CN108113983A - Application of the natural small molecule compounds in ubiquitin chain synthesis reaction is inhibited - Google Patents
Application of the natural small molecule compounds in ubiquitin chain synthesis reaction is inhibited Download PDFInfo
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- CN108113983A CN108113983A CN201711240534.1A CN201711240534A CN108113983A CN 108113983 A CN108113983 A CN 108113983A CN 201711240534 A CN201711240534 A CN 201711240534A CN 108113983 A CN108113983 A CN 108113983A
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- Prior art keywords
- ubiquitin
- small molecule
- natural small
- reaction
- synthesis reaction
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Links
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- 102000044159 Ubiquitin Human genes 0.000 title claims abstract description 72
- -1 small molecule compounds Chemical class 0.000 title claims abstract description 29
- 238000003786 synthesis reaction Methods 0.000 title claims abstract description 24
- 102100020695 Ubiquitin-conjugating enzyme E2 N Human genes 0.000 claims abstract description 25
- 101710192923 Ubiquitin-conjugating enzyme E2 N Proteins 0.000 claims abstract description 25
- 230000002401 inhibitory effect Effects 0.000 claims abstract description 12
- 230000000694 effects Effects 0.000 claims abstract description 11
- 230000001404 mediated effect Effects 0.000 claims abstract description 8
- MWDZOUNAPSSOEL-UHFFFAOYSA-N kaempferol Natural products OC1=C(C(=O)c2cc(O)cc(O)c2O1)c3ccc(O)cc3 MWDZOUNAPSSOEL-UHFFFAOYSA-N 0.000 claims description 38
- REFJWTPEDVJJIY-UHFFFAOYSA-N Quercetin Chemical compound C=1C(O)=CC(O)=C(C(C=2O)=O)C=1OC=2C1=CC=C(O)C(O)=C1 REFJWTPEDVJJIY-UHFFFAOYSA-N 0.000 claims description 34
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- ZVOLCUVKHLEPEV-UHFFFAOYSA-N Quercetagetin Natural products C1=C(O)C(O)=CC=C1C1=C(O)C(=O)C2=C(O)C(O)=C(O)C=C2O1 ZVOLCUVKHLEPEV-UHFFFAOYSA-N 0.000 claims description 17
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/12—Ketones
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
- A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a kind of application of natural small molecule compounds in ubiquitin chain synthesis reaction is inhibited.Present invention firstly discovers that the natural small molecule compounds of specific structure can inhibit ubiquitin chain synthesis reaction, which has the characteristics of IC50 values are low, and efficiency is high, and specificity is good；In addition, the present invention also proves that natural small molecule compounds can inhibit the ubiquitin ammonolysis reaction that desmoenzyme Ubc13 is mediated by inhibiting the activity of ubiquitin binding enzyme Ubc13 by the external ubiquitination of parsing step by step reaction and intracellular ubiquitination reaction experiment.
Description
Technical Field
The invention relates to the field of biotechnology and medicine, in particular to application of natural small molecular compounds in inhibition of ubiquitin chain synthesis reaction.
Background
Ubiquitination is achieved by the Ubiquitin protein itself as a signal molecule via the Ubiquitin activating enzyme (Ubiquitin-
The ubiquitination modification of the protein is achieved by covalently modifying the lysine residues of the substrate protein by a cascade of activating Enzyme) E1, Ubiquitin-conjugating Enzyme (Ubiquitin-conjugating Enzyme) E2 and Ubiquitin Ligase (Ubiquitin ligand) E3.
A single ubiquitin molecule modifies a single lysine residue on a substrate protein, resulting in a monoubiquitination modification. Multiple ubiquitin molecules modify multiple lysine residues on the substrate protein, which can form polyubiquitination modification. The ubiquitin protein body has 7 lysine residues of K48, K63, K11, K6, K29, K27 and K33, and the lysine residues of the ubiquitin molecule which is covalently modified on the substrate protein can also continue to undergo ubiquitination modification to form an ubiquitin chain. In addition, the free amino group at the N terminal of the ubiquitin molecule can be connected with the carboxyl group at the C terminal of another ubiquitin molecule through ubiquitination reaction to form a linear ubiquitin chain.
