WO2001096372A2

WO2001096372A2 - Zinc transporters proteins and their use in medicinal preparations

Info

Publication number: WO2001096372A2
Application number: PCT/GB2001/002597
Authority: WO
Inventors: Kathryn Mary Taylor; Helen Elizabeth Morgan; Robert Ian Nicholson
Original assignee: University College Cardiff Consultants Ltd
Priority date: 2000-06-13
Filing date: 2001-06-13
Publication date: 2001-12-20
Also published as: WO2001096372A3; AU2001264099A1

Abstract

A family of protein sequences has been identified, the function of which is to regulate zinc levels in cells. Methods of treatment and diagnosis of diseases selected from those involving disorders of zinc homeostasis are provided, the methods comprising the administration of therapeutically effective amounts of one or more polypeptides comprising the consensus regions of the proteins, or homologues thereof, or antibodies generated against the regions or the proteins. The particular diseases concerned may include neurodegenerative disorders and oestrogen responsive metastatic breast cancer. Also provided are methods for the selection of test compounds for use in the treatment of zinc-homeostasis related disorders, and recombinant proteins containing one or more of the consensus sequences of the family of proteins.

Description

Zinc Transporters

This invention relates to the field of treatment and diagnosis of oncological and neurological disease and, in particular, though not exclusively, it relates to the use of proteins in cancer treatment and diagnosis.

Investigation of the structure of the oestrogen-regulated gene LIN-1, associated with metastatic breast cancer (1), has revealed a histidine-rich transmembrane protein (2) with the potential to bind and/or transport Zn²⁺ ions. Many proteins and DΝA require Zn²⁺-binding for function (3). Zn²⁺ needs to be transported across biological membranes and its uptake and efflux must be tightly regulated as it is both essential and potentially toxic to animal cells (4-6). The mechanisms of Zn²⁺ efflux and of Zn ⁺ induced cell death are currently unknown. Mammalian Zn²⁺ transporters can impart Zn²⁺ resistance to cells (4). However it is unclear if they actually transport Zn²⁺ across membranes or whether they act as cofactors for other molecules such as ion channels (4, 7).

In US 5693465, there is described a method for determining the risk of metastasis of a breast tumour, the method comprising determining whether a tissue sample from the tumour expresses a substantial portion of LIV-1.

It has now been found that LIN-1 and other proteins form the basis of a new family with the potential to bind and transport Zn²⁺ across cell membranes and it is an object of the present invention to provide methods of using this finding in the medical, especially oncological, field.

Reference will be made to the accompanying Figure 1, which shows the sequences of the identified proteins of the LIN-1 family. Other proteins may be identified by following the methods described herein. The sequences have been aligned, with spaces (dashes) inserted where necessary to allow a more clear alignment of consensus regions. The bars (I to NIII) indicate the approximate locations of the assigned transmembrane regions, whilst the "+" signs indicate the approximate location of the metalloprotease motif. LIN-1 is referred to in Figure 1 as U41060; the terms are used interchangeably herein.

Accordingly, one aspect of the present invention provides the use of a polypeptide comprising one or more of the consensus regions of the proteins shown in Figure 1, or a functional homologue thereof, in the preparation of a medicament for the treatment of a disease selected from those involving disorders of zinc homeostasis. The consensus regions are defined by the stretches of matching or similar residues when the sequences are aligned with each other. The terms 'similar' or 'similarity' as used herein refer to the similarity of amino acid residues as defined according to the following scheme: aromatic residues, F, H W and Y; charged residues, D, E, H, K and R; hydrophobic residues A, C, F, G, H, I, L, M, N, and Y; aliphatic residues I, L and N; tiny residues A, G and S; polar residues D, E, H, K, Ν, Q, R, S and T; small residues A, C, S, T, D, Ν, N, G and P;

residues with high β-turn-forming propensity A, C, S, T, D, E, Ν, N, G and P; alcohol

