Mutants of usp14 with high activity and uses thereof

Abstract

The application provides a USP14 mutant with high activity and uses thereof, the mutant comprising a mutation based on a reference sequence, the amino acid sequence of the reference sequence being shown as SEQ ID NO: 1. The mutant described in the application has high activity and is dose-dependent, the mutant can be used for drug screening as a unique target, and a tumor cell line, a tumor model or a neurodegenerative disease model can be prepared by expressing the mutant in cells.

Description

Technical Field

[0001] This application relates to the field of bioengineering technology, specifically to a highly active USP14 mutant and its uses. Background Technology

[0002] The ubiquitin-26S proteasome system (UPS) is crucial for maintaining intracellular protein homeostasis in eukaryotic cells. The 26S proteasome is a large molecular machine composed of a 19S regulatory particle (RP) and a 20S core particle (CP). The 19S regulatory particle is responsible for deubiquitinizing substrate proteins, unfolding them into linear polypeptide chains, and transporting them to the 20S core particle. The 20S core particle then degrades the unfolded protein through ATP hydrolysis. Ubiquitination refers to the covalently linking of ubiquitin chains to substrate proteins via isopeptide bonds through a cascade reaction of E1 activating enzymes, E2 conjugating enzymes, and E3 ligases. Deubiquitination is the reverse process of ubiquitination, catalyzed by deubiquitinases (DUB). DUBs can hydrolyze the ester, peptide, or isopeptide bonds at the C-terminus of ubiquitin, detaching ubiquitin molecules from ubiquitinated proteins or precursor proteins and maintaining the homeostasis of ubiquitin and proteins. The coordinated action of ubiquitination and deubiquitination, two reversible processes, allows for precise regulation of the type and degree of protein ubiquitination, influencing the function and fate of corresponding proteins and playing crucial roles in numerous life activities such as cell division, differentiation, and signal transduction.

[0003] USP14 is a DUB that binds to the 26S proteasome and can be recruited to the 19S particle for activation. It prevents the proteasome from recognizing and degrading protein substrates by cleaving ubiquitin chains. Correspondingly, inhibition of USP14 can increase proteasome degradation activity. Recent studies have shown that USP14 is amplified and overexpressed in various types of cancer. For example, in liver cancer, its expression in tumor tissue is significantly higher than in adjacent normal liver tissue; inhibition of USP14 can inhibit liver cancer cell proliferation, alter the cell cycle, and induce apoptosis. In breast cancer, USP14 expression is significantly increased in breast cancer tissue; knockdown can inhibit breast cancer cell proliferation and metastasis and promote apoptosis. In non-small cell lung cancer (NSCLC), both the mRNA and protein levels of USP14 in cancerous tissue are significantly higher than in normal lung cells; downregulation of USP14 can arrest the tumor cell cycle and inhibit cell proliferation. Furthermore, USP14 is associated with the development and progression of neurodegenerative diseases. Studies have shown that USP14 is a key protein for maintaining the level of local synaptic monomeric ubiquitin and the opening of the neuromuscular junction, and mice deficient in USP14 develop motor disorders such as ataxia. In a hippocampal model of Alzheimer's disease (AD), loss of USP14 leads to decreased ubiquitin levels and temporary synaptic plasticity deficits.

[0004] USP14 has been recognized as a novel target for cancer treatment and a potential target for treating neurodegenerative diseases. Research on its inhibitors is receiving increasing attention. Several USP14 inhibitors have been developed. β-AP15 blocks the deubiquitination activity of USP14 without inhibiting proteasome activity. In xenograft mouse models, β-AP15 exhibited antitumor activity and overcame tumor cell resistance to protease inhibitors. The analogue VLX1570, derived from this compound, possesses higher potency and solubility and has been approved by the FDA for clinical trials. In 2014, a combination of VLX1570 and dexamethasone was used in a Phase I / II clinical trial for MM. However, this trial is currently paused due to dose-limiting toxicities observed with VLX1570. Furthermore, in 2010, Daniel Finely et al. from Harvard Medical School first reported the selective USP14 inhibitor IU1. Subsequent studies showed that IU1 treatment can reduce neuronal damage caused by ischemic stroke. Mice treated with IU1 showed increased survival, reduced infarct volume, and decreased neuronal loss compared to control mice. Furthermore, IU1 can promote the degradation of some neurodegenerative disease-related proteins (such as tau, TDP-43, and abnormal prion proteins) through the UPS pathway. Overall, research on inhibitors targeting USP14 is ongoing, aiming to obtain inhibitors with high activity, high selectivity, and clinical efficacy, offering new possibilities for the treatment of tumors, neurodegenerative diseases, and more.

