Chimeras degrading card domain protein aggregates and uses thereof

Abstract

The application discloses a chimeric body degrading CARD domain protein aggregates and application thereof, and the chimeric body is composed of a light-controlled targeting protein and a light-controlled degradation protein; the light-controlled targeting protein is connected by a MAVS CARD domain, a flexible connecting peptide and a photosensitive protein pMag in sequence; and the light-controlled degradation protein is connected by a photosensitive protein nMag, a flexible connecting peptide and a RING domain of TRIM21 in sequence. The application constructs a light-controlled activated CARD-RING chimeric body, and when a CARTAC produces toxic side effects, or in cells with an abnormally activated RIG-I / MDA5 signal path, the activity of the CARTAC can be effectively controlled or the autoimmune diseases caused by the abnormal RIG-I / MDA5 can be inhibited.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to a chimera that degrades CARD domain protein aggregates and its applications. Background Technology

[0002] In traditional antiviral therapies, most drugs and vaccines target specific viral proteins. However, if the virus mutates, these drugs can become ineffective or escape the immune system. CARTAC's advantage lies in targeting the viral RNA genome itself, as well as the RIG-I / MDA5-MAVS pathway that allows cells to sense the virus. Regardless of viral mutations, as long as the virus is an RNA virus and activates this pathway, it can be recognized and attacked by CARTAC.

[0003] Although CARTAC is a broad-spectrum antiviral treatment, its long-term presence may cause problems such as immunotoxicity. Moreover, overactivation of RIG-I / MDA5 is an important mechanism in many autoimmune diseases.

[0004] It should be noted that the information disclosed in the background section above is only used to enhance the understanding of the background of this disclosure, and therefore may include information that does not constitute prior art known to those skilled in the art. Summary of the Invention

[0005] The technical objective of this application is to address the above-mentioned shortcomings by providing a chimera that degrades CARD domain protein aggregates and its application. This application constructs a light-activated CARD-RING chimera. When CARTAC produces toxic side effects, or in cells with abnormal activation of the RIG-I / MDA5 signaling pathway, MCARD-pMag can be recruited to MAVS aggregates. Blue light irradiation induces pMag and nMag to interaggregate, thereby recruiting a large number of nMag-RING to the complex. After the RING domain aggregates, it can activate its E3 ubiquitin ligase activity, causing the MAVS aggregates to undergo ubiquitination modification, and finally be degraded through the proteasome pathway, thereby effectively controlling the activity of CARTAC or inhibiting autoimmune diseases caused by abnormal RIG-I / MDA5.

[0006] To achieve the above objectives, this application provides the following technical solution:

[0007] According to one aspect of this application, a chimera of CARD domain protein aggregates is provided, the chimera comprising a light-controlled targeting protein and a light-controlled degradation protein; the light-controlled targeting protein is sequentially linked by a MAVS CARD domain, a flexible linker peptide, and a photosensitizing protein pMag, and the light-controlled degradation protein is sequentially linked by a photosensitizing protein nMag, a flexible linker peptide, and a TRIM21 RING domain.

[0008] In some embodiments, the amino acid sequence of the MAVS CARD domain is shown in SEQ ID NO:3;

[0009] In some embodiments, the amino acid sequence of the photosensitive protein pMag is shown in SEQ ID NO:5; the amino acid sequence of the photosensitive protein nMag is shown in SEQ ID NO:6.

[0010] In some embodiments, the RING domain of TRIM21 is a domain that forms a heterodimer with pMag after blue light irradiation, and the amino acid sequence of the RING domain of TRIM21 is shown in SEQ ID NO:7.

[0011] In some embodiments, the amino acid sequence of the flexible linker peptide is shown in SEQ ID NO:4.

[0012] In some embodiments, the amino acid sequence of the light-controlled targeting protein is shown in SEQ ID NO:1, and the amino acid sequence of the light-controlled degradation protein is shown in SEQ ID NO:2.

[0013] According to another aspect of this application, a chimera that degrades CARD domain protein aggregates is provided for use in the preparation of products for degrading CARD domain protein aggregates.

