Nanobret-based protein ubiquitination degradation promoting drug screening system and method
阅读说明：本技术 基于nanoBRET的促蛋白泛素化降解药物筛选系统及方法 (Nanobret-based protein ubiquitination degradation promoting drug screening system and method ) 是由 王自峰 刘强 唐照 雷鑫星 ***·卡姆兰·拉贾 刘芳 刘美玲 张海亮 于 2019-10-23 设计创作，主要内容包括：本发明公开了基于nanoBRET的促蛋白泛素化降解药物筛选系统及方法。筛选系统包括：荧光蛋白修饰的泛素表达质粒,用于表达荧光蛋白修饰的泛素；内源性泛素基因UBB及UBC敲除的细胞系；以及靶蛋白-荧光素酶融合蛋白。本发明的一些实例,避免了每次检测前18小时加入小分子荧光底物,同时也避免了小分子荧光底物对细胞造成的毒性影响,使用方便,可以实时监测活细胞中靶蛋白的泛素化水平,结果更加可靠,可以直观反映待筛选化合物对靶蛋白的影响,利于促蛋白泛素化降解药物的高通量筛选。(The invention discloses a screening system and a screening method of a protein ubiquitination degradation promoting drug based on nanoBRET. The screening system includes: a fluorescent protein modified ubiquitin expression plasmid for expressing fluorescent protein modified ubiquitin; cell lines with endogenous ubiquitin gene UBB and UBC knockouts; and a target protein-luciferase fusion protein. According to some embodiments of the invention, the addition of the small-molecule fluorescent substrate 18 hours before each detection is avoided, and the toxic influence of the small-molecule fluorescent substrate on cells is also avoided, so that the method is convenient to use, can monitor the ubiquitination level of the target protein in living cells in real time, has a more reliable result, can visually reflect the influence of the compound to be screened on the target protein, and is beneficial to high-throughput screening of the protein ubiquitination degradation promoting drug.)
1. A screening system for a protein ubiquitination degradation drug based on bioluminescence resonance energy transfer comprises:
a fluorescent protein modified ubiquitin expression plasmid for expressing fluorescent protein modified ubiquitin;
cell lines with endogenous ubiquitin gene UBB and UBC knockouts; and
target protein-luciferase fusion protein.
2. The screening system for a pro-protein ubiquitination degradation drug according to claim 1, wherein: the fluorescent protein is a long stokes shift protein LSSmorange.
3. The screening system for a pro-protein ubiquitination degradation drug according to claim 1, wherein: the ubiquitin includes wild-type and mutant ubiquitin.
4. The screening system for a pro-protein ubiquitination degradation drug according to claim 3, wherein: the mutant ubiquitin comprises ubiquitin of K6, K11, K27, K29, K33, K48 and K63 types.
5. The screening system for a pro-protein ubiquitination degradation drug according to claim 1, wherein: the target protein-luciferase fusion protein is introduced as an expression plasmid.
6. The system for screening a drug for promoting ubiquitination degradation of protein according to claim 1 or 5, wherein: the luciferase is
7. The screening system for a pro-protein ubiquitination degradation drug according to any one of claims 1 to 5, wherein: the protein ubiquitination degradation drug screening system is also added with a proteasome inhibitor.
8. The screening system for a pro-protein ubiquitination degradation drug according to claim 1, wherein: the proteasome inhibitor is selected from MG 132.
9. The screening system for a pro-protein ubiquitination degradation drug according to any one of claims 1 to 5, wherein: the fluorescent protein is modified at the N-terminus of ubiquitin.
10. A screening method of a drug for promoting ubiquitination degradation of protein, wherein the screening system is as defined in any one of claims 1 to 9, and the screening step comprises:
1) transferring the ubiquitin expression plasmid modified by the fluorescent protein, the target protein-luciferase fusion protein or the expression plasmid thereof into an endogenous ubiquitin gene UBB and a UBC knockout cell line and expressing;
2) adding a compound to be screened and a luciferase substrate, and detecting the fluorescence value after the action;
3) comparing the difference in fluorescence values of the different treatments to determine whether the compound and/or the target protein can promote ubiquitination degradation of the protein.
Technical Field
The invention relates to a drug screening system and a drug screening method.
