Anne Uimari
email: anne.uimari@uku.fi

Tuomo A Keinänen

Anne Karppinen

Patrick Woster

Pekka Uimari

Juhani Jänne

Leena Alhonen
email: leena.alhonen@uku.fi

A I Virtanen

Spermine analogs regulated expression of spermidine/spermine N 1acetyltransferase and its effects on depletion of intracellular polyamine pools in mouse fetal fibroblasts

Keywords: , spermine analogs, polyamines, gene expression, enhanced green fluorescent protein (EGFP) acetyltranferase, EGFP, enhanced green fluorescent protein, DENSpm,

Spermine analogs regulated expression of spermidine/spermine N1-acetyltransferase and its effects on depletion of intracellular polyamine pools in mouse fetal fibroblasts

INTRODUCTION

The polyamines putrescine, spermidine and spermine are essential in central cellular processes including proliferation and differentiation . The intracellular polyamine pools are tightly regulated by means of biosynthesis, catabolism, uptake and export. In catabolic pathway spermidine/spermine N 1acetyltransferase (SSAT) catalyzes the acetylation of spermine and spermidine making them suitable substrates for the next enzyme in the pathway, polyamine oxidase, or for the polyamine export machinery. Multiple factors regulate SSAT activity by controlling the gene expression at one or several levels. For example elevated intracellular polyamine concentrations regulate SSAT by increasing gene transcription, stabilizing mRNA, enhancing mRNA translation and increasing protein stability . Similarly, polyamine analogs, the mimics of natural polyamines, can effect SSAT expression at multiple levels. Several types of N-alkylated polyamine analogs have been synthetized. They vary in their ability to displace the natural polyamines in the cell as well as to fulfil the biological functions of natural polyamines. In addition, the analogs in general are poor substrates for the polyamine catabolic enzymes. Because of the stability and the functional ability to regulate polyamine metabolic enzymes as well as by the ability to deplete intracellular polyamine pools, the analogs produce a long-term effect which disrupts polyamine metabolism. As a consequence, disturbed cellular functions and cytotoxicity may result. The development of polyamine-based molecules for therapeutic purposes is an ongoing research field and for example the N-terminally alkylated polyamine analog N 1 , N 11 -bis(ethyl)norspermine (DENSpm) has been in clinical trials against several human solid tumors . Antiproliferative action of DENSpm seems at least in some cell lines to be connected to the induction of SSAT . DENSpm increases SSAT activity by thousands of folds and effects hnRNA splicing, increases SSAT mRNA level, enhances translation and effects protein stability . In addition to symmetrically substituted alkylpolyamine DENSpm, several unsymmetrically substituted alkylpolyamine analogs such as CPENSpm (N 1 -ethyl-N 11 -((cyclopropyl)methyl)-4,8diazaundecane) and CHENSpm (N 1 -ethyl-N 11 -((cycloheptyl)methyl)-4,8-diazaundecane) have been studied with the respect to their cytotoxicity and ability to induce SSAT. Like DENSpm, CPENSpm is an effective superinducer and inhibitor of SSAT and has antitumor action in vitro . Interestingly, CHENSpm is highly cytotoxic, inhibits SSAT but does not effectively elevate SSAT activity . In the present work, the spermine analog-mediated regulation of SSAT in non-tumour cells was studied. We produced a SSAT-EGFP (enhanced green fluorescent protein) fusion construct to facilitate the use of detectable marker protein. The expression, localization, activity and function of SSAT-EGFP in response to spermine analogs in transiently transfected SSAT-deficient mouse fetal fibroblats (devoid of endogenous SSAT activity) were followed. Addition of relatively stable EGFP at the carboxyl terminus of SSAT abolished neither activity nor low abundance of the SSAT enzyme. For comparison the wild type mouse fetal fibroblasts were used to analyze the regulation of genome-encoded SSAT. The analog-affected changes in SSAT activities of SSAT-EGFP and endogenous SSAT were very similar, although, as expected, the regulation of genome-encoded SSAT by the analogs was shown to be more complex. Interestingly, the levels of intracellular polyamine pools followed the degree of SSAT activity but, as shown in the SSATdeficient cells, were decreased also by mechanisms independent of the SSAT function.

EXPERIMENTAL Materials

DENSpm was synthesized essentially as described by Rehse et al. . CHENSpm and CPENSpm were synthesized as previously described . Generation of mice with disrupted SSAT-gene is described previously .

Syngenic and SSAT-deficient primary fetal fibroblasts were isolated according to the previously described method , except that the fetuses were taken on day 16 of pregnancy. The cells were seeded in six well plates at a density of 3.5 x 10 5 cells/well and grown in DMEM (Sigma) supplemented with 10% FBS and 50 µg/ml of gentamycin at 37°C in 7.5% CO 2 atmosphere. Polyamine analogs were used in 10 µM concentrations.

Production of SSAT-EGFP fusion construct

The open reading frame region of the mouse SSAT gene was amplified from the cloned cDNA with the primers 5'-CTAAAGCTTCAAAAGACGAAAATGGC-3' (forward, the start codon underlined) and 5'-GATGGATCCTCCTCTGCTGCCATTTTTTAG-3' (reverse, the last codon underlined). The PCR product was digested with BamHI and HindIII restriction enzymes and ligated in frame with the EGFP gene into the pEGFP-N1 vector (Clonetech). The structure of the constructed plasmid was confirmed by automated sequencing. Plasmid DNAs for transfection were prepared using QIAFilter Maxi kit (Qiagen) following the manufacturer's protocol.

Transfection

Cells were transfected with calcium phosphate as follows. The cells were seeded in six well plates at a density of 3.5 × 10 5 cells/well and incubated for 24 hours. Two micrograms of plasmid DNA was mixed with 40 µl of 0.25 M CaCl 2 . Then 40 µl of 2 x BBS (50 mM BES, pH 6.95, 280 mM NaCl, 1.5 mM Na 2 HPO 4 ) was added and the mixture was incubated for 30 minutes at RT. The medium was removed and the cells were washed twice with DMEM. The wells were covered with 1.5 ml of DMEM and 80 µl of plasmid preparation was added into each well. The plates were kept for 2.5 hours in the incubator, after which the medium was removed and the cells were washed as described above. Growth medium without or with the analogs was added and the cells were incubated for 24 hours.

Analysis of ODC and SSAT activities and intracellular polyamine contents

The activities of ODC and SSAT were measured according to the published methods. Natural polyamines were determined by HPLC as described previously . Polyamine analogs were measured using a previously described method .

Western blot analysis

Trypsinized and washed (PBS) cells were lysed in a buffer containing 25 mM Tris (pH 7.4), 0.1 mM EDTA, 0.1% Triton-X-100, 1 mM dithiotreitol, and 1 × Complete EDTA-Free protease inhibitor cocktail (Roche). Samples were centrifuged (15 min, 10 000g, +4°C) and protein contents from the supernatants were measured by Bio-Rad Protein Assay kit. Western blot analysis was performed with standard protocols using 30 µg of protein and GFP antibody (BV living colors A.v. peptide antibody, Clonetech) or SSAT antibody. SSAT antibody was affinity-purified from the serums of rabbits injected with the E. coliproduced recombinant mouse SSAT protein. Ponceau staining of the membrane and detection of β-actin (Sc1616, Santa Cruz Biotechnology) was used to confirm the equal loading.

Reverse transcription-PCR

Total RNA from the cells was extracted by TRIzol Reagent (Invitrogen) and treated with Dnase I (Dnafree, Ambion) according to the manufacturer's instructions. One microgram of Dnase-treated RNA was used for first-strand cDNA synthesis using oligo-dT primers and AMV reverse transcriptase (Promega) in a total volume of 25 µl. PCR reaction contained 2 µl of the first-strand cDNA, 1x PCR buffer with MgCl 2 (Finnzymes), dNTP mix (200 µM each), 25 pmol forward and reverse primers and 0.5 U DyNAzyme DNA polymerase (Finnzymes) in a total volume of 50 µl. The used primers were as listed below. SSAT: 5'-TACGTCGACATGGCTAAATTTAAGATCCG-3' (forward), 5'-TACGTCGACGATACAGCAACTTGCCAATC-3' (reverse); EGFP: 5'-CCCTGAAGTTCATCTGCAC -3' (forward), 5'-GTTGTACTCCAGCTTGTG-3' (reverse); SSAT-X: 5'-TACGTCGACATGGCTAAATTTAAGATCCG-3' (forward), 5'-TACGTCGACCAATGCTATGTCCTCATTTAT -3' (reverse); 18S RNA: 5'-CTACCACATCCAAGGAAG-5' (forward), 5'-CTCAGCTAAGAGCATCGAG-3' (reverse). The program consisted one cycle of 95°C for 3 min, 24 (18S RNA), 25 (SSAT, EGFP) or 29 (SSAT-X) cycles of 95°C for 30 seconds, 58°C for 40 seconds, 72°C for 40 seconds followed by one cycle of 72°C for 5 min. PCR products were separated on a 0.9% agarose gel and scanned by Typhoon 9400 (GE Healthcare). The images were analyzed by the Image Quant TL program (GE Healthcare).