[0014] In some embodiments, the method for degrading CARD domain protein aggregates includes constructing the chimera into a plasmid vector, transfecting the plasmid vector into target cells, and irradiating the target cells with blue light.

[0015] In some embodiments, the wavelength of the blue light is 470±20 nm, and the illuminance of the blue light is 3 mW / cm². 2 ,

[0016] In some embodiments, the irradiation time is 1 minute.

[0017] In some embodiments, the plasmid vector is PCDNA3.1.

[0018] According to another aspect of this application, a chimera that degrades CARD domain protein aggregates is provided for use in the preparation of products for the diagnosis and / or treatment of diseases related to CARD domain protein aggregates.

[0019] According to another aspect of this application, a chimera of degraded CARD domain protein aggregates is provided for use in the preparation of a medicament for treating autoimmune diseases caused by RIG-I / MDA5 abnormalities.

[0020] Compared with the prior art, the advantages and positive effects of this application are as follows: This application constructs a light-controlled activated CARD-RING chimera. When CARTAC produces toxic side effects, or in cells with abnormal activation of the RIG-I / MDA5 signaling pathway, MCARD-pMag can be recruited to MAVS aggregates. Blue light irradiation induces pMag and nMag to aggregate, thereby recruiting a large number of nMag-RING to the complex. After the RING domain aggregates, it can activate its E3 ubiquitin ligase activity, promote the ubiquitination modification of MAVS aggregates, and finally be degraded through the proteasome pathway, thereby effectively controlling the activity of CARTAC or inhibiting autoimmune diseases caused by abnormal RIG-I / MDA5. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This is a schematic diagram illustrating the photo-controlled degradation of CARD aggregates or activation of RIG-I in the embodiments of this application.

[0023] Figure 2 The bimolecular fluorescence complementation experiment in Example 2 of this application demonstrates that the chimera degrades the CARD aggregate complex via light control.

[0024] Figure 3 This illustrates the changes in fluorescence signals analyzed by flow cytometry in Embodiment 2 of this application.

[0025] Figure 4 The experiment shown in Example 3 of this application illustrates the analysis of the chimera via photocontrolled degradation of the MAVS aggregated complex using a splitting luciferase complementation assay.

[0026] Figure 5 This illustrates how, in Example 4 of this application, Western blot was used to detect the photo-controlled degradation of MAVS aggregated complexes of chimeras.

[0027] Figure 6 This is a bar chart of RNase activity after blue light treatment in Example 5 of this application. Detailed Implementation

[0028] To better understand the above-mentioned objectives, features, and advantages of this application, the application will be further described below with reference to the accompanying drawings and embodiments. It should be noted that, unless otherwise specified, the embodiments and features described in the embodiments of this application can be combined with each other.

[0029] The present application will be further described below with reference to the accompanying drawings and specific embodiments.

[0030] Figure 1 This is a schematic diagram illustrating the photo-controlled degradation of CARD aggregates or activation of RIG-I in embodiments of this application. Figure 1 As shown, RNA viruses infect host cells or cause gain-of-function mutations in RIG-I, thereby activating RIG-I-like receptors (RLRs), releasing the self-inhibitory state of their CARD domains, and recruiting CARTAC chimeras and M... CARD -pMag is transferred to RLRs aggregates. When cells are treated with blue light at a wavelength of 460 nm, pMag and nMag dimerize, causing a large number of TRIM21 RING (RING) domains to be recruited to the MAVS aggregate complex, activating RING E3 ubiquitin ligase activity, and ultimately degrading the complex via the ubiquitin-proteasome pathway.

[0031] Example 1: Construction of a chimeric carrier for photo-controlled degradation of MAVS aggregate complexes.

[0032] The chimera consists of two parts, namely the light-controlled targeting protein (M) CARD -pMag) and light-controlled degradation protein (nMag-RING). The specific vector construction strategy is as follows:

[0033] Light-controlled targeting protein (M) CARD Construction of the pMag eukaryotic expression plasmid: The MVS CARD domain (1-100 amino acids) nucleic acid sequence and the pMag gene sequence were constructed into the PCDNA3.1+ eukaryotic expression vector using a seamless cloning kit, and named PCDNA3.1-M. CARD -pMag.

