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  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present invention relates to a regulator of Calcineurin and uses of this regulator.
• Calcineurin is a serine/threonine protein phosphatase, which is a heterodimer composed of a catalytic subunit (Calcineurin A) and a regulatory subunit (Calcineurin B).
• the catalytic subunit is about 60 and the regulatory subunit is about 18 kDa.
• This protein phosphatase is under the control of calcium and calmodulin, wherein binding of calcium and calmodulin is necessary for enzymatic activity.
• both subunits, the catalytic subunit and the regulatory subunit, occur in different isoforms which are encoded by different genes.
• three distinct genes (A- ⁇ , A- ⁇ , A- ⁇ ) for the catalytic subunit have been characterized.
• this subunit has five distinct domains: the N- terminal domain of sofar unknown function, the catalytic domain, the B- subunit binding domain, the calmodulin-binding domain and the auto- inhibitory domain.
• Calcineurin plays an important role in the coupling of calcium signals to cellular responses. Although it is ubiquitously expressed, distinct tissue expression patterns have been described for various isoforms of each subunit. Now Calcineurin is discussed in various contextes. For example, Calcineurin has been implicated in neuronal signalling pathways but the neuronal function is only poorly understood. Furthermore, Calcineurin is discussed in the context of immunosuppression, wherein Calcineurin acts via the transcription factor NFAT (Nuclear Factor Of Activated T-Cells) on the T-cell response. Regarding the regulation of Calcineurin activity it is known that this enzyme is regulated by calcium and calmodulin (Rusnak, F. and Mertz, P. (2000), Physiol. Rev.
• Calcineurin expression, activity and tissue distribution have been described in a variety of disorders ranging from ischemia/reperfusion injury to cardiac hypertrophy, heart failure, stroke, amyotrophic lateral sclerosis, a degenerative motor neuron disease, skeletal muscle diseases and epilepsy.
• Calcineurin plays a crucial role in widespread diseases like heart failure and stroke, there is a special interest in the regulatory mechanisms concerning the activity of Calcineurin. The detailed knowledge of the regulation mechanisms would provide a possibility to influence the activity of Calcineurin, especially in pathological situations.
• the invention has the object to provide suited targets for influencing the activity of this crucial enzyme and to provide active agents for the treatment of diseases, which are connected with a disturbance of Calcineurin activity.
• the invention comprises a regulator of Calcineurin, which is based on a splice variant of the catalytic subunit of Calcineurin.
• this regulator lacks the complete subunit, the catalytic and the Calcineurin B binding domain, therefore it possesses no phosphatase activity on its own. It retains part of the N-terminal domain, and the complete calmodulin-binding domain as well as the autoinhibitory domain.
• This inventive regulator is able to stimulate the phosphatase activity of Calcineurin, especially at low calcium concentrations.
• the splice variant itself binds calcium. Therefore the stimulating activity of the splice variant is especially surprising, because it should be assumed, that the splice variant and Calcineurin compete for calcium.
• the splice variant forms a protein complex with Calcineurin, especially with the catalytic Calcineurin holoenzyme.
• a protein complex with Calcineurin and superoxide dismutase is especially preferred.
• the stimulating effect of the splice variant is dependent on low calcium concentrations.
• these calcium concentrations are below 1 mM.
• a putative function of the splice variant under natural conditions is to render Calcineurin into a more calcium sensitive enzyme thus triggering Calcineurin linked signalling pathways at even lower calcium concentrations.
• the inventive splice variant provides an additional regulatory mechanism that ensures Calcineurin activity even at low calcium concentrations.
• the regulator of Calcineurin is based on a splice variant of the ⁇ -form, ⁇ -form and/or ⁇ - form of the catalytic subunit.
• the splice variants of the different isoforms occur each in two forms (a) and (b), wherein one form (b) lacks 30 nucleotides respectively 10 amino acid residues as depicted in figures 1 to 6.
• the regulator is characterized by an amino acid sequence at least 70 % and preferably at least 80 %, especially at least 90 % identical to the amino acid sequence of the splice variant of the ⁇ -form (SEQ ID NO 7, 8), the ⁇ form (SEQ ID NO 9, 10) and/or the ⁇ -form (SEQ ID NO 11 , 12).
• the inventive regulator is coded by a nucleotide sequence at least 70 % and preferably at least 80 %, especially at least 90 % identical to the nucleotide sequence coding for the splice variant of the ⁇ -form (SEQ ID NO 1 , 2), the ⁇ -form (SEQ ID NO 3, 4) and/or the ⁇ -form (SEQ ID NO 5, 6).