Microscopy

To examine the fluorescence intensity, the cells were cultured on coverslips. After incubation the cells were washed with PBS and fixed with 4 % paraformaldehyde for 30 min. After that the cells were washed twice for 10 min with PBS. For propidium iodide (PI) staining, the cells were permeabilized with 0.3% Triton-X-100 (Fluka) in PBS for 15 min at room temperature and washed twice for 10 min with PBS. Then the cells were treated with RNase (Boehringer Mannheim Biochemicals) at concentration of 100 µg/ ml in PBS for 20 min at room temperature and washed three times with PBS. The cells were counterstained with PI (Sigma) in PBS at final concentration of 500 ng/ml for 5 minutes and washed three times with PBS. The coverslips were mounted on glass slides and examined under Olympus AX 70 fluorescence microscopy or under Biorad Radiance 2100 confocal laser scanner. An argon ion laser emitting at 488 and 543 was used to excite EGFP and PI respectively. During scanning, a 40 x oil immersion objective was used. Serial optical slices of 1 µm or less were taken.

Flow cytometry analysis

Transfected cells in the six well assay plates were trypsinized, washed (PBS) and suspended in 0.5 ml of PBS. To fix the cells, 0.5 ml of 4 % PFA was added. Proportion of EGFP positive cells was analyzed with flow cytometry (FACScalibur, Becton Dickinson). EGFP expression level was obtained by dividing the mean fluorescence intensity (FL1 channel) of the EGFP positive population by the mean fluorescence intensity of the EGFP negative population.

Determination of SSAT and SSAT-EGFP half-lives

The syngenic and SSAT-deficient cells were seeded in six well plates at a density of 3.5 x 10 5 cells / well. SSAT-decifient cells were transfected with SSAT-EGFP as described above and both the syngenic and transfected cells were grown without or with 10 µM concentration of the analogs for 24 hours. The media was replaced with media containing cycloheximide at final concentration of 10 µg / ml without or with the analogs. Each treatment was repeated three times (experiments). Samples in triplicate were collected from each treatment in four or five timepoints ranging from 0 minutes to 10 hours. SSAT activity of the samples was detected as described above. Average SSAT activity values were calculated for the

Statistical analysis

Data are expressed as mean ± SD where applicable. GraphPad Prism 4.03 (GraphPad software) software package was used to perform one way ANOVA and Dunnett's multiple comparison test. Linear regression analyses were performed using GLM procedures of the SAS v.9.1 -statistical package (SAS Institute Inc.).

RESULTS

Accumulation of SSAT-EGFP in untreated and spermine analogs treated cells

In order to study the regulation and intracellular localization of SSAT, a CMV promoter driven fusion construct containing the protein coding region of SSAT at the amino terminus of EGFP was made. SSATdeficient mouse fetal fibroblasts were transfected with the SSAT-EGFP fusion construct or an EGFP control as described in experimental procedures. Cells were grown for 24 hours either without or with 10µM DENSpm, CPENSpm or CHENSpm. During that time, no cytotoxicity was observed (N1/N0 value ≥1). The cells were fixed with 4% paraformaldehyde and studied under fluorescence microscopy. The control cells grown without or with the analogs strongly expressed EGFP and were easily visualized by the microscope. In contrast, the SSAT-EGFP-transfected cells grown without the analogs or treated with CHENSpm contained no detectable signal. However, the intensity of fluorescence in the fusion construct containing cells was enhanced by DENSpm and CPENSpm treatments and could be observed under fluorescence microscopy. The differences in fluorescence intensities of the samples were confirmed by measuring the values for relative fluorescence by flow cytometry. As shown in Figure 1A, the fluorescence of EGFP-expressing control cells was decreased by the analogs, however still remaining several times higher than the fluorescence in SSAT-EGFP-expressing cells. As expected, significant increase in the fluorescence intensity was detected in the SSAT-EGFP-expressing cells grown with DENSpm or CPENSpm compared with the cells without the analog treatment. Fluorescence in the fusion construct-expressing cells grown with CHENSpm was undistinguishable from that of the untreated cells.

To further visualize the presence of fluorescent proteins a western blot analysis was performed from the protein samples of the cells grown under similar conditions as described above. The EGFP control protein was easily detected in high amounts in all EGFP-transfected samples (data not shown). In contrast, in the SSAT-EGFP transfected cells the GFP and SSAT antibodies detected a very low amount of the fusion protein only in the homogenates from the DENSpm-treated cells (data not shown). For comparison, the abundance of SSAT protein in the samples of analog-treated wild-type mouse fetal fibroblasts was analyzed with a western blot. It is known that SSAT is expressed in a very low level and as seen in figure 1B only DENSpm treatment increased the SSAT amount into a detectable level in the experimental set-up and with that particular SSAT antibody used. However, the results supported the view obtained from the cells expressing the SSAT-EGFP fusion protein. Thus, the intensity of the observed fluorescence correlated with the abundance of EGFP and SSAT-EGFP proteins.

Intracellular localization of SSAT-EGFP

To study the intracellular localization of SSAT-EGFP, the same samples which were used for fluorescence microscopy were also analyzed with confocal microscopy. In the case of EGFP control A c c e p t e d M a n u s c r i p t expressing cells, the fluorescent protein localized strongly both in the cytoplasm and the nucleus as shown in the merged images of EGFP and propidium iodide -stained nuclei (Fig. 2). Studied analogs had no effect on the localization of EGFP. Because of the higher intensity of the excitation light used in confocal microscope, it was possible to visualize the SSAT-EGFP protein both in untreated and analog-treated cells. In all samples, SSAT-EGFP was localized both in the cytoplasm and the nucleus (Fig. 2). However, occasionally cells in which the analog treatment reduced or fragmented the nuclear localization of SSAT-EGFP were found (data not shown). Whether this reflects the action of SSAT-EGFP or the analog induced changes in the physiology and condition of the treated cells in general remains to be resolved.

Analog-affected SSAT activities and intracellular polyamine levels

Spermine analogs DENSPm, CPENSpm and CHENSpm all act as inhibitors of SSAT in vitro . However, only DENSpm and CPENSpm superinduce the enzyme in living cells. To study the function of SSAT-EGFP fusion protein the SSAT activity and the intracellular polyamine levels in transfected cells grown without or with the analogs were measured. As a control, EGFP transfected cells were subjected to similar analysis. SSAT-deficient mouse fetal fibroblasts were used because they lack endogenous SSAT activity. As shown in table 1, the presence of SSAT-EGFP led to nine fold induction in SSAT activity in transfected untreated cells when compared with the cells transfected with EGFP. However, the changes in intracellular polyamine levels were only minor. In contrast, treatments with polyamine analogs caused significant changes in SSAT activities and in intracellular polyamine levels. In DENSpm-treated cells, the activity of SSAT-EGFP was thirty times higher than in the cells grown without the analog and the intracellular spermidine and spermine levels were significantly depleted. CPENSpm and CHENSpm activated SSAT-EGFP by more than sixteen and three folds, respectively, and also significantly decreased spermidine and spermine levels. Also significant differences in the accumulation of putrescine were found: DENSpm depleted and CHENSpm increased the amount of putrescine. The function of SSAT-EGFP protein in transfected cells was also confirmed by the accumulation of N 1 -acetylspermidine which is considered as an indicator of SSAT activity. DENSpm treatment caused the highest enzyme activity and protein accumulation of SSAT-EGFP compared with CPENSpm and especially with CHENSpm. Thus, in the case of SSAT-EGFP the observed SSAT activities correlated with the fluorescence and abundance of the fusion protein detected by microscopy, flow cytometry and western blot analysis. Instead, surprising results were obtained from the SSAT-deficient cells transfected only with the EGFP gene. In all samples there were only a low background level of SSAT activity (cells have deficient SSAT gene). However, all tested analogs depleted the intracellular spermidine and spermine levels significantly. In addition, DENSpm and CHENSpm significantly affected the putrescine levels. Interestingly, the changes in polyamine pools in EGFP expressing cells were exactly similar than those in the cells transfected with SSAT-EGFP. Therefore, it seems that all tested analogs induce also a SSAT-independent mechanism to reduce intracellular polyamines in mouse fetal fibroblasts.

In order to compare the SSAT activities of the SSAT-EGFP fusion protein and the endogenous SSAT protein, as well as to study the intracellular polyamine levels, the wild-type fetal fibroblasts were subjected to similar analog treatments as described above. As shown in table 2, the SSAT activities and polyamine pools observed in the wild-type cells with and without the analog treatments were very similar as the ones obtained from the SSAT-EGFP transfected cells deficient of endogenous SSAT.

ODC activity in spermine analog -treated cells

To see how the spermine analogs affect to the production of polyamines, the activity of ornithine decarboxylase (ODC), the key enzyme in the polyamine biosynthetic pathway, was measured in the SSAT-deficient and wild-type mouse fetal fibroblasts. The results are shown in table 3. DENSpm significantly reduced the ODC activity in both cell lines. Interestingly, CPENSpm significantly increased the activity in wild-type cells and significantly decreased the activity in SSAT deficient cells. However, CHENSpm did not change the ODC activity in either line. Thus, the decreased intracellular polyamine pools in the analog-treated cells do not totally correlate with the changes observed in biosynthesis.