residues S and T. A functional homologue is one having the ability to bind and transport Zn²⁺ across cell membranes and comprising the metalloprotease motif shown in Figure 1. Preferably, the polypeptide or homologue comprises two or more such consensus regions. The polypeptide or homologue preferably includes at least one of the regions I-NIII. Preferably, the polypeptide or homologue comprises consensus regions N, NI and the metalloprotease motif. More preferably, the polypeptide or homologue has a degree of similarity of 30 to 90% with the proteins of Figure 1 or 70-90% with the consensus regions. Most preferably, the polypeptide or homologue has the sequence of one of the proteins shown in Figure 1. A further aspect of the present invention provides the use of a nucleic acid molecule encoding a polypeptide comprising one or more of the consensus regions of the proteins shown in Figure 1, or a functional homologue thereof, in the preparation of a medicament for the treatment of a disease selected from those involving disorders of zinc homeostasis.

Preferably, the disease may be breast cancer, especially oestrogen responsive metastatic breast cancer.

Preferably, the polypeptide does not have the same sequence as LIN-1 or U41060.

In another aspect, the invention provides a method of selecting compounds for use in the treatment of a disease selected from those involving disorders of zinc homeostasis, the method comprising determining the effect of a test compound on a function of a polypeptide comprising one or more of the consensus regions of the proteins shown in Figure 1, or a functional homologue thereof.

Suitable methodologies are well known to those skilled in the art, and can be found in Combinatorial Chemistry; synthesis, analysis and screening, ed. Jung G, 1999, John Wiley and sons.

The compounds may be selected for use in the treatment of breast cancer, especially oestrogen-responsive metastatic breast cancer. Accordingly, in yet another aspect, the invention provides compounds for the treatment of a

disease selected from those involving disorders of zinc homeostasis, the compounds having

been identified by using the method of the present invention as defined above.

The function of the protein to be affected by the test compounds is preferably its zinc

regulating ability. The testing is preferably performed in cell culture. Suitable methods

include FACS analysis as described herein..

In a further aspect, the invention provides recombinant proteins including at least one of

the consensus sequences of the proteins shown in Figure 1, the consensus sequences being defined by the stretches of matching or similar residues when the protein sequences are

aligned with each other. Preferably, the consensus sequence includes at least one of the

regions I to NIII from Figure 1. More preferably, the consensus sequence also includes the

metalloprotease motif of Figure 1. Most preferably, the consensus sequence comprises region N, the metalloprotease motif and region NI.

A recombinant protein is one which is not endogenously produced but is produced by

synthetic methods.

Alternatively, or additionally, the consensus sequence includes that corresponding with

residues 285 to 298 of U41060.

The invention also provides an antibody molecule having affinity for one or more of the proteins listed in Figure 1. Preferably, the antibody molecule has affinity for one or more of the consensus sequences described above. The term antibody molecule includes antigen-binding fragments such as Fv, Fab and Fab'. The invention also provides a kit of antibody molecules directed against at least two different proteins selected from those listed in Figure 1, or against at least two of their consensus sequences as described above.

In yet another aspect, the invention provides a method for identifying proteins involved in zinc homeostasis, the method comprising comparing a test protein with one or more of the proteins listed in Figure 1, wherein the involved proteins are identified by having similarity with the sequences given in Figure 1. Preferably, the test protein sequences are compared with two, more preferably five or more, sequences selected from Figure 1.

Also provided is a method of diagnosis of a zinc homeostasis-related condition in a subject, the method comprising contacting a sample from that subject with an antibody molecule of the present invention and measuring the binding of that antibody to the sample.

In another aspect, a method of treatment of a zinc homeostasis-related condition in a subject is provided, the method comprising contacting the subject with an antibody molecule of the present invention.

In a further aspect, the invention provides a method of treatment of a disease selected from those involving disorders of zinc homeostasis, the method comprising the administration, to a subject in need of such treatment, of a therapeutically effective amount of a polypeptide comprising one or more of the consensus regions of the proteins listed in Figure 1, or a functional homologue thereof. Preferably, the polypeptide or homologue comprises at least two of those consensus regions. The polypeptide or homologue

preferably includes at least one of the regions I- VIII, and may comprise the whole sequence

of a protein selected from those listed in Figure 1. The disease may be breast cancer,

especially oestrogen responsive metastatic breast cancer.

