Use of quinines or synthetic bacteria in the treatment of diseases associated with mutations in tyrosyl-tRNA synthetases

Abstract

The application provides application of quinofine or a synthetic bacterium thereof in treatment of diseases related to mutation of tyrosyl-tRNA synthetase. The quinofine or the synthetic bacterium thereof can restore defects caused by mutation of tyrosyl-tRNA synthetase, and thus can be used for treatment of diseases related to mutation of human tyrosyl-tRNA synthetase, such as Charcot-Marie-Tooth disease type C and infantile onset multi-system neurologic, endocrine and pancreatic disease type 2, or reproduction defects of Caenorhabditis elegans.

Description

Technical Field

[0001] This invention relates to the application of quinine or its synthetic bacteria in the treatment of diseases related to tyrosyl-tRNA synthetase mutations, and belongs to the field of biomedicine. Background Technology

[0002] Aminoacyl-tRNA synthetase is responsible for linking specific amino acids to corresponding transfer RNAs (tRNAs) during protein synthesis, participating in protein translation. Aminoacyl-tRNAs, as carriers of amino acids, are fundamental molecules in protein synthesis. Organisms contain approximately 20 types of tRNAs, and tRNAs are the RNA molecules with the highest modification density and the most diverse range of chemical modifications within cells. Among them, tRNAs... tyr tRNA his tRNA asn and tRNA asp Four types of tRNAs can be mediated by the QTRT1 / 2 enzyme complex, utilizing queuine taken from the environment to replace the G at position 34 with a Q. Previous studies have shown that Q modification of tRNAs regulates the accuracy and efficiency of protein translation. Furthermore, cognitive and neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, and schizophrenia are associated with the loss of Q modification in tRNAs.

[0003] Mutations in aminoacyl-tRNA synthetase in organisms can lead to motor neuron diseases. Specifically, mutations in tyrosyl-tRNA synthetase can cause Charcot-Marie-Tooth Disease (MIM608323), an intermediate type C disease, and multisystem neuroendocrine and pancreatic disease type 2 (IMNEPD2, MIM608323), which has an infancy-onset stage. 619418). Peroneal muscular atrophy (PFMA) was included in the first batch of rare diseases catalogs jointly formulated by five departments including the National Health Commission in 2018. PFMA is a group of diseases in which motor and / or sensory peripheral nerves are affected, leading to muscle weakness and atrophy, as well as sensory loss. Symptoms first occur in the distal legs and later in the hands. Due to abnormalities in nerve axons or the insulating layer (myelin sheath) around the axons, nerve cells in people with this disease cannot send electrical signals normally. Infantile multisystem neuroendocrine-pancreatic disease (IMNEPD) is a rare genetic syndrome, mainly manifested as multisystem involvement of the neuroendocrine-pancreatic glands. This disease involves systemic abnormalities, including neurological, endocrine, and pancreatic dysfunction. It is usually diagnosed in infancy or early childhood and is characterized by global developmental delay, microcephaly, intellectual disability, ataxia, sensorineural hearing loss, and pancreatic exocrine insufficiency. In addition, patients may exhibit hypotonia, growth retardation, peripheral demyelinating neuropathy, facial deformities, and other endocrine abnormalities. Brain imaging may show progressive cerebellar atrophy in some patients. Furthermore, numerous studies have reported significantly altered expression patterns of aminoacyl-tRNA synthases (ARSs) in cancerous tissues compared to normal tissues, suggesting a close association with cancer development and progression.

[0004] Currently, there are no effective treatments or drugs for peroneal muscular atrophy and multisystem neurological, endocrine, and pancreatic diseases that occur in infancy.

[0005] In other species, such as nematodes, defects caused by mutations in tyrosine-tRNA synthetase also exist, such as reduced reproductive capacity, and there are currently no relevant treatments. Summary of the Invention

[0006] This invention provides the use of quinine or its synthetic bacteria in the preparation of medicaments for treating diseases related to tyrosine-tRNA synthetase mutations.

[0007] In some implementations, in subjects suffering from the aforementioned tyrosyl-tRNA synthase mutation-related disease, their tyrosyl-tRNA synthase gene... yars-1 The mutations 1893 C>T and / or 1947 C>T exist.

[0008] In some implementations, in subjects suffering from the aforementioned tyrosyl-tRNA synthetase mutation-related disease, the tyrosyl-tRNA synthetase contains mutations S152F and / or P170L.

[0009] In some embodiments, the synthetic bacteria synthesize quinine or its precursors in amounts higher than those of Escherichia coli OP50.

[0010] In some implementations, the precursor is preQ0, preQ1, or quinine-containing tRNA.

[0011] In some embodiments, the synthetic bacteria are selected from Enterobacter holmieae and Pseudomonas aeruginosa.

[0012] In some embodiments, the synthetic bacteria are selected from Enterobacter holmieae CEN2ent1 and Pseudomonas aeruginosa MYB11.

[0013] In some implementations, the subject is a human being, and the tyrosine-tRNA synthetase mutation-related disease is selected from peroneal muscular dystrophy type C and infancy-onset multisystem neuroendocrine and pancreatic diseases type 2.

