
5.88
119
10/19
23
vvv
3802 3804 4503 5302 5303 5602 6002 6003 7003 7102
gi|2119726 gi|2119726 gi|51772115 gi|56385 gi|56385 gi|38649320 gi|204074 gi|204074 gi|204074 gi|34327777
73.984 73.984 296.086 71.112 71.112 51.736 11.468 11.468 11.468 34.856
5.87 5.87 5.87 5.43 5.43 6.70 6.74 6.74 6.74 9.10
106 151 73 108 74 237 159 96 48 116
8/10 11/12 11/18 10/17 6/11 19/32 8/9 7/14 5/18 12/27
13 20 3b 20 13 42 53 52 26 18
vvv vv VV VV VV
7301 8006 9101
gi|24159086 gi|443161 gi|229252
Gastrotropin (Ileal lipid-binding protein) heat shock 70kD protein 5 similar to ubiquitin-protein ligase heat shock 60 vitamin D-binding protein precursor glucose regulated protein, 58 kDa (similar to PDI ERp61) glucose regulated protein, 58 kDa; ER-60 protease dnaK-type molecular chaperone grp75 dnaK-type molecular chaperone grp75 Filamin, alpha Hsc70-ps1 Hsc70-ps1 enolase liver fatty acid binding protein p14 liver fatty acid binding protein p14 liver fatty acid binding protein p14 heterogeneous nuclear ribonucleoprotein A3 variant a Enoyl-CoA Hydratase, Chain F Chain A, retinol binding protein II RNase
28.498 15.857 14.541
6.41 5.51 8.64
80 129 71
5/8 6/7 5/12
21 39 31
a
regulationa FeD
V VVV
v VVV VV VVV v
V
VV
v vvv
Arrows indicate ranges of fold change: v from 1, 5 to 2, 0; vv from 2, 0 to 2, 5; vvv > 2, 5. b A fragment of the protein has probably been detected.
Table 2. Functions and Cellular Localizations of the Proteins Regulated by the Dietary Treatments spot
protein
5602
Enolase
3702
Vitamin D-binding protein precursor Filamin A Ribonucleoprotein A3 Rnase Chain A, Enoyl-CoA Hydratase Gastrotropin (Ileal lipid-binding protein) o I-BABP Liver fatty acid binding protein o L-FABP
4503 7102 9101 7301 2002
6002 6003 7003 8006 2803 3703 3705 3701 3802 3804 2905 5302
subcellular localization
function
Catalyzes the interconvertion of 2-phosphoglycerate and phosphoenolpyruvate in the glycolytic way Binds vitamin D sterols and actin monomers
regulation CuD
Cytosol
UP
Secreted
UP
Actin filaments cross-linking protein Ribonucleoproteosome component Endonuclease Catalyzes hydration of enoyl-CoA in 3-L-hydroxy-acil-CoA in fatty acids oxidation Stimulates gastric acid and pepsinogen secretion. Binds bile salts and bilirubins.
Cytosol Nucleus Secreted Mitochondrial matrix
DOWN UP
Cytoplasmic
UP
Binds free fatty acids and a large number of hydrophobic molecules
Cytoplasmic
DOWN
Chain A, retinal binding protein II Grp78 PDI, ER60
Involved in the intracellular transport of retinol Molecular chaperone Catalyzes formation and isomerization of disulfide bonds.
Cytoplasmic
DOWN
Endoplasmic reticulum Endoplasmic reticulum
UP UP
Heat shock protein 60 kDa Grp75
Molecular chaperone
Mitochondrial matrix
UP
Molecular chaperone
Mitochondrial matrix
UP
Involved in the process of ubiquitinilation of proteins Molecular Chaperone
Cytosol
UP
Cytosol
DOWN
Ubiquitin-protein ligase Heat shock protein 8 o HSC70
regulation FeD
DOWN UP
UP DOWN
UP
DOWN
5303
usually follows a severe Cu depletion.19 On the other hand, a copper supplement lowers cholesterol and triglyceride serum levels, while increases the level of phospholipids and nonesterified fatty acids, concomitantly decreasing the hepatic concentrations of iron and zinc.20 In rats, a moderate iron
deficiency lowers cholesterol concentrations in liver and serum lipoproteins and also depresses the serum phospholipid levels.21 At the moment, the greatest part of the molecular events correlating the levels of micronutrients with the metabolism of lipids and cholesterol are still largely unknown. The only Journal of Proteome Research • Vol. 4, No. 5, 2005 1785
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Figure 3. (A) Metabolism of cholesterol and its relationships with proteome changes. (B) Metabolism of fatty acids and its relationships with proteome changes. Effects in CuD and FeD animals are indicated by full and empty arrows, respectively. LCFA, Long Chain Fatty Acids; TG, Triglycerides; PL, phospholipids; CE, Cholesterol Esters; TAG, Triacilglycerols; VLDL, Very Low-Density Lipoprotein.