Thus, monoubiquitination, polyubiquitination, and 8 different forms of homoubiquitin chain modification can occur in a cell. The complexity of ubiquitin chains is further increased if one ubiquitin chain binds to a different lysine residue of ubiquitin itself, forming a mixed or branched ubiquitin chain.
In most cases, the synthesis of K63-linked polyubiquitin chains requires the formation of a heterodimer of another protein subunit Uev with ubiquitin-binding enzyme Ubc 13. E1 catalyzes the transthioesterification reaction to covalently attach donor ubiquitin to the cysteine thiol group of the active site of Ubc13, after which a second ubiquitin monomer is non-covalently bound to Uev with the lysine (K63) side chain at position 63 near the thioester bond formed between the donor ubiquitin and Ubc 13. K63 is thus able to attack thioester bonds to transfer donor ubiquitin from the active site of Ubc13 to lysine side chain 63 of recipient ubiquitin, forming isopeptide bonds linking two ubiquitin molecules, and through multiple cycles, K63 linked ubiquitin chains can be formed. The K63 ubiquitin chain is mainly involved in non-proteolytic signal pathways such as regulation of DNA damage repair, mitochondrial inheritance, regulation of ribosome and NF-kB signal pathways and the like in mammalian cells, and is related to hereditary Parkinson's disease. Abnormal regulation of the K63 ubiquitin chain can cause various diseases.
In recent years, the importance of ubiquitination modification in life activities has been increasingly focused and demonstrated. However, as the research progresses, more biological problems of ubiquitin become apparent, and the research is awaited by the scientific families of multiple disciplines. In the future, all ubiquitin modifying enzymes are expected to be identified, molecular signals involved in ubiquitination and biological functions thereof are disclosed, and a tandem interaction network of ubiquitination and other post-translational modifications is established, so that ubiquitination targets of key proteins related to certain diseases are found, single-target or multi-target medicines are developed, and the method contributes to human overcoming of diseases which cannot be treated at present.
Currently, only proteasome inhibitor PS34(Valcade) and inhibitor lenalidomide or thalidomide of E3 ubiquitin ligase CRL4CRBN enter clinical application, but the drugs affect more cell signal pathways, are not ideal in specificity and have higher cytotoxicity.
Thus, the development of highly effective clinical drugs against ubiquitination systems is a current challenge.
Disclosure of Invention
The invention provides an application of a natural small molecular compound in inhibition of ubiquitin chain synthesis reaction, the natural small molecular compound can inhibit ubiquitin chain synthesis reaction, and the natural small molecular compound has the characteristics of low IC50 value, high efficiency and good specificity.
The invention provides an application of a natural small molecular compound in inhibiting ubiquitin chain synthesis reaction, wherein the structural formula of the natural small molecular compound is as follows:
wherein R is1Is H or OH; r2Is H or OH; r3Is H or OH; r4Is H or OH; r5Is H or OH;
or,
wherein R is6Is H or OH; r7Is H or OH; r8Is H or OH; r9Is H or OH; r10Is H or OH;
or,
wherein R is11Is H or OH; r12Is H or OH;
or,
preferably, the natural small molecule compound is quercetin, luteolin, myricetin, fisetin, morin, kaempferol, apigenin, butein, chrysoeriol chalcone, anthocyanin or epigallocatechin gallate.