SSAT mRNA expression in analog treated cells

As the amount of SSAT-EGFP protein was clearly increased by DENSpm and CPENSpm, studies to analyze the steady state level of the fusion construct mRNA were conducted. Reverse transcriptase PCR was run with the primers for the EGFP part, the SSAT part or the junction part of the fusion construct and they all gave similar results. Figure 3 shows the accumulation of EGFP and SSAT-EGFP mRNA-derived signals amplified with EGFP specific primers. Relative optical density (ROD) of the mRNA concentration using target gene / 18S rRNA indicated that CPENSpm slightly increased the transcription of EGFP in the pEGFP-N1 control plasmid-transfected cells. Instead, transcription of SSAT-EGFP fusion construct was not significantly affected by the analog treatments. If anything, DENSpm slightly decreased the amount of SSAT-EGFP mRNA. Thus, the increased amount of SSAT-EGFP protein detected in the DENSpm and CPENSpm exposed cells was not a consequence of enhanced transcription.

It is known that DENSpm stabilizes wild-type SSAT mRNA and also fine-tunes the expression of SSAT by regulated unproductive splicing and translation (RUST) . However, the effects of CPENSpm and CHENSpm have not been published. In order to see how the analogs affect transcription of the genomic SSAT gene and splicing of the SSAT pre-mRNA, RT-PCR from the analog-treated wild-type mouse fetal fibroblasts was performed. The primers detecting the normal SSAT mRNA and the primers amplifying the alternative splice variant, SSAT-X, were used. The results are shown in figure 4. Significantly increased amount of SSAT mRNA was present in the cells treated with DENSpm and CPENSpm. In contrast, CHENSpm did not change the amount of SSAT mRNA pool. In accordance with the previous report , significant decrease in the accumulation of SSAT-X, the unproductive splice variant, in DENSpm treated cells was observed when compared with the amount present in the untreated cells. Interestingly, the unproductive splicing of SSAT pre-mRNA was enhanced by CHENSpm, the analog not able to superinduce SSAT. On the other hand, CPENSpm did not affect the SSAT pre-mRNA splicing compared with the untreated cells. Differences in analog-affected transcriptional regulation between the endogenous SSAT and the transfected SSAT-EGFP fusion construct reflect the important influence of SSAT promoter and the whole genomic sequence, including introns, for the regulation and expression of the gene.

Effects of the analogs on the half-lives of SSAT and SSAT-EGFP proteins

In order to study how the analogs affect the stability of SSAT and SSAT-EGFP the half-lives of SSAT and SSAT-EGFP without or with the analogs were determined by analysing the changes in SSAT activity after protein synthesis inhibitor cycloheximide addition. The results are presented in Table 4. The halflives of SSAT and SSAT-EGFP were lowest in the untreated cells and were comparable to each other. Both DENSpm and CPENSpm increased the half-lives of SSAT and SSAT-EGFP. The extended halflives of the proteins were similar compared with each other. However, DENSpm extended the half-lives more intensively than CPENSpm. CHENSpm had only a minor effect on the SSAT half-life, but interestingly, seemed to increase the half-life of SSAT-EGFP quite extensively. Altogether, the results suggest that the wild-type SSAT and SSAT-EGFP have similar half-lives which are extended by spermine analogs.

DISCUSSION

We have generated a SSAT-EGFP fusion construct which is expressed in low amounts but with detectable SSAT activity in transiently transfected SSAT-deficient mouse fetal fibroblasts. The enzyme activity and intracellular accumulation of the fusion protein is significantly enhanced by spermine analogs A c c e p t e d M a n u s c r i p t the fusion product. The studied analogs cause similar changes also in the wild-type cells expressing endogenous SSAT. In addition, in all cases the enhanced SSAT activity correlates with the decreased polyamine levels. Thus, the expression of either endogenous SSAT or the transfected SSAT-EGFP can lead to similar cellular changes in mouse fetal fibroblasts. However, when the regulation of SSAT and SSAT-EGFP expression by spermine analogs is analyzed at different levels some differences can be found. We have shown that the SSAT-EGFP mRNA levels in cells grown without or with the analogs are not significantly different (Fig. 3). Thus, transcription of the fusion construct containing only the coding region of SSAT (no 3' or 5' UTRs) is not enhanced by the analogs and hence the increased transcription is not the reason for protein accumulation. It has been reported previously that the transcription of the FLAG-SSAT construct containing only the protein encoding sequence was not increased, but decreased, by DENSpm . On the other hand, in our study a significant increase in the SSAT mRNA level was obtained in the wild-type mouse fetal fibroblasts showing the ability of DENSpm to induce the SSAT transcript production from the genomic SSAT. This also is the case in vivo since the amount of SSAT transcript in the liver samples isolated from DENSpm-treated mice is higher than in the samples from untreated mice . In addition, we showed that the splicing of SSAT pre-mRNA is directed towards the variant transcript, SSAT-X, by CHENSpm and as reported previously production of the normal SSAT mRNA is enhanced by DENSpm. Thus, the regulation of genome-encoded SSAT is differentially finetoned by spermine analogs. Altogether, our findings confirm that the complete regulation of endogenous SSAT gene expression happens at multiple levels and involves the promoter and the exonic and intronic sequences as well as the 3' and 5' UTRs.

Because of the low expression level, the intracellular localization of SSAT has been difficult. Under DENSpm-induced conditions using immunohistochemistry and immunocytochemistry, SSAT has been detected in the cytoplasm of human adenocarcinoma A549 cells and in the cytoplasm, nucleus and mitochondria of the human breast cancer L56Br-C1 cells, respectively . We were not able to detect the SSAT-EGFP under the fluorescent microscopy unless DENSpm or CPENSpm was added to the growth medium. However, confocal microscopy studies facilitated the detection of the fusion protein in all conditions. SSAT-EGFP localized both in the cytoplasm and in the nucleus in untreated and analog treated samples. The other cell organelles were not studied in detail for the presence of SSAT-EGFP. Since EGFP is widely used to determine the subcellular localization of other proteins SSAT-EGFP could offer a tool to follow the intracellular localization of the low abundant SSAT in different types of cells, including nontumor cells (in normal growth conditions) like used in this study.

The low basal activity of SSAT-EGFP could result from very rapid turnover rate of the protein, like is the case of SSAT itself, or it can be caused by the inefficient translation of the mRNA. In recent studies with the FLAG-tagged SSAT and SSAT fused to Renilla luciferase, it was suggested that DENSpm regulates SSAT mRNA translational efficiency by inhibiting a repressor protein from binding to SSAT transcript . In that study, the half-life of the SSAT-luciferase fusion protein in HeLa cells was not affected by DENSpm; only the translation was more effective. Whether DENSpm and CPENSpm-induced accumulation of SSAT-EGFP is affected by the enhanced translation remains to be analyzed. On the other hand, it has been shown earlier that the SSAT protein stability is enhanced by the presence of DENSpm . Studies to reveal the important regions or amino acids of SSAT protein in terms of proteosomal degradation have indicated the importance of carboxyl-terminal glutamic acids at position 170 and 171 (the two last amino acids of SSAT) . Changing these amino acids to either glutamine or lysine or adding two extra lysine residues at the end of the protein rendered the SSAT protein stable. In our study, EGFP was added at the carboxyl-terminus of the protein and thus, could influence the proteosomal degradation of the protein. However, determination of SSAT and SSAT-EGFP half-lives, based on SSAT activity, suggested that in mouse fetal fibroblasts the half-lives of endogenous SSAT and plasmid expressed SSAT-EGFP are very similar and spermine analogs DENSpm and CPENSpm stabilize the both proteins. Interestingly, also CHENSpm seemed to clearly extent the half-live of the fusion protein in the transfected SSAT-deficient mouse fetal fibroblasts, but did not highly affect the half-life of the SSAT protein in the wild-type cells. The reason why CHENSpm do not highly induce the SSAT activity of SSAT-EGFP although it extends the half-life of the protein is not known. One possible explanation can be that the A c c e p t e d M a n u s c r i p t configuration of the fusion protein allows CHENSpm to perform stabilization but at the same time the activity of SSAT-EGFP is limited. However, the total protein amounts of SSAT-EGFP and SSAT are not increased by CHENSpm as shown in this study and in addition, the production of variant transcript SSAT-X is increased by the analog in the wild-type cells. In general, it is interesting to realize that EGFP is a relatively stable protein with a half-life of 26 hours. The half-life of SSAT-EGFP being close to the halflife of the wild-type SSAT protein makes SSAT-EGFP an unstable variant of EGFP fusions.