Background
Ubiquitin-proteasome system (UPS) is the major pathway for intracellular protein degradation, involving more than 80% of intracellular protein degradation. The system comprises ubiquitin (Ub), ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2), ubiquitin ligase (E3), proteasome and its substrate (protein). Wherein:
① ubiquitin contains 76 amino acid residues, has molecular weight of about 8.5kDa, and is widely present in eukaryotic cells, the combination of ubiquitin chains and protein substrates forms a recognition signal degraded by proteasomes, and in addition, ubiquitination has a target localization function in protein endocytosis and exocytosis.
The ubiquitin molecule contains 7 lysine sites (K6, K11, K27, K29, K33, K48 and K63) in its entire length, 1 glycine (Gly) site at the C-terminus and a methionine (Met1) site at the N-terminus. According to the existing research, in both intracellular environment and extracellular reaction system, ubiquitination can occur at each lysine site of ubiquitin itself and methionine (Met1) site at N-terminal to extend ubiquitin chain. Among them, polyubiquitination at positions K48 and K63 is the most widely studied, while other types of ubiquitinated chains are less studied and are considered to be atypical ubiquitination. However, with the intensive study on the assembly, recognition and hydrolysis of ubiquitinated chains, the function and significance of atypical ubiquitination is gradually gaining importance.
② ubiquitin activating enzyme E1 ubiquitin in the E1-ubiquitin intermediate can be transferred to several E2 by the thioester bond of a cysteine residue with a C-terminal activated glycine residue of ubiquitin.
③ ubiquitin conjugated enzyme E2, which acts in the form of ubiquitin conjugated enzyme, with cysteine as the active site, part of the E2 members play a role in cell-specific processes, but the full role of E2 is not clear.
④ ubiquitin ligase E3, a key element of the selective degradation mechanism of the ubiquitin-proteasome system, recognizes degraded proteins and ligates ubiquitin to a substrate.
⑤ proteasome (2.5MDa), a barrel structure composed of 2 19S and 1 20S subunits, 19S is a regulatory subunit, located at both ends of the barrel structure, recognizing polyubiquitinated protein and unfolding it, a deubiquitinated isopeptidase is also located on the 19S subunit, the substrate deubiquitinating 20S is a catalytic subunit, located in the middle of the two 19S subunits, and its active site is located on the inner surface of the barrel structure, which can avoid the influence of cell environment.
Degradation of proteins by the ubiquitin-proteasome system is a multi-step process involving a variety of different proteins. Proteins that need to be degraded by the proteasome are first tagged with ubiquitin, i.e., a covalent link is formed between a lysine on the protein and ubiquitin. This process is a three-enzyme cascade, i.e., a series of reactions need to occur catalyzed by three enzymes, and the entire process is called the ubiquitination signaling pathway. In the first reaction step, ubiquitin activating enzyme (also known as E1) hydrolyzes ATP and adenylates one ubiquitin molecule. Ubiquitin is then transferred to the cysteine residue of the active center of E1, accompanied by adenylation of a second ubiquitin molecule. The adenylated ubiquitin molecule is then transferred to the cysteine residue of a second enzyme, ubiquitin cross-linking enzyme (E2). Finally, a member of the highly conserved ubiquitin ligase (E3) family (which varies depending on the substrate protein) recognizes specific target proteins that need to be ubiquitinated and catalyzes the transfer of ubiquitin molecules from E2 to the target proteins. The target protein must be labeled with at least four ubiquitin monomer molecules (in the form of polyubiquitin chains) before being recognized by the proteasome. Thus, it is E3 that makes this system substrate specific. The amounts of E1, E2 and E3 proteins depend on the organism and cell type, and a large number of different E3 proteins are present in humans, suggesting that the ubiquitin-proteasome system can act on a large number of target proteins. The ubiquitin receptor protein has a ubiquitin-like domain at the N-terminus and one to more ubiquitin binding domains. The ubiquitin-like domain can be recognized by the 19S regulatory particle, while the ubiquitin-binding domain can bind ubiquitin by forming a triple helix bundle. These receptor proteins may be able to bind polyubiquinated proteins and carry them to the proteasome for degradation.
The ubiquitin-proteasome system is an important regulatory means of a series of life processes in cells. In cells, most proteins are broken down by the ubiquitin-proteasome system. The ubiquitin-proteasome system plays two main roles: one is by breaking down abnormal or damaged proteins to maintain the quality of the cells; secondly, the basic life activity of the cells is controlled by decomposing proteins with specific functions; both of them finally ensure the normal function of tissues and organs. The ubiquitin-proteasome system participates in the growth, differentiation, DNA replication and repair, cell metabolism, immune reaction and other important physiological and biochemical processes of cells.
