Histamine H3 receptor protein mutant with bias, gene and application

Abstract

The invention provides a biased histamine H3 receptor protein mutant, a gene and application. The amino acid sequence of the histamine H3 receptor protein mutant is as shown in SEQ ID NO. 1. According to the invention, two independent downstream signal channel G protein and repressor protein of a histamine H3 receptor are detected, and the receptor I225W with complete G protein bias is constructed through artificial modification. Signal transduction experiments show that the modified protein shows good G protein bias, can be used for researching a biased signal transduction mechanism of the histamine H3 receptor, and provides a theoretical basis and a model for developing targeted H3 receptor drugs. Certain application prospects are realized in the field of medicines.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a histamine H3 receptor protein mutant with bias, a gene and an application thereof. Background Art

[0002] Histamine is a widely distributed biogenic amine in the human body. It was first discovered to mediate various inflammatory reactions in the body. The existence of central histamine was first discovered in 1943, and it was later proved that histamine is an important neurotransmitter and neuromodulator in the central nervous system. Most of histamine is produced by histaminergic neurons in the tuberomammillary nucleus of the hypothalamus, and is widely and diffusely projected to the cortex, thalamus, pons and other locations, and is involved in various brain functions such as learning and memory, sleep and wakefulness. Brain histamine acts on different histamine receptors on the presynaptic membrane or postsynaptic membrane to exert its function.

[0003] In the central nervous system, there are three main types of histamine receptors: histamine type 1 receptor (H1R), histamine type 2 receptor (H2R) and histamine type 3 receptor (H3R), all of which are members of the monoamine subfamily of rhodopsin G protein-coupled receptors. H1R and H2R are located postsynaptically; while H3R is mainly located on presynaptic histaminergic neurons and other neurons, negatively regulating the synthesis and release of histamine and other neurotransmitters. In the basal ganglia, especially the dorsal and ventral striatum, most H3Rs are located on the postsynaptic membrane and have been shown to be involved in neurobehavioral disorders such as Alzheimer's disease, attention deficit hyperactivity disorder, epilepsy, stress, anxiety, Parkinson's disease, etc. It has been reported that H3R antagonists can relieve narcolepsy, inhibit epilepsy and improve cognitive ability. At present, the H3R antagonist tiplisin has been approved for marketing in many countries and regions for the treatment of narcolepsy and excessive sleepiness.

[0004] Recent studies have shown that H3R antagonists reduce neuronal apoptosis and cerebral infarction volume in the acute phase after cerebral ischemia, but their effects are unrelated to histaminergic neurons. At the same time, H3R antagonists can promote angiogenesis after focal cerebral ischemia, which is unrelated to histaminergic neurons but is related to H3R in vascular endothelial cells, involving the regulation of Annexin A2 (ANXA2). Therefore, H3R has the potential to become a new target for the treatment of ischemic stroke. Since H3R is mainly distributed in the central nervous system and related drugs have few peripheral side effects, H3R is a target with great clinical application prospects.

[0005] H3R can mediate downstream Gi protein and repressor protein signaling pathways respectively. Current studies have shown that different signaling pathways can be involved in different physiological functions. For example, the repressor protein pathway of NTSR1 mediates the therapeutic effect of reducing addiction, while its G protein pathway mediates the side effects of hypothermia, hypotension and movement disorders. Therefore, the development of a biased H3R receptor will help to further distinguish the functions of different H3R signaling pathways and facilitate the development of drugs with high efficacy and low toxicity and side effects.

[0006] If researchers artificially modify H3R through molecular biological methods and develop a biased exogenous protein, it can be used to study the relationship between different effector protein signaling pathways and diseases such as Alzheimer's disease, attention deficit hyperactivity disorder, epilepsy, stress, anxiety, Parkinson's disease, etc. In the future, H3R receptors can be modified through gene editing, which has extremely high therapeutic potential. Summary of the invention

[0007] The present invention provides an artificially modified exogenous protein with bias. An expression vector containing the exogenous protein gene is obtained by in vitro amplification. The protein is expressed in tumor cells by liposome transfection, and then a signal transduction experiment is used to detect and prove that the downstream signal of the exogenous protein has bias.

