Use of ring finger protein 114 in preparation of medicine for treating cataract
By targeting and degrading CRYAA with cyclic finger protein 114 and recombinant protein TAT-RNF114, the problem of lens opacity in cataracts was solved, achieving effective treatment of cataracts and restoration of lens transparency, providing a biosafe drug solution.
Patent Information
- Application Number
- CN202410484102.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-04-22
- Publication Date
- 2026-01-06
- Estimated Expiration
- 2044-04-22
AI Technical Summary
Current technologies are insufficient to effectively reverse lens opacity, and postoperative loss of accommodation and complications cannot be overcome, failing to meet the need for healthy vision throughout the entire process. The pathogenesis of cataracts has not been thoroughly explored, and there is a lack of economical and effective targeted therapies.
Using cyclic finger protein 114 (RNF114) and its recombinant protein TAT-RNF114, a pharmaceutically acceptable formulation was prepared by targeting the ubiquitination and degradation of abnormally aggregated CRYAA or CRYAA mutants in lens cells and utilizing cell-penetrating peptides to promote the entry of RNF114 into cells to exert its effects.
It effectively reverses lens opacity, improves the pathological process of cataracts, reduces CRYAA aggregation, and increases lens transparency. It is suitable for various types of cataracts and has high biocompatibility.
Smart Images
Figure CN118542932B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedical technology, specifically to the pharmaceutical use of cyclic finger protein 114 in the preparation of drugs for treating cataracts. Background Technology
[0002] Cataracts are the leading cause of blindness worldwide, with their prevalence increasing significantly with age. Cataracts primarily manifest as clouding of the lens of the eye, clinically presenting as decreased vision or even blindness. Currently, surgery is the only effective treatment for cataracts; however, postoperative loss of accommodation, surgically induced aberrations, postoperative endophthalmitis, and posterior capsule opacification remain intractable problems. Surgery alone is far from sufficient to meet patients' needs for restoring healthy vision throughout their lives. Therefore, in-depth exploration of the pathogenesis of cataracts and the search for novel strategies to reverse lens opacity, especially the development of cost-effective targeted therapies, are crucial for cataract prevention and treatment.
[0003] Crystallins are responsible for maintaining the transparency of the lens and are the main soluble structural proteins of the vertebrate lens, accounting for approximately 95% of the total lens proteins. CRYAA, a subtype of the α-crystallin family, is highly abundant in the lens and plays a crucial molecular chaperone role in stabilizing the correct folding of other lens proteins. Abnormal crystallin homeostasis can easily lead to cataracts and various eye diseases. Disruption of lens protein homeostasis caused by various factors triggers abnormal protein aggregation within the lens, resulting in irreversible lens opacity, a common pathogenesis of cataracts. Oxidative stress is a key factor in the development and progression of cataracts, and the homeostasis of the redox state within the lens is fundamental to maintaining the lens's elastic structure and optical properties. Studies have shown that the lens of a cataract has a higher level of lipid peroxidation than the normal lens. Hydrogen peroxide, as an important oxidizing substance in the eye, and high concentrations of reactive oxygen species affect protein folding, modification and aggregation by promoting apoptosis of lens cells, denaturation of lens proteins and reduced solubility of lens proteins. This disrupts the homeostasis of lens proteins, thus leading to lens opacity.
[0004] The homeostasis and functional integrity of proteins within the lens require a complex protein quality control network comprised of multiple intracellular molecular mechanisms and pathways, which work synergistically to maintain protein folding, assembly, degradation, and repair. Among these, the ubiquitin-proteasome system (UPS) and autophagy are primarily responsible for degrading misfolded or aggregated proteins within the cell. However, in most regions of the lens, only UPS activity exists, without autophagy. Within the lens, UPS selectively targets abnormally structured crystallins, including oxidized, glutathioneized, thermally denatured, and truncated forms, mediating their ubiquitination and rapid hydrolysis to maintain the lens's protein turnover balance and quality control.
