CN111748531B - Application of USP33 as medication target in preparation of medicines - Google Patents

Application of USP33 as medication target in preparation of medicines Download PDF

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CN111748531B
CN111748531B CN201910252833.XA CN201910252833A CN111748531B CN 111748531 B CN111748531 B CN 111748531B CN 201910252833 A CN201910252833 A CN 201910252833A CN 111748531 B CN111748531 B CN 111748531B
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赵永良
牛凯峰
方洪波
魏迪
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Beijing Institute of Genomics of CAS
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Abstract

The invention provides an application of USP33 as a drug target in preparation of drugs, belonging to the field of protein engineering. The invention discovers deubiquitinase USP33 interacting with Parkin by using co-immunoprecipitation and mass spectrometry, and further discovers that USP33 is positioned on the outer membrane of mitochondria and can be used for modifying E3 ligase Parkin by deubiquitination. Knocking down USP33 can promote the translocation of Parkin to mitochondria, thereby enhancing mitophagy and removing damaged mitochondria, and proves that USP33 regulates and controls mitophagy by influencing the deubiquitination of Parkin, USP33 can inhibit the apoptosis of nerve cells under MPTP treatment, USP33 can be used as a medication target to prepare medicines, and has good application potential and value.

Description

Application of USP33 as medication target in preparation of medicines
Technical Field
The invention belongs to the field of protein engineering, and particularly relates to application of USP33 as a drug target in preparation of drugs.
Background
USP33(VDU1) is a deubiquitinase having 942 amino acids and a molecular size of 107kD, capable of removing ubiquitin molecules from a substrate and ubiquitinating in a pVHL (von Hippel-Lindau (VHL) disease-dependent manner. Structural biology reveals: the N-terminal contains ZnF UBP structure, similar structure is also found in deubiquitinase HDAC6, and the C-terminal has DUSP structure. The ZnF UBP structure has three zinc ions, and the 221-arginine mutation of USP33 can affect the combination with 75-glycine of ubiquitin molecule. USP33 has at least three variants, variant 1 is full length, with 25 exons; variant 2 lacks exon 2, translation is from the start codon of exon 3 lacking 31 amino acids; variant 3 is variant in which exon 15 is alternatively spliced out and exon 22 is later spliced out. All three variants have a distribution in the endoplasmic reticulum, with variant 3 also being distributed on the golgi apparatus. In the cell, E3 ubiquitin ligase beta-TrCP can ubiquitinate USP33 to promote the ubiquitination degradation thereof, in the process, DSG (XX) of USP332+nThe structure is indispensable.
More reports are that USP33 is closely related to the occurrence of cancer, and after tissue section analysis of colon cancer patients, the expression level of USP33 in colon cancer is low, which indicates that the tumor has stronger metastatic capacity. Under the induction stimulation of SDF-1, the beta-arrestin 2 protein is combined with USP33, the ubiquitination level of the beta-arrestin 2 is inhibited, the protein level is not influenced, the endocytosis process of CXCR4 is further inhibited, the ERK is continuously activated by the low expression of USP33, and the occurrence and development of rectal cancer are further influenced. USP33 is involved in regulating the Slit-Robo pathway in breast, lung and colorectal cancer. The nerve guidance factor Slit is a secretory glycoprotein which can inhibit leukocyte chemotaxis induced by chemokine SDF and tumor angiogenesis, and USP33 inhibits tumor migration by regulating the activity of Slit protein, and stabilizes the protein level by inhibiting ubiquitination of Robo1, which is necessary for inhibiting Slit protein in tumor migration. Recently, it has also been shown that MicroRNA-365 also promotes lung cancer proliferation by down-regulating the USP33-Slit2-Robo1 pathway.
USP33 is widely distributed in the brain, the loss of Slit protein easily causes the abnormal accumulation of axons of cross neurons and axons of a longitudinal conduction path at the central nerve midline, and the Slit-Robo pathway plays an important role in mediating the growth direction of the axons and the formation of a projection path. USP33 interacts with Robo1 and knock-down USP33 causes mouse embryonic DiI-labelled axons to cross the midline barrier, similar to the mouse phenotype of the Slit1/Slit2/Slit3 mutations, indicating that USP33 has an important role in axon-guided developmental processes.
Role of USP33 in cell cycle regulation. The central body protein CP110 can regulate the replication of the central body, inhibit the elongation of the central body, and the over-expression of CP110 can cause the over-replication of the central body and the ciliation. USP33 is located on the centrosome during the S phase and G2/M phase and interacts with CP110, removing ubiquitin molecules of CP110 and stabilizing proteins, promoting centrosome replication and mitosis, further regulating cell cycle.
Mitochondria are extremely important organelles in eukaryotes and play important roles in metabolism, energy supply and apoptosis, but mitochondria can also generate a large amount of harmful substances such as active oxygen in the process of oxidative phosphorylation, so that mitochondrial proteins and DNA are damaged, and then apoptosis is caused (Baker et al, 2014). In order to maintain homeostasis of the cell itself, it is critical to be able to clear damaged mitochondria in time. After mitochondrial damage and senescence, cells are selectively and efficiently cleared in time using an autophagy mechanism, a process known as mitophagy. The Pink1-Parkin pathway is a key mechanism for mediating mitochondrion autophagy, Parkin is E3 ubiquitin ligase, mainly mediates substrate ubiquitination, regulatory protein degradation, signal transduction and the like, is widely expressed in various tissues and organs, is rich in the nervous system, skeletal muscle and the like, and often causes diseases such as neurodegenerative diseases, tumors, leukemia and the like due to abnormal functions. Pink1 mediates its transfer to mitochondria by phosphorylating Parkin, and activated Parkin ubiquitinates mitochondrial outer membrane proteins.
Parkin is an E3 ubiquitin ligase encoded by the PARK2 gene. As ubiquitin ligase, ubiquitin molecules can be covalently linked with substrates to form substrate polyubiquitinated chains, so that proteases can recognize the substrates and degrade the substrates or regulate signal pathways. It is also located downstream of the PINK1 induction pathway, mediating the clearance of damaged mitochondria. Parkin is mainly distributed in the cytoplasm and has also been reported to be located on mitochondria. The N-terminus of Parkin has a UBL domain (N-terminal ubiquitin-like domain) and two RING domains, which function to recruit ubiquitin binding to the E2 enzyme. When mitochondria are damaged, the permeability of mitochondria to protons is increased, membrane potential difference and PH difference on two sides of an inner membrane are eliminated, PINK1 cannot cross the inner membrane of the mitochondria due to the loss of the potential of the inner membrane of the mitochondria, MTS signal peptide cannot be cut, full-length PINK1 is stable and enriched on the outer membrane of the mitochondria, and in combination with TOM (mitochondrial outer membrane protein), adjacent PINK1 in a complex are mutually phosphorylated so as to be highly activated, promote translocation of Parkin and ubiquitin UB in cytoplasm to the mitochondria, the Parkin is phosphorylated, the activated Parkin is combined with the phosphorylated ubiquitin, further ubiquitinate and degrade substrate proteins, such as TOM20, mfn1 and VDAC (visual desktop computer), receptor protein p62 recognizes ubiquitinated protein, mitochondrial ubiquitinated protein is recognized by p62, so that the mitophagemigration vesicle is wrapped, and occurrence of mitochondrial autophagy is induced. The Parkin is not only a pathogenic cause of neurodegenerative diseases such as parkinsons and the like, but also one of pathogenic factors for the occurrence and development of various tumors, leukemia and the like. Disorders of mitophagy are also closely related to the above diseases, and Parkin is an important molecule for regulating mitophagy. As an E3 ligase, Parkin can attach a highly conserved ubiquitin UB chain to a target protein for degradation or signal transduction. Parkin can ubiquitinate not only substrate proteins but also itself.
Studies have shown that PARK2 is an anti-cancer gene. Abnormal expression of the Parkin protein is found in many cancers, such as liver cancer, gastric cancer, breast cancer, rectal cancer, lung cancer and the like. Parkin also plays an important role in the antiviral immune mechanisms, 21 days after m.tubericalis virus infection in PARK 2-deficient mice, the number of viruses in the lungs, spleen and liver of the mice was 10-fold that of the control group, and the PARK 2-deficient mice died of the virus infection at 85 days of infection, while the control group was all alive. After l.monocytoenes infection in drosophila, loss of function of LC3 in bak 2 reduces the loss causing autophagy and reduces survival of drosophila.
The ubiquitin-proteasome degradation pathway is a pathway which is researched more in cells, mainly degrades proteins marked by ubiquitin molecules in cells, has a non-degradation function, regulates the location and activity of cell proteins, and participates in processes such as cell differentiation, cell cycle, signal transduction, DNA repair and the like. The general process is as follows: in cells, ATP molecules can activate E1 protein, ubiquitin molecules can be activated under the action of E1 and are transferred to E2 to form a new structure, namely an E2-ubiquitin intermediate, meanwhile, E3 ubiquitin ligase recognizes target protein to be degraded, glycine residues at the C end of ubiquitin are combined with lysine residues of the target protein to form isopeptide bonds, and according to the process, the ubiquitin molecules can be continuously connected with each other to finally form polyubiquitinated chains to guide the recognition of protease phase of the protein to be degraded so as to be degraded. Ubiquitin molecules are proteins which are composed of 76 amino acids, have a molecular weight of about 8.5kD, are widely distributed in eukaryotic cells, are highly conserved evolutionarily, and are often covalently bound to a variety of proteins to play a role. Finding and determining the substrates of Parkin and the ubiquitination chains that Parkin participates in formation is very important for understanding the molecular mechanism of mitophagy.
Disclosure of Invention
The invention uses USP33 as a target to search the protein with the interaction, so as to discover the application of USP33 as a drug target and the application of the USP33 as the drug target in preparing drugs, and provide a treatment basis for treating related diseases mediated by USP33 action protein.