Wherein the structural formula of quercetin, luteolin, myricetin, fisetin, morin, kaempferol and apigenin belongs to formula (I); structural formulas of butein and chamomile chalcone belong to formula (II); the structural formula of the anthocyanin belongs to the formula (III); the structural formula of the epigallocatechin gallate is shown as a formula (IV). Specifically, the structural formula of quercetin is as follows:the structural formula of luteolin is as follows:
the structural formula of myricetin is as follows:
the structural formula of fisetin is:
the structural formula of morin is as follows:
the kaempferol has a structural formula as follows:
the structural formula of apigenin is as follows:
the structural formula of the butein is as follows:
the structural formula of the calliopsis chalcopyrite chalcone is as follows:
the structural formula of the anthocyanin is as follows:
the structural formula of epigallocatechin gallate is as follows:
experiments show that the natural small molecule compound realizes the inhibition of K63 ubiquitin chain synthesis reaction mediated by ubiquitin conjugated enzyme Ubc13 by inhibiting the activity of ubiquitin conjugated enzyme Ubc 13.
Therefore, further, the ubiquitin chain synthesis reaction is a K63 ubiquitin chain synthesis reaction mediated by ubiquitin conjugating enzyme Ubc 13.
Further, in the ubiquitin chain synthesis reaction system, the concentration of the ubiquitin conjugated enzyme Ubc13 is 0.1-1 mu M; the concentration of the natural small molecular compound is 0.1-100 mu M.
Specifically, the concentration of the natural micromolecular compound belonging to the formulas (I-III) is 1-100 mu M; and the concentration of the natural small molecular compound belonging to the formula (IV) is 0.1-3 mu M.
Further, the natural small molecule compound inhibits K63 ubiquitin chain synthesis reaction by inhibiting ubiquitin conjugating enzyme Ubc13 mediated ammonolysis reaction.
The invention proves that the natural small molecular compound can inhibit the activity of ubiquitin conjugating enzyme Ubc13 and further inhibit ubiquitin ammonolysis reaction mediated by the conjugating enzyme Ubc13 through in-vitro step-by-step analytic ubiquitination reaction and intracellular ubiquitination reaction experiments.
The invention also provides an application of the natural small molecule compound in inhibiting the activity of ubiquitin conjugated enzyme Ubc13, wherein the structural formula of the natural small molecule compound is shown as follows:
wherein R is1Is H or OH; r2Is H or OH; r3Is H or OH; r4Is H or OH; r5Is H or OH;
or,
wherein R is6Is H or OH; r7Is H or OH; r8Is H or OH; r9Is H or OH; r10Is H or OH;
or,
wherein R is11Is H or OH; r12Is H or OH;
or,
further, the natural small molecule compound is quercetin, luteolin, myricetin, fisetin, morin, kaempferol, apigenin, butein, chamomile chalcone, anthocyanidin or epigallocatechin gallate. The structural formula of the natural small molecule compound is presented in the content.
Compared with the prior art, the invention has the following beneficial effects:
the invention discovers for the first time that the natural small molecular compound with a specific structure can inhibit the ubiquitin chain synthesis reaction, and the natural small molecular compound has the characteristics of low IC50 value, high efficiency and good specificity; in addition, the invention also proves that the natural small molecule compound can inhibit ubiquitin ammonolysis reaction mediated by a conjugating enzyme Ubc13 by inhibiting the activity of ubiquitin conjugating enzyme Ubc13 through in vitro step-by-step analytic ubiquitination reaction and intracellular ubiquitination reaction experiments.
Drawings
FIG. 1 is the electrophoresis chart of the ubiquitin chain synthesis reaction detecting ubiquitin protein in example 2.
FIG. 2 is a graph showing the results of Surface Plasmon Resonance (SPR) measurement of the interaction between quercetin and ubiquitin-binding enzyme in example 2;
wherein A is an interaction curve measured by an SPR instrument; and B is a curve which is subjected to nonlinear fitting by using single-point reading and a corresponding obtained binding dissociation equilibrium constant KD.
FIG. 3 is a graph showing the results of Surface Plasmon Resonance (SPR) detection of the interaction of luteolin with ubiquitin binding enzyme in example 2;
wherein A is an interaction curve measured by an SPR instrument; and B is a curve which is subjected to nonlinear fitting by using single-point reading and a corresponding obtained binding dissociation equilibrium constant KD.
FIG. 4 is a graph showing the results of Surface Plasmon Resonance (SPR) detection of the interaction between morin and ubiquitin-binding enzyme in example 2.
