Analysis of HIF-prolyl hydroxylases binding to substrates Manuel O. Landa ´zuri b , Alicia Vara-Vega b , Mariano Vito ´n b , Yolanda Cuevas a , Luis del Peso a, * a Departamento de Bioquı ´ mica, Instituto de Investigaciones Biome ´ dicas ‘Alberto Sols’, Consejo Superior de Investigaciones Cientı ´ficas-Universidad Auto ´ noma de Madrid, Arturo Duperier 4, 28029 Madrid, Spain b Servicio de Inmunologı ´ a, Hospital de la Princesa-Universidad Auto ´ noma de Madrid, Madrid, Spain Received 19 September 2006 Available online 16 October 2006 Abstract Hypoxia inducible transcription factors (HIF) are mainly regulated by a group of proline hydroxylases (EGLNs) that, in the presence of oxygen, target HIF for degradation. HIFa contains two independent oxygen degradation domains (N-ODD and C-ODD) that are substrates for these enzymes. In this work, we employed the yeast two-hybrid assay to study the sequence determinants required for the binding of EGLN1 and 3 to HIF1a in a cellular context. Our results demonstrate that, while EGLN1 is able to recognize both ODDs within full length HIF1a protein, EGLN3 only binds to CODD. The analysis of the residue substitutions within CODD uncovered novel critical determinants for EGLN1 and 3 binding. In addition, our results show that both enzymes have a very similar, albeit not identical, residue preference at specific positions in their substrate sequences. Ó 2006 Elsevier Inc. All rights reserved. Keywords: HIF; EGLN; PHD; Hypoxia; Proline-hydroxylation; VHL In response to a decrease in oxygen supply, cells react by inducing a characteristic gene expression program through the activation of the hypoxia inducible factor (HIF) tran- scription factors [1]. HIFs are heterodimers of two sub- units: an oxygen-regulated a subunit (HIF1a,2a, or 3a) and HIFb. The gross activity of these transcription factors is mainly determined by the stability of the a subunit. In the presence of oxygen, specific HIFa proline residues become hydroxylated [2,3]. This post translational modifi- cation directs the recruitment of pVHL, the substrate rec- ognition subunit of an E3-ubiquiting ligase complex, that targets HIFa for proteosomal degradation [4]. HIFa hydroxylation is mediated by the activity of a family of 2-oxoglutarate (2OG)-dependent hydroxylases termed PHDs (proline hydroxylase domain; PHD1, PHD2, and PHD3) or EGLNs (EGL Nine homologues; EGLN2, EGLN1, and EGLN3, respectively). These enzymes require molecular oxygen for the reaction [5,6], consequently the hydroxylation of HIFa, and hence its degradation, is com- promised under low oxygen tension. Thus, the sequential activity of EGLNs [5–7] and pVHL [4] explains the oxy- gen-dependent regulation of HIFa protein levels. In the presence of oxygen, human HIF1a protein is hydroxylated at two independent proline residues, Pro 402 and Pro 564 , [8] that lie in a conserved LXXLAP motif [2,3]. In addition, point mutation of specific HIF1a residues within this motif prevented HIF hydroxylation [6,9], suggesting its importance for EGLN activity. How- ever, following studies suggested the absence of a rigid consensus sequence for EGLN binding to this motif [9]. A recent study [10] further investigated the sequence requirements within LXXLAP for EGLN activity and found that Leu and, to lesser extent, Ala residues within this motif can be replaced by many residues without sig- nificantly affecting the substrate properties of the sequence. On the other hand, residues downstream this 0006-291X/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.09.170 * Corresponding author. Fax: +34 91 585 4401. E-mail address: [email protected](L. del Peso). www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 351 (2006) 313–320 BBRC
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www.elsevier.com/locate/ybbrc
Biochemical and Biophysical Research Communications 351 (2006) 313–320
BBRC
Analysis of HIF-prolyl hydroxylases binding to substrates
Manuel O. Landazuri b, Alicia Vara-Vega b, Mariano Viton b, Yolanda Cuevas a,Luis del Peso a,*
a Departamento de Bioquımica, Instituto de Investigaciones Biomedicas ‘Alberto Sols’, Consejo Superior de Investigaciones Cientıficas-Universidad Autonoma
de Madrid, Arturo Duperier 4, 28029 Madrid, Spainb Servicio de Inmunologıa, Hospital de la Princesa-Universidad Autonoma de Madrid, Madrid, Spain
Received 19 September 2006Available online 16 October 2006
Abstract
Hypoxia inducible transcription factors (HIF) are mainly regulated by a group of proline hydroxylases (EGLNs) that, in the presenceof oxygen, target HIF for degradation. HIFa contains two independent oxygen degradation domains (N-ODD and C-ODD) that aresubstrates for these enzymes. In this work, we employed the yeast two-hybrid assay to study the sequence determinants required forthe binding of EGLN1 and 3 to HIF1a in a cellular context. Our results demonstrate that, while EGLN1 is able to recognize both ODDswithin full length HIF1a protein, EGLN3 only binds to CODD. The analysis of the residue substitutions within CODD uncovered novelcritical determinants for EGLN1 and 3 binding. In addition, our results show that both enzymes have a very similar, albeit not identical,residue preference at specific positions in their substrate sequences.� 2006 Elsevier Inc. All rights reserved.