• the invention comprises a nucleotide sequence, which is at least 70 % and preferably at least 80 %, especially at least 90 % identical to a nucleotide sequence according to figure 1 (SEQ ID NO 1 , 2), which represents the sequence of the splice variant of the ⁇ -form, and/or which is at least 70 % and preferably at least 80 %, especially at least 90 % identical to a nucleotide sequence according to figure 2 (SEQ ID NO 3, 4), which represents the sequence of the ⁇ -form of the splice variant.
• these nucleotide sequences code for a regulator of Calcineurin.
• the invention comprises an amino acid sequence, which is at least 70 % and preferably at least 80 %, especially at least 90 % identical to an amino acid sequence according to figure 4 (SEQ ID NO 7, 8), which represents the amino apid sequence of the ⁇ -form of the splice variant, and/or according to figure 5 (SEQ ID NO 9, 10), which represents the amino acid sequence of the ⁇ -form of the splice variant.
• amino acid sequences form a regulator for Calcineurin as described above.
• the invention comprises an active agent especially for the treatment of diseases, wherein the active agent is the regulator of Calcineurin as described above. Since the inventive regulator is able to stimulate the phosphatase activity of Calcineurin, the active agent is especially useful for the treatment of diseases, which are accompanied by reduced Calcineurin activity, e.g. Alzheimers disease or amyotrophic lateral sclerosis.
• an active agent for the treatment of diseases which influences the regulator of Calcineurin as it is described above.
• said active agent increases the activity of the regulator of Calcineurin.
• Such an active agent is especially useful for the treatment of diseases, which are accompanied by low Calcineurin activities.
• the active agent reduces the activity of the regulator of Calcineurin.
• Such an active agent is especially useful for the treatment of diseases which are accompanied by an increased Calcineurin activity.
• the active agent is a substance, which interacts with the regulator thereby blocking its stimulating activity.
• the active agent is an inactive variant of the regulator, which competes for substrates with the functional regulator, and/or influences the forming of functional protein complexes.
• Such functional protein complexes may be built of Calcineurin and the regulator and/or superoxide dismutase, and/or other components.
• diseases which could be effectively treated with an active agent, which reduces the activity of the regulator of Calcineurin, are cardiovascular diseases. For example in tissue of heart failure organs it is described, that Calcineurin is about four times higher than in normal tissue.
• the part of the regulator of Calcineurin is stimulation of Calcineurin, which results in the closing of calcium channels in a feedback mechanism, which terminates the activity of Calcineurin, because the necessary calcium influx is stopped.
• an inventive agent which reduces the activity of the regulator, i.e. the stimulator of Calcineurin
• the feedback mechanism is stopped. Consequently, the calcium channels stay open and the calcium influx continues, resulting in a maintenance of Calcineurin activity. Consequently it depends on the certain circumstances of the disease to be treated, if an active agent is chosen, which increases the activity of the regulator of Calcineurin or an active agent is chosen, which reduces said activity.
• cardiovascular diseases and/or neurodegenerative diseases like epilepsy or amyotrophic lateral sclerosis and other diseases like skeletal muscle diseases, immunological diseases, inflammation diseases and/or cancer, for example, are preferred candidates for the treatment with an active agent according to the invention.
• all CNAs can be used to influence apoptosis.
• activators of the CNAs and the CNAs by their own
• inhibitors of the CNAs and modified CNAs by their own
• activators of CNAs can be used e.g. as therapeutical agents (for instance as therapeutical proteins) to treat e.g. cancer, immune diseases, neurological diseases, heart and cardiovascular diseases and/or inflammation diseases by induction of apoptosis in problematic cells.
• therapeutical agents for instance as therapeutical proteins
• CNAs and/or the CNAs by their own can be used as activator of apoptosis.
• inhibitors of the CNAs can be used as therapeutical agents to treat e.g. cancer, immune diseases, neurological diseases, heart and cardiovascular diseases and/or inflammation diseases by prevention of apoptosis in problematic cells.
• Such inhibitors of the inventive matter can be, in addition to the already described, for instance antisense sequence(s) of the CNAs and/or antibodies against the CNAs.
• the invention comprises the use of an active agent as described above for the treatment of diseases, and a method for the treatment of diseases, wherein at least one inventive active agent as described above is administered.
• an active agent as described above for the treatment of diseases
• a method for the treatment of diseases wherein at least one inventive active agent as described above is administered.
