JP4487056B2 - Ubiquitin-conjugating enzyme immobilized protein chip - Google Patents

Description

  The present invention relates to a ubiquitin-conjugating enzyme-immobilized protein chip for screening a regulator capable of regulating a ubiquitination pathway and a screening method for a ubiquitination pathway regulator.

  Ubiquitin is a protein that exists universally in eukaryotes consisting of 76 amino acid residues, and ubiquitination of proteins is a post-translational modification of proteins and is known to be a proteolytic labeling reaction (for example, , See Non-Patent Document 1.)

FIG. 1 is a diagram schematically illustrating a ubiquitination pathway. The ubiquitination reaction includes three types of enzymes, namely, a ubiquitin activating enzyme (hereinafter sometimes abbreviated as E1), a ubiquitin-binding enzyme (hereinafter sometimes abbreviated as E2), and a ubiquitin ligase (hereinafter referred to as E2). , Which may be abbreviated as E3) (see, for example, Non-Patent Document 2). E1 is a single molecule, while E2 and E3 are known to have molecular diversity. For example, in yeast, 10 or more types of E2 have been identified.
E2 is usually thought to interact with multiple E3s, which interact with multiple different substrate proteins with similar recognition sites.

  In the ubiquitination of proteins, first, ubiquitin is activated by E1 accompanied by hydrolysis of ATP, and thioester-bonds to the active SH group of E1. Next, ubiquitin is transferred to the active SH group of E2, and the ε-amino group of the lysine side chain of the target protein recognized by E3 and the carboxyl group of ubiquitin are isopeptide-bonded. A protein in which a multi-ubiquitin chain is bound by polyubiquitination in which a cycle in which a second ubiquitin is bound to a first ubiquitin that is isopeptide-bound to the protein becomes a target protein of the 26S proteasome that is an ATP-dependent protease Recognized and disassembled.

Targeted proteolysis by the ubiquitination pathway from protein ubiquitination to proteasome proteolysis is the homeostasis of metabolism, transcription and translation in protein synthesis, signal transduction, cell cycle, tumor formation, etc. (See, for example, Non-Patent Document 3.)
Examples of intracellular target proteins that can be degraded by this ubiquitination pathway include oncoprotein, which is a protein that cancerizes cells.
Therefore, when this ubiquitination pathway is disrupted (rapid degradation of target protein, reduction of target protein degradation), neurodegenerative diseases such as cancer, muscle atrophy, Alzheimer's disease and Parkinson's disease, immune and inflammatory response diseases It is thought to cause various diseases such as (see, for example, Non-Patent Document 4).
In other words, if it is a substance that can regulate this ubiquitination pathway (for example, a substance that regulates the ubiquitination pathway so that proteins harmful to the human body can be normally disrupted), various diseases such as the above-mentioned cancers, neurodegenerative diseases, etc. It can be a drug that treats and prevents. Therefore, in recent years, searching for such a regulatory substance (new drug candidate substance) has been performed.
Examples of points that can regulate the ubiquitination pathway include the following three (see FIG. 1).
(1) Transition from E1 to E2 (2) Target protein recognition by E3 (3) Target protein recognition of proteasome

In order to search for the above-mentioned regulator (new drug candidate substance), conventionally, an electrophoresis technique has been employed.
For example, a case where a regulatory substance (new drug candidate substance) at the regulation point (1) is searched for based on the conventional method will be described below.
(A) When E1, E2, and ubiquitin (hereinafter sometimes abbreviated as Ub) are reacted, an E1-Ub complex and an E2-Ub complex are formed, and five types including E1, E2, and Ub The protein (complex) is present. When these proteins (complexes) are electrophoresed according to a conventional method, the five proteins (complexes) have different mobilities, so that they can be separated in a gel and the bands can be visualized and confirmed.
(B) Next, in addition to E1, E2, Ub, a new drug candidate substance (organic compound, protein, etc.) is reacted, and this is confirmed by electrophoresis in the same manner as in (A).
At this time, compared with the band pattern of electrophoresis obtained in (A), compared to the case of reacting under the condition where there is no new drug candidate substance in (A), the E1-Ub complex or E2-Ub If it is judged that the amount of complex produced is different, this new drug candidate substance is recognized as a substance capable of regulating the binding of E1 and Ub and E2 and Ub, that is, a substance capable of regulating the ubiquitination pathway. It is done.

  At the regulation points (2) and (3) above, a search for regulators (new drug candidate substances) of the ubiquitin-proteasome pathway was performed using the above-described electrophoresis technique.

Wilkinson, K.D., et al. “Ubiquitin is the ATP-dependent proteolysis factor of rabbit reticulocytes.” 1980 J. Biol. Chem. 255, 7529-7532) Scheffner, M., et al. “Protein ubiquitination involving an E1-E2-E3 enzyme ubiquitin thioester cascade.” 1995 Nature 373, 81-83) hershko, A., et al. “The ubiquitin system.” 2000 Nature medicine Volume 6, Number 10, 1073-1081) Ciechanover, A., “The ubiquitin-proteolytic system and pathogenesis of human diseases: a novel platform for mechanism-based. drug targeting.) "2003 Biochem Soc Trans, 31 (2): 474-481

As described above, since E2 and E3 have molecular diversity, in order to search for a new drug candidate substance using the electrophoresis method, for example, a combination of the type of E2 and the number of new drug candidate substances is used. It was necessary to perform a corresponding electrophoresis experiment.
In addition, since complicated steps such as a step of reacting E1, E2, and Ub and an electrophoresis step are required, it takes time and effort, and it is difficult to obtain experimental results quickly.

In addition, since the effect of the drug may differ depending on each user due to factors such as constitution due to the difference in gene level of the drug user, in recent years, it has been desired to prepare a drug (tailor-made drug) suitable for the user. ing. In this case, it is preferable that a drug suitable for each user can be quickly prepared because it can be administered before the disease progresses.
Therefore, it is desired to efficiently search for new drug candidate substances suitable for each user.

  Accordingly, an object of the present invention is to provide means for efficiently searching for a substance capable of regulating the ubiquitination pathway and a screening method for a ubiquitination pathway regulator using the means.

  As a result of intensive studies to solve the above problems, the present inventors have succeeded in constructing an E2 immobilized protein chip in which E2 is immobilized on a substrate without impairing the biological activity of E2. As a result of further earnest research, the present inventors have completed the present invention by finding a strategy that can detect the biological activity of E2 with high sensitivity and exhaustiveness.

That is, the first characteristic configuration of the present invention for achieving the above object is to screen a plurality of different ubiquitin-binding enzymes (E2) at a plurality of different positions on a substrate in order to screen for a modulator capable of regulating the ubiquitination pathway. The ubiquitin-binding enzyme (E2) -immobilized protein chip was placed on the substrate and the substrate on which the enzyme was immobilized was subjected to a blocking treatment with a blocking agent containing 10 to 50 mM DTT .

In order to achieve the above object, the second characteristic configuration of the present invention includes a protein printing step of producing an E2 immobilized protein chip by arranging a plurality of different ubiquitin-binding enzymes (E2) at a plurality of different positions on a substrate,
Wherein the E2 immobilized Protein chip, biotinylated labeling ubiquitin in the presence of ubiquitination reagent containing a ubiquitin activating enzyme (E1) (UBM), the contact step of contacting a test substance,
A washing step of washing the E2-immobilized protein chip to remove the ubiquitination reagent (UBM) after the reaction on the E2-immobilized protein chip and a test substance;
A detection step of detecting a signal of labeled ubiquitin bound to the ubiquitin-conjugating enzyme (E2) on the E2-immobilized protein chip,
Compared with the signal in the ubiquitin-conjugating enzyme (E2) not brought into contact with the test substance, the change in signal intensity by the test substance with respect to each of the plurality of different ubiquitin-conjugating enzymes (E2) was observed, and ubiquitin was converted into the ubiquitin-activating enzyme (E1). A screening method for a ubiquitination pathway regulator, which is used for screening a predetermined regulatory substance, by examining characteristics as a regulatory substance that regulates the ubiquitination pathway by controlling the transfer from) to ubiquitin-binding enzyme (E2) It is in the point.