In response to a decrease in oxygen supply, cells react byinducing a characteristic gene expression program throughthe activation of the hypoxia inducible factor (HIF) tran-scription factors [1]. HIFs are heterodimers of two sub-units: an oxygen-regulated a subunit (HIF1a, 2a, or 3a)and HIFb. The gross activity of these transcription factorsis mainly determined by the stability of the a subunit. Inthe presence of oxygen, specific HIFa proline residuesbecome hydroxylated [2,3]. This post translational modifi-cation directs the recruitment of pVHL, the substrate rec-ognition subunit of an E3-ubiquiting ligase complex, thattargets HIFa for proteosomal degradation [4]. HIFahydroxylation is mediated by the activity of a family of2-oxoglutarate (2OG)-dependent hydroxylases termedPHDs (proline hydroxylase domain; PHD1, PHD2, andPHD3) or EGLNs (EGL Nine homologues; EGLN2,
0006-291X/$ - see front matter � 2006 Elsevier Inc. All rights reserved.
EGLN1, and EGLN3, respectively). These enzymes requiremolecular oxygen for the reaction [5,6], consequently thehydroxylation of HIFa, and hence its degradation, is com-promised under low oxygen tension. Thus, the sequentialactivity of EGLNs [5–7] and pVHL [4] explains the oxy-gen-dependent regulation of HIFa protein levels.
In the presence of oxygen, human HIF1a protein ishydroxylated at two independent proline residues,Pro402 and Pro564, [8] that lie in a conserved LXXLAPmotif [2,3]. In addition, point mutation of specific HIF1aresidues within this motif prevented HIF hydroxylation[6,9], suggesting its importance for EGLN activity. How-ever, following studies suggested the absence of a rigidconsensus sequence for EGLN binding to this motif[9]. A recent study [10] further investigated the sequencerequirements within LXXLAP for EGLN activity andfound that Leu and, to lesser extent, Ala residues withinthis motif can be replaced by many residues without sig-nificantly affecting the substrate properties of thesequence. On the other hand, residues downstream this
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motif have been shown to be required for EGLN bindingand activity [9–11], but sequence requirements outsidethis region have been less extensively studied.
Regardless of this apparent lack of a strict consensussequence for target selection, each EGLN shows a differentpreference for the sequence containing HIF1a Pro402
(NODD) or HIF1a Pro564 (CODD) residues [10,12,13].Thus, EGLNs have different substrate preferences, both in
vivo and in vitro, yet the sequence determinants responsiblefor this substrate discrimination remain unclear.
In this work, we employ the yeast two-hybrid system toassay the interaction of the EGLNs to their target sequenc-es on HIFa. Since HIF signalling is not conserved in yeast,this assay allows studying the interaction between theEGLNs and HIF, in a cellular context, without interfer-ence with the endogenous regulatory machinery. With aidof this assay, we identified residue substitutions on theHIF1a sequence that allowed EGLN binding and also cal-culated the frequency of appearance for each amino acidon a particular position.
Materials and methods
Cells and reagents. For the yeast two-hybrid assays described in thiswork, we used the reporter yeast strain AH109 from BD Biosciences (PaloAlto, CA). Yeast growth media reagents (YPD, SD amino acids, andX-gal) were from BD Biosciences.