In order to achieve the purpose of the invention, the invention starts by researching the angle of which factors are regulated and controlled by Parkin in the process of generating the mitochondrial autophagy, optimizes and improves the co-immunoprecipitation experimental process and the mass spectrometry detection technology, the mass spectrometry result shows that the USP33 has interaction with the Parkin, then the relevant documents since the discovery of the Parkin gene in 1998 are consulted, the interaction protein of the Parkin is particularly concerned, and the article report on the interaction of the Parkin and the USP33 is not discovered. The invention then improves and optimizes the prior technical means and experimental scheme, researches the subcellular localization of USP33, and proves that USP33 and mitochondria have co-localization through immunoblotting experiments.
The invention utilizes proteinase K degradation experiments to confirm that USP33 is positioned on the outer membrane of mitochondria. The hydrophobic structure and analysis of USP33 show that amino acid at position 549-569 is a transmembrane domain, the deletion of which can cause USP33 to be dispersed in cytoplasm, and the cell separation component shows that no USP33 protein exists in the mitochondria, thereby further confirming that USP33 is positioned in mitochondria, which is the first time that USP33 is found to be the deubiquitinase positioned on the outer membrane of the mitochondria.
The present inventors found that knockdown of USP33 promoted GFP-Parkin translocation, but USP33 was not found to translocate under CCCP treatment. CCCP treatment of cells can cause depolarization of mitochondria to cause damage, and mitochondrial autophagy is caused to the cells to remove damaged mitochondria, in an experiment, CCCP treatment of the cells is used for 12 hours, immunofluorescence and immunoblotting are used for analyzing disappearance of the mitochondria, and compared with a control group, USP33 knocking down can accelerate removal of the damaged mitochondria. Treatment with Oligomycin and Antimycin A also causes an increase in ROS in the mitochondria, damaging mitochondrial DNA. Mitochondrial DNA clearance is also an important indicator for assessing the occurrence of mitochondrial autophagy, and USP33 knockdown also results in accelerated clearance of damaged mitochondrial DNA. Indicating that USP33 is involved in regulation both early and late in the development of mitophagy.
The invention constructs 7 single-point mutated UB plasmids, detects the ubiquitination condition of the Parkin protein after the over-expression of USP33, and finds that ubiquitin chains connected with K6, K11, K27 and K48 can be obviously reduced. To further clarify the role played by the 4 ubiquitinated chains, the present inventors performed cycloheximide experiments and found that USP33 knock-down significantly extended the half-life of the Parkin protein, promoting protein stability, which explains why USP33 knock-down promotes Parkin translocation and mitochondrial damage clearance.
The structure of the Parkin is analyzed for the first time, ubiquitination modification is generally carried out at lysine residue sites of protein, the inventor finds 18 lysine residue sites altogether, mutation is carried out one by one, after mutation of K48 and K435 sites of the Parkin is found, ubiquitination of the Parkin is obviously reduced, after USP33 is knocked down, ubiquitination of wild type Parkin and K48 mutants is obviously increased, ubiquitination of K435 mutants of the Parkin is not changed greatly, and the K435 is an important site for de-ubiquitination of the Parkin of USP 33.
MPTP treatment of cells can damage mitochondria to oxidize respiratory chain, cause apoptosis of cells, and induce the occurrence of Parkinson disease after long-term treatment in vivo. In the invention, MPTP is used for treating nerve cell SH-SY5Y, and compared with a control group, the USP33 knock-down obviously inhibits apoptosis and protects nerve cells, so that the USP33 mediated mitochondrion autophagy plays an important role in apoptosis and plays an indispensable role in the occurrence of Parkinson diseases.
Through systematic experimental study, the invention finally determines that USP33 has important function in preparing the medicament as a medicament target by progressive and ring-and-ring deduction, and can regulate and control the action mechanism of mitochondrion autophagy in related diseases by influencing the deubiquitination of Parkin.
Based on the research result of the invention, the invention provides the application of USP33 (the amino acid sequence is shown as SEQ ID NO. 1) as a medication target in preparing a medicament, wherein the medicament is any one or more of the following:
(1) drugs for treating neurodegenerative diseases;
(2) a medicament for treating tumors;
(3) drugs for the treatment of cardiovascular diseases;
(4) drugs for the treatment of immune system disorders;
(5) a medicine for treating diseases caused by virus infection.
The invention provides an application of USP33 in preparing a medicine for treating a Parkin protein-mediated mitophagy-related disease. The amino acid sequence of the USP33 is shown in SEQ ID NO. 1.
The invention discovers that USP33 acts on the site of the Parkin protein K435 with an amino acid sequence shown as SEQ ID NO.2 by being positioned on the outer membrane of mitochondria to regulate the level of mitochondrial autophagy.
Furthermore, the invention discovers that USP33 reduces the ubiquitination level of the Parkin protein by acting on the K435 site of the Parkin protein with the amino acid sequence shown as SEQ ID No.2 to de-ubiquitinate and modify the Parkin protein, thereby weakening or preventing the mitophagy.
The invention provides application of USP33 in preparing a medicine for counteracting, preventing or delaying the ubiquitination process of Parkin protein.
In addition, USP33 counteracts, prevents or delays the ubiquitination process of the Parkin protein by acting on the K435 site of the Parkin protein with the amino acid sequence shown as SEQ ID NO. 2.
The invention provides application of USP33 in preparing a medicine for reducing the stability of Parkin protein.
The application specifically comprises that USP33 is positioned on the outer membrane of mitochondria and acts on the site K435 of the Parkin protein with the amino acid sequence shown as SEQ ID NO.2, so that the stability of the Parkin protein is reduced.
The invention provides any one of the following applications of the USP33 expression promoter,
(1) the application in preparing the medicine for treating the Parkin-mediated mitochondrial autophagy related diseases;
(2) application in preparing medicine for lowering the stability of Parkin protein.
The invention provides any one of the following applications of an inhibitor of USP33 expression,
(1) the application in preparing the medicine for treating the Parkin-mediated mitochondrial autophagy related diseases;
(2) the application in preparing the medicine for promoting the ubiquitination process of the Parkin protein;
(3) the application of the derivative in preparing a medicine for promoting the stability of the Parkin protein and prolonging the half-life period of the Parkin protein.
Further, the USP33 expression inhibitor can promote mitophagy by knocking down or inhibiting the expression of USP33 and the deubiquitination level of K435 site of Parkin protein with an amino acid sequence shown as SEQ ID NO. 2.
The invention also provides a Parkin protein mutant, wherein the 435 th amino acid K of the sequence shown in SEQ ID NO.2 is mutated into R.
The invention provides any one of the following applications of the Parkin protein mutant,
(1) the application in preparing the medicine for regulating and controlling the level of mitochondrial autophagy;
(2) the application of the compound in preparing medicines for weakening or reducing the level of mitochondrial autophagy;
(3) the application in preparing the medicine for treating the diseases related to the mitophagy.
The invention also provides a preparation for treating the Parkin protein mediated mitochondrial autophagy related diseases, which contains the USP33, the USP33 expression promoter or the USP33 expression inhibitor, and the preparation is a medicine, a pharmaceutical composition, a health product, a food or an additive.
Furthermore, the preparation acts on a K435 site of the Parkin protein with an amino acid sequence shown as SEQ ID No.2 through USP33, de-ubiquitinates and modifies the Parkin protein, reduces or promotes the ubiquitination level of the Parkin protein, regulates the level of mitophagy, and further treats diseases related to mitophagy.
In the preparation, the disease is neurodegenerative disease, tumor, cardiovascular disease, immune system disease or disease caused by virus infection.
In the application of the invention, the disease is neurodegenerative disease, tumor, cardiovascular disease, immune system disease or disease caused by virus infection.
The neurodegenerative disease comprises Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, spinocerebellar ataxia, Pick's disease, cerebral ischemia, brain injury, epilepsy;
the tumor comprises breast cancer, prostatic cancer, lung cancer, gastric cancer and liver cancer;
the cardiovascular diseases comprise coronary heart disease, angina pectoris, hypertension, hyperglycemia, hyperlipemia, and pulmonary heart disease;
the immune system diseases comprise systemic lupus erythematosus, rheumatoid arthritis, autoimmune hemolytic anemia, various skin diseases and chronic liver diseases.
More than 100 deubiquitinating enzymes have been discovered so far in the USP family, and play an important role in life activities. In the research of adjusting and controlling mitochondrial autophagy by deubiegol, particularly for adjustment and control research of Parkin protein, related research is slow, and in the research of Baris Bingol and the like in 2014, Yuqing and the like in 2015, screening is respectively carried out by using a human-derived deubiquitinase cDNA library (human DUB complementary DNalibrary), the adjustment and control effect of USP33 on Parkin is not found, and meanwhile, in the research of Thomas M Durcan and the like in 2014, large-scale siRNA reduction screening technology is used again, and related information is not found, so that the relation between the two cannot be found by a simple verification experiment, technical obstacles existing in the prior art are reflected from the side, and the relation between USP33 and Parkin is not found by scientific research workers all the time. In order to research the mechanism, the invention adopts the previous quite different research thinking, does not proceed from the perspective of researching the regulation of the deubiquitinase on the Parkin, but starts from the perspective of researching which factors the Parkin is regulated by in the process of the occurrence of the mitochondrial autophagy, firstly looks up the relevant documents since the discovery of the Parkin gene in 1998, particularly focuses on the interacting protein of the Parkin, and does not discover the article reporting the interaction of the Parkin and the USP 33. And optimizing and improving the co-immunoprecipitation experimental process and the mass spectrum detection technology, finally finding the interaction between USP33 and Parkin, and finally determining the action mechanism of USP33 for regulating and controlling the mitophagy in related diseases by influencing the deubiquitination of Parkin.
