JP2019064968A - Angiogenesis controlling agent and method for utilizing same - Google Patents

Abstract

To provide cell adhesion controlling agents, angiogenesis controlling agents, or preventing or treating agents for diseases attributed to the aberrant of cell adhesion or angiogenesis.SOLUTION: The present invention provides an agent for controlling the cell adhesion to an extracellular matrix, an angiogenesis controlling agent, or a preventing or treating agent for diseases attributed to the aberrant of cell adhesion or angiogenesis, characterized by comprising a substance controlling the ubiquitin ligase signaling associated protein of the following (A) or (B), or a gene thereof. (A) At least one BTB protein selected from the group consisting of ANKFY1, KCTD18, BTBD7, CCIN, KCTD2, KLHL18, KLHL34, ZBTB6. (B) At least one protein selected from the group consisting of SNX9, SNX 11, SNX 17.SELECTED DRAWING: None

Description

The present invention relates to an angiogenesis control agent, a method of using the same, and the like.

[Angiogenesis]
The "angiogenesis" includes a broad sense "angiogenesis" and a narrow sense "angiogenesis".

The narrow sense “angiogenesis” refers to so-called “Angiogenesis” in which new blood vessels branch from existing blood vessels.
In the broad sense of “angiogenesis”, in addition to “angiogenesis” in a narrow sense, so-called “vasculogenesis” in which new blood vessels are formed from endothelial precursor cells (angioblasts) in the absence of existing blood vessels ) Is included.
The term "angiogenesis" as used herein refers to "angiogenesis" in a broad sense, including both "(in a narrow sense) angiogenesis" and "vasculogenesis".

By the way, while angiogenesis is an important physiological phenomenon required in the process of wound healing, it is also involved in the development of chronic inflammation and malignancy.

Therefore, the control of angiogenesis is considered as an important tool that is useful for the treatment of various diseases.

Therefore, the detailed mechanism of angiogenesis is gradually being elucidated. For example, in the early stage of angiogenesis, for example, the extracellular domain of the membrane protein Integrin expressed on the cell membrane of vascular endothelial cells is fibronectin, By adhering to extracellular matrix (hereinafter sometimes referred to as "ECM") containing glycoproteins such as collagen, laminin and fibrinogen as main components, it is possible for cell extension and vascularization beyond that. It is known to be involved.

[Angiogenesis regulator]
So far, various angiogenesis control agents have been studied, and the present inventors have also found various angiogenesis control agents related to ubiquitin ligase (E3) utilizing the ubiquitin system described below (patent Documents 1 to 3). And all of those E3s contained BTB domain-containing protein as a part of component (adapter).
Therefore, the ubiquitin system will be described below.

[Ubiquitin system]
The ubiquitin system is a protein consisting of 76 amino acids, such as ubiquitin (hereinafter sometimes referred to as "Ub"), which is used to modify other proteins, thereby degrading proteins, controlling signal transduction, DNA It is a phenomenon involved in various life phenomena such as repair.

Modification of other proteins means addition of Ub to the target protein (monobylation, diubilation, polyubilation, etc.).
Ubylation is caused by the amide bond between the amino group of the side chain of lysine (K) possessed by the target protein and the carboxyl group of glycine (G) at the C-terminus of Ub; In the case, the next Ub binds to lysine in Ub.

The following three enzymes are used in this ubiquitin system.
E1: Ubiquitin activating enzyme
E2: Ubiquitin conjugating enzyme
E3: Ubiquitin transfer enzyme (ubiquitin ligase)

Therefore, these enzymes are also attracting attention in that they are useful for the prevention, treatment and the like of diseases associated with the above-mentioned life phenomena, and various Ub-related enzymes are being searched.

[Ubiquitin ligase (E3)]
By the way, among these enzymes, E3 which acts at the final stage of Ubization is known to be a complex consisting of a plurality of components having various roles as described below.

Cullin (CUL) proteins such as [Cullin1 (CUL1), Cullin 2 (CUL2), Cullin 3 (CUL3)], etc. that have {1} ligase activity and also play a role of a platform to connect other components.
Adapter site that recognizes the {2} substrate (the protein targeted for Ub conversion)
{3} Other sites (Rbx1, Nedd8 (N8, Neural Precursor Cell Expressed, Developmentally Down-Regulated 8), etc.)

And, by changing the adapter site with substrate specificity of {2}, it is possible to realize Ubization for various proteins.

[E3 adapter]
It is known that the proteins corresponding to this adapter site include types such as F-box-containing proteins, BC-box-containing proteins, and BTB domain-containing proteins, which have different binding sites with {1} (non-patent document) 1, 2 etc.).

On the other hand, 183 types of human proteins having a BTB domain are already known from the gene level.

However, there are still not many examples that could be suggested to be an E3 adapter after actually showing some data for a protein having a BTB domain (hereinafter sometimes referred to as "BTB protein") .

The present inventors have now identified new BTB proteins that can function as adapters in E3, and found that their regulators can be angiogenesis regulators.

It is to be noted that the control mechanism of angiogenesis by controlling the BTB protein of the present invention is based on the control of adhesion of membrane protein "Integrin" to "ECM", which is an early stage of angiogenesis, and further, the adhesion control to this ECM Prior to, we also found that the intracellular localization of "Integrin" was controlled.

“Integrin” is a type of membrane protein having a function of cell adhesion molecule, and its structure is a heterodimer composed of two subunits of “α chain” and “β chain”. Since each of the α chain and β chain has a plurality of types, various combinations of these subunits enable adhesion to various types of ECM.

At this time, eight BTB proteins found by the present inventors, when controlled by their siRNA etc., have almost the same phenotype as in the control of CUL3 (change in intracellular behavior of Integrin, change in cell adhesion to ECM) It is very likely that the E3 adapter constitutes E3 together with CUL3 because it shows changes in angiogenesis, or the like.

And of these eight types of BTB proteins, for KCTD18, SNX9 (probably targeted for mono-Ub conversion) has been identified as an E3 substrate, and for SNX9 and two other SNX proteins, “Integrin β1” It has been confirmed that it is involved in the control of intracellular localization.

Patent No. 5665213 Unexamined-Japanese-Patent No. 2015-126722 Japanese Patent Application No. 2017-110363

Natural Cell Biology vol.5 No.11, P.950-951, November 2003 Current Biology, vol.14, R59-R61, January 20, 2004

Therefore, an object of the present invention is to provide an angiogenesis regulator and the like using a novel E3 and its signaling related protein or a regulator of its gene.

The above-mentioned object is achieved by the following first to sixth inventions.

<First invention>
An agent for controlling cell adhesion to an extracellular matrix, comprising a substance that controls the ubiquitin ligase signaling-related protein of (A) or (B) below or a gene thereof.
(A) at least one BTB protein selected from the group consisting of ANKFY1, KCTD18, BTBD7, CCIN, KCTD2, KLHL18, KLHL34, ZBTB6 (B) at least one kind selected from the group consisting of SNX9, SNX11, SNX17 protein

<Second invention>
An agent for controlling angiogenesis, comprising a substance that controls the ubiquitin ligase signaling-related protein of the following (A) or (B) or a gene thereof.
(A) at least one BTB protein selected from the group consisting of ANKFY1, KCTD18, BTBD7, CCIN, KCTD2, KLHL18, KLHL34, ZBTB6 (B) at least one kind selected from the group consisting of SNX9, SNX11, SNX17 protein

<Third Invention>
A preventive or therapeutic agent for a disease caused by abnormality in cell adhesion or angiogenesis, which comprises a substance that controls the following (A) or (B) ubiquitin ligase signaling-related protein or gene thereof.
(A) at least one BTB protein selected from the group consisting of ANKFY1, KCTD18, BTBD7, CCIN, KCTD2, KLHL18, KLHL34, ZBTB6 (B) at least one protein selected from the group consisting of SNX9, SNX11, SNX17

<Fourth invention>
An ubiquitin ligase component comprising at least one BTB protein or a derivative thereof selected from the group consisting of ANKFY1, KCTD18, BTBD7, CCIN, KCTD2, KLHL18, KLHL34, ZBTB6.

<Fifth Invention>
A ubiquitin ligase comprising CUL3 as a ligase activity body, wherein the adapter site is at least one BTB protein selected from the group consisting of ANKFY1, KCTD18, BTBD7, CCIN, KCTD2, KLHL18, KLHL34, ZBTB6. , Ubiquitin ligase.

<Sixth Invention>
A screening method of a control agent for cell adhesion to extracellular matrix or an angiogenesis control agent, characterized by using the control ability of the following (I) or (II) as an index.
(I) Interaction between the following (A) and CUL3 (II) Ubiquitination of the following (B) (A) ANKFY1, KCTD18, BTBD7, CCIN, KCTD2, KLHL18, KLHL34, ZBTB6 at least selected from the group consisting of At least one protein selected from the group consisting of one BTB protein (B) SNX9, SNX 11, SNX 17

The various control agents of the present invention, novel ubiquitin ligase components, ubiquitin ligases, etc. can be used as various experimental / research reagents, and also by controlling angiogenesis through control of cell adhesion by Integrin β1, various It can be used for the prevention or treatment of diseases and the like.

