Method for inhibiting the SWAP-70 protein

Abstract

The invention relates to the field of biomedical and pharmacological research, in particular in the field of immunology, allergies, cancers, bone diseases and autoimmune diseases. The invention is based on the recent finding that SWAP-70 dimerizes, that the dimerization takes place via a specific, largely unique and limited region of the protein, and that this dimerization is central to the function of the protein (and probably the stability thereof). The invention provides a screening method which makes it possible to identify new active ingredients which, by accumulating at the dimerization domain and inhibiting SWAP-70 activity, suppress the supporting function of SWAP-70 in tumorigenesis, tumor cell migration and invasion, bone-degrading osteoclast activity, and the allergic reaction, as well as in autoimmune diseases. The object is achieved by a method for identifying a substance which inhibits the activity of SWAP-70, wherein the method comprises the following: contacting at least one test substance with SWAP-70, detecting the degree of dimerization of SWAP-70, selecting a test substance which inhibits the dimerization of SWAP-70.

Claims

1. A method for identifying a substance which inhibits dimerization of switch-associated protein-70 (SWAP-70), wherein the method comprises the following: (i) contacting: (a) a test substance with (b) a dimerization domain of SWAP-70 consisting of the sequence QDEETVRKLQARLLEEESSKRAELEKWHLEQQQAIQTTEAEKQELE QQRVMKEQALQEAMAQLEQLELERKQALEQYEGVKKKLE (SEQ ID No. 1) or a partial sequence thereof having at least 70 contiguous amino acid residues of SEQ ID NO: 1; (ii) detecting the degree of dimerization of the SWAP-70 dimerization domain in a sample containing the SWAP-70 dimerization domain and said test substance and in a sample containing the SWAP-70 dimerization domain that does not contain test sample, and (iii) identifying the test substance as an inhibitor of the dimerization of SWAP-70 when dimerization in the sample containing said test substance is decreased compared to dimerization in the sample that does not contain test sample.

2. The method of claim 1, wherein a FRET-based assay is performed to detect the degree of dimerization.

3. The method of claim 1, wherein dimerization is measured in vitro or in situ.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) The invention will be described in greater detail below by means of figures and examples, without being limited thereto:

(2) FIG. 1 shows schematically the structure of a SWAP-70 protein. The SWAP-70 protein consists of the N-terminal EF hand domain, a pleckstrin homology domain (PH) which binds PIP.sub.3 and is responsible for the localisation of the membrane, a three-part coiled coil domain (CC1, CC2, CC3) and a C-terminal F-actin binding site (AB), by which SWAP-70 binds specifically to non-muscular actin (but not to muscular actin). The dimerisation domain is identified by a black bar and is located predominantly in the CC3 domain.

(3) FIG. 2 shows the results of the gel electrophoresis from Example 1. It is clear that GST-SWAP-70 and His-SWAP-70 interact with one another. In the top part of the figure it can be seen that His-SWAP-70 is bound to GST-SWAP-70, visualised by addition of anti-His. If only GST is present, however, and not GST-SWAP-70, His-SWAP-70 has no binding partner, is washed out and does not provide a binding site for anti-His. Controls without His-SWAP-70 are shown in the bottom part of the figure.

(4) FIG. 3 shows the results of the gel electrophoresis from Example 3. S=supernatant, P=pellet.

(5) FIG. 4 shows the results of the gel electrophoresis from Example 4. S=supernatant, P=pellet.

EXAMPLE 1: PULL-DOWN ASSAY FOR DIMERISATION

(6) A pull-down assay with purified recombinant GST-SWAP-70 and SWAP-70 demonstrated the interaction between these two proteins.

(7) For the in vitro pull-down assay, 0.4 μM purified GST-SWAP-70 and His-SWAP-70 or GST for control were mixed in 20 mM Tris, pH 7.5, 100 mM NaCl, 10% glycerol, 1 mM EDTA, 25 mM CaCl.sub.2, 0.1% NP40 and 1 mM DTT were mixed and shaken for 1 hour at 4° C. Glutathione agarose beads were then added and the samples were incubated for 30 minutes, washed and resuspended in SDS-Page loading buffer. The samples were then subjected to SDS-Page and immunoblotting.