FIG. 5 is a graph showing the results of Surface Plasmon Resonance (SPR) detection of the interaction between epigallocatechin gallate and ubiquitin conjugating enzyme in example 2.
FIG. 6 is a graph showing the results of measuring the semi-inhibitory concentration (IC50) of quercetin in the binding-to-epiubiquitination reaction in example 2;
wherein, A is the result of detecting ubiquitination reaction products by Western Blot; and B is a curve obtained by fitting calculation after quantitative analysis is carried out on the Western Blot result graph and a corresponding IC50 value.
FIG. 7 is a graph showing the results of determining the half inhibitory concentration (IC50) of luteolin in the binding-ubiquitination reaction of example 2;
wherein, A is the result of detecting ubiquitination reaction products by Western Blot; and B is a curve obtained by fitting calculation after quantitative analysis is carried out on the Western Blot result graph and a corresponding IC50 value.
FIG. 8 is a graph showing the results of determining the half inhibitory concentration (IC50) of epigallocatechin gallate in the case of the conjugation-deubiquitination reaction in example 2;
wherein, A is the result of detecting ubiquitination reaction products by Western Blot; and B is a curve obtained by fitting calculation after quantitative analysis is carried out on the Western Blot result graph and a corresponding IC50 value.
FIG. 9 is a graph showing the results of Western Blot analysis of the products of the ammonolysis reaction after addition of quercetin in example 3;
wherein, A is the result of Western Blot detection of the ammonolysis reaction product catalyzed only by Ubc 13; b is the result of Western Blot detection of the product of the ammonolysis reaction catalyzed by Ubc13 in the presence of E3 ubiquitin ligase TRAF 6.
FIG. 10 is a graph showing the Western Blot detection of the aminolysis reaction product after addition of luteolin in example 3;
wherein, A is the result of Western Blot detection of the ammonolysis reaction product catalyzed only by Ubc 13; b is the result of Western Blot detection of the product of the ammonolysis reaction catalyzed by Ubc13 in the presence of E3 ubiquitin ligase TRAF 6.
FIG. 11 shows the Western Blot detection result of the aminolysis reaction product catalyzed by Ubc13 in the presence of E3 ubiquitin ligase TRAF6 after addition of epigallocatechin gallate in example 3.
FIG. 12 is a graph showing the results of Western Blot detection using non-reducing SDS-PAGE after quercetin treatment in example 3.
FIG. 13 is a graph showing the results of Western Blot detection by non-reducing SDS-PAGE after treatment with epigallocatechin gallate in example 3.
Detailed Description
EXAMPLE 1 preparation of each starting Material in ubiquitin chain Synthesis reaction
1. Purification of ubiquitin (Ub)
The unlabeled ubiquitin cDNA was constructed on prokaryotic expression vector pET14 b. In transformation of Escherichia coli
(BL21(DE3)/pJY2 strain), expression was induced with 0.5mM IPTG at 37 ℃ for 4 hours. 1L of E.coli was collected, centrifuged, the supernatant was discarded, resuspended in 20ml of buffer (50mM Tris-Cl pH7.6,1mM PMSF, 0.02% (v/v) NP-40,0.4mg/ml lysozyme), and subjected to ultrasonic lysis. After 20 minutes of centrifugation at 20000g, the supernatant is transferred to a clean container, 70% perchloric acid is slowly added dropwise under stirring at 4 ℃, most of the impure proteins are denatured and precipitated, and stirring is continued until the pH reaches about 4.0. After 30 minutes, 20000g were centrifuged for 20 minutes and the supernatant was collected. Dialyzed twice against 100 volumes of ammonium acetate buffer, pH 4.5. Then, after the cation exchange column was equilibrated with ammonium acetate buffer solution of pH4.5, the dialyzed protein supernatant was passed through and bound to the column, followed by gradient elution with ammonium acetate buffer solution of pH4.5 containing 0 to 500mM NaCl. Collecting ubiquitin protein-containing fraction, dialyzing in 100 times volume of deionized water for 2-3 times, ultrafiltering, concentrating to appropriate volume, packaging, and storing at-20 deg.C.