In another aspect, the invention provides a method of treatment of a disease selected from

those involving disorders of zinc homeostasis, the method comprising the administration,

to a subject in need of such treatment, of a therapeutically effective amount of a nucleic

acid molecule encoding a polypeptide comprising one or more of the consensus regions of

the proteins shown in Figure 1, or a functional homologue thereof.

Apoptosis, programmed cell death, a physiological process for killing cells, is critical for

the normal development and function of multicellular organisms, aberrations of which have

been linked to a variety of diseases (27). Abnormalities of apoptosis can contribute to a number of diseases such as cancer (28), autoimmunity (29) and degenerative disorders

(30). Signalling for apoptosis occurs through multiple pathways originating both inside and

outside the cell. Accordingly, the diseases to be diagnosed or treated according to the

present invention may also be those involving disorders of apoptosis. The invention also

provides a method of modifying apoptosis in vitro or in vivo, the method comprising contacting cells with a polypeptide comprising one or more of the consensus regions of the

proteins having the sequences listed in Figure 1, or a functional homologue thereof. The term modifying refers to increasing or decreasing, preferably increasing, the level of apoptosis. The polypeptide or homologue preferably comprises two or more consensus regions. The polypeptide or homologue may include at least one of the regions I to NIII and may comprise the whole sequence of a protein selected from Figure 1.

The invention will now be described in more detail by way of example only and with reference to the accompanying drawings, of which:

Figure 1 shows the sequences of the identified proteins of the LIN-1 family, as described above;

Figure 2 shows the individual, non-aligned sequences of the proteins listed in Figure 1, together with their species of origin and alternative accession numbers;

Figure 3 is a schematic diagram of the domain organisation of the LIN-1 family of proteins;

Figure 4 shows the results of an experiment looking at multimeric complex formation in two of the proteins listed in Figure 1. CAA20238 and Q 15043 were expressed in CHO cells and then analysed by SDS-PAGE under both reducing (R) and non-reducing (ΝR) conditions;

Figure 5 is a schematic diagram of the zinc-binding environment of the metzincins (18), altered to suggest potential zinc co-ordinating residues in the metalloprotease motif of the LIV-l family. Shaded residues are known to co-ordinate zinc, square boxes represent conserved residues unique to the LIN-1 family and dotted lines suggest zinc co-ordination ability; Figure 6 shows the results of experiments looking at cellular import of zinc in cells

expressing proteins of the LIV-l family;.

Figure 7 shows the results of further experiments looking at zinc import into cells

expressing LIV-l family proteins;

Figure 8 shows the effect of some of the LIN-1 family members on apoptosis. The results

are expressed as the % of cells taking up the dye YO-PRO-1 (which enters cells only if

they are apoptotic);

Figure 9 shows the effect of the apoptotic changes of Figure 8 on cell growth curves; and

Figure 10 shows a fluorescence micrograph of apoptosis in cells expressing a LIN-1 family

member. The occurrence of numerous 'vacuole-like' structures can be seen in enlarged

CHO cells 22 hours after transfection with CAA20238 with a Green Fluorescent Protein

C-terminal tag.