[0014] In some implementations, the subject is *C. elegans*, and the tyrosyl-tRNA synthetase mutation-related disease is a reproductive defect.

[0015] In some implementations, the subject has normal TGT complex function.

[0016] Quinine or its synthetic bacteria can restore the defects caused by mutated tyrosine-tRNA synthetase, and thus can be used to treat diseases related to tyrosine-tRNA synthetase mutations, such as peroneal muscular dystrophy type C and multisystem neurological, endocrine and pancreatic diseases type 2 with onset in infancy. Attached Figure Description

[0017] Figure 1 The reproductive capacity, relevant gene mutation sites, and corresponding amino acid mutation sites of wild-type and mutant nematodes are shown. (A) Reproductive capacity of wild-type *Caenorhabditis elegans* and nematodes with point mutations in the tyrosyl-tRNA synthetase gene (gk440720 and cas8272); (B) Reproductive capacity of the tyrosyl-tRNA synthetase gene ( yars-1 (C) Point mutation information) Schematic diagram; Amino acid mutation sites are conserved across different species.

[0018] Figure 2A A bar chart showing the recovery of reproductive capacity in mutant nematodes (gk440720) after feeding with different bacteria.

[0019] Figure 2BBar chart showing the recovery of reproductive capacity in mutant nematodes (gk440720) after feeding with CEN2ent1, MYB11 strains or their supernatants.

[0020] Figure 2C Bar chart showing the recovery of reproductive capacity of mutant nematode (gk440720) by CEN2ent1, MYB11 and OP50 strains and their supernatants at different dilutions.

[0021] Figure 2D These are results from non-target metabolomics analysis.

[0022] Figure 2E Peak diagram analysis results of preQ0, a key metabolite identified by non-target mass spectrometry, in different strains.

[0023] Figure 2F The structures of quinine and its precursor molecules preQ0 and preQ1 and the synthetic pathway of quinine in bacteria are shown.

[0024] Figure 2G Bar chart showing the effect of different concentrations of quinine and its precursor molecules preQ0 and preQ1 on the recovery of reproductive capacity in mutant nematodes (gk440720).

[0025] Figure 2H Bar chart showing the recovery of reproductive capacity in mutant nematodes (gk440720) with and without live bacteria OP50, based on the addition of quinine and its precursor molecules preQ0 and preQ1.

[0026] Figure 2I Bar chart showing the recovery of reproductive capacity of CEN2ent1 strain or quinine in mutant nematodes (cas8272).

[0027] Figure 2J The study demonstrated the effect of SLC35F2 knockdown on the reproductive capacity of quinine-restored mutant nematodes.

[0028] Figure 3A Northern blot results show the Q modification level in wild-type and mutant nematodes (gk440720).

[0029] Figure 3B The bar chart shows the percentage of Q-modified tRNAs in wild-type and mutant nematodes (gk440720).

[0030] Figure 3C Total tRNA relative to internal control 5S RNA in wild-type and mutant nematodes (gk440720) tyr Horizontal bar chart.

[0031] Figure 3D The bar chart shows the reproductive capacity of wild-type and mutant nematodes (gk440720) after TGT complex gene knockdown. In the figure, EV: empty vector, plasmid empty vector (negative control).

[0032] Figure 3E This shows the Q-modified tRNA after tgt-1 gene knockdown. tyr Horizontal Northern blot results.

[0033] Figure 3F This shows the Q-modified tRNA after tgt-1 gene knockdown. tyr Horizontal percentage bar chart.

[0034] Figure 3G Tyrosine tRNA was shown tyr and empty tRNA tyr Horizontal Northern blot results.

[0035] Figure 3H Tyrosine tRNA was shown tyr Percentage bar chart.

[0036] Figure 3I Western blot results to illustrate protein synthesis rates in wild-type and mutant nematodes (gk440720).

[0037] Figure 3J A bar chart showing the protein synthesis rates of wild-type and mutant nematodes (gk440720). Detailed Implementation

[0038] Unless otherwise stated, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art.

[0039] In this article, "queuine-synthetic bacteria" refers to bacteria that possess enzymes capable of synthesizing queuine, and can synthesize queuine or its precursors, such as preQ0, preQ1, or tRNA containing queuine (which degrades to produce queuine). The synthesized queuine can be located within the bacterial cell, secreted outside the cell, or both. Preferably, the queuine-synthetic bacteria mentioned herein have a higher synthesis capacity than *Escherichia coli* (e.g., OP50), for example, the queuine content (wt) within a single bacterium (including queuine-containing tRNA) is more than twice that of *E. coli* (e.g., 2, 5, or 10 times more), or the concentration of queuine (including queuine-containing tRNA) within the bacterium is more than twice that of *E. coli* (e.g., 2, 5, or 10 times more).