2. Purification of E2 ubiquitin-conjugating enzyme Ubc13-Uev2
His-tagged Ubc13, Uev2 protein cDNA was constructed on pET15b prokaryotic expression vector. After transformation of E.coli (BL21(DE3)/pLys strain), respectively, expression was induced with 0.2mM IPTG overnight at room temperature. After 1L of E.coli was centrifuged and the supernatant was discarded, it was resuspended in 20ml of buffer I (20mM Tris-Cl pH8.0,300mM NaCl,1mM PMSF, 0.5% (V/V) NP-40,20mM imidazole) and subjected to ultrasonication. After 20min centrifugation at 20000g, the supernatant was transferred to a new 50ml centrifuge tube. 1mL of Ni-NTA beads (from Thermo Fisher Scientific) were added and bound on a 4 ℃ tumbling shaker for 1-2 hours, the mixture containing the beads was loaded into an empty chromatography column of appropriate size, and after the cell lysate had flowed out, the beads were left in the column, followed by washing with buffer I, buffer II (20mM Tris-Cl pH8.0,300mM NaCl,1mM PMSF,20mM imidazole) for 30 column volumes in sequence, and finally eluted with an eluent (20mM Tris-Cl pH8.0,300mM NaCl,1mM PMSF,250mM imidazole).
Equimolar amounts of His-Ubc13 and His-Uev2 protein were mixed and left at 4 ℃ for 1 hour. After the cation exchange column was equilibrated with buffer III (20mM HEPES pH7.4, 10% (v/v) glycerol), the Ubc13-Uev2 protein mixture was passed through and bound to the column, followed by gradient elution with buffer III containing 0-300mM NaCl. Fractions containing the Ubc13-Uev2 binary protein complex were collected. Finally, ultrafiltering and concentrating to a proper volume, subpackaging and storing at-80 ℃.
3. Purification of E1 ubiquitin activating enzyme and E3 ubiquitin ligase TRAF6
His-tagged E1 and TRAF6 protein cDNAs were constructed on pFastBac-HT-A vector. Protein expression was carried out in SF-9 cells by a baculovirus expression system (baculovirus expression system). 100ml of SF-9 cells were centrifuged to collect the supernatant, which was then resuspended in 20ml of buffer I (20mM Tris-Cl pH8.0,300mM NaCl,1mM PMSF, 0.5% (V/V) NP-40,20mM imidazole) and subjected to ultrasonic lysis. After centrifugation at 20000g for 20 minutes, the supernatant was filtered through a 0.45 μm pore filter and transferred to a new 50ml centrifuge tube. 1mL of Ni-NTA beads (from Thermo Fisher Scientific) were added, and the mixture was loaded onto an empty chromatography column of appropriate size, after the cell lysate had flowed out, the beads were left in the column for 1-2 hours by tumbling on a shaker at 4 ℃, followed by washing 30 column volumes with buffer I and buffer II (20mM Tris-Cl pH8.0,300mM NaCl,1mM PMSF,20mM imidazole), and then eluting with an eluent (20mM Tris-Cl pH8.0,300mM NaCl,1mM PMSF,250mM imidazole). The protein-containing eluate is ultrafiltered and replaced into preservation buffer (20mM HEPES pH7.4, 10% (v/v) glycerol) to dilute the original elution buffer by over 200 times, and finally ultrafiltered and concentrated to appropriate volume, subpackaged, and preserved at-80 deg.C.