[0034] Light-controlled targeting protein (M) CARD -pMag), which includes the MAVS CARD domain that targets and binds to MAVS aggregates and the light-controlling protein pMag connected by a flexible linker peptide.

[0035] The amino acid sequence of the MAVS CARD domain is (SEQ ID NO:3): MPFAEDKTYKYICRNFSNFCNVDVVEILPYLPCLTARDQDRLRATCTLSGNRDTLWHLFNTLQRRPGWVEYFIAALRGCELVDLADEVASVYQSYQPRTS.

[0036] The amino acid sequence of the flexible linker peptide is (SEQ ID NO:4): GSAGSAAGSGGS.

[0037] The amino acid sequence of the light-controlled protein pMag is (SEQ ID NO:5):

[0038] MGHTLYAPGGYDIMGYLRQIRNRPNPQVELGPVDTSCALILCDLKQKDTPIVYASEAFLYMTGYSNAEVLGRNCRFLQSPDGMVKPKSTRKYVDSNTINTIRKAIDRNAEVQVEVVNFKKNGQRFVNFLTIIPVRDETGEYRYSMGFQCETE.

[0039] In other words, M CARD The specific amino acid sequence of -pMag is as follows (SEQ ID NO:1): M CARD -Linker-pMag;

[0040] MPFAEDKTYKYICRNFSNFCNVDVVEILPYLPCLTARDQDRLRATCTLSGNRDTLWHLFNTLQRRPGWVEYFIAALRGCELVDLADEVASVYQSYQPRTSGSAGSAAGSGGSMGHTLYAPGGYDIMGYLRQI RNRPNPQVELGPVDTSCALILCDLKQKDTPIVYASEAFLYMTGYSNAEVLGRNCRFLQSPDGMVKPKSTRKYVDSNTINTIRKAIDRNAEVQVEVVNFKKNGQRFVNFLTIIPVRDETGEYRYSMGFQCETE.

[0041] Construction of eukaryotic expression plasmid for light-controlled degradation protein (nMag-RING): The nMag gene sequence and the TRIM21 RING (RING) domain sequence were constructed into the PCDNA3.1+ eukaryotic expression vector using a seamless cloning kit and named PCDNA3.1-nMag-RING.

[0042] The photosensitive protein nMag-RING consists of the photosensitive protein nMag, which forms a heterodimer with pMag after blue light irradiation, and the TRIM21 RING domain, which is linked by a flexible linker peptide.

[0043] The amino acid sequence of the photosensitive protein nMag is (SEQ ID NO:6): MGHTLYAPGGYDIMGYLDQIGNRPNPQVELGPVDTSCALILCDLKQKDTPIVYASEAFLYMTGYSNAEVLGRNCRFLQSPDGMVKPKSTRKYVDSNTINTIRKAIDRNAEVQVEVVNFKKNGQRFVNFLTIIPVRDETGEYRYSMGFQCETEGS.

[0044] The amino acid sequence of the flexible linker peptide is shown in SEQ ID NO:4.

[0045] The amino acid sequence of the TRIM21 RING domain is (SEQ ID NO:7): MASAARLTMMWEEVTCPICLDPFVEPVSIECGHSFCQECISQVGKGGGSVCPVCRQRFLLKNLRPNRQLANMVNNLKEISQEARE.

[0046] In other words, the specific amino acid sequence of nMag-RING is as follows (SEQ ID NO:2): nMag-Linker-RING;

[0047] MGHTLYAPGGYDIMGYLDQIGNRPNPQVELGPVDTSCALILCDLKQKDTPIVYASEAFLYMTGYSNAEVLGRNCRFLQSPDGMVKPKSTRKYVDSNTINTIRKAIDRNAEVQVEVVNFKKNGQRF VNFLTIIPVRDETGEYRYSMGFQCETEGSAGSAAGSGGSMASAARLTMMWEEVTCPICLDPFVEPVSIECGHSFCQECISQVGKGGGSVCPVCRQRFLLKNLRPNRQLANMVNNLKEISQEARE.