[0040] In this article, "tyrosyl-tRNA synthetase mutation" refers to an amino acid sequence change relative to the wild type in the corresponding species. This change includes one or more amino acid substitutions, deletions, or insertions. For amino acid substitution, this article refers to the replacement of one amino acid (the original amino acid) at a specific position in the amino acid sequence with another amino acid (the substituted amino acid). For example, if the 152nd amino acid residue in the wild-type protein is changed from serine (S) to phenylalanine (F), an amino acid substitution can be considered present in the mutant protein. For amino acid substitution, this article uses the following nomenclature: original amino acid, position, and substituted amino acid, and uses the IUPAC-defined single-letter abbreviations for amino acid names. For the example described above, it can be represented as S152F. Corresponding to protein mutations, the encoding gene also exhibits nucleotide mutations relative to the wild-type gene. For nucleotide substitution, this article uses the following nomenclature: nucleotide position, original nucleotide > substituted nucleotide. For example, the gene mutation corresponding to the S152F mutation described above can be represented as 1893 C>T. The amino acid positions in this article were determined by the reference protein sequence (NCBI Reference Sequence: NP_740947.2), and the nucleotide positions were determined by the reference gene sequence (NCBI gene ID: 173311).

[0041] In this article, "TGT complex" refers to tRNA-guanine transglycosylase, an RNA-modifying enzyme whose core function is to catalyze base exchange on transfer RNA (tRNA) molecules: replacing guanine (G) with a Q (quinine) base. In humans, the TGT complex is a heterodimeric complex composed of a catalytic subunit (QTRT1) and a non-catalytic subunit (QTRT2); in nematodes, it is composed of a catalytic subunit (TGT-1) and a non-catalytic subunit (TGT-2).

[0042] In this article, "treatment" includes curative, alleviating, or preventative effects. Therefore, therapeutic and preventative treatments can include improving symptoms of the disorder or preventing or otherwise reducing the risk of developing specific symptoms. Treatments can be provided to delay, slow, or reverse the progression of the disease and / or one or more of its symptoms. "Therapeutic" can also refer to reducing the severity of existing symptoms. The subject of treatment can be any person, including humans, other mammals, and even nematodes.

[0043] In this document, "or" refers to a single element among the listed optional elements, unless the context explicitly indicates otherwise. The term "and / or" refers to any one, any two, any three, any more, or all of the listed optional elements.

[0044] In this document, the terms “contains,” “comprising,” “having,” and similar expressions do not exclude elements not listed. These terms also include cases where the document consists only of the listed elements.

[0045] This invention mainly provides the application of quinine or its synthetic bacteria in the treatment of diseases related to tyrosyl-tRNA synthetase mutations, and the main technical solutions involved are as follows: 1. Mutations in tyrosyl-tRNA synthetase can lead to human diseases such as Charcot-Marie-Tooth disease and infantile-onset multisystem neurologic, endocrine, and pancreatic diseases. We have found that quinine can treat diseases caused by these gene mutations by restoring the function of tyrosyl-tRNA synthetase.

[0046] 2. Supplementing with beneficial bacteria rich in quinine can achieve the same therapeutic effect.

[0047] 3. Use quinine precursors (such as preQ0, preQ1) or similar substances to achieve the same therapeutic effect.

[0048] 4. Since quinine can simultaneously modify histidyl-tRNA, asparaginyl-tRNA, and aspartyl-tRNA, quinine, its precursor molecules, and beneficial bacteria rich in quinine and its precursor molecules can also have therapeutic effects on human diseases related to mutations in the corresponding acyl-tRNA synthetase genes.

[0049] 5. Due to the significant alteration in the expression patterns of aminoacyl-tRNA synthases (ARSs) in cancer tissues, the above treatment methods, used alone or in combination with existing drugs, may have better efficacy in treating specific cancers.

[0050] The technical solution of the present invention will be further described below with reference to embodiments and accompanying drawings. The advantages and features of the present invention will become clearer with the description. However, it should be understood that the embodiments are merely exemplary and do not constitute a limitation on the scope of the present invention. It should be noted that, unless otherwise specified, the experimental methods used in the following embodiments are conventional methods in the art.

[0051] Example 1 We investigated the effects of tyrosyl-tRNA synthetase (yars-1) deficiency on physiological reproduction in a Caenorhabditis elegans model. We found that, compared to wild-type controls, two independent point mutations in the tyrosyl-tRNA synthetase gene (gk440720 and cas8272) resulted in reproductive defects in the nematodes, manifested as a significant reduction in the number of offspring. Figure 1 A). The two point mutations are a C-to-T mutation at position 1893 of the gene sequence (NCBI gene ID: 173311) (gk440720) or a C-to-T mutation at position 1947 (cas8272). Figure 1 B). Ultimately, this results in the protein sequence (NCBI Reference Sequence: NP_740947.2) changing from the evolutionarily conserved serine (S) to phenylalanine (F), or from prolyl (P) to leucine (L) at position 170. Figure 1 C) Both mutation sites are located within the enzyme's catalytic domain. Currently reported mutations in tyrosine-tRNA synthetase causing human diseases are concentrated in this catalytic domain, consistent with the mutation location in this study. This indicates that defects in the protein's catalytic function are the primary cause of related diseases. Our subsequent research also demonstrated that quinine achieves its therapeutic effect by improving the function of this protein.