[0005] However, wild-type USP14 alone has very low activity. Human USP14 protein is functionally divided into two domains: a ubiquitin-like domain (UBL) and a catalytic domain (CAT). The UBL domain can bind to its catalytic domain, meaning USP14 exhibits intramolecular self-inhibition. Furthermore, the catalytic domain of USP14 alone is also inactive. Structural studies of USP14 have shown that near the active site of wild-type USP14, two loops (BL1 and BL2) block the entry of substrates into the active site, representing another form of intramolecular self-inhibition. This self-inhibition of USP14 can be relieved during functional performance through different mechanisms: 26S proteasome binding or phosphorylation by kinases such as Akt. High-throughput screening of compound libraries requires USP14 with deubiquitinating enzyme activity. However, both 26S proteasome binding and phosphorylation by kinases such as Akt pose significant challenges to drug screening. Summary of the Invention

[0006] To address the technical problems existing in the prior art, this application provides a USP14 mutant with high activity, which can be used as a sole target for screening small molecule drugs and can be used to prepare tumor models, tumor cell lines or neurodegenerative disease models.

[0007] The specific technical solution of this application is as follows:

[0008] 1. A USP14 mutant comprising a mutation based on a reference sequence, the amino acid sequence of which is shown in SEQ ID NO:1.

[0009] 2. The USP14 mutant according to claim 1, wherein the amino acids of the mutant contain an amino acid mutation corresponding to at least one of R293 and E335 of SEQ ID NO:1, preferably containing an amino acid mutation corresponding to R293 or E335 of SEQ ID NO:1.

[0010] 3. A USP14 mutant comprising an amino acid sequence as shown in SEQ ID NO:2 or 4.

[0011] 4. A nucleic acid molecule comprising a sequence encoding the USP14 mutant as described in any one of items 1-3.

[0012] 5. The nucleic acid molecule according to claim 4, wherein the nucleic acid molecule comprises the sequence shown in SEQ ID NO:3 or 5, or the sequence shown in SEQ ID NO:3 or 5.

[0013] 6. An expression vector comprising the nucleic acid molecule described in any one of items 4-5.

[0014] 7. The expression vector according to item 6, wherein the expression vector is a plasmid, granule, bacteriophage, or viral vector, preferably a plasmid.

[0015] 8. A host cell containing the expression vector described in item 6 or 7.

[0016] 9. The host cell according to item 8, wherein the host cell is a eukaryotic cell, a prokaryotic cell or a bacterial cell, preferably a prokaryotic cell, and more preferably Escherichia coli.

[0017] 10. The use of the USP14 mutant of any one of items 1-3, the USP14 mutant encoded by the nucleic acid molecule of any one of items 4-5, the USP14 mutant expressed by the expression vector of any one of items 6-7, or the USP14 mutant produced by the host cell of any one of items 8-9 in drug screening, preparation of tumor models, tumor cell lines, or neurodegenerative disease models.

[0018] The effects of the invention

[0019] The mutant described in this application has high activity and is dose-dependent. The mutant can be used as a single target for drug screening in vitro, and its expression in cells can be used to prepare tumor cell lines, tumor models, or neurodegenerative disease models. Attached Figure Description

[0020] Figures 1 to 11 This is a sequencing diagram of plasmids made using the R293L, R293A, E264S, V329A, F278A, E335S, K300A, K300L, R307E, R307S, and S432E point mutation genes.

[0021] Figure 12 This is a schematic diagram of the in vitro deubiquitinating enzyme activity of wild-type and two mutants.

[0022] Figure 13 This is a schematic diagram of the in vitro deubiquitinating enzyme activity of wild-type and different mutants. Detailed Implementation

[0023] The present application will now be described in detail with reference to the described embodiments. Although specific embodiments of the present application are shown, it should be understood that the present application can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this application will be thorough and complete, and will fully convey the scope of the present application to those skilled in the art.

[0024] It should be noted that certain terms are used in the specification and claims to refer to specific components. Those skilled in the art will understand that different terms may be used to refer to the same component. This specification and claims do not distinguish components based on differences in terminology, but rather on differences in function. The terms "comprising" or "including" used throughout the specification and claims are open-ended and should be interpreted as "comprising but not limited to." The following descriptions in the specification are preferred embodiments for carrying out this application; however, these descriptions are for the purpose of understanding the general principles of the specification and are not intended to limit the scope of this application. The scope of protection of this application shall be determined by the appended claims.