The invention has the beneficial effects that: the invention provides a new application of USP 33. The USP family members are numerous, but each member has a similar deubiquitinase functional region, and other gene regions are completely different. The invention develops a new method, the Flag-Parkin interaction protein is enriched by a co-immunoprecipitation method, an SDS-PAGE gel electrophoresis is used for separating an enriched protein sample, then Coomassie brilliant blue G250 is used for dyeing detection, and protein mass spectrum analysis is carried out, so that the interaction between USP33 and Parkin is found, the USP33 and the previously reported USP8 are creatively proved to participate in regulating and controlling the function of the Parkin gene, but the USP33 and the USP8 have opposite effects, and only the USP33 plays a protective effect on cells after being knocked down. The invention further discovers that USP33 is located on the outer membrane of mitochondria for the first time and can de-ubiquitinate and modify E3 ligase Parkin. In the process of occurrence of mitochondrial autophagy, USP33 can remove ubiquitination chains of K6, K11, K27 and K48 connected with Parkin protein, mutation of K435 site of Parkin can prevent change of Parkin ubiquitination level after USP33 is knocked down, the mutation is a key site of interaction between USP33 and Parkin, and meanwhile, knocking down USP33 can promote occurrence of mitochondrial autophagy. Completely different from the mechanism regulation of the deubiquitinating enzyme related to the mitophagy which is discovered in the past. The invention discloses a molecular mechanism of deubiquitinase USP33 participating in regulation of a mitochondrial autophagy process by regulating a Parkin-PINK1 pathway for the first time. The study also finds that the knocking-down USP33 has a protective effect on MPTP-treated nerve cells SH-SY5Y, which indicates that USP33 is a potential drug target for treating Parkinson's disease, and shows that USP33 can be used as a new target for treating mitochondrial autophagy-related diseases including Parkinson's disease and tumors, and has good application potential and value.
Drawings
FIG. 1 is a diagram showing the identification result of the Parkin interacting protein, wherein, a diagram is that a Flag-Parkin plasmid is transfected in U2OS cells, and G418 is used for screening the cells to obtain cells stably expressing the Flag-Parkin for immunoblotting analysis. And B, performing amplification culture on a cell line No.4 stably expressing Flag-Parkin, collecting cells, incubating a whole cell lysate with anti-Flag beads, and separating an enriched protein sample by using SDS-PAGE gel.
FIG. 2 is a graph showing the results of direct interaction between Parkin and USP33 and no interaction with USP20, wherein panel A transfects Flag-Parkin or Flag-GFP plasmid in HEK293 cells, collects cell lysates, incubates with anti-Flag Beads, and detects endogenous USP 33. B for transfer of Flag-HA-USP33 or Flag-GFP into HEK293 cells, the same procedure as A, endogenous Parkin was detected in Pull Down complex. Panel C is an in vitro GST pull-down experiment that verifies the direct interaction between Parkin and USP 33. Panel D shows the transfection of Flag-USP20 or Flag-GFP plasmids in HEK293 cells, following the same procedure as A, with no endogenous Parkin seen in the Flag-USP20 protein pull down complex. Flag-GFP served as a negative control.
Fig. 3 is a graph showing the interaction result between USP33 and different domains of Parkin, and a is a fragmented schematic diagram of Parkin. Panel B shows the co-transfection of different functional domain fragments of HA-USP33 and Flag-Parkin in HEK293 cells. HA-USP33 was co-immunoprecipitated with Flag-Parkin protein (lane 2 of IP samples), and only Flag-UBL-linker, Flag-RING1 and Flag-RING2 were detected to have an interaction with HA-USP 33. Flag-GFP served as a negative control.
Fig. 4 is a graph showing the interaction results between Parkin and different functional domains of USP33, and a is a fragmented schematic diagram of USP 33. Panel B shows the co-transfection of different functional domain fragments of HA-Parkin and Flag-USP33 in HEK293 cells. HA-Parkin was co-immunoprecipitated with Flag-USP33 protein (lane 2 of IP samples), and only Flag-USP was detected to interact with HA-Parkin. Flag-GFP served as a negative control.
FIG. 5 is a graph showing that USP33 is located in the outer mitochondrial membrane. Wherein A is immunofluorescence, indicating that USP33 is co-localized with mitochondria. U2OS cells were fixed in 4% paraformaldehyde for 30 minutes, then treated with 0.1% Triton X-100 for 15 minutes, immunized with anti-USP33 antibody overnight at 4 ℃ and then incubated with fluorescent secondary antibody for 2 hours at 37 ℃. The staining results were observed using a confocal microscope. B is an isolated fraction of HEK293 cells, endogenous USP33 was detected in cytoplasm and mitochondria. C is proteinase K cleavage experiment result, indicating that USP33 is located in outer mitochondrial membrane. Mitochondria isolated from HEK293 cells were treated with 100ng/ml proteinase K and 1% TritonX-100 and analyzed by immunoblotting. SMAC is a mitochondrial inner membrane protein and VDAC is a mitochondrial outer membrane protein.
FIG. 6 shows transmembrane domain 549-569(TM) which is a key representation of the localization of USP33 to mitochondria, panel A is the prediction of the transmembrane domain of USP33 using the online tool TMpred. Regions scoring greater than 500 were transmembrane domains. Panel B shows the transfection of GFP-USP33 or GFP-USP33- Δ TM plasmids in HEK293 cells. Immunoblot analysis showed that GFP-USP33 was distributed in both cytoplasm and mitochondria, but GFP-USP33- Δ TM was found only in cytoplasm. Panel C shows that GFP-USP33 or GFP-USP33- Δ TM plasmids are also transfected in U2OS cells, and immunofluorescence assay also shows that deletion of amino acid residue at position 549-569 causes GFP-USP33 distribution and cytoplasm, and no obvious co-localization with mitochondria.
FIG. 7 is a graph showing the results of the GFP-Parkin translocation changes in CCCP treatments at different concentrations, in which GFP-Parkin stably expressing cells were treated with 10, 20 and 40 μ M CCCP for 2 hours, fixed in 4% paraformaldehyde for 30 minutes, and observed in an inverted microscope for GFP-Parkin translocation. With increasing concentration of CCCP, the amount of GFP-Parkin translocated to mitochondria increased.
FIG. 8 is a graph showing that knocking down USP33 promotes GFP-Parkin translocation to mitochondria. Panel A shows the transfection of USP33 siRNA into U2OS cells stably expressing GFP-Parkin, followed by treatment of the cells with 20. mu.M CCCP for various periods of time, harvesting of the cells for 30 minutes with 4% paraformaldehyde and 15 minutes with 0.1% Triton X-100, and visualization using confocal laser microscopy. Panel B is the ratio of GFP-Parkin translocation to mitochondria (the ratio of cells co-localized with the GFP-Parkin and mitochondrial protein TOM20 to all cells in the microscopic field) at different times of CCCP treatment in the control and USP33 knockdown groups. At least 200 cells were counted per time point and the results showed the average of three independent experiments. Standard error,. p < 0.05. Panel C USP33 knockdown efficiency assay. Cells were transfected with siRNA from USP33 for 60 hours before detection.
FIG. 9 is a graph showing the results of CCCP-induced mitochondrial autophagy promoted by knockdown of USP33, detected after transfection of siUSP33 or siControl48 hours in cells stably expressing GFP-Parkin. Panel A shows CCCP treatment of cells for 12 hours, cells were harvested and treated with 4% paraformaldehyde for 30 minutes, then 0.1% Triton X-100 for 15 minutes, and analyzed by confocal microscopy. Panel B shows U2OS-GFP-Parkin cells treated with 20. mu.M CCCP for 12 hours, and after collection of the cells for lysis, changes in protein expression of USP33, TOM20 and GFP-Parkin were detected by immunoblot analysis. The bar graph is a quantitative plot of the fluorescence of the mitochondrial protein TOM20 for the different treatments in panel a. For data from three independent experiments, no less than 100 cells were counted in each case. Standard error, # p < 0.01.
FIG. 10 is a schematic representation of the promotion of the clearance of damaged mitochondrial DNA by knock-down of USP33, panel A showing the initial knock-down of the gene of interest using adenovirus shUSP33 in GFP-Parkin stably expressed cells, treatment of the cells with oligomycin/antimycin A, collection of the cells at 6 and 12 hours, fixation with 4% paraformaldehyde for 30 minutes, and permeabilization of the cells at 0.1% Triton X-100 for 15 minutes, and confocal microscopy of the cells. The USP33 knockdown cells cleared more mtDNA than the control 12 hours after oligomycin/antimycin A treatment. Panel B shows the construction of recombinant adenovirus vectors capable of knocking down USP33 using homologous recombination technology. Immunoblot analysis knockdown efficiency detection. The histogram is a quantification of mitochondrial DNA fluorescence (total DNA fluorescence-fluorescence of nuclei) for the different treatments of Panel A.
FIG. 11 is a graph showing the results of deubiquitinase USP33 deubiquitinating Parkin, Panel A shows co-transfection of Flag-Parkin, HA-UB and GFP-USP33 in HEK293 cells, followed by treatment of the cells with MG132 for 3 hours, incubation of cell lysates with anti-Flag beads, and immunoblot analysis. Panel B shows the transfection of USP33 iRNA in U2OS cells stably expressing Flag-Parkin, followed by immunoblot analysis after 48 hours.
FIG. 12 is a graph showing the results of mutations USP33 that did not affect ubiquitination of Parkin, co-transfecting Flag-Parkin, HA-UB, GFP-USP33 or GFP-USP33-C194S-H683Q in HEK293 cells, treating the cells with MG132 for 3 hours, incubating the cell lysates with anti-Flag resin, and performing immunoblot analysis.