[0052] The specific experimental procedures involved in this embodiment are as follows. Wild-type *C. elegans* and two independent mutants were cultured in NGM culture dishes containing the standard bacterium *E. coli* OP50. After three days of culture, the nematodes reached adulthood, and then the number of offspring produced by each nematode was counted. The counting process lasted approximately four days. Compared with the normal wild type, the two independent *C. elegans* tyrosyl-tRNA synthetase gene mutants (gk440720 and cas8272) exhibited reproductive defects, producing only a very small number of offspring. Gene sequencing revealed that each of the two independent *C. elegans* tyrosyl-tRNA synthetase genes, yars-1, contained a point mutation. Figure 1 Table B shows schematic diagrams and mutation site information for two independent *C. elegans* tyrosyl-tRNA synthetase genes, yars-1. YARS-1 protein structure and information on the two mutation sites are shown in [the diagram]. Figure 1 (C, top row), after NCBI Blast sequence alignment analysis, it was shown that the two mutation sites are highly conserved evolutionarily. Figure 1 (C bottom row).

[0053] Example 2 To investigate whether gut microbiota and its metabolites can inhibit functional defects caused by tyrosyl-tRNA synthetase mutations, we screened 15 different bacterial strains (from the Caenorhabditis Genetics Center (CGC)) and examined whether each bacterium had a therapeutic effect on reproductive defects associated with the tyrosyl-tRNA synthetase mutant (yars-1 (gk440720)). The 15 bacterial strains are as follows: Escherichia coli ( Escherichia coli OP50; Xiangfang Enterobacter ( Enterobacter xiangfangensis CEN2ent1; pale yellow pseudomonas ( Pseudomonas lurida ) MYb11; Sphingosomalidone ( Sphingomonas molluscorum JUb134; Escherichia coli ( Escherichia coli K-12; ammonia-producing Lelitone ( Lelliottia amnigena JUb66; Berkeley Pseudomonas ( Pseudomonas berkeleyensis MSPm1; Acinetobacter gemiformis ( Acinetobacter guillouiae MYb10; Fish tufted ... Comamonas piscis BIGb0172; Animal pale bacillus ( Ochrobactrum pecoris MYb71; Sphingosine mononitrate ( Sphingobacterium sp. BIGb0170; Chrysogenum ( Chryseobacterium sp .) JUb44; Stenotrophomonas maltophilia ( Stenotrophomonas maltophilia JUb19; Escherichia coli ( Escherichia coli Ht115; Pantothecin (a nematicide) Pantoea nemavictus ) BIGb0393.

[0054] We found that feeding the bacteria with two strains (CEN2ent1) Enterobacter xiangfangensis ) and MYB11 Pseudomonas lurida It can completely restore the reproductive capacity of mutant nematodes, producing the same number of offspring as wild-type nematodes. Figure 2AMeanwhile, we also found that adding the culture supernatant of bacterial strain CEN2ent1 alone to mutant nematodes could also inhibit reproductive defects caused by tyrosine tRNA synthetase mutations, while the culture supernatant of another strain, MYB11, had no effect. Figure 2B This indicates that CENent1 bacteria can secrete beneficial metabolites into the culture medium, while MYB11 cannot. To further demonstrate that the reproductive defect caused by the tyrosyl-tRNA synthetase mutation can be restored by beneficial metabolites, we diluted the CEN2ent1 culture supernatant and added it to the mutant nematodes. The results showed that even after a 10-fold dilution, the CEN2ent1 culture supernatant (CEN sup) could still effectively rescue the reproductive capacity of the mutant. Figure 2C ).