For example, the reaction characteristics of a plurality of different E2s as modulators that can regulate the ubiquitination pathway by contacting (reacting) the test substance once with an E2-immobilized protein chip on which different E2s are arranged. Can be examined. That is, it is not necessary to perform many experiments according to the combination of the type of E2 and the number of regulators, unlike the conventional method using electrophoresis.
Therefore, the experimental result can be obtained quickly.

  Furthermore, as described in the examples described later, the E2 immobilized protein chip of the present invention and the screening method for a ubiquitination pathway regulator using the protein chip include immobilization of a protein on a substrate, cost reduction, In addition, it has been recognized that optimum conditions can be studied and good results can be obtained in order to reduce background noise and the like.

Specifically, in the ubiquitin-conjugating enzyme (E2) -immobilized protein chip, the substrate is preferably functionalized with hydrogel N-hydroxysuccinimide or aldehyde.
Hydrogel N-Hydroxysuccinimide or E2 immobilized protein that retains E2 enzyme activity in a detectable state while immobilizing E2 enzyme activity by immobilizing E2 on a substrate functionalized with aldehyde Chips can be provided.

In the present invention, in the ubiquitin-conjugated enzyme (E2) -immobilized protein chip, the substrate on which the enzyme is immobilized is subjected to a blocking treatment with a blocking agent containing 10 to 50 mM DTT .
By performing a blocking treatment with a blocking agent containing 10 to 50 mM DTT, it is possible to effectively reduce the background noise by suppressing non-specific binding to the non-printing region while effectively retaining the enzyme activity of E2. An E2 immobilized protein chip can be provided.

And in the screening method of a ubiquitination pathway regulatory substance, it is preferable that the substrate is functionalized with hydrogel N-hydroxysuccinimide or aldehyde.
Hydrogel N-Hydroxysuccinimide or E2 immobilized protein that retains E2 enzyme activity in a detectable state while immobilizing E2 enzyme activity by immobilizing E2 on a substrate functionalized with aldehyde Since a chip can be provided, it is possible to provide a highly sensitive and reliable screening method for a ubiquitination pathway regulator.

In the screening method of the ubiquitination pathway modulators of the present invention uses a biotinylated labeled ubiquitin as a standard識化ubiquitin.
The use of biotinylated labeled ubiquitin makes it possible to detect signals with high sensitivity and accuracy, and to provide a highly sensitive and reliable screening method for a ubiquitination pathway regulator. .

Then, it is particularly preferable to add 1 to 20 μM of biotinylated labeled ubiquitin, and it is preferable to add 2.5 to 250 nM of E1.
By adding E1 and ubiquitin in the above concentration range, background noise can be reduced and detection of a sufficiently observable signal can be achieved, and more sensitive and reliable regulation of the ubiquitination pathway It becomes possible to provide a screening method for substances.

In the screening method for a ubiquitination pathway regulator of the present invention, the washing step is preferably performed in the presence of SDS (sodium dodecyl sulfate).
By washing in the presence of SDS, labeled ubiquitin not bound by thiol ester bonds with E2 immobilized on the substrate can be surely removed, and by reducing background noise, It is possible to provide a screening method for a ubiquitination pathway regulator with high sensitivity and reliability.

Furthermore, in the screening method for a ubiquitination pathway regulator of the present invention, after the protein printing step, a blocking step of adding a blocking agent is applied to the substrate before the contact step, and the blocking agent is 10 to 50 mM. It is preferable to contain DTT.
By using a blocking agent containing 10-50 mM DTT (dithiothreitol), it is possible to effectively retain the enzyme activity of E2, to detect the activity, and to suppress nonspecific binding to the non-printing region. Thus, background noise can be reduced, and a highly sensitive and reliable screening method for a ubiquitination pathway regulator can be provided.

  Since the E2-immobilized protein chip of the present invention can directly and comprehensively evaluate substances capable of regulating the ubiquitination pathway, it is effective for screening for modulators capable of regulating a predetermined ubiquitination pathway.

  In addition, if the modulator is a drug, it is possible to quickly prepare a drug suitable for each user in the manufacture of a tailor-made drug that mixes the drug suitable for the user, and the drug is administered before the disease progresses. It becomes possible.

  The present invention can provide an E2-immobilized protein chip that can support E2 on a substrate while maintaining the biochemical activity of E2, and can detect the biological activity with high sensitivity. E2 has a property rich in molecular diversity, but by using the protein chip of the present invention, high-throughput, comprehensive and direct evaluation of each biological activity of a plurality of E2 isoforms is possible. can do. Since E2 is an enzyme involved in the ubiquitin pathway, the protein chip of the present invention can provide direct and comprehensive information at a high throughput as a means of knowing a modulator that can regulate the ubiquitination pathway. . Thus, the present invention also provides a high-throughput screening method for modulators that can modulate the ubiquitination pathway. The substance that affects the biological activity of E2 screened by the present invention is useful for the treatment and prevention of cancer, neurodegenerative diseases and the like as a modulator that can regulate the ubiquitination pathway.

  Moreover, according to the present invention, an E3 immobilized protein chip can be constructed for E3 as well as E2, and the protein chip has a wide range of utility values.

Embodiments of the present invention will be described below with reference to the drawings.
The ubiquitination pathway for the target protein by the proteasome is known from the ubiquitination of the protein, but when this ubiquitination pathway is disrupted and the specific degradation of the target protein is not performed normally, various types of cancer, neurodegenerative diseases, etc. It is thought to cause illness.
In other words, if there is a substance that can regulate this ubiquitination pathway (for example, a substance that regulates the ubiquitination pathway so that proteins harmful to the human body can be normally disrupted), various diseases such as cancer and neurodegenerative diseases can be treated. Can be a medicine to do. Therefore, in recent years, searching for such a regulatory substance (hereinafter sometimes referred to as a new drug candidate substance) has been performed.

On the other hand, in recent years, great efforts have been made to realize protein chips. Similar to DNA microarrays, this protein chip is considered to be highly useful as a low-cost, quick and simple screening technique because of the low consumption of expensive and valuable samples and reagents. Therefore, it is considered that an efficient screening system can be established if a protein chip is applied in the search for the modulator (new drug candidate).
The key to the practical use of protein chips is to solve problems such as immobilization of proteins on a substrate, cost reduction, and reduction of background noise.

  Therefore, to achieve the goal of establishing a screening system that can efficiently search for modulators that can regulate the ubiquitination pathway using protein chips, it is important to study the optimal experimental conditions. . If such a screening system is established, it is expected that a medicine suitable for the user (tailor-made medicine) can be efficiently produced and various diseases can be rapidly treated.

  The present invention provides a protein chip capable of efficiently searching for a substance capable of regulating the ubiquitination pathway, and a screening method for a substance capable of regulating the ubiquitination pathway using the protein chip. This will be described in detail below.

  In one aspect of the present invention, an E2 immobilized protein chip is provided. The protein chip in the present invention means a chip in which a plurality of proteins or peptide fragments are fixed on the surface of a substrate, and means a chip that can efficiently analyze protein-protein, protein-drug, etc. interactions. In the E2-immobilized protein chip in the invention, E2 is immobilized on the surface of the substrate.