The inhibition of ANKFY1 gene using two kinds of siANKFY (# 1, # 2) shows the effect of HUVEC (human umbilical vein endothelial cells) on cell spreading and cell area on basement membrane by antibody staining of Integrin β1 FIG. It is a figure showing the average value of each cell size (area) of FIG. 1-1A. FIG. 5 is a view showing the effect on angiogenesis by the inhibition of ANKFY1 gene using siANKFY1 # 1 by staining of F-actin with fluorescently labeled phalloidin. It is a figure which shows the result of having confirmed that ANKFY1 and CUL3 interact in the presence of Nedd8 by the immunoprecipitation (IP) and Western blotting which used the antibody with respect to the label | marker (HA) of ANKFY1. The effect of HUVEC on cell spreading and cell area on basement membrane by inhibition of CUL3 gene (siCUL3 # 1) is shown by antibody staining of paxillin or vinculin, which is a kind of backing protein of adhesion site between cell membrane and ECM. FIG. It is a figure showing the average value of each cell size (area) of the one which carried out antibody staining of vinculin among FIGS. 1-4A. FIG. 6 shows the influence of HUVEC adhesion to various extracellular matrix proteins by inhibition of CUL3 gene (siCUL3 # 1). It is a figure which showed the influence on the localization position of Integrin alpha 2 and Integrin beta 1 in HUVEC by inhibition (siCUL3 # 1) of CUL3 gene by antibody staining of each Integrin protein. It is a figure which shows the result of having confirmed Integrin alpha 2 expression level in HUVEC by Western Blotting 72 hours after CUL3 gene inhibition (siCUL3 # 1). Western Blotting effects of ANKFY1 and Integrinβ1 expression in HUVEC by transfection with siANKFY1 (# 3) alone or by cotransfection of siANKFY1 (# 3) and RNAi resistant ANKFY1 (ANKFY1-HA) It is a figure which shows the confirmed result. The effect on the localization of ANKFY1 and Integrinβ1 in HUVEC by transfection with siANKFY1 (# 3) alone or by cotransfection of siANKFY1 (# 3) and RNAi resistant ANKFY1 (ANKFY1-HA) FIG. 6 shows the results of antibody staining for ANKFY1) or Integrin β1. It is the figure which confirmed the fall of the amount of KCTD18 expression by gene inhibition of KCTD18 using three types of siKCTD18 (# 1, # 2, # 3) by measurement of mRNA of KCTD18 by quantitative PCR. It is a figure which shows the result of having confirmed the influence of inhibition of KCTD18 gene using three types of siKCTD18 (# 1, # 2, # 3) on the protein mass of Integrin beta 1 and SNX9 protein in HUVEC by Western Blotting. FIG. 6 is a view showing the effect of HUVECs on cell spreading and cell area on basement membrane by inhibition of KCTD18 gene using three kinds of siKCTD18 (# 1, # 2, # 3) by antibody staining of Integrin β1. . FIG. 6 shows the effect on angiogenesis by the inhibition of the KCTD18 gene using three siKCTD18 (# 1, # 2, # 3) by staining of F-actin with fluorescently labeled phalloidin. It is a figure which shows the result of having confirmed the influence on the localization position of Integrin beta 1 in HUVEC by the inhibition of KCTD18 gene using three types of siKCTD18 (# 1, # 2, # 3) by the antibody staining of Integrin beta 1. The inhibition of the SNX9 gene using three types of siSNX9 (# 1, # 2, # 3) shows the effect of HUVEC on the basement membrane on cell spreading and cell area by staining of F-actin with fluorescently labeled phalloidin FIG. FIG. 7 shows the effect on angiogenesis by the inhibition of the SNX9 gene using three types of siSNX9 (# 1, # 2, # 3) by staining of F-actin with fluorescently labeled phalloidin. It is a figure which shows the result of having confirmed the influence on the protein mass of Integrin (beta) 1 and SNX9 protein in HUVEC by inhibition of SNX9 gene using three types of siSNX9 (# 1, # 2, # 3) by Western Blotting. It is a figure which shows the result of having confirmed the influence on the localization position of Integrin beta 1 in HUVEC by the inhibition of SNX9 gene using three types of siSNX9 (# 1, # 2, # 3) by the antibody staining of Integrin beta 1. It is a figure which shows the result of having confirmed the influence on the localization position of Integrin beta 1 in HUVEC by inhibition of various SNX proteins other than SNX9 by antibody staining of Integrin beta 1. It is a figure which shows the result of having confirmed the influence on the localization position of Integrin beta 1 in HUVEC by inhibition of other various BTB proteins using siRNA by the antibody staining of Integrin beta 1.

Hereinafter, the present invention will be described in detail.

[Various control agents of the present invention]
The "regulator of cell adhesion to extracellular matrix", "angiogenesis regulator" of the present invention is characterized in that it contains a substance that regulates the ubiquitin ligase signaling related protein of the following (A) or (B): It is a thing.

(A) at least one BTB protein selected from the group consisting of ANKFY1, KCTD18, BTBD7, CCIN, KCTD2, KLHL18, KLHL34, ZBTB6 (B) at least one kind selected from the group consisting of SNX9, SNX11, SNX17 protein

Here, (A) is considered to be a protein which plays a role as an adapter protein of E3, since it has a BTB structure and its inhibition exhibits a phenotype equivalent to that at the time of CUL3 inhibition.

Further, (B) is considered to be a protein which plays a role as a Ub-forming substrate of E3 because SNX9 interacts with the E3 adapter protein (KCTD18) of (A) and the like.
Furthermore, inhibition of the E3 component, such as siCUL3 and siKCTD18, resulted in no significant change in the intracellular expression level of its target substrate, SNX9 (Fig. 2-1B), and SNX9, SNX 11, SNX 17 The protein of (B) is related to the localization of Integrin β1 (Fig. 3-3 B, Fig. 4), etc., as described in Patent Documents 1 to 3 (which are later degraded by proteasome). It is considered not to be a "ubiquitinated substrate" but to be a "mono-ubiquitinated substrate" that is involved in the control of the intracellular localization of proteins.

In the present invention, "control" includes "inhibition (suppression) or promotion (increase)" of "amount or function".

That is, in the present invention, “control of a protein or its gene” means:
"Reduction (reduction) or promotion (increase) of the amount of the protein or its gene in the administration subject (living body, cells, experimental samples, etc.)"
Or "inhibition or promotion of the function of the protein or its gene in the subject of administration"
Means.

Therefore, "the ubiquitin ligase signaling related protein of (A) or (B) (hereinafter, may be simply referred to as" (A) or (B) protein "or" (A) or (B) ") or the same. Examples of the substance that controls a gene include "a substance that inhibits (suppresses) or promotes (increases) the amount or function of (A) or (B) protein or its gene" in an administration subject.

(A substance that inhibits (suppresses) the amount or function of (A) or (B) protein or its gene)
As “a substance that inhibits (suppresses) the amount or function of (A) or (B) protein or its gene”,
"A substance that suppresses the expression level of (A) or (B) protein or its gene"
"A substance that degrades (A) or (B) protein or its gene"
"A substance that inhibits the function of (A) or (B) protein or its gene"
Etc.

Specifically, the following may be mentioned, but the present invention is not necessarily limited thereto.

(I) Various nucleic acid substances used for gene expression control or the like, various nucleic acids, including siRNA genes for the genes of (A) or (B), shRNA genes, miRNA genes, antisense RNA, nucleic acid aptamers or ribozymes Degradation enzymes, and various low molecular weight compounds etc.
(Ii) Various peptides or proteins used to control proteins, including antibodies against (A) or (B), peptide aptamers, cyclic macropeptides, or various proteases, and various low molecular weight compounds Compound etc.

The siRNA gene, shRNA gene, miRNA gene, antisense RNA, nucleic acid aptamer, ribozyme, etc. for the gene of (i) (A) or (B) can be appropriately designed based on known findings, As the siRNA gene, for example, one described in Table 1 described later can be purchased, and it is also possible to request design from Sigma or the like.
Nucleic acid aptamers are generally obtained by screening from a known large library or the like consisting of random sequences.

The antibody to (A) or (B) of (ii) may be used to immunize a suitable host using (A) or (B) as an antigen according to a known method, artificial synthesis according to a conventional method, etc. These antibodies can also be purchased as known antibodies listed in Table 2.

In the present invention, the antibody may be any of a monoclonal antibody (mAb), a polyclonal antibody (pAb), or a chimeric antibody or a humanized antibody, and may be a phage antibody.

In the present invention, when the antibody is used for various "regulatory agents" or "prophylactic or therapeutic agents", monoclonal antibodies are preferable because the control target is clear.
However, when using for the screening method of this invention mentioned later, a polyclonal antibody may be sufficient.

In addition, it is common to obtain a peptide aptamer also by screening from a known large library etc. like the above-mentioned nucleic acid aptamer.