• the invention comprises a pharmaceutical composition, which comprises at least one active agent as described above in an effective amount and at least one pharmaceutical carrier.
• the pharmaceutical composition is intended for topical or systemical administration, for example.
• the invention cpmprises the use of a regulator of Calcineurin for the diagnosis of diseases, wherein the frequency of said regulator in the organism is analysed.
• a regulator of Calcineurin for the diagnosis of diseases, wherein the frequency of said regulator in the organism is analysed.
• several disorders are accompanied by an increased or decreased activity of Calcineurin in comparison with non-pathological conditions.
• this altered Calcineurin activity is caused by a changed activity and/or frequency of the regulator of Calcineurin, which is based on a splice variant as described above.
• the inventive splice variant of the ⁇ -form of Calcineurin A is considerably stronger expressed than in normal animals, as described in further detail in the examples below. Therefore the invention comprises the use of the regulator for diagnosis of the respective diseases, especially in the early diagnosis of the diseases.
• at least one substance is used, which interacts with the regulator of Calcineurin.
• the regulator of Calcineurin is preferably a regulator as described above.
• said interacting substance is an antibody, which interacts with the regulator, preferably on the level of protein-protein or protein-peptide interaction.
• any antibody specific for the regulator is suitable in the inventive manner, for example polyclonal antibodies and/or monoclonal antibodies, as will be obvious for an expert in the art.
• the specific antibody is placed on an array, which permits a fast and easy performance of the diagnostic test, for example the antibody is placed on a dip stick as it is known in the field of immunodiagnosis.
• the interacting substance is a nucleic acid molecule, which hybridizes with at least a portion of a nucleic acid sequence coding for the regulator.
• This embodiment is especially preferred, because such hybridisation probes are cheap and easy to obtain and the diagnostic test can be performed by common methods.
• the hybridisation probe i.e. the nucleic acid molecule, which hybridizes with at least a portion of the nucleic acid sequence coding for the regulator, is placed on a DNA chip, and the complete diagnostic reaction is preferably performed in an automated manner.
• the inventive use is especially useful for fast diagnosis of different diseases.
• said diseases are connected with a disturbance of Calcineurin activity
• said diseases are neurodegenerative diseases like amyotrophic lateral sclerosis and/or said diseases are cardiovascular diseases like heart failure.
• said diseases are also immunological diseases, inflammation diseases and/or cancer are examples for diseases connected with a disturbance of calcineurin activity.
• the invention comprises a kit, which comprises at least one substance, which is able to interact with the regulator of Calcineurin.
• This kit is especially useful for diagnosis as described above.
• the kit comprises at least one specific antibody for the regulator.
• the kit comprises at least one nucleic acid molecule, which is able to hybridize with at least a portion of a nucleic acid sequence coding for the regulator.
• the invention comprises a method for searching substances, which influence the activity of Calcineurin.
• This method is characterized in that a splice variant of the catalytic subunit of Calcineurin is used to identify and/or isolate substances, which are able to interact with the splice variant itself and/or with Calcineurin.
• the activity of Calcineurin is measured in vitro, whereby the influence of the regulator is analysed with or without (as control) additional substances, which possibly influence the activity of Calcineurin via the regulator.
• the complex formation between at least Calcineurin, the regulator and the substance possibly influencing the activity of Calcineurin is used as read-out system.
• the invention comprises an active agent for the treatment of diseases, which is identified and/or isolated by a method as described above.
• Figure 1 Nucleotide sequence of the inventive splice variant of the ⁇ - form of Calcineurin A, a further variant of this splice variant lacks nucleotides 268 to 297,
• Figure 2 Nucleotide sequence of the inventive splice variant of the Y- form of Calcineurin A, a further variant of this splice variant lacks nucleotides 232 to 261 ,
• Figure 3 Nucleotide sequence of the inventive splice variant of the ⁇ - form of Calcineurin A, a further variant of this splice variant lacks nucleotides 244 to 273
• Figure 4 Amino acid sequence of the inventive splice variant of the ⁇ - form of Calcineurin A, a further variant of this splice variant lacks amino acid residues 90 to 99,
• Figure 5 Amino acid sequence of the inventive splice variant of the ⁇ form of Calcineurin A, a further variant of this splice variant lacks amino acid residues 78 to 87,
• Figure 6 Amino acid sequence of the inventive splice variant of the ⁇ - form of Calcineurin A, a further variant of this splice variant lacks amino acid residues 82 to 91 ,
• FIG. 7 PCR amplification of Calcineurin A- ⁇ . Calcineurin cDNAs were amplified by nested PCR and the amplicons were separated on a 0,8 % agarose gel. In addition to the known transcripts at 1.6 kb, an additional splice variant of about 450 bp was detected,
• Figure 8 Domain and exon structure of Calcineurin (CN) A ⁇ 16K. The sequence consists of exons 1 , 11 , 12, 13 and 14 and contains part of the N-terminal domain, the calmodulin-binding domain and the autoinhibitory domain. Catalytic domain (exon 2 - 8) and Calcineurin B-binding domain (exon 9 + 10) are missing.