Fig. 13 shows that defubiquitinase USP33 removes ubiquitinated chains linked to Parkin proteins K6, K11, K48 and K63. Different ubiquitinated chains are covalently bound to substrate proteins, mediating different signaling pathways, e.g., the K48 chain primarily mediates protein degradation and the K63 chain primarily activates downstream signaling pathways. HA-UB, Flag-Parkin, HA-UB and GFP-USP33 at different mutation sites were co-transfected in HEK293 cells, and then cells were treated with MG132 for 3 hours and cell lysates were incubated with anti-Flag resin. Binding indicates that USP33 affects self-ubiquitination of Parkin protein through K6, K11, K48 and K63 ubiquitinated chains.
FIG. 14 is a graph showing the results of USP33 regulating the stability of Parkin protein, knocking down USP33 in U2OS cells, then treating the cells with Cycloheximide (CHX), collecting the cells at different time points, and analyzing the trend of USP33 and Parkin with the treatment time. The results indicate that the half-life of Parkin protein is significantly extended in USP33 knockdown cells, indicating that the knockdown energy of USP33 enhances the protein stability of Parkin. The right panel is a quantification plot of the Parkin protein of the left panel.
FIG. 15 is a verification diagram of USP33 for promoting deubiquitination of Parkin protein in vitro, wherein A is a graph showing that GST-Parkin, HA-UB, E1 and E2-UBCH7 proteins purified in vitro are added into a reaction system in an in vitro reaction, reacted at 37 ℃ for 2 hours, added with 1 XSDS loading buffer solution and boiled for 5 minutes, and then subjected to immunoblotting analysis. Ponceau staining revealed GST-Parkin. Panel B shows the in vitro reaction procedure in which purified GST-Parkin, GST-USP33, HA-UB, E1 and E2-UBCH7 proteins are added to the reaction system. Ponceau staining revealed GST-Parkin and GST-USP33 (arrows).
FIG. 16 is a schematic diagram showing the action of USP33 deubiquitination on the site of Parkin K435, co-transfection of Flag-Parkin-K48R or Flag-Parkin-K435R plasmid and HA-UB into HEK293 cells, transfection of siControl and siUSP33, collection of cells, incubation of Flag resin with cell lysis solution, and immunoblot analysis.
FIG. 17 is a graph of the results of knocking down USP33 to enhance Parkin self-ubiquitination, after transfection of siControl and siUSP33 in a Flag-Parkin over-expressed stable cell line, and 48 hours later, treatment of the cells with CCCP, and collection of the cells at different time points. Cells were directly subjected to immunoblot analysis. Knock-down of USP33 resulted in an increased level of ubiquitination of Parkin, consistently above the control, with increasing CCCP treatment time.
FIG. 18 is a graph showing the results of suppressing SH-SY5Y cell apoptosis by knocking down USP33 after MPTP treatment, and transfecting siRNA of USP33 in SH-SY5Y cells, followed by treating the cells with MPTP drug. After the cells were collected, they were analyzed by apoptosis kit and flow cytometer. The left panel is a graph of the detection result of the flow cytometer, and the right panel is a quantitative graph of three experiments, a control graph under MPTP treatment at different concentrations and a quantitative graph of an experiment of the apoptosis of a USP33 knockdown group. After MPTP treatment, the apoptosis rate of SH-SY5Y of nerve cells of a USP33 knock-down group is obviously lower than that of a control group, which indicates that the occurrence of mitochondrial autophagy is promoted by knocking-down USP33, so that the apoptosis of the cells is reduced, and the normal life activity of the cells is protected.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. The sequences of the biomaterials, reagents, and primers used in the examples are shown below.
TABLE 1 cell line information
Figure BDA0002012822630000101
Figure BDA0002012822630000111
TABLE 2 plasmid information
Figure BDA0002012822630000112
TABLE 3 antibody information
Figure BDA0002012822630000113
Figure BDA0002012822630000121
TABLE 4 primer sequences
Figure BDA0002012822630000122
Figure BDA0002012822630000131
TABLE 5 Main reagents and kits
Figure BDA0002012822630000132
The apoptosis detection adopted by the embodiment of the invention is as follows:
1) old cell culture medium was aspirated into a 15ml centrifuge tube for use. After the cells are washed once by PBS, pancreatin is added into the culture dish, the time for the pancreatin to digest the cells needs to be well held, and the detection of cell apoptosis is interfered because false positive of cell necrosis is generated due to easy damage of cell membranes.
2) Adding the cell culture solution collected in the step 1), slightly blowing and collecting the cells, adding the cells into the centrifuge tube again, centrifuging for 5min at room temperature of 1,000g, completely sucking the supernatant by using a vacuum pump, re-suspending the cell sediment by using new PBS, sucking 10 mu l of cell suspension, putting the cell suspension on a cell counting plate, and calculating the number of the cells.
3) Aspirate 5 to 10 million resuspended cells into a new 1.5ml EP tube and centrifuge at 1,000g room temperature for 5min, a procedure that may be performed primarily on a common laboratory bench without the need for a clean bench top procedure. Resuspend cells in 195. mu.l binding solution. Note that the following steps all need to be protected from light.
4) Add 2. mu.l Annexin V-FITC solution, taking care to mix the reagents quickly and gently with the resuspended cells.
5) Add 5. mu.l PI staining solution and mix gently.
6) The EP tube is wrapped by aluminum foil paper and is protected from light, the tube is incubated for 20 minutes at room temperature in the protected light, and cells are continuously resuspended for 2-3 times in the middle to facilitate staining.
7) If the cell suspension is used for flow cytometry detection, the cell suspension is transferred to a special tube for the flow cytometer, placed on ice and immediately subjected to flow cytometry detection. If used for fluorescence microscopy, the cells were collected by centrifugation at 1,000g for 5min, resuspended in 100. mu.l Annexin V-FITC conjugate, and visualized by smear.
Unless otherwise specified, the methods used in the embodiments of the present invention, including RNA extraction methods, reverse transcription reactions, plasmid construction, cell culture, cell transfection, construction of stably expressed cell lines, proteinase K treatment experiments, immunofluorescence experiments, western blot experiments, extraction and purification of prokaryotic expression proteins, apoptosis detection experiments, etc., are all conventional and well-known techniques. The mitochondrial extraction kit used in the examples was the QIangen Qproteome mitochondria isolation kit. The invention is not limited to the kit, and those skilled in the art can select other mitochondria extraction kits to participate in the technical scheme of the invention to realize the technical effect of the invention.
The experiments performed in the embodiment of the invention are performed in parallel for 3-10 independent experiments, and the data difference of the experiment results conforms to statistics of P <0.05 and P <0.01, and the difference is obvious. The result data in the embodiment are average values after multiple independent experiments, the result is objective, the repeatability is good, and the statistical significance is achieved.
Example 1 Co-immunoprecipitation and Mass Spectrometry techniques to identify the interaction of Parkin with Deubiquitinase USP33
1. The invention utilizes the technology of co-immunoprecipitation combined with mass spectrum to research the interaction protein network of Parkin. In U2OS cells, pcDNA3-Flag-Parkin plasmid was transfected and cells were screened through 400ug/ml G418 to obtain a cell line stably expressing Flag-Parkin protein SEQ ID NO.2 (FIG. 1, panel A).
Selecting the number 4 cell with higher expression quantity for amplification culture, collecting the whole cell lysate, incubating anti-Flag Beads with the cell lysate, enriching Flag-Parkin interaction protein by using a co-immunoprecipitation method, separating the enriched protein sample by using SDS-PAGE gel electrophoresis (figure 1B), then staining and detecting by using Coomassie brilliant blue G250, and carrying out protein mass spectrometry. The control group was transfected with pcDNA3-Flag-vector plasmid. And analyzing a mass spectrum result, and mainly selecting proteins capable of influencing self-ubiquitination or substrate ubiquitination of the Parkin in consideration of the fact that the Parkin plays a role in mitochondrion autophagy and is mainly realized through ubiquitination regulation. Two deubiquitinases, USP33 and USP15, were found in the mass spectrum. USP15 has been reported to have no effect on ubiquitination of Parkin itself. Whether the deubiquitinase USP33 acts on Parkin or not and whether the deubiquitinase participates in the regulation of mitochondrial autophagy are not reported.
2. This example followed transient overexpression of pcDNA3-Flag-Parkin plasmid in HEK293 cells, collection of cell lysates after 36 hours, co-immunoprecipitation of Flag-Parkin protein, detection with USP33 antibody by Western blotting experiments, and experimental confirmation of the interaction of Parkin with endogenous USP33 (FIG. 2, panel A).
pcDNA3-Flag-HA-USP33 plasmid was also transiently transfected into HEK293 cells, cells were lysed after 36 hours, cell lysates were incubated with anti-HAbeads and detection was performed using the Parkin antibody, confirming that USP33 also interacts with endogenous Parkin (FIG. 2, panel B). This is in contrast to USP15, where exogenous USP15 had an interaction with exogenous Parkin, but no interaction was detected between endogenous proteins. The deubiquitinase USP20 is a homologue of USP33, the deubiquitinase USP20 is a homologue of the USP33, the deubiquitinase USP33 and the deubiquitinase USP33 are cooperatively involved in regulating and controlling multiple pathways such as hypoxia induction, endocytosis pathways and the like in cells, Flag-USP20 expression plasmids are cloned and constructed, HEK293 cells are transfected, and the interaction between the USP20 and endogenous Parkin is found by utilizing co-immunoprecipitation and Western blot analysis. There is an article reporting that the knock-down of USP20 has no effect on the occurrence of mitophagy, and therefore the inventors speculated that USP20 is not involved in the process of mitophagy (see fig. 2, panel D). The results provide basis for later detection of functional relation between USP33 and Parkin.
GST Pull Down experiment: and expressing and purifying the GST fusion protein. It should be noted that the purified GST fusion protein can be used in the next experiment without elution. Protein lysates were prepared, a portion was left as Input, and the remainder was mixed with the GST fusion protein. Rotate at 4 ℃ for 3 h. Centrifuge at 500g for 2min and wash 3 times with pre-cooled PBS. Adding 1 xSDS Loading buffer, and boiling at 80 deg.C for 10 min. The sample is used for detection.
