JP4101104B2 - Methods relating to diseases related to abnormal secretion of hormones secreted from the pituitary gland or / and pineal gland using novel interactions between proteins - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a disease-related disease-related technique for hormones secreted from the pituitary gland or / and pineal gland utilizing a novel interaction between proteins. More specifically, the present invention relates to a screening method for a therapeutic agent for an abnormality-related disease of growth hormone (GH) or other hormones secreted from the pituitary gland or / and pineal gland.
[0002]
[Prior art]
In the post-sequence era, large-scale gene function analysis is urgently needed. The gene function analysis so far has mainly been disruption experiments and overexpression experiments. In other words, knockout and vice versa, and the function of the gene has been predicted from the “phenotype” that is the resulting cell change.
[0003]
In addition, as one of the efforts of post-genome sequencing, a technique for predicting the function from the three-dimensional structure of the post-translational product of a gene with unknown function, that is, the three-dimensional structure analysis of a protein has been born. This technology called structural genomics is based on identifying the function of a protein from its “form / shape”, but predicting its function only from the three-dimensional structure of the protein is the function of the protein. Is not necessarily directly identified, and therefore cannot always be an effective means.
[0004]
For this reason, as the most direct functional analysis method of gene function analysis, interaction analysis between proteins, which are products after gene translation, has begun to attract attention. This is because, if it is possible to know what kind of interaction between a protein whose function is unknown and a protein whose function is known, detailed gene function information can be obtained most directly. In addition, the interaction between proteins means the chemical bond or dissociation including the noncovalent bond between proteins, and a covalent bond.
[0005]
Here, the ELKL protein (ELKL motif kinase) is considered to be a protein that is closely involved in the secretion of hormones secreted from the pituitary gland or / and pineal gland (Non-patent Document 1).
[0006]
In addition, the “14-3-3 protein family” is known as a protein that significantly increases in the cerebrospinal fluid of patients with Creutfeld-Jakob disease. It was also found that the 3-3 antigen was detected at a high level (Non-patent Document 2). As described above, the 14-3-3 protein has been found to be useful as a marker for knowing the state of a brain disease, but there are still many unknown parts in its function.
[0007]
[Non-Patent Document 1]
Development Biology 214, 87-101 (1999), Stephanie Bessone et al., “EMK Protein Kinase-Null Mice: Dwarfism and Hyperfertility Associated with Affiliation in the World.”
[Non-Patent Document 2]
Hiroshi Kido et al., Research on the dynamics of 14-3-3 protein associated with AIDS encephalopathy, [on line], [March 5, 2003 search], Internet, URL: http://www.acc.go.jp /kenkyu/hiyorimi/2000hiyorimi/20.htm
[0008]
[Problems to be solved by the invention]
Based on the interaction analysis between proteins, which is the most direct function analysis method of gene function analysis, comprehensive analysis of completely new interactions between proteins enables the analysis of proteins with unknown functions and specific diseases. Discover new interactions with proteins known to be involved in pathogenesis. It is an object of the present invention to provide a screening method for a substance having a function of regulating the interaction based on the new knowledge about the interaction.
[0009]
[Means for Solving the Problems]
In the present invention, firstly, from the pituitary gland or / and pineal gland characterized by utilizing an interaction between a protein belonging to the 14-3-3 protein family and an ELKL protein (ELKL motif kinase). A screening method for a secretory regulator of a secreted hormone is provided.
[0010]
The amino acid sequences of proteins belonging to the 14-3-3 protein family are shown in “SEQ ID NOs: 1 to 7”, and the amino acid sequences of ELKL proteins (ELKL motif kinase) are shown in the attached “SEQ ID NO: 8” and “SEQ ID NO: 9”, respectively. .
[0011]
In the present invention, in the present invention, proteins belonging to the 14-3-3 protein family are not limited to the proteins shown in the amino acid sequences shown in “SEQ ID NOs: 1 to 7”. And a protein in which a part of the amino acid sequence shown in “SEQ ID NOS: 1 to 7” is deleted, substituted, added or inserted. Further, the ELKL protein is not limited to the proteins shown in the amino acid sequences shown in SEQ ID NO: 8 and SEQ ID NO: 9, but physiological functions that interact with proteins belonging to the 14-3-3 protein family. And a protein in which a part of the amino acid sequence shown in SEQ ID NO: 8 or SEQ ID NO: 9 has been deleted, substituted, added or inserted is also included.