[0048] Example 2: Bimolecular fluorescence complementation experiment verifies that the chimera degrades CARD aggregates by light control.

[0049] The MAVS CARD domain (amino acids 1-100) was constructed into bimolecular fluorescence complementary (BIFC) vectors pBiFC-VN173 and pBiFC-VC155 using a seamless cloning kit (Novozymes, ClonExpressII One Step Cloning Kit-C112). These vectors were then combined with M overexpression... CARDThe pCDNA3.1+ vectors of pMag and nMag-RING were co-transfected into HEK293T cells.

[0050] The specific transfection steps are as follows: pBiFC-VN173, pBiFC-VC155, and PCDNA3.1-M are transfected... CARD 1 μg of each of the four plasmids—pMag, PCDNA3.1-nMag-RING, etc.—were mixed in an EP tube, and an equal volume of diluted transfection reagent was added. After standing for 20 min, the mixture was added to serum-free HEK293T cells. The control group's nMag-RING plasmid was replaced with a plasmid expressing only nMag (this plasmid was constructed by directly inserting the nMag nucleic acid sequence into the pCDNA3.1+ vector using the aforementioned seamless cloning kit). After inducing MAVS aggregation complex formation via poly(I:C) or VSV, green fluorescence was emitted due to the proximity of the VN and VC fragments of the Venus protein. Cells were further treated with blue light (470±20 nm, 3 mW cm⁻¹). -2 (1 minute).

[0051] Figure 2 The bimolecular fluorescence complementation experiment in Example 2 of this application demonstrates that the chimera degrades the CARD aggregated complex via light-controlled degradation. For example... Figure 2 As shown, the green fluorescence was significantly reduced after blue light treatment, indicating a significant reduction in aggregate proteins. These results confirm the feasibility of the proposed method.

[0052] The cells treated above were further digested with trypsin, washed three times with PBS, and a single-cell suspension was prepared with a cell concentration controlled at 5 × 10⁻⁶. 6 ~1×10 7 / mL, using flow cytometry, laser selection: 488nm, FITC channel, voltage adjusted according to the negative control (non-transfected group) to place the negative cell population in the lower left of the scatter plot, and the intensity of Venus fluorescence signal was detected.

[0053] Figure 3 This illustrates the changes in fluorescence signals analyzed by flow cytometry in Embodiment 2 of this application. For example... Figure 3 As shown, after blue light treatment, the green fluorescence was significantly reduced at the quantitative level, indicating a significant reduction in aggregate proteins.

[0054] Example 3: Splitting luciferase complementation experiment analysis of the chimera via light-controlled degradation of MAVS aggregated complex.

[0055] The N-terminus and C-terminus fragments of Gaussian luciferase were constructed from the C-terminus of the MAVS CARD domain, respectively. The above vector was then combined with M... CARDThe pMag and nMag-RING vectors were co-transformed into HEK293T cells. The control group's nMag-RING plasmid was replaced with a plasmid expressing only nMag. After treatment with poly(I:C) or VSV for 1 h to induce MAVS aggregation complex formation, the culture medium was discarded, cells were washed with PBS, and lysis buffer was added to lyse the cells. The cells were incubated at 4°C for 10 min, centrifuged at 13000 rpm for 5 min, and the supernatant was collected. 50 μL of the sample was pipetted into a microtiter plate (96-well plate), and an equal volume of Gaussian luciferase working solution (50 μL / well) was added to each well. The cells were incubated at room temperature for 10-15 min in the dark, and the fluorescence intensity was read using a luminescent microplate reader. The principle of luciferase activation is based on the proximity of the N-terminal and C-terminal fragments of Gaussian luciferase. The cells were further treated with blue light.

[0056] Figure 4 The assay shown in Example 3 of this application illustrates the analysis of the chimera's photocontrolled degradation of the MAVS aggregated complex via luciferase complementation. The results are as follows... Figure 4 As shown, blue light treatment significantly reduced phosphatase activity, indicating a significant decrease in aggregate proteins. These results further confirm the feasibility of this approach.