exception is represented by the expression of stearoyl coenzyme A desaturase and fatty acid synthase. Stearoyl coenzyme A desaturase, involved in fatty acid desaturation, contains one atom of iron and its activity is decreased in iron deprivation.22 Recent observations showed that iron overload increases the mRNA expression and the activity of this enzyme in mouse liver.23 On the other hand dietary copper deficiency was shown to increase sterol regulatory element binding protein-1 (SREBP1), a stronger enhancer of fatty acid syntase promoter activity, thereby accelerating hepatic lipid synthesis.24 In our experiments, we observed an up-regulation in the levels of Enoyl-Coa Hydratase in copper-deficient rats, and different regulations of three proteins belonging to the fatty acid binding protein superfamily either in the CuD or in the FeD samples. The intracellular lipid binding protein family includes low molecular weight proteins that share a similar general structure. These soluble proteins are present in a wide variety of tissues, but their exact function and physiological roles remains poorly understood. The small intestine contains three different types of lipid binding proteins: the Liver Fatty Acid Binding protein (L-FABP), present also in liver and kidneys, the Intestinal Fatty Acid Binding Protein (I-FABP), strictly confined to the small intestine and the Ileal Bile Acid Binding Protein (I-BABP). L-FABP and I-FABP bind long chain fatty acid (LCFA) in the gut, while I-BABP seems to be involved in the ileal fate of the bile acids (BAs).25 I-BABP is up-regulated in CuD, but down-regulated in FeD rats. Primary bile acids are synthesized from cholesterol in the liver, then conjugated and secreted into bile. Two carriers, the ileal bile acid transporter and the I-BABP mediate the transport of BAs in the intestine. Most of the BAs are backward cycled to the liver before their new secretion into the bile, and the maintenance of their circulation is then essential to ensure a correct cholesterol balance. As copper deficiency enhances cholesterol levels in serum, it can be hypothesized that, according to our results, an over-production of biliary acids stimulates I-BABP synthesis in rat intestine. On the other hand, a decrease of cholesterol levels, as a consequence of iron 1786
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deficiency, could explain the down regulation in rats fed on low iron diet, in agreement with the observed alteration of the biliary acids levels in both cases (Figure 3A). Moreover, we observed a down-regulation of L-FABP levels in the intestine of copper-deficient rats. L-FABP constitutes a low capacity, high affinity system that predominates at low substrate concentration, while passive diffusion is effective when LCFA intake is abundant. A copper-deficient status, associated with higher hepatic liver lipid synthesis,24 could imply a lowered demand of dietary fatty acids and a subsequent down regulation of transporters such as L-FABP (Figure 3B). We also detected altered levels of enoyl coA hydratase, which is involved in the process of fatty acid oxidation, and retinal binding protein, belonging to the fatty acid binding protein superfamily. These findings underlines once more the involvement of the two micronutrients in the metabolisms of fatty acids, although data present in the literature do not provide yet more close physiological and/or molecular correlations with the metabolism of iron and copper. Molecular Chaperones. A group of molecular chaperones, namely Hsc70, Grp78, PDI, Hsp60, and Grp75, are differently regulated in CuD rats. Among them, all but Hsc70 resulted to be up- regulated by copper deficiency. Several authors reported the correlation of heat shock protein expression and copper levels,26-28 but it is not easy to build up a rational scheme of the cell response, being the effects of copper levels dependent on tissue or cell type and/or experimental conditions. Considering the molecular chaperones found regulated in this analysis, it can be pointed out that they arelocalized into the endoplasmic reticulum (Grp78 and PDI) and into the mitochondria (Hsp60 and Grp75), thus indicating a compartmentalized stressing effect of the metal deficiency. All of these proteins are involved in protein folding processes, and a first hypothesis cannot rule out a possible alteration of protein folding processes in these sites. In a recent paper Nittis and Gitlin,29 demonstrated that hephaestin, a multicopper oxidase essential for iron homeostasis is post-translationally regulated in colon carcinoma cells subjected to copper chelation. Hephaestin is a N-linked gly-