  Here, the ubiquitination-binding enzyme (E2) immobilized on the substrate refers to an enzyme belonging to the family of ubiquitination-binding enzymes (E2). E2 is an enzyme having molecular diversity, and specifically includes human-derived UbcH6, UbcH5b, UbcH5b, UbcH5c, UbcH2b, UbcH3, UbcH4, UbcH7 and the like, but is not limited thereto. Moreover, E2 to which a tag is added, such as an oligohistidine tag, can also be suitably used, and can be prepared by adding a desired tag when expressing a protein. By immobilizing the protein on the substrate via the tag, it is possible to align the orientation of the protein on the substrate, and the immobilization reaction to the substrate occurs at a location separate from the protein body. The influence on the activity can be minimized.

  The E2-immobilized protein chip of the present invention is produced by printing and immobilizing E2 on a suitable substrate. Immobilization of E2 on the substrate is performed while maintaining the three-dimensional structure of the protein (that is, the secondary structure and tertiary structure) and the biological activity of E2. Also, ideally, in order to ensure the identity of the immobilized protein reactivity, the protein is immobilized on the substrate with its orientation aligned. As described above, the ability to immobilize functional proteins is an important point for protein chip development.

  Here, the substrate used for immobilization is preferably flat glass, and a slide glass having a 12 × 4 subarray divided by a Teflon mask used in the examples described below can also be preferably used.

  Protein printing is performed on a suitable substrate, preferably using an ink jet microarrayer. A microarray robot or the like for printing at a high density is commercially available, and a commercially available product such as High Precision ink-jet microarrayer (manufactured by Cartesian Technologies) can be used. However, simply adsorbing protein to the substrate surface is problematic in terms of mechanical strength, so the surface of the substrate must be chemically treated in advance to chemically immobilize E2 on the substrate via a covalent bond or the like. Specifically, the functionalization by hydrogel N-hydroxysuccinimide (hereinafter sometimes abbreviated as NHS) and the functionalization by aldehyde are particularly suitable as the substrate processing method in the protein chip of the present invention. Is available.

  Next, a method of using the E2-immobilized protein chip will be described, but the scope of the present invention is not limitedly interpreted according to the mode of use.

  The E2 immobilized protein chip can be used to screen for a modulator that can regulate the ubiquitination pathway. Therefore, in another embodiment of the present invention, a method for screening for a modulator capable of regulating the ubiquitination pathway is also encompassed by the present invention. Since ubiquitin is involved in many biological reactions such as carcinogenesis and cell cycle, viral infection, and neurodegenerative diseases in vivo, regulators that can regulate the ubiquitination pathway include, for example, cancer and nerves. It can be a therapeutic drug for degenerative diseases or the like, or a preventive drug.

  In the present invention, screening is performed by immobilizing E2 on a substrate and reacting the immobilized E2 in the presence of E1 and labeled ubiquitin. Therefore, the regulatory substance to be screened is ubiquitin. It is a substance that can control the transfer from E1 to E2. Therefore, a modulator is a substance that inhibits or delays the transfer of ubiquitin from E1 to E2 (inhibitor), and a substance that promotes the transfer of ubiquitin from E1 to E2 (activator). Shall mean. Examples of the inhibitor include ubiquitin-like molecules (hereinafter sometimes abbreviated as Ubls) and the like. In this case, the binding reaction between ubiquitin and E2 and the binding reaction between ubiquitin-like molecule and E2 compete. As a result, ubiquitin is inhibited from transferring from E1 to E2. The screening method of the present invention searches for and identifies a novel such substance.

One embodiment of the screening method for a modulator capable of regulating the ubiquitination pathway according to the present invention is shown in detail below (see FIG. 2). This screening method
1. A protein printing step in which different ubiquitin-binding enzymes (E2) are arranged at a plurality of different positions on a substrate to produce a ubiquitin-binding enzyme-immobilized protein chip;
2. A blocking step for performing a blocking treatment to avoid non-specific reaction on the substrate,
3. A contacting step in which a test substance is brought into contact with the protein chip in the presence of a ubiquitination reagent (hereinafter sometimes abbreviated as UBM) containing labeled ubiquitin and ubiquitin-activating enzyme (E1);
4). A washing step of washing the E2 immobilized protein chip to remove the ubiquitination reagent (UBM) after the reaction on the E2 immobilized protein chip, and a test substance;
5). A detection step of detecting a signal of labeled ubiquitin bound to the ubiquitin-conjugating enzyme (E2) on the protein chip;
6). An analysis step for quantifying and analyzing the detected signal is included.
Below, each process is demonstrated in detail.

1. Protein printing step The protein printing step is a step of printing and immobilizing E2 on a substrate. The printing and fixing of E2 has already been described above.

2. Blocking step The blocking step generates a high background noise because non-specific binding to the non-printing region generates, for example, blocking in the region where E2 is not immobilized on the substrate. This is done by using an agent to provide a substrate surface that is resistant to non-specific binding, which can reduce background noise. The blocking agent used in the blocking step needs to have a low background and is not reactive with any component of the ubiquitin pathway, specifically, 10 to 50 mM DTT, particularly It has been found in the study examples described below that 2% BSA (Bovine Serum Albumin) and 10 mM DTT-containing PBS (Phosphate Buffered Saline) (pH 7.6) can be suitably used. . However, the present invention is not limited to this. Based on this finding, it was found that a good blocking effect was obtained by containing 10-50 mM DTT, particularly 10 mM DTT. It is possible to appropriately modify the composition of the blocking agent. However, the presence of a high concentration of DTT exceeding 50 mM is not preferable because the signal derived from E2 to be detected is significantly affected. The blocking agent commonly used in Western blots is milk, but since milk contains a small amount of biotin, its use should be avoided when using biotinylated labeled ubiquitin.

3. Contacting step In the contacting step, E2 immobilized on the substrate is brought into contact with a test substance which is a modulator candidate compound capable of regulating the ubiquitination pathway in the presence of a labeled ubiquitin and an ubiquitination reagent containing E1. The specific method is not particularly limited. For example, the substrate on which E2 is immobilized is immersed in a test solution in which a test substance is dissolved in an appropriate solvent, or the test solution is spotted on a region on the substrate where E2 is immobilized. Is done. The contact time can be appropriately changed according to the type of E2 to be immobilized.

  As a test substance used in the screening method of the present invention, an organic compound synthesized organically, a protein library, or a component derived from animals, plants, and microorganisms can be used.

  The ubiquitination reagent is a reaction in which ubiquitin in the ubiquitination pathway such as E1, ubiquitin, ATP, etc. is activated by E1 along with ATP hydrolysis and thioester-bonds to the active SH group of E1, and the subsequent reaction , And ubiquitin bound to E1 means a reagent necessary for successfully carrying out a reaction in which ubiquitin is transferred to the active SH group of E2.

Ubiquitin is labeled to facilitate detection of E2 ubiquitination. It is necessary to use a label that does not inhibit the ubiquitination reaction of E2, and specifically, it is preferable to label with biotin. In addition, the biotinylated label is preferably an N-terminal label, but a homogenous label can also be suitably used. Depending on the type of E2 to be detected for ubiquitination (for example, UbcH2b), a low signal can be obtained with a homogenous label, whereas a strong signal can be detected with an N-terminal label. is there. This is thought to be due to the fact that the 48th lysine of ubiquitin is involved in the binding to UbcH2b, but at present, such a phenomenon has been observed only for UbcH2b. Since even detection is possible in some cases, homogenous labels can also be suitably used.
The direct ubiquitin labeling with a fluorescent dye such as Cy3 label decisively affects the reaction process, and since the fluorescent dye has a charge, it becomes an interfering substance in detecting the activity. In addition, since it is necessary to add a color former when detecting the use of an enzyme label or the like, it causes contamination with adjacent spots, which is not preferable.