[0005] In the UPS process, the E3 ubiquitin ligase, a key component of the ubiquitination cascade, determines substrate specificity and diversity. CRYAA's E3 ubiquitin ligase, RING finger114 (RNF114), is a member of the zinc-binding protein family. Its gene is located on chromosome 20q13.13, with a molecular weight of 25694 Da. It contains C2H2, C2HC, and RING finger zinc-binding domains. It binds to ubiquitin through the C-terminal ubiquitin interaction motif (UIM) and exerts its E3 ligase activity through the N-terminal RING domain. Studies on the heart, liver, and kidney have reported that RNF114 protein typically functions as an E3 ligase in the ubiquitin-proteasome system, playing a crucial role in substrate ubiquitination and various cellular physiological processes. However, no related research has been previously reported in the ophthalmology field. Summary of the Invention
[0006] The purpose of this invention is to provide a drug that can effectively reverse lens opacity and alleviate abnormal pathological changes in cataracts, thereby broadening the means of cataract prevention and treatment.
[0007] To achieve the above objectives, the present invention adopts the following technical solution:
[0008] This invention provides the use of ring finger 114 (RNF114) in the preparation of drugs for treating cataracts, the amino acid sequence of which is shown in SEQ ID NO.1.
[0009] This invention demonstrates that overexpression of RNF114 in cataract lens cells or administration of an effective dose of RNF114 can effectively reverse lens opacity and improve the abnormal pathological progression of cataracts.
[0010] Furthermore, the manifestations of cataracts include abnormal aggregation of lens proteins or mutations in lens proteins within the lens cells.
[0011] Furthermore, RNF114 improves the cataract pathological process by targeting the ubiquitination and degradation of abnormally aggregated CRYAA or CRYAA mutants in lens cells.
[0012] Furthermore, the cataract is either hypothermic cataract or oxidative stress-induced cataract. Hypothermia or oxidative stress can induce cataracts. In the drug provided by this invention, RNF114 enters lens cells to mediate the ubiquitination of CRYAA and effectively degrades CRYAA aggregates, thereby maintaining lens protein homeostasis. It is worth noting that the drug provided by this invention is not limited to the two disease types mentioned above.
[0013] Furthermore, the carboxyl terminus of cyclic finger protein 114 in the drug is fused with a cell-penetrating peptide. Cell-penetrating peptides (CPPs) are a class of short peptides capable of crossing cell membranes or tissue barriers. They can transport biomolecules such as proteins into cells through mechanisms such as endocytosis and direct penetration to exert their effector functions. Compared to other non-natural chemical molecules, CPPs have advantages such as good biocompatibility, low cytotoxicity, degradability after cellular transport, and the ability to directly fuse with bioactive proteins for recombinant expression.
[0014] Furthermore, the cell-penetrating peptide is a transcription transactivator peptide (TAT). Studies have shown that fusion expression of TAT proteins can increase yield and solubility, and be effectively introduced into the cell membrane, regardless of cell type. Due to the polypeptide properties of TAT, it can be normally degraded after entering the cell, exhibiting good biocompatibility and low cytotoxicity.
[0015] This invention provides the application of recombinant protein TAT-RNF114 in the preparation of cataract treatment drugs. The recombinant protein TAT-RNF114 includes cyclic finger protein 114 with an amino acid sequence as shown in SEQ ID NO.1 and a TAT motif fused to its C-terminus, the amino acid sequence of which is shown in SEQ ID NO.2.
[0016] The TAT motif with the amino acid sequence YGRKKRRQRRR is derived from the transcriptional transactivator of human immunodeficiency virus (HIV-1). This invention fuses the TAT motif with RNF114, effectively promoting the entry of RNF114 into human lens epithelial cells and animal lenses, without exhibiting significant cytotoxicity.
[0017] This invention demonstrates that administering an effective dose of recombinant protein TAT-RNF114 to the lens of a cataract patient can effectively improve the pathological phenotype of cataracts, providing important evidence for the application of RNF114 in the treatment of cataracts and other diseases.