[0055] To identify which beneficial metabolites exerted the beneficial effect, we extracted total metabolites from the CEN2ent1 culture supernatant and performed non-target metabolomics analysis. The non-target metabolomics analysis revealed that, compared to the negative control strain OP50, 46 metabolites in the CEN2ent1 culture supernatant were at concentrations more than 5 times higher than their corresponding metabolites in OP50. Figure 2D Importantly, the metabolite quinine precursor molecule preQ0 was detected only in the supernatant of CEN2ent1 medium, while it was completely absent in the negative control OP50 strain. Figure 2E (Each strain was tested in triplicate). Quinine is a metabolite produced exclusively by bacteria, synthesized from GTP as a starting compound through a multi-step metabolic pathway that modifies bacterial tRNA. tyr tRNA his tRNA asp and tRNA asn (Q-tRNA), this synthesis process produces two intermediate metabolites - preQ0 and preQ1 ( Figure 2F These four tRNAs produce quinine molecules during degradation. Figure 2F Animals, as hosts, cannot synthesize quinine molecules themselves; they can only absorb quinine molecules from gut bacteria and food and use them to modify their four aminoacyl-tRNAs. To verify that quinine or its precursor molecules indeed played a beneficial role in restoring the reproductive capacity of the tyrosyl-tRNA synthetase mutant, we added quinine and its precursor molecules preQ0 and preQ1, respectively. The results showed that all three molecules could restore the reproductive capacity of the tyrosyl-tRNA synthetase mutant (gk440720) in a concentration-dependent manner. Figure 2GDo these three bacterial metabolites all act directly on the host mutant? Or do the precursor molecules preQ0 and preQ1 require further metabolism by live bacteria to produce quinine, which then affects the mutant host? Therefore, we added the three metabolites (quinine, preQ0, and preQ1) to inactivated OP50 bacteria. The results showed that only quinine maintained its beneficial effect in the absence of live bacteria, while the other two precursor molecules (preQ0 and preQ1) lost their beneficial effect in the absence of live bacteria. Figure 2H The results showed that quinine is a beneficial metabolite that directly acts on tyrosyl-tRNA synthetase mutants, while the precursor molecules preQ0 and preQ1 require metabolism by live bacteria to exert their effects. Furthermore, supplementing another tyrosyl-tRNA synthetase mutant (cas8272) with quinine also restored its reproductive capacity. (Beneficial bacteria CEN2ent1 or supplementation with 10 μM quinine also restored the reproductive capacity of another tyrosyl-tRNA synthetase gene mutant (cas8272).) Figure 2I This indicates that quinine and the beneficial bacterium CEN2ent1 have broad therapeutic effects on tyrosyl-tRNA synthetase mutations. These results suggest that bacterial quinine, and its metabolized precursor molecules, can restore physiological functions associated with different types of tyrosyl-tRNA synthetase mutations. These beneficial bacteria and their metabolite quinine can be used to treat diseases caused by tyrosyl-tRNA synthetase mutations. The host can transport quinine into the cell via the transporter protein SLC35F2 to exert its function. Knockdown of the nematode homolog Y73E7A.3 (RNAi knockdown experiment, where the constructed Y73E7A.3 RNAi plasmid was transferred into iOP50 competent bacteria to obtain a strain capable of producing double-stranded interfering RNA, which was then fed to nematodes to achieve target gene knockdown) inhibited the beneficial effects of quinine, demonstrating that nematodes also exert their function by transporting quinine via the SLC35F2 homolog protein. Figure 2J ).

[0056] The specific experimental procedures involved in this embodiment are as follows. The tyrosine-tRNA synthetase gene mutant (gk440720) was cultured and screened on 15 different bacterial strains (each strain was cultured in an NGM culture dish). After 48 hours of culture, the number of progeny produced by the mutant under each bacterial culture condition was counted. The statistical results showed that two strains (CEN2ent1 ( Enterobacter xiangfangensis ) and MYB11 ( Pseudomonas luridaThe tyrosyl-tRNA synthetase gene mutant (gk440720) could be completely restored, and the number of offspring produced was basically the same as that of wild-type nematodes under OP50 bacterial culture conditions. After culturing CEN2ent1 and MYB11 in liquid LB medium at room temperature for 48 hours, the bacterial culture was centrifuged at 5000g, the supernatant was retained, and the precipitated bacterial cells were discarded. The retained supernatant was then filtered through a 0.22µm filter to remove any potential residual bacteria, ensuring sterility. 200µl of the obtained supernatant was then added to a culture dish containing OP50 bacteria to feed the tyrosyl-tRNA synthetase gene mutant (gk440720). The results showed that although both CEN2ent1 and MYB11 bacteria could restore the mutant's reproductive capacity, only the supernatant from CEN2ent1 could still function, indicating that only CEN2ent1 could secrete beneficial substances into the culture medium. Adding 200 μL of CEN2ent1 culture supernatant diluted 10-fold to a culture dish containing OP50 bacteria to feed mutant nematodes still showed efficacy. Metabolite extraction of the culture supernatant (80% methanol extraction for 1 hour, centrifugation at 12000g for 20 minutes, rotary evaporation to obtain bacterial supernatant metabolites, which were then sent to the company for non-target metabolomics analysis) identified a total of 1723 metabolites, of which only 46 were present in CEN2ent1 at concentrations higher than those in OP50. Non-target mass spectrometry analysis showed that the quinine precursor molecule preQ0 was present only in the CEN2ent1 culture supernatant, and not in the OP50 culture supernatant. The metabolic synthesis pathway of quinine and its precursor molecules exists only in bacteria. Experiments involving the addition of quinine and its precursor molecules preQ0 and preQ1 showed that all three molecules, when added to live OP50 bacteria, could restore the reproductive capacity of the yars-1 mutant in a concentration-dependent manner. The minimum effective concentration of preQ0 was 10 nM, with the maximum beneficial effect observed at 10 μM; the minimum effective concentration of preQ1 was 1 nM, with the maximum beneficial effect observed at 10 μM; quinine showed slightly weaker effects, but 100 nM-10 μM could still effectively restore the mutant's reproductive capacity (subsequent studies indicated that the poor effect was due to the added quinine being consumed by the OP50 bacteria, leading to a decrease in the effective concentration). When the three molecules were added to inactivated OP50 bacteria (200 μL of 10 μM solution was added to the surface of a culture dish containing OP50), only quinine exhibited a beneficial effect, indicating that only quinine can directly exert a beneficial effect in the mutant host, while the other two precursor molecules (preQ0 and preQ1) require metabolism by live bacteria to function.