  The addition concentration of E1 and labeled ubiquitin increases the background noise because the increase in the addition amount of E1 and the increase in the addition amount of ubiquitin leads to an increase in the background noise. To be optimized. E1 is preferably added at 2.5 to 250 nM, in particular 100 nM. Ubiquitin is preferably added at 1 to 20 μM, more preferably 1 to 10 μM, particularly 1 to 5 μM.

  Moreover, what was made to react in presence of a ubiquitination reagent, without making an immobilized E2 spot contact with a test substance can be utilized suitably as control. By comparing the signal intensity detected in the control with the signal intensity detected in the contact spot with the test substance due to the presence of the control spot, the ability of the test substance to regulate ubiquitination can be evaluated.

  In addition, in order to confirm the effectiveness of the reaction on the chip, a negative control printed with BSA on the substrate can be used, and when this BSA spot does not emit a signal, it is detected in a later detection step. It can be shown that the signal produced is not due to ubiquitin bound non-specifically on the substrate surface.

4). Washing process The washing process here means washing immediately after the contact reaction between the test substance and the ubiquitination reagent, and the presence of labeled ubiquitin not bound by the thiol ester bond with E2 immobilized on the substrate is high. In order to generate ground noise, in order to remove the labeled ubiquitin that does not form a thiol ester bond with E2 on the substrate, and E1 and the test substance, for example, the substrate is immersed in a cleaning solution. The washing solution used in the washing step has a low background like the blocking agent and has no reactivity with any component of the ubiquitin pathway. It is necessary to be able to remove non-covalently bound molecules present on the substrate surface that cause background noise without being cleaved. Specifically, the washing is preferably performed in the presence of 0.1% or more SDS, particularly 0.1 to 10% SDS, and 0.1% SDS-containing 20 mM Tris buffer (pH 5.7) is used. It has been found in the study examples described below that it can be suitably used. However, the present invention is not limited to this, and the knowledge that a good cleaning effect is exhibited by the presence of 0.1% SDS, particularly 0.1 to 10% SDS, and on the substrate Since the knowledge that the binding between E2 and ubiquitin exhibits stability was obtained, the composition of the cleaning solution can be appropriately modified based on this knowledge.

5). Detection step The detection step detects labeled ubiquitin bound to E2 immobilized on the protein chip. Detection is performed according to the type of ubiquitin label.
For example, when ubiquitin is labeled with biotin, streptavidin phycoerythrin (hereinafter sometimes abbreviated as SAPE) is used for detection. That is, a staining solution containing SAPE is brought into contact with the protein chip after the washing step, and after washing, a signal emitted from the label is detected using a microscanner or the like. The detection can be performed, for example, using a commercially available microarray scanner such as White light Scanner-Array Work (manufactured by Applied Precision).

6). Analysis step The detected signal is quantified using, for example, commercially available software such as IMAGENE ^™ software, and the data is analyzed. Here, the detected signal corresponds to the amount of ubiquitin covalently bound to E2, and the test substance that changes the signal intensity as compared with the signal (control) in E2 in which the test substance is not contacted, The ubiquitination pathway is selected as a regulatable regulator by controlling the transfer from E2 to E2. That is, when the labeled ubiquitin is not detected at the position where E2 is fixed on the substrate, or when the detection amount is decreased as compared with the control, the test substance reacts to transfer ubiquitin from E1 to E2. By inhibiting, it is determined as an inhibitor that inhibits the ubiquitin pathway, and this can be a potent therapeutic drug or preventive drug for cancer, neurodegenerative diseases and the like. On the other hand, when the detected amount is increased compared to the control, the test substance is determined to be an activator that activates the ubiquitin pathway by activating the reaction of transferring from E1 to E2, which is a cancer, It can be a potent therapeutic drug or preventive drug for neurodegenerative diseases and the like.

  Furthermore, the E2 immobilized protein chip of the present invention can be used for various applications. Below, the aspect of utilization other than the above is demonstrated. These forms of use are also included in the present invention.

  The ability to form thiol ester bonds with ubiquitin can be used to screen proteins and enables high-throughput processing, so it is an alternative to today's standard technologies such as SDS-PAGE and Western blotting. Can be presented as For example, E2 immobilized protein chips can be used to check the functionality of expression-purified E2 in quality inspections at recombinant protein production sites. In addition, since new E2s are continuously discovered, the E2 immobilized protein chip is used to screen unknown proteins for their ability to bind ubiquitin and to identify new family members. It can be used in basic research. In this case, the protein to be investigated is printed on a substrate and an El catalyzed ubiquitin binding reaction is performed in the presence of labeled ubiquitin. Furthermore, it can be used to elucidate the substrate specificity of E2 and the involvement of different pathways in the ubiquitin pathway.

  Since the construction of mutants by site-directed mutagenesis is often used for structural studies to elucidate the mechanism of activity of proteins, including enzymes, E2 immobilized protein chips have been constructed. It can be used as a useful tool for screening the activity level of E2 mutants and for the isolation of dominant negative mutants of E2.

  In addition, the E2 immobilized protein chip can be used for investigation of ubiquitination of a target protein. A given protein is immobilized on a microarray and ubiquitination is investigated by conducting a ubiquitination reaction in parallel with the various E2s immobilized together. This can then contribute to the determination of specific pathways involving the binding of ubiquitin to the target protein.

  Furthermore, the E2-immobilized protein chip of the present invention can be used as a tool for determining the ubiquitination profile of a large number of proteins in a biological sample, and shows the correlation between ubiquitin system dysfunction and disease. Can be elucidated, which can provide a starting point for identifying targets in drug development. That is, the effect of E2 on ubiquitination is confirmed by detecting labeled ubiquitin by bringing immobilized E2 into contact with a biological sample such as blood or urine.

(Preparation Experiment Example 1)
Preparation of E1 and E2 Enzyme origin E1 uses rabbit-derived E1, E2 uses human-derived UbcH2b, UbcH3, UbcH4, UbcH7, UbcH6, UbcH5b, (C85A) UbcH5b, UbcH5c, which are added with His-Tag It is. UbcH6, UbcH5b, (C85A) UbcH5b, UbcH5c were purchased from Cosmo Bio. UbcH2b, UbcH3, UbcH4 and UbcH7 were obtained by expression and purification. 5 cDNA containing 6His-Tag at the N-terminus was cloned into pT7-7 vector (provided by MYAMOTO) downstream of the T7 promoter.

2. E. Expression in E. coli The above five enzymes (E1, UbcH2b, UbcH3, UbcH4, UbcH7) are recombinant BL21 E. coli. It was expressed using E. coli and purified. 10 ng of plasmid DNA was transferred to 20 μl of BL21 (DE3) E. E. coli competent cells (Stratagene) were added, and the mixture was incubated on ice for 30 minutes, followed by heat treatment at 42 ° C. for 30 seconds. After keeping the cells on ice for 2 minutes, 180 μl of SOC medium (Stratagene) was added and shaken at 37 ° C. for 1 hour. Subsequently, the mixture was allowed to stand on an LB agar medium (manufactured by Nacalai Tesque) containing 50 μg / mL carbenicillin (manufactured by Sigma Aldrich) and then incubated overnight at 37 ° C. Half of the colonies on the plate were collected and inoculated into 200 mL of LB medium (Nacalai Tesque) containing 50 μg / mL carbenicillin. Bacterial cells were grown at 37 ° C. for about 3 hours at 200 rpm until OD _600nm = 0.6, then transferred to a shaker at 26 ° C. and incubated for about 30 minutes until OD _600nm = 0.8. Protein expression was induced by the addition of 20 μM IPTG (Nacalai Tesque) and incubated at 26 ° C. and 200 rpm for about 12-14 hours. Transformed E. coli E. coli cells were collected, centrifuged at 8300 rpm for 10 minutes at 4 ° C., and the cell pellet obtained by discarding the supernatant was stored at −80 degrees until purification as described below.