[0008] Specifically, the present invention provides a histamine H3 receptor protein mutant with bias, and the amino acid sequence of the histamine H3 receptor protein mutant is shown in SEQ ID NO.1.

[0009] The present invention also provides a gene encoding the histamine H3 receptor protein mutant.

[0010] Preferably, the nucleotide sequence of the gene is shown as SEQ ID NO.2.

[0011] The invention also provides an expression box, a recombinant vector, a recombinant microorganism or a transgenic cell line containing the gene.

[0012] The present invention also provides an expression vector, the expression vector comprising the gene. In the present embodiment, plasmid pcDNA3.1 is selected as an example.

[0013] The present invention also provides the use of the histamine H3 receptor protein mutant, or the gene, or the expression box, recombinant vector, recombinant microorganism or transgenic cell line containing the gene in the preparation of a biased H3 receptor drug.

[0014] When used, the gene is introduced into cells to express the histamine H3 receptor protein mutant.

[0015] The present invention also provides a biased H3 receptor drug, the active ingredient of which includes the histamine H3 receptor protein mutant, or the gene, or an expression box, a recombinant vector, a recombinant microorganism or a transgenic cell line containing the gene.

[0016] The present invention utilizes the independent signal pathway downstream of the H3 receptor protein and constructs a variant H3 receptor I225W with G protein bias through artificial modification. Signal transduction experiments show that the modified protein exhibits good G protein bias, can be used to study the mechanism of histamine H3 receptor biased signal transduction, and provides a theoretical basis and model for the development of drugs targeting H3 receptors. It has certain application prospects in the field of medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the plasmid map of pcDNA3.1-H3R-WT, on which a biased receptor was obtained through modification; and it serves as a positive control for G protein activation in response to histamine.

[0018] Figure 2 The plasmid map of pcDNA3.1-H3R-WT-I225W is used to detect G protein activation in response to histamine.

[0019] Figure 3 and Figure 5 Plasmid maps of pcDNA3.1-H3R-WT-RLUC8 and pcDNA3.1-H3R-V2R-RLUC8, respectively, were used as positive controls for the recruitment of the repressor protein in response to histamine.

[0020] Figure 4 and Figure 6 Plasmid maps of pcDNA3.1-H3R-WT-I225W-RLUC8 and pcDNA3.1-H3R-V2R-I225W-RLUC8, respectively, were used to detect the recruitment of repressor proteins in response to histamine.

[0021] Figure 7 The figure is a comparison of the membrane surface receptor expression of each group of cells. Each group of cells was transfected with H3R-WT and H3R-I225W and the corresponding functional plasmids, and the expression of H3R membrane on the cell surface was detected by ELISA after 48 hours, and the receptors were highly expressed.

[0022] Figure 8 and Fig. 9The following are the curves of H3R-V2R mutant and H3R-WT recruiting repressor proteins. BRET assay was performed 48 hours after transfection, and the maximum effect of H3R-I225W recruiting repressor proteins was calculated to be 54.55% of WT, or the specific ratio could not be calculated due to the obvious weakening of the agonistic effect. This indicates that the I225W mutation has a significant effect on the downstream repressor protein signal of H3R.

[0023] Fig.10 The curves of G protein activation by H3R-WT mutants are shown respectively. NanoBit assay was performed 48 hours after transfection, and the maximum effect of H3R-I225W on G protein activation was calculated to be 96.30% of WT, indicating that the I225W mutation had no significant effect on the downstream G protein signal of H3R. DETAILED DESCRIPTION

[0024] Example 1: Cloning and construction of H3 receptor protein expression vector pcDNA3.1-H3R-WT

[0025] Gene synthesis was performed at GENEWISE according to the complete gene sequence of the H3 receptor protein (ENSG00000101180). The nucleotide sequence of the gene is shown in SEQ ID NO.13, and the amino acid sequence is shown in SEQ ID NO.14. The H3 receptor sequence was constructed into the pcDNA3.1 vector by homologous recombination. The sequences of the primers used in the construction are shown in Table 1.

[0026] Table 1 Primers

[0027]

[0028] The pcDNA3.1-H3R-WT plasmid DNA fragment was obtained by PCR method. The PCR reaction system is shown in Table 2.