Generation of plasmid constructs. Constructs expressing full lengthHIF1a, HIF1a(392–414), and HIF1a(554–576) as Gal4AD fusion proteinswere generated by PCR amplification of the indicated coding regions fromhuman HIF1a cDNA and cloned into the pGAD-T7 plasmid (BD Bio-sciences). The constructs expressing Gal4DBD fused to EGLN-1, EGLN-2, and EGLN-3 were generated by PCR amplification and cloned into thepBRIDGE plasmid (BD Biosciences). EGLN cDNAs were generouslyprovided by Steven L. McKnight [6] and Peter Ratcliffe [3]. Libraries ofHIF1a mutants were generated by PCR using the pGAD-HIF1a(554–576)construct as template and primers in which the codon for the position tobe mutated was substituted by the random sequence NNS. An indepen-dent library for each position was generated, except for positionsLeu562Ala563, Tyr565Ile566, and Asp569Asp570Asp571, that were mutage-nized simultaneously. The identity of all constructs was confirmed bysequencing.
Yeast transformation and interaction assays. Freshly prepared com-petent yeast cells were transformed with 0.1 lg of each of the indicatedplasmids by a modified version of the lithium acetate method [14]. Forthe library screening experiments, yeast transformed with EGLN1constructs were selected on plates lacking Leu, Trp, and His (-Leu-Trp-His plates), and those transformed with EGLN3 were selected onstringent plates lacking Leu, Trp, His, and adenine (-Leu-Trp-His-Ad-enine plates). After selection, colonies were used as template for PCRamplification and amplicons were sequenced in order to determine thesequence identity of the pGAD-HIF1a construct. For the semi-quan-titative interaction experiments, transformed yeast were plated onminimal SD media plates in the absence of Leu and Trp. Subsequently,equal number of colonies from each transformation was transferred tosaline solution (0.9% NaCl) and subjected to serial dilutions. Aliquotsof each cell suspension (typically 20 ll) were plated on different strin-gency plates.
Calculation of information content. Information content in the EGLNbinding site was calculated as previously described by Schneider et al. [15]including the correction for sampling error. For its application to ami-noacid sequences, sampling uncertainty was calculated by a modificationof the approximate method [15].
Results
Characterization of EGLN1/3 binding to HIF1a by the yeast
two-hybrid assay
We employed the yeast two-hybrid system [16,17] toinvestigate the interaction between the EGLNs and theODDs. For these assays, the coding sequences of EGLN1,EGLN2, and EGLN3 were cloned in-frame with the DNABinding Domain from the yeast Gal4 transcription factorand used to transform yeast in combination with a plasmidencoding for Gal4-Activation Domain-HIF-1a fusion pro-tein. The DBD-EGLN1 construct used in this assay derivesfrom an EGLN1 PCR variant (DEGLN1) that lacks 100residues in its N-terminal region, from Gly76 to Gly177.This variant has been extensively used to study EGLN1biochemistry [6,10,12,18]. Yeast transformed withAD-HIF1a in combination with either DBD-DEGLN1 orDBD-EGLN3 were able to grow in highly restrictive media(Fig. 1A, bottom panel), demonstrating a direct and stronginteraction of EGLN1 and EGLN3 with full length HIF1a.In contrast, transformation with DBD-EGLN2 did notsupport yeast growth in restrictive media (data not shown).This result was not due to an inefficient expression ofDBD-EGLN2, as judged by inmunoblot (data not shown),but it might be explained by incorrect folding/subcellularlocalization. Hence, the rest of the experiments in this workwere performed with EGLN1 and 3 enzymes only. Theinteraction of EGLN1 and 3 with HIF1a was specific, sinceno detectable growth was observed when DBD-DEGLN1/EGLN3 plasmids were co-transformed with a Gal4-ADconstruct not fused to HIF1a (data not shown). In addi-tion, mutation of HIF1a Pro402 and Pro564 to Ala residues(P402A, P564A) resulted in a complete lack of binding toeither EGLN1 or 3 (Fig. 1A). Note that, in the case ofEGLN3, the binding to the proline double mutant was lostonly under high stringent conditions (Fig. 1A, bottom pan-el), which is probably an indication of the strong interac-tion between enzyme and substrate. Since the Pro residueis an absolute requirement for EGLN binding and activity[9,12], we used this Pro to Ala mutant as a control forbackground binding. Thus, only those interactionsobserved in conditions where the interaction with thismutant was lost or strongly reduced were considered aspositive.