3. Protein Purification E1 was separated and purified on Sepharose-ubiquitin. Specifically, it was performed as follows. The cell pellet was resuspended in 20 mL of 50 mM Tris buffer (pH 7.6: manufactured by Wako Pure Chemical Industries, Ltd.), and then sonicated 3 times on ice for 3 minutes. The cell lysate was centrifuged at 10,000 rpm for 15 minutes at 4 ° C. and the supernatant was filtered. The Sepharose-ubiquitin column was prepared using a 1 mM ubiquitin solution (Sigma Aldrich) according to the instructions of the resin manufacturer (NHS activation-sepharose ^™ 4 Fast Flow: manufactured by Amersham Biosciences), and 10 mM MgCl ₂ (Wako Pure Chemical Industries, Ltd.). Equilibrated with 50 mM Tris buffer (pH 7.6). The supernatant containing 10 mM MgCl ₂ and 5 mM ATP (Sigma Aldrich) was loaded onto the column. The column was washed with 20 mL of 0.5 mM KCl-containing 50 mM Tris buffer (pH 7.6) and 10 mL of 50 mM Tris buffer (pH 7.6), and E1 protein was contained in 50 mM Tris buffer containing 10 mM DTT (manufactured by Wako Pure Chemical Industries). eluting at pH 7.6) and collecting 15 fractions (0.5 mL each). The column was finally washed with 20 mL of 50 mM Tris buffer (pH 9) containing 1 M KCl and 20 mL of 50 mM Tris buffer (pH 7.6).

  E2 was separated and purified on NTA resin. Specifically, it was performed as follows. The cell pellet was resuspended in 10 mL of cold PBS (pH 7.6: manufactured by Nacalai Tesque), then sonicated 3 times for 3 minutes on ice, and the cell lysate was collected at 10,000 rpm at 4 ° C. For 15 minutes. The supernatant was applied to a 1 mL Ni-NTA column (Qiagen) pre-equilibrated with 20 mL of PBS, and washed with 25 mL of PBS (pH 7.6) containing 20 mM imidazole (Wako Pure Chemical Industries). . The E2 protein was then eluted with PBS containing 0.3M imidazole (pH 7.6) and 10 fractions (0.5 mL each) were collected.

  The presence of purified protein in each fraction was assessed using the Bradford method (protein assay reagent: Bio-Rad). In order to check the purity of the purified enzyme, the flow-through, washing, and protein-containing fractions were analyzed by SDS-PAGE. The protein-containing fraction is 50 mM Tris buffer (pH 7.6) in the case of E1, and Spectra / Por regenerated cellulose dialysis membrane MWCO 3500 (manufactured by Spectrum Laboratories) against PBS (pH 7.6) in the case of E2. Was dialyzed 3 times at 4 ° C for 3 hours. The protein concentration was measured by the Bradford method as described above.

(Examination example 1)
Parameter analysis experiment (preliminary study in solution)
Elucidation of some important parameters was examined by reacting E1, ubiquitin, and E2 in a liquid phase and then loading the gel to detect the intensity of the ubiquitination signal of E2.

In vitro ubiquitination of E2 (UbcH2b) was performed in a reaction mixture containing 3 μM E2, 10 mM MgCl ₂ , 2 mM ATP in 50 mM Tris buffer (pH 7.6). At this time, the molar concentration of E1 was set to 6 nM to 3 μM, and the molar concentration of biotinylated ubiquitin was set to various molar concentrations of 3 μM to 100 μM. The reaction mixture was incubated at 37 ° C. for 30 minutes or 5 hours. After each incubation, 4 μL of SDS loading buffer 5X (Bio-Rad) was added to 16 μl of each reaction treatment solution, and the obtained sample was heat-treated at 95 ° C. for 5 minutes and then separated on Biorad readygel J. After that, it was stained with Coomassie blue and decolorized with 7% methanol and 10% acetic acid.

The results are shown in FIG. A band corresponding to the covalent complex formed between UbcH2b and ubiquitin is surrounded by a box. Observation of a band representing UbcH2b near 20 kD under all tested conditions revealed that the majority of E2 was not ubiquitinated even when E1 was incubated with equimolar UbcH2b. A range study at E1 concentration confirmed that complex formation decreased at a constant UbcH2b concentration. This represents a progressive decrease in UbcH2 thiol ester formation. It was also found that when the ubiquitin concentration was increased, an equilibrium state was reached when the ubiquitin concentration was 10 times the E2 concentration. Furthermore, it was confirmed that the incubation time had no difference between 30 minutes and 5 hours, so that the thiol ester binding reaction of UbcH2b-ubiquitin in the liquid phase reached the saturation point in 30 minutes. Turned out to reach. It is recognized that as the first approach to monitor ubiquitin transfer from E1 to E2, this experiment points out the effects of important parameters that must be further considered in on-chip experiments. .
Therefore, from the results of this experiment, the E1 concentration can be reduced to a ratio of 1: 5 without affecting the transthiolation efficiency, and the increase in the ubiquitin concentration has a slight effect on thiol ester formation, On the other hand, it has been found that even if the reaction time is prolonged, the effect is not affected at all and the reaction cannot be further pushed up from the equilibrium point.

(Examination example 2)
Experiments on optimization of washing conditions In the on-chip ubiquitination of E2, various washing conditions for removing non-covalent molecules existing on the substrate surface after contacting UBM with immobilized E2 are various. We compared cleaning solutions and tried to construct optimal cleaning conditions.
Three types of E2 (UbcH2b, UbcH3 UbcH7) were fixed on a slide glass, reacted by adding UBM (E1 +, E1-), washed with 0.1% SDS-containing buffer, and washed with PBS. The case was compared. Then, a positive control in which biotinylated ubiquitin was printed on the substrate (control + Ub-biotin) and a negative control in which BSA was spotted on the substrate (control-BSA) were prepared.

  The results are shown in FIGS. 4A and 4B. It was found that when the reaction was performed in the absence of E1, only the SDS-containing solution removed all signals, but the other solutions could not be removed. Initially, E2-ubiquitin binding was weak and it was expected that the use of highly stringent wash solutions would not be suitable, but contrary to the original expectation, the thiol bond formed between immobilized E2 and ubiquitin Has been found to have surprising stability on the substrate surface. This emphasizes the difference between the liquid phase and on-chip. From the above results, it was found that non-covalent molecules can be effectively removed by washing in the presence of SDS. The SDS concentration is preferably 0.1% or more as examined here, and the upper limit is about 10% from the viewpoint of maintaining the thiol bond formed between E1 and ubiquitin. The conclusion is desirable.

  The above results also indicate that the positive control showed a high signal, whereas the negative control did not detect a signal. From this, it can be confirmed that the signal detected on the region where E2 is immobilized is a signal derived from biotinylated ubiquitin bound to the immobilized E2. Therefore, it proves that ubiquitination on E2 of E2 is feasible.

  Next, the following three types of reaction solutions (-E1-ubiquitinated biotin, -E1-ATP + ubiquitinated biotin 1 μl, -E1 + ATP + ubiquitinated biotin 1 μl, ) Was added to react with the immobilized E2, and then the signal intensity was compared in the case of washing with PBS.

