Preparation method and application of tau-derived minimal functional peptide targeting cul3-rhoa axis

Abstract

The application discloses a preparation method and application of a Tau-derived minimum functional peptide targeting a CUL3-RhoA axis, and an amino acid sequence of the Tau-derived minimum functional peptide is shown as SEQ ID NO. 4: GNIHHKPGGGQVEVKSEKLD. The application of the Tau-derived minimum functional peptide targeting the CUL3-RhoA axis in the preparation of a drug for treating diseases related to over-activation of osteoclasts belongs to the technical field of biological medicines, and the drug plays a pharmacological effect by blocking RhoA ubiquitination through CUL3, activating a RhoA-ROCK-MLC2 tension axis, and destroying a closed loop of osteoclasts. The Tau-derived minimum functional peptide can avoid application defects of full-length proteins, has low toxicity, high selectivity and physiological friendliness.

Description

Technical Field

[0001] This invention relates to a method for preparing and applying a Tau-derived minimal functional peptide targeting the CUL3-RhoA axis, belonging to the field of biomedical technology. Background Technology

[0002] Osteoporosis is a systemic skeletal disease characterized by a significant reduction in bone mass and degenerative damage to the microstructure of bone tissue. Its direct consequences include significantly increased bone fragility and a dramatically increased risk of fractures, making it a major public health problem that seriously threatens the health of middle-aged and elderly people. From a pathological perspective, the essence of osteoporosis is an imbalance in bone remodeling homeostasis: in the bone remodeling cycle, osteoclasts, responsible for bone resorption, are abnormally activated, with their functional activity significantly exceeding that of osteoblasts, responsible for bone formation. This ultimately leads to an imbalance between bone formation and bone resorption, resulting in a continuous loss of net bone mass.

[0003] Current clinical treatments for osteoporosis primarily rely on bisphosphonates and RANKL (receptor activator of nuclear factor kappa B ligand) inhibitors. These drugs broadly inhibit excessive bone resorption by inducing osteoclast apoptosis and inhibiting osteoclast differentiation and maturation, thus slowing bone loss to some extent. However, these traditional intervention strategies are mostly non-specific and broad-spectrum, which, while blocking pathological bone resorption, can also severely interfere with normal physiological bone remodeling processes, leading to a series of serious adverse reactions, including osteonecrosis of the jaw, atypical fractures, and problems such as the accumulation of bone micro-damage and decreased bone repair capacity due to excessive inhibition of bone turnover. Therefore, how to precisely inhibit excessive osteoclast activation while preserving physiological bone remodeling function to the greatest extent has become a key scientific problem and clinical translational bottleneck in the field of bone metabolic diseases.

[0004] In recent years, an increasing number of studies have suggested that Tau, a microtubule-associated protein, is not only an important neuronal skeletal protein in the central nervous system, but also plays a crucial metabolic regulatory role in peripheral bone tissue. The vast majority of existing research focuses on the pathological role of Tau protein aberrant phosphorylation and accumulation in neurodegenerative diseases such as Alzheimer's disease, seriously neglecting its physiological functions and regulatory mechanisms in maintaining bone metabolic homeostasis. Related research is still in its early stages.

[0005] However, it is worth noting that full-length Tau protein has inherent defects such as large molecular weight and complex spatial structure. If it is directly used as a drug target for transformation and development, it will face multiple challenges such as high preparation cost, low in vivo delivery efficiency and poor tissue specificity. At the same time, there is also a potential risk of central nervous system toxicity, which greatly limits its clinical application prospects.

[0006] Based on this, screening and identifying the smallest functionally active peptide in Tau protein that can specifically bind to RhoA (a member of the Ras homologous gene family A) and independently mediate the anti-osteoporosis effect is of great theoretical significance and outstanding clinical translational value for avoiding the application defects of full-length proteins and developing novel bone resorption inhibitors that are low in toxicity, highly selective, and physiologically friendly. Summary of the Invention

[0007] To address the aforementioned technical problems, the first objective of this invention is to provide a Tau-derived minimal functional peptide targeting the CUL3-RhoA axis; the second objective is to provide its preparation method; and the third objective is to provide its applications. It avoids the application drawbacks of full-length proteins, exhibits low toxicity, high selectivity, and is physiologically friendly.

