CN112194729B - Biological probe for detecting degradation activity of ubiquitin-proteasome system - Google Patents
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Abstract
The invention discloses a biological probe for detecting degradation activity of a ubiquitin-proteasome system, and belongs to the technical field of biology. The biological probe is formed by fusing ubiquitin and cpeGFP, and the amino acid sequence is shown as SEQ ID NO. 1. The method comprises the steps of carrying out cyclic rearrangement on GFP to obtain cpeGFP serving as a luminescent group, associating structural change of ubiquitin with the cpeGFP, enabling the ubiquitin to enter a contraction state after phosphorylation, enabling the ubiquitin to interact with the cpeGFP to enhance fluorescence, and reflecting structural change of the ubiquitin by detecting fluorescence intensity change; the-SH at the N terminal of the cpEGFP is used as a linker to be connected with the C terminal of ubiquitin, so that the response to phosphorylation of the ubiquitin is more sensitive. The probe can be used for relevant basic research and can also be used for screening ubiquitin kinase activators and proteasome inhibitors.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to a fluorescent probe for realizing the detection of cell level ubiquitin phosphorylation and ubiquitin dependent proteasome activity.
Background
The ubiquitin-proteasome system is the major pathway for degradation of intracellular proteins, involving more than 80% of proteins in cells, which are labeled with ubiquitin (polypeptide) and then recognized and degraded by proteasomes to maintain intracellular protein homeostasis. The proteasome targeted drugs have important research and development values in resisting tumors (generally requiring inhibitors) and neurodegenerative diseases (requiring development enhancers).
The method for detecting ubiquitin-dependent proteasome activity can be used for basic research and drug screening. In 2000, researchers have designed a probe Ub-linker-GFP for detecting ubiquitin-dependent proteasome activity, and ubiquitin (Ub) in the probe can be connected with more ubiquitin to form ubiquitin chains, which are degraded by proteasomes, and the probe has more applications in basic research. In addition, there are some probes based on deubiquitination, such as Ub-R-GFP, in which ubiquitin is hydrolyzed by deubiquitinase to form R-GFP, which is ubiquitinated at Lys of GFP and then degraded by proteasome. These probes have high fluorescence intensity and high background fluorescence due to the use of GFP.
The preliminary study of the subject groups shows that ubiquitin can be phosphorylated, and the representation after phosphorylation is also the inhibition of ubiquitin-dependent proteasome. Ubiquitin-dependent proteasome degradation proteins involve two processes, (1) ubiquitination of the substrate to be degraded; (2) ubiquitin chains bind to the proteasome. When ubiquitin is phosphorylated, the ubiquitin structure will appear in both a contracted and a relaxed state, with 50% of each state at neutral pH. The diastolic state can be further ubiquitinated to form a ubiquitin chain and can be combined with proteasome, once the ubiquitin is in the systolic state, the ubiquitin chain can not be extended, and the ubiquitin which has formed the ubiquitin chain is in the systolic state and can not be combined with proteasome. However, the GFP of the above probes does not have a function of sensing structural changes of proteins, and thus phosphorylation detection of ubiquitin cannot be achieved.
The cpEGFP is a process in which the original N-terminal and C-terminal of GFP are linked and cleaved at a new site to form new N-terminal and C-terminal, so that 11 β fragments of GFP are rearranged such that the fluorescent luminophore is covered by a cap that is loosely closed and open dynamically at the N-terminal, and opened (decreased fluorescence intensity) and closed (increased fluorescence intensity) as the structure of the protein to which the N-terminal is linked changes. The cpEGFP has the advantages of low fluorescence intensity and weak background fluorescence, and is mainly characterized in that the cover can be opened and closed to reflect the structural change of the related protein, so that the cpEGFP is generally used for detecting the interaction between protein domains or between proteins.
Therefore, how to effectively bind ubiquitin to cpEGFP to correlate the fluorescence intensity of cpEGFP with the structural change of ubiquitin is a problem to be solved by those skilled in the art.
Disclosure of Invention
The invention aims to provide a fluorescent probe capable of detecting ubiquitin phosphorylation of an ubiquitin-proteasome system, and the sensitivity of the fluorescent probe on the ubiquitin phosphorylation detection is improved.