  The results are shown in FIG. 4C. Some signals were detected when incubated on the surface of the E2 immobilized substrate in the presence of only biotinylated ubiquitin (ie -E1), so that ubiquitin binds non-covalently to E2. There was found. This result is consistent with the view that there is an additional site on UbcH2b for weak interaction with ubiquitin detected by previous NMR studies.

(Examination example 3)
Experiments on optimization of blocking agents In on-chip ubiquitination, the effects of blocking agents on the signal indicating the E2 enzyme activity and the background signal were compared and examined for various blocking agents described below. Tried to build.

The reaction conditions for examination are as follows.
E2: UbcH7
Substrate: Hydrogel-NHS functionalized glass slide (Jap-NHS)
E1: 100 nM
Ubiquitin: homogenous biotinylated ubiquitin 1 μM
Reaction time: 5 hours

The blocking agent examined this time will be described. The blocking agents used in the study are as follows.
Block Ace, Milk, 2% BSA, 2% BSA and 50 mM DTT, 2% BSA and 10 mM DTT

  The examination results are shown in FIG. In the blocking agent, when 10 mM DTT was present, a background signal was clearly reduced as compared to the case of Block Ace, milk and BSA alone, and an increase in the signal derived from E2-ubiquitin was also observed. From the results of FIG. 5, it was found that it is preferable to perform blocking in the presence of DTT in the application of the present invention. Here, as a factor that the background signal is reduced in the presence of DTT, the detection of the background signal is based on whether the ubiquitin species, that is, ubiquitin alone or ubiquitinated E1 is present in the non-protein printing region on the substrate surface. This is due to the fact that DTT can be covalently bonded to the NHS activated surface, so that DTT reacts with E1-ubiquitin to cleave the thiol ester bond, thereby releasing ubiquitin from E1 and One possible contribution is to reduce the ground signal. On the other hand, the increase in E2-ubiquitin-derived signal in the presence of DTT indicates that E2 is isolated and purified from the living body in an acidic state and then immobilized on the substrate. This is considered to explain that a reducing action by a reducing agent is required. However, it was also observed that the signal from E2 was significantly affected by the presence of high concentrations of DTT (greater than 50 mM). Presumably, during printing of E2 and subsequent incubation, some of the immobilized E2 is oxidized to thiol-crosslinked dimers, but DTT in the blocking agent increases the concentration of monomeric E2 at the opposite of this reaction. Let Therefore, E2 molecules covalently bound to the substrate surface due to high concentration DTT may be released into the reaction solution, and the presence of high concentration DTT denatures the protein and deactivates enzyme activity. It is thought to reduce the efficiency of the chemical reaction. From the above examination results, it is concluded that blocking in the presence of 10 to 50 mM DTT is suitable for application of the present invention.

(Examination example 4)
Experiment on optimization of contact reaction time with UBM In the on-chip ubiquitination of four types of E2 (UbcH2b, UbcH3, UbcH4, UbcH7), the effect of reaction time with UBM on ubiquitin transthiolation efficiency over time In addition, signal intensity and background intensity were detected and examined to identify the optimal contact reaction time.

The reaction conditions for examination are as follows.
E1: 100 nM
Ubiquitin: homogenous biotinylated ubiquitin 1 μM

  The results are shown in FIG. In the case where the ubiquitination reaction of E2 was carried out in the liquid phase (see Study Example 1), the equilibrium state was reached by the reaction for 30 minutes, but E2 was immobilized on a chemically functionalized glass slide. When done, the incubation time was clearly found to affect the reaction efficiency. As a result, UbcH2b, UbcH3, and UbcH4 have the highest signal detected when the contact reaction time is 5 hours, and UbcH7 has the highest signal detected when the contact reaction time is 4 hours, but the contact reaction time is 5 It was observed that a high level of signal was detected even in the case of time. From this result, it was concluded that the contact reaction in 5 hours was optimal to detect all the E2 species used in this experiment well, but depending on the E2 species used. It is preferable to set appropriately.

(Examination example 5)
Experiments to observe the activity of the enzyme in a heterogeneous on-chip 4 E2 (UbcH7, UbcH4, UbcH3, UbcH2b) have 60% amino acid sequence identity and behave similarly in solution The reactivity was confirmed to be quite different when fixed on a glass slide (see FIGS. 6 and 7).
These results were also sufficient to conclude that UbcH3, in particular UbcH2b, showed lower transthiolation efficiency compared to the other two E2. Since the development of chip-based screening systems requires robust assays, the detected signal should be as high as possible over background noise. In order to achieve a ubiquitination profile similar to the four immobilized E2, it is necessary to obtain higher signals of UbcH3 and UbcH2b. Therefore, it is necessary to search for a condition that allows high signal observation, low background, and detection, and studies were conducted to achieve optimization of E1 and ubiquitin concentrations.

(Examination example 6)
Optimization examination of E1 and ubiquitin concentrations The effects of E1 and ubiquitin concentrations on signal and background noise in on-chip ubiquitination of UbcH2b and UbcH4 were examined.
The above E2 is immobilized on a hydrogel-NHS functionalized slide glass (Matsunami Glass Industrial Co., Ltd.). The ubiquitin concentrations are 1 μM and 10 μM, and the E1 concentrations with respect to the ubiquitin at each concentration are 0.025, 0.1, and 0.25. , Each was reacted with immobilized E2, and a signal and a background signal were detected.

The results are shown in FIG. When the E1 concentration was increased at a constant biotinylated ubiquitin concentration, it was confirmed that a strong signal was detected in a concentration-dependent manner (FIGS. 8A and 8B). This was expected to increase the detection signal as a result of increasing the E1 transthiolation efficiency, but it was also found that increasing the E1 concentration increased background noise. In particular, since the signal corresponding to UbcH2b-ubiquitin thiol ester is rather low, an increase in background will give less favorable results compared to UbcH4.
Moreover, although the result performed by the high biotinylated ubiquitin density | concentration is shown to FIG. 8C and FIG. 8D, it turned out that the increase in a ubiquitin density | concentration also becomes a factor of a high background. Thus, it has been found that increasing E1 and ubiquitin concentrations are not a suitable means to achieve high transthiol efficiency on-chip. Next, a more detailed study was conducted for setting the optimum addition concentration of E1 and ubiquitin.

  The optimization of the E1 concentration was examined by setting the E1 concentration to 2.5 nM, 25 nM, 250 nM, and 2500 nM.

  The examination results are shown in FIG. From FIG. 9, it was recognized that the background signal was effectively suppressed and the signal derived from ubiquitin bound to E2 could be observed well when the E1 concentration was in the range of 2.5 to 250 nM. Further, it was found that the optimum result was derived when the E1 concentration was set to 100 nM by the optimization protocol found in the examples described below.

  Subsequently, optimization of the ubiquitin concentration was examined by setting the ubiquitin concentration to 10 nM, 100 nM, 1 μM, 5 μM, and 10 μM.

  The examination results are shown in FIG. From FIG. 10, it was recognized that the background signal was effectively suppressed and the signal derived from ubiquitin bound to E2 could be observed well in the range of ubiquitin concentration of 1 to 10 μM, particularly 1 to 5 μM. Here, although not specifically shown, it is understood that a good signal similar to that obtained when the concentration of ubiquitin is 20 μM can be observed even when the concentration of ubiquitin is 1 to 10 μM. Setting the concentration is preferable in the application of the present invention.

(Examination example 7)
Experiments on optimization of ubiquitin labeling method The effects of ubiquitin labeling on E2 enzyme activity in the on-chip ubiquitination of four types of E2 (UbcH2b, UbcH3, UbcH4, UbcH7) are described below. We compared labeling methods and tried to construct an optimal labeling method.