[0057] Example 4: Western blot detection of chimera-mediated degradation of MAVS aggregated complexes.

[0058] The MAVS CARD domains were constructed into bimolecular fluorescence complementary (BIFC) vectors, and the above vectors were then combined with M... CARDThe pMag and nMag-RING vectors were co-transformed into HEK293T cells. The control group's nMag-RING plasmid was replaced with a plasmid expressing only nMag. After inducing MAVS aggregation complex formation with poly(I:C) or VSV, the cells were treated with blue light at a wavelength of 460 nm. After 1 h of treatment, the cells were collected, RIPA lysis buffer was added, and the cells were lysed on ice for 30 minutes. The protein concentration was determined using the BCA method. 5× Loading Buffer was added at a 4:1 ratio, and the cells were heated at 100 °C for 5-10 minutes to denature the protein. A 15% protein gel was prepared, and the protein sample and marker were loaded into the gel wells. Electrophoresis was performed at 80 V for approximately 25-35 minutes, followed by electrophoresis at 120 V until the bromophenol blue reached the bottom of the gel. The PVDF membrane was activated by soaking in methanol for 30 seconds, equilibrated with transfer buffer, and filter paper and a sponge pad were prepared and soaked in transfer buffer. The "sandwich" structure was assembled in the following order: sponge → filter paper → gel → PVDF membrane → filter paper → sponge. Transfer the membrane at a constant current of 300mA for 90-120 minutes. Block with 5% skim milk powder (prepared with TBST) at room temperature for 1 hour. Dilute the primary antibody according to the instructions and incubate overnight at 4°C on a shaker. Wash the membrane 3 times with TBST. Dilute the HRP-labeled secondary antibody at a ratio of 1:5000-1:10000 and incubate at room temperature on a shaker for 1 hour. Wash the membrane 3 times with TBST for 5-10 minutes each time. Mix ECL chemiluminescence solution A and solution B at a 1:1 ratio. Acquire the signal using a chemiluminescence imager and adjust the exposure time.

[0059] Figure 5 This illustrates how, in Example 4 of this application, Western blot analysis was performed to detect the photo-controlled degradation of MAVS aggregated complexes of chimeras. For example... Figure 5 As shown, the protein expression levels of each component in the MAVS aggregate complex were significantly reduced after blue light irradiation.

[0060] Example 5: RNase activity after blue light treatment.

[0061] CARTAC plasmid with M CARD The pMag and nMag-RING vectors were co-transfected into HEK293T cells. The control group's nMag-RING plasmid was replaced with a plasmid expressing only nMag. CARTAC activation was induced by poly(I:C) or VSV. After 24 hours of culture, the cells were further treated with blue light. After 20 minutes of treatment, the cells were lysed and purified by immunoprecipitation using Anti-Flag Beads. RNase activity was measured using an RNase activity fluorescence detection kit (Beyotime, P0347M).

[0062] CARTAC plasmid was generated by CARTAC N plasmids and CARTAC CThe plasmid composition utilizes a split-RNase strategy, activating oligomers through the MAVSCRAD domain. The CARTAC plasmid induces complementary activation of split-RNase, leading to in-situ targeted cleavage of viral RNA. Long-term use of this method can induce immunotoxic side effects. Furthermore, overactivation of RIG-I / MDA5 is a crucial mechanism in various autoimmune diseases. Gain-of-function mutations in RIG-I (such as DDX58 R109C) can weaken its autoinhibitory capacity, leading to persistent activation of the type I interferon signaling pathway and inducing systemic lupus erythematosus (SLE) manifestations such as lupus nephritis. Similarly, abnormal activation of MAVS can cause macrophage overactivation, inducing macrophage activation syndrome. This syndrome is often associated with autoimmune diseases such as systemic juvenile idiopathic arthritis or SLE, clinically manifesting as fever, hepatosplenomegaly, and pancytopenia. In microglia, overactivation of MAVS can promote the release of inflammatory factors such as IFN-β, TNF-α, and IL-6, which is closely related to the development of neurodegenerative diseases such as Parkinson's disease.