In order to achieve the purpose, the invention adopts the following technical scheme:
a biological probe for detecting degradation activity of a ubiquitin-proteasome system is formed by fusing ubiquitin and enhanced green fluorescent protein with changed cyclic sequence;
the construction method of the enhanced green fluorescent protein with the changed circular sequence comprises the following steps: connecting the N-terminal and the C-terminal of the amino acid sequence of the green fluorescent protein, and breaking between the 144 th position and the 145 th position of the green fluorescent protein to form a new N-terminal and a new C-terminal, and deleting the tyrosine residue and the aspartic acid residue at the new N-terminal;
the serine residue at the N terminal of the enhanced green fluorescent protein with the changed cyclic sequence is connected with the C terminal of ubiquitin.
The working principle of the biological fluorescent probe Ub-cpeGFP constructed by the invention is as follows:
the bioluminescent probe Ub-cpeGFP can be degraded by ubiquitin-dependent proteasomes. When ubiquitin (Ub) subunit is phosphorylated by ubiquitin kinase to generate pUb-cpeGFP, pUb is in a contraction state, the cpeGFP subunit is also subjected to conformational change, the exposure degree of a fluorescent group in an aqueous solution is changed, pUb interacts with cpeGF, on one hand, the ratio of the contraction state is increased, and on the other hand, the fluorescence intensity of the cpeGFP is enhanced. Once ubiquitin enters the contracted state, the probe is rendered incapable of degradation by ubiquitin-dependent proteasomes, resulting in an increase in fluorescence intensity. The probe is also not capable of being degraded and exhibits increased fluorescence intensity when the proteasome degradation activity is reduced under some drugs or disease conditions.
Therefore, the degradation of Ub-cpeGFP is influenced by kinase phosphorylation and ubiquitin-dependent proteasome activity, and therefore, the ubiquitin phosphorylation and the ubiquitin-dependent proteasome degradation protein activity can be detected by detecting the fluorescence intensity and the Ub-cpeGFP level at a cell level, and the method is not only used for related basic research, but also used for screening ubiquitin kinase, proteasome activator and inhibitor.
The invention relates to a method for preparing a cPEPGFP (cyclic mutation GFP) by molecular cloning, carrying out circular rearrangement on a classical Green Fluorescent Protein (GFP), combining an original N-terminal with an original C-terminal, cutting at an N144 site of the GFP to obtain a new N-terminal and a new C-terminal, and deleting a tyrosine residue and an aspartic acid residue at the N-terminal. Research shows that the background fluorescence of the modified cpEGFP is weakened, and the serine residue is used as a new N terminal to be connected with ubiquitin (Ub), so that the probe is more sensitive to the phosphorylation response of ubiquitin.
Furthermore, the amino acid sequence of the biological probe is shown as SEQ ID NO. 1. The 1 st to 76 th positions in the sequence are ubiquitin (Ub), and the G at the 76 th position is mutated into V, so that the ubiquitin can be prevented from being hydrolyzed by deubiquitinase; 77-319 is cpEGFP, i.e. the original N-terminal and C-terminal of GFP are linked by adding GGTGGSG, and the N144 of GFP is broken to form a new C-terminal, the new N-terminal is deleted for YN to form a new N-terminal with-SH, and the new N-terminal is linked to the C-terminal of ubiquitin.
The coding sequence of the biological probe is shown in SEQ ID NO. 2.
The invention provides a kit for detecting degradation activity of a ubiquitin-proteasome system, which comprises: a recombinant plasmid containing a biological probe coding sequence with a nucleotide sequence shown as SEQ ID NO. 2.
The recombinant plasmid is introduced into cells to express a biological fluorescent probe Ub-cpeGFP, and when ubiquitin phosphorylation substances or substances inhibiting ubiquitin-dependent proteasome activity exist, the degradation of a ubiquitin-proteasome system to the biological probe is reduced, and the characteristic of fluorescence intensity enhancement is shown along with the accumulation of the biological probe.
The original vector of the recombinant plasmid can adopt a plasmid which is commonly used for cell transfection in the field, such as a PCDNA3.1 plasmid.
The invention provides application of the biological probe in preparation of a kit for detecting ubiquitin phosphorylation. When ubiquitin is phosphorylated, the biological probe shows an increase in fluorescence intensity, and thus can be used to determine whether the test compound causes ubiquitin phosphorylation.
For example, ubiquitin kinase phosphorylates ubiquitin, and thus, the biological probe can be applied to screening for activators or inhibitors of ubiquitin kinase.
The invention also provides application of the biological probe in preparation of a kit for detecting ubiquitin-dependent proteasome activity. When the ubiquitin-dependent proteasome activity is inhibited, the bioprobe exhibits an increase in fluorescence intensity, and thus, can be used for screening inhibitors or activators of ubiquitin-dependent proteasomes.