[0012]
Specifically, the screening method according to the present invention can be used to screen for compounds that can change (inhibit or enhance) the binding characteristics of the interaction between the target proteins. Such a compound can be a candidate for a therapeutic or prophylactic agent for a disease (disease) associated with a protein-protein interaction of interest.
[0013]
Such a screening method includes a method of adding a candidate compound at an appropriate concentration to the reaction conditions for detecting the interaction and examining the influence on the interaction. This method can be screened as a method similar to the conventional enzyme-labeled immunosorbent assay (ELISA method). Then, 14-3-3 or ELKL is bound and immobilized on a 96-well plate, a molecule to be screened is added thereto, and further ELKL (14-3-3 if ELKL is immobilized), labeled antibody, and substrate in that order. To determine how much the ELKL binding changes with 14-3-3 in the presence of the substance to be screened. The stronger the color of the substrate, the stronger the binding between the two.
[0014]
Drug screening can also be performed by single-molecule direct observation using NMR spectroscopy, X-ray crystal analysis, electron microscope, or the like. Screening can also be performed efficiently using a surface plasmon resonance sensor (SPR). Either 14-3-3 or ELKL is also immobilized on an SPR sensor chip, and ELKL (ELKL is added by adding a molecule to be screened). In the case of the liquid, 14-3-3) is fed onto the sensor chip. A binding curve is observed in real time by surface plasmon resonance, and the binding curve increases when the binding between the two increases, and decreases or disappears when the binding decreases.
[0015]
Among the substances screened, in the case of a substance that promotes the secretion of hormones secreted from the pituitary gland or / and pineal gland, for example, growth hormone (GH), it develops due to insufficient or decreased secretion of growth hormone. It can be used as a therapeutic agent for dwarfism (pituitary dwarfism) in which the height is significantly lower than that of the elderly. On the other hand, in the case of a substance that suppresses the secretion of growth hormone, it can be used as a therapeutic agent for giantism, acromegaly, etc. that develops due to excessive secretion of the hormone. Melanin is secreted from the pineal gland and is related to the biological clock of the organism, and can be used to treat and prevent insomnia and jet lag. Hormones such as FSH, LH, and prolactin secreted from the pituitary gland are important for the function of the ovary, and can be used for the treatment and prevention of irregular menstrual or infertility.
[0016]
The second means provided by the present invention is the production of an ELKL protein (ELKL) belonging to the 14-3-3 protein family for the manufacture of a therapeutic agent for diseases related to abnormal secretion of hormones secreted from the pituitary gland or / and pineal gland. use of interaction with motif kinase).
[0017]
This means a means by which the interaction is used directly or indirectly in the process of producing a therapeutic agent for diseases related to abnormal secretion of hormones. If a specific example is given, the target substance will be selected using the screening method, and this substance can be used for the production of a therapeutic agent for diseases related to abnormal secretion of hormones. Alternatively, the interaction can be used when examining whether or not a substance that is a candidate for a hormonal secretion abnormality-related disease therapeutic agent has a correct activity (quality inspection).
[0018]
The third means provided by the present invention is the diagnosis of diseases related to abnormal secretion of hormones secreted from genes encoding proteins belonging to the 14-3-3 protein family or pituitary gland or / and pineal gland comprising the proteins. It is a marker for. The base sequences of genes encoding proteins belonging to the 14-3-3 protein family are shown in “SEQ ID NOs: 10 to 18”.
[0019]
That is, between the protein belonging to the 14-3-3 protein family and the ELKL protein (ELKL motif kinase) that is clearly involved in diseases related to abnormal secretion of hormones secreted from the pituitary gland or / and pineal gland. On the basis of completely new knowledge obtained as a result of intensive studies by the inventors of the present invention that a mutual reaction occurs, a protein belonging to the 14-3-3 protein family or a gene encoding the protein is secreted by the hormone. It was found to be involved in the development of abnormalities related diseases.