EXAMPLES

Materials and Methods

Expression of recombinant proteins in CHO cells

DΝA constructs for expression in CHO cells were engineered by PCR with primers F2A5 (5'): CCCATCGGAT CCGGCACAAT GGCCAGAGGC CTGGGG, and F2A3 (3'): CCCATCCTTA TCGTCATCGT CGTACAGATC CTCAAGGTGG GCAATCAGCA CC for CAA20238, and FamA5 (5'): CCCCACACCA TGAAGCTGCT GCTGCTGCAC CC, and famaA3 (3'): CCCATCCTTC CTTTCATCCT C, for Q15043, and 5'-CCCATCGGAT CCGGCACAAT GGCGAGGAAG TTATC-3' and 5'-GAAATTTATA CGAAACACGA TTTTATG-3' for LIV-l. Constructs were inserted into pcDNA3.1/V5/HisTOPO or pcDNA3.1/CT-GFP-TOPO by TOPO-TA cloning (Invitrogen) to produce C-terminal fusion proteins of V5 or GFP respectively. CHO cells plated at 1.1 x lOVml the previous day were transiently transfected with plasmid using Transfast (Promega) and harvested between 22-40 hours. Mock-transfected cells are designated CHO and cells transfected with the LacZ gene and expressing the control cytosolic protein β-Galactosidase are designated LacZ. Cells harvested by scraping in the presence of lysis buffer (0.5% NP-40, EDTA in Krebs Ringer Hepes buffer (KRH, 120mM NaCl, 25mM HEPES, 4.8mM KCl, 1.2mM KH₂PO₄, 1.2mM MgSO₄ and 1.3mM CaCl₂, pH 7.4) and protease inhibitor cocktail (1/10, Sigma) were separated by 10% SDS-PAGE, before immunoblotting onto 0.2μm nitrocellulose and probing with V5 or GFP antibody (Invitrogen, 1/5000) for 1 hour, goat anti-mouse HRP conjugated antibody (Amersham, 1/5000) for 30 min and chemiluminescent development with West Femto reagent (Pierce). FACS analysis for apoptosis and zinc determination

Transfected CHO cells were harvested with 3mM EDTA in PBS and resuspended at lxlOVml in KRH buffer or medium. Cells were incubated with or YO-PRO-1 (Molecular Probes) and 1.5 μM propidium iodide for 20 mins on ice in KRH or newport green diacetate (Molecular Probes) for lhour in medium at 37°C. For zinc analysis, 25μM Zinc chloride and/or lOμM cell permeant zinc chelator TPEN (Molecular Probes) were added for 15mins prior to testing. Cells were resuspended in KRH prior to fluorescent measurements using a Becton Dickinson Flow Cytometer.

Methods for the preparation of antibodies against regions of the proteins can be found in US 5693456 and Antibodies, a laboratory manual, ed. Harlow E and Lane D, 1988 Cold Spring Harbor Laboratory, New York. RESULTS

Alignment of thirty-six proteins from fourteen different species emphasises their sequence similarity (Figure 1). They code for 6-8 transmembrane domain proteins with a long

N-terminus and short C-terminus (see Figure 3). These proteins have three interesting features. First is the identity similarity between sequences in the transmembrane regions, especially II and V (up to 88% and up to 80%, respectively, Figure 1) and conserved extramembrane charged residues, implying ion transporting ability (8, 9). A conserved histidine in transmembrane domain II of this family could act as an intrachannel metal binding site in the same manner as the conserved histidine in transmembrane domain V of conventional zinc transporters (10, 11, 12) . Two of the human sequences (CAA20238 and

Q 15043) transiently expressed in CHO cells produce high molecular weight bands in

non-reducing conditions (>400kDa) on SDS-PAGE, which are disrupted in reducing

conditions (see Figure 4). This implies oligomer formation in membranes, a requirement of ion channel formation (9).

Second is the presence of a new well-conserved motif spanning 74 residues (shown by bold dashes in Figure 1) and including the hydrophobic transmembrane domains V and VI. Adjacent to this latter domain and included in the new motif is the consensus sequence of the catalytic Zri^-binding site of the zincin and PDF groups of metalloproteases, HEXXHXXG, which is essential for their activity (13-15). The LIV-l family motif contains novel conserved proline and glutamic acid residues (underlined), HEXPHEXGD, and is adjacent to a transmembrane domain (Figure 1). Both are previously unprecedented in mammals. In conventional metalloproteases, Zri^ co-ordinates with both histidines, a water molecule from the first glutamic acid and other residues downstream, depending on the family sub group (13, 16 17). The LIV-l family has no Met-turn of metzincins (18), but there are conserved residues downstream that may act as ligands.