[0025] definition

[0026] Ubiquitin-specific protease 14 (USP14) is a ubiquitinating deubiquitinating enzyme found throughout eukaryotes, playing a crucial role in editing ubiquitin chain length and maintaining the stability of the free ubiquitin library. It can regulate the degradation of target proteins through deubiquitination and participate in the development and progression of tumors and the nervous system through multiple signaling pathways. Therefore, USP14 holds promise as a highly promising target for the treatment of related diseases.

[0027] As used herein, “homology,” “identity,” and “similarity” refer to the sequence similarity between two nucleic acid molecules. “Homology,” “identity,” or “similarity” can be determined by comparing positions in each sequence, and the sequences can be aligned for comparison purposes. When equivalent positions in the compared sequences are occupied by the same bases, the molecules are identical at that position; when equivalent sites are occupied by the same or similar amino acid residues (e.g., similar in spatial or electrical properties), the molecules can be considered homologous (similar) at that position. The expression of homology / similarity or identity percentage refers to the number of identical or similar amino acids at shared positions in the compared sequences. “Irrelevant” or “non-homologous” sequences share less than 40% identity with the sequences of this application, preferably less than 25%. The absence or presence of extra residues (amino acids or nucleic acids) also reduces identity and homology / similarity when comparing two sequences. In specific implementations, for two or more sequences or subsequences, determined by using the BLAST or BLAST 2.0 sequence comparison algorithm with the default parameters described below, or by manual alignment and visual inspection provided online, for example, by the National Center for Biotechnology Information (NCBI), if their sequences exhibit approximately 60% identity in the specified region, or approximately 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher, when compared and aligned for maximum correspondence within a comparison window or specified region, they can be considered substantially or significantly homologous, similar, or identical. This definition also relates to or can be used to test sequence complements. Therefore, to the extent permitted by the context of this paper, for example, if a nucleotide sequence can be predicted to be naturally present in a DNA duplex, or can be naturally present as one or both of the complementary strands, then a nucleotide sequence complementary to the specified target sequence or a variant thereof is itself considered "similar" to the target sequence, and when "similar" nucleic acid sequences are involved, this includes single-stranded sequences, their complementary sequences, double-stranded strand complexes, sequences capable of encoding the same or similar polypeptide products, and any permissible variants of any of the foregoing. Similarity must be limited to analyses of single nucleic acid strand sequences, which may include, for example, the detection and quantification of the expression of a specific RNA sequence or coding sequence in a cell. This definition also includes sequences with deletions and / or additions, as well as sequences with substitutions.In the implementation scheme, identity or similarity may be in regions of at least about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 10, 21, 22, 23, 24, 25 or more nucleotides, or in regions of more than about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or more nucleotides.

[0028] As used herein, the term "nucleotide" refers to naturally occurring nucleotides, as well as synthetic nucleotide analogs that can be recognized by cellular enzymes.

[0029] As used herein, the term "expression vector" refers to any naturally occurring or artificially constructed expression vector containing a nucleic acid molecule that can be catalyzed by cellular transcriptases and / or translatases. Exemplary expression vectors include plasmids, viruses (including bacteriophages), granules, artificial chromosomes, or transposons. In some embodiments, the expression vector is a plasmid.

[0030] As used herein, the term "host cell" refers to any biological cell that can be cultured in a culture medium and used to express recombinant genes. These host cells can be eukaryotic or prokaryotic cells, microbial cells such as bacterial cells, or cells derived from a cell line (such as an immortalized mammalian cell line), etc. In some embodiments, the host cell is a prokaryotic cell, such as Escherichia coli.

[0031] As used in this article, the term "recombinant" refers to nucleic acid molecules or polypeptides that are produced through artificial intervention in a non-natural environment.

[0032] USP14 mutant

[0033] This application provides a USP14 mutant comprising a mutation based on a reference sequence, the amino acid sequence of which is shown in SEQ ID NO:1.