The E2 was immobilized on a substrate, and the enzyme activity of E2 was measured. The reaction conditions for examination are as follows.
E1: 100 nM
Labeled ubiquitin: 1 μM
Reaction time: 5 hours

  The ubiquitin labeling method examined this time is shown below.

Biotinylated label-N-terminal labeled ubiquitin (hereinafter sometimes abbreviated as NtermUbb)
-Homogenous labeled ubiquitin (hereinafter sometimes abbreviated as Ubb)
25 mg of ubiquitin (manufactured by Sigma) was dissolved in 1 mL of carbonate buffer (0.1 M KCO ₃ , 250 mM NaCl), and 4.1 mg of biotincaproyl-NHS (manufactured by Sigma) was added to this solution. Incubated for 3 hours. Biotinylated ubiquitin was precipitated three times with 100% ethanol, dissolved in distilled water, and then the protein concentration was measured.

Cyanine 3 labeling (Ub-Cy3)
0.1 mg of ubiquitin (manufactured by Sigma) was labeled with cyanine 3 using a FluoroLink ^™ MAb Cy3 labeling kit (manufactured by Amersham Biosciences). The degree of purification and the amount of labeled ubiquitin were evaluated using UV-Visible spectroscopy according to the manufacturer's protocol.

  The results are shown in FIG. It was confirmed that the transthiolation efficiency was lowest when the four types of E2 examined this time were reacted with cyanine 3-labeled ubiquitin. Direct ubiquitin labeling with fluorescent dyes has been found to critically affect the reaction process. In addition, N-terminally labeled biotinylated ubiquitin was able to obtain a higher signal in UbcH2b than homogenous labeled biotinylated ubiquitin. This interferes with the hypothesis that the 48th lysine residue of ubiquitin is involved in the interaction with UbcH2b. Since this phenomenon was not observed with other enzymes, it was assumed that other residues of ubiquitin are involved in the interaction with other E2. Although the signal level was low in UbcH2b, homogenous-labeled biotinylated ubiquitin did not affect the transthiolation process in the detection of enzymes other than UbcH2b, and more residues were biotinylated in the detection step. Since it was recognized by streptavidin, it was confirmed that a high signal could be obtained.

(Examination example 8)
Experiment on optimization of surface treatment of substrate In the on-chip ubiquitination of 7 types of E2 (UbcH2b, UbcH3, UbcH4, UbcH7, UbcH5b, (C85A) UbcH5b, UbcH5c), the effect of the immobilized surface on the enzyme activity of E2 Various chemical surface treatment methods described below were compared and an attempt was made to construct an optimum substrate surface treatment method.

  The substrate used was a glass slide having a 12 × 4 subarray divided by a Teflon mask and manufactured by either Matsunami Glass Industry or ERI Scientific (USA).

The reaction conditions for examination are as follows.
E1: 100 nM
Ubiquitin: homogenous biotinylated ubiquitin 1 μM
Reaction time: 5 hours

  The surface treatment method studied this time will be described below.

Surface treatment with aldehyde (sometimes abbreviated as aldehyde)
The slide glass made by ERIE scientific was subjected to aldehyde treatment by NoAb Bioscience.
A typical protein displays a large number of lysines on its surface. By functionalizing the surface of the substrate with aldehyde, the lysine residue of the protein and the aldehyde group on the substrate surface are amine-coupled to form a substrate. The protein is immobilized on.

Surface treatment with hydrogel-NHS (may be abbreviated as NHS)
The slide glass was hydrogel coated by NoAb Bioscience (USA-NHS, Jap-NHS, respectively). Hydrogel is a hydrophilic polymer of a water-swelling structure, and in an aqueous medium, hydrogel forms a swollen three-dimensional structure, reducing the possibility of protein denaturation and producing a printed protein. In contrast, it provides a solution-like environment and the three-dimensional structure of the hydrogel can achieve a higher protein density than the plane.

Surface treatment with BSA-NHS (may be abbreviated as BSA-NHS)
PBS (pH 7.6) containing 15 μL 2% BSA (manufactured by Wako Pure Chemical Industries, Ltd.) was applied onto each subarray of hydrogel-NHS functionalized slide glass (NoAb Bioscience, Matsunami Glass Industrial Co., Ltd.) and incubated overnight at room temperature did. The slides were rinsed twice in PBS and twice with distilled water at a later date, then centrifuged at 2000 rpm for 5 minutes to dry the surface, and then placed in a desiccator for 1 hour to completely dry the surface. I put it. A solution containing 100 mM N, N-Disuccinimidyl carbonate (manufactured by Sigma Aldrich) and 100 mM N, N-Diisopropylylamine (manufactured by Wako Pure Chemical Industries, Ltd.) in an array glass as a slide glass Applied and incubated at room temperature for 3 hours. The slide glass was rinsed three times in 100% ethanol (manufactured by Wako Pure Chemical Industries, Ltd.), centrifuged at 2000 rpm for 5 minutes, and stored in a desiccator at room temperature until use. It has been reported that the use of a glass slide functionalized with BSA-NHS (BSA-N-hydroxysuccinimide, BSA: bovine serum albumin) as a substrate has succeeded in producing a protein chip spotted with more than 10,000 proteins. (See Macbeath et al. Science 289: 1760-1763, 2000). This is due to the fact that NHS-activated lysine, aspartic acid, and glutamic acid residues on BSA can react rapidly with primary amines on the printed protein to form a covalent amide bond. The functionalization of BSA-NHS makes it possible to keep the substrate surface hydrated and prevent the spotted protein from being denatured, and the spotted protein can be immobilized by a chemical reaction.

Surface treatment with nickel (may be abbreviated as nickel)
A solution containing 16 mM ABNTA (Aminobutyrophilic Acid: manufactured by Dojin) dissolved in 15 μL of PBS (pH 8) was applied onto each subarray of hydrogel-NHS functionalized glass slide (NoAb Bioscience, manufactured by Matsunami Glass Industrial Co., Ltd.). Incubate overnight at room temperature. Later, it was rinsed 3 times with distilled water and immersed in 0.5 M NiCl (Wako Pure Chemical Industries, Ltd.) for 24 hours at room temperature. The glass slides were rinsed 3 times in distilled water and immersed in 100% ethanol and then centrifuged at 2000 rpm for 5 minutes to dry the surface and stored in a desiccator at room temperature until use. The nickel coating immobilizes proteins on a substrate surface-modified with a nickel complex via an oligohistidine tag, and enables the protein to be aligned and immobilized on the substrate.

The examination results are shown in FIGS. 12A and 12B. FIG. 12A shows a comparison result of enzyme activity when a glass slide manufactured by Matsunami Glass Industry is subjected to surface treatment with hydrogel-NHS, BSA-NHS, and nickel, and FIG. 12B shows ERIE Scientific. The comparison result of the enzyme activity at the time of performing the surface treatment by hydrogel-NHS and an aldehyde to the glass slide manufactured by the company is shown.
From the results of FIG. 12A, it was found that high enzyme activity can be retained when functionalized with hydrogel-NHS, compared to the case where surface treatment was performed with BSA-NHS and nickel. In addition, according to FIG. 12B, when the surface treatment was performed with hydrogel-NHS and when the surface treatment was performed with artehydride, the same enzyme activity was observed in both cases.
Therefore, it has been found that in application in the present invention, it is preferable to functionalize the substrate with hydrogel-NHS or aldehyde.

(Example)
The optimization protocol was clarified from the results of the study in the above study example. The optimization protocol is shown below.