It is proposed that the second glutamic acid or a histidine adjacent to transmembrane domain III are involved in zinc co-ordination (Figure 5). The conserved proline, which can stabilise and disrupt helices (19, 20), may also be significant. The hydrophobic transmembrane domains V and VI and the Zn²⁺-binding metalloprotease motif constitute a new motif that is well conserved in all species studied, suggesting a pore domain.

Third is the presence of multiple histidine-rich repeats, similar to those in zinc transporters (HX)_n (10). These regions are important for binding and transporting Zn²⁺ (21) and are more plentiful in most of the new family than in other Zn²⁺ transporters (see Table 1, below, showing a comparison of histidine-rich repeats in 30 of the new family of proteins (Table 1 A) with those in the known zinc transporters (Table IB)). It is noteworthy that the occurrence of these histidine-rich repeats in this new family are predominantly in three different areas of the sequence, the N-terminus, the second transmembrane loop and the third transmembrane loop (Figs 1 and 3). This positions them uniquely on opposing membrane sides unlike conventional zinc transporters.

Interestingly,, there appears to be a further conserved region, at least in some of the human, mammalian and drosophila sequences (see the first 17 sequences in fig. 1, excluding AW186578). This region appears to stretch from residue 285 to 298 of U41060 (and corresponding residues in the other sequences) but may include further residues up and downstream. The presence of cysteine and proline residues in this region may indicate disulphide bonding and conformational bending features, respectively.

EXAMPLE 1

The new family of proteins can increase intracellular zinc

Zn²⁺ is a co-factor of over 300 enzymes, involved in protein, nucleic acid, carbohydrate and lipid metabolism, as well as the control of gene transcription, growth, development and differentiation (3). Zn²⁺ deficiency stunts growth and causes serious metabolic disorders (4) while increased levels of Zn²⁺ can have a beneficial effect on cell growth (33 .

Figure 6 shows the differences of intracellular zinc content between CHO cells expressing a family member and control cells as measured by loading cells with Newport Green Diacetate. Approximately 20-25% of control cells had high levels of intracellular zinc compared with 40-45% in test samples. These were transient transfections which routinely transfected only 20-30% of cells, suggesting that cells expressing the test proteins have higher intracellular zinc levels. The addition of 25 μM extracellular zinc increased the number of cells with high intracellular zinc further without altering the controls. This result indicates that the transfected cells import the extracellular zinc.

Figure 7 shows a different experiment where addition of 25μM extracellular zinc causes a 75-90% increase in the number of transfected test cells with increased intracellular zinc levels within 15 mins, whereas the control cells showed only small changes. Addition of the cell-permeant zinc chelator TPEN for a further 15min reversed this effect for LIV-l and CAA20238. The addition of TPEN to Q15043 had little effect, suggesting that Q15043 may use a different mechanism.

EXAMPLE 2

The new family of proteins can increase cell apoptosis

Apoptosis, programmed cell death, a physiological process for killing cells, is critical for the normal development and function of multicellular organisms, aberrations of which have been linked to a variety of diseases (27). Abnormalities of apoptosis can contribute to a number of diseases such as cancer (28), autoimmunity (29) and degenerative disorders (30). Signalling for apoptosis occurs through multiple pathways originating both inside and outside the cell. Amongst the earliest changes in cells undergoing apoptosis is plasma membrane blebbing (31) and a loss of membrane phospholipid asymmetry, resulting in the exposure of phosphatidylserine at the surface of the cell (32).

Alteration of the intracellular pool of Zn²⁺ has been well documented to cause apoptosis in many different cell types (34, 35, 36, 37) and labile Zn²⁺ has been proposed to direct tissue growth and death by regulating both mitosis and apoptosis (37).

CHO cells expressing the recombinant proteins were tested for apoptosis associated with loss of membrane potential by incubating with the fluorescent dye YO-PRO-1, which enters apoptotic cells. It is clear from Figure 8 that 20% more cells transfected with CAA20238 and 10% more cells transfected with Q 15043 are positive for apoptosis. However, cells expressing U41060 exhibit the same amount of apoptosis as the controls, presumably due to the low expression level of LIV-l in cells.