(A substance that promotes (increases) the amount or function of (A) or (B) protein or its gene)
As "the substance that promotes the amount or function of (A) or (B) protein or its gene",
"A substance that increases the expression level of (A) or (B) protein or its gene"
"(A) or (B) protein or its gene" itself "(A) or (B) substance promoting the function of the protein or its gene"
Etc.

Specifically, the following may be mentioned, but the present invention is not necessarily limited thereto.

(I) A gene of (A) or (B) or a variant thereof (ii) (A) or (B) a protein or derivative thereof

In the present invention, “gene” in (i) means purine bases such as adenine (A) and guanine (G), pyrimidine bases such as thymine (T), uracil (U) and cytosine (C), and modifications thereof It is a polynucleotide containing a base as a component, and is single-stranded or double-stranded DNA, cDNA, single-stranded or double-stranded RNA, a hybrid consisting of single-stranded DNA and single-stranded RNA, RNA and DNA Contains a single-stranded chimera.
(I) “variant” is a nucleic acid sequence in which one to several nucleic acids are “deleted”, “replaced”, “added” or “inserted” to the extent that the function as the gene is not impaired In addition to their own modified products, those that have been subjected to known modifications used for nucleic acid drugs and the like, such as "methylation", "phosphorylation", "amino group modification" and the like, are included.

In the present invention, the gene can be used in the form of a plasmid, a virus vector or the like, and in this case, it may be single stranded or double stranded.

These genes are artificially synthesized using a DNA synthesizer or a transformed cell according to a conventional method, and a part of the base of the naturally occurring extracted polynucleotide for extracting a naturally occurring polynucleotide is deleted. The polynucleotides obtained by these methods are synthesized by using reverse transcriptase, DNA polymerase, RNA polymerase or the like to synthesize the target sequence by using a sequence complementary to the target sequence, substituting, adding, inserting. It can be produced by a method such as modifying a base.

The (A) or (B) protein of (ii) of the present invention is any known protein.

Protein of (A)

ANKFY1: Ankyrin repeat and FYVE domain containing 1 or alias: Rabankyrin-5, ANKHZN, BTBD23, ZFYVE14 etc. A known protein consisting of about 1200 amino acids, for example, as "FHC02023" manufactured by Promega, etc. It is available from the market.

KCTD18: A known protein consisting of about 400 amino acids, also called potassium channel tetramerization domain containing 18, or alias: 6530404F10Rik etc. For example, it is available from the market as "FHC21268E" manufactured by Promega.

BTBD7: BTB (POZ) domain containing 7 or alias: known protein consisting of about 1100 amino acids also called FUP1 etc. For example, it is available from the market as “FHC01173” manufactured by Promega.

It is a known protein consisting of about 600 amino acids, which is also called CCIN: calicin or alias: BTBD20, KBTBD14 etc. For example, it is available from the market as "FHC08991" produced by Promega.

KCTD2: A known protein consisting of about 250 amino acids called potassium channel tetramerization domain containing 2, and it is available from the market as, for example, "FHC11364" manufactured by Promega.

KLHL 18: A known protein consisting of about 600 amino acids called kelch like family member 18 and is commercially available, for example, as "FHC 05762" manufactured by Promega.

KLHL34: A known protein of about 650 amino acids called kelch like family member 34, which is commercially available, for example, as "FHC 27326" manufactured by Promega.

ZBTB6: A known protein consisting of about 400 amino acids, also called zinc finger and BTB domain containing 6, or alias: ZID, ZNF 482, etc. For example, it is available from the market as "FHC03234" manufactured by Promega. .

Protein of (B)

SNX9: sorting nexin 9, or another name: SDP1, SH3PX1, SH3PXD3A, or a known protein consisting of about 600 amino acids, which is also available from the market as, for example, "FHC03587" manufactured by Promega. .

SNX 11: A known protein of about 250 amino acids called sorting nexin 11, which is available from the market as, for example, “FHC26087M” manufactured by Promega.

SNX 17: A known protein consisting of about 500 amino acids called sorting nexin 17 and is commercially available, for example, as “FHC00391” manufactured by Promega.

The term "derivative" in (ii) includes precursors of each protein, those in which the reactive functional group is protected, and those in which the side chain is modified with various substituents, sugar chains and the like.

The sequences of the gene and amino acid of (A) or (B) are described in the following databases and the like.
"NCBI CCDS database"
(Http://www.ncbi.nlm.nih.gov/CCDS/CcdsBrowse.cgi)

Similar to the ubiquitin ligase component or ubiquitin ligase of the present invention, the various control agents of the present invention can be used as a preventive or therapeutic agent for diseases caused by abnormalities in cell adhesion or angiogenesis as well as in the laboratory. It is very useful as an experimental / research reagent in, for example, a part of a reagent used in the screening method of the present invention.

In addition to the above-mentioned components, solvents and other various additives generally used for the experimental and research reagents can be contained as the experimental and research reagents.

[Disease preventive or therapeutic agent of the present invention]
The agent for the prophylaxis or treatment of a disease of the present invention is characterized in that it comprises the above-mentioned control agent of the present invention and can prevent or treat a disease caused by abnormality in cell adhesion or angiogenesis.

(Other ingredients)
The preventive or therapeutic agent of the present invention can contain other components in addition to the above-mentioned "control agent" of the present invention as long as the object of the present invention is not inhibited. Be

Excipients, lubricants, binders, disintegrants, stabilizers, flavoring agents, diluents, surfactants, emulsifiers, solubilizers, absorption enhancers, moisturizers, adsorbents, fillers, fillers, Moisturizer, preservative etc.
Examples of the excipient include organic excipients and inorganic excipients.

(Dosage form)
The dosage form of the prophylactic or therapeutic agent of the present invention includes, for example, forms such as tablets, capsules, granules, powders, pills, troches, or syrups, solutions for injection and catheters, and the like.

(Content of active ingredient)
The content of the active ingredient (the various controlling agents of the present invention) in the preventive or therapeutic agent of the present invention varies depending on the dosage form, and can not be generally limited, within the range where various dosage forms are possible. For example, in the case of a liquid preparation, it is preferably 0.0001 to 10 (w / v%), more preferably 0.001 to 5 (w / v%), particularly in the case of injections , Preferably 0.0002 to 0.2 (w / v%), more preferably 0.001 to 0.1 (w / v%), and in the case of a solid preparation, preferably 0.01 to 50 (w / w) %, More preferably 0.02 to 20 (w / w%), etc., but it is not necessarily limited to this range.

(Production method)
The prophylactic or therapeutic agent of the present invention can be formulated by known methods using the above-mentioned components.

<< Other combination agent >>
The preventive or therapeutic agent of the present invention can be used as a combination with other known cell adhesion control agents, angiogenesis control agents and the like.

(Administration route)
The administration route of the prophylactic or therapeutic agent of the present invention includes systemic administration and local administration, which may be either oral administration, intravenous administration such as intravenous injection, intravenous injection such as intravenous injection, intramuscular administration such as intramuscular injection, catheter administration Transdermal administration, intranasal administration, intradermal administration, subcutaneous administration, intraperitoneal administration, rectal administration, mucosal administration, inhalation, intracapsular administration, etc. It can be selected.

(Method of administration)
When the preventive or therapeutic agent of the present invention is in the form of a gene therapeutic agent containing siRNA etc., for example, when using a plasmid, a method of directly administering the expression plasmid into a muscle (DNA vaccine method), liposome The method includes lipofection method, microinjection method, calcium phosphate method, electroporation method and the like, and DNA vaccine method and liposome method are particularly preferable.

When using a viral vector, it can be carried out by incorporating a target gene into a virus according to a known method.

Examples of viruses used for viral vectors include nontoxic, retrovirus (such as lentivirus), adenovirus, adeno-associated (associated) virus, herpes virus, Sendai virus, vaccinia virus, pox virus, polio virus, Sindbis virus And various DNA viruses or RNA viruses such as SV (simian virus) 40 and immunodeficiency virus (HIV).

Among viruses, retrovirus, adenovirus, adeno-associated virus, vaccinia virus and the like are preferable, and particularly, adenovirus having high infection efficiency is preferable.

In order to actually cause the gene to act as a pharmaceutical, in addition to the “in vivo method” in which the gene is directly introduced into the body, the gene is introduced into cells collected from humans and then the transfected cells are returned to the body There are "ex vivo methods" and the like.

The “in vivo method” is preferable in that it is simple, with little cost and labor, and the “ex vivo method” is preferable in that the efficiency of gene introduction into cells is good.

In the case of administration by the "in vivo method", an appropriate administration route can be selected according to the disease, symptoms and the like of the therapeutic purpose. The administration route includes, for example, vein, artery, subcutaneous, intradermal, intramuscular, stomach (via catheter) and the like.

When administered by the "in vivo method", for example, a formulation such as a liquid drug can be taken. In general, a form such as an injection containing a gene is preferable, and various components commonly used for injections and the like can be added, if necessary.

In addition, in a liposome or a membrane fusion liposome (such as Sendai virus (HVJ) -liposome) containing a gene, it can be used in the form of a liposome preparation such as a suspending agent, a freezing agent, and a centrifugation concentration freezing agent.