In all, several N-alkylated polyamine analogs have been synthesized and evaluated as antitumor agents. Also the analogs used in this study have anti-proliferative or cytotoxic effects. In general, polyamine analogs inhibit cell growth in different ways. One suggested route is the superinduction of SSAT followed by the depletion of intracellular polyamines. As reported previously, the spermine analogs vary in their ability to induce SSAT. In three different human prostate cancer cell lines DENSpm strongly induced SSAT whereas CPENSpm induced it less than DENSpm but clearly more than CHENSpm which gave only a low induction of SSAT activity . Also the intracellular spermidine and spermine levels were less affected by CHENSpm in all three cell lines. In our study, the main purpose was to analyze the regulation of SSAT expression and thus a 24 hours treatment time was used to minimize the growth inhibition or death of the cells by the analogs. However, it needs to be emphasized, that even though cytotoxicity was not detected the reduced EGFP control protein expression in analog-treated cells may reflect decreased cellular functions caused, at least partly, by reduction of intracellular polyamines occurring within this 24 hour treatment time. The reduction of spermidine and spermine pools by all analogs and the slight accumulation of putrescine in CHENSpm and CPENSpm-treated cells as well as the decrease of putrescine level in DENSpm-treated samples were similar in wild-type cells and in the SSAT-EGFP-expressing cells. Interesting results in terms of intracellular polyamine levels in response to the analogs came from the cells expressing EGFP alone. There were no changes in SSAT activity (as expected because the endogenous SSAT is disrupted) but the intracellular polyamine levels were similar to those of wild-type SSAT and SSAT-EGFP expressing cells treated with the analogs. Lower N 1 -acetyl-spermidine levels in the EGFP than in the SSAT-EGFP expressing cells showed the absence of SSAT function and, in the case of DENSpm-treated EGFP transfected cells, the lower value of N 1 -acetyl-spermidine compared with the untreated and CHENSpm and CPENSpm treated EGFP expressing cells again suggested the strong ability of DENSpm to remove the intracellular polyamines. The loss of intracellular polyamines is interesting since the back-conversion of polyamines needs a functional SSAT protein. Although, spermine oxidase can convert spermine to spermidine it is not able to oxidase spermidine to putrescine . Whether there are still some unknown enzymes in the catabolic pathway of polyamines able to catalyze the back conversion remains to be seen. Earlier it has been shown that DENSpm depletes intracellular polyamines in the mouse embryonic stem cells in which the SSAT gene is disrupted .On the other hand, DENSpm treatment of the SSAT-deficient mouse does not lead to depletion of higher intracellular polyamines in several studied tissues, though the animals are more sensitive to the toxic effects of the analog than their syngenic littermates . In addition of stimulating the catabolic enzymes, the analogs can inhibit polyamine biosynthetic enzymes, thus further lowering the intracellular polyamine pools. In this study the ODC activity was clearly reduced by DENSpm and by CPENSpm in SSAT-deficient cells. However, in wildtype cells only DENSpm decreased ODC activity and, in fact, CPENSpm increased it. We did not study the activity of S-adenosylmethionine decarboxylase, AdoMetDC , the other key enzyme in the biosynthesis of polyamines in this study. However, it has been shown earlier that DENSpm and CPENSpm reduce the activity of AdoMetDC, while CHENSpm does not decrease the enzyme activity . Thus, the low intracellular polyamine levels in DENSpm-and CPENSpm-treated cells could partly reflect the reduced biosynthesis but in the case of CHENSpm some additional mechanisms including direct replacement from binding sites and the transport system need to be considered.

In conclusion, the SSAT-EGFP introduced in this paper is encoding a protein with many functional properties similar to SSAT and thus, can be applied to polyamine research. In mouse fetal fibroblasts, the spermine analogs DENspm, CHENSpm and CPENSpm distinctly and systematically regulate the expression of SSAT at multiple levels, including the splicing of pre-mRNA. In addition, these analogs also induce SSAT-independent mechanisms to reduce intracellular polyamines levels.

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  Biochemical Journal Immediate Publication. Published on 27 May 2009 as manuscript BJ20090411 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090411 A c c e p t e d M a n u s c r i p t

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  Biochemical Journal Immediate Publication. Published on 27 May 2009 as manuscript BJ20090411 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090411 A c c e p t e d M a n u s c r i p t triplicates. Based on these average activity values on different time points a linear regression line was fitted to each experiment within a treatment. The half-lives for SSAT and SSAT-EGFP were estimated from the regression line as: half-life = -0.5 * (a/b), where a is the intercept and b is the regression coefficient of the estimated regression line. For each treatment the mean half-life over the three experiments was calculated.

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  Biochemical Journal Immediate Publication. Published on 27 May 2009 as manuscript BJ20090411 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090411 A c c e p t e d M a n u s c r i p t

  DENSpm and CPENSpm. The spermine analog CHENSpm only slightly increases the SSAT activity of 8 Biochemical Journal Immediate Publication. Published on 27 May 2009 as manuscript BJ20090411 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090411

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  Biochemical Journal Immediate Publication. Published on 27 May 2009 as manuscript BJ20090411 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090411

Fig. 1 .

 1 FIGURE LEGENDSFig. 1. Effects of spermine analogs on the accumulation of SSAT protein in the mouse fetal fibroblasts. A) Mouse SSAT-deficient fetal fibroblasts expressing either EGFP or SSAT-EGFP were grown without or with 10 µM analogs for 24 hours. The proportion of fluorescent protein positive cells was determined with flow cytometry. The expression level was obtained by dividing the mean fluorescence intensity of the EGFP-positive population by the mean fluorescence intensity of the EGFP-negative population. Representative experiment of two is presented. Data are shown as mean ± S.D., where n = 3. *p < 0.05, **p < 0.01 compared with the corresponding untreated control groups. B) The cell lysates of wild-type mouse fetal fibroblasts grown without or with 10 µM analogs for 24 hours were analyzed for the presence of SSAT by western blot. Three independent samples of each treatment are shown. ß-actin was used as a loading control.

Fig. 2 .Fig. 3 .Fig. 4 . 14 Biochemical

 23414 Fig. 2. Intracellular localization of EGFP and SSAT-EGFP in analog-treated cells. Control pEGFP-N1 and SSAT-EGFP fusion construct transfected cells were treated with analogs for 24 hours, fixed and counterstained with propidium iodide (PI) to allow nuclei to be visualized. Confocal images were obtained from the same field for detection of EGFP (green) or PI (red) fluorescence. Images were merged to obtain the co-localization of the EGFP and PI (yellow). Note that also the nuclei of untransformed cells are visible. Fig. 3. Transcription of SSAT-EGFP and EGFP in analog treated cells. (Upper figure) RT-PCR with EGFP derived primers recognizing EGFP and SSAT-EGFP in pEGFP-N1 or SSAT-EGFP construct transfected cells, respectively. The lanes represent independent samples derived from transfected cells grown without or with 10 µM analogs for 24 hours. 18S rRNA was amplified as an internal control. (Lower figure) the intensity of the RT-PCR bands was quantified by densitometry and ROD was determined by dividing the EGFP (representing EGFP or SSAT-EGFP) value by the 18S rRNA value. Data are shown as mean ± S.D., were n = 3. *p < 0.05 compared with corresponding untreated control group. Fig. 4. Accumulation of SSAT and SSAT-X mRNAs in analog treated cells. (Upper figure) SSAT and SSAT-X were amplified by RT-PCR from samples of untreated wild-type mouse fetal fibroblasts or cells treated with 10 µM analogs for 24 hours. 18S rRNA was amplified as an internal control. The lanes represent independent samples. (Lower figure) intensity of the RT-PCR products in the gel was quantified by densitometry. ROD was determined by dividing the SSAT and SSAT-X values by the 18S rRNA value. Data are shown as mean ± S.D. (n = 3). *p < 0.05 ,**p < 0.01 compared with untreated control group.

Biochemical

  Journal Immediate Publication. Published on 27 May 2009 as manuscript BJ20090411 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090411 A c c e p t e d M a n u s c r i p t TABLE 2 Intracellular polyamine pools of wild-type mouse fetal fibroblasts. Cells were grown for 24 hours without or with 10 µM spermine analogs. Data are expressed as mean ± S.D. (n = 30.05, b p < 0.01, statistical significance of difference between no analog and analog treated cells NA, not applicable 16 Biochemical Journal Immediate Publication. Published on 27 May 2009 as manuscript BJ20090411 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090411 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

  wild-type and SSAT-deficient mouse fetal fibroblasts. Cells were grown for 24 hours without or with 10 µM spermine analogs. Data are expressed as mean ± S.D. (n = 3). Treatment ODC activity (pmol/30 min/mg protein) a a p < 0.05, b p < 0.01, statistical significance of difference between no analog and analog treated cells 17 Biochemical Journal Immediate Publication. Published on 27 May 2009 as manuscript BJ20090411 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090411 A c c e p t e d M a n u s c r i p t TABLE 4 Half-lives of SSAT and SSAT-EGFP detected in the wildtype and in the SSAT-EGFP transfected SSAT-deficient mouse fetal fibroblasts. Mean ± S.D. of three independent experiments performed in triplicates is shown. Publication. Published on 27 May 2009 as manuscript BJ20090411 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090411 A c c e p t e d M a n u s c r i p t

Figure 1 . 19 Biochemical

 119 Figure 1.

Figure 2 . 20 Biochemical

 220 Figure 2.

Figure 3 . 21 Biochemical

 321 Figure 3.

Figure 4 . 22 Biochemical

 422 Figure 4.

6

  Biochemical Journal Immediate Publication. Published on 27 May 2009 as manuscript BJ20090411 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090411

TABLE 1

 1 Intracellular polyamine pools of EGFP and SSAT-EGFP transfected SSAT-deficient mouse fetal fibroblasts. Cells were grown for 24 hours without or with 10 µM spermine analogs. A representative of two experiments performed in triplicates is shown. Data are expressed as mean ± S.D. (n = 3).