• CN B Calcineurin B-binding domain
• CaM calmodulin-binding domain
• Al autoinhibitory domain
• Figure 9 Immunoblot of Calcineurin.
• 1 bovine brain Calcineurin (Sigma)
• 2 bovine brain Calcineurin pulled down with Cu/Zn superoxide dismutase (Cu/Zn SOD was bound to NiNTA-magnetic beads prior to incubation with bovine brain CNA, Sigma).
• 3 human recombinant SOD.
• 4-9 murine spinal cord protein extracts, 4 th , 6 th and 8 th lane: cytosolic fraction, 5 th , 7 th and 9 th lane: pellet fraction, 4 th - 7 th lane: wild-type. 8 th and 9 th lane: mice transgenic for human mutated Cu/ZnSOD.
• Figure 12 Direct interaction of Calcineurin A ⁇ 16K and standard Calcineurin.
• 1 Calcineurin positive control.
• 2 bacterial control eluate was bound to Ni-NTA magnetic beads and used to pull down bovine brain CN (Sigma).
• 3 pull-down of Calcineurin with CNA ⁇ 16K (CNA ⁇ 16K was bound to Ni-NTA-magnetic beads prior to incubation with bovine brain CN, Sigma).
• 4 pull-down of spinal cord homogenate with CNA ⁇ 16K (CNA ⁇ 16K was bound to NiNTA-magnetic beads prior to incubation with murine brain homogenate).
• 5 bacterial control eluate was bound to Ni-NTA-magnetic beads and used to pull down spinal cord homogenate. Examples:
• the inventive splice variants are able to enhance the susceptibility of cells to calcium- influx and therefore are at least partially responsible for influences on calcium-linked signalling in amyotrophic lateral sclerosis as well as in apoptosis of neurons, stroke and heart failure, for example.
• the stimulating effect of the inventive splice variants increases when a splice variant, is preincubated with Calcineurin. This shows a time-dependent mechanism, which is also strenghtened by the detection of a direct physical interaction between the splice variant and standard Calcineurin.
• the effect of the inventive splice variants works via calmodulin, e.g. increasing the activating effect of calmodulin by stabilizing the Calcineurin-calmodulin complex.
• Calcineurin A ⁇ was amplified by a nested PCR from human spinal cord poly-A + -RNA from Clontech, Palo Alto, California. PCRs were conducted in a 25 ⁇ l volume. They contained 2.5 ⁇ l 10x PCR-buffer (Stratagene), 0.25 ⁇ l 25mM dNTP-mix, 0.25 ⁇ l CNA-sl primer (10 ⁇ M), 0.25 ⁇ l CNA- as1 primer (10 ⁇ M), 2.5 ⁇ l cDNA, 0.25 ⁇ l Taq Polymerase (Promega), 0.25 ⁇ l Pfu polymerase and distilled water ad 25 ⁇ l. PCR was started by a first heating step at 95°C for 3 min.
• the amplification conditions were 35 cycles of 40 sec at 95 °C for denaturing, 40 sec at 55 °C for annealing and 3 min at 72 °C for elongation. PCR was terminated with a 10 min extension step at 72 °C.
• the nested PCR was performed with 2.5 ⁇ l of the first amplification reaction with the primers CNA-s2 and CNA-as4 as above.
• the amplification conditions were 25 cycles of 40 sec at 95 °C for denaturing, 40 sec at 55 °C for annealing and 3 min at 72 °C for elongation. PCR was terminated with a 10 min extension step at 72 °C.
• CNA-S1 ATC TGC TCA GAC GAT GAA CTR GG (SEQ ID NO 13)
• CNA-as1 GGC ATC CTC TCG, TTA ATT CGG (SEQ ID NO 14)
• the resulting amplicons were digested with Bam HI and Xma I, electrophoresed and purified after gel electrophoresis with the Qiaquick gel extraction kit (Qiagen, Hilden, Germany) according to the manufacturers recommendations.