Next, we investigated the binding of the EGLNs to eachof the two target sequences on HIF1a (NODD andCODD). As shown in Fig. 1A, mutation of Pro402 to Ala(P402A) within full length HIF1a did not prevent the bind-ing of EGLN1 or EGLN3. In the case of EGLN1, a mod-est decrease in the interaction strength was consistentlyobserved, as compared to the binding to HIF1a wild type(Fig. 1A, bottom panel). However, when Pro564 (CODD)was mutated to Ala (P564A) the binding of EGLN3 toHIF1a was completely abrogated, while that of EGLN1remained unaffected (Fig. 1A, bottom panel). These resultssuggest that, while EGLN1 bound both target sequences
Fig. 1. Differential binding of EGLN1 and EGLN3 to the HIF1a degradation domains. Yeast cells were transformed with the indicated constructs,expressing full length HIF (A), or isolated ODDs (B), in combination with a plasmid encoding for DEGLN1 or EGLN3. Serial dilutions of transformedclones were grown on plates lacking the indicated nutrients. WT, full length wild type HIF1a; P402A, HIF1a P402A mutant; P564A, HIF1a P564Amutant; PP, HIF1a P402A/P564A mutant; NODD, pGAD-HIF1a(392–414); CODD, pGAD-HIF1a(554–576); CODD*, pGAD-HIF1a(554–576)P564A.The results shown are representative of at least four independent experiments.
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within full length HIF1a, the binding of EGLN3 to NODDwas undetectable. In addition, this observation indicatesthat EGLN1 binding to NODD is independent of a func-tional CODD sequence. In order to verify whether this sub-strate preference pattern was determined by the sequencesurrounding the prolines or by the conformational cluesconferred by the location of the prolines within the fulllength protein, we investigated the binding of EGLNs toisolated NODD and CODD sequences (Fig. 1B). For thispurpose, constructs encoding for Gal4-AD fused to a 22residue peptide derived from NODD (EPDALTLLAPAAGDTIISLDFG, underlined is Pro402) or CODD(TDLDLEMLAPYIPMDDDFQLRS, underlined isPro564) were generated. A construct encoding for a CODDsequence containing the mutation P564A was used as anegative binding control.
As shown in Fig. 1B, both EGLN1 and EGLN3 wereable to bind to Gal4 AD-CODD. Interestingly, the bind-ing of EGLN1 to isolated CODD was weaker than thatof EGLN3, as demonstrated by the lack of growth of theformer under high stringent conditions (Fig. 1B, bottompanel). In contrast, only EGLN1, but not EGLN3, wasable to bind to NODD (Fig. 1B). Therefore, the sub-strate preference of each EGLN primarily depends onthe sequence from �9 to +12 relative to the targetproline.
Characterization of the sequence determinants on EGLN1/3
substrates
The previous results demonstrate that EGLN1 andEGLN3 show distinct substrate specificities (Fig. 1B). Withthe aim of identifying the critical residues for the differen-tial binding of each EGLN to their targets, we mutatedeach individual position from �6 to +10 (relative to pro-line 564) in CODD to any of the 20 naturally occurringamino acids. The resulting mutants were tested for theirability to bind each EGLN. To this end, libraries of ran-dom mutants for each one of the 17 individual positionsfrom �6 to +10 within CODD were generated and usedin combination with the EGLN1 or EGLN3 fusion con-structs to transform yeast. Only yeast cells transformedwith a library clone that contained an amino acid substitu-tion that retained binding to EGLNs were able to growunder stringent conditions. PCR amplification andsequencing of the pGAD-CODD construct from these col-onies allowed us to identify substitutions within CODDthat permitted EGLN interaction (Table 1). A graphicalrepresentation of the screening results for EGLN-1 andEGLN-3 is shown in Fig. 2. A color code is used to repre-sent the frequency of clones containing a specific aminoacid (upper graphs) or groups of amino acids (lowergraphs) for a given position on HIF1a sequence. The
Table 1Results of EGLN1 and EGLN3 screening of mutant HIF1a libraries
Residue position (Relative to target proline/Residue in HIF1a)
The values represent the absolute number of clones identified that encode a specific amino acid residue.