[0034] The amino acid sequence of SEQ ID NO:1 is as follows:

[0035] MPLYSVTVKWGKEKFEGVELNTDEPPMVFKAQLFALTGVQPARQK

[0036] VMVKGGTLKDDDWGNIKIKNGMTLLMMGSADALPEEPSAKTVFVEDM

[0037] TEEQLASAMELPCGLTNLGNTCYMNATVQCIRSVPELKDALKRYAGALR

[0038] ASGEMASAQYITAALRDLFDSMDKTSSSIPPIILLQFLHMAFPQFAEKGEQ

[0039] GQYLQQDANECWIQMMRVLQQKLEAIEDDSVKETDSSSASAATPSKKKS

[0040] LIDQFFGVEFETTMKCTESEEEEVTKGKENQLQLSCFINQEVKYLFTGLKL

[0041] RLQEEITKQSPTLQRNALYIKSSKISRLPAYLTIQMVRFFYKEKESVNAKV

[0042] LKDVKFPLMLDMYELCTPELQEKMVSFRSKFKDLEDKKVNQQPNTSDK

[0043] KSSPQKEVKYEPFSFADDIGSNCGYYDLQAVLTHQGRSSSSGHYVSWV

[0044] KRKQDEWIKFDDDKVSIVTPEDILRLSGGGDWHIAYVLLYGPRRVEIMEE

[0045] ESEQ

[0046] The above sequence is the amino acid sequence of wild-type USP14.

[0047] In some embodiments, the mutant contains an amino acid mutation at at least one of the sites R293 and E335 of SEQ ID NO:1, preferably an amino acid mutation at the site R293 or E335 of SEQ ID NO:1.

[0048] In this application, the sites mentioned above are counted starting from the N-terminus. For example, R293 refers to the 293rd amino acid mutation starting from the N-terminus of SEQ ID NO:1.

[0049] The term "corresponds" has the meaning commonly understood by those skilled in the art. Specifically, "corresponds" means the position in one sequence that corresponds to a specified position in another sequence after two sequences have been aligned for homology or sequence identity.

[0050] In this application, the mutant may be an R at position 293 of SEQ ID NO:1 that is mutated to leucine L or an E at position 335 of the sequence that is mutated to serine S.

[0051] In this application, no restrictions are placed on the method of mutation. Mutation can be carried out using conventional methods in the art, such as directed mutagenesis, random mutagenesis, or construction of synthetic oligonucleotides. The mutated DNA sequence is then expressed in a host cell to obtain a mutant with amino acid substitution, insertion, and / or deletion. In this application, the mutant is obtained by single-point mutation of synthetic oligonucleotides.

[0052] The mutant described in this application has high deubiquitinase activity.

[0053] In this application, no restrictions are placed on the method for determining the activity of deubiquitinating enzymes. Conventional methods can be used, such as the method disclosed by Dang et al. See Dang LC, Melandri FD, Stein RL. Kinetic and mechanistic studies on the hydrolysis of ubiquitin cterminal 7-amido-4-methylcoumarin by deubiquitinating enzymes. Biochemistry, 1998, 37(7): 1868-1879.

[0054] In this application, the amino acid sequence of the 293rd position R mutated to L is as shown in SEQ ID N:2.

[0055] The amino acid sequence of SEQ ID NO:2 is as follows:

[0056] MPLYSVTVKWGKEKFEGVELNTDEPPMVFKAQLFALTGVQPARQK

[0057] VMVKGGTLKDDDWGNIKIKNGMTLLMMGSADALPEEPSAKTVFVEDM

[0058] TEEQLASAMELPCGLTNLGNTCYMNATVQCIRSVPELKDALKRYAGALR

[0059] ASGEMASAQYITAALRDLFDSMDKTSSSIPPIILLQFLHMAFPQFAEKGEQ

[0060] GQYLQQDANECWIQMMRVLQQKLEAIEDDSVKETDSSSASAATPSKKKS

[0061] LIDQFFGVEFETTMKCTESEEEEVTKGKENQLQLSCFINQEVKYLFTGLKL

[0062] LLQEEITKQSPTLQRNALYIKSSKISRLPAYLTIQMVRFFYKEKESVNAKV

[0063] LKDVKFPLMLDMYELCTPELQEKMVSFRSKFKDLEDKKVNQQPNTSDK

[0064] KSSPQKEVKYEPFSFADDIGSNCGYYDLQAVLTHQGRSSSSGHYVSWV

[0065] KRKQDEWIKFDDDKVSIVTPEDILRLSGGGDWHIAYVLLYGPRRVEIMEE

[0066] ESEQ

[0067] In this application, the amino acid sequence of E at position 335 being mutated to S is as shown in SEQ ID N:4.