[0008] To achieve the aforementioned first objective, the technical solution of the present invention is: a Tau-derived minimal functional peptide targeting the CUL3-RhoA axis, characterized in that: its amino acid sequence is as shown in SEQ ID NO.4: GNIHHKPGGGQVEVKSEKLD.

[0009] The application of a Tau-derived minimal functional peptide targeting the CUL3-RhoA axis in the preparation of a drug for treating osteoclast overactivation-related diseases is described. By binding to CUL3 to block RhoA ubiquitination, it activates the RhoA-ROCK-MLC2 tension axis and disrupts the osteoclast closure loop to exert its therapeutic effect.

[0010] Specifically, the osteoclast overactivation-related diseases are osteoporosis, peri-implantitis, or periodontitis.

[0011] Another objective of this invention is achieved as follows: a method for preparing the Tau-derived minimal functional peptide targeting the CUL3-RhoA axis, characterized in that it is prepared using an Escherichia coli prokaryotic expression system, including the steps of constructing a recombinant expression vector, transforming the host bacterium, inducing expression, cell disruption, and purification by high-performance liquid chromatography.

[0012] In the above scheme, Rosetta (DE3) strain is used as the host cell.

[0013] In the above scheme: IPTG inducer was used for expression induction, with a concentration of 0.1-1 mM and a temperature of 30℃.

[0014] In the above scheme, cell disruption is performed using ultrasound.

[0015] In the above scheme, the specific steps of high performance liquid chromatography purification are as follows: the expression product is filtered through a 0.22 μm filter membrane and loaded onto a Ni-NTA affinity chromatography column. The supernatant protein solution is loaded at a flow rate of 1 mL / min. The column is washed with NTA-0 buffer until the eluent is free of protein. Impurities are eluted with buffers containing 20 mM and 60 mM imidazole in sequence. Finally, the target peptide is eluted with buffer containing 500 mM imidazole.

[0016] The present invention has the following beneficial effects: (1) Small molecular weight and high specificity: The peptide contains only 20 amino acid residues and is the smallest functional unit in Tau protein that inhibits osteoclast activity. It retains the key binding site of the full-length Tau protein with CUL3 / RhoA, and can accurately target the ubiquitination regulation mechanism in osteoclasts, avoiding the potential complex biological risks of the full-length protein. (2) Unique "braking" mechanism: Unlike traditional drugs that inhibit bone resorption by killing cells, the Tau-peptide4 of the present invention stabilizes RhoA protein through a "double locking" mechanism (interfering with the binding of CUL3 to RhoA and protecting RhoA), and physically destroys osteoclast function by utilizing cytoskeleton tension overload, thereby achieving "functional braking" of osteoclasts rather than "cell killing", which is conducive to maintaining the physiological balance of the bone microenvironment. (3) High safety: In vivo experiments have confirmed that the smallest functional peptide effectively alleviates bone loss without causing sleep structure disorder, motor coordination disorder or anxiety-like behavior in mice, and no obvious neurotoxicity was detected, demonstrating good biosafety. (4) Broad application prospects: The preparation process of this polypeptide is mature and the cost is low. It not only provides a new candidate drug for osteoporosis, but also provides a new strategy for the treatment of diseases such as peri-implantitis and periodontitis accompanied by excessive osteoclast activation. Attached Figure Description

[0017] Figure 1 The following is a flowchart of the design and screening process for Tau-peptide4 and the results of GST pull-down validation: (A) Molecular docking results of Tau-RhoA; (B) GST pull-down validation of direct binding between the two in vitro; (C) COIP binding point mutation experiment validation of the specificity of Tau-RhoA interaction in vivo; (D) Schematic diagram of Tau truncated variant design; (E) GST pull-down experiment validation of the binding ability of Tau-peptide4 to RhoA; (F) Western Blot detection of the molecular weight of purified Tau-peptide4 recombinant protein.

[0018] Figure 2To validate the in vitro function of Tau-peptide4, (A) the effect of Tau-peptide4 on the stability of RhoA protein; (BC) the effect of Tau-peptide4 on the ubiquitination level of RhoA; and (DE) TRAP staining and detection of osteoclast-related gene expression.