[0018] Furthermore, the cataract disease mentioned includes, but is not limited to, age-related cataracts, congenital cataracts, and hypothermic cataracts.
[0019] Furthermore, the drug comprises an effective dose of recombinant protein TAT-RNF114 and a pharmaceutically acceptable carrier. The drug is prepared with recombinant protein TAT-RNF114 as the main active ingredient and a pharmaceutically acceptable carrier, and can be formulated according to pharmaceutically available formulation preparation methods.
[0020] Terminology Explanation:
[0021] The term "comprising" is an open-ended expression, meaning it includes the contents specified in this invention, but does not exclude other aspects.
[0022] The term "treatment" in some embodiments refers to improving a disease or condition (i.e., slowing, stopping, or alleviating the development of a disease or at least one of its clinical symptoms). In other embodiments, it refers to alleviating or improving at least one bodily parameter, including bodily parameters that may not be perceptible to the subject. In still other embodiments, it refers to regulating a disease or condition physically (e.g., stabilizing perceptible symptoms) or physiologically (e.g., stabilizing bodily parameters) or both. In still other embodiments, it refers to preventing or delaying the onset, flare-up, or worsening of a disease or condition.
[0023] The term "effective dose" refers to the amount of a compound that is sufficient to treat a disease when administered to a subject. The effective dose can vary depending on the severity of the disease and the physical condition, age, weight, and sex of the subject to be treated.
[0024] The term "pharmaceutically acceptable carrier" refers to any formulation or carrier medium capable of delivering an effective dose of the active substance of the present invention without interfering with the biological activity of the active substance and without toxic side effects on the host or subject.
[0025] The beneficial effects of this invention are as follows:
[0026] This invention discloses for the first time the function of RNF114 in the process of cataract reversal. Studies have shown that RNF114 can target the ubiquitination and degradation of abnormally aggregated proteins in human lens cells, effectively improving the pathological progression of cataracts. Therefore, RNF114 can be used to develop and prepare therapeutic drugs for cataracts. Furthermore, this invention utilizes the fusion of cell-penetrating peptides with RNF114 to prepare recombinant proteins, which can promote the effective entry of RNF114 into lens tissues and cells to exert its effects, and has high biocompatibility. Attached Figure Description
[0027] Figure 1To transform GFP-CRYAA or GFP-CRYAA(Y118D) plasmids into human lens epithelial cells, and to observe the intracellular distribution of green fluorescently labeled CRYAA protein in the cells by immunofluorescence microscopy at 24 or 48 hours after transfection.
[0028] Figure 2 RNF114 or inactivated RNF114ΔC plasmid was transfected into human lens epithelial cells and then treated at 4°C for 24 or 48 hours. The intracellular distribution of CRYAA protein indicated by green fluorescence was observed by immunofluorescence microscopy.
[0029] Figure 3 The image shows the intracellular CRYAA aggregation fluorescent spots in a mutant protein cataract cell model at 0h and 24h after overexpression of RNF114 or inactivation of RNF114 (RNF114ΔC). In the image, A is an image of intracellular CRYAA aggregation fluorescent spots taken using live cell imaging; B is the count of fluorescent spots.
[0030] Figure 4 The protein levels of CRYAA(Y118D) in mutant protein cataract cell models were measured by overexpressing RNF114 or by overexpressing RNF114 and simultaneously treating cells with the proteasome inhibitor MG132. In A, the protein levels of CRYAA(Y118D) in each treatment cell were detected by Western blotting; and in B, the protein levels of CRYAA(Y118D) and RNF114 were statistically analyzed.
[0031] Figure 5 To overexpress or knock down RNF114 in HLECs and treat them at low temperature (4°C) for different durations, the cell viability of HLECs was detected by the CCK8 assay.
[0032] Figure 6 This is for constructing the recombinant plasmid pET28a-His-RNF114-TAT-HA vector. This vector contains the coding sequence of the promoter RNF114 gene and the HA protein tag.