[0068] The amino acid sequence of SEQ ID NO:4 is as follows:

[0069] MPLYSVTVKWGKEKFEGVELNTDEPPMVFKAQLFALTGVQPARQK

[0070] VMVKGGTLKDDDWGNIKIKNGMTLLMMGSADALPEEPSAKTVFVEDM

[0071] TEEQLASAMELPCGLTNLGNTCYMNATVQCIRSVPELKDALKRYAGALR

[0072] ASGEMASAQYITAALRDLFDSMDKTSSSIPPIILLQFLHMAFPQFAEKGEQ

[0073] GQYLQQDANECWIQMMRVLQQKLEAIEDDSVKETDSSSASAATPSKKKS

[0074] LIDQFFGVEFETTMKCTESEEEEVTKGKENQLQLSCFINQEVKYLFTGLKL

[0075] RLQEEITKQSPTLQRNALYIKSSKISRLPAYLTIQMVRFFYKSKESVNAKV

[0076] LKDVKFPLMLDMYELCTPELQEKMVSFRSKFKDLEDKKVNQQPNTSDK

[0077] KSSPQKEVKYEPFSFADDIGSNCGYYDLQAVLTHQGRSSSSGHYVSWV

[0078] KRKQDEWIKFDDDKVSIVTPEDILRLSGGGDWHIAYVLLYGPRRVEIMEE

[0079] ESEQ

[0080] Those skilled in the art will also understand that the mutant is not limited to the specific sequences listed above. The sequence of the mutant should encompass sequences that contain one, two, or three or more nucleotide mutations compared to the sequence shown in SEQ ID NO:2 or 4, but are substantially functionally identical to it, and also include sequences that have 95%, 96%, 97%, 98%, or 99% sequence identity compared to the sequence shown in SEQ ID NO:2 or 4.

[0081] This application provides a USP14 mutant containing an amino acid sequence as shown in SEQ ID NO:2 or 4.

[0082] Nucleic acid molecules, expression vectors, and host cells

[0083] This application provides a nucleic acid molecule comprising a sequence encoding the USP14 mutant as claimed in any of the preceding claims. In some embodiments, the nucleic acid molecule comprises the sequence shown in SEQ ID NO: 3 or 5, or a sequence as shown in SEQ ID NO: 3 or 5.

[0084] In some embodiments, the nucleotide sequence encoding the aforementioned USP14 mutant is codon-optimized. Typically, codon optimization involves balancing the percentage of selected codons with the abundance of publicly available human transfer RNA, ensuring that neither is overloaded or restricted. In some cases, this may be necessary because most amino acids are encoded by more than one codon, and codon usage varies from organism to organism. Differences in codon usage between the transfected gene and the host cell can affect protein expression and immunogenicity of the nucleic acid construct. Typically, for codon optimization, codons are selected to balance with human usage frequencies. Typically, amino acid codon redundancy ensures that different codons encode one amino acid. In some embodiments, when selecting codons for substitution, it may be necessary that the resulting mutation be a silent mutation so that the codon change does not affect the amino acid sequence. Typically, the last nucleotide of the codon can remain unchanged without affecting the amino acid sequence.

[0085] The sequence of SEQ ID NO:3 is as follows:

[0086] ATGCCGCTCTACTCCGTTACTGTAAAATGGGGAAAGGAGAAATTT

[0087] GAAGGTGTAGAATTGAATACAGATGAACCTCCAATGGTATTCAAGGC

[0088] TCAGCTGTTTGCGTTGACTGGAGTCCAGCCTGCCAGACAGAAAGTTAT

[0089] GGTGAAAGGAGGAACGCTAAAGGATGATGATTGGGGAAACATCAAA

[0090] ATAAAAAATGGAATGACTCTACTAATGATGGGGTCAGCAGATGCTCT

[0091] TCCAGAAGAACCCTCAGCCAAAACTGTCTTCGTAGAAGACATGACAG

[0092] AAGAACAGTTAGCATCTGCTATGGAGTTACCATGTGGATTGACAAAC

[0093] CTTGGTAACACTTGTTACATGAATGCCACAGTTCAGTGTATTCGTTCT

[0094] GTGCCTGAACTCAAAGATGCCCTTAAAGGTATGCAGGTGCCTTGAG

[0095] AGCTTCAGGGGAATGGCTTCAGCGCAGTATATTACTGCAGCCCTTAG

[0096] AGATTTGTTTGATTCCATGGATAAAACTTCTTCCAGTATTCCACCTATT

[0097] ATTCTACTGCAGTTTTTGCACATGGCTTTCCACAGTTTGCCGAGAAA

[0098] GGTGAACAAGGACAGTATCTTCAACAGGATGCTAATGAATGTTGGAT

[0099] ACAAATGATGCGAGTATTGCAACAGAAATTGGAAGCAATAGAGGATG

[0100] ATTCTGTTAAAGAGACAGACTCCTCATCTGCATCGGCAGCGACACCTT

[0101] CTAAAAAAAGAAGTTTAATCGATCAGTTCTTCGGTGTTGAGTTTGAAA

[0102] CTACCATGAAATGTACAGAATCTGAAGAAGAAGGAAGTCACCAAAGGA

[0103] AAGGAAAATCAACTTCAGCTTAGCTGTTTTATCAATCAGGAAGTCAAG

[0104] TATCTTTTTACAGGACTTAAATTGCTTCTTCAGGAAAAATCACCAAA

[0105] CAGTCTCCAACGTTGCAAAGAAATGCCTTGTATATCAAATCTTCCAAG

[0106] ATCAGCCGGCTGCCTGCTTACTTGACCATTCAGATGGTTCGATTTTTTTT

[0107] ATAAAGAGAAGGAATCTGTGAATGCCAAAGTTCTTAAGGATGTTAAA

[0108] TTTCCTCTTATGTTGGATATGTATGAACTGTGTACACCAGAACTTCAA

[0109] GAGAAAATGGTGTCTTTTCGATCCAAATTCAAGGATCTAGAAGATAA

[0110] AAAAGTGAATCAGCAGCCAAATACAAGTGACAAAAAGAGTAGTCCCC

[0111] AGAAAGAAGTTAAGTATGAACCCTTTTCTTTTGCTGATGATATTGGCT

[0112] CCAATAATTGTGGATACTATGACTTACAAGCAGTACTAACACACCAG

[0113] GGAAGGTCTAGTTCTTCAGGTCATTATGTATCATGGGTGAAAAGGAA

[0114] ACAAGATGAATGGATTAAGTTTGATGATGACAAAGTCAGCATCGTAA

[0115] CACCAGAAGATATCTTACGGCTTTCTGGTGGTGGAGACTGGCATATCG

[0116] CTTACGTTCTACTCTATGGGCCTCGCAGAGTTGAAATAATGGAAGAGG

[0117] AAAGTGAACAGTAA

[0118] The sequence of SEQ ID NO:5 is as follows:

[0119]

[0120] This application provides an expression vector comprising the nucleic acid molecules described above. In some embodiments, the expression vector is a plasmid, granule, bacteriophage, or viral vector, preferably a plasmid.

[0121] For example, the nucleic acid molecule encoding the mutant described above can be cloned into a suitable expression vector. This expression vector can be any suitable recombinant expression vector and can be used to transform or transfect any suitable host. Suitable vectors include those designed for propagation and amplification, or for expression, or both, such as plasmids and viruses. In some embodiments, the expression vector is a plasmid.

[0122] In this application, the expression vector may contain regulatory sequences (such as transcription and translation start and stop codons) that are specific to the type of host to which the vector is to be introduced (e.g., bacteria, fungi, plants, or animals), depending on whether the vector is DNA-based or RNA-based. The vector may also contain a non-natural promoter operatively linked to a nucleotide sequence encoding the aforementioned mutant. The promoter may be a non-viral or viral promoter, such as the cytomegalovirus (CMV) promoter, the SV40 promoter, the RSV promoter, and promoters found in long terminal repeat sequences of mouse stem cell viruses, as well as other promoters known to those skilled in the art. In this application, the recombinant vector comprises, in the 5' to 3' direction: a promoter, a GST tag, a target gene, and a terminator. Furthermore, the recombinant vector should also include a selection marker, such as an antibiotic resistance gene.

[0123] This application provides a host cell that includes the expression vector described above.

[0124] In this application, an expression vector is transformed into a host cell for further expression or cloning within the host cell. In some embodiments, a method for preparing a USP14 mutant is provided, the method comprising culturing a host cell containing nucleic acid encoding the USP14 mutant as described above under conditions suitable for expression of the USP14 mutant, optionally inducing the expression using an inducing agent, and recovering the USP14 mutant from the host cell (or host cell culture medium). In some embodiments, the host cell is a eukaryotic cell, a prokaryotic cell, or a bacterial cell, preferably a prokaryotic cell, and more preferably *Escherichia coli*.

[0125] The host cell refers to a cell in which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include transformants and transformed cells, which include primary transformed cells and their derived progeny, regardless of passage number. Progeny cells may not be entirely identical to parent cells in terms of nucleic acid content, but may contain mutations.