(8) In the gel figure (FIG. 2), it can clearly be seen that GST-SWAP-70 and His-SWAP-70 interact with one another.

EXAMPLE 2: GEL FILTRATION FOR DIMERISATION

(9) For further testing of the oligomerisation, recombinant, purified SWAP-70, SWAP-70w/oCC (mutant in which the dimerisation domain is missing) and SWAP-70 C-term (shortened SWAP-70 with actin binding domain and a portion of the dimerisation region) as well as protein standards (gel filtration marker kit for proteins, molecular weight 29-700 kDa, Sigma) were analysed individually by means of gel filtration at a throughput of 60 ml/h on a Superdex 200 HPLC column (Amersham Pharmacia Biotech). The elution volumes of the protein standards were fitted to a curve in relation to their theoretical molecular weight. This curve was used to determine the experimental molecular weights.

(10) The experimentally determined molecular mass of SWAP-70 was 151±17.6 kDa, approximately twice the theoretical molecular mass of SWAP-70 (71.6 kDa), which suggests that dimers have formed.

(11) The experimentally determined molecular mass of SWAP-70 C-term was 79±3.5 kDa, which, compared with the theoretical molecular mass of 19 kDa, suggests tetramerisation.

(12) The experimentally determined molecular mass of SWAP-70w/oCC was 50±3 kDa, which corresponds approximately to the theoretical molecular mass of 46 kDa and shows that SWAP-70 mutants in which the dimerisation domain is missing do not dimerise.

(13) The results are listed briefly below in table form:

(14) TABLE-US-00003 Theoretical molecular Experimentally determined Protein mass [kDa] molecular mass [kDa] SWAP-70 71.6 151 ± 17.6 SWAP-70 C-term 19 79 ± 3.5 SWAP-70w/oCC 46 50 ± 3.sup. 

EXAMPLE 3: SWAP-70 BUNDLES ACTIN FILAMENTS BY MEANS OF ITS C-TERMINAL REGION

(15) The actin bundling activity of SWAP-70 was tested by means of low-speed centrifugation. In the same manner, the actin bundling activity of SWAP-70 C-term was tested.

(16) Actin (3 μM) was first polymerised. This was followed by mixing with SWAP-70, or with SWAP-70 C-term, incubation at room temperature for 30 minutes and centrifugation at 13,000 rpm and 4° C. for 30 minutes. A control was treated in the same manner without the addition of SWAP-70 or SWAP-70 C-term. The supernatants were removed and precipitated with ice-cold acetone. The pellets, which contained the crosslinked F-actin, were washed and resuspended in 1×SDS-Page loading buffer. The samples were analysed on Coomassie-stained gel. The protein bands were quantified by means of densitometry using Fiji software.

(17) While significantly more actin was present in the supernatant than in the pellet in the control (left-hand part), this ratio was displaced in favour of the pellet by the addition of SWAP-70 and SWAP-70 C-term. Because SWAP-70 C-term is capable, because of tetramerisation, of bundling more actin filaments than SWAP-70, the corresponding band on the gel figure (on the far right) is stronger than the actin band in the pellet upon addition of SWAP-70 (middle).

EXAMPLE 4: THE DIMERISATION OF SWAP-70 BRINGS ABOUT ACTIN BUNDLING

(18) The actin bundling activity of SWAP-70w/oCC was tested by means of low-speed centrifugation.

(19) Actin (3 μM) was first polymerised. This was followed by mixing with SWAP-70w/oCC, incubation at room temperature for 30 minutes and centrifugation at 13,000 rpm and 4° C. for 30 minutes. A control was treated in the same manner without the addition of SWAP-70w/oCC. The supernatants were removed and precipitated with ice-cold acetone. The pellets, which contained the crosslinked F-actin, were washed and resuspended in 1×SDS-Page loading buffer. The samples were analysed on Coomassie-stained gel. The protein bands were quantified by means of densitometry using Fiji software.

(20) An additional band was detected in the sample that contained SWAP-70w/oCC as compared with the control, which shows that SWAP-70w/oCC had not bound to actin. Because SWAP-70w/oCC lacks the dimerisation domain, this suggests that actin bundling takes place with the involvement of the dimerisation domain.