[0033] Figure 7 To detect the fluorescence signal indicating HA tags in cells and lens tissue sections after incubating TAT-RNF114 recombinant protein with HLECs and isolated rat lenses for 30 minutes, fixation and slide preparation were performed.
[0034] Figure 8 The dynamic changes of CRYAA fluorescent spots accumulated in the cytoplasm of HLECs with TAT-RNF114 and GFP-CRYAA(Y118D) knocked in for 0-4 hours are shown in Figure A, where A represents the dynamic changes of CRYAA fluorescent spots accumulated in the cytoplasm observed by live cell imaging; and B represents the statistical analysis of the fluorescent spots.
[0035] Figure 9 The image shows the changes in lens transparency in a low-temperature cataract lens tissue model treated with TAT-RNF114. Image A shows the changes in lens transparency observed through microscopic images, while image B shows the statistical analysis of lens transmittance.
[0036] Figure 10 The image shows the changes in lens transparency in a zebrafish cataract model induced by H2O2 treatment with TAT-RNF114. Image A is a microscopic image; image B shows the changes in the severity of cataracts in live zebrafish as assessed using the LOCS III scoring system. Detailed Implementation
[0037] The present invention will be further described below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.
[0038] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified. Example 1: Investigating the role of RNF114 in the pathogenesis of cataracts caused by protein homeostasis disruption.
[0039] 1. Construct a cataract model using human lens cells (HLECs) by introducing mutant lens proteins or by cryogenics.
[0040] 1.1 For the cataract cell model induced by the expression of mutant lens proteins, a CRYAA WT plasmid (constructed by inserting the CRYAA-encoding gene fragment with the nucleotide sequence shown in SEQ ID NO.3 into the NheI / HindIII site of the PEGFP-N1 plasmid) carrying a GFP green fluorescent tag and a CRYAA (Y118D) mutant plasmid (constructed by inserting the CRYAA (Y118D)-encoding gene fragment with the nucleotide sequence shown in SEQ ID NO.4 into the NheI / HindIII site of the PEGFP-N1 plasmid) specifically causing cataracts was lysed and encapsulated using Lipofectamine 3000 (Thermo Fisher Scientific). These plasmids were then transfected into HLECs cells (SRA01 / 04) that had grown to 70% confluence according to the manufacturer's instructions. The transfection efficiency of the cell lines was detected by Western blotting.
[0041] like Figure 1As shown, clustered fluorescent spots were observed in HLECs under an immunofluorescence microscope to indicate that the cells successfully expressed the mutant CRYAA (Y118D) in an aggregated state.
[0042] 1.2 For the low-temperature construction of a human lens cell (HLEC) cataract model, the HLEC cell line (SRA01 / 04) was incubated at 4°C for 24 or 48 hours in Hibernate-A medium (Thermo Fisher Scientific, MA, USA) supplemented with 10% FBS and 1% penicillin-streptomycin. For the rewarming treatment, the cells were first washed three times with phosphate-buffered saline (PBS), and then incubated at 37°C in preheated F12 medium supplemented with 10% FBS and 1% penicillin-streptomycin. After 30 minutes of incubation, the cells were washed again with PBS and placed in preheated extracellular solution (Beyotime) for subsequent detection.
[0043] like Figure 2 As shown, after 24 or 48 hours of low-temperature-rewarming treatment, CRYAA primary antibody and corresponding fluorescent secondary antibody were fixed by immunofluorescence, and the slides were mounted. Under an immunofluorescence microscope, CRYAA fluorescent spots aggregated in the HLECs control group were visible.