[0019] Figure 3 To assess the in vivo therapeutic effect of Tau-peptide4, (A) a schematic diagram of the treatment regimen in a mouse osteoporosis model induced by retinoic acid; (B) a three-dimensional reconstruction image using Micro-CT; and (C) quantitative analysis of bone volume fraction (BV / TV) and the number and thickness of trabeculae (Tb.N, Tb.Th).

[0020] Figure 4 To assess the in vivo therapeutic effect of Tau-peptide4, H&E staining and TRAP staining were used to observe pathological changes in bone tissue.

[0021] Figure 5 For the assessment of the neurological safety of Tau-peptide4, (A) the movement trajectory of mice in the cross maze; (B) the results of AT8 immunohistochemistry and Nissl staining. Detailed Implementation

[0022] The present invention will be further described below with reference to the embodiments and accompanying drawings:

[0023] Example 1

[0024] Screening and design of Tau-peptide 4

[0025] Based on the molecular docking results of the Tau-RhoA interaction interface, the Tau protein mainly binds to RhoA and CUL3 through its C-terminus. Figure 1 The AC of the Tau protein was designed in this embodiment. Four candidate short peptides were designed: Peptide1 (sequence: KCGSKDNIKHVPGGGSVQIV, corresponding to Tau582-602), Peptide2 (sequence: GGSVQIVYKPVDLSKVTSKC, corresponding to Tau594-614), Peptide3 (sequence: DLSKVTSKCGSLGNIHHKPG, corresponding to Tau606-625), and Peptide4 (sequence: GNIHHKPGGGQVEVKSEKLD, corresponding to Tau618-637), which cover different segments of the Tau protein and correspond to SEQ ID NO.1-SEQ ID NO.4 in the sequence listing, respectively. The binding ability of the peptides to RhoA was verified by GST pull-down experiments. The results showed that Peptide4 retained a significant in vitro binding ability to RhoA, and its binding strength was not significantly different from that of the full-length Tau protein. Figure 1 (DE). Therefore, Peptide4 was selected as the minimal functional peptide of this invention.

[0026] Example 2

[0027] Preparation of Tau-peptide4

[0028] Tau-peptide4 was prepared using an Escherichia coli (E. coli) prokaryotic expression system (via Wuhan Jinkairui Biosynthesis).

[0029] Expression: Rosetta (DE3) strain was used as the host cell. The target gene was constructed into a pET vector (containing a His-tag), and positive clones were selected for kanamycin resistance after transformation. Single colonies were picked and inoculated into LB medium, and cultured at 37°C with shaking until the OD600 reached approximately 0.6. IPTG inducer (final concentration 0.1-1 mM) was added and expression was induced at 30°C.

[0030] Disruption and isolation: Collect bacterial cells, resuspend in NTA-0 buffer (pH 8.0), and sonicate (200W, 3s / 4s on / off). After centrifugation at 16000rpm, if the target peptide is in the supernatant (soluble expression, existing in the supernatant as a soluble protein), collect the supernatant directly; if it is in the precipitate (the peptide forms inclusion bodies in E. coli expression), denature and dissolve it using 6M guanidine hydrochloride or 8M urea solution, and then refold it by serial dilution or dialysis.

[0031] Purification: The expression product was filtered through a 0.22 μm filter and then loaded onto a Ni-NTA affinity chromatography column. The supernatant protein solution was loaded at a flow rate of 1 mL / min; the column was washed with NTA-0 buffer (pH 8.0) until the eluent was free of protein; contaminating proteins were eluted sequentially with buffers containing 20 mM and 60 mM imidazole, and finally the target peptide was eluted with buffer containing 500 mM imidazole. The column was washed with 3 column volumes of deionized water and sealed with 20% ethanol; the purity of the collected eluent was determined by SDS-PAGE, and was ≥80%.

[0032] Identification: Detected by SDS-PAGE electrophoresis and Western blotting. Figure 1 The recombinant protein (F) had a molecular weight that met expectations (estimated molecular weight of approximately 47.78 kDa) and high purity, and was confirmed as the target product.