The present invention also provides a method of screening for inhibitors or activators of ubiquitin-proteasome system activity, said method comprising the steps of:
(1) cloning a DNA fragment with a nucleotide sequence shown as SEQ ID NO.2 into a PCDNA3.1 plasmid to obtain a recombinant plasmid;
(2) introducing the recombinant plasmid into HEK293 cells through transfection, and enabling the cells to express a biological probe Ub-cpeGFP;
(3) when screening inhibitors (including proteasome direct inhibitors or ubiquitin kinase PINK1 activators), adding a compound to be tested into transfected cells, acting for 3-24 h, collecting the cells, detecting the fluorescence intensity of a biological probe, and comparing with a control, such as the fluorescence intensity is enhanced, indicating that the compound to be tested inhibits the activity of a ubiquitin-proteasome system;
when an activator (including a proteasome direct activator or a ubiquitin kinase PINK1 inhibitor) is screened, a known inhibitor is added into transfected cells to improve fluorescence background, or recombinant plasmid expressing PINK1 is co-transfected to enhance the action of PINK1 and improve fluorescence background, a compound to be detected is added, after the action is performed for 3-24 hours, the cells are collected, the fluorescence intensity of a biological probe is detected, and if the fluorescence intensity is weakened compared with a control, the compound to be detected activates the activity of the ubiquitin-proteasome system.
In the step (3), the change of Ub-cpeGFP is detected by adopting flow cytometry detection, fixed cell imaging, fluorescence spectrum detection and immunoblotting biochemical detection technologies.
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention carries out circular rearrangement on green fluorescent protein GFP to obtain the most applicable cpeGFP which is fused with ubiquitin (Ub), associates the structural change of the ubiquitin in the contraction state and the relaxation state with the cpeGFP, and reflects the structural change of the ubiquitin by detecting the change of fluorescence intensity; the-SH at the N terminal of the cpeGFP is used as a linker to be connected with the C terminal of ubiquitin, so that the ratio of the phosphorylated ubiquitin to enter a contracted state can be increased, on one hand, the contracted ubiquitin can just interact with the cpeGFP, the cover at the N-terminal is closed, fluorescence is enhanced, and on the other hand, the ratio of the contracted state is increased, and the degradation of ubiquitin-dependent proteasomes is more favorably realized.
(2) When the fluorescent probe provided by the invention is used for detecting the cell level, the fluorescent probe basically has no background fluorescence, the cpeGFP fluorescence is weak, and under normal conditions, Ub-cpeGFP is rapidly degraded by a ubiquitin-dependent proteasome pathway.
(3) The fluorescent probe provided by the invention is convenient to use, and can be used for detecting the cell level by transfecting the probe, or establishing a stable cell strain, or transfecting based on viruses.
(4) The fluorescent probe provided by the invention has high sensitivity, and the fluorescence intensity begins to increase 1 hour after 10 mu M MG132 is added.
(5) The fluorescent probe provided by the invention has excitation and emission wavelength peak values basically consistent with those of Green Fluorescent Protein (GFP), and can be detected by a conventional fluorescence microscope and a fluorescence spectrometer.
Drawings
FIG. 1 shows the sequence (A) and excitation (B) and emission spectra (C) of a Ub-cPEGFP biological probe used in the detection according to the present invention.
FIG. 2 is a graph showing the inhibition of ubiquitin-dependent proteasome degradation proteins by MG132 using biological probes as detected by flow cytometry (A) and immunoblotting (B) in example 2.
FIG. 3 shows the detection of co-expressed Ub-cpeGFP and ubiquitin kinase PINK1 by fluorescence microscopy in example 3, with the probes emitting light.
FIG. 4 shows luminescence detected by fluorescence microscopy for probes of different linkage lengths between Ub and cpEGFP in comparative example 1.
FIG. 5 shows luminescence of the probe in comparative example 2 in which different amino acid residues were bonded between Ub and cpEGFP as detected by fluorescence microscopy.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to specific embodiments, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Plasmids, cells, reagents and medicaments mentioned in the following examples are all commercial products.
Example 1
Construction of a bioluminescent Probe Ub-cpeGFP
The amino acid sequence of the designed biological fluorescent probe Ub-cpeGFP is shown as SEQ ID NO.1, and the coding sequence is shown as SEQ ID NO. 2. The nucleotide sequence was synthesized by the same company, and then molecularly cloned between EcoRI and XhoI multiple cloning sites of the PCDNA3.1 plasmid to obtain a PCDNA3.1/Ub-cPEGFP plasmid. Sequencing identifies the correctness of the inserted sequence.