The fact that CAA20238 expression causes apoptosis of cells is reflected in the observed growth of cells (Figure 9) during the course of a transfection experiment and by the large number of 'vacuole-like' structures that are evident 22 hours post transfection (see figure 10) which precede apoptotic death. This decrease in cell number is not an effect on cell growth but a direct result of this apoptotic death. Cells transfected with Q 15043, exhibiting less apoptosis, lie between CAA20238 and control cells. Cells expressing LIV-l appear to have a 10% growth advantage 30 hours after transfection.

The 36 sequences given in Figure 1 form a new protein family with a novel motif, HEXPHEXGD, and a possible ion channel structure. By similarity to other Zn"^" transporters and metalloproteases, and based on the experimental data described above these proteins are likely to bind and/or transport Zn"^" across membranes.

Oestrogen-regulated LIV-l has been implicated in metastatic breast cancer (2). The other proteins may have a role in cancer and/or other diseases. Multiple human tissue array analysis of LIV-l, CAA20238 and Q 15043 suggest that they are predominantly present in hormonal tissues. BAA86579 occurs principally in brain (22) where aberrant Zn^ levels have been linked to apoptosis and neuronal disease (23). These proteins form an exciting new family, which have the potential to control Zn^ homeostasis in cells, failure of which has been linked with cancer and disease (24, 25).

The LIV-l family of transmembrane proteins are capable of causing Zn²⁺-related apoptosis. These sequences have a motif HEXPHEXGD fitting the consensus sequence for the Zn²⁺-binding site of the PDF and zincins group of metalloproteases (21) yet containing unique proline and glutamic acid residues (bold) previously unprecedented in an HEXXH motif. This motif forms part of a highly conserved region of 74 residues including two potential transmembrane domains (Fig 1), which are suggested to be the signature for this new family. Interestingly these sequences also show similarity to conventional mammalian Zn²⁺ transporters, containing 6 or 8 transmembrane domains and histidine-rich repeats of the form (HX)₃-β (6). However, unlike conventional Zn²⁺ transporters, these histidine-rich repeats occur in three areas of the proteins, on both sides of the membrane (Fig 1) and in greater numbers, containing as many as 51 histidine residues in total compared to 4-11 for mammalian Zn²⁺ transporters (Table 1). The ZIP (ZRT, IRT-like protein) family of metal transporters (6, 10, 38) show some similarity to this new family of proteins in the conserved residues present in the transmembrane domain V. However, they neither contain the conserved 74-residue motif comprising the HEXXH motif (Fig 1) or histidine-rich motifs in three areas of the sequence (Fig 1). The transmembrane domains of the new family are well-conserved, especially transmembrane domain II and those adjacent to the HEXXH motif (Fig 1), with conserved charged residues, either in the transmembrane domains or closely adjacent, suggesting pore-forming proteins capable of transporting ions (9). Transient expression of both CAA20238 and Q15043 showed high molecular weight bands in non-reducing conditions which disappeared in reducing conditions (Fig 4) and a band corresponding to possible trimer formation, similar to the stable intermediate of the GABA receptor ion channel (8). Both these facts suggest an ability to form membrane complexes with a capacity for ion channel transport.

The discovery of this new family has important implications for the control of the life and death of cells due to the close connection between intracellular Zn²⁺ and apoptosis.

LIV-l has been implicated in metastatic breast cancer, yet we have observed its expression to be over 10,000 fold reduced compared to the other family member proteins examined so far suggesting a possible growth advantage (see figure 9) to breast cancer cells. These proteins have the potential to control apoptosis of cells by the transport of intracellular Zn²⁺. This function has important implications not only for the growth control of healthy cells but also for a number of significant diseases such as cancer and neurodegeneration. An added bonus for future drug intervention is the fact that Zn²⁺ is relatively non-toxic to cells, therefore offering a potential method of therapy.

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TABLE 1-A

Claims

1. The use of a polypeptide comprising one or more of the consensus regions of the proteins shown in Figure 1, or a functional homologue thereof, in the preparation of a medicament for the treatment of a disease selected from those involving disorders of zinc homeostasis.