				Polyamine pools (pmol/µg protein)
	Treatment / Transgene SSAT activity
		(pmol/10 min/mg protein)	Put	N 1 -AcSpd	Spd	Spm	Analog
	No analog
	EGFP	39 ± 16	1.5 ± 0.10	0.82 ± 0.05	17.8 ± 0.42	5.6 ± 0.19	NA
	SSAT-EGFP	347 ± 112	2.0 ± 0.26	0.88 ± 0.10	15.9 ± 0.16	5.0 ± 0.23	NA
	DENSpm
	EGFP	19 ± 19	0.9 ± 0.08 b	0.65 ± 0.09 a	5.4 ± 0.38 b	2.1 ± 0.17 b	16.3 ± 1.15
	SSAT-EGFP	10564 ± 2853 b	1.1 ± 0.12 b	0.95 ± 0.11	5.3 ± 0.49 b	2.4 ± 0.19 b	17.3 ± 0.98
	CHENSpm
	EGFP	59 ± 28	2.4 ± 0.30 b	0.79 ± 0.05	13.4 ± 0.22 b	4.4 ± 0.02 b	10.3 ± 0.92
	SSAT-EGFP	1185 ± 197	2.8 ± 0.09 b	1.44 ± 0.06 a	12.6 ± 0.66 b	4.5 ± 0.26 b	10.4 ± 0.15
	CPENSpm
	EGFP	45 ± 1	1.7 ± 0.07	0.79 ± 0.04	10.2 ± 0.42 b	3.4 ± 0.14 b	12.8 ± 1.77
	SSAT-EGFP	5805 ± 51 b	2.3 ± 0.06	1.05 ± 0.10	9.6 ± 0.13 b	3.4 ± 0.07 b	13.2 ± 1.25
	a p < 0.05, b p < 0.01, statistical significance of difference between no analog and analog treated cells
	NA, not applicable
			15

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

© 2009 The Authors Journal compilation © 2009 Portland Press Limited

ACKNOWLEDGEMENTS

We gratefully acknowledge Ms Tuula Reponen for HPLC analysis and Ms Arja Korhonen and Ms Sisko Juutinen for their skilful technical assistance.

FUNDING

This work was supported by the Academy of Finland.


Lucia Banci

Ivano Bertini
email: ivanobertini@cerm.unifi.it

Vito Calderone

Nunzia Della-Malva

Isabella Caterina Felli

Sara Neri

Anna Pavelkova

Antonio Rosato

Prof Ivano Bertini

Copper(I)-mediated protein-protein interactions result from suboptimal interaction surfaces

Keywords: HAH1, ATP7A, ATP7B, Menkes disease, metal homeostasis

come

Introduction

Many functional processes in the cell occur through weak, transient interactions among proteins. For these cases, the interaction is associated to a small, negative change in free energy. Indeed, if the free energy change were large and negative, the complex would be stable and nontransient, whereas if it were positive, no interaction would occur. The study of these interactions is crucial for the thorough understanding of the mechanisms at the basis of Life and of their alterations caused by mutations. Metal homeostasis in cells, which includes uptake, controlled distribution to metalloproteins, storage, and excretion of metal ions, is a process involving many transient interactions.

Copper is utilized as a cofactor in a variety of proteins [START_REF] Linder | Biochemistry of Copper[END_REF], but it can be potentially toxic in vivo and thus its intracellular concentration is presumably strictly controlled [START_REF] O'halloran | Metallochaperones: An Intracellular Shuttle Service for Metal Ions[END_REF][START_REF] Harrison | Intracellular copper routing: the role of copper chaperones[END_REF][START_REF] Puig | Molecular mechanisms of copper uptake and distribution[END_REF]. Its traffic and homeostasis occur through copper-mediated protein-protein interactions [START_REF] Banci | The Atx1-Ccc2 complex is a metal-mediated proteinprotein interaction[END_REF], where copper(I) is bound to ligands from both proteins of the complex. The free energy of formation of copper(I)mediated protein-protein adducts is the outcome of the balance of the metal-donor(s) bond energies, of the hydrophobic and hydrophilic interaction energies at the interface and of the entropic implications related to the establishment of the above interactions.

In order to better understand the processes of metal binding and transfer, we have focused on copper trafficking from the cytoplasm to the trans-Golgi network (TGN), where copper is inserted in a number of enzymes [START_REF] Yuan | The Menkes/Wilson disease gene homologue in yeast provides copper to a ceruloplasmin-like oxidase required for iron uptake[END_REF]. In the cytoplasm, copper(I) is bound to a metallochaperone which delivers the metal to a variable number of soluble metal-binding domains (MBDs) of specific ATPases located at the TGN membrane [START_REF] O'halloran | Metallochaperones: An Intracellular Shuttle Service for Metal Ions[END_REF]. In yeast, the metallochaperone is called Atx1 and the ATPase is Ccc2 [START_REF] Lin | A role for the Saccharomyces cerevisiae ATX1 gene in copper trafficking and iron transport[END_REF]. In humans, the metallochaperone is called Atox1 or HAH1 and two target ATPases exist: ATP7A and ATP7B [START_REF] Hamza | Interaction of the copper chaperone HAH1 with the Wilson disease protein is essential for copper homeostasis[END_REF]. Both ATP7A and ATP7B have six soluble MBDs. The present research aims at shedding light on the nature of the interaction between the metallochaperone and a MBD of the ATPases in the general frame of understanding the energetics of copper(I)-mediated interactions. With this in mind, we addressed the study of the copper(I)mediated complex between HAH1 and the first MBD of ATP7A (MNK1). We also investigated whether introducing cadmium(II) instead of copper(I) had an impact on the interaction. The structures of the copper(I)-and cadmium(II)-mediated adducts are here reported, the former in solution and the latter as a crystal (whose existence in solution was validated by 113 Cd NMR). We probed some of the key interactions observed in the structures by site-directed mutagenesis.

Experimental

Sample preparation

We cloned and expressed the first soluble metal-binding domain of ATP7A (MNK1), corresponding to amino acids 5-77 of the full-length protein, using essentially the same protocol adopted for other individual MNK domains [START_REF] Banci | Solution structure and backbone dynamics of the Cu(I) and apo-forms of the second metal-binding domain of the Menkes protein ATP7A[END_REF]. WT-and mutant HAH1 protein samples were produced as previously reported [START_REF] Anastassopoulou | Solution structure of the apo-and copper(I) loaded human metallo-chaperone HAH1[END_REF]. Further details are supplied in the Supplementary Information section.

Structural analysis

All NMR experiments were collected at 298K except on Bruker Avance spectrometers equipped with a triple resonance cryoprobe. The experiments for the sequence-specific assignment of resonances were acquired on the samples in which only one of the partners were 13 C, 15 N labelled, while the other was unlabeled. The 1D 113 Cd experiment was acquired with a BBO probe at 600 MHz. Tables S1, S2 report details on the NMR experiments performed. The dynamics of the protein backbone was investigated through 15 N relaxation [START_REF] Ishima | Protein dynamics from NMR[END_REF] and allowed us to estimate the overall rotational correlation time of the molecules (Table S3). Combined NMR chemical shift variations were calculated from the experimental 1 H and 15 N chemical shift variations (Dd( 1 H) and Dd( 15 N), respectively) measured between corresponding peaks, through the following equation [START_REF] Garrett | Identification by NMR of the binding surface for the histidine-containing phosphocarrier protein HPr on the N-terminal domain of enzyme I of the escherichia coli phosphotransferase system[END_REF].

( ) ( ) ( ) ( ) 2 N 25 1 H 2 15 2 1 d d d D + D = D combined
The intramolecular cross-peaks used for structure calculations were extracted from 3D NOESY-HSQC spectra acquired on samples where the apo-form of one of the partners, enriched in 15 N and 13 C, was mixed with a 1.5 molar excess of the unlabeled copper(I)-form of the other partner. The intermolecular cross-peaks were integrated in w 1 -13 C-edited, w 2 -13 C-separated, w 3 -13 C, 15 N-filtered NOESY experiments acquired on samples containing 1:1:1 mixture of copper(I):enriched partner:unlabeled partner.

Crystals of HAH1-Cd(II)-MNK1 belonging to the spacegroup P2 1 2 1 2 1 were obtained using the sitting drop vapor diffusion method at 16° C from a solution containing 0.1 M sodium citrate, 20% PEG-6000, 0.8mM complex concentration, at pH 4.7.

Structural models of all MBDs of the MNK protein were built using the program Modeller v9.5 [START_REF] Sali | Comparative protein modelling by satisfaction of spatial restraints[END_REF], and then used in protein-protein docking calculations with the program HADDOCK [START_REF] Dominguez | HADDOCK: a protein-protein docking approach based on biochemical or biophysical information[END_REF].