[0044] 2. Detection of CRYAA aggregate protein by overexpression of RNF114 in a human lens cell (HLECs) cataract model
[0045] 2.1 In HLECs, cells were lysed and encapsulated using Lipofectamine 3000 (Thermo Fisher Scientific) and transformed into the RNF114 WT plasmid (constructed by inserting the RNF114 encoding gene fragment, as shown in SEQ ID NO.5, into the HindIII / BamHI site of the pCDNA3.1 plasmid, with the amino acid sequence of the encoded RNF114 protein shown in SEQ ID NO.1), or an inactivated RNF114 (RNF114ΔC) plasmid (constructed by inserting the RNF114ΔC encoding gene fragment, as shown in SEQ ID NO.6, into the HindIII / BamHI site of the pCDNA.1 plasmid), using the same method as above. 24 hours after transfection, the cells were treated at 4°C for 24 or 48 hours and then rewarmed for 30 minutes, as described above. Immunofluorescence was used to image the CRYAA aggregated fluorescent spots in the RNF114 overexpression group and the control group, and the number of fluorescent spots was counted.
[0046] like Figure 2As shown, the number of CRYAA aggregation fluorescent spots in cells overexpressing RNF114 after 24 and 48 hours of cryo-rewarming treatment was less than that in cells overexpressing RNF114ΔC and the control group. This indicates that exogenous RNF114 has a significant effect on reducing endogenous CRYAA aggregation under low temperature.
[0047] 2.2 In the mutant protein cataract cell model, RNF114 WT or inactivated RNF114 (RNF114ΔC) plasmid was transformed into the cell, using the same method as above. Live-cell imaging was used to capture images of CRYAA aggregation fluorescent spots in designated cells at 0h and 24h of overexpression, and the number of fluorescent spots was counted.
[0048] like Figure 3 As shown, 24 hours after transfection with the RNF114 WT plasmid, the number of fluorescent spots aggregated in the mutant protein cataract cell model was significantly reduced, and the fluorescence intensity was weakened. However, there was no significant difference in the intensity of CRYAA(Y118D) fluorescent spots aggregated in cells between the 0h and 24h groups in the RNF114ΔC transfection group. Figure 3 B).
[0049] 2.3 While transforming the RNF114 WT plasmid into the mutant protein cataract cell model, the proteasome inhibitor MG132 100 nM was added to the culture medium to treat the cells for 24 hours. The protein level of CRYAA (Y118D) in each treatment cell was detected by Western blotting.
[0050] like Figure 4 As shown, the level of CRYAA(Y118D) protein was significantly reduced in cells overexpressing RNF114 WT, while co-treatment with MG132 inhibited the reduction of CRYAA(Y118D) protein level. This indicates that RNF114 with E3 enzyme activity can effectively promote the degradation of aggregated CRYAA.
[0051] The above data provide a theoretical basis for exploring the role of RNF114 in the pathogenesis of cataracts caused by protein homeostasis disruption, suggesting that exogenous RNF114 plays an important role in preventing protein aggregation-related cataracts.
[0052] Example 2: Regulation of RNF114 cytotoxicity, particularly exogenous RNF114, in HLECs
[0053] Using the Lipofectamine 3000 (Thermo Fisher Scientific) transfection kit, RNF114 WT plasmid or RNF114 siRNA (sequence GUGGAACACUGCAAAUUAUTT) was transfected into HLECs, or RNF114WT plasmid and RNF114 siRNA were co-transfected. 24 hours after transfection, the cells were treated at 4°C for 48 hours. The cell viability of HLECs before and after low-temperature treatment was detected by CCK8 assay. The cells transfected with the empty vector plasmid served as a control.
[0054] like Figure 5 As shown, compared with the control group, overexpression of RNF114 WT or knockdown of RNF114 did not significantly affect the viability of HLEC cells before or after low-temperature treatment, indicating that regulation of RNF114 in HLECs did not cause significant cytotoxicity.
[0055] Example 3: Construction of recombinant protein TAT-RNF114
[0056] To facilitate the delivery of RNF114 to HLECs and lens tissues and increase the effective drug concentration within the tissues, this embodiment constructs a recombinant protein RNF114-TAT by conjugating the TAT motif to prepare a lens drug. The TAT motif (amino acid sequence YGRKKRRQRRR, SEQ ID NO.2) is derived from the transcriptional transactivator (TAT) of human immunodeficiency virus (HIV-1) and is a classic cell-penetrating peptide.