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Dietary Effects of Copper and Iron Deficiency
cosylated polypeptide that enters the secretory pathway to reach the basolateral surface of the enterocyte. These authors demonstrated the presence of a control quality system in the late secretory pathway that, in conditions of copper deprivation, promotes a retrotranslocation to the endoplasmic reticulum, the ubiquitination and the proteasomal degradation of the protein. On the other hand, ceruloplasmin, another multicopper oxidase, was shown to be retained in the endoplasmic reticulum, when expressed in a form not able to bind copper.30 Finally, the assembly of the multisubunit mitochondrial enzyme cytochrome c oxidase was shown to be impaired in copperdeficient rats.31 These observations show that many copper-proteins might undergo a misfolding process in the absence of a sufficient supply of their cofactor. As a consequence, the accumulation of misfolded products might elicit the induction of molecular chaperones that resides in the endoplasmic reticulum and in the mitochondria, where many cupro-enzyme are located. A parallel up-regulation of an ubiquitin protein ligase may address to the proteasome the irretrievable fraction of misfolded proteins.
Conclusions Most of people suffering iron- or copper-deficiencies, due to dietary unbalances or nutritional disorders, are often asymptomatic patients who can potentially develop sickness related to the impairment of vital functions. Molecular genetic tools able to easily detect developing illnesses could help in elucidating many uncharacterized aspects of iron and copper metabolism, and could be of great interest for diagnosis and prevention of the subtle consequences of metals deficiencies. To this aim, a differential display proteome analysis was carried out to fish out proteins characterizing an animal model made mildly deficient in both metals. Only Comassie stainable protein spots were selected, to isolate easily traceable proteins whose levels were the most sensitive to the variations compatible with dietary metal content. These data integrate the previous results obtained at transcriptional level in the same experimental system.4 The majority of the significantly changed proteins had close relations to fatty acid metabolism and to protein folding, and was mainly influenced by copper deficiency. The first data are in good agreement with published reports, while less is known about correlations between copper and molecular chaperones. Copper and iron deficiency are able to influence the abundance of proteins involved in fatty acid metabolism, but correlation of trends and intensities of changes is hard to explain. Moreover, our data suggest that a common mechanism of posttranscriptional control might exist in cells subjected to copper restrictions. In this context, the misfolding process of copperbinding proteins would elicit the induction of molecular chaperones in specific compartments and promote, in this way, a proteasome-mediated degradation. Taken together, our results represent a first attempt to highlight the intestinal proteome changes elicited by iron or copper dietary restriction, proposing new tools to define preand/or sub-clinical states of metal deficiency. Future developments of metabolomics will help to better correlate our findings with other metabolic pathways. Abbreviations: ACTH, adrenocorticotropic hormone (clip 18-39); BAs, bile acids; C, control group; CuD, copper-deficient group; DDRT-PCR, differential display reverse transcription
polymerase chain reaction; 2-DE, two-dimensional electrophoresis; DMT1, divalent metal transporter 1; FeD, irondeficient group; IDA, iron deficiency anemia; IEF, iso-electricfocusing; I-BABP, ileal bile acid binding protein; I-FABP, intestinal fatty acid binding protein; IPG, immobilized pH gradient; LCFA, long chain fatty acid; L-FABP, liver fatty acid binding protein; MALDI-TOF-MS, matrix assisted laser desorption ionization-time-of-flight-mass spectrometry; PMF, peptide mass fingerprinting; VDBP, vitamin D binding protein.
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