The experimental structures of HAH1-Cu(I)-MNK1 and HAH1-Cd(II)-MNK1 are available from the PDB (2K1R, including chemical shift data, and 3CJK, respectively). The coordinates of the modelled MBD:HAH1 structures are available from http://www.cerm.unifi.it/modstruc/MNKdomains_HAH1modeladducts.zip. Further details on structure determination and analysis are given in the Supplementary Information.

Results and Discussion

Formation of the HAH1-Cu(I)-MNK1 and HAH1-Cd(II)-MNK1 adducts

An equimolar mixture of HAH1 and of the first domain of ATP7A (MNK1) formed a metalmediated adduct with 1:1:1 stoichiometry when presented with Cu + . The complex was in fast (on the NMR timescale) exchange with the free proteins. A similar adduct is presumably formed by HAH1 and MNK4 [15;16], whereas this does not happen with domains 2, 5, 6 of ATP7A [17;18]. By measuring the reorientation time for molecular tumbling in solution of MNK1 (5.5 ns) and of HAH1 (4.6 ns), and comparing it to the corresponding value for the complex (8.9 ns), it can be deducted that ca. 88% of the protein is involved in the intermolecular complex. In addition, it is known that the relative affinity for copper(I) of the various MBDs of ATP7A is similar and approximately ten times that of HAH1 [START_REF] Banci | An atomic level investigation of the disease-causing A629P mutant of the Menkes protein ATP7A[END_REF][START_REF] Banci | An NMR study of the interaction between the human copper(I) chaperone and the second and fifth metal-binding domains of the Menkes protein[END_REF][START_REF] Yatsunyk | Copper(I) binding and transfer by the Nterminus of the Wilson disease protein[END_REF]. From these data, it can be estimated that the constant for the equilibrium Cu(I)-MNK1 + HAH1 D MNK1-Cu(I)-HAH1 is close to 10 5 M -1 .

We additionally investigated the interaction of the HAH1:MNK1 mixture, prepared in the absence of metal ions, with Zn 2+ and Cd 2+ . Addition of Zn 2+ led to extensive sample precipitation, whereas Cd 2+ induced formation of an adduct at essentially 100% completion, as shown by the HSQC spectra of both partners in the mixture. 1D 113 Cd NMR spectra were recorded to obtain hints on the chemical identity and number of the donor atoms. A shift of 680 ppm was measured, which is consistent [START_REF] Oz | NMR spectroscopy studies of I=1/2 metal ions in biological systems[END_REF] with the 113 Cd environment seen in the crystal structure. The adduct was in slow rather than fast equilibrium with the free proteins. HAH1 and MNK6 are known not to form an adduct when mixed at 1:1 ratio in the presence of equimolar copper(I) [START_REF] Banci | An atomic level investigation of the disease-causing A629P mutant of the Menkes protein ATP7A[END_REF]. Also with Cd 2+ , an adduct between HAH1 and MNK6 could not be detected. In addition, we did not observe the formation of Cu + or Cd 2+ -mediated HAH1 dimers, which have been reported under different experimental conditions [21;22].

The structures of HAH1 and MNK1 within the HAH1-Cu(I)-MNK1 adduct

The structure of the HAH1-Cu(I)-MNK1 complex was solved using a combination of: i) intramolecular NOEs and dihedral angle restraints measured on protein samples containing the apoform of one of the two partners enriched in 15 N, 13 C in the presence of a 1.5 molar excess of the copper(I)-form of the unlabeled partner; ii) intermolecular NOEs measured on protein samples containing the same proteins as for i) but at a 1:1 ratio; iii) distance restraints between the Sg atoms of the cysteines and the copper(I) ion based on the results of mutagenesis experiments (see section on metal coordination). The data from i) allowed us to obtain the solution structure of each protein when 100% in the complex, whereas the distance restraints from ii) originate solely from the complex. This procedure allowed us to confidently assess the structural rearrangements due to complex formation, which has not been possible for the Atx1-Cu(I)-Ccc2a adduct [START_REF] Banci | The Atx1-Ccc2 complex is a metal-mediated proteinprotein interaction[END_REF]. The structure of the HAH1-Cd(II)-MNK1 complex was solved at 1.8 Å. Statistics for the two structures are given in Tables S4 andS5, respectively.

The solution structure of copper(I)-HAH1 (PDB entry 1TL4) [START_REF] Anastassopoulou | Solution structure of the apo-and copper(I) loaded human metallo-chaperone HAH1[END_REF] could be superimposed very well to the present structure of HAH1 in the HAH1-Cu(I)-MNK1 adduct (Figure 1), with a backbone RMSD between the two mean structures of 0.95 Å (Table S6). Significant differences could be noted at the end of the first b strand for residues 8-10, located before the metal-binding loop and for the region of loop V, encompassing residues 57-60. Other changes regarded the orientation of side chains at the protein-protein interface, e.g. the side chain of Arg21 experienced a significant rearrangement to accommodate the partner. In HAH1 alone, the side chain of Lys60 rearranges upon metallation, pointing toward the loaded metal site [START_REF] Anastassopoulou | Solution structure of the apo-and copper(I) loaded human metallo-chaperone HAH1[END_REF]; in the present structures, this side chain points towards the partner molecule, with a conformation similar to that observed in apo-HAH1. The NMR structure of copper(I)-MNK1 (PDB entry 1KVJ) [START_REF] Desilva | Solution structures of the reduced and Cu(I) bound forms of the first metal binding sequence of ATP7A associated with Menkes disease[END_REF] had a backbone RMSD to the present one of 1.4 Å (Table S7). Note that 1KVJ had five amino acids at the Nterminus more than the present one and was solved under different experimental conditions. Significant differences were observed for the backbone conformation of loops I and V; in addition, a reorientation of helix a 1 was apparent. A rearrangement of the same helix has been reported for yeast Ccc2a upon interaction with its partner [START_REF] Banci | The Atx1-Ccc2 complex is a metal-mediated proteinprotein interaction[END_REF]. interface being smaller and slightly rotated with respect to HAH1-Cd(II)-MNK1. A list of contacts across the interface for the two complexes is given in Table 1. Most contacts, except those in the metal-binding loop I, are conserved in both structures.

The structures of HAH1-Cu(I)-MNK1 and HAH1-Cd(II)-MNK1

Table 2 reports an analysis of the protein-protein interface for HAH1-Cd(II)-MNK1, HAH1-Cu(I)-MNK1 (shown in Figure 3), yeast Atx1-Cu(I)-Ccc2a, and Cd-HAH1 2 . Some consistent features are observable: i) the total interface area is between 800 and 1000 Å 2 , i.e. 400-500 Å 2 per partner, covering slightly more than 10% of the total surface; ii) the fraction of non-polar atoms at the interface is, on average, between 54% and 64%; iii) the propensity score of the interfaces is negative. The propensity score is calculated on the basis of the residue composition of the interfaces, using the pre-computed propensity of each amino acid [START_REF] Bahadur | A dissection of specific and non-specific protein-protein interfaces[END_REF]. The latter represents a measure of how much more (positive propensity) or less (negative propensity) frequently a given amino acid is at a protein-protein interface than just on the protein surface. The interface areas were smaller than "standard" areas for protein-protein complexes, which are typically at least 1000 Å 2 [24;25]. However, because these are relatively small proteins, the interface/total area ratio is within the lower end of the distribution of values from complexes in the PDB. The fraction of non-polar atoms at the interface was also relatively low. The negative propensity score indicates that the interfaces analysed here were composed mainly by residues that, when at the protein surface, are more likely not to be at the interaction interface. This is due to an over-representation of Gly, Ala and charged residues at the interface, in particular in HAH1 (Table 2). For example in HAH1-Cd(II)-MNK1 the core of the interface comprised Cys12, Gly13, Lys57, Thr58, Gly59 of HAH1, all of which have negative propensities, and Met65, Gly66, Phe67 and Asp68 of MNK1, of which only Met and Phe are favoured at interfaces. Cumulatively, these data provide a consistent, general description of how the metallochaperone/ATPase pair(s) is designed to prevent the formation of stable adducts in the absence of the metal ion.

We then performed molecular modelling followed by protein-protein flexible docking to create models of the structures of the adducts of all six MBDs of ATP7A with HAH1. We did not include the metal explicitly, as we wanted to evaluate the different energetic properties of the protein-protein interface under the assumption of a constant contribution from copper(I) coordination. Table 3 shows a comparison of the calculated energies of interaction at the end of the docking simulation. Note that to enable a direct comparison, also the structure of the MNK1-Cu(I)-HAH1 was recalculated with the same strategy. The cumulative HADDOCK scores identified the first, fourth and fifth MBDs as those for which the interaction should be most favourable. Of these three, only for the first and fourth MBDs the convergence rate [START_REF] Dominguez | HADDOCK: a protein-protein docking approach based on biochemical or biophysical information[END_REF] was greater than 50%. When looking at the calculated energies, it appeared that the largest significant difference among all the complexes formed by the six MBDs was in the predicted electrostatic energy of interaction, which was more favourable for the first and fourth MBDs by 150 to 300 kcal mol -1 (Table 3). The differences among other energetic terms were within 10-20 kcal mol -1 . This constitutes a demonstration of the key role of electrostatics in domain selection.