[0063] Among them, CARTAC N The plasmid amino acid sequence is (SEQ ID NO:8):MPFAEDKTYKYICRNFSNFCNVDVVEILPYLPCLTARDQDRLRATCTLSGNRDTLWHLFNTLQRRPGWVEYFIAALRGCELVDLADEVASVYQSYQPRTSGSLEASPSNPGASNGSMKPPQFTWAQWFETQHINMTSQQCTNAMQVINNYQRRCKNQNTFLLTTFANVVNVCGNPNMTCPSNKT.

[0064] CARTAC C The plasmid amino acid sequence is (SEQ ID NO:9): MPFAEDKTYKYICRNFSNFCNVDVVEILPYLPCLTARDQDRLRATCTLSGNRDTLWHLFNTLQRRPGWVEYFIAALRGCELVDLADEVASVYQSYQPRTSGSLEASPSNPGASNGSRKNCHHSGSQVPLIHCNLTTPSPQNISNCRYAQTPANMFYIVACDNRDQRRDPPQYPVVPVHLDRII.

[0065] Figure 6 This is a bar chart showing the RNase activity after blue light treatment in Example 5 of this application. Figure 6As shown, blue light treatment significantly reduced the RNase activity of CARTAC. These results confirm that the RNase activity of CARTAC can be turned off through photocontrol.

[0066] A chimera that broadly inhibits the replication of different RNA viruses by targeting and degrading viral RNA has demonstrated a relatively ideal antiviral effect and can specifically activate RNases after viral infection. However, the chimera's persistence after its antiviral effect has been exhausted may lead to off-target or toxic side effects. To address these issues, this study developed a photo-controlled chimera that degrades the MAVS aggregation complex. This chimera can not only conditionally shut down the RNase activity of CARTAC, but can also be used to treat various autoimmune diseases caused by overactivation of RIG-I.

[0067] The photo-activated CARD-RING chimera in this application is made by M CARD It consists of two parts: pMag (the fusion of the CARD domain of MAVS with the light-sensitive protein pMag) and nMag-RING (the fusion of the light-sensitive protein nMag with the RING domain of TRIM21). When CARTAC produces toxic side effects, or in cells with abnormal activation of the RIG-I / MDA5 signaling pathway, M... CARD -pMag can be recruited to MAVS aggregates; blue light irradiation induces pMag and nMag to inter-aggregate, thereby recruiting a large number of nMag-RING to the complex. After the RING domain aggregates, it can activate its E3 ubiquitin ligase activity, causing ubiquitination modification of MAVS aggregates, which are eventually degraded through the proteasome pathway, thereby effectively controlling CARTAC activity or inhibiting autoimmune diseases caused by RIG-I / MDA5 abnormalities.

[0068] Through the above specific embodiments, those skilled in the art can easily implement this application. However, it should be understood that this application is not limited to the specific embodiments described above. Based on the disclosed embodiments, those skilled in the art can arbitrarily combine different technical features to achieve different technical solutions.

Claims

1. A chimera that degrades CARD domain protein aggregates, characterized in that, The chimera is composed of a light-controlled targeting protein and a light-controlled degradation protein; the light-controlled targeting protein is sequentially linked by a MAVS CARD domain, a flexible linker peptide, and a photosensitizing protein pMag, and the light-controlled degradation protein is sequentially linked by a photosensitizing protein nMag, a flexible linker peptide, and a TRIM21 RING domain. The chimera is capable of photo-controlled degradation of CARD domain protein aggregates under pathological conditions such as viral infection or abnormal activation of the RIG-I / MDA5 pathway. The amino acid sequence of the MAVS CARD domain is shown in SEQ ID NO:3; The amino acid sequence of the photosensitive protein pMag is shown in SEQ ID NO:5; the amino acid sequence of the photosensitive protein nMag is shown in SEQ ID NO:6; The RING domain of TRIM21 is the domain that forms a heterodimer with pMag after blue light irradiation, and the amino acid sequence of the RING domain of TRIM21 is shown in SEQ ID NO:

7. The amino acid sequence of the flexible linker peptide is shown in SEQ ID NO:4.

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