In this example, pGEX-6p-GST-USP33 and pET-28b-His-Parkin prokaryotic expression plasmids were constructed, respectively, and the two plasmids were overexpressed in E.coli BL21, respectively, and GST-USP33 and His-Parkin proteins were purified using anti-GST agarose and Ni-NTA agarose, respectively. Incubation of both proteins with anti-GST agarose mixed with each other, no Parkin was detected in the two protein alone samples, while Parkin was detected in the two mixed samples, precluding the interaction between the proteins and agarose, demonstrating the direct interaction between USP33 and Parkin (FIG. 2, panel C).
3. This example truncates pcDNA3-Flag-Parkin and constructs expression plasmids containing different functional domains: Flag-UBL-Linker, Flag-RING0, Flag-RING1, Flag-IBR and Flag-RING 2. The method comprises the steps of transiently transfecting pcDNA3-HA-USP33 and expression plasmids of different functional domains in HEK293 cells, collecting the cells for lysis, incubating anti-Flag agarose and cell lysate for 3 hours, using a co-immunoprecipitation method to obtain a Pull-down protein complex, and analyzing by a Western blotting experiment. As shown in FIG. 3, the interaction of Flag-UBL-Linker and Flag-RING2 with USP33 was strong, whereas the interaction of Flag-RING1 with USP33 was weak, and other areas had no interaction. Structural analysis of Parkin shows that the number of lysine residues in the Flag-UBL-Linker and Flag-RING2 regions is large, and that the UBL region is a ubiquitin-like structure and likely to cause ubiquitination.
Meanwhile, the invention also truncates USP33, constructs Flag-Zinc finger, Flag-USP, Flag-DUSP1 and Flag-DUSP2 expression plasmids respectively, cotransfects pcDNA3-HA-Parkin plasmid and the plasmids into HEK293 cells, and also utilizes the co-immunoprecipitation method. As shown in FIG. 4, only Flag-USP interacts with Parkin. The USP domain is the core domain of USP33, and comprises two short and conserved structures, a histidine box and a lysine box, which can remove ubiquitin molecules from the action protein. Indicating that the USP functional domain is the key domain of the interaction of USP33 with Parkin.
Example 2 mitochondrial localization of USP33 protein
1.USP33 is located in the outer mitochondrial membrane
The localization of endogenous USP33 in cells was confirmed by immunofluorescence experiments, and in U2OS cells, mitochondria were labeled with Tom20 (mitochondrial outer membrane protein) antibody, and U2OS cells were labeled with USP33 antibody, as shown in panel a of fig. 5, and as a result, significant co-localization of endogenous USP33 and mitochondrial protein Tom20 was found, indicating that USP33 protein is localized on mitochondria. Next, the HEK293 cell was subjected to cell fraction separation, as shown in the B-diagram of fig. 5, no contamination of the nucleus Lamin B and the cytoplasm Tubulin was detected in the mitochondrial fraction, indicating that the purity of the separated mitochondria was good and that it could be used in the next experiment. Meanwhile, Western blotting experiments confirmed that USP33 is present in mitochondria, which is consistent with the observation in panel A of FIG. 5 on immunofluorescence. The separated cellular components, nucleus, are labeled with Lamin B, cytoplasm with Tubulin, and mitochondria with VDAC.
To further confirm which part of mitochondria USP33 is located, the present invention performed proteinase K degradation experiments, where proteinase K degrades outer membrane proteins and protects inner membrane proteins and membrane gap proteins. Mitochondrial fractions were isolated from HEK293 cells, purified mitochondria were diluted to a concentration of 1ug/ul, placed on ice, and mitochondria treated with 100ng/ml proteinase K for 30min, normally with degradation of mitochondrial outer membrane proteins and no degradation of the transmembrane and inner membrane proteins, which experiments showed that USP33 and the outer membrane protein VDAC were degraded, but neither the mitochondrial outer membrane protein VDAC nor the transmembrane gap protein SMAC were degraded when treated with 1% Triton-100. When both treated mitochondria, the protein of the gap of the membrane SMAC is degraded, and the result shows that USP33 is positioned at the outer membrane of mitochondria, which is shown in a graph C of figure 5.
2. The transmembrane domain 549-569AA (TM) is key to the localization of mitochondria by USP33
Normally, the mitochondrially localized precursor protein will have at its N-terminus a cleavable signal sequence, called a mitochondrially localized signal peptide, which binds to a receptor on the outer mitochondrial membrane and facilitates transport of the protein into the mitochondria. The invention firstly uses computer prediction software TargetP to analyze and predict the mitochondrial subcellular localization of USP33, the score is lower, and the mitochondrial localization signal is presumed not to exist. The software TMpred (http:// www.ch.embnet.org/software/TMPRED _ form. html) was then used to predict the transmembrane domain of USP33, as shown in Panel A of FIG. 6, USP33 has a transmembrane domain at 549 569AA, referred to as the TM (transmembrane domain) sequence of USP 33. In order to verify the importance of the TM sequence on the distribution of USP33 in mitochondria, GFP-USP33 and GFP-USP 33-delta TM plasmid with deletion of the TM sequence are respectively constructed, HEK293 cells are transiently transfected, and cell components are separated. The results showed that the USP33 mutant protein lacking the TM sequence was distributed only in the cytoplasm compared to the control wild-type USP33 both in the cytoplasm and mitochondria (FIG. 6, panel B). While U2OS cells were also transiently transfected with GFP-USP33 and GFP-USP33- Δ TM plasmids, immunofluorescence revealed that wild-type GFP-USP33 co-localized with mitochondria, but TM sequence-deleted USP33 appeared in diffuse distribution in the cells (FIG. 6, panel C). Combining the results of Western blotting experiments and immunofluorescence experiments, the TM sequence (549-569AA) is necessary for the localization of USP33 to mitochondria.
Example 3 knockdown of USP33 promotes GFP-Parkin translocation to mitochondria
When CCCP (carboxyl cyanine 3-chlorophenylhydrazone, inhibitor of mitochondrial oxidative phosphorylation) is used for treating cells, mitochondria are damaged to depolarize, a large amount of Parkin protein is recruited to mitochondria, and mitochondrial autophagy is started. To investigate whether the knockdown of USP33 affected the process of mitochondrial autophagy initiation, the GFP-Parkin plasmid was constructed and transfected into U2OS cells to establish a GFP-Parkin stably expressing cell line.
After treating the GFP-Parkin stably expressing cells with different concentrations of CCCP for 2 hours, and observing the GFP-Parkin translocation, as shown in FIG. 7, the GFP-Parkin was aggregated and translocated in most cells at concentrations of 20. mu.M and 40. mu.M compared to the control group, whereas the GFP-Parkin was aggregated and translocated only rarely at 10. mu.M, the inventors selected CCCP at a concentration of 20. mu.M for the next experiment.
siControl and siUSP33 siRNAs were transfected separately into GFP-Parkin stably expressing cells (see corresponding siRNA sequences in Table 4), and after 36 hours the transfected cells were plated, mostly plated and a small portion left for knock-out detection. Cells were treated with CCCP and collected at various time points for immunofluorescence detection. As shown in fig. 8, protein expression levels of USP33 were significantly reduced in USP33 knockdown cells. The co-localization of green fluorescent-tagged Parkin and red fluorescent-tagged TOM20 was found in USP33 knockdown cells at around 15 minutes of CCCP treatment, indicating that GFP-Parkin has translocated to mitochondria (ratio of about 7.38%), which continued for up to about 80 minutes, the proportion of cells translocating GFP-Parkin protein was significantly higher than the control, and after 90 minutes, the proportion of cells translocating GFP-Parkin protein was not much different (USP33 knockdown proportion of cells 83.68%, control proportion of cells 83.43%). The above results indicate that knocking down the protein USP33 promotes the recruitment of GFP-Parkin to mitochondria, promoting the development of early mitophagy.
Example 4 knockdown of USP33 promotes mitophagy
To investigate whether USP33 affected the clearance of mitochondria from the end-stage of mitophagy injury, the inventors first transfected siControl and siUSP33 sirnas in GFP-Parkin stably expressed cells, respectively, and collected protein samples 12 hours after 20 μ M CCCP treatment of the cells. As shown in fig. 9, CCCP treatment decreased TOM20 protein in cells of the control group, indicating that mitophagy could occur under CCCP treatment, whereas TOM20 protein decreased more than in cells with USP33 knockdown, and immunofluorescence was performed while marking mitochondria with the mitochondrial outer membrane protein TOM20, and cells treated with CCCP for 12 hours, which was statistically found to have about twice as many cells cleared from the control group compared to the knockdown group of USP 33. The results show that knockdown of USP33 accelerates the rate of cell clearance damaging mitochondria.
In order to better study the effect of USP33 on mitophagy, the inventors constructed recombinant adenovirus vectors capable of knocking down USP33 in Escherichia coli BJ5183 bacteria by using a homologous recombination technology.
The construction process of the adenovirus shRNA comprises the following steps: designing a target site according to the experiment requirements, and connecting the target site with the pshuttle _ U2 plasmid to construct a required vector. The plasmid pshuttle _ U2 was linearized with a single cut of PmeI. Preparing electric transformation, firstly melting the pAdEasy-containing BJ5183 on ice, adding the linearized plasmid into the competent cells, mixing and standing for 5-10min, performing electric transformation, wherein after the electric transformation, an nonreactive LB culture medium is quickly added, culturing for 1h in a shaker at 37 ℃, centrifuging for 6,000rpm for 2min, plating, and picking out monoclonal culture for enzyme digestion identification the next day. And transferring the obtained positive recombinant adenovirus plasmid into DH5 alpha again, carrying out amplification culture to extract the plasmid, and purifying. Recombinant adenovirus positive plasmids were extracted and linearized by restriction with PacI. HEK293 cells were prepared 24h in advance and plated in 6-well plates to ensure that the next day cell concentrations were 80-90%. The linearized plasmid was transfected into HEK293 cells using Lipo 2000. After culturing for 10-14 days, most cells are subjected to plaque, and even most cells are shed. Directly blowing up the adherent cells, putting the cells into a 15ml centrifuge tube, and repeatedly freezing and thawing for 3 times at the temperature of liquid nitrogen-37 ℃. Centrifuging at 12,000g for 5min, collecting supernatant, storing at-80 deg.C, and labeling for one generation. The first generation virus solution was added to a60 mm petri dish and cultured for approximately 3-5 days until-70% of the cells were detached. Repeating the steps 10) and 11) to mark as the second generation, and then expanding and culturing the first generation to obtain the knock-down effect of the detection gene of the infection target cell.