[0020]
From this, by examining the expression state of the protein, mutation of the gene, single nucleotide polymorphism, etc., it can be used for diagnosis of diseases related to abnormal secretion of the hormone. That is, a protein belonging to the 14-3-3 protein family or a gene encoding the protein is useful as a marker for diagnosis of diseases related to abnormal secretion of hormones secreted from the pituitary gland or / and pineal gland.
[0021]
The fourth means provided by the present invention is a method for treating diseases associated with abnormal secretion of hormones secreted from the pituitary gland or / and pineal gland using a protein belonging to the 14-3-3 protein family or a gene encoding the protein. This is a risk assessment method.
[0022]
To examine the expression state of a protein belonging to the 14-3-3 protein family involved in the hormone secretion abnormality-related disease via ELKL protein (ELKL motif kinase), the mutation of the gene encoding the protein, and the single nucleotide polymorphism. This makes it possible to evaluate the risk of developing diseases related to abnormal secretion of hormones secreted from the pituitary gland or / and pineal gland in the future.
[0023]
As described above, the present invention relates to ELKL protein (ELKL motif kinase), which is known as a protein that is clearly involved in the development of diseases related to abnormal secretion of hormones secreted from the pituitary gland or / and pineal gland, As a result of the interaction with a protein belonging to the 14-3-3 protein family, the interaction itself as well as the protein belonging to the 14-3-3 protein family or the gene encoding the protein are It has the technical significance of being useful for drug discovery technology, diagnostic technology, testing technology, risk assessment technology, etc., for the treatment of diseases related to secretion disorders.
[0024]
【Example】
The inventors of the present application used a protein (tyrosine 3-monooxygenase / tryptophan 5-monooxygenase activation protein, beta polypeptide) belonging to the 14-3-3 protein family and having the amino acid sequence shown in “SEQ ID NO: 1”.
[0025]
A partner protein that recognizes this target protein (hereinafter referred to as “14-3-3 protein”) and shows an interaction was efficiently searched for by the following method. The amino acid sequences of all proteins belonging to the 14-3-3 protein family are shown in “SEQ ID NOs: 2 to 7”.
[0026]
Next, a partner protein that recognizes and interacts with the 14-3-3 protein was efficiently searched for by the following method. A 14-3-3 protein gene (SEQ ID NO: 10) for the purpose of finding a partner protein was prepared as cDNA. The gene for the 14-3-3 protein was purchased from GeneStor of Invitrogen Corp. and incorporated into an expression vector for mammalian cells having a CMV promoter.
[0027]
To this, a tag (epitope tag) that does not affect the structure and function of the protein was selected and fused. Specifically, “FLAG tag” was selected as the epitope tag. For the tag fusion, the inverted PCR method was used (article: Imai, Y. et al., Nucleic Acids Res., 1991, 19, 2785). A PCR primer for an expression vector into which cDNA was incorporated was designed and amplified so that a tag was attached to the carboxyl terminus of the 14-3-3 protein, and the target clone was obtained.
[0028]
Subsequently, the 14-3-3 protein gene fused with the tag was introduced into cultured cells (transfected). HEK293T cells were used as cultured cells for gene introduction, and PolyFect from Qiagen Co., Ltd. was used as a transfection reagent. The introduction procedure followed the protocol of the product.
[0029]
The gene introduced into the cultured cell is expressed in the cell as a recombinant protein fused with the tag, and interacts with a partner protein present in the cell to form a complex. The tag is added to this complex. For this reason, the cells were extracted from the cells by a known “immunoprecipitation method” using an antibody against the tag.
[0030]
The extraction method is as follows. The transfected cells were solubilized using a solubilization buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 50 mM NaF, 1 mM Na ₃ VO ₄ , 1 mM PMSF, 1% Triton X100). After the solubilization buffer was added, the cells were scraped and collected in a centrifuge tube and ultracentrifuged (55,000 rpm, 4 ° C., 20 minutes).