[0033] Example 3

[0034] In vitro biological function evaluation of Tau-peptide4 ( Figure 2 )

[0035] RhoA stability testing:

[0036] Osteoclasts treated with tau-peptide 4 (10 nM) were collected, and an Input group and a homologous IgG negative control group were set up. Total protein was extracted using non-denaturing lysis buffer containing protease inhibitors, MG132, and deubiquitinase inhibitor (NEM). Equal volumes of protein supernatant were collected, and anti-CUL3 antibody or anti-RhoA specific antibody was added, respectively. The mixture was incubated overnight at 4°C, followed by the addition of Protein A / G magnetic beads to enrich the protein complex. After thorough washing with lysis buffer and boiling elution, the RhoA or CUL3 levels in the precipitate complex were detected by Western blot. Immunoprecipitation assay (IP:RhoA) showed the interaction between endogenous RhoA and CUL3. Figure 2 The interaction was weakened in the presence of the peptide Tau-peptide4. Western blot results showed that the abundance of RhoA protein was significantly increased in the Tau-peptide4-treated group compared with the control group. Actinomycin (CHX) tracking experiments showed that Tau-peptide4 significantly slowed down the degradation rate of RhoA (RhoA). Figure 2 (C).

[0037] Ubiquitination inhibition assay: Before osteoclast collection, pretreatment with MG132 was performed to enrich ubiquitinated proteins. Cells were then collected, and total protein was extracted using a non-denaturing lysis buffer containing protease inhibitors, MG132, and NEM. RhoA protein was then enriched by co-immunoprecipitation with an anti-RhoA antibody, followed by SDS-PAGE and Western blot analysis. The ubiquitination level and immunoprecipitation efficiency of RhoA were detected using anti-ubiquitin and anti-RhoA antibodies, respectively, while the expression changes of total RhoA protein in whole-cell lysate (WCL) were simultaneously detected. Co-immunoprecipitation (Co-IP) results showed that Tau-peptide4 effectively weakened the binding of CUL3 to RhoA, reducing the ubiquitination level of RhoA. Figure 2 RhoA immunoprecipitation-ubiquitination in the presence of the proteasome inhibitor MG132 showed proteasome-dependent degradation of RhoA, and Tau-peptide4 further reduced RhoA ubiquitination under these conditions. Figure 2 B).

[0038] Osteoclast differentiation inhibition assay: Under aseptic conditions, mouse femurs and tibias were harvested, the epiphysis was removed, and the medullary cavity was flushed with α-MEM medium containing antibiotics until pale. The suspension was collected and cultured for 24 h. Adherent cells were discarded, and 5 mL of lymphocyte separation medium was added to a 15 mL centrifuge tube. The suspended cells were spread evenly along the tube wall on the upper layer of the separation medium. The tube was then transferred to a pre-chilled 4°C benchtop centrifuge and centrifuged at 1400 rpm for 22 minutes with a gradient of 5 units. The white, cloudy mononuclear cell layer at the mid-layer boundary was aspirated using a pipette and transferred to a new centrifuge tube. 10 mL of pre-chilled PBS was added to the collected cells to resuspend them. The tube was centrifuged at 1400 rpm for 5 min, and the supernatant was discarded. The cells were washed twice to remove the lymphocyte separation medium. The cells were resuspended in complete medium (α-MEM + 10% FBS + 1% antibiotics). The cell count was adjusted to 4 × 10⁶ cells / mL. 5 Cells / mL were seeded into 24-well plates, and RANKL (100 ng / mL) and M-CSF (44 ng / mL) were added for induction culture. Differentiation was maintained at 37℃ and 5% CO2. Tau-peptide 4 (100 nM) was added to the BMMS-derived osteoclast differentiation induction system. TRAP staining results of BMMS-derived osteoclast cultures induced with or without Peptide 4 (- indicates no Peptide 4, + indicates Peptide 4) showed that Peptide 4 treatment reduced the number of TRAP-positive multinucleated osteoclasts (typical large vesicle structure), meaning the number of multinucleated osteoclasts formed in the experimental group was significantly reduced. Furthermore, cytoskeleton staining showed disruption of the closed loop structure. Figure 2 D). Figure 2 E represents the Western Blot analysis results of TRAP protein in osteoclast cultures with or without Peptide 4 (- indicates no Peptide 4, + indicates Peptide 4); Hsp90 was used as an internal control. Western Blot results showed that the expression of osteoclast marker proteins (such as TRAP) was significantly downregulated. Figure 2 E).