[0029] Table 2 PCR amplification system

[0030] Template DNA 1μl Forward primer (10 μM) 1.5μl Reverse primer (10 μM) 1.5μl High-fidelity Taq enzyme 15μl Double distilled water 10μl DMSO 1μl Total volume 30μl

[0031] PCR reaction conditions: pre-denaturation at 95°C for 2 min; denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 2 min, for a total of 18 cycles; extension at 72°C for 10 min.

[0032] The obtained DNA fragment was transformed into competent cells DH5α for amplification, and positive colonies were extracted and sequenced for verification. The eukaryotic expression vector pcDNA3.1-H3R-WT was obtained, and its map is shown in Figure 1 shown.

[0033] Example 2: Cloning and construction of H3 receptor protein expression vector pcDNA3.1-H3R-WT-I225W

[0034] Deletion mutation primers were designed based on pcDNA3.1-H3R-WT to mutate the 225th position of the H3 receptor protein from tryptophan to isoleucine. The sequences of the mutation primers are shown in Table 3.

[0035] Table 3 Mutation primers

[0036]

[0037] Using the pcDNA3.1-H3R-WT plasmid prepared in Example 1 as a template, the pcDNA3.1-H3R-WT-I225W plasmid DNA fragment was obtained by mutation PCR method. The PCR reaction system and PCR reaction conditions were the same as those in Example 1.

[0038] The obtained DNA fragment was transformed into competent cells DH5α for amplification, and positive colonies were extracted and sequenced for verification. The eukaryotic expression vector pcDNA3.1-H3R-WT-I225W (map as shown in Figure 2 The mutant obtained by mutation of H3 receptor was named as H3R-I225W protein, the amino acid sequence of H3R-I225W protein was shown in SEQ ID No.1; the gene sequence of H3R-I225W was shown in SEQ ID No.2.

[0039] Example 3: Detection of membrane expression of receptors

[0040] The flag tag was fused to the N-terminus of H3R-WT and H3R-1225W proteins. 48 hours after plasmid transfection, ELISA experiments were performed using cells plated on 96 white plates to detect the membrane expression of the receptor. The cells were fixed with 4% formaldehyde for 5 minutes at room temperature and then washed once with PBS. After fixation, the cells were blocked with blocking buffer (1% BSA in PBS solution) for 2 hours at room temperature. After that, they were incubated with a 1:10,000 dilution of monoclonal anti-FLAG M2-peroxidase (HRP) antibody (Sigma Aldrich) in blocking buffer at room temperature for 0.5 hours. After careful washing, 80 μL of SuperSignal ELISAFemto Maximum Sensitivity Substrate (ThermoFisher Scientific) solution was added to each well. The brightness signal was measured using an interval of 500 milliseconds. The experiment showed that the H3R-I225W receptor was highly expressed ( Figure 7 ).

[0041] Example 4: Biased effect of H3R-I225W on downstream signaling

[0042] (1) H3R-I225W significantly affects the recruitment of repressor proteins

[0043] Histamine family receptors, as G protein-coupled membrane receptors, can be phosphorylated by GRK, thereby recruiting repressor proteins. In the BRET experiment, Rluc (sequence as shown in SEQ ID NO.5) and Venus (sequence as shown in SEQ ID NO.6) sequences are respectively carried on H3R and repressor proteins. When these two recombinant proteins interact, the energy emitted by luciferase can be transferred to the fluorescent protein, thereby detecting a yellow light peak in the luminescence spectrum. The ability of H3R to recruit repressor proteins is determined by the BRET experiment.

[0044] In the BRET experiment, the plasmids containing H3R-WT-Rluc8 / H3R-WT-I225W-Rluc8 / H3R-V2R-Rluc8 / H3R-V2R-I225W-Rluc8 (the plasmid map of pcDNA3.1-H3R-WT-RLUC8 is as shown in Figure 3 The plasmid map of pcDNA3.1-H3R-WT-I225W-RLUC8 is shown in Figure 4 The plasmid map of pcDNA3.1-H3R-V2R-RLUC8 is shown in Figure 5 The plasmid map of pcDNA3.1-H3R-V2R-I225W-RLUC8 is shown in Figure 6 The plasmids of 1:4 were transfected into HEK293T cells, and about 24 hours later, they were plated into white opaque 96-well plates coated with adhesion factors. After 24 hours, the cells were rinsed twice with PBS, and then 45 microliters of buffer (HBSS, 5mM HEPES pH 7.4 and 0.1% BSA) were added and incubated for 30 minutes. 10μM coelenterazine 400a was added as a fluorescent substrate, incubated at room temperature for 5 minutes, and fluorescence was detected by a multifunctional microplate reader, with an excitation light of 410nm and an emission light of 515nm; after adding histamine with a gradient dilution, 15 cycles were measured again.