The recombinant adenovirus vector has the advantages of capability of large-scale amplification in HEK293 cells, high transfection efficiency, wide infected cell range, high virus titer and the like. The constructed shControl (SEQ ID NO.3) and shUSP33(SEQ ID NO.4 and SEQ ID NO.5) virus solutions are respectively added into U2OS cells, and the knockdown effect is detected after 48 to 72 hours, as shown in B in figure 10, compared with a control group, the USP33 protein expression quantity of cells infected with shUSP33 adenovirus is obviously reduced, USP33 protein is very effectively knocked down, the siRNA knockdown efficiency is not greatly different from that of USP33, and the USP33 adenovirus construction success is shown and can be used for the next experiment.
Another indicator to assess the efficiency of mitophagy development is qualitative and quantitative analysis of mtDNA. The mitophagy-inducing drug Oligomycin is an ATP synthase inhibitor that inhibits oxidative phosphorylation and ATP-dependent responses. Antiminin A can bind to the mitochondrial electron transport chain, severely blocking the electron transport of CoQ to cytochrome C. When Oligomycin and Antimycin A treat cells, mitochondria are damaged to oxidize respiratory chains, so that the ROS level of the mitochondria is increased, and mitochondrial DNA mutation is increased, thereby inducing the occurrence of mitochondrial autophagy. As shown in A in FIG. 10, shUSP33 and shControl virus solution are respectively added into GFP-U2OS cells, the cells are plated after 48 hours, Oligomycin and Antimycin A medicines are added after cell climbing, the cells are collected at different time points for immunofluorescence observation, the longer the medicine treatment time is, the more DNA in mitochondria disappears, and the DNA disappearance speed in mitochondria is obviously higher than that in the control group in USP33 knocked-down cells. The results further demonstrate that the knockdown of USP33 can promote the clearance of damaged mitochondria, suggesting that USP33 plays a key role in the development of mitophagy.
Example 5 Deubiquitinase USP33 is able to modulate the ubiquitination modification of Parkin
As an E3 ubiquitin ligase, the Parkin-mediated ubiquitin-proteolytic enzyme system plays an important role in the development of mitophagy. Example 1 demonstrates the direct interaction of USP33 with Parkin by co-immunoprecipitation experiments, to reveal how deubiquitinase USP33 interacts with Parkin to regulate the mechanism of mitochondrial autophagy, the inventors co-transferred GFP-USP33 or GFP-vector and Flag-Parkin and HA-UB plasmids in HEK293 cells, collected whole cell fractions 48 hours after transfection, co-immunoprecipitated with anti-Flag agarose, and detected the ubiquitination level of Parkin by HA antibody. As shown in fig. 11, USP33 was able to significantly promote deubiquitination of Parkin protein. On the other hand, an overexpression stable cell line of Flag-Parkin is constructed, siControl and siUSP33 are transfected in the cell respectively, and detection for 48 hours shows that the knocking energy of USP33 can obviously inhibit the de-ubiquitination of Parkin and the ubiquitination level of Parkin is obviously increased. The above results indicate that the regulation of Parkin by USP33 is achieved by ubiquitination modification.
Example 1 demonstrates that USP33 interacts directly with Parkin cells primarily through the USP domain. Mutations at C194S and H683Q of the USP domain cause loss of deubiquitinating enzyme activity of USP 33. In this example, the inventors constructed GFP-USP33-C194S-H683Q double mutant plasmid, transiently transfected into HEK293 cells with Flag-Parkin and HA-UB plasmids, and found that Parkin co-transfected with wild-type USP33 could be significantly de-ubiquinated, while Parkin co-transfected with double mutant USP33 could not be de-ubiquinated, further demonstrating that USP33 is a de-ubiquitinase of Parkin and that USP domain is essential for the interaction of both (fig. 12).
Example 6 Deubiquitinase USP33 removes the ubiquitinated chains of the Parkin proteins K6, K11, K48 and K63 linked
To investigate which ubiquitination chain the Parkin itself can be modified and which ubiquitination chain USP33 can affect, this example first single mutation of the 7 lysine residues of HA-UB to arginine residues and a full mutation (all of the 7 lysine residues are mutated to arginine residues) respectively followed by transfection of the mutated HA-UB plasmid and the wild-type HA-UB plasmid in HEK293 cells, transient transfection of Flag-Parkin, GFP-USP33 or HA-GFP plasmid, and collection of samples for testing after 48 hours. As shown in fig. 13, the Parkin plasmid can be modified by 7 ubiquitinated chains, with more modifications of the K11, K29, K48, and K63 chains. USP33 overexpression can significantly affect wild type, K6, K11, K48 and K63 linked ubiquitinated chains with little effect on other ubiquitin chain modifications.
Example 7 knockdown of USP33 promotes Parkin protein stability
Previous experiments have demonstrated that USP33 acts on multiple polyubiquitinated chains of Parkin, and in order to investigate whether polyubiquitination modification affects Parkin stability, Cycloheximide (CHX) experiments were performed in this example. shUSP33 and shControl virus liquid are respectively added into U2OS cells, a ribosome inhibitor cycloheximide is added after 24 hours, the cells are collected at different time points, and the expression quantity of the Parkin protein is detected. The results found that the half-life of Parkin protein was significantly prolonged in USP33 knockdown cells relative to the control, indicating that the knockdown of USP33 was able to enhance the stability of Parkin protein (fig. 14).
Example 8 USP33 promotes Deubiquitination of the Parkin protein in vitro
Parkin is an E3 ubiquitin ligase that is capable of ubiquitinating not only substrate proteins but also self-ubiquitination. Previous findings indicate that USP33 is able to de-ubiquitinate and modulate Parkin in vivo, and that the knockdown of USP33 promotes stability of Parkin protein. To further validate the results of the above studies, this example performed in vitro ubiquitination experiments.
In experimentsIn the experiment, the protein GST-Parkin and GST-USP33 are respectively overexpressed by using escherichia coli BL21, and GST-USP33 and GST-Parkin protein are respectively purified by using anti-GST agarose, and then an in-vitro ubiquitination experiment is carried out by using the purified recombinant protein and an enzyme compound, wherein Parkin is E3 ubiquitin ligase and can carry out self ubiquitination without adding E3 ligase. First, ubiquitin ligation reaction system (25mM Tris-HCl pH7.4, 2mM ATP, 0.4mM DTT, 1mM MgCl) was prepared2) Next, purified GST-Parkin and GST-USP33, HA-UB, E1 and E2 were put into the reaction system. The reaction was carried out at 37 ℃ for 2 hours, after the reaction, 1 XSDS loading Buffer was added to terminate the reaction, boiling was carried out at 80 ℃ for 10 minutes, and Western blotting was carried out on protein samples using 8% SDS PAGE gel. As shown in fig. 15, Parkin is able to self-ubiquitinate in the presence of UB without the need for additional E3 ubiquitin ligase. Purified GST-USP33 protein was then added to the in vitro ubiquitination system, and the ubiquitination level of Parkin was reduced. The results indicate that USP33 enables deubiquitination modification of Parkin both in vivo and in vitro.
Example 9K 435 site important for Parkin ubiquitination modification
Several studies have shown that the E3 ubiquitin ligase Parkin can self-ubiquitinate, but it is not known which specific lysines can ubiquitinate, whether USP33 acts at these sites. To investigate the above problem, the Parkin amino acid sequence was first analyzed in this example, and 18 lysine sites were found in total. And then sequentially constructing plasmids with single lysine site mutation, transiently transfecting the constructed mutant Parkin plasmids or wild Parkin plasmids and HA-UB into HEK293 cells, collecting the cells after 36-48 hours for lysis, and analyzing by using co-immunoprecipitation and Western blotting experiments. When the K48 and K435 sites of Parkin are mutated, the ubiquitination level of Parkin is obviously reduced, and other sites are not changed or are not obviously changed.
The Flag-Parkin-K48R or Flag-Parkin-K435R plasmid and HA-UB were co-transfected into HEK293 cells, siControl and siUSP33 were transfected respectively, and knocking down USP33 by co-immunoprecipitation and Western blotting experimental analysis, the ubiquitination levels of wild type and K48R mutant Parkin were both increased and differed little, while the ubiquitination level of K435R mutation was significantly lower than that of wild type Parkin and differed little from that of USP33 untaken group (FIG. 16). The experiment proves that USP33 deubiquitination acts on the K435 site of Parkin.
Example 10 USP33 is able to regulate the self-ubiquitination of Parkin during the development of mitophagy
The present examples have demonstrated that USP33 knockdown promotes GFP-Parkin translocation to mitochondria during the early stages of mitophagy initiation. In the early days, Parkin was involved by ubiquitination-modified forms, and it was speculated that USP33 could be involved in the development of mitophagy by modulating Parkin ubiquitination-modified forms. To test this hypothesis, this example transfects siControl and siUSP33 in a stable cell line with Flag-Parkin overexpression, divides the cells into six well plates for 24 hours, treats the cells with CCCP, and collects the cells at different time points. As shown in FIG. 17, the ubiquitination level of Parkin in control cells increased with increasing CCCP treatment time, consistent with literature reports (Durcan et al, 2014; Chan et al, 2011). Compared with the cells of the control group, the USP33 knockdown group has higher ubiquitination level of Parkin than the control group. The results indicate that USP33 can be involved in the development of mitophagy by modulating the Parkin ubiquitination-modified form.