The whole ubiquitin-proteasome system relates to a plurality of control nodes, and when the control nodes are in a normal state, the decomposition of various proteins in cells is always maintained in a dynamic equilibrium state on the principle of ensuring the efficient exertion of various functions of the body. The functional disorder of ubiquitin-proteasome system refers to the most intrinsic disorder of a series of external diseases caused by the imbalance of intracellular protein metabolism after the dynamic balance is broken. Drug design directed to the ubiquitin-proteasome system may bring new advantages for drug development.
In basic research and drug development, it is often necessary to detect the level of ubiquitination modification of intracellular proteins, including wild-type ubiquitination modification (WT), and various specific ubiquitination modifications (K6, K11, K27, K29, K33, K48, and K63). The existing detection technology mainly adopts a Western blot technology to carry out semi-quantitative detection. The technology needs to crack cells to extract protein, then carries out electrophoresis and immune hybridization, and has long time consumption and low detection flux. The prior art lacks an effective screening system for promoting protein ubiquitination degradation drugs.
Bioluminescence Resonance Energy Transfer (BRET) is a technique for detecting intracellular protein interactions. When two protein molecules interact, the spectrum of a luminescent signal generated by the protease reaction of the donor luciferase expressed by fusion of one protein is overlapped with the excitation spectrum of the fluorescent group of the receptor marked by the other protein, so as to induce the fluorescence of the receptor molecule.
The traditional BRET platform uses renilla luciferase (RLuc) as an energy donor and Yellow Fluorescent Protein (YFP) as an energy acceptor. Compared with the Fluorescence Resonance Energy Transfer (FRET) technology, the BRET technology has the advantages of no need of exciting light, no need of bleaching, less damage to cells and wider data window for detecting protein interaction. However, this system has two disadvantages: 1) RLuc light output is too low, resulting in low BRET efficiency and difficulty in detection. 2) RLuc and YFP are very close in spectrum (i.e., high spectral overlap) and therefore produce a high background that increases detection noise and also reduces sensitivity and dynamic range.
In view of the above disadvantages, the BRET process was greatly improved by Promega. The method mainly comprises the following steps: 1) use of
Despite the great advances in NanoBRET, there are also significant disadvantages: nanobretTMThe 618 fluorophore is a proprietary patent of promega corporation and is expensive. And the technique needs to add NanoBRET 18 hours ahead of timeTM618 fluorophore substrate, with some toxicity to cells.
Disclosure of Invention
The invention aims to provide a system and a method for screening a protein ubiquitination degradation promoting drug based on fluorescence.
The technical scheme adopted by the invention is as follows:
in a first aspect of the present invention, there is provided:
a screening system for a protein ubiquitination degradation drug based on bioluminescence resonance energy transfer comprises:
a fluorescent protein modified ubiquitin expression plasmid for expressing fluorescent protein modified ubiquitin;
cell lines with endogenous ubiquitin gene UBB and UBC knockouts; and
target protein-luciferase fusion protein.
In some examples of a profibiquitinizing degradation drug screening system, the fluorescent protein is the long stokes shift protein lssmoorange.
In some examples of the profibiquitinization degradation drug screening system, the ubiquitin includes wild-type and mutant ubiquitin.
In some examples of the screening system for protein ubiquitination degradation drugs, the mutant ubiquitins include ubiquitin types K6, K11, K27, K29, K33, K48, and K63.
In some examples of the profibiquitinization degradation drug screening system, the target protein-luciferase fusion protein is introduced as an expression plasmid.
In some examples of the profibiquitinizing degradation drug screening system, the luciferase isluciferase。
In some examples of the screening system for a pro-protein ubiquitination degradation drug, the screening system for a pro-protein ubiquitination degradation drug is further added with a proteasome inhibitor.
In some examples of the screening system for a profibiquitinizing degradation drug, the proteasome inhibitor is selected from MG 132.
In some examples of the profibiquitinization degradation drug screening system, the fluorescent protein is modified at the N-terminus of ubiquitin.