• the digested amplicons were ligated into pQE30 vector (Qiagen, Hilden, Germany) digested with Bam HI and Xma I. Resulting clones were sequenced completely.
• the resulting plasmid pQE30K16 was transformed into M15 bacteria, positive clones were inoculated into LB medium containing 50 ⁇ g/ml Ampicillin at 37°C overnight with vigorous shaking. Next morning 0.5 ml bacteria were inoculated into 1 liter LB medium containing 100 ⁇ g/ml ampicillin, after the bacteria reached a OD 6 oo Of 0.5 to 0.7, IPTG was added to the culture to a final concentration of 1 ⁇ mol/l. After 4 hours the bacteria were pelleted by centrifugation at 5,000g for 20 min and the pellet was frozen at -80 °C over night.
• the frozen pellet was thawed in ice for 15-30 min and the cells were resuspended in lysis buffer (50mM NaH 2 P0 4 , 300mM NaCI (pH8.0)) at 2.5 ml per gram wet weight. Lysozyme was added for a final concentration of 1 mg/ml and the cells were incubated on ice for 30 min, then the cells were sonicated on ice 6 times for 10 seconds. Cellular debris was pelleted by a centrifugation of 30 min at 10,000g and 4 °C and the supernatant was mixed with 1 ml of a 50 % slurry of Ni-NTA. The mixture was incubated at 4 °C with gentle shaking for 60 min and loaded into a column.
• lysis buffer 50mM NaH 2 P0 4 , 300mM NaCI (pH8.0)
• wash buffer 50mM NaH 2 P0 4 , 300mM NaCI, 50mM imidazole (pH8.0)
• the protein was eluted with 1.6 ml elution buffer (50mM NaH 2 P0 4) 300mM NaCI, 500mM imidazole (pH8.0)).
• a buffer exchange was performed by ultrafiltration (centrex UF-2, Schleicher & Schuell, Dassel).
• Calcineurin activity was assayed with the Calcineurin Activity Assay kit (Calbiochem, Palo Alto, USA).
• Calcineurin Activity Assay kit Calbiochem, Palo Alto, USA.
• 5 ⁇ l purified Calcineurin Sigma Aldrich, Deisenhofen, Germany
• 25 ⁇ l 2x assay buffer 100mM NaCI, 50mM Tris, 6mM MgCI 2 , 0.5mM CaCI 2 , 0.5mM DTT, 0.25% NP- 40, 0.25 ⁇ M Calmodulin
• 10 ⁇ l substrate solution 150 ⁇ M Rll Phosphopeptide
• 5 ⁇ l CNA ⁇ 16K solution 1.5 ⁇ g/ ⁇ l
• the beads were washed 2 times for 30 minutes with 500 ⁇ l of buffer A at constant shaking (1000 rpm, 4 °C) and elution was performed with 100 ⁇ l of buffer C buffer (50mM NaH 2 P0 4 , 300mM NaCI, 500mM imidazole, pH8.0).
• buffer C buffer 50mM NaH 2 P0 4 , 300mM NaCI, 500mM imidazole, pH8.0.
• the calcineurin was detected by western blotting.
• CNA ⁇ K16 calcineurin splice variant was amplified from procaryotic expression vector pQE30K16 using the following primers:
• 16kfw2 ggg gac aag ttt gta caa aaa age agg ctt aga agg aga tag aac c - ATG GCC GCC CCG GAG CCG GCC CGG GCT GCA (without histidine-tag)
• the amplicons were inserted into the eucaryotic expression vector pDEST 12.2 by recombination according to the manufacturers recommendations.
• the resulting clones were sequenced and named pEXP12-K101 for the His-tagged and pEXP12-K105 or the non-tagged form.
• TNF induced apoptosis in Hela cells was used as a model for apoptosis in cancer cells, but which is in principle also important in nerve cells, immune cells, heart muscle cells and other tissues [Cell 1996 Nov. 1 ; 87 (3): 565-76, Dissection of TNF receptor 1 effector functions: JNK activation is not linked to apoptosis while NF- kappaB activation prevents cell death. Liu ZG, Hsu H, Goeddel DV, Karin M.].
• Hela cells were tranfected with either CNA ⁇ 16K containing plasmids (pEXP12-K101 or pEXP12-K105) or as an control with an empty pCDNA3 vector or no plasmid at all.