The excitation and emission spectra of the fluorescent probe are shown in FIG. 1.
Example 2
Detecting the inhibition effect of a proteasome inhibitor MG132 on ubiquitin-dependent proteasome activity by a flow cytometer and an immunoblotting method
a. Transfection of PCDNA 3.1/Ub-cPEPGFP plasmid in HEK293 cells: 40 ten thousand HEK293 cells were plated in 6-well plates and 24 hours later transfected with lipo3000 liposomes to express Ub-cPEGFP.
b. 24 hours after transfection, 10. mu.M MG132 (proteasome inhibitor) was added, and 0,1,2,3,4,5 hours after addition, cells were harvested by trypsinization, 1000 rpm, and centrifugation for 5 minutes, respectively.
c. The received cells were resuspended in phosphate buffer, and then the fluorescence intensity of cpEGFP was measured by flow cytometry, the average fluorescence intensity of the cells was calculated, and the inhibitory effect of MG132 on proteasome was analyzed.
d. After flow detection, cells are recovered, the cells are cracked by using a cell cracking kit, SDS gel electrophoresis detection is carried out, then the level of Ub-cpeGFP is detected by using an anti-GFP antibody, and the degradation condition of the Ub-cpeGFP by the ubiquitin-dependent protease system is analyzed.
As shown in FIG. 2, the biological probe Ub-cpeGFP has weak fluorescence when the MG132 in Panel A does not start to act, and the fluorescence intensity of the probe can be obviously increased in a time-dependent manner when 10 μ M MG132 acts for 1-5 hours; in panel B, Ub-cpeGFP levels increased with the time of action of MG132, indicating that degradation of Ub-cpeGFP by the ubiquitin-dependent proteasome system is inhibited by MG 132.
Example 3
Detecting the effect of a proteasome inhibitor MG132 or a co-expressed ubiquitin kinase PINK1 on inhibiting ubiquitin-dependent proteasome degradation probes by a fluorescence microscopy method
a. Separately transfected PCDNA3.1/Ub-cpEGFP or separately transfected PCDNA3.1/Ub-EGFP in HEK293 cells, or co-transfected two plasmids of PCDNA3.1/Ub-cpEGFP and PCDNA3.1/sPINK1, or co-transfected two plasmids of PCDNA3.1/Ub-EGFP and PCDNA3.1/sPINK 1: HEK293 cells were plated at a density of 3 ten thousand per well in 24-well cell culture plates with round cover slips on the bottom using lipofection.
b. 24 hours after transfection, cells transfected with either PCDNA3.1/Ub-cPEGFP or PCDNA3.1/Ub-EGFP alone were treated with 10. mu.M MG132 for 5 hours. All cells were fixed with freshly prepared 4% paraformaldehyde.
c. The expression of green fluorescence was observed using a confocal microscope.
As shown in FIG. 3, weak green fluorescence was observed when both probes were transfected separately, and when MG132 was activated for 5 hours, the intensity of green fluorescence was increased, and when sPINK1 was co-transfected, the green fluorescence was also increased, but the fluorescence of PCDNA 3.1/Ub-cPEPGFP was more significantly increased.
Comparative example 1
This comparative example compares different linkage lengths between Ub and cpeGFP, uses ubiquitin L67S mutation to convert all ubiquitin structures to a contracted state, and compares Ub-SH-cpeGFP, Ub Δ (G75, G76) -SH-cpeGFP deleting two Gly at the end of Ub (i.e., G75 and G76), and Ub Δ (G75, G76) -cpeGFP further deleting-SH-.
The results of plasmid construction and cell transfection carried out by the methods of examples 1 and 2 and fluorescence microscope detection are shown in FIG. 4, and the Ub-SH-cPEPGFP probe constructed by the invention has no obvious fluorescence when fused with wild-type ubiquitin, and has strong fluorescence when fused with ubiquitin with L67S mutation. Deletion of two Gly at the end of Ub (G75 and G76) resulted in strong fluorescence expression for both the fused wild type and mutant probes. After further deletion of-SH-, neither the probe fused with the wild type nor the mutant type emits light.
Comparative example 2
This comparative example compares different amino acid linkage patterns between Ub and cpeGFP, and replaces the-SH-linkage with the-LE-linkage to construct Ub-LH-cpeGFP.
The results of plasmid construction and cell transfection performed according to the methods of examples 1 and 2 and fluorescence microscopy show that, as shown in FIG. 5, no fluorescence was observed after HEK293 cells were transfected with both plasmids, whereas both were fluorescent when the proteasome inhibitor MG132 was added, but the fluorescence intensity of the-SH-linked probe was stronger and the fluorescence intensity of the-LE-linked probe was weaker.