In a second aspect of the present invention, there is provided:
a screening method of a drug for promoting ubiquitination degradation of protein, the screening system of the invention is described in the first aspect, and the screening step comprises:
1) transferring the ubiquitin expression plasmid modified by the fluorescent protein, the target protein-luciferase fusion protein or the expression plasmid thereof into an endogenous ubiquitin gene UBB and a UBC knockout cell line and expressing;
2) adding a compound to be screened and a luciferase substrate, and detecting the fluorescence value after the action;
3) comparing the difference in fluorescence values of the different treatments to determine whether the compound and/or the target protein can promote ubiquitination degradation of the protein.
The invention has the beneficial effects that:
according to some embodiments of the invention, the addition of the small-molecule fluorescent substrate 18 hours before each detection is avoided, and the toxic influence of the small-molecule fluorescent substrate on cells is also avoided, so that the method is convenient to use, can monitor the ubiquitination level of the target protein in living cells in real time, has a more reliable result, can visually reflect the influence of the compound to be screened on the target protein, and is beneficial to high-throughput screening of the protein ubiquitination degradation promoting drug.
According to some embodiments of the invention, better BRET imaging effect can be obtained by introducing the target protein-luciferase fusion protein expression plasmid and the ubiquitin expression plasmid modified by the fluorescent protein into the endogenous ubiquitin gene UBB and the UBC knockout cell line.
Some embodiments of the invention may further enhance BRET imaging by adding proteasome inhibitors, such as MG 132.
Drawings
FIG. 1 shows the result of the functional verification of OgUb ubiquitination. Western Blot experiment shows that OgUb/OgUb-K48O can effectively polyubiquitinate and modify target protein. B. Immunofluorescence staining experiments show that target proteins modified by polyubiquitination of OgUb/OgUb-K48O are positioned to lysosomes for degradation;
figure 2 shows the results of the QmodiUb-NanoBRET system test. A. Will be provided with
Detailed Description
A screening system for a protein ubiquitination degradation drug based on bioluminescence resonance energy transfer comprises:
a fluorescent protein modified ubiquitin expression plasmid for expressing fluorescent protein modified ubiquitin;
cell lines with endogenous ubiquitin gene UBB and UBC knockouts; and
target protein-luciferase fusion protein.
In some examples of a profibiquitinizing degradation drug screening system, the fluorescent protein is the long stokes shift protein lssmoorange. The protein has large difference of excitation wavelength and wavelength, and is easy to detect. Of course, other similar proteins may be used.
In some examples of the profibiquitinization degradation drug screening system, the ubiquitin includes wild-type and mutant ubiquitin.
In some examples of the screening system for protein ubiquitination degradation drugs, the mutant ubiquitins include ubiquitin types K6, K11, K27, K29, K33, K48, and K63.
Thus, the influence of different compounds on the degradation of the ubiquitination of the protein can be detected respectively.
In some examples of the profibiquitinization degradation drug screening system, the target protein-luciferase fusion protein is introduced as an expression plasmid. This approach tends to go relatively low cost.
In some examples of the profibiquitinizing degradation drug screening system, the luciferase is
Thus can
In some examples of the screening system for a pro-protein ubiquitination degradation drug, the screening system for a pro-protein ubiquitination degradation drug is further added with a proteasome inhibitor.
In some examples of the screening system for a profibiquitinizing degradation drug, the proteasome inhibitor is selected from MG 132.
By adding a proteasome inhibitor, the BRET efficiency can be further improved.
In some examples of the profibiquitinization degradation drug screening system, the fluorescent protein is modified at the N-terminus of ubiquitin.
In a second aspect of the present invention, there is provided:
a screening method of a drug for promoting ubiquitination degradation of protein, the screening system of the invention is described in the first aspect, and the screening step comprises:
1) transferring the ubiquitin expression plasmid modified by the fluorescent protein, the target protein-luciferase fusion protein or the expression plasmid thereof into an endogenous ubiquitin gene UBB and a UBC knockout cell line and expressing;
2) adding a compound to be screened and a luciferase substrate, and detecting the fluorescence value after the action;
3) comparing the difference in fluorescence values of the different treatments to determine whether the compound and/or the target protein can promote ubiquitination degradation of the protein.