[0057] Specifically, firstly, a eukaryotic expression vector expressing the recombinant RNF114 protein was constructed. The DNA coding sequence of RNF114 was obtained by PCR amplification and cloned into the pET28a expression vector. A histidine (His) tag for protein affinity purification was inserted at the amino terminus of the RNF114 DNA coding sequence, and a TAT (47-58) motif was inserted at the carboxyl terminus. Subsequently, a hemagglutinin (HA) tag was ligated, thus constructing the recombinant plasmid pET28a-His-RNF114-TAT-HA vector. Figure 6As shown in the figure. The recombinant protein TAT-RNF114 has 256 amino acids, and its amino acid sequence is shown in SEQ ID NO.7. It has a molecular weight of 29 kDa and an isoelectric point pI of 8.52. The plasmid was transfected into *E. coli* BL21(DE3). The supernatant from the bacterial culture was collected, and the filtered supernatant was passed through a GE His-tagged purification column to bind the protein. Using an AKATA protein purifier, the protein was eluted with a gradient of imidazole solutions to obtain the recombinant TAT-RNF114 protein. Subsequent ultrafiltration and dialysis were performed to obtain the purified TAT-RNF114 recombinant protein. The final protein concentration was then determined.
[0058] Subsequently, 50 μM of recombinant TAT-RNF114 protein was incubated with HLECs and isolated rat lenses for 30 minutes, respectively. Cells and lens tissues were then fixed and incubated with HA primary antibody and corresponding red fluorescent secondary antibody. Immunofluorescence was used to detect the red fluorescent signal indicating the HA tag in cells and tissue sections. This data can determine the efficiency of TAT-RNF114 recombinant protein entering cells and lens tissues, ensuring sufficient drug concentration at the target site to exert its efficacy.
[0059] like Figure 7 As shown, after incubating HLECs and isolated rat lenses with TAT-RNF114 recombinant protein for 30 minutes, they were fixed and prepared into slides, incubated with primary and secondary antibodies, and mounted. Under a fluorescence microscope, red fluorescence could be detected in the cytoplasm or inside the lens of the sample, indicating that the recombinant protein has effective permeability to cells and lens tissues.
[0060] Example 4: Evaluation of the therapeutic effect of TAT-RNF114 recombinant protein on cataracts using a mutant protein cataract cell model.
[0061] The TAT-RNF114 recombinant protein prepared in Example 3 was incubated with HLECs knocked into GFP-CRYAA(Y118D) constructed in Example 1 for 4 hours, and the dynamic changes of CRYAA fluorescent spots aggregated in the cytoplasm were observed by live cell imaging.
[0062] like Figure 8 As shown, in cells incubated with TAT-RNF114 for 2 hours and 4 hours, the number of CRYAA(Y118D) aggregated fluorescent spots was significantly reduced, and the fluorescence intensity was decreased.
[0063] Example 5: Evaluation of the therapeutic effect of TAT-RNF114 recombinant protein on cataracts using a cryogenic cataract lens tissue model and an H2O2-induced cataract zebrafish animal model.
[0064] 1. Constructing a low-temperature cataract lens tissue model and an H2O2-induced cataract zebrafish animal model.
[0065] 1.1 For the construction of a hypothermic cataract lens tissue model, rat lenses were incubated at 4°C for 24 or 48 hours in Hibernate-A medium (Thermo Fisher Scientific, MA, USA) supplemented with 10% FBS and 1% penicillin-streptomycin. For rewarming, the lenses were first rinsed three times with phosphate-buffered saline (PBS), and then incubated at 37°C in preheated F12 medium supplemented with 10% FBS and 1% penicillin-streptomycin. After 30 minutes of incubation, the lenses were washed again with PBS and placed in preheated extracellular solution (Beyotime) for subsequent analysis.