4. Purification of reaction intermediates Ub-Ubc 13/Uev2
1ml of the reaction mixture (20mM HEPES pH7.4, 5mM magnesium chloride, 2mM ATP, 0.5mM DTT, 0.2. mu. M E1, 5. mu.M Ubc13/Uev2, 10. mu.M Ub) was prepared, 200. mu.l of the reaction mixture was dispensed into a 1.5ml ion tube, and after reaction at 30 ℃ for 10min, 5 volumes of 4 ℃ precooled buffer A (20mM HEPES pH7.4, 10% glycerol) were added, and the mixture was centrifuged at 20000g for 20min to remove the precipitate, and the supernatants were combined. In a cold chamber at 4 ℃ in GEThe reaction product was bound to a 1ml HiTrap Q HP anion exchange column under the control of a conventional rapid protein liquid chromatograph of Purifier, followed by continuous gradient elution using buffer A and buffer B (20mM HEPES pH7.4, 10% glycerol, 1M sodium chloride), and fractions containing reaction intermediates Ub to Ubc13/Uev2 were collected, concentrated by ultrafiltration at 4 ℃ and dispensed, and stored at-80 ℃.
Example 2
1. Ubiquitin chain Synthesis reaction
The following reaction system was prepared:
setting a control group and a 11 group sample group; the control group also contained 5% DMSO, and the sample groups also contained natural small molecule compounds (purchased from Selleck, inc., dissolved in DMSO) that were quercetin (sample group 1), luteolin (sample group 2), myricetin (sample group 3), fisetin (sample group 4), morin (sample group 5), kaempferol (sample group 6), apigenin (sample group 7), anthocyanidin (sample group 8), butein (sample group 9), parthenolide (sample group 10), and epigallocatechin gallate (sample group 11), respectively.
After the sample group and the control group were reacted at 30 ℃ for 30 minutes, SDS-PAGE sample buffer was added to terminate the reaction, followed by electrophoresis and Western Blot to detect ubiquitin proteins, the detection results are shown in FIG. 1.
2. Surface Plasmon Resonance (SPR) detection of the interaction of natural small molecule compounds with ubiquitin-conjugating enzymes was detected in a Biacore T200 instrument from GE at 25 ℃. Ubc13-Uev2 complex protein was first immobilized on a matched CM7 chip according to the protocol provided by GE.
The native small molecule compound (this experiment) was dissolved in working buffer (10mM HEPES pH7.4,150 mM NaCl,3mM EDTA, 0.05% Surfactant P20and 5% DMSO) to make up a total of 8 concentrations of buffer from 100. mu.M to 0.78. mu.M in a 2-fold dilution gradient. Buffers containing different concentrations of small molecule compounds were flowed through the detection cell at a flow rate of 30 μ l/min according to the time program of 60 second pre-run, 60 second binding, and 180 second dissociation.
At the end of each cycle, regeneration was performed with regeneration buffer (Glycine-HCl pH 2.2). The detection result is recorded by matched software and processed by data. The detection results of natural small molecule compounds quercetin, luteolin, morin and epigallocatechin gallate are shown in fig. 2-5.
3. Determination of semi-inhibitory concentration IC50 of Natural Small molecule Compounds in combination with in vitro ubiquitination reactions
Refer to the first step of the example for the implementation of the reaction of ubiquitin chain synthesis. The control group also contained 5% DMSO, and the sample group also contained a series of concentration gradients of natural small molecule compounds (purchased from Selleck corporation, dissolved in DMSO) which were quercetin (sample group 1), luteolin (sample group 2), or epigallocatechin gallate (sample group 3).
After the sample group and the control group were left to react at 30 ℃ for 30 minutes, SDS-PAGE sample buffer was added to terminate the reaction, followed by electrophoresis and Western Blot to detect ubiquitin proteins. Results of Western blot signal intensity of immunoblots was quantified using Image J software, followed by nonlinear fitting (log (inhibitor) vs. normalized response — Variable slope option) using GraphPad Prism software with the compound concentration as the abscissa and the immunoblot signal intensity as the ordinate, thereby calculating the half inhibitory concentration of each set of natural small molecule compound samples. The results are shown in FIGS. 6 to 8.