(Dose)
Although the dose of the prophylactic or therapeutic agent of the present invention varies depending on the administration route, symptoms, patient's age, body weight, form of the prophylactic or therapeutic agent, etc., for example, the amount of the active ingredient in the prophylactic or therapeutic agent Preferably 0.005 to 500 mg, more preferably 0.1 to 100 mg per kg of the subject body weight required, provided that the lower limit is preferably 0.01 mg (more preferably 0.1 mg) per day for adults. The upper limit is preferably 20 g (more preferably 2000 mg, more preferably 500 mg, particularly preferably 100 mg), and it is desirable to administer it once or several times depending on the condition.

In the case where the active ingredient in the preventive or therapeutic agent of the present invention is a gene, for example, 0.0001 to 100 mg, preferably 0.001 to 10 mg, etc. as a gene, about once every several days to several months etc. It is preferred to administer.

Target disease
The disease to be prevented or treated of the prophylactic or therapeutic agent of the present invention is a disease caused by abnormal cell adhesion or angiogenesis.

Diseases resulting from abnormal cell adhesion include, for example, cancer cell metastasis, leukocyte adhesion failure (leukocyte adhesion failure to vascular endothelial cells), platelet asthenia gravis (platelet adhesion failure to vascular endothelial cells), and various other diseases. Inflammatory diseases (impairment of vascular endothelial cell adhesion) and the like.

The diseases caused by abnormal angiogenesis include, for example, “cancer”, “diabetes”, “arteriosclerosis”, “myocardial infarction”, “ocular neovascular disease (diabetic retinopathy, age-related macular degeneration, lens Posterior fibroplasia, corneal neovascularization, neovascular glaucoma), "articular rheumatism", "psoriasis vulgaris", "sclerotic atherosclerosis", "ischemic heart disease", "Alzheimer's disease" and the like.

[Ubiquitin ligase component of the present invention]
The ubiquitin ligase component of the present invention includes the following (A) or a derivative thereof.

It turned out that (A) is a protein which functions as an adapter component of ubiquitin ligase which makes CUL3 a ligase active body.

(A) at least one BTB protein selected from the group consisting of ANKFY1, KCTD18, BTBD7, CCIN, KCTD2, KLHL18, KLHL34, ZBTB6

The ubiquitin ligase component of the present invention may be used alone or in combination with other components (CUL3, Rbx1, Nedd8, etc.) as the above-mentioned regulatory agent of the present invention, or as a prophylactic or therapeutic agent for diseases. it can.

[Ubiquitin ligase of the present invention]
The ubiquitin ligase of the present invention is characterized by including the ubiquitin ligase component of the present invention described above.
That is, it contains (A) as an adapter site and CUL3 as a ligase activity main body.

The ubiquitin ligase of the present invention also interacts with E2 and contributes to Ub formation by bringing Ub protein close to a substrate (Rbx1 etc.), or a substance that plays a role in switching ubiquitin ligase activity (Nedd8 etc.) Etc. can be included.

[The screening method of the present invention]
The screening method of the present invention is characterized by screening “control agent of cell adhesion to extracellular matrix” or “angiogenic control agent” using the control ability of the following (I) or (II) as an index. It is a thing.

(I) Interaction between the following (A) and CUL3 (II) Ubiquitination (Ub) of the following (B)

(A) at least one BTB protein selected from the group consisting of ANKFY1, KCTD18, BTBD7, CCIN, KCTD2, KLHL18, KLHL34, ZBTB6 (B) at least one group selected from SNX9, SNX11, SNX17 protein

“Use the controllability of (I) or (II) as an index” means that addition of a candidate substance is a candidate by examining whether “(I) or (II)” is inhibited or promoted. It is meant to determine the choice of the substance.

How to confirm (I):
The “interaction between (A) and CUL3” can be confirmed by a conventional method capable of confirming protein interaction. For example, in addition to the yeast two-hybrid method, co-immunoprecipitation method, pull-down assay method, the following test AlphaScreen ^TM system like that used in an example like.

(AlphaScreen ^TM system)
The AlphaScreen ^TM system, a PerkinElmer Inc. known system is mainly used the principles and methods as follows.
(For details, refer to "http://www.perkinelmer.com/category/alpha-kits" etc.)

(principle)
Two proteins for which the interaction is to be confirmed are bound to the following beads (1) and (2), respectively.
Only when the two proteins interact (bind) and the (1) and (2) beads are brought into proximity within 200 nm, (2) luminescence of the thioxine derivative inside occurs.
By detecting this luminescence signal, protein interactions can be confirmed.

(Used beads)
(1) Donor Beads (manufactured by PerkinElmer): beads containing phthalocyanine (photosensitizer, which excites ambient oxygen by receiving light at 680 nm and converts it to singlet oxygen).
(2) Acceptor Beads (manufactured by PerkinElmer): beads containing a thioxine derivative (which emits light at 520 to 620 nm depending on energy received from singlet oxygen).

(Binding of each test protein and beads)
For example, the following relationship can be used.
"Streptavidin"-"Biotin"
"Antigen"-"antibody" (e.g. ("FLAG tag" and "anti-FLAG antibody")

(Detection of luminescence signal)
Detection is possible using EnVision Reader (manufactured by PerkinElmer) or the like.
Incidentally, according to the AlphaScreen ^TM system, the value of the luminescent signal, if it exceeds 10 times the background value, it can be determined that are directly bonded.

How to check (II):
“B-formation of (B)” can be confirmed, for example, by reproducing the addition reaction of Ub to (B) using a recombinant Ub protein in a test tube, but detecting the number of added Ub proteins "Type of Ub (mono, di, poly Ub etc)" can also be confirmed by doing.

That is, by using the confirmation method of (I) or (II) as described above, by examining the influence on the confirmation result of "(I) or (II)" by the addition of the candidate substance, It is possible to screen for “cell adhesion regulator or angiogenesis regulator”.

[Example 1: E3 Component]
The E3 component of the present invention is produced using the above-mentioned BTB protein of (A) or a derivative thereof.
By using these E3 components, the generation of E3 is promoted, the localization of Integrinβ1 to the cell membrane is promoted through the Ubization of E3 substrate, and the promotion of angiogenesis through the adhesion of Integrinβ1 to the extracellular matrix is promoted. Thus, it is considered possible to prevent or treat a disease caused by hypoangiogenesis.

Example 2 Various Control Agents of the Present Invention
The above-mentioned protein (A) or (B) or a derivative thereof, their gene or variant thereof, siRNA molecule for each gene, shRNA gene, miRNA gene, or the like using the conventional method for producing protein preparations, nucleic acid drugs etc. Antisense RNA, Nucleic Acid Aptamers, Ribozymes, Various Nucleic Acid Degrading Enzymes, Various Low Molecular Weight Compounds, or Antibodies to Proteins of (A) or (B) Obtained by Known Immunization Methods etc., Peptide Aptamers, Cyclic Macropeptides And various control agents of the present invention including various proteases, various low molecular weight compounds and the like.
More specifically, for example, (A) or (B) proteins, their genes, various siRNA genes listed in Table 1, or various antibodies listed in Table 2 or the like are used.

These various regulatory agents control the subcellular localization of Integrinβ1 through the regulation of Ubization of E3 substrates such as SNX protein, and control of angiogenesis through the control of adhesion of Integrinβ1 to the extracellular matrix, and thus cells It is considered possible to prevent or treat a disease caused by an abnormality in adhesion or angiogenesis.

[Test example]
The effects of the various control agents and the like of the present invention were confirmed by the following test examples.

In addition, the siRNA used in each test example, the isolation | separation / detection method of a protein, the antibody for detection, etc. are as follows.

<< Materials and method: Material and method >>
(SiRNA reagent)
The sequences of the siRNA reagents used in each test example except for the control (siCONT) described below are as shown in Table 1 and the sequence listing.

(Control siRNA):
siCONT # 1: MISSION siRNA Universal Negative Control # 1 (Model No .: SIC-001)
siCONT # 2: Stealth RNAi siRNA Negative Control, Med GC (Model No .: 12935300)

(Separation of protein)
Proteins in the test sample were separated by SDS-PAGE electrophoresis with a 7.5%, 15% gel, or 10-20% gradient gel, and used for protein detection by the following method.

(Protein detection: Western Blotting)
After transferring a protein from a gel used in SDS-PAGE electrophoresis to a PVDF membrane (manufactured by Bio-Rad), Western blotting using primary antibody, secondary antibody, secondary antibody labeling, etc. shown in Table 2 Detection of protein by

As a luminescence substrate for secondary antibody labeling, ECL Prime Western Blotting Detection Reagent (manufactured by GE Healthcare Biosciences) was used.

(Detection equipment in Western Blotting)
CCD camera type image analyzer:
Image Quant LAS-4000 (manufactured by GE Healthcare Biosciences)

(Detection of proteins: immunostaining in cells)
The cells are fixed with 4% PFA-PBS, permeabilized with 0.1% Triton-PBS, blocked with 3% BSA-PBS, and labeled with the primary antibody, secondary antibody, and secondary antibody described in Table 2. , Visualization of proteins in cells by immunostaining was performed.