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Fig. 2. Frequencies of residue substitution found in the screening of mutant HIF1a(554–576) libraries. The library screening (see text for details) resultsfor EGLN1 (left panels) and EGLN3 (right panels) are represented as density graphs of each amino acid frequency (Y-axis) at a given position (X-axis).The color code indicates the frequency intervals as shown. The label on the X-axis shows the sequence of the wild type HIF1a protein. On the Y-axis the 20amino acids (single letter code) and the stop signal (asterisk) are shown. In the bottom panels, the frequency of groups of amino acids is represented. Ar,aromatic/bulky side chains (Phe, Tyr, Trp, His); Al, aliphatic side chains (Val, Leu, Ile, Met); Sm, small side chains (Gly, Ala, Cys); Pl, uncharged polarside chains (Ser, Thr, Gln, Asn); Ba, basic side chains (Lys, Arg, His); Ac, acidic side chains (Asp and Glu); P, Pro. (For interpretation of the references tocolor in this figure legend, the reader is referred to the web version of this paper.)
Fig. 3. EGLN binding site information content. Information contentfor each position (relative to target Pro) was calculated from the data inTable 1.
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screening results indicated that the only residue that cannotbe replaced by any other amino acid is the target proline(Pro564 in HIF1a CODD). All the interacting clonesobtained after the yeast transformation with a library ofrandom nucleotides at codon 564 encoded for Pro(Table 1 and Fig. 2 upper graphs, frequency equal to 1).This restricted and strong preference for a single type ofresidue reflects the large information content of this posi-tion for EGLN binding (Fig. 3). No other position showedsuch a restrictive amino acid preference and, even thosepositions with a clear bias toward a specific residue (suchas Ala at position �1) had some variation among the inter-acting clones (Table 1). In fact, the screening revealed thatmost of the positions within the �6 to +10 region showed alarge degree of tolerance to substitutions, as previouslyobserved for the LXXLAP motif [10]. In particular thepositions �7, �3, and +4 admitted a wide range of residues(Table 1 and Fig. 2) resulting in a low information content(Fig. 3). The results shown in Fig. 2 (upper graph) andTable 1 indicated that both EGLNs had a strong prefer-
ence for Ala residues at position �1 relative to Pro564. Incontrast, no clear preference was observed for Leu residuesat positions 559 or 562 (Table 1 and Fig. 2, upper graphs).Instead, there was a highly significant tendency to aliphaticnonpolar residues at these positions (Table 1 and Fig. 2,lower graphs). We also found an unexpected tolerancefor arginine residues at position �2, in particular in the
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case of EGLN1 (Table 1). These results are in agreementwith a recent report that described the effect of substitu-tions within the LXXLAP motif on EGLN kinetic param-eters [10]. The screening also revealed a previouslyunnoticed preference of EGLNs for aliphatic residues atposition +2 and for aliphatic/small non polar residues at+9 and +10 (Fig. 2, bottom panel, and Table 1). Theseresults were confirmed by the lack of binding of EGLN1to I566A and L574A mutants (Fig. 4A). In addition, bothEGLNs showed a strong preference towards an asparticresidue at position +7 (Fig. 2, upper graphs), in agreementwith previous reports showing that the mutation of this res-idue had a profound impact on EGLN/VHL binding toHIFa [9,19]. In close proximity to Asp571 there are twoother aspartic residues (Asp569 and Asp570). Our data indi-cated that EGLNs preferred an acidic residue at position+6, but had no clear preference at position +5 (Fig. 2, low-er graphs). Finally, no stop codons were identified at anyposition of the interacting constructs, suggesting a lowEGLN affinity for substrate peptides shorter than 20 aminoacids.
EGLN1/3 show differential substrate preferences
Despite the similar substrate requirements of EGLN1/3,a closer examination of data showed some significant dif-
Fig. 4. EGLN binding to selected CODD mutants. Yeast were transformed wEGLN3 fusion proteins, together with pGAD-CODD plasmids encoding differegrown on plates lacking the indicated nutrients. WT: wild type; SGD: D569S Dshown are representative of at least two independent experiments.
ferences. EGLN1 had a strong preference for aromatic res-idues at +1 that was not matched by EGLN3 (Table 1 andFig. 2). On the other hand, EGLN3 showed a strong pref-erence for Pro residues at position +3 that was not sharedby EGLN1 (Table 1 and Fig. 2). Additionally, the numberof clones encoding for acid residues at position +6 (D570in HIF1a sequence) was much higher in the EGLN3screening (Table 1 and Fig. 2). These differences are reflect-ed in the information content at positions +1, +3, and +6(Fig. 3).