We further characterized the MNK1-Cu(I)-HAH1 complex by mutating some amino acids of HAH1 at the protein-protein interface. Arg21, Val22 and Thr58 of HAH1 were targeted. Arg21 is conserved or conservatively substituted by Lys in eukaryotic metallochaperones [START_REF] Arnesano | Metallochaperones and metal transporting ATPases: a comparative analysis of sequences and structures[END_REF], and is involved in an intermolecular salt bridge with Asp68 of MNK1 (Table 1). A negatively charged residue is commonly, but not strictly, found at position 68 of eukaryotic MBDs. We mutated Arg21 to Gln in order to remove its positive charge. Val22 is present or is conservatively replaced by Ile or Leu in the large majority of all metallochaperones. It is replaced by Ala in the homologue protein from C. elegans. The side chain of Val22 points towards the partner and makes contacts with the backbone of amino acids 64-66 of MNK1. We mutated Val22 to Ala. Finally, Thr58 is fairly conserved in eukaryotic metallochaperones. Its side chain makes hydrogen bonds with the backbone of Leu55, whereas the backbone atoms are involved in intermolecular contacts. We mutated Thr58 to Ala. All three mutants could bind copper(I) and were capable of interacting with MNK1. The mutations of Arg21 and Val22 had little impact on the structure, as shown by the high similarity of the HSQC spectra of the WT and mutant proteins. The strength of the interaction for the WT and mutant HAH1 systems could be straightforwardly evaluated by analysing the combined chemical shift variations obtained at the same HAH1 and MNK1 concentrations (Figure 4). Both the Arg21Gln and Val22Ala mutants featured a weaker interaction with the partner; the decrease of the combined chemical shift variations was similar for the two mutants. For Arg21Gln, this effect can be ascribed to the loss of the electrostatic interaction. In the case of Val22Ala, the reduced interaction can be ascribed to the loss of the favourable contribution due to the desolvation of the hydrophobic side chain of Val22 upon complex formation. Instead, the HSQC spectrum of the Thr58Ala mutant was significantly perturbed, and showed the occurrence of multiple protein conformations. In addition, several peaks were broadened beyond detection at the first addition of MNK1. Despite these complications, it appeared that the combined chemical shift variations upon interaction with Cu(I)-HAH1 were as large as that of the wild-type protein, but the maxima did not correspond to the same amino acids (Figure 4). The HAH1-Cu(I)-MNK1 adduct was thus formed by the Thr58Ala mutant to an extent similar to that of the WT protein, but possibly with a distorted conformation.

Metal coordination

We used site-directed mutagenesis to investigate the role of individual cysteines in the formation of the HAH1-Cu(I)-MNK1 adducts. To this end, we observed through HSQC spectra whether the metal-mediated adduct was formed upon addition of the metal ion (at equimolar ratio) to a 1:1 mixture containing the wild-type (WT) form of one protein and a mutant of the other protein lacking one of the two copper(I)-binding cysteines. Only three cysteines were needed for the formation of the HAH1-Cu(I)-MNK1 adduct. The two cysteines of each partner that are more Nterminal in sequence (Cys12 of HAH1 and Cys15 of MNK1) were strictly required for the interaction, whereas the third cysteine could be either Cys15 of HAH1 or Cys18 of MNK1.

The crystal structure of HAH1-Cd(II)-MNK1 (determined at pH 4.7) provides independent information on Cd 2+ coordination. The metal ion was tetra-coordinated by the side chains of the two cysteines of MNK1 and of Cys12 of HAH1. The fourth ligand was provided by the hydroxyl of Thr14 of MNK1, leading to an elongated tetrahedral geometry. Thus, MNK1 provided three ligands and HAH1 only one. The Cd 2+ -Og-Cb angle was about 150°, suggesting that the hydroxyl group may be deprotonated. Site-directed mutagenesis showed that both cysteines of MNK1 were necessary for the formation of the complex. In the Cd-HAH1 2 structure [START_REF] Wernimont | Structural basis for copper transfer by the metallochaperone for the Menkes/Wilson disease proteins[END_REF], the Cd 2+ ion is instead at the center of an essentially regular tetrahedron formed by the four Sg atoms of the metal-binding cysteines.

In both adducts, the net charge at the metal site is -2. Instead, the charge at the more solventexposed site in isolated Cu(I)-HAH1 is -1. In addition, upon complex formation the distance from the metal site of the Ne of Lys60 of HAH1 increased. These effects could be compensated by the favourable orientation of the dipole moment of the first helix of the incoming MNK1 molecule.

Concluding remarks

The whole body of the data on the interaction between metallochaperones and their partner ATPases, as well as studies with copper(I) chelators [START_REF] Hussain | Conserved residues modulate copper release in human copper chaperone Atox1[END_REF], demonstrate that metal homeostasis in cells involves a subtle balance between the control of metal reactivity and the capability to transfer the metal between proteins with an appropriate kinetics. The use of metallochaperones to deliver copper to specific intracellular targets has been likened to the action of an enzyme lowering the activation barrier for copper transfer to their specific partners [START_REF] Huffman | Energetics of Copper Trafficking Between the Atx1 Metallochaperone and the Intracellular Copper-transporter, Ccc2[END_REF]. Additionally, the fine tuning of the identity of the amino acids at interface may allow the metallochaperone-based system to select among different metal ions. The selection can result from the complete abrogation of the formation of the metal-mediated adduct or from a kinetic trap in which the adduct gets locked [START_REF] Liu | Multiple metal binding domains enhance the Zn(II) selectivity of the divalent metal ion transporter AztA[END_REF]. The present mutagenesis data indicate that it is not a single amino acidic substitution that plays a decisive role. Instead, the network of Van der Waals and electrostatic interactions taking place among the interface residues as well as, presumably, the variation of the conformational space available to the same residues determine the configuration of the adduct and its energy of formation. Structural modelling suggested that for the various MBDs of ATP7A, the electrostatic contribution is crucial to determine the energetics of the interaction with the metallochaperone.

Protein recognition takes place only in the presence of the copper(I) ion, as a result of the small interface area and low propensity of the surface of HAH1 for forming a stable intermolecular complex. This, together with the fact that the affinity for copper(I) of the MBDs of ATP7A is approximately a ten-fold higher than that of HAH1 [START_REF] Banci | An atomic level investigation of the disease-causing A629P mutant of the Menkes protein ATP7A[END_REF][START_REF] Banci | An NMR study of the interaction between the human copper(I) chaperone and the second and fifth metal-binding domains of the Menkes protein[END_REF][START_REF] Yatsunyk | Copper(I) binding and transfer by the Nterminus of the Wilson disease protein[END_REF], suggests that upon interaction the metal ion is effectively transferred to the ATPase. However, for some MBDs within the soluble tail, such as MNK1, an intermolecular adduct is formed at detectable levels, involving one cysteine residue from HAH1. While the latter can be regarded as a weakly trapped intermediate along the metaltrafficking pathway, its formation might also be instrumental to determine rearrangements of the inter-domain interactions of the ATPase. The latter inter-domain interactions and their variations during the catalytic cycle are important for the enzymatic function of the protein [30;31], as well as to determine the intracellular localization of the ATPase [START_REF] Hamza | Essential role for Atox1 in the coppermediated intracellular trafficking of the Menkes ATPase[END_REF]. The formation of adducts between HAH1 and the first or fourth MBD of ATP7A can thus modulate the interaction of the N-terminal cytosolic tail with the other protein domains, ultimately constituting a means for the cell to modulate enzyme turnover [START_REF] Mandal | Functional roles of metal binding domains of the Archaeoglobus fulgidus Cu(+)-ATPase CopA[END_REF].

Tables Table 1 Interprotein contacts in the present structures. For the NMR structure, short distances (< 4.0 Å) occurring in >50% of the family members are reported. The backbone and side chains (as of Table 1) of the partner are also shown.

  Biochemical Journal Immediate Publication. Published on 19 May 2009 as manuscript BJ20090422 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090422 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