[0057] Example 3 Quinine derived from gut bacteria affects host tRNA tyr tRNA his tRNAasp and tRNA asn The 34-G modification produces Q. Does this mean that the quinine produced by the beneficial bacteria CEN2ent1 is mediated through the inhibition of host tRNA? tyr How can we modify and restore the function of the mutant? We first set up four experimental groups and tested the nematode tRNA in different groups. tyr The level of Q modification at position 34. Four groups were identified: wild-type nematodes fed with quinine-deficient bacteria (WT on OP50), tyrosyl-tRNA synthetase mutant nematodes fed with quinine-deficient bacteria (yars-1(gk440720) on OP50), tyrosyl-tRNA synthetase mutant nematodes fed with quinine-rich bacteria (yars-1(gk440720) on CEN2ent1), and tyrosyl-tRNA synthetase mutant nematodes directly supplemented with quinine (yars-1(gk440720) on OP50+queuine). APB gel separation experiments showed that the tRNA levels of wild-type nematodes fed with OP50 bacteria and tyrosyl-tRNA synthetase mutant nematodes (yars-1(gk440720)) were significantly lower than those of queuine. tyr The level of Q modification at position 34 was very low, while the tRNA levels of tyrosine-tRNA synthetase mutant nematodes fed with CEN2ent1 or directly supplemented with quinine were significantly lower. tyr The level of Q modification in the 34-bit region is significantly increased. Figure 3A and 3B Meanwhile, tRNA in the tyrosyl-tRNA synthetase mutant nematode (yars-1(gk440720)) lacking quinine... tyr Overall levels decreased, while tRNA levels decreased after quinine supplementation. tyr The overall level has been restored. Figure 3C So, does quinine work directly by affecting tRNA? tyr Could a Q-modification at position 34 restore the function of tyrosine-tRNA synthetase mutants? tyr The Q modification at position 34 is catalyzed by the TGT1 / 2 protein complex. In a quinine-supplemented tyrosine-tRNA synthetase mutant, we knocked down two subunits of the TGT1 / 2 protein complex (nematode homologs: tgt-1 and tgt-2), and found that TGT complex knockdown indeed eliminated the beneficial effects of quinine. Figure 3D This indicates that wild-type nematodes do not require quinine supplementation and can develop and reproduce "normally" even with TGT1 / 2 complex knockdown. Mutants, however, are significantly affected by TGT1 / 2 complex knockdown. Knocking out any subunit of the TGT1 / 2 complex prevents added quinine from being used for Q modification of tRNA and thus from restoring reproductive capacity. This suggests that quinine likely works by modifying tRNA... tyrQ-modification was performed to rescue the function of tyrosine-tRNA synthetase mutants.

[0058] APB gel separation experiments demonstrated that knockdown of the tgt-1 gene resulted in a decrease in the level of Q-modified tRNAtyr, inhibiting the beneficial effects of quinine (Figures E and F). Acid gel assays were used to detect tyrosyl-tRNA levels, and the results showed a significant decrease in the tyrosyl-tRNAtyr ratio in the yars-1 mutant, indicating abnormal tyrosyl-tRNA synthetase function. However, CEN2ent1 bacteria or quinine supplementation restored tyrosyl-tRNAtyr levels (Figures G and H).

[0059] Tyrosyl-tRNA synthetase is used to synthesize tyrosine and the corresponding tRNA. tyr Tyrosyl-tRNA synthetase is used for protein translation and synthesis. Mutations in tyrosyl-tRNA synthetase reduce enzyme activity and decrease the production of the corresponding tyrosyl-tRNA, thus affecting protein translation and synthesis efficiency. Consistent with this, compared to wild-type nematodes (WT), the tyrosyl-tRNA synthetase mutant (yars-1(gk440720)) showed a significant decrease in protein synthesis efficiency when fed with quinine-deficient bacteria (OP50). Figure 3I and 3J Importantly, feeding the probiotic CEN2ent1 rich in quinine or directly supplementing with quinine can effectively restore protein synthesis. Figure 3I and 3J The above results indicate that quinine derived from beneficial bacteria can improve the activity and function of mutant tyrosyl-tRNA synthetase, thereby restoring protein translation and synthesis in mutants. Therefore, we believe that diseases caused by tyrosyl-tRNA synthetase mutations can be treated using quinine or its precursor molecules and corresponding beneficial bacteria.

[0060] The specific experimental procedures involved in this embodiment are as follows.