Sequence listing
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<120> a bioprobe for detecting degradation activity of ubiquitin-proteasome system
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Leu Gly Met Asp Glu Leu Tyr Lys Gly Gly Thr Gly Gly Ser Gly Val
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Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu
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attcagaaag aaagcaccct gcatctggtt ctgcgcctgc gcggcgtgag ccataatgtg 240
tatattatgg ccgataaaca gaaaaatggc attaaggcca attttaaaat ccgtcataac 300
attgaagacg gcggcgttca gctggcagat cattatcagc agaatacccc gattggcgat 360
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ctgggcatgg atgaactgta taaaggcggt accggtggta gcggcgtgag caaaggtgaa 540
gaactgttta ccggtgtggt tccgattctg gtggaactgg atggtgacgt gaatggtcat 600
aaattttcag tgagcggcga aggtgaaggc gatgcaacct atggcaaact gaccctgaaa 660
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aaaggtattg attttaaaga ggatggtaac attctgggcc ataaactgga atataattaa 960
Claims (8)
1. A biological probe for detecting the degradation activity of ubiquitin-proteasome system is characterized in that the amino acid sequence of the biological probe is shown in SEQ ID NO. 1.
2. The biological probe for detecting degradation activity of ubiquitin-proteasome system according to claim 1, wherein the coding sequence of the biological probe is shown in SEQ ID No. 2.
3. A kit for detecting the degradation activity of the ubiquitin-proteasome system, comprising: a recombinant plasmid containing a biological probe coding sequence with a nucleotide sequence shown as SEQ ID NO. 2.
4. The kit for detecting degradation activity of ubiquitin-proteasome system according to claim 3, wherein the primary vector is PCDNA3.1 plasmid.
5. Use of the biological probe according to claim 1 or 2 for the preparation of a kit for the detection of ubiquitin phosphorylation.
6. Use of the biological probe of claim 1 or 2 for screening ubiquitin kinase inhibitors or activators.
7. Use of a biological probe according to claim 1 or 2 for the preparation of a kit for detecting ubiquitin-dependent proteasome activity.
8. A method of screening for inhibitors or activators of ubiquitin-proteasome system activity, comprising the steps of:
(1) cloning a DNA fragment with a nucleotide sequence shown as SEQ ID NO.2 into a PCDNA3.1 plasmid to obtain a recombinant plasmid;
(2) introducing the recombinant plasmid into HEK293 cells through transfection, and enabling the cells to express a biological probe Ub-cpeGFP;
(3) when the inhibitor is screened, adding a compound to be detected into transfected cells, acting for 3-24 h, collecting the cells, detecting the fluorescence intensity of a biological probe, and comparing with a control, if the fluorescence intensity is enhanced, indicating that the compound to be detected inhibits the activity of the ubiquitin-proteasome system;
when the activator is screened, a known inhibitor is added into transfected cells to improve the fluorescence background, or recombinant plasmids expressing ubiquitin kinase are co-transfected to enhance the effect of the ubiquitin kinase and improve the fluorescence background, a compound to be detected is added to act for 3-24 h, then the cells are collected, the fluorescence intensity of the biological probe is detected, and if the fluorescence intensity is weakened compared with a control, the compound to be detected activates the activity of the ubiquitin-proteasome system.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001081427A1 (en) * | 2000-04-27 | 2001-11-01 | Karolinska Innovations Ab | A reporter protein construct, partly comprising gpf |
| CN104844704A (en) * | 2014-02-17 | 2015-08-19 | 华东师范大学 | Ub-Nanoluc reporter gene system and Ub-Ub-GS-Nanoluc reporter gene system, constructions and applications thereof |
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| US20130017554A1 (en) * | 2011-06-23 | 2013-01-17 | College Of William And Mary | Sumo-specific affinity tag |
| CN111154785B (en) * | 2020-01-20 | 2022-01-04 | 浙江大学 | Method for modifying ubiquitin and inhibiting ubiquitination pathway |
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| CN104844704A (en) * | 2014-02-17 | 2015-08-19 | 华东师范大学 | Ub-Nanoluc reporter gene system and Ub-Ub-GS-Nanoluc reporter gene system, constructions and applications thereof |
| CN108136197A (en) * | 2015-09-15 | 2018-06-08 | 斯坦福大学托管董事会 | Optical Response polypeptide and its application method |
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