2. Use according to claim 1 wherein the polypeptide or homologue comprises two or more such consensus regions.

3. Use according to claim 1 wherein the consensus region includes at least one of the regions I to VIII.

4. Use according to claim 2 or claim 3 wherein the polypeptide or homologue comprises consensus regions N, NI and the metalloprotease motif.

5. Use according to any preceding claim wherein the polypeptide or homologue has the sequence of one of the proteins shown in Figure 1.

6. Use according to any preceding claim wherein the disease is selected from breast cancer, neurodegenerative disorders and apoptotic disorders.

7. A method of selecting compounds for use in the treatment of a disease selected from those involving disorders of zinc homeostasis, the method comprising determining the effect of a test compound on a function of a polypeptide comprising one or more of the consensus regions of the proteins shown in Figure 1, or a functional homologue thereof.

8. A method according to claim 7 in which the compounds are selected for use in the treatment of breast cancer, neurodegenerative disorders or apoptotic disorders.

9. Compounds for the treatment of a disease selected from those involving disorders of zinc homeostasis, the compounds having been identified using the method of claim 8.

10. Compounds according to claim 9 identified by determining their effect on the zinc regulating ability of the selected protein.

11. Recombinant proteins including at least one of the consensus sequences of the proteins shown in Figure 1.

12. Synthetic proteins according to claim 11 wherein the consensus sequence includes at least one of the regions I to NIII from Figure 1.

13. Synthetic proteins according to claim 11 or claim 12 wherein the consensus sequence also includes the metalloprotease motif of Figure 1.

14. Synthetic proteins according to any of claims 11 to 13 wherein the consensus sequence comprises regions N, VI and the metalloprotease motif.

15. An antibody molecule having affinity for one or more of the proteins listed in Figure 1.

16. An antibody molecule according to claim 15 having affinity for one or more of the consensus sequences of the proteins listed in Figure 1.

17. A kit of antibody molecules directed against at least two different proteins selected from those listed in Figure 1.

18. A kit of antibody molecules according to claim 17 wherein the antibodies are directed against at least two of the consensus sequences of the proteins listed in Figure 1.

19. A method for identifying proteins involved in zinc homeostasis, the method comprising comparing a test protein sequence with one or more of the proteins listed in Figure 1, wherein the involved proteins are identified by having similarity with the sequences of Figure 1.

20. A method of diagnosis of a zinc homeostasis-related condition in a subject, the method comprising contacting a sample from that subject with an antibody molecule according to claim 15 or claim 16, and measuring the binding of that antibody to the sample.

21. A method of treatment of a zinc homeostasis-related condition in a subject, the method comprising contacting the subject with an antibody molecule according to claim 15 or claim 16.

22. A method of treatment of a disease selected from those involving disorders of zinc homeostasis, the method comprising the administration, to a subject in need of such treatment, of a therapeutically effective amount of a polypeptide comprising one or more of the consensus regions of the proteins listed in Figure 1, or a functional homologue thereof.

23. A method according to claim 22 wherein the polypeptide or homologue comprises at least two of the consensus regions.

2.4. A method according to claim 22 wherein the polypeptide or homologue comprises at least one of the regions I to NIII.

25. A method according to any of claims 22 to 24 wherein the polypeptide comprises the whole sequence of a protein selected from those listed in Figure 1.

26. A method according to any of claims 22 to 25 wherein the disease is selected from breast cancer, apoptotic disorders and neurodegenerative disorders.

27. A method of treatment of a disease selected from those involving disorders of zinc homeostasis, the method comprising the administration, to a subject in need of such treatment, of a therapeutically effective amount of a nucleic acid molecule encoding a polypeptide comprising one or more of the consensus regions of the proteins shown in Figure 1 , or a functional homologue thereof.

28. A method of modifying apoptosis in vitro or in vivo, the method comprising contacting cells with a polypeptide comprising one or more of the consensus regions of the proteins having the sequences listed in Figure 1, or a function homologue thereof.