• Cell apoptosis was induced by TNF (10 ng/ml, 48 h post transfection). In every case cells were cotransfected with an CMV- ⁇ -plasmid expressing LacZ. Therefore, cell survival can be tested by counting LacZ positive (blue) cells. Reduction of LacZ positive cell, tested by X-Gal staining 24 h post-transfection, indicates cell death by apoptosis.
• Frozen spinal cord tissues were transferred into 2x homogenization buffer (500 mM Sucrose, 4mM EDTA, 0.02 % ⁇ -mercaptoethanol; 500 ⁇ l/100 mg tissue).
• 2x homogenization buffer 500 mM Sucrose, 4mM EDTA, 0.02 % ⁇ -mercaptoethanol; 500 ⁇ l/100 mg tissue.
• PMSF 10 mg/ml
• Leupeptin 1 mg/ml
• the tissues were homogenized with a glass homogenizer in this solution and then transferred into cups. These cups were centrifuged for 3 hours at 4 °C and 14,000 x g to separate soluble from particle (non-soluble) containing fractions. The soluble fraction was filled in a new cup after centrifugation.
• the particle containing cup was filled up with an adequate volume of 2x homogenization buffer. Protein content of both fractions was determined by the BioRad protein assay reagent, with bovine serum albumin as the standard.
• Calcineurin A ⁇ splice variants were amplified by a nested PCR from human spinal cord poly-A + -RNA (Clontech, Palo Alto, USA).
• a strong band at 1.6 kb indicated the transcripts for the Calcineurin A ⁇ splice variants.
• Cloning and sequencing indeed revealed that the 1.6 kb band consisted of two splice variants, differing by 30 base pairs by alternative splicing of exon 13. In addition, a smaller band was obtained at about 450 bp that was subsequently isolated and cloned (fig. 7).
• this cDNA represents a cloning artefact or whether it is a novel Calcineurin A ⁇ splice variant that is transcribed into protein
• western blot analysis on purified, commercially available bovine Calcineurin (Sigma Aldrich, Deisenhofen, Germany) were perfomed. Although a smaller immunoreactive band was visible in the range of 35 kD with an anti-Calcineurin A antibody, immunoreactivity in the range of 16 kD was not detectable.
• the cDNA was cloned into the pQE30 expression vector, resulting in a hypothetical protein of 158 amino acids and a calculated molecular weight of 16,921.13 Da, with an N-terminal HlS-tag for quick purification from bacterial lysates.
• Induction with IPTG and purification with Ni-NTA agarose indeed yielded a protein of 17 kDa.
• Expression efficiency was low, from a 500 ml culture about 600 ⁇ g of Calcineurin A ⁇ 16K protein was yielded.
• Western-blotting of affinity column eluates revealed positive immunoreactivity with the anti-Calcineurin A antibody.
• CNA ⁇ 16K exhibited no phosphatase activity in a Calcineurin activity assay using phospho-RII peptide as a substrate.
• CNA ⁇ 16K exhibited no phosphatase activity in a Calcineurin activity assay using phospho-RII peptide as a substrate.
• CNA ⁇ 16K was investigated by incubating purified bovine Calcineurin (Sigma, Deisenhofen) with and without CNA ⁇ 16K.
• bacteria were transformed with pQE30, induced, harvested and the purification process was repeated exactly as in the case of the Calcineurin A ⁇ 16K. Comparable elution fractions served as controls for the CNA ⁇ 16K. As seen in fig.
• a pull down assay with CNA ⁇ 16K as binding protein was performed, to verify if there is a direct interaction in vitro between CNA ⁇ 16K and purified Calcineurin.
• CNA ⁇ 16K was bound to magnetic Ni- NTA beads and incubated with purified bovine Calcineurin as well as murine spinal cord eluates. Again, the corresponding eluates from pQE30-transformed and Ni-NTA agarose purified bacteria served as controls.
• strong binding of Calcineurin to immobilized histidine-tagged CNA ⁇ 16K could be seen either using purified bovine brain Calcineurin or murine spinal cord homogenate (fig. 12, lanes 3 and 4). In contrast, no detectable binding was seen where the control eluate was used (fig. 12, lanes 2 and 5).
• CNA ⁇ 16K is an important regulator of neuronal apoptosis, since it is neuron specific.
• the CNAs are important in apoptosis, in particular cancer, brain, heart and immune system, since calcineurin and therefore the regulators are involved in regulation of cell- cyclus and immune system [Rusnak F (2000), Physiological reviews 80, 1483-1521], and in cancer, as is illustrated by the results with the Hela cancer cells.

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