[0061] 1. APB gel separation and Northern blot experiments Total RNA was extracted from 100 μL of nematode sample using an RNA extraction kit. 3 μg of total RNA from each sample was added to a microcentrifuge tube containing 4.5 μL of water. 0.5 μL of 1 M Tris-HCl (pH 9.0) was added to each tube and mixed thoroughly. The tubes were incubated at 37°C for 30 minutes to deacylate the tRNA. After brief centrifugation, 5 μL of 2× acidic RNA loading dye (8 M urea, 0.1 M HOAc / NaOAc, pH 4.8, 0.05% bromophenol blue, 0.05% xylenecyanide) was added to each tube. All samples (10 μL per tube) were loaded onto a pre-electrophoretic 6% (0.4 mm thick) acidic denaturing PAGE gel (1× TBE, 0.1 M NaOAc / HOAc, pH 4.8). In a 4°C cold room, use acidic TAE electrophoresis buffer (1× TAE, 0.1 M NaOAc / HOAc, pH 4.8) at 18W for approximately 1 hour until the xylene blue staining band is near the bottom. Gel transfer and Western blot: Transfer the gel containing the target RNA to a Hybond-XL membrane (GE Healthcare, RPN303S) and treat under vacuum at 80°C for 4 hours using a gel desiccant (Bio-Rad, 1651745). Remove the gel from the membrane by immersing the gel and membrane in distilled water. Perform two UV crosslinkings (254 nm, 1200 mJ) and block twice with hybridization buffer (20 mM phosphate, pH 7, 300 mM NaCl, 1% SDS) for 30 minutes each time. In a UVP hybridization oven (Analytik Jena 95-0030-01), the membrane was incubated with a 3 pmol / ml biotinylated tRNA probe at 60°C for 16 hours. The membrane was washed twice for 30 minutes each time with 50 mL of washing buffer (20 mM phosphate, pH 7, 300 mM NaCl, 2 mM EDTA, 0.1% SDS). Subsequently, the membrane was incubated with streptavidin-HRP conjugate (Genscript M00091) in 30 mL of hybridization buffer (1:5000–1:10000 dilution) at room temperature for 30 minutes. The membrane was washed three times for 5 minutes each time with 25 mL of washing buffer. The membrane was transferred onto plastic wrap, RNA side up. Peroxidase detection reagents 1 and 2 (Bio-Rad 1705061) were mixed (0.1 mL / cm² membrane) and pipetted onto the top surface of the membrane. Incubate the membrane with the probe and reagent mixture for 5 minutes using a northern blot. Transfer the membrane to a fresh plastic wrap. Scan the membrane using a ChemiDoc imaging system (Bio-Rad).The results showed that wild-type nematodes fed with OP50 (quinine-deficient) bacteria or tyrosyl-tRNA synthetase gene mutants had very low levels of Q modification, while mutant nematodes fed with CEN2ent1 or directly supplemented with quinine had significantly increased levels of Q modification. This indicates that CEN2ent1 does indeed provide the host with quinine molecules and modulates tRNA. tyr Q-modification was performed. The tRNA of the tyrosyl-tRNA synthetase gene mutant nematode. tyr When levels drop, CEN2ent1 or quinine supplementation can restore tRNA levels. tyr level.

[0062] 2. RNAi knockdown experiment The constructed tgt-1 and tgt-2 RNAi plasmids were transferred into competent bacteria at iOP50 cells, resulting in a strain capable of producing double-stranded interfering RNA. This strain was then fed to nematodes, thereby achieving knockdown of the target genes. Following TGT complex gene knockdown (tgt-1 and tgt-2 RNAi knock-down), quinine supplementation failed to effectively restore the mutant's reproductive capacity. This indicates that quinine supplementation does indeed restore the mutant's reproductive capacity through Q modification of the TGT complex.

[0063] 3. Acidic gel and Northern blot experiments Take 100 μL of nematode sample and extract total RNA using a sodium acetate buffer system at pH 4.5–5.2. Take 3 μg of total RNA from each sample. Take one sample for deacylation treatment as a negative control: in 5 μL of 0.1 M Tris-HCl (pH 9.0) buffer, incubate the RNA sample at 37°C for 30–45 minutes. Add 2× RNA loading buffer (containing 8 M urea, 0.05% bromophenol blue, and 0.05% xylenecyanide) to each sample (untreated group and deacylated group), and centrifuge to mix. Prepare a 6.5% acidic denaturing polyacrylamide gel (containing 8 M urea, 0.1 M sodium acetate, pH 5.0–5.2), and pre-run at 18 W constant power for 30 minutes in a 4°C cold room using 0.1 M sodium acetate (NaOAc) buffer (pH 5.0–5.2) as the electrophoresis buffer. All samples were loaded into a gel and electrophoresed at 18 W at 4°C for approximately 1 hour until the xylene cyanide staining band migrated to the bottom of the gel. After electrophoresis, the gel was transferred to a nylon membrane, fixed by UV crosslinking, and then subjected to Northern blot hybridization to detect the aminoacylation and deaminoacylation bands of the target tRNA.

[0064] 4. Protein synthesis rate experiment After culturing synchronized nematodes for 3 days, they were added to a medium containing 0.5 mg / ml puromycin and treated for 4 hours. The newly synthesized proteins were then labeled with puromycin. Nematode samples were then collected, sonicated for 2 minutes, and analyzed by Western blot. The results showed that the total protein translation expression level of tyrosyl-tRNA synthetase gene mutant nematodes fed with OP50 (quinine-deficient) bacteria was decreased, while the total protein translation expression of the mutant nematodes could be restored by beneficial bacteria CEN2ent1 or quinine supplementation.