Example 3
1. The experiment for synthesizing ubiquitin chain by using in vitro ubiquitin ammonolysis reaction shows that the natural small molecular compound inhibits the enzyme activity of Ubc13
The following reaction system was prepared:
setting a control group and a sample group; wherein the control group further contains 5% DMSO, and the sample group further contains natural small molecule compounds (dissolved in DMSO), which are quercetin (sample group 1), luteolin (sample group 2), and epigallocatechin gallate (sample group 3). The same group of samples are set with or without 10nM TRAF6 protein, and reacted at 30 deg.c for 5 min to 4 hr, and the reaction product is separated through SDS-PAGE electrophoresis and then WesternBlot is used to detect ubiquitin protein. The results are shown in FIGS. 9 to 11.
As shown in FIGS. 9-11, quercetin, luteolin and epigallocatechin gallate all can inhibit the enzyme activity of Ubc 13.
2. Non-reducing SDS-PAGE is utilized to show that the natural small molecular compound inhibits the intracellular Ubc13 ubiquitin ammonolysis reaction activity
Adherent cultured cells were seeded onto the culture plate. mu.M quercetin or 150. mu.M epigallocatechin gallate was added to the medium and maintained for 0 to 12 hours. After the small molecule compound treatment, the cells were collected at the indicated time points, lysed with cell lysis buffer (50mM Tris-Cl pH7.4,150 mM NaCl, 10% glycerol, 1% NP-40), centrifuged at 20000g for 10 minutes, the supernatant collected, and SDS-PAGE sample buffer (without reducing agent) added. After separation by SDS-PAGE, the samples were examined by Western Blot for Ubc13, UbcH7, UbcH5c and beta-Actin. The results are shown in FIGS. 12 and 13.
As shown in fig. 12 and 13, quercetin and epigallocatechin gallate both specifically inhibited the ubiquitin aminolysis activity of intracellular Ubc13, while other E2 ubiquitin conjugating enzymes such as UbcH7 and UbcH5c were unaffected.
Claims (7)
1. The application of a natural small molecule compound in inhibiting ubiquitin chain synthesis reaction is characterized in that the structural formula of the natural small molecule compound is as follows:
wherein R is1Is H or OH; r2Is H or OH; r3Is H or OH; r4Is H or OH; r5Is H or OH;
or,
wherein R is6Is H or OH; r7Is H or OH; r8Is H or OH; r9Is H or OH; r10Is H or OH;
or,
wherein R is11Is H or OH; r12Is H or OH;
or,
。
2. the use of claim 1, wherein the natural small molecule compound is quercetin, luteolin, myricetin, fisetin, morin, kaempferol, apigenin, butein, camomile chalcone, anthocyanins, or epigallocatechin gallate.
3. The use according to any of claims 1 or 2, wherein the ubiquitin chain synthesis reaction is the K63 ubiquitin chain synthesis reaction mediated by ubiquitin conjugating enzyme Ubc 13.
4. The use according to claim 3, wherein in the ubiquitin chain synthesis reaction system, the concentration of the ubiquitin conjugating enzyme Ubc13 is 0.1-1 μ M; the concentration of the natural small molecular compound is 0.1-100 mu M.
5. The use according to claim 3, wherein the natural small molecule compound inhibits the K63 ubiquitin chain synthesis reaction by inhibiting the ubiquitin conjugating enzyme Ubc13 mediated aminolysis reaction.
6. The application of a natural small molecule compound in inhibiting the activity of ubiquitin conjugated enzyme Ubc13 is characterized in that the structural formula of the natural small molecule compound is as follows:
wherein R is1Is H or OH; r2Is H or OH; r3Is H or OH; r4Is H or OH; r5Is H or OH;
or,
wherein R is6Is H or OH; r7Is H or OH; r8Is H or OH; r9Is H or OH; r10Is H or OH;
or,
wherein R is11Is H or OH; r12Is H or OH;
or,
。
7. the use of claim 6, wherein the natural small molecule compound is quercetin, luteolin, myricetin, fisetin, morin, kaempferol, apigenin, butein, camomile chalcone, anthocyanins, or epigallocatechin gallate.
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