[0066] Isolated rat lenses were placed in culture dishes containing extracellular solution and photographed using a Nikon SMZ18 microscope to assess changes in lens transparency. For lens transmittance measurements, an untreated transparent lens was placed in the center of the microscope's field of view, the bottom light source was turned on, and image A was captured. Subsequently, the light source was turned off, and image B was captured. Image B was used to correct for ambient light interference. Then, the bottom light source was turned on, the treated lens was placed directly above the bottom light source, and image C was captured. The grayscale values of the lens region were analyzed using ImageJ, and the transmittance was calculated using the following formula: Lens transmittance = (CB) / (AB) × 100%.
[0067] 1.2 For the construction of the H2O2-induced cataract zebrafish animal model, 4% H2O2 was injected into the anterior chamber of zebrafish using a microsyringe. 72 hours later, the zebrafish lens was photographed using a Nikon SMZ18 microscope to determine whether cataracts had formed in the injected eye. Visual analysis was used to qualitatively assess the improvement in transparency, and a cataract grading system adapted from LOCS III was used: Grade 0, clear lens; Grade 1, loss of normal appearance of the anterior lens, lens nucleus, and posterior pole of the lens, and prominent Y-shaped sutures; Grade 2, discrete anterior changes with significant nuclear opacity; Grade 3, increased involvement of the lens nucleus and cortex, accompanied by increased opacity; Grade 4, fully mature cataract involving the cortex and nucleus.
[0068] 2. Evaluation of the therapeutic effect of TAT-RNF114 recombinant protein on cataracts using a cryogenic cataract lens tissue model and a H2O2-induced cataract zebrafish animal model.
[0069] 2.1 The low-temperature cataract lens tissue model was treated with TAT-RNF114. The changes in lens transparency were observed by taking pictures under a microscope, and the changes in lens transparency were quantified by lens transmittance.
[0070] like Figure 9 As shown, rat isolated lenses treated with TAT-RNF114 showed significantly improved transmittance and reduced nuclear turbidity after low-temperature rewarming.
[0071] 2.2 A zebrafish cataract model was induced by treating H2O2 with TAT-RNF114. Microscopic images were taken and the severity of cataracts in live zebrafish was assessed using the LOCSIII scoring system.
[0072] like Figure 10 As shown, treatment of live zebrafish with TAT-RNF114 significantly reduced H2O2-induced cataracts, but had no significant effect in the inactivated RNF114 group and the TAT group alone.
Claims
1. Use of a cyclophilin 114 for the manufacture of a medicament for the treatment of cataract, characterized in that, The amino acid sequence of the RING finger protein 114 is shown as SEQ ID NO.
1.
2. Use according to claim 1, wherein The manifestations of the cataract include abnormal aggregation of crystallin in lens cells or mutation of crystallin.
3. Use according to claim 2, wherein the compound is ###0002### The RING finger protein 114 improves the pathological process of the cataract by targeting ubiquitination degradation of the abnormal aggregation of alpha-crystallin subtype CRYAA or CRYAA mutant in lens cells.
4. The use according to claim 1, wherein The cataract is oxidative stress cataract.
5. The use according to claim 1, wherein the compound is ###0002### The carboxy-terminal of the RING finger protein 114 in the drug is fused with a cell-penetrating peptide.
6. The use according to claim 5, wherein the compound is ###0002### The cell-penetrating peptide is a transcriptional transactivator peptide.
7. Use of the recombinant protein TAT-RNF114 in the preparation of a drug for the treatment of cataracts, characterized in that, The recombinant protein TAT-RNF114 comprises the RING finger protein 114 with the amino acid sequence shown as SEQ ID NO. 1 and a TAT motif fused at the C-terminal of the RING finger protein 114, and the amino acid sequence of the TAT motif is shown as SEQ ID NO. 2; the amino acid sequence of the recombinant protein TAT-RNF114 is shown as SEQ ID NO.
7.
8. Use according to claim 7, wherein the compound is ###0002### The cataract disease is at least one of age-related cataract and congenital cataract.
9. The use according to claim 7, wherein the compound is ###00003### 8 or ###00004### 9. The drug comprises an effective dose of the recombinant protein TAT-RNF114 and a pharmaceutically acceptable carrier.