Figures 1

 1 Figures1 and 2show respectively the structures of HAH1-Cu(I)-MNK1 and HAH1-Cd(II)-MNK1 determined in this work. The two structures were similar, with the HAH1-Cu(I)-MNK1 interface being smaller and slightly rotated with respect to HAH1-Cd(II)-MNK1. A list of contacts across the interface for the two complexes is given in Table1. Most contacts, except those in the metal-binding loop I, are conserved in both structures.Table2reports an analysis of the protein-protein interface for HAH1-Cd(II)-MNK1, HAH1-Cu(I)-MNK1 (shown in Figure3), yeast Atx1-Cu(I)-Ccc2a, and Cd-HAH1 2 . Some consistent features are observable: i) the total interface area is between 800 and 1000 Å 2 , i.e. 400-500 Å 2 per partner, covering slightly more than 10% of the total surface; ii) the fraction of non-polar atoms at the interface is, on average, between 54% and 64%; iii) the propensity score of the interfaces is negative. The propensity score is calculated on the basis of the residue composition of the interfaces, using the pre-computed propensity of each amino acid[START_REF] Bahadur | A dissection of specific and non-specific protein-protein interfaces[END_REF]. The latter represents a measure of how much more (positive propensity) or less (negative propensity) frequently a given amino acid is at a protein-protein interface than just on the protein surface. The interface areas were smaller than "standard" areas for protein-protein complexes, which are typically at least 1000 Å 2[24;25]. However, because these are relatively small proteins, the interface/total area ratio is within the lower end of the distribution of values from complexes in the PDB. The fraction of non-polar atoms at the interface was also relatively low. The negative propensity score indicates that the interfaces analysed here were composed mainly by residues that, when at the protein surface, are more likely not to be at the interaction interface. This is due to an over-representation of Gly, Ala and charged residues at the interface, in particular in HAH1 (Table2). For example in HAH1-Cd(II)-MNK1 the core of the interface comprised Cys12, Gly13, Lys57, Thr58, Gly59 of HAH1, all of which have negative propensities, and Met65, Gly66, Phe67 and Asp68 of MNK1, of which only Met and Phe are favoured at interfaces. Cumulatively, these data provide a consistent, general description of how the metallochaperone/ATPase pair(s) is designed to prevent the formation of stable adducts in the absence of the metal ion.We then performed molecular modelling followed by protein-protein flexible docking to create models of the structures of the adducts of all six MBDs of ATP7A with HAH1. We did not include the metal explicitly, as we wanted to evaluate the different energetic properties of the protein-protein interface under the assumption of a constant contribution from copper(I) coordination. Table3shows a comparison of the calculated energies of interaction at the end of the docking simulation. Note that to enable a direct comparison, also the structure of the MNK1-Cu(I)-HAH1 was recalculated with the same strategy. The cumulative HADDOCK scores identified the first, fourth and fifth MBDs as those for which the interaction should be most favourable. Of these three, only for the first and fourth MBDs the convergence rate[START_REF] Dominguez | HADDOCK: a protein-protein docking approach based on biochemical or biophysical information[END_REF] was greater than 50%. When looking at the calculated energies, it appeared that the largest significant difference among all the complexes formed by the six MBDs was in the predicted electrostatic energy of interaction, which was more favourable for the first and fourth MBDs by 150 to 300 kcal mol -1 (Table3). The differences among other energetic terms were within 10-20 kcal mol -1 . This constitutes a demonstration of the key role of electrostatics in domain selection.We further characterized the MNK1-Cu(I)-HAH1 complex by mutating some amino acids of HAH1 at the protein-protein interface. Arg21, Val22 and Thr58 of HAH1 were targeted. Arg21 is conserved or conservatively substituted by Lys in eukaryotic metallochaperones[START_REF] Arnesano | Metallochaperones and metal transporting ATPases: a comparative analysis of sequences and structures[END_REF], and is involved in an intermolecular salt bridge with Asp68 of MNK1 (Table1). A negatively charged residue is commonly, but not strictly, found at position 68 of eukaryotic MBDs. We mutated Arg21 to Gln in order to remove its positive charge. Val22 is present or is conservatively replaced by Ile or Leu in the large majority of all metallochaperones. It is replaced by Ala in the homologue protein from C. elegans. The side chain of Val22 points towards the partner and makes contacts with the backbone of amino acids 64-66 of MNK1. We mutated Val22 to Ala. Finally, Thr58 is fairly conserved in eukaryotic metallochaperones. Its side chain makes hydrogen bonds with the backbone of Leu55, whereas the backbone atoms are involved in intermolecular contacts. We mutated Thr58

Biochemical

  Journal Immediate Publication. Published on 19 May 2009 as manuscript BJ20090422 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090422 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

Biochemical

  Journal Immediate Publication. Published on 19 May 2009 as manuscript BJ20090422 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090422 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

Table 3 .

 3 Statistics for the structures of the adducts between each of the six MBDs of ATP7A and HAH1 calculated with the docking program HADDOCK. Only the best cluster of solutions was taken into account. 7 ± 5.7 -65.4 ± 3.0 -57.2 ± 4.7 -103.8 ± 3.2 -91.2 ± 7.3 -48.9 ± 3.7 RMSD from the overall lowest-energy structure (Å) 1.2 ± 0.9 0.7 ± 0.5 1.2 ± 0.7 0.8 ± 0.5 0.6 ± 0.4 1.0 ± 0.7 Van der Waals Energy (kcal mol -1 ) -28.1 ± 6.8 -36.1 ± 2.8 -36.4 ± 3.5 -27.7 ± 0.8 -46.9 ± 5.5 -26.7 ± 2.0 Electrostatic Energy (kcal mol -1 ) -391 ± 43 -242 ± 17 -89 ± 13 -382 ± 12 -206 ± 20 -246 ± 20 Desolvation Energy (kcal mol -1 ) 1.3± 4.1 18.4 ± 3.5 -3.6 ± 3.3 -0.7 ± 0.9 -6.6 ± 2.3 26.3 ± 6.3 Restraints violation Energy (kcal mol -1 ) 13 ± 11 7.4 ± 3.6 5.4 ± 3.5 9.6 ± 2.5 35 ± 27 7.1 ± 2.6 Biochemical Journal Immediate Publication. Published on 19 May 2009 as manuscript BJ20090422 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090422 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

Figure 1 .

 1 Figure 1. Comparison of the solution structures of A) HAH1-Cu(I)-MNK1; B) Cu(I)-HAH1 (PDB code: 1TL4); C) Cu(I)-MNK1 (PDB code: 1KVJ). The side chains of the metal-binding cysteines are shown in green. The fourth cysteine, which does not bind the metal in the present structure, is shown in grey.

Figure 2

 2 Figure 2Crystal structure of HAH1-Cd(II)-MNK1 (same orientation as in Figure1A). The side chains of the metal-binding cysteines are shown in green; the side chain of metal-binding threonine is in dark green. The fourth cysteine is shown in grey.

Figure 3

 3 Figure 3Electrostatic potential surfaces of left) MNK1 and right) HAH1 in the HAH1-Cu(I)-MNK1. The backbone and side chains (as of Table1) of the partner are also shown.

Figure 4 .

 4 Figure 4. Combined chemical shift variations of selected residues for WT-HAH1 and the three interface mutants. Not all peaks could be detected in the Thr58Ala mutant.

Table 2 .

 2 Properties of the protein-protein interface of various metal-mediated complexes (calculated on the experimental structures with the program Proface[START_REF] Saha | ProFace: a server for the analysis of the physicochemical features of protein-protein interfaces[END_REF])

		HAH1-Cd(II)-MNK1	HAH1-Cu(I)-MNK1	Yeast Atx-Cu(I)-Ccc2a		HAH1 dimer
		HAH1 MNK1 Total HAH1 MNK1 Total Atx1 Ccc2a Total HAH1 (Chain A) HAH1 (Chain B) Total
	Interface Area (Å 2 )	449	434	883	527	490	1017	416	426	842	494	478	972
	Interface Area / Surface	0.11	0.10	0.10	0.12	0.11	0.12	0.1	0.11	0.11	0.12	0.12	0.12
	Area
	Number of atoms	50	44	94	54	56	110	46	51	97	51	52	103
	Number of residues	15	14	29	16	14	30	15	15	30	15	15	30
	Fraction of non-polar	0.70	0.64	0.67	0.61	0.54	0.57	0.59	0.49	0.54	0.59	0.58	0.58
	atoms
	Non-polar interface area	253	219	472	249	210	459	185	161	345	223	218	441
	(Å 2 )
	Fraction of fully buried	0.16	0.18	0.17	0.28	0.25	0.26	0.28	0.29	0.29	0.22	0.17	0.19
	atoms
	Residue Propensity Score -1.87	0.46	-1.41	-2.18	-0.21 -2.39 -2.32	-0.17 -2.49	-1.61	-1.67	-3.28
	Local Density	32.76	28.95		35.04	33.86		29.91 33.53		31.84	30.92

Biochemical Journal Immediate Publication. Published on 19 May 2009 as manuscript BJ20090422 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090422 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

Funding

Financial support from MIUR (project PRIN 2005), from the EC (contract 031220, SPINE2-COMPLEXES) and from the Ente Cassa di Risparmio di Firenze (Projects "Basi Molecolari di patologie umane correlate a disfunzioni della catena respiratoria" and "Relazione varianti proteiche strutturali-malattie genetiche") is acknowledged. The HADDOCK web server is supported by the EC through the eNMR project (contract 213010).

Suppelementary Information Available:

Supplementary Table S1: Acquisition parameters for NMR experiments performed on HAH1-Cu(I)-MNK1 containing 13 C, 15 N-MNK1 Supplementary Table S2: Acquisition parameters for NMR experiments performed on HAH1-Cu(I)-MNK1 containing 13 C, 15 N-HAH1 Supplementary Table S3: Acquisition parameters for NMR experiments performed on the equimolar mixture of HAH1:MNK1. S4: Measured values of the correlation time (t c ) for protein reorientation in solution for MNK1 and HAH1 isolated and in the HAH1-Cu(I)-MNK1 complex. S5: Statistical analysis of the solution structure of HAH1-Cu(I)-MNK1. S8 Backbone RMSD values for MNK1 in the various structures available.

Supplementary Table

Supplementary Table

Supplementary Table S6: X-ray data collection and refinement statistics

Supplementary Table S7 Backbone RMSD values for HAH1 in the various structures available

Supplementary Table