Example 11 USP33 knockdown inhibits SH-SY5Y apoptosis following MPTP treatment
MPTP (1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine) is neurotoxin, MPTP is nontoxic, but can penetrate blood brain barrier, and is metabolized into toxic MPP + under the action of monoamine oxidase B (monoamine oxidase B) generated by glial cells, and MPP + can kill dopaminergic neurons. In addition, MPP + can interfere NADH dehydrogenase, so that a large amount of free radicals are accumulated, mtDNA mutation is increased, mitochondrial dysfunction is caused, and apoptosis is caused to generate symptoms similar to Parkinson's disease. SH-SY5Y cell is a human neuroblastoma cell line and is a better cell model for researching neurodegenerative diseases. Research reports that mitophagy plays an important role in the occurrence and development of Parkinson. The inventors speculate that USP 33-mediated mitophagy prevents the onset of parkinson's disease by clearing damaged mitochondria. To confirm this hypothesis, the present example first transfected siControl and siUSP33 into SH-SY5Y cells, added 0.5mM MPTP 24 hours later, followed by flow-based detection of apoptosis rate using the apoptosis kit Annexin V/PI staining. As shown in fig. 18, the apoptosis rate of the control group was 10.41% and that of the USP33 knock-down group was 7.56% in the absence of MPTP treatment, and the apoptosis rate of the control group was increased by 23.06% after the drug treatment, and the apoptosis rate of the USP33 knock-down group was 12.67% lower than that of the control group. Indicating that USP33 knockdown can reduce SH-SY5Y apoptosis after MPTP treatment, suggesting that USP 33-mediated mitophagy plays a role in the development of parkinson's disease.
Sequence listing
<110> Beijing institute of genomics of Chinese academy of sciences
Application of USP33 serving as medication target in preparation of medicines
<130> KHP191110297.1
<160> 45
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Met Thr Gly Ser Asn Ser His Ile Thr Ile Leu Thr Leu Lys Val Leu
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Pro His Phe Glu Ser Leu Gly Lys Gln Glu Lys Ile Pro Asn Lys Met
20 25 30
Ser Ala Phe Arg Asn His Cys Pro His Leu Asp Ser Val Gly Glu Ile
35 40 45
Thr Lys Glu Asp Leu Ile Gln Lys Ser Leu Gly Thr Cys Gln Asp Cys
50 55 60
Lys Val Gln Gly Pro Asn Leu Trp Ala Cys Leu Glu Asn Arg Cys Ser
65 70 75 80
Tyr Val Gly Cys Gly Glu Ser Gln Val Asp His Ser Thr Ile His Ser
85 90 95
Gln Glu Thr Lys His Tyr Leu Thr Val Asn Leu Thr Thr Leu Arg Val
100 105 110
Trp Cys Tyr Ala Cys Ser Lys Glu Val Phe Leu Asp Arg Lys Leu Gly
115 120 125
Thr Gln Pro Ser Leu Pro His Val Arg Gln Pro His Gln Ile Gln Glu
130 135 140
Asn Ser Val Gln Asp Phe Lys Ile Pro Ser Asn Thr Thr Leu Lys Thr
145 150 155 160
Pro Leu Val Ala Val Phe Asp Asp Leu Asp Ile Glu Ala Asp Glu Glu
165 170 175
Asp Glu Leu Arg Ala Arg Gly Leu Thr Gly Leu Lys Asn Ile Gly Asn
180 185 190
Thr Cys Tyr Met Asn Ala Ala Leu Gln Ala Leu Ser Asn Cys Pro Pro
195 200 205
Leu Thr Gln Phe Phe Leu Asp Cys Gly Gly Leu Ala Arg Thr Asp Lys
210 215 220
Lys Pro Ala Ile Cys Lys Ser Tyr Leu Lys Leu Met Thr Glu Leu Trp
225 230 235 240
His Lys Ser Arg Pro Gly Ser Val Val Pro Thr Thr Leu Phe Gln Gly
245 250 255
Ile Lys Thr Val Asn Pro Thr Phe Arg Gly Tyr Ser Gln Gln Asp Ala
260 265 270
Gln Glu Phe Leu Arg Cys Leu Met Asp Leu Leu His Glu Glu Leu Lys
275 280 285
Glu Gln Val Met Glu Val Glu Glu Asp Pro Gln Thr Ile Thr Thr Glu
290 295 300
Glu Thr Met Glu Glu Asp Lys Ser Gln Ser Asp Val Asp Phe Gln Ser
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Cys Glu Ser Cys Ser Asn Ser Asp Arg Ala Glu Asn Glu Asn Gly Ser
325 330 335
Arg Cys Phe Ser Glu Asp Asn Asn Glu Thr Thr Met Leu Ile Gln Asp
340 345 350
Asp Glu Asn Asn Ser Glu Met Ser Lys Asp Trp Gln Lys Glu Lys Met
355 360 365
Cys Asn Lys Ile Asn Lys Val Asn Ser Glu Gly Glu Phe Asp Lys Asp
370 375 380
Arg Asp Ser Ile Ser Glu Thr Val Asp Leu Asn Asn Gln Glu Thr Val
385 390 395 400
Lys Val Gln Ile His Ser Arg Ala Ser Glu Tyr Ile Thr Asp Val His
405 410 415
Ser Asn Asp Leu Ser Thr Pro Gln Ile Leu Pro Ser Asn Glu Gly Val
420 425 430
Asn Pro Arg Leu Ser Ala Ser Pro Pro Lys Ser Gly Asn Leu Trp Pro
435 440 445
Gly Leu Ala Pro Pro His Lys Lys Ala Gln Ser Ala Ser Pro Lys Arg
450 455 460
Lys Lys Gln His Lys Lys Tyr Arg Ser Val Ile Ser Asp Ile Phe Asp
465 470 475 480
Gly Thr Ile Ile Ser Ser Val Gln Cys Leu Thr Cys Asp Arg Val Ser
485 490 495
Val Thr Leu Glu Thr Phe Gln Asp Leu Ser Leu Pro Ile Pro Gly Lys
500 505 510
Glu Asp Leu Ala Lys Leu His Ser Ser Ser His Pro Thr Ser Ile Val
515 520 525
Lys Ala Gly Ser Cys Gly Glu Ala Tyr Ala Pro Gln Gly Trp Ile Ala
530 535 540
Phe Phe Met Glu Tyr Val Lys Arg Phe Val Val Ser Cys Val Pro Ser
545 550 555 560
Trp Phe Trp Gly Pro Val Val Thr Leu Gln Asp Cys Leu Ala Ala Phe
565 570 575
Phe Ala Arg Asp Glu Leu Lys Gly Asp Asn Met Tyr Ser Cys Glu Lys
580 585 590
Cys Lys Lys Leu Arg Asn Gly Val Lys Phe Cys Lys Val Gln Asn Phe
595 600 605
Pro Glu Ile Leu Cys Ile His Leu Lys Arg Phe Arg His Glu Leu Met
610 615 620
Phe Ser Thr Lys Ile Ser Thr His Val Ser Phe Pro Leu Glu Gly Leu
625 630 635 640
Asp Leu Gln Pro Phe Leu Ala Lys Asp Ser Pro Ala Gln Ile Val Thr
645 650 655
Tyr Asp Leu Leu Ser Val Ile Cys His His Gly Thr Ala Ser Ser Gly
660 665 670
His Tyr Ile Ala Tyr Cys Arg Asn Asn Leu Asn Asn Leu Trp Tyr Glu
675 680 685
Phe Asp Asp Gln Ser Val Thr Glu Val Ser Glu Ser Thr Val Gln Asn
690 695 700
Ala Glu Ala Tyr Val Leu Phe Tyr Arg Lys Ser Ser Glu Glu Ala Gln
705 710 715 720
Lys Glu Arg Arg Arg Ile Ser Asn Leu Leu Asn Ile Met Glu Pro Ser
725 730 735
Leu Leu Gln Phe Tyr Ile Ser Arg Gln Trp Leu Asn Lys Phe Lys Thr
740 745 750
Phe Ala Glu Pro Gly Pro Ile Ser Asn Asn Asp Phe Leu Cys Ile His
755 760 765
Gly Gly Val Pro Pro Arg Lys Ala Gly Tyr Ile Glu Asp Leu Val Leu
770 775 780
Met Leu Pro Gln Asn Ile Trp Asp Asn Leu Tyr Ser Arg Tyr Gly Gly
785 790 795 800
Gly Pro Ala Val Asn His Leu Tyr Ile Cys His Thr Cys Gln Ile Glu
805 810 815
Ala Glu Lys Ile Glu Lys Arg Arg Lys Thr Glu Leu Glu Ile Phe Ile
820 825 830
Arg Leu Asn Arg Ala Phe Gln Lys Glu Asp Ser Pro Ala Thr Phe Tyr
835 840 845
Cys Ile Ser Met Gln Trp Phe Arg Glu Trp Glu Ser Phe Val Lys Gly
850 855 860
Lys Asp Gly Asp Pro Pro Gly Pro Ile Asp Asn Thr Lys Ile Ala Val
865 870 875 880
Thr Lys Cys Gly Asn Val Met Leu Arg Gln Gly Ala Asp Ser Gly Gln
885 890 895
Ile Ser Glu Glu Thr Trp Asn Phe Leu Gln Ser Ile Tyr Gly Gly Gly
900 905 910
Pro Glu Val Ile Leu Arg Pro Pro Val Val His Val Asp Pro Asp Ile
915 920 925
Leu Gln Ala Glu Glu Lys Ile Glu Val Glu Thr Arg Ser Leu
930 935 940
<210> 2
<211> 465
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Met Ile Val Phe Val Arg Phe Asn Ser Ser His Gly Phe Pro Val Glu
1 5 10 15
Val Asp Ser Asp Thr Ser Ile Phe Gln Leu Lys Glu Val Val Ala Lys
20 25 30
Arg Gln Gly Val Pro Ala Asp Gln Leu Arg Val Ile Phe Ala Gly Lys
35 40 45
Glu Leu Arg Asn Asp Trp Thr Val Gln Asn Cys Asp Leu Asp Gln Gln
50 55 60
Ser Ile Val His Ile Val Gln Arg Pro Trp Arg Lys Gly Gln Glu Met
65 70 75 80
Asn Ala Thr Gly Gly Asp Asp Pro Arg Asn Ala Ala Gly Gly Cys Glu
85 90 95
Arg Glu Pro Gln Ser Leu Thr Arg Val Asp Leu Ser Ser Ser Val Leu
100 105 110
Pro Gly Asp Ser Val Gly Leu Ala Val Ile Leu His Thr Asp Ser Arg
115 120 125
Lys Asp Ser Pro Pro Ala Gly Ser Pro Ala Gly Arg Ser Ile Tyr Asn
130 135 140
Ser Phe Tyr Val Tyr Cys Lys Gly Pro Cys Gln Arg Val Gln Pro Gly
145 150 155 160
Lys Leu Arg Val Gln Cys Ser Thr Cys Arg Gln Ala Thr Leu Thr Leu
165 170 175
Thr Gln Gly Pro Ser Cys Trp Asp Asp Val Leu Ile Pro Asn Arg Met
180 185 190
Ser Gly Glu Cys Gln Ser Pro His Cys Pro Gly Thr Ser Ala Glu Phe
195 200 205
Phe Phe Lys Cys Gly Ala His Pro Thr Ser Asp Lys Glu Thr Ser Val
210 215 220
Ala Leu His Leu Ile Ala Thr Asn Ser Arg Asn Ile Thr Cys Ile Thr
225 230 235 240
Cys Thr Asp Val Arg Ser Pro Val Leu Val Phe Gln Cys Asn Ser Arg
245 250 255
His Val Ile Cys Leu Asp Cys Phe His Leu Tyr Cys Val Thr Arg Leu
260 265 270