(Detection equipment in immunostaining)
As a microscope, an A1R confocal microscope (manufactured by Nikon) was used.

(AlphaScreen ^TM)
Interaction between "CUL3" (ligase activity body) and "BTB protein (A) (E3 adapter)" interactions and the like of adapter proteins and candidate substrate proteins was confirmed using known AlphaScreen ^TM system described above .

Note that the AlphaScreen ^TM system, using two kinds of beads below.

(Biotin tag binding protein capture beads)
Streptavidin bound to Donor Beads (manufactured by PerkinElmer) described in (1) above.

(Beads for capturing FLAG tag binding proteins)
A product obtained by binding an anti-FLAG antibody (anti-DYKDDDDK antibody, manufactured by Wako) to the Acceptor Beads (manufactured by PerkinElmer) of (2) described above via Protein A.

One of the proteins (FLAG tag binding protein) whose interaction is to be confirmed was dispensed into the wells of the 384-AlphaScreen plate.

The other protein (biotin tag binding protein) is dispensed into the well, and after addition of the above-mentioned Acceptor Beads and Donor Beads (Total volume: 25 μL, Buffer composition: 100 mM Tris-HCl, pH 7.5, 1 mg / mL bovine serum albumin, 0.1% Tween 20, 100 mM NaCl), and incubated at room temperature for 1 hour.
The FlexDrop dispenser (made by PerkinElmer) was used for the dispensing.

Luminescent signal was detected using EnVision Reader (manufactured by PerkinElmer).

(In the above sequence, lower case represents RNA and upper case represents DNA.)

[Test Example 1: Use of ANKFY1 gene regulatory substance]
The effects of various regulators of the present invention using siANKFY1, which is one of ANKFY1 gene regulatory substances, were confirmed.

Test Example 1-1: Control of Cell Adhesion to Extracellular Matrix by si ANKFY 1
<Experimental procedure: Protocol>
(Knockdown of ANKFY1)
Control siRNA (siCONT # 2) or siANKFY1 (# 1, # 2) described in Table 1 is transfected into HUVEC (EGM-2 medium, Lonza, Inc.) using Lipofectamine RNAiMAX (Thermo Fisher Scientific), respectively. The cells were seeded on a basement membrane (BD Matrigel (registered trademark)) containing extracellular matrix proteins.

(Confirmation of cell spreading)
The state of extension after adhesion of cells to the basement membrane one hour was observed using the cell membrane protein Integrin β 1 as an index.
Integrin β1 was detected by the above-described detection instrument by immunostaining using the anti-Integrin β1 antibody described in Table 2 or the like. The results are shown in FIG. 1-1A (bars in the figure: 20 μm).

(Measurement of cell area)
The area of the cells detected in FIG. 1-1A was measured and is shown in FIG. 1-1B. Data represent mean values measured on 70 or more cells in 3 independent experiments.
Data show mean ± standard error of the mean (***: p <0.001).

<result>
By inhibiting the expression of ANKFY1 gene, spreading of HUVEC on the basement membrane (adhesiveness of Integrinβ1 to ECM) was inhibited (Fig. 1-1A), and the cell area was also reduced (Fig. 1-1B). .

<Discussion>
From the above, it has been found that the ANKFY1 gene regulator can be a regulator of cell adhesion via regulation of adhesion of Integrinβ1 to ECM.

Test Example 1-2: Regulation of angiogenesis by si ANKFY1
<Experimental procedure: Protocol>
(Knockdown of ANKFY1 or CUL3)
Control siRNA (siCONT # 2), siCUL3 # 1 described in Table 1 or siANKFY1 # 1 were respectively transfected into HUVEC (EGM-2 medium, Lonza Co., Ltd.) using Lipofectamine RNAiMAX (Thermo Fisher Scientific).

(Confirmation of angiogenesis)
The above HUVEC were cultured using the collagen sandwich method as follows.

Specifically, 100 μL of collagen gel (Cellmatrix Type IA (manufactured by Nitta Gelatin Co., Ltd.)) is spread and solidified on the bottom of each well of 12-wells dish, each HUVEC is seeded thereon, and 100 μL of collagen gel is again applied. Then, angiogenesis was induced by adding VEGF-A (manufactured by R & D) to a concentration of 50 ng / ml in the medium.
The presence or absence of angiogenesis was stained by adding a 1000-fold diluted solution of Rhodamine phalloidin (manufactured by Thermo Fisher Scientific) specifically binding to F-actin to the culture medium after 66 hours of stimulation with VEGF-A. After 60 minutes, the fluorescence photograph of F-actin present in was observed by photographing with an A1R confocal microscope (manufactured by Nikon) (FIG. 1-2: bar: 200 μm).

<result>
As in the case of siCUL3 transfection, angiogenesis was inhibited by the inhibition of the expression of the ANKFY1 gene.

<Discussion>
From the above, it has been found that the ANKFY1 gene regulator can be a regulator of angiogenesis.

Test Example 1-3 Confirmation of Interaction between ANKFY1 and CUL3
<Experimental procedure: Protocol>
(Transfection)
In order to confirm that ANKFY1 and CUL3 actually interact in cells, the efficiency of gene transfer is good and easy to handle, human fetal kidney epithelial cells HEK293T cells (DMEM medium, manufactured by Wako Co., Ltd.), lipofection reagent Transfect FLAG-labeled CUL3, HA-labeled ANKFY1 (ANKFY1-HA or HA-ANKFY1) or Myc-labeled Nedd8 in a combination as shown in FIG. 1-3 using “GeneJuice” (manufactured by Millipore), and force It was expressed.

(Recovery of cell lysate)
The cultured cells were treated with Buffer (1% NP40 (Nonidet p-40), 150 mM NaCl, 25 mM Tris-HCl, 1 mM EDTA, 5% glycerol, pH 7.4), and the cell lysate was recovered. .

(Input sample)
After centrifuging at 10000 g and collecting the supernatant, a portion was used as an "Input" sample.

(IP (HA) sample)
The anti-HA antibody (rabbit anti-HA pAb) described in Table 2 was added to the supernatant remaining after the “Input” sample was collected, and HA-labeled ANKFY1 was immunoprecipitated to be an “IP (HA)” sample.

(Separation and detection of protein)
For “Input” and “IP (HA)” samples, detection of proteins by the above-mentioned SDS-PAGE (using 10-20% gradient gel), Western blotting, using the primary antibody, secondary antibody etc. in Table 2. The results of performing the test are shown in Figure 1-3.
What is indicated by "*" is Nedd8-modified CUL3.
The band directly below “*” and detected by “anti-CUL3 antibody” is free CUL3 immunoprecipitated together with Nedd8-conjugated CUL3 (alone), as a heterodimer and other multimers with Nedd-8-conjugated CUL3, It is considered to be one separated by SDS-PAGE treatment or the like, and one from which Nedd8 of a part of Nedd8-conjugated CUL3 has fallen off.

In addition, “heavy chain” and “light chain” in the figure are considered to be those of the above-mentioned anti-HA antibody (rabbit anti-HA pAb) whose three-dimensional structure was destroyed by the denaturing agent used in Western blotting. Be
The “heavy chain” and “light chain” of “rabbit anti-HA pAb” were simultaneously detected on the Myc-labeled Nedd8 detection gel because the sequences of mouse IgG and rabbit IgG were very similar. It is considered that the secondary antibody for Myc detection described in 2 (goat anti-mouse IgG mAb) has reacted to "rabbit anti-HA pAb"("heavychain","lightchain").

Furthermore, as a result of detection of “Input” sample prior to immunoprecipitation, it is considered that endogenous Nedd8 is the reason that Nedd8-modified CUL3 (* mark) is detected also in the three lanes on the left without Myc-Nedd8 addition. Be
As can be seen from the fact that the proportion of CUL3 by endogenous Nedd8 is lower than that of free CUL3, the balance between Nedd8 conversion and Nedd8 deintercalation is strictly limited by known enzyme groups in cells, and always Nedd8 conversion CUL3 It is known that the amount of is kept low.

<result>
As shown in the figure, in the "IP (HA)" sample, CUL3 was clearly detected only when Nedd8 was forcibly expressed in addition to FLAG-labeled CUL3 and HA-labeled ANKFY1 (Fig. 1-3, " 2 lanes at the right end of IP (HA)).

When a similar experiment was performed with the addition of a Nedd8 inhibitor (MLN-4924, manufactured by Boston Biochem), CUL3 was not detected even when Myc-labeled Nedd8 was forcibly expressed (not shown). .

<Discussion>
From the above, it was confirmed that ANKFY1 interacts with CUL3.
That is, it turned out that ANKFY1 is a component of E3 with CUL3 as an adapter for Ub-izing a substrate.

At the same time, it was also found that the E3 cofactor such as Nedd8 is essential for the interaction between ANKFY1 and CUL3.

Both ANKFY1-HA with HA tag added at the N-terminus and HA-ANKFY1 with C-terminus were both found to interact with CUL3, but the expression efficiency in cells is better with ANKFY1-HA Since it was high, it was found that ANKFY1-HA is suitable as a confirmation reagent for the interaction.