[0031]
Agarose beads (purchased from SIGMA) to which the FLAG peptide antibody was immobilized were added to the cell extract (supernatant) after centrifugation, and the mixture was stirred at 4 ° C. for 3 hours. The stirred beads are collected by centrifugation (1,000 rpm, 4 ° C., 1 minute), washed with a solubilizing buffer, and then a 14-3-3 protein bound to the beads is eluted by adding a buffer containing a FLAG peptide. The protein complex containing the 14-3-3 protein was recovered.
[0032]
After the extraction, the protein that is the partner of the interaction with a known tandem mass meter (MS / MS) is converted into a known method called “mass tag method” (Yodosha “Proteome analysis method”, Toshiaki Isobe, Identified by Nobuhiro Takahashi, P129-P142).
[0033]
Specifically, a sample extracted before analysis was first digested and digested with “trypsin” or “lysyl endopeptidase” which is an enzyme that recognizes and cleaves a specific amino acid. In this experiment, lysyl endopeptidase was used.
[0034]
Specifically, the collected protein complex was concentrated by centrifugation and then dissolved in an enzyme reaction buffer (100 mM Tris, pH 8.8). Lysyl endopeptidase was added so that the enzyme substrate ratio (weight ratio) became 1/100 to 1/50, and the mixture was reacted at 37 ° C. for 12 hours to obtain a digest.
[0035]
After the digestion and degradation work, the mass value and internal amino acid sequence information of each degraded peptide were obtained using tandem mass spectrometry.
[0036]
Using the mass values of the enzymatically digested peptide fragments, candidate amino acid sequences were automatically searched and selected from the database, and the mass value set when the sequence was fragmented with each amino acid was calculated.
[0037]
The database is a protein database “SwissProt” (Internet address: ftp.ebi.ac.uk/pub/databases/sp_tr_nrdb/fasta/sprot.fas.Z) and a nucleic acid database. NCBI RefSeq ”(Internet address: ftp.ncbi.nih.gov/refseq/H_sapiens/mRNA_Prot/hs.faa.gz) was used.
[0038]
The partner protein was identified by comparing this calculated value with the measured fragment ion (MS / MS spectrum), and it was involved in diseases related to abnormal secretion of hormones secreted from the pituitary gland or / and pineal gland. Was found to be known "ELKL".
[0039]
Thereby, it turned out that 14-3-3 protein and ELKL show interaction. In addition, the sequence of the digested peptide (2 peptide) of the enzyme (lysyl endopeptidase) of the ELKL protein detected in this analysis is as follows (1) and (2).
[0040]
(1)
FRQIVSAVQYCHQK
(Phe-Arg-Gln-Ile-Val-Ser-Ala-Val-Gln-Tyr-Cys-His-Gln-Lys)
(2)
VLDANSCQSELHEK
(Val-Leu-Asp-Ala-Asn-Ser-Cys-Gln-Ser-Glu-Leu-His-Glu-Lys)
[0041]
The amino acid sequences of ELKL proteins identified based on the amino acid sequences of these peptides are shown in the attached “SEQ ID NO: 8” and “SEQ ID NO: 9”. In addition, the nucleotide sequences of genes encoding ELKL proteins are shown in “SEQ ID NO: 19” and “SEQ ID NO: 20”.
[0042]
【The invention's effect】
As described above, the present invention relates to the ELKL protein (ELKL motif), which is known as a protein that is clearly involved in the development of diseases related to abnormal secretion of growth hormone and other hormones secreted from the pituitary gland or / and pineal gland. kinase) interacted with proteins belonging to the 14-3-3 protein family. For this reason, the interaction and the protein belonging to the 14-3-3 protein family or the gene encoding the protein are particularly useful for the drug discovery technology of the disease-related medicine.
[0043]
[Sequence Listing]

Claims (1)

Pituitary gland characterized by utilizing interaction between 14-3-3 protein family protein tyrosine 3-monooxygenase / tryptophan 5-monooxygenase activation protein, beta polypeptide and ELKL protein (ELKL motif kinase) / And screening method for secretion regulators of hormones secreted from the pineal gland.