[0065] Currently, there are no effective treatments or drugs for human diseases related to tyrosyl-tRNA synthetase (Tyr-tRNA synthetase) gene mutations. Through the above examples, this invention discovers that supplementing with quinine or quinine-producing bacteria can serve as a method and drug for treating diseases related to tyrosyl-tRNA synthetase mutations. This discovery represents the first successful drug treatment for diseases related to this gene mutation.

[0066] Mutations in the tyrosyl-tRNA synthetase gene can lead to reproductive defects in *C. elegans*, and these two mutation sites are evolutionarily conserved. Figure 1 (C) While there are currently no clinical reports of natural mutations at this specific site in the human population, numerous reports have confirmed that functional defects in human tyrosyl-tRNA synthetase can lead to Charcot-Marie-Tooth disease type C and multisystem neuroendocrine and pancreatic diseases type 2 with onset in infancy. Based on the conservation of gene function and homologous pathogenic mechanisms, it is reasonable to infer that mutations at these conserved functional sites can disrupt the normal structure and physiological function of tyrosyl-tRNA synthetase, thereby causing related diseases in the human body, although the symptoms may differ from those in nematodes. Currently reported mutations are concentrated in the catalytic domain of the protein, consistent with the mutation location in this study. This indicates that catalytic function defects in this protein are the main cause of related diseases. Furthermore, since this invention demonstrates that functional defects in tyrosyl-tRNA synthetase caused by mutations at different sites can be restored by supplementing with quinine or quinine-producing bacteria, we can expect that functional defects caused by other mutation sites in tyrosyl-tRNA synthetase can also be restored by supplementing with quinine or quinine-producing bacteria.

[0067] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0068] References: National Health Commission, Ministry of Science and Technology, Ministry of Industry and Information Technology, etc. Notice on the Publication of the First Batch of Rare Disease Catalogues.

[0069] Mammalian Queuosine tRNA Modification Impacts Translation to EnhanceCell Proliferation and MHC-II Expression, Olivia NP Zbihley, KatherineJohnson, Luke R. Frietze 1, Wen Zhang 1, Marcus Foo, Hoang Anh V. Tran, Nicolas Chevrier, Tao Pan, JMB , 2025. Queuine: A Bacterial Nucleobase Shaping Translation in Eukaryotes, AnnE Ehrenhofer-Murray, JMB , 2025. Disease association and therapeutic routes of aminoacyl-tRNAsynthetases, Ina Yoon, Uijoo Kim, Jaeyoung Choi, Sunghoon Kim, Trends in Molecular Medicine , 2023. Aminoacyl-tRNA synthetases in human health and disease, Alexandra KTurvey, Gabriella A Horvath, André RO Cavalcanti, Front. Physio , 2022. Two microbiome metabolites compete for tRNA modification to impact mammalian cell proliferation and translation quality control, nature cellbiology, Wen Zhang, et al. , 2025. Queuosine is incorporated into tRNA precursor before splicing, WeiGuo, Igor Kaczmarczyk, Kevin Kopietz, Florian Flegler, Stefano Russo, EgeCigirgan, Andrzej Chramiec-Głąbik, Łukasz Koziej, Cansu Cirzi, JirkaPeschek, Klaus Reuter, Mark Helm, Sebastian Glatt & Francesca Tuorto, Nature communication 2025.

Claims

1. The use of quinine, its precursors, or the bacteria that synthesize them in the preparation of a medicament for treating diseases related to tyrosine-tRNA synthetase mutations in subjects, characterized in that, The precursor substance is preQ0, preQ1, or quinine-containing tRNA. The synthetic bacteria are selected from the following strains or any combination thereof: Enterobacter xiangfangense CEN2ent1; Pale yellow pseudomonas MYb11; Sphingosomalidone JUb134; Escherichia coli K-12; ammonia-producing Lelitone JUb66; Berkeley Pseudomonas MSPm1; Acinetobacter gemcitabine MYb10; Fish clump bacillus BIGb0172; Animal paleobacterium MYb71; Sphingomyelin BIGb0170; Chlorella vulgaris JUb44; Stenotrophomonas maltophilia JUb19; Escherichia coli Ht115; Pantothecin BIGb0393, a nematicide bacterium; The subject is a human being, and the disease is a tyrosyl-tRNA synthetase mutation-related disease selected from Charcot-Marie-Tooth disease type C and multisystem neuroendocrine and pancreatic disease type 2 with onset in infancy; or the subject is a Caenorhabditis elegans worm, and the disease is a reproductive defect.

2. The application according to claim 1, characterized in that, The mutated tyrosine-tRNA synthetase gene yars-1 contains mutations 1893 C>T and / or 1947 C>T.

3. The application according to claim 1, characterized in that, The tyrosine-tRNA synthetase was mutated to S152F and / or P170L.

4. The application according to claim 2 or 3, characterized in that, The subjects had normal TGT complex function.

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