Test Example 1-4: Phenotype by siCUL3
<Experimental procedure: Protocol>
(CUL3 knockdown)
Control siRNA (siCONT # 1) or siCUL3 # 1 described in Table 1 is transfected into HUVEC (EGM-2 medium, Lonza Co., Ltd.) using Lipofectamine RNAiMAX (Thermo Fisher Scientific), respectively, and basement membrane (BD Matrigel) It seeded on the registered trademark.

(Confirmation of cell spreading)
The state of extension after 1 hour adhesion of cells to the basement membrane was observed by immunostaining with the antibodies described in Table 2 against focal adhesion markers (paxillin) and vinculin. The results are shown in Fig. 1-4A (bar: 20 μm).

(Confirmation of cell area)
Moreover, the average value of the cell area observed with vinculin among FIG. 1-4A is shown to FIG. 1-4 B (***: p <0.001).

(Confirmation of adhesion to ECM)
The adherence of the siCUL3 # 1-transfected HUVECs to various ECM proteins was confirmed using the CytoSelect® 48-well cell adhesion assay kit as described below.

(Transfection)
72 hours after siCUL3 transfection HUVEC were treated with a solution containing trypsin and then recovered in EGM-2 medium.

(Contact with ECM proteins)
Next, the above-described transfected cells were seeded at 1 × 10 ⁴ cells / well in CytoSelect (registered trademark) 48-well to which various ECM proteins were previously applied in each well.
Then, it incubated at 37 degreeC for 1 hour.

(Staining of cells)
After washing 4 times with PBS to remove cells not adhered to ECM protein, add 200 μl of Cell Stain Solution (CELL BIOLABS) to each well, and incubate at room temperature for 10 minutes to obtain ECM protein-adherent cells. It was taken into

(Measurement of absorbance)
Add 200 μl of Extraction Solution (CELL BIOLABS) to each well and incubate for 10 minutes at room temperature to destroy the cells, collect the dye-extracted solution (150 μl), and measure its absorbance (OD 560) It was used as an indicator of the number of cells attached to ECM proteins.

The larger the absorbance, the greater the number of cells attached to each ECM protein.

The results are shown in FIGS. 1-4C (**: p <0.01).

(CUL3 knockdown)
Control siRNA (siCONT # 1) or siCUL3 # 1 described in Table 1 was transfected to HUVEC (EGM-2 medium, Lonza, Inc.) using Lipofectamine RNAiMAX (Thermo Fisher Scientific) in the same manner as described above.

(Intracellular localization of Integrin α2 and Integrin β1)
Integrinα2 and Integrinβ1 in the transfected cells were detected using the anti-Integrinα2 antibody and the anti-Integrinβ1 antibody described in Table 2 (FIG. 1-4 D / bar: 20 μm).
The third column from the left in Fig. 1-4D is the result of "merging" the two left column photographs (the detection results of "Integrinα2" and "Integrinβ1"), and the rightmost column is the "merge". It is an enlarged view of the framed portion in the photograph.

(Intragrin α2 intracellular expression level)
Control siRNA (siCONT # 1) or siCUL3 # 1 described in Table 1 was transfected into HUVEC (EGM-2 medium, Lonza Co., Ltd.) using Lipofectamine RNAiMAX (Thermo Fisher Scientific), respectively, and the primary antibody of Table 2 The results of detection of Integrin α2 and CUL3 by the above-described SDS-PAGE (using 7.5% gel) and Western Blotting using a secondary antibody etc. are shown in FIG. 1-4E.
In addition, GAPDH (15% gel was used in SDS-PAGE) was used as a control.

<result>
Cells in which CUL3 was knocked down did not show lamellipodia like control cells, and the cells remained almost circular (Fig. 1-4A), and the cell area was also about 30% of the control (Fig. 1-4A). 1-4B).
In addition, it was found that the CUL3 knockdown particularly reduced the adhesion of cells to type IV collagen (a type of ECM) (Fig. 1-4C).
In control cells, Integrinα2 is mainly expressed on the plasma membrane as in Integrinβ1, whereas in cells in which CUL3 is knocked down, Integrinα2 (the other α of the Integrin protein that forms heterodimers) The chain subunit) was also found to be localized in the cell along with Integrin β1, as seen in the small spots in the cell (Fig. 1-4D: arrows).
Since total protein expression levels in Integrinα2 cells were not altered by CUL3 knockdown (Fig. 1-4 E), localized Integrinα2 was transferred from the plasma membrane into the cells I understand.

<Discussion>
From the results of FIGS. 1-4A to E described above and the middle of FIG. 1-2, CUL3 mediates intracellular localization of Integrin proteins such as Integrinα2 and Integrinβ1 to form type IV collagen (ECM) in cells. It has been found that it inhibits the adhesion of the protein, which in turn inhibits angiogenesis.

Test Example 1-5 Subcellular Localization and Rescue Test of Integrin β1 by si ANKFY1
<Experimental procedure: Protocol>
(Knockdown of ANKFY1)
Control siRNA (siCONT # 2) or siANKFY1 # 3 described in Table 1 was transfected into HUVEC (EGM-2 medium, Lonza Co., Ltd.) using Lipofectamine RNAiMAX (Thermo Fisher Scientific), respectively, and the primary antibody of Table 2 Detection of Integrin β1, HA, and ANKFY1 was performed by the above-mentioned SDS-PAGE (using 10-20% gradient gel) and Western blotting, using a secondary antibody etc., and the results are shown in FIG. 1-5A.

In addition, as a rescue test of knockdown of ANKFY1, protein detection was similarly performed also about what introduce | transduced ANKFY1-HA made into siANKFY1 resistance with lentivirus with siANKFY1 (FIG. 1-5 A: lane of right end).
"SiANKFY1-resistant ANKFY1-HA" was introduced into HUVEC by lentivirus using ANKFY1 cDNA which does not contain a sequence corresponding to the knockdown target site of siANKFY1 # 3 (the 3 'end untranslated region of ANKFY1 mRNA).
As a control, GAPDH (using 15% gel in SDS-PAGE) was used.

(Subcellular localization of Integrin β1 and ANKFY1-HA)
As described above, the intracellular localization of Integrinβ1 and ANKFY1-HA was confirmed by immunostaining for HUVEC subjected to ANKFY1 knockdown and rescue, the results of which are shown in FIGS. 1-5B (bar: 20 μm).
The third column from the left in Fig. 1-5B is the result of "merging" the two left column photographs (detection results of "ANKFY1-HA" and "Integrin β1"), and the rightmost column is the It is the enlarged view of the framed part in a "merge" photograph.

<result>
As in the case of knocking down endogenous ANKFY1 with siANKFY1 # 3, when ANKFY1 resistant to siANKFY1 is simultaneously introduced with knockdown, there is no change in the total expression level of Integrinβ1 compared to the control (siCONT # 2) However, intracellular localization of Integrinβ1 by siANKFY1 was restored (Fig. 1-5B).
As in the case of siANKFY1 # 3, there is no change in the total expression level in cells as well as Integrinβ1 and Integrinα2 by knockdown by siANKFY1 # 1 and # 2, also by Western Blotting. Confirmed (not shown).
Furthermore, in experiments in which only Integrinβ1 was detected on the cell surface using Alexa488-labeled TS2 / 16 (anti-Integrinβ1 antibody), in the siANKFY1 (# 1, # 2) treatment, cell surface Integrinβ1 was 40% each of siCONT treatment, It has also been confirmed by quantification of the fluorescence intensity of immunostaining that it has fallen to about 20% (not shown).

<Discussion>
The above demonstrated that the intracellular localization of Integrinβ1 by siANKFY1 is not simply a side effect of siRNA introduction.

Summary of Test Example 1
As these FIGS. 1-4A to E show, inhibition of CUL3 also causes a phenotype similar to that of ANKFY1 inhibition (reduction of cell spreading (cell area in basement membrane), adhesion inhibition to ECM (type IV collagen) , Integrin β 1 (cellular release) was shown.
These are the results of inhibition of angiogenesis by CUL3 inhibition in the middle of Fig. 1-2 mentioned above, interaction of ANKFY1 and CUL3 in Fig. 1-3, intracellular localization of Integrin β1 by ANKFY1 of Fig. 1-5A, B, etc. As a result, we found that the control of cell adhesion and angiogenesis by ANKFY1 gene regulatory substance, the control of Integrinβ1's behavior, etc. (Fig. 1-1A, B, Fig. 1-2 lower) indicated the control of E3. That is, it turns out that ANKFY1 was a kind of adapter of E3.

[Test Example 2: Use of KCTD18 Gene Regulatory Substance]
The effects of various regulators of the present invention using siKCTD18, which is a type of KCTD18 gene regulator, were confirmed.

Test Example 2-1 Control of Cell Adhesion to Extracellular Matrix by siKCTD 18
<Experimental procedure: Protocol>
(KCTD18 knockdown)
Control siRNA (siCONT # 1) or siKCTD18 (# 1 to # 3) described in Table 1 are transfected into HUVEC (EGM-2 medium, Lonza Co., Ltd.) using Lipofectamine RNAiMAX (Thermo Fisher Scientific). The results of measuring the mRNA expression level of KCTD18 by quantitative PCR are shown in FIG.