In view of these results, we tested the effect of specificmutations at +1, +3, and +6 on EGLN1/3 binding toCODD (Fig. 4). In agreement with the above data, substi-tution of Tyr at position +1 (Y565) by other residues, suchas Cys or Glu, had a strong effect on EGLN1, but notEGLN3, binding (Fig. 4B). Interestingly, Y565A substitu-tion had only a modest effect (Fig. 4B), in accord with thefrequency of Ala at this particular position (Table 1). Incontrast to the strong effect of specific single mutationson EGLN1 binding (Fig. 4A and B), mutations at positions+3 or +6 had no detectable effect on EGLN3 binding(Fig. 4A). These results raise the possibility that theEGLN3-CODD interphase comprises multiple residues,whose interactions cooperate in the generation of a highaffinity binding that is largely unaffected by individualsubstitutions.
ith pBRIDGE plasmid, encoding for Gal4DBD-DEGLN1 or Gal4DBD-nt mutants of HIF1a(554–576). Serial dilutions of transformed clones were570G double mutant. Other symbols and labels are as in Fig. 1. The results
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Discussion
Previous works demonstrated that EGLNs showed adifferential enzymatic activity toward isolated ODDs[12,13]. The results reported herein strongly suggest thatthose differences in activity are due to specific binding pref-erences of each EGLN. Our data also indicate that bindingto a specific ODD, within full length HIFa, does notrequire the presence of an additional ODD in the samemolecule. In contrast to our results, a recent report [20],using hydroxylation specific antibodies, found that hydrox-ylation of the CODD sequence within HIF1a precedes andis required for that of NODD. Moreover, contrary to ourobservations and those of other groups [12,13], EGLN3was found to hydroxylate Pro402 in vivo efficiently [20].Therefore, further studies are required to fully elucidatethe complex regulation of HIFa by EGLNs.
The differential binding of EGLN1/3 to each ODD wasalso observed when the interaction was tested against theisolated ODDs. Thus, the main sequence requirementsfor EGLN should be contained in a small region (from�9 to +12) around the target proline. However, it shouldbe noted that, in the case of EGLN1, the binding to the iso-lated ODDs is weaker than to the sequences within the fulllength protein. Thus, interactions with residues outside thisminimal ODD are likely required for the optimal bindingof EGLN1 to HIFa. In agreement with this conclusion, awork was published [18] during the writing of this manu-script showing that the activity of EGLNs toward ODD-derived sequences, in particular that of EGLN1, is stronglyaffected by peptide length.
In order to identify the residues required for the differen-tial ODD preference, we investigated the sequence determi-nants in CODD for the binding of EGLN1 and EGLN3.Although other studies have previously addressed the rele-vance of specific residues within ODD for EGLN binding/activity [9–11,19], most of these works based their conclu-sions on mutation of the wild type residue to a single differ-ent amino acid. In contrast, in our strategy, we tested eachposition for all potential mutations, hence obtaining moredetailed information about sequence requirements for sub-strate binding. In the work by Li et al. [10] a complete anal-ysis of selected positions within the LXXLAP motif wascarried out. However, positions outside this motif werenot investigated in such detail. In the present work weinvestigated positions ranging �6 to +10 and found posi-tions outside the LXXLAP region that were relevant forbinding. Thus, our results indicate that both enzymes havevery similar, albeit not identical, residue preferencestowards their substrate sequence. These differences maybe exploited in the design of specific inhibitors for each iso-form. Finally, unlike previous works [10], the strategy usedherein provides quantitative data regarding residue prefer-ence at each position. Thus, these results may be of help forthe identification of putative EGLN substrates. In fact, adatabase search with a position-specific scoring matrixbased on the data on Table 1 was able to identify HIFa
proteins among the potential substrates with the highestscore (data not shown).
Acknowledgments
This work was supported by grants from Fondo deInvestigaciones Sanitarias (FIS01/0264 to L.P.), from Min-isterio de Ciencia y Tecnologıa (SAF2002-02344 andSAF2005-00180 to L.P. and SAF2004-00824 to M.O.L.),and from Red Cardiovascular (RECAVA).
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