Asn Asp Arg Gln Phe Val His Asp Pro Gln Leu Gly Tyr Ser Leu Pro
275 280 285
Cys Val Ala Gly Cys Pro Asn Ser Leu Ile Lys Glu Leu His His Phe
290 295 300
Arg Ile Leu Gly Glu Glu Gln Tyr Asn Arg Tyr Gln Gln Tyr Gly Ala
305 310 315 320
Glu Glu Cys Val Leu Gln Met Gly Gly Val Leu Cys Pro Arg Pro Gly
325 330 335
Cys Gly Ala Gly Leu Leu Pro Glu Pro Asp Gln Arg Lys Val Thr Cys
340 345 350
Glu Gly Gly Asn Gly Leu Gly Cys Gly Phe Ala Phe Cys Arg Glu Cys
355 360 365
Lys Glu Ala Tyr His Glu Gly Glu Cys Ser Ala Val Phe Glu Ala Ser
370 375 380
Gly Thr Thr Thr Gln Ala Tyr Arg Val Asp Glu Arg Ala Ala Glu Gln
385 390 395 400
Ala Arg Trp Glu Ala Ala Ser Lys Glu Thr Ile Lys Lys Thr Thr Lys
405 410 415
Pro Cys Pro Arg Cys His Val Pro Val Glu Lys Asn Gly Gly Cys Met
420 425 430
His Met Lys Cys Pro Gln Pro Gln Cys Arg Leu Glu Trp Cys Trp Asn
435 440 445
Cys Gly Cys Glu Trp Asn Arg Val Cys Met Gly Asp His Trp Phe Asp
450 455 460
Val
465
<210> 3
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
gaagaggaca cgccttagac t 21
<210> 4
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
gatcatgtgg cgaagcata 19
<210> 5
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
gtgcaataag attaataaag taa 23
<210> 6
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
atgatagtgt ttgtcaggtt caac 24
<210> 7
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
cacgtcgaac cagtggtc 18
<210> 8
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
atgacaggat caaattcaca c 21
<210> 9
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
caaagaccga gtttctac 18
<210> 10
<211> 34
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
cagcatcttc cagctcaggg aggtggttgc taag 34
<210> 11
<211> 34
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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cttagcaacc acctccctga gctggaagat gctg 34
<210> 12
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
caaggaggtg gttgctaggc gacagggggt tc 32
<210> 13
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
gaaccccctg tcgcctagca accacctcct tg 32
<210> 14
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
gtgattttcg cagggaggga gctgaggaat gac 33
<210> 15
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
gtcattcctc agctccctcc ctgcgaaaat cac 33
<210> 16
<211> 35
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
cagagaccgt ggagaagagg tcaagaaatg aatgc 35
<210> 17
<211> 35
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
gcattcattt cttgacctct tctccacggt ctctg 35
<210> 18
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
cacactgaca gcaggaggga ctcaccacca gc 32
<210> 19
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
gctggtggtg agtccctcct gctgtcagtg tg 32
<210> 20
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
gtattgcaga ggcccctgtc aaagagtgca gccgggaaaa ctcag 45
<210> 21
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
ctgagttttc ccggctgcac tctttgacag gggcctctgc aatac 45
<210> 22
<211> 35
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
caaagagtgc agccgggaag actcagggta cagtg 35
<210> 23
<211> 35
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
cactgtaccc tgagtcttcc cggctgcact ctttg 35
<210> 24
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
gactagtgca gaatttttct ttagatgtgg agcacacc 38
<210> 25
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
ggtgtgctcc acatctaaag aaaaattctg cactagtc 38
<210> 26
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
ctttaaatgt ggagcacacc ccacctctga cagggaaaca tc 42
<210> 27
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
gatgtttccc tgtcagaggt ggggtgtgct ccacatttaa ag 42
<210> 28
<211> 35
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
gtcccaactc cttgattaga gagctccatc acttc 35
<210> 29
<211> 35
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
gaagtgatgg agctctctaa tcaaggagtt gggac 35
<210> 30
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
ctgccgggaa tgtagagaag cgtaccatg 29
<210> 31
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
catggtacgc ttctctacat tcccggcag 29
<210> 32
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
cgttgggaag cagcctccag agaaaccatc 30
<210> 33
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
gatggtttct ctggaggctg cttcccaacg 30
<210> 34
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
ccaaagaaac catcaggaaa accaccaagc cctgtccccg c 41
<210> 35
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
gcggggacag ggcttggtgg ttttcctgat ggtttctttg g 41
<210> 36
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
gcctccaaag aaaccatcaa gagaaccacc aagccctg 38
<210> 37
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
cagggcttgg tggttctctt gatggtttct ttggaggc 38
<210> 38
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
caagaaaacc accaggccct gtccccgctg cc 32
<210> 39
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
ggcagcgggg acagggcctg gtggttttct tg 32
<210> 40
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
catgtaccag tggaaagaaa tggaggctgc atg 33
<210> 41
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 41
catgcagcct ccatttcttt ccactggtac atg 33
<210> 42
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 42
ggctgcatgc acatgaggtg tccgcagccc cag 33
<210> 43
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 43
ctggggctgc ggacacctca tgtgcatgca gcc 33
<210> 44
<211> 19
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 44
caauguuaau ucaggauga 19
<210> 45
<211> 19
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 45
uucuccgaac gugucacgu 19

Claims (6)

  1. The application of USP33 in preparing a medicine for treating the Parkin protein mediated mitochondrial autophagy related diseases; the USP33 acts on a K435 site of the Parkin protein with an amino acid sequence shown as SEQ ID NO.2 by locating on the outer membrane of mitochondria to regulate the level of mitochondrial autophagy.
  2. 2. The use according to claim 1, wherein USP33 reduces the ubiquitination level of the Parkin protein by acting on the K435 site of the Parkin protein having the amino acid sequence shown in SEQ ID No.2 to de-ubiquitinate and modify the Parkin protein, thereby reducing or preventing mitophagy.
  3. The application of USP33 in preparing a medicine for counteracting, preventing or delaying the ubiquitination process of the Parkin protein; the USP33 counteracts, prevents or delays the ubiquitination process of the Parkin protein by acting on the K435 site of the Parkin protein with the amino acid sequence shown as SEQ ID NO. 2.
  4. The application of USP33 in preparing a medicine for reducing the stability of the Parkin protein; the USP33 is positioned on the outer membrane of mitochondria and acts on the K435 site of the Parkin protein with the amino acid sequence shown as SEQ ID NO.2, so that the stability of the Parkin protein is reduced.
  5. The application of the USP33 expression promoter in preparing the drug for reducing the stability of the Parkin protein;
    the USP33 expression promoter improves or promotes the deubiquitination level of K435 site of Parkin protein with an amino acid sequence shown as SEQ ID NO.2 by improving or promoting the expression of USP33, thereby reducing the mitochondrion autophagy.
  6. Use of an inhibitor of USP33 expression, characterized in that,
    (1) the application in preparing the medicine for treating the Parkin-mediated mitochondrial autophagy related diseases;
    (2) the application in the preparation of the medicine for promoting the stability of the Parkin protein and prolonging the half-life period of the Parkin protein;
    the USP33 expression inhibitor inhibits the deubiquitination level of K435 site of Parkin protein shown in SEQ ID NO.2 through knocking down or inhibiting the expression of USP33, thereby promoting mitophagy.
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"NCBI Reference Sequence: NP_004553.2";匿名;《GenBank》;20190228;第1-3页 *
"NCBI Reference Sequence: NP_055832.3";匿名;《GenBank》;20190223;第1-3页 *
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