In order to screen the drugs for promoting protein ubiquitination degradation, the inventor designs a set of technology and tools capable of detecting the ubiquitination level of protein in living cells in real time, and names the technology and tools as QmodiUb-NanoBRET (quantized modified ubiquitination based on NanoBRET assay). Preferably, the long Stokes shift protein LSSmorange (maximum excitation) is selectedAnd an emission wavelength of 437nm and 572nm, respectively) instead of the conventional NanoBRETTM618 fluorescent group, which avoids adding small-molecule fluorescent substrate 18 hours before each detection and avoids the toxic effect of the small-molecule fluorescent substrate on cells. Furthermore, the inventor fuses LSSmorange with wild type and various mutant ubiquitin (called OgUb fusion protein for short) to construct a novel NanoBRET system capable of directly detecting various ubiquitin modifications.
The specific experimental procedures were as follows:
construction of fusion expression plasmid OgUb of LSSmOrange fluorescent protein and different types of ubiquitin mutants
The characteristics and synthesis of the OgUb series plasmid comprise:
designing based on a pLVX lentivirus expression vector;
resistance is Amp and Blastidin;
ubiquitin molecule Ub is fused with the end C of LSSmorange fluorescent protein;
the ubiquitin molecule Ub comprises 8 types, in particular wild type, Ub-K6O, Ub-K11O, Ub-K27O, Ub-K29O, Ub-K33O, Ub-K48O and Ub-K63O.
Lssmorage was linked to Ub as follows: the LSSmorange fluorescent protein is positioned at the N end of the Ub protein, and the LSSmorange fluorescent protein and the Ub protein are connected through GSSGGSlinker.
Construction of endogenous ubiquitin gene UBB and UBC knockout cell line
One sgRNA was designed for each of the intron regions flanking the UBB and the first exon of the UBC gene, and loaded into the PX459 vector. The design is as follows:
target sequence
Target point position
SEQ ID NO.
sgUBB-up
CTGATTGGTGGGGGACGCGGTGG
chr17:16284030-16284060
1
sgUBB-dn
CAAGACCATCACTCTGGAGGTGG
chr17:16285706-16285736
2
sgUBC-up
TTGGCACATAACCTAACCAGTGG
chr12:125395409-125395439
3
sgUBC-dn
GGTGACGTCACGACACGACGAGG
chr12:125398679-125398709
4
Cloning the sgRNA into a PX459 vector according to the PX459 use instruction, transiently transfecting HEK293T cells, and screening for 3 days by using puromycin antibiotics after 24 hours;
digesting the cells and planting monoclonals on a 96-hole cell culture class;
after the monoclone grows for 5-7 days, digesting and taking part of cells for identification, and detecting whether the first exons of UBB and UBC are knocked out. The detection primers are as follows:
primer name
Sequence of
SEQ ID NO.
dsgUBB-F
TCAAGAAAAAAAAGGAAAGACCCC
5
dsgUBB-R
TCAACATTAAGTACCTGCAAGCCA
6
dsgUBC-F
AACTTTAAGCGAGAGAAGAGGGAGT
7
dsgUBC-R
AGAAGGACATTTTAGGACGGGACT
8
BERT test experiment
Will be provided with
After 42 hours, the cells were divided into two groups, one with MG132 and one without MG132
After 48 hours, add per well
To verify the feasibility of the QmodiUb-NanoBRET system, the inventors first examined whether the OgUb fusion protein could efficiently ubiquitinate the target protein. Experimental results show that OgUb can be efficiently linked to a target protein for polyubiquitination modification (fig. 1A). Immunofluorescence experiments also showed that proteins modified by OgUb ubiquitination were carried to lysosomes for degradation (fig. 1B).
Finally, the inventors will
To increase BRET sensitivity, the inventors first knocked out endogenous Ub (major knockouts UBB and UBC) by CRISPR/Cas9, and the stable cell line thus obtained would more efficiently use OgUb to modify the target protein. The inventor shall provide
Therefore, by adding the compound to be detected into the screening system and observing BRET fluorescence change, the influence of the compound or target protein thereof on target protein ubiquitination degradation can be well determined, and high-throughput screening of the protein ubiquitination degradation promotion medicine is facilitated.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
SEQUENCE LISTING
<110> Zhongshan university tumor prevention and treatment center (Zhongshan university affiliated tumor hospital, Zhongshan university tumor research institute)
<120> screening system and method for protein ubiquitination degradation promoting drug based on nanoBRET
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