EXAMPLE 5: DETECTION OF DIMERISATION IN VIVO BY FRET

(21) 10 μg of Venus-SWAP-70 and Cerulean-SWAP-70 (Venus is an improved yellow fluorescent protein and Cerulean an improved cyan fluorescent protein) are transfected with Lipofectamin 2000 (Invitrogen) in NIH 3T3 cells which had been grown for 16 hours on coverslips coated with poly-(L-lysine) (100 μg/ml). The serum was then removed from the cells for a period of 2 hours, whereupon they are stimulated with 15 nM EGF (PeproTech) in Dulbecco's modified Eagle's medium (DMEM) and immediately fixed for 10 minutes with 4% polyformaldehyde (PFA), PBS (phosphate buffered saline). Acceptor Photobleaching FRET Makro, LAS software (Leica) is used to record images. The fluorescence intensities are detected before and after the bleaching of a region of interest with a 510 nm laser (100% power). The FRET efficiency is determined with the pbFRET v1 plugin (Mike Lorenz, MPI-CBG, Germany) using ImageJ (NIH) or Fiji. The samples are analysed at room temperature using a Leica TCS SP5 confocal laser scanning microscope (Leica, Germany) and an HCX PL APO 63X 1.4 NA by means of oil objective. The images are captured in the linear region of the reaction of the detectors, pixel saturation being prevented with LAS AF software (Leica), and analysed by Fiji.

EXAMPLE 6: DETERMINATION OF THE DIMERISATION IN VITRO BY DIFFUSION ANALYSIS AND FLUORESCENCE CROSS CORRELATION SPECTROSCOPY

(22) In each case 1 μg of SWAP-70 protein is labelled either with the fluorescent dyes Alexa 488 or with Atto 655, approximately from 1 to 2 molecules of the dye in question being bound per molecule of SWAP-70. The SWAP-70 protein so labelled is brought in a confocal microscope in solution into a measuring chamber and excited by means of one laser (labelling with one dye) or two lasers (combination of the two differently labelled SWAP-70 molecules). The diffusion of a labelled SWAP-70 is measured over time and correlates with the monomeric, dimeric or multimeric state of the protein, which can accordingly be derived therefrom. The reciprocal influencing of the two different labellings that occurs as a result of the dimerisation is determined as fluorescence cross correlation. Both methods confirm the existence of SWAP-70 dimers. A shortened (42 kDa based on gel filtration and SDS gel electrophoresis) protein, SWAP-70w/oCC, in which the above-mentioned dimerisation sequence is missing, proved to be a monomer in these tests too.

(23) The following tables show examples of results of mass calculations based on diffusion times. The diffusion time of a molecule is directly proportional to the spatial root of the molecular mass, which accordingly can approximately be determined according to the formula below. The molecular mass of the dyes is used for calibration.

(24) $\frac{{\tau }_d^{S70}}{{\tau }_d^{\mathrm{dye}}}=\frac{\sqrt[3]{M{W}^{S70}}}{\sqrt[3]{M{W}^{\mathrm{dye}}}}$

(25) The measured diffusion times of the dyes Atto 655 and Alexa 488 were used according to this formula to calculate the molecular mass of SWAP-70 or of the above-mentioned SWAP-70w/oCC fragment. Table 1 shows the diffusion times.

(26) TABLE-US-00004 TABLE 1 Diffusion time 42 kDa 42 kDa SWAP-70 SWAP-70 frag- frag- Diffusing red green ment ment molecule (Alexa) (Atto) Atto 655 Alexa 488 red green Diffusion 247 ± 5 207 ± 7 37 ± 1.4 33 ± 0.6 161 ± 3 130 ± 2 time (μs)
Table 2 shows the number of monomers per molecule, obtained from the molecular mass derived from the diffusion time, for either red or green dyed SWAP-70, based on the individual dyes used as standards or the SWAP-70w/oCC fragment. The results show a very good approximation to a dimeric molecular structure and thus confirm the results of the gel filtration showing the above-mentioned dimeric SWAP-70.

(27) TABLE-US-00005 TABLE 2 Number of SWAP-70 monomers per SWAP-70 molecule, based on the indicated standards. No. of monomers No. of monomers Standard SWAP-70 red SWAP-70 green 42 kDa fragment 2.1 ± 0.3 2.6 ± 0.5 Atto 655 or Alexa 488 2.6 ± 0.6 2.3 ± 0.4