(Intragrin expression level of Integrin β1 and SNX9)
Control siRNA (siCONT # 1), siCUL3 # 1, or siKCTD18 (# 1 to # 3) described in Table 1 are transfected in the same manner as described above, and the primary antibody, secondary antibody etc. of Table 2 are used. The results of detection of Integrin β1 and SNX9 by SDS-PAGE (using a 7.5% gel) and Western blotting described above are shown in FIG.
In addition, GAPDH (15% gel was used in SDS-PAGE) was used as a control.

(Confirmation of cell spreading)
Cells transfected with siCONT # 1 or siKCTD18 (# 1- # 3) as described above were seeded onto basement membrane (BD Matrigel®) containing extracellular matrix proteins.
The state of extension after adhesion of cells to the basement membrane one hour was observed using the cell membrane protein Integrin β 1 as an index.
Integrin β1 was detected by the above-described detection instrument by immunostaining using the anti-Integrin β1 antibody described in Table 2. The results are shown in Figure 2-1C.

<result>
Depending on the type of siKCTD18, although the expression level of Integrinβ1 and SNX9 may be affected somewhat (Fig. 2B), extension of HUVEC on the basement membrane by any siKCTD18 (Integrinβ1 adhesion to ECM) Was inhibited (Fig. 2-1C).

<Discussion>
From the above, it was found that the KCTD18 gene regulator can be a regulator of cell adhesion via regulation of adhesion of Integrinβ1 to ECM.

Test Example 2-2 Regulation of Angiogenesis by si KCTD 18
<Experimental procedure: Protocol>
(KCTD18 knockdown)
Control siRNA (siCONT # 1) or siKCTD18 (# 1 to # 3) described in Table 1 were transfected into HUVEC (EGM-2 medium, Lonza, Inc.) using Lipofectamine RNAiMAX (Thermo Fisher Scientific). .

(Confirmation of angiogenesis)
The above HUVEC were cultured using the collagen sandwich method as follows.

Specifically, 100 μL of collagen gel (Cellmatrix Type IA (manufactured by Nitta Gelatin Co., Ltd.)) is spread and solidified on the bottom of each well of 12-wells dish, each HUVEC is seeded thereon, and 100 μL of collagen gel is again applied. Then, angiogenesis was induced by adding VEGF-A (manufactured by R & D) to a concentration of 50 ng / ml in the medium.
The presence or absence of angiogenesis was stained by adding a 1000-fold diluted solution of Rhodamine phalloidin (manufactured by Thermo Fisher Scientific) specifically binding to F-actin to the medium 66 hours after stimulation with VEGF-A. After 60 minutes, the fluorescence photograph of F-actin present in the inside was confirmed by photographing with an A1R confocal microscope (manufactured by Nikon). The results are shown in Figure 2-2.

<result>
Inhibition of the expression of the KCTD18 gene inhibited angiogenesis (Figure 2-2).

<Discussion>
From the above, it has been found that the KCTD18 gene regulator can be a regulator of angiogenesis.

Test Example 2-3: Intracellular Localization of Integrin β 1 by si KCTD 18
<Experimental procedure: Protocol>
(KCTD18 knockdown)
Control siRNA (siCONT # 1) or siKCTD18 (# 1 to # 3) described in Table 1 were transfected into HUVEC (EGM-2 medium, manufactured by Lonza, Inc.) using Lipofectamine RNAiMAX (Thermo Fisher Scientific).

(Subcellular localization of Integrin β1)
Integrin β1 in the transfected cells was detected using the anti-Integrin β1 antibody described in Table 2 or the like. The results are shown in Figure 2-3.

<result>
In all of the cells in which KCTD18 was knocked down, Integrin β1 was found to be localized intracellularly (FIG. 2-3).

<Discussion>
From the above, it has been found that KCTD18 regulates cell adhesion and thus regulates angiogenesis by mediating intracellular localization of Integrin β1.

Summary of Test Example 2
The phenotypes shown in FIGS. 2-1A to C, FIGS. 2-2, and 2-3 are similar to the above-described inhibition of CUL3 (reduction of cell spreading (cell area) in basement membrane, angiogenesis Inhibition of Integrin β1 (subcellular release).
That is, it is understood that the control of cell adhesion and angiogenesis by the KCTD18 gene regulator, the control of Integrin β1 behavior and the like mean the control of E3, that is, KCTD18 was also a kind of adapter of E3.

[Test Example 3: Use of SNX9 Gene Regulatory Substance]
The effects of various regulators of the present invention using siSNX9, which is a type of SNX9 gene regulator, were confirmed.

Test Example 3-1 Control of Cell Adhesion to Extracellular Matrix by siSNX 9
<Experimental procedure: Protocol>
(SNX9 knockdown)
Control siRNA (siCONT # 1) or siSNX9 (# 1 to # 3) described in Table 1 were transfected into HUVEC (EGM-2 medium, Lonza, Inc.) using Lipofectamine RNAiMAX (Thermo Fisher Scientific). The cells were seeded on a basement membrane (BD Matrigel®) containing extracellular matrix proteins.

(Confirmation of cell spreading)
The state of extension after 1 hour of adhesion of cells to the basement membrane was observed using F-actin forming the intracellular skeleton of blood vessels as an index.
The presence or absence of cell spreading was stained by adding a 1000-fold diluted solution of Rhodamine-phalloidin (manufactured by Thermo Fisher Scientific) that specifically binds to F-actin to the basement membrane seeded with cells (in blood vessels) ) F-actin was confirmed by detection with the detection device described above. The results are shown in Figure 3-1.

<result>
By inhibiting the expression of the SNX9 gene, spreading of HUVEC on the basement membrane was inhibited (Fig. 3-1).

<Discussion>
From the above, it was found that the SNX9 gene regulator can be a regulator of cell adhesion.

Test Example 3-2 Regulation of Angiogenesis by siSNX 9
<Experimental procedure: Protocol>
(SNX9 knockdown)
Control siRNA (siCONT # 1) or siSNX9 (# 1- # 3) described in Table 1 were transfected into HUVEC (EGM-2 medium, Lonza Co., Ltd.) using Lipofectamine RNAiMAX (Thermo Fisher Scientific). .

(Confirmation of angiogenesis)
The above HUVEC were cultured using the collagen sandwich method as follows.

Specifically, 100 μL of collagen gel (Cellmatrix Type IA (manufactured by Nitta Gelatin Co., Ltd.)) is spread and solidified on the bottom of each well of 12-wells dish, each HUVEC is seeded thereon, and 100 μL of collagen gel is again applied. Then, angiogenesis was induced by adding VEGF-A (manufactured by R & D) to a concentration of 50 ng / ml in the medium.
The presence or absence of angiogenesis was stained by adding a 1000-fold diluted solution of Rhodamine phalloidin (manufactured by Thermo Fisher Scientific) specifically binding to F-actin to the culture medium after 66 hours of stimulation with VEGF-A. After 60 minutes, the fluorescence photograph of F-actin present in was observed by photographing with an A1R confocal microscope (manufactured by Nikon). The results are shown in Figure 3-2.

<result>
Inhibition of the expression of the SNX9 gene inhibited angiogenesis (FIG. 3-2).

<Discussion>
From the above, it has been found that the SNX9 gene regulator can be a regulator of angiogenesis.

Test Example 3-3: Intracellular Localization of Integrin β1 by siSNX 9
<Experimental procedure: Protocol>
(SNX9 knockdown)
Control siRNA (siCONT # 1) or siSNX9 (# 1 to # 3) described in Table 1 were transfected into HUVEC (EGM-2 medium, manufactured by Lonza, Inc.) using Lipofectamine RNAiMAX (Thermo Fisher Scientific).

(Intragrin expression level of Integrin β1 and SNX9)
Integrin β1 and SNX9 were detected by Western blotting using the above-described SDS-PAGE (using 7.5% gel) and the anti-Integrin β1 antibody described in Table 2. The results are shown in Figure 3-3A.
As a control, GAPDH (SDS-PAGE uses a 15% gel) was used.

(Subcellular localization of Integrin β1)
Moreover, the same transfection as above was performed, and Integrin β1 in the transfected cells was detected using the antibodies described in Table 2 and the like. The results are shown in Figure 3-3B.

<result>
As can be seen from FIGS. 3-3A and 3-3B, in the cells in which SNX9 was knocked down, it was revealed that Integrinβ1 was localized in the cells, although there was no particular change in the amount of Integrinβ1. did.

<Discussion>
From the above, the inhibition of the cell adhesion of Test Example 3-1 described above by SNX9 is caused by the intracellular localization of Integrin β1, as in the case of ANKFY1 of Test Example 1 and KCTD 18 of Test Example 2 and the like. found.

Test Example 3-4 Confirmation of Interaction between SNX 9 and KCTD 18
<Experimental procedure: Protocol>
By the aforementioned AlphaScreen ^TM system, was confirmed the interaction between SNX9 and KCTD18, the value of the luminescent signal was 144,870 (not shown).

