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In vitro control of protein synthesis in mammalian cells by IP3 receptor-binding protein

外国特許コード F110003826
整理番号 I018P009WO
掲載日 2011年7月5日
出願国 アメリカ合衆国
出願番号 29368107
公報番号 20100167314
公報番号 8252547
出願日 平成19年3月20日(2007.3.20)
公報発行日 平成22年7月1日(2010.7.1)
公報発行日 平成24年8月28日(2012.8.28)
国際出願番号 JP2007056529
国際公開番号 WO2007108557
国際出願日 平成19年3月20日(2007.3.20)
国際公開日 平成19年9月27日(2007.9.27)
  • 特願2006-077607 (2006.3.20) JP
  • 2007JP056529 (2007.3.20) WO
発明の名称 (英語) In vitro control of protein synthesis in mammalian cells by IP3 receptor-binding protein
発明の概要(英語) The present invention provides a composition comprising an IP3 receptor-binding protein (IRBIT), a nucleic acid that controls the expression and translation of IRBIT, or an antibody against IRBIT for controlling at least one intracellular biological function selected from the group consisting of (1) protein synthesis, (2) phosphatidylinositol metabolism, and (3) intracellular pH.
When phosphatidylinositol 4,5-bisphosphate is hydrolyzed through the activation of a receptor on a cell membrane, inositol 1,4,5-trisphosphate (IP3), which is an intracellular second messenger, is generated.
IP3 binds to an IP3 receptor (IP3R), thereby inducing Ca2+ release from organelles for intracellular calcium storage (mainly, the endoplasmic reticulum).
In this IP3/Ca2+ signaling pathway, the IP3 receptor plays a role in converting the IP3 signal into Ca2+ signal (M. J. Berridge, Nature (1993) 361: 315-325; M. J. Berridge et al., Nat. Rev. Mol. Cell. Biol. (2000) 1: 11-21; T. Furuichi and K. Mikoshiba, J. Neurochem. (1995) 64: 953-960).
The IP3 receptor is a tetrameric intracellular IP3-gated Ca2+ release channel.
In mammals, there exist 3 different types of IP3 receptors (i.e., type 1, type 2, and type 3) (T. Furuichi et al., Nature (1989) 342: 32-38; T. Sudhof et al., EMBO J. (1991) 10: 3199-3206; 0.
Blondel et al., J. Biol. Chem. (1993) 268: 11356-11363).
Of them, the type 1 IP3 receptor (IP3R1) is expressed at high levels in the central nervous system and particularly in the cerebellum (P. F. Worley et al., Nature (1987) 325: 159-161; T. Furuichi et al., Recept. Channels (1993) 1: 11-24).
Mouse IP3R1 comprises 2749 amino acids and has 3 functionally different regions.
Specifically, an IP3-binding domain is present in the vicinity of the N-terminus, a channel-forming domain having a six-transmembrane region is present in the vicinity of the C terminus, and a control region is present between the two regions.
The deletion mutant analysis of the IP3-binding domain revealed that the amino acids 226-578 of the IP3 receptor was a minimum region required for specific and high-affinity binding of a ligand.
This region is referred to as the IP3 binding core.
With an increase of cytoplasmic Ca2+ concentration by activation of the IP3 receptor, the activities of a wide variety of downstream target molecules are controlled.
These downstream target molecules play important roles in wide-ranging cellular responses including fertilization, development, proliferation, secretion, synaptic plasticity, and the like.
The present inventors have previously discovered a novel IP3 receptor-binding protein and named it "IRBIT" (IP3R-binding protein released with inositol 1,4,5-trisphosphate) (JP2004-129612A).
The IP3 receptor is widely distributed in various tissues and cells of mammals such as humans and mice (e.g., in the brain, heart, liver, kidney, pancreas, and thymus gland).
Accordingly, IRBIT is inferred to be present also in such tissues or cells.
The amino acid and nucleotide sequences of mouse IRBIT have been determined by the present inventors (JP2004-129612A, H. Ando et al., J. Biol. Chem. (2003) 278: 10602-10612).
Such IRBIT comprises 530 amino acids.
Human IRBIT and mouse IRBIT share 100% identity.
The region for binding to the IP3 receptor is present in the N-terminal region of IRBIT, corresponding to amino acids 1-104 in a human or a mouse.
IRBIT is characterized in that: (1) IRBIT is a neutral protein (presumed pI: 6.48) in which the N-terminal region is relatively acidic (presumed pI: 4.98); (2) a plurality of phosphorylation sites are localized in a concentrated manner in the N-terminal region, so that phosphorylation is predicted to be necessary for interaction with IP3R1; (3) the lysine residue at position 508, which is essential for the binding of IP3R1 to IP3, is also essential for interaction with IRBIT; (4) IRBIT is dissociated by IP3 from interaction with IP3R1; and (5) because IRBIT is dissociated from IP3R1 and is extracted from crude microsome fractions by high salt, it is inferred that its interaction with IP3R1 takes place due to electrostatic binding, for example (JP2004-129612A).
IRBIT has the property that it binds to the IP3 binding region of the IP3 receptor and is dissociated in vitro from the IP3 receptor by IP3.
Therefore, it has also been revealed that IRBIT has a function of suppressing the activity of the IP3 receptor by suppressing the binding of IP3 to the IP3 receptor (JP2004-129612A).
The present inventors have now found target molecules of IRBIT and important biological in vivo functions of IRBT as a tertiary messenger, as described below.

An object of the present invention is to provide a composition and a method that make it possible to control intracellular biological functions with the use of interaction between IRBIT and its molecular targets.
Another object of the present invention is to provide a method for screening for a substance that makes it possible to control the above biological functions by suppressing or enhancing the binding between IRBIT and its molecular targets within cells.
This time, the present inventors have conducted concentrated studies to achieve the above objects.
The present inventors have thus found that targets of IRBIT are a molecule which controls a protein synthesis, a molecule which controls phosphatidylinositol, and a molecule which controls a pH within cells.
Moreover, the present inventors have now proved that IRBIT has a role as a tertiary messenger (particularly, as an important molecule for controlling intracellular metabolism) for controlling the functions of binding to the 3 types of target molecules discovered in connection therewith, thereby controlling the functions of the target molecules.

特許請求の範囲(英語) [claim1]
1. A method for using an IP3 receptor-binding protein (IRBIT) to conduct in vitro or ex vivo control of protein synthesis involved in cytoplasmic mRNA polyadenylation mediated by cleavage/polyadenylation specificity factor (CPSF) within mammalian cells, comprising: introducing a vector capable of expressing DNA encoding IRBIT into the cells to modulate the binding of the IRBIT to the CPSF within the cells, thereby controlling the protein synthesis within the cells,
wherein the IRBIT is from a mammal; and
wherein the ex vivo control comprises removing the cells from a patient prior to the introducing step and returning the cells after the introducing step.
2. The method according to claim 1, wherein the IRBIT is a protein comprising an amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 or a protein which comprises an amino acid sequence having 98% or more identity with said amino acid sequence and has an ability to suppress the protein synthesis by binding CPSF.
3. The method of claim 1, wherein the control is in vitro control and the mammalian cell is selected from the group consisting of CHO, COS, HEK293, HeLa and NIH3T3 cells.
4. The method of claim 1, wherein the control is ex vivo control and the mammalian cell is a tumor cell.
5. The method according to claim 1, wherein the mammalian cell is a neuronal cell.
  • 発明者/出願人(英語)
  • 435/7.8
  • 435/455
  • 514/1.1
  • 530/300
  • 536/23.5