[0039] Example 4

[0040] Evaluation of the efficacy and safety of Tau-peptide4 in vivo therapy

[0041] Animal model construction and drug administration: such as Figure 3 As shown in Figure A, C57BL / 6 mice were selected to establish an early osteoporosis model by continuous gavage administration of retinoic acid. Mice were randomly divided into a model group and a treatment group. Mice in the treatment group received daily intraperitoneal injections of tau-peptide 4 (dosage adjusted according to body weight) for 4 consecutive weeks. A normal control group and a model control group (injected with physiological saline) were also established concurrently.

[0042] Bone mineral density and microstructure analysis: After treatment, Micro-CT scans were performed on the femur and tibia of mice. Figure 3 B). Three-dimensional reconstruction images and quantitative analysis showed that, compared with retinoic acid treatment alone, the bone volume fraction (BV / TV) of the tibia and femur, as well as the number and thickness of trabeculae (Tb.N, Tb.Th), were significantly increased in the Tau peptide4 treatment group, and the trabecular sparsity was significantly reduced in three-dimensional reconstruction. Figure 3 (C).

[0043] Histological analysis: H&E and TRAP staining combined with Ctsk / p-MLC2 double-labeled immunofluorescence indicated that Tau peptide4 significantly reduced TRAP. + The number of osteoclasts was reduced and bone resorption characteristics were decreased, accompanied by decreased Ctsk signaling and increased p-MLC2 signaling. Figure 4 ).

[0044] Example 5

[0045] Neurological safety assessment of Tau-peptide4

[0046] Neurobehavioral safety testing: The elevated cruciate maze (EPM) test and sleep electroencephalography (EEG) were performed on the experimental mice. Results showed that mice treated with Tau-peptide4 did not exhibit significant differences from normal mice in open-arm dwell time, total movement distance, and sleep-wake cycle, and no obvious anxiety-like behaviors or neurotoxic reactions were observed. Figure 5 A). Representative AT8 immunohistochemical and Nissl staining results of brain sections from the same cohort (the boxed area is a high-magnification image), indicating that administration of Tau-peptide4 did not cause abnormal Tau protein phosphorylation or significant neuronal cell structural damage. Figure 5 B).

[0047] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. The application of a Tau-derived minimal functional peptide targeting the CUL3-RhoA axis in the preparation of a medicament for treating osteoclast overactivation-related diseases, characterized in that: The amino acid sequence of the Tau-derived minimal functional peptide targeting the CUL3-RhoA axis is shown in SEQ ID NO.4: GNIHHKPGGGQVEVKSEKLD. It exerts its therapeutic effect by binding to CUL3 to block RhoA ubiquitination, activating the RhoA-ROCK-MLC2 tension axis, and disrupting the osteoclast closure loop. The osteoclast overactivation-related disease is osteoporosis.

2. A method for preparing the Tau-derived minimal functional peptide targeting the CUL3-RhoA axis as described in claim 1, characterized in that: The preparation was carried out using an Escherichia coli prokaryotic expression system, including the steps of constructing a recombinant expression vector, transforming the host bacteria, inducing expression, cell disruption, and purification by high-performance liquid chromatography.

3. The method for preparing the Tau-derived minimal functional peptide targeting the CUL3-RhoA axis according to claim 2, characterized in that: Rosetta (DE3) strain was used as the host cell.

4. The method for preparing the Tau-derived minimal functional peptide targeting the CUL3-RhoA axis according to claim 3, characterized in that: Expression was induced using IPTG inducer at a concentration of 0.1-1 mM and a temperature of 30°C.

5. The method for preparing the Tau-derived minimal functional peptide targeting the CUL3-RhoA axis according to claim 4, characterized in that: Cell disruption was performed using ultrasound.

6. The method for preparing the Tau-derived minimal functional peptide targeting the CUL3-RhoA axis according to claim 5, characterized in that, The specific steps for purification by high performance liquid chromatography are as follows: After filtering the expression product through a 0.22 μm filter membrane, it is loaded onto a Ni-NTA affinity chromatography column, and the supernatant protein solution is loaded at a flow rate of 1 mL / min; the column is washed with NTA-0 buffer until the eluent is free of protein; impurities are eluted with buffers containing 20 mM and 60 mM imidazole in sequence, and finally the target peptide is eluted with buffer containing 500 mM imidazole.

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