<result>
It was confirmed that SNX9 interacts with KCTD18 because the value of the luminescence signal far exceeds 10 times the background value (1176), which is one of the criteria for interaction.

<Discussion>
From the above, it was found that SNX9 is very likely to be an U3 substrate of E3 with KCTD18 as an adapter.

Summary of Test Example 3
The knockdown phenotype of SNX9 shown in FIGS. 3-1 to 3-3 etc. is similar to the above-mentioned knockdown of CUL3, ANKFY1, or KCTD18 (cell spreading (cell area in basement membrane)). Reduced, intracellular localization of Integrin β1 (release from membrane)).
That is, regulation of cell adhesion and angiogenesis by the SNX9 gene regulator, regulation of behavior of Integrin β1, etc. were also associated with regulation of E3.

Although SNX9 is considered to be a substrate of E3 having KCTD18 as an adapter as shown in the above-mentioned Test Example 3-4, according to FIG. 2-1B, expression of SNX9 is caused by knockdown of CUL3 or KCTD18. SNX9 is considered to be a "mono-Ubylated substrate" rather than a "poly-Ubylated substrate" of this E3, as there is no significant change in amount.

[Test Example 4: Use of Other SNX Protein Gene Regulators]
The effects of various regulators of the present invention using siSNX, which is one of the regulators of various SNX protein genes, were confirmed.

<Experimental procedure: Protocol>
(Knockdown of various SNX proteins)
Control siRNA (siCONT # 1) or siSNX1, siSNX5, siSNX6, siSNX10, siSNX11, siSNX16, siSNX17, siSNX20, siSNX33 described in Table 1 respectively, using Lipofectamine RNAiMAX (Thermo Fisher Scientific) for HUVEC (EGM-2 medium, Lonza Transfection was carried out.

(Subcellular localization of each SNX protein)
Integrin β1 in the transfected cells was detected using the anti-Integrin β1 antibody described in Table 2 or the like. The results are shown in FIG.

<result>
Among the 9 types of SNX proteins tested, SNX 11 and 17 also showed intracellular localization of Integrin β1 like SNX 9 (FIG. 4).

<Discussion>
SNX11 and SNX17 are also considered to be substrates of E3 in which some BTB protein is used as an adapter, as in SNX9.
However, it was found that SNX11 and 17 do not interact with KCTD18 (see the above-mentioned Test Example 3-4) which is an adapter of SNX9 which is the same SNX protein (not shown).
Therefore, as the adapter for SNX 11 and 17, among the 175 types of BTB proteins in Test Example 5 below, the other 6 types of BTB proteins that are found to be involved in the intracellular localization of Integrin β 1 are potent It can be considered as a candidate.
The 175 types of BTB proteins used for confirming the subcellular localization of Integrin β1 in Test Example 5 described later have already known substrates among 183 types of BTB proteins currently known at the gene level. Since most of them are covered except for those mentioned above, among them, the test example was extracted by treatment with siRNA (which is extracted as one showing intracellular localization of Integrin β1 similar to siCUL3, siSNX11, siSNX 17 etc., This is because it is reasonable to consider that one of the six BTB proteins of 5 is an E3 adapter using SNX11, 17 as a substrate.

[Test Example 5: Use of Other BTB Protein Gene Regulators]
The effect of “the various control agents of the present invention” using BTB protein siRNA, which is one of the control substances of various BTB protein genes, was confirmed.

<Experimental procedure: Protocol>
(Knockdown of various BTB proteins)
Control siRNA (siCONT # 1) or siRNA (siRNA Libraries (Thermo Fisher Scientific)) targeting 175 types of BTB proteins, respectively, using Lipofectamine RNAiMAX (Thermo Fisher Scientific) respectively, HUVEC (EGM-2 medium, Lonza stock) Transfection).

(Subcellular localization of Integrin β1)
The results of detection of Integrin β1 in the transfected cells using the anti-Integrin β1 antibody described in Table 2 and the like are shown in FIG. 5 (bar: 20 μm).

<result>
As can be seen from FIG. 5, among the 175 types tested, the siRNA for 6 BTB proteins (BTBD7, CCIN, KCTD2, KLHL18, KLHL34, ZBTB6), as with siCUL3 and siANKFY1 # 2, is the intracellular localization of Integrin β1. Presented.

On the other hand, depending on the suppression of expression of "BAZF" (patent document 1) and "SPOP" (patent document 2), which are known E3 adapters that control VEGFR2 (VEGF receptor 2) signal and Notch signal in HUVEC, There was no intracellular localization (comparative control).

Among the six types of proteins, for KLHL18 and KLHL34, the value of the emission signal by analysis using known AlphaScreen ^TM system described above are each a 68242,61800 (which is one of the criteria for interaction It was confirmed that “the interaction with CUL3”, which is one of the proof of being an adapter of E3 (not shown), which sufficiently exceeds 10 times the background value (1078)).

In addition, as for the other BTB proteins (BTBD7, CCIN, KCTD2 and ZBTB6) described above, as in the case of ANKFY1 shown in Test Example 1-3, mutual interaction with CUL3 can be achieved by further adding a factor such as Nedd8. It is thought that the action can be confirmed.

<Discussion>
From the above results, the regulators of these six BTB protein genes can also be cell adhesion regulators and angiogenesis regulators, like the regulators of the ANKFY1 gene (Test Example 1) and the KCTD18 gene (Test Example 2). I think that the.
Further, as shown in the discussion of Test Example 4, it is considered that any of these six types of BTB proteins is an adapter of E3 in which SNX11, SNX17 and the like of Test Example 4 are Ub-ized substrates.

From the results of the comparative example described above, even if the present angiogenesis control system based on the localization control of Integrin β1 is an angiogenesis control system through the same action of E3, Patent Document 1 (BTB protein: BAZF It has been confirmed that the regulation of VEGFR2 signal and Notch signal such as in the patent document 2 (BTB protein: SPOP) is a completely different route control system.

[Summary]
By combining the results of Test Examples 1 to 5, the E3 component of the present invention, E3 and E3 control agents, respectively, control agents for cell adhesion to extracellular matrix, and control agents for angiogenesis, and thus cell adhesion It has been found that it can be used as a preventive or therapeutic agent for diseases caused by sexual or angiogenic abnormalities.

In addition, from these results, it is possible to screen a control agent for cell adhesion to extracellular matrix and an angiogenesis control agent by the screening method of the present invention using the control ability of (I) or (II) below as an index. It became clear.
(I) Interaction between the following (A) and CUL3 (II) Ubiquitination of the following (B)

The various regulatory agents of the present invention, the novel ubiquitin ligase component and ubiquitin ligase are useful as test / research reagents in the laboratory, and also through the control of intracellular localization of Integrin β1 and the control of cell adhesion, and in turn, the regulation of angiogenesis. Therefore, it can be used for the prevention or treatment of various diseases and the like.

Claims (6)

An agent for controlling cell adhesion to an extracellular matrix, comprising a substance that controls the ubiquitin ligase signaling-related protein of (A) or (B) below or a gene thereof.
(A) at least one BTB protein selected from the group consisting of ANKFY1, KCTD18, BTBD7, CCIN, KCTD2, KLHL18, KLHL34, ZBTB6 (B) at least one kind selected from the group consisting of SNX9, SNX11, SNX17 protein An agent for controlling angiogenesis, comprising a substance that controls the ubiquitin ligase signaling-related protein of the following (A) or (B) or a gene thereof.
(A) at least one BTB protein selected from the group consisting of ANKFY1, KCTD18, BTBD7, CCIN, KCTD2, KLHL18, KLHL34, ZBTB6 (B) at least one kind selected from the group consisting of SNX9, SNX11, SNX17 protein A preventive or therapeutic agent for a disease caused by abnormality in cell adhesion or angiogenesis, which comprises a substance that controls the following (A) or (B) ubiquitin ligase signaling-related protein or gene thereof.
(A) at least one BTB protein selected from the group consisting of ANKFY1, KCTD18, BTBD7, CCIN, KCTD2, KLHL18, KLHL34, ZBTB6 (B) at least one protein selected from the group consisting of SNX9, SNX11, SNX17 An ubiquitin ligase component comprising at least one BTB protein or a derivative thereof selected from the group consisting of ANKFY1, KCTD18, BTBD7, CCIN, KCTD2, KLHL18, KLHL34, ZBTB6. A ubiquitin ligase comprising CUL3 as a ligase activity body, wherein the adapter site is at least one BTB protein selected from the group consisting of ANKFY1, KCTD18, BTBD7, CCIN, KCTD2, KLHL18, KLHL34, ZBTB6. , Ubiquitin ligase. A screening method of a control agent for cell adhesion to extracellular matrix or an angiogenesis control agent, characterized by using the control ability of the following (I) or (II) as an index.
(I) Interaction between the following (A) and CUL3 (II) Ubiquitination of the following (B) (A) ANKFY1, KCTD18, BTBD7, CCIN, KCTD2, KLHL18, KLHL34, ZBTB6 at least selected from the group consisting of At least one protein selected from the group consisting of one BTB protein (B) SNX9, SNX 11, SNX 17