Four types of E2 (UbcH2b, UbcH3, UbcH4, UbcH7), 0.5-2 mg / ml on a slide glass at room temperature using a high-precision inkjet microarrayer (Cartesian Technologies: USA) Microprinting was performed in a 100% humidity atmosphere. As the slide glass, a hydrogel-NHS functionalized glass slide (manufactured by Matsunami Glass Industrial Co., Ltd.) having a 12 × 4 subarray divided by a Teflon mask was used, and E2 was printed on each subarray. The distribution volume was 10 nL per spot, and spotting was performed with an average spot diameter of 250 to 500 μm and an average protein weight of 5 ng / spot (0.25 pmol / spot). After printing, the slides were incubated for 1 hour at room temperature in a water saturated chamber. Next, in order to avoid non-specific binding to the glass substrate surface, a blocking agent (PBS containing 2% BSA, 10 mM DTT (pH 7.6)) was applied to each subarray, incubated at room temperature for 2 hours, and then slide glass Was washed 4 times in PBS (pH 7.6). After washing, 15 μL of UBM (containing 100 nM rabbit E1, 1 μM labeled ubiquitin, 10 mM MgCl ₂ , 2 mM ATP, 0.5 mg / mL BSA) in 50 mM Tris (pH 7.6) is applied to each slide array. Then, it was incubated at 37 ° C. for 5 hours, reacted with E2 immobilized on a glass slide, and ubiquitin was bound to E2 via a thioester bond. As the labeled ubiquitin, biotinylated labeled ubiquitin was used. After the reaction, in order to avoid non-covalently bound molecules on the glass surface, the slide glass was washed by shaking in 50 mL of 50 mM Tris buffer (pH 5.7) containing 0.1% SDS and rinsed 4 times in distilled water. And dried under vacuum. Then, 15 μl of a staining solution containing 20 μg / mL streptavidin-phycoerythrin (manufactured by Molecular Probe) / 5% BSA-containing PBS (pH 7.6) was applied to each subarray, and then incubated at room temperature for 30 minutes. Thereafter, the glass slide was rinsed in 50 mL of PBS (pH 7.6) and washed by shaking for 2 minutes in PBS (pH 7.6) containing 0.05% Tween 20 (ICN Biomed, Inc.). did. And after rinsing carefully in distilled water, it was centrifuged for 5 minutes at 20,000 rpm for uniform drying of the surface. The glass slide was scanned at 550 nm using a WHITE-LIGHT scanner (Applied Precision), the signal was quantitated by IMAGENE ^™ software, and the data was analyzed. The results are shown in FIG.

Looking at FIG. 13, a significant signal relative to background was observed in all E2 families studied. In addition, when the above experiment was performed with a UBM reagent without addition of E1, no signal was detected (FIG. 13, left), so that the covalently bound ubiquitin on E2 is E1-dependent. The signal detected on E2 is confirmed to be due to ubiquitin transferred from E1. Thus, it has been found that this assay can be suitably used to investigate the ubiquitination level of the E2 family.
Although not shown here, no signal was detected in the case of UBM reagent without addition of ATP, so it was confirmed that the ubiquitin activation mechanism of E1 is ATP-dependent.
Then, when the E2-immobilized protein chip is contacted with the UBM, a test substance that may be able to regulate the ubiquitination pathway is added to control the transfer of the test substance from E1 to E2 of ubiquitin. By doing so, it is possible to investigate whether or not it can be used as a modulator that can regulate the ubiquitination pathway. The substance selected and identified as a regulatory substance capable of regulating the ubiquitination pathway can be a therapeutic drug or a preventive drug for diseases associated with abnormalities in the ubiquitination pathway.

(Another embodiment)
Different E2s are placed at different locations on the substrate, but different desired target proteins can be placed at different locations on the substrate.

  Furthermore, it is possible to arrange a plurality of different ubiquitin ligases (E3) at a plurality of different positions on the substrate. (In the case where E3 is included, the result of examining the optimization of E2 is disclosed in this embodiment. However, it is considered necessary to disclose the grounds that this strategy can be applied to E3.) Thus, like the E2-immobilized protein chip, it has a wide utility value such as screening for a preparation that can regulate the ubiquitin pathway.

Diagram showing the ubiquitin pathway The figure which shows the general principle of the analysis method using the protein chip of this invention Diagram showing the results of parameter analysis experiments in the liquid phase Figure showing the results of an optimization study experiment on cleaning conditions Graph showing the results of optimization study on blocking agents Graph showing the results of the study for optimization of contact reaction time The figure which shows the enzyme activity in on-chip ubiquitination of four different E2 The graph which shows the result of optimization examination experiment of E1 and ubiquitin concentration The graph which shows the result of optimization examination experiment of E1 concentration Graph showing the results of an optimization study of ubiquitin concentration Graph showing the results of an experiment for optimizing the labeling method of ubiquitin FIG. 12A is a graph showing the results of an examination for optimizing the substrate surface treatment method, FIG. 12A is a graph showing the results of examination with Matsunami Glass Industrial glass, and FIG. 12B is a graph showing the results of examination with ERIE glass. The figure which shows the result of E2 ubiquitination experiment of an Example

Claims (8)

In order to screen for modulators capable of regulating the ubiquitination pathway, different ubiquitin-binding enzymes (E2) are separately arranged at different positions on the substrate ,
A ubiquitin-binding enzyme (E2) -immobilized protein chip, wherein the enzyme-immobilized substrate is subjected to a blocking treatment with a blocking agent containing 10 to 50 mM DTT .   The ubiquitin-binding enzyme (E2) -immobilized protein chip according to claim 1, wherein the substrate is functionalized with hydrogel N-hydroxysuccinimide or aldehyde. A protein printing step in which a plurality of different ubiquitin-conjugating enzymes (E2) are arranged at a plurality of different positions on a substrate to produce an E2 immobilized protein chip;
A contacting step in which a test substance is brought into contact with the E2 immobilized protein chip in the presence of a biotinylated labeled ubiquitin and a ubiquitination reagent (UBM) containing a ubiquitin activating enzyme (E1);
A washing step of washing the E2-immobilized protein chip to remove the ubiquitination reagent (UBM) after the reaction on the E2-immobilized protein chip and a test substance;
A detection step of detecting a signal of labeled ubiquitin bound to the ubiquitin-conjugating enzyme (E2) on the E2-immobilized protein chip,
Compared with the signal in the ubiquitin-conjugating enzyme (E2) not brought into contact with the test substance, the change in the signal intensity by the test substance for each of the different ubiquitin-conjugating enzymes (E2) is seen. A method for screening a ubiquitination pathway regulatory substance, which is used for screening a predetermined regulatory substance by examining characteristics as a regulatory substance that regulates the ubiquitination pathway by controlling transfer from) to the ubiquitin-binding enzyme (E2). The method for screening a ubiquitination pathway regulator according to claim 3, wherein the substrate is functionalized with hydrogel N-hydroxysuccinimide or aldehyde. The method for screening a ubiquitination pathway regulator according to claim 3 or 4, wherein 1 to 20 µM of the biotinylated labeled ubiquitin is added. The method for screening a ubiquitination pathway regulator according to any one of claims 3 to 5, wherein 2.5 to 250 nM of the ubiquitin activating enzyme (E1) is added. The screening method for a ubiquitination pathway regulator according to any one of claims 3 to 6, wherein the washing step is performed in the presence of SDS. The blocking process which adds a blocking agent is given to the board | substrate before giving the said contact process after the printing process of the said protein, The said blocking agent contains DTT of 10-50 mM in any one of Claims 3-7. Screening method for ubiquitination pathway regulators.