[0045] Figure 8 and Fig. 9 The following are the curves of H3R-V2R and H3R-WT mutants recruiting repressor proteins. BRET assay was performed 48 hours after transfection, and the maximum effect of H3R-I225W recruiting repressor proteins was calculated to be 54.55% of WT, or the specific ratio could not be calculated due to the obvious weakening of the agonistic effect. This indicates that the I225W mutation has a significant effect on the downstream repressor protein signal of H3R.

[0046] (2) H3R-I225W does not affect G protein activation

[0047] Histamine family receptors, as G protein-coupled membrane receptors, can activate G proteins, causing the Gα, Gβ and Gγ subunits of G proteins to dissociate and activate downstream signaling molecules. In the NanoBit experiment, SmBit (sequence shown in SEQ ID NO.8) and LgBit sequences (sequence shown in SEQ ID NO.9) are attached to Gα and Gγ, respectively. When the spatial distance between the two is close enough, they will have hydrolase activity and hydrolyze the fluorescent substrate to emit fluorescence. The NanoBit experiment is used to measure the ability of H3R to dissociate Gi proteins.

[0048] In the NanoBit experiment, we used LIPO transfection reagent to transfect plasmids containing H3R-WT / H3R-WT-I225W, Gαi-SmBit (sequence as shown in SEQ ID NO.10), Gβ (sequence as shown in SEQ ID NO.11), and Gγ-LgBit (sequence as shown in SEQID NO.12) gene fragments into HEK293T cells at a ratio of 6:2:1:1. After about 24 hours, the cells were plated in a white-bottomed opaque 96-well plate coated with adhesion factors. After 24 hours, the cells were rinsed twice with PBS, and then 10μM coelenterazine h was added as a fluorescent substrate (the buffer was HBSS, 5mM HEPES pH 7.4 and 0.1% BSA were added). Incubated at room temperature for 1 hour, luminescence was measured with a multifunctional microplate reader; after adding gradient dilutions of histamine, 15 cycles were measured again.

[0049] Fig.10 The curve of G protein activation by H3R-WT mutant. NanoBit assay was performed 48 hours after transfection, and the maximum effect of H3R-I225W on G protein activation was calculated to be 96.30% of WT, indicating that the I225W mutation had no significant effect on the downstream G protein signal of H3R.

[0050] In summary, the H3R receptor protein truncation we constructed: H3R-I225W protein, has a G protein-biased downstream signal.

Claims

1. A histamine H3 receptor protein mutant having a bias, characterized in that: The amino acid sequence of the histamine H3 receptor protein mutant is shown in SEQ ID NO.

1.

2. A gene encoding the histamine H3 receptor protein mutant according to claim 1.

3. The gene according to claim 2, characterized in that The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

4. An expression cassette, recombinant vector, recombinant microorganism or transgenic cell line containing the gene according to claim 2 or 3.

5. An expression vector, characterized in that: The expression vector comprises the gene according to claim 2 or 3.

6. Use of the histamine H3 receptor protein mutant according to claim 1, or the gene according to claim 2 or 3, or an expression cassette, a recombinant vector, a recombinant microorganism or a transgenic cell line containing the gene according to claim 2 or 3 in the preparation of a biased H3 receptor drug.

7. The use according to claim 6, characterized in that When used, the gene is introduced into cells to express the histamine H3 receptor protein mutant.

8. A H3 receptor-biased drug, characterized in that: The active ingredient of the biased H3 receptor drug includes the histamine H3 receptor protein mutant according to claim 1, or the gene according to claim 2 or 3, or an expression cassette, recombinant vector, recombinant microorganism or transgenic cell line containing the gene according to claim 2 or 3.

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