[0126] use

[0127] This application provides the use of the USP14 mutant described in any of the above claims, the USP14 mutant encoded by the nucleic acid molecule described in any of the above claims, the USP14 mutant expressed by the expression vector described in any of the above claims, or the USP14 mutant generated by the host cell described in any of the above claims in drug screening, preparation of tumor models, tumor cell lines, or neurodegenerative disease models.

[0128] The USP14 mutant provided in this application can be used as the sole target of USP14 for drug screening in vitro, and expressing the mutant in cells can prepare tumor models, tumor cell lines or neurodegenerative disease models.

[0129] Example

[0130] This application provides a general and / or specific description of the materials and test methods used in the experiments. In the following examples, unless otherwise specified, % represents wt%, i.e., weight percentage. Reagents or instruments used, unless otherwise specified, are all commercially available conventional reagent products.

[0131] Example 1: Construction of wild-type USP14 recombinant expression plasmid

[0132] Based on the DNA sequence SEQ ID NO.:1 of wild-type USP14, it was synthesized by Beijing Liuhe BGI Genomics Co., Ltd., and cloned into the pGEX-4T-2 plasmid vector to obtain the recombinant plasmid USP14-pGEX-4T-2.

[0133] Example 2: Construction of a high-activity USP14 mutant expression plasmid

[0134] Design R293L point mutation primers using the wild-type USP14 DNA sequence SEQ ID NO:1 as a template:

[0135] Upstream primer: CTTAAATTGCTTCTTCAGGAAGAAATC (SEQ ID NO:6)

[0136] Downstream primer: TTCCTGAAGAAGCAATTTAAG (SEQ ID NO:7)

[0137] Design R293A point mutation primers using the wild-type USP14 DNA sequence SEQ ID NO:1 as a template:

[0138] Upstream primer: CTTAAATTGGCACTTCAGGAAGAAATC (SEQ ID NO:8) Downstream primer: TTCCTGAAGTGCCAATTTAAG (SEQ ID NO:9)

[0139] Design E264S point mutation primers using the wild-type USP14 DNA sequence SEQ ID NO:1 as a template:

[0140] Upstream primer: GAAGAAGAAAGCGTCACCAAAGGAAAG (SEQ ID NO:10)

[0141] Downstream primer: TTTGGTGACGCTTTCTTCTTC (SEQ ID NO:11)

[0142] Design V329A point mutation primers using the wild-type USP14 DNA sequence SEQ ID NO:1 as a template:

[0143] Upstream primer: ATTCAGATGGCACGATTTTTTTATAAA (SEQ ID NO:12) Downstream primer: AAAAAATCGTGCCATCTGAAT (SEQ ID NO:13)

[0144] Design F278A point mutation primers using the wild-type USP14 DNA sequence SEQ ID NO:1 as a template:

[0145] Upstream primer: CTTAGCTGTGCAATCAATCAGGAAGTC (SEQ ID NO:14)

[0146] Downstream primer: CTGATTGATTGCACAGCTAAG (SEQ ID NO:15)

[0147] Design E335S point mutation primers using the wild-type USP14 DNA sequence SEQ ID NO:1 as a template:

[0148] Upstream primer: TTTATAAAAGCAAGGAATCTGTGAAT (SEQ ID NO:16) Downstream primer: AGATTCCTTGCTTTTATAAAA (SEQ ID NO:17)

[0149] Design K300A point mutation primers using the wild-type USP14 DNA sequence SEQ ID NO:1 as a template:

[0150] Upstream primer: GAAATCACCGCACAGTCTCCAACGTTG (SEQ ID NO:18) Downstream primer: TGGAGACTGTGCGGTGATTTC (SEQ ID NO:19)

[0151] K300L point mutation primers were designed using the wild-type USP14 DNA sequence SEQ ID NO:1 as a template:

[0152] Upstream primer: GAAATCACCCTTCAGTCTCCAACGTTG (SEQ ID NO:20) Downstream primer: TGGAGACTGAAGGGTGATTTC (SEQ ID NO:21)

[0153] Design R307E point mutation primers using the wild-type USP14 DNA sequence SEQ ID NO:1 as a template:

[0154] Upstream primer: ACGTTGCAAGAGAATGCCTTGTATATC (SEQ ID NO:22) Downstream primer: CAAGGCATTCTCTTGCAACGT (SEQ ID NO:23)

[0155] Design R307S point mutation primers using the wild-type USP14 DNA sequence SEQ ID NO:1 as a template:

[0156] Upstream primer: ACGTTGCAAAGCAATGCCTTGTATATC (SEQ ID NO:24) Downstream primer: CAAGGCATTGCTTTGCAACGT (SEQ ID NO:25)

[0157] Design S432E point mutation primers using the wild-type USP14 DNA sequence SEQ ID NO:1 as a template:

[0158] Upstream primer: AGGTCTAGTGAATCAGGTCATTATGTATC (SEQ ID NO:26)

[0159] Downstream primer: ATGACCTGATTCACTAGACCTTC (SEQ ID NO:27)

[0160] PCR reaction system and procedure:

[0161]

[0162] 95℃ for 30 seconds, (95℃ for 15 seconds, 55℃ for 15 seconds, 72℃ for 3 minutes) × 30 cycles, 72℃ for 3 minutes.

[0163] The PCR product was purified using a DNA gel extraction kit, transformed into DH5α expression host bacteria, inoculated into sterile antibiotic-free LB medium, and incubated at 37°C with shaking for 40 minutes before being plated (containing 100 μg / cm² of ampicillin). 2After overnight culture, single clones were picked and cultured in 200 μl of sterile culture medium (containing 100 μg / ml ampicillin), and then sent to Beijing Liuhe BGI Genomics Co., Ltd. for sequencing. The results are as follows: Figures 1-11 As shown, this allows for the identification of plasmids containing the mutant. The plasmid is returned after successful sequencing.

[0164] Example 3: Expression and purification of highly active USP14 mutant

[0165] The plasmid obtained in Example 2 was used to transform the BL21(DE3) expression host bacteria, and the culture was inoculated into sterile antibiotic-free LB medium. After shaking culture at 37°C for 40 minutes, the culture was spread on plates (containing 100 μg / cm² of ampicillin). 2 After overnight culture, single colonies were picked and cultured in 1000 ml of sterile culture medium (containing 100 μg / ml ampicillin) at 37°C with shaking until OD reached. 600 Once the pH reached approximately 0.6, IPTG (final concentration 0.5 mM) was added, and the temperature was adjusted to 22°C for 12 hours of induction expression. The bacteria were harvested (4°C, 4000 rpm, centrifuged for 12 minutes), the waste liquid was discarded, and the cells were resuspended in lysis buffer (25 mM Tris, 150 mM NaCl, 2 mM DTT, pH 7.5). The cells were sonicated, and then centrifuged again at 4°C, 14000 rpm for 45 minutes to separate the supernatant from the precipitate. The supernatant was collected and conjugated to an affinity column (GE Healthcare: GS4B). Subsequently, PPase enzyme (10 mg / ml, 50 μL) was used for digestion at room temperature for two hours, followed by elution with Lysis buffer.

[0166] The eluted protein was sequentially passed through an anion exchange column (GE pre-packed column Source 15Q) and a gel filtration chromatography column (GE pre-packed column Superdex 200Increase 10 / 300GL) to obtain highly pure and active USP14 mutants.

[0167] Example 4: Identification of deubiquitinating enzyme activity in highly active USP14 mutants

[0168] Dang et al. developed a general method for determining the activity of deubiquitinating enzymes based on the substrate ubiquitin C-terminal 7-amido-4-methylcoumarin (Ub-AMC) (Dang LC, Melandri FD, Stein RL. Kinetic and mechanistics studies on the hydrolysis of ubiquitin cterminal 7-amido-4-methylcoumarin by deubiquitinating enzymes. Biochemistry, 1998, 37(7): 1868-1879). When the deubiquitinating enzyme reacts with the deubiquitinating enzyme fluorescent probe (Ub-AMC), AMC is hydrolyzed and released, thus the increase in fluorescence can be measured. The activity of the deubiquitinating enzyme is measured by detecting the increase in fluorescence. The reaction system consisted of 50 mM Tris (pH 7.5), 1 mM EDTA, 1 mM ATP, 5 mM MgCl2, and 1 mM DTT. After adding the USP14 mutant, 1 μM Ub-AMC was rapidly added. The hydrolysis of Ub-AMC was monitored at emission wavelengths of 380 nm and 460 nm. Each experiment was repeated three times. The results are as follows: Figure 12 and Figure 13 As shown.

[0169] from Figure 12 and Figure 13 It can be seen that after adding the USP14 mutant described in this application, the fluorescence increase was high after 15 minutes of reaction, and from Figure 12 It can be seen that the activity of the USP14 mutant increases with increasing concentration.

[0170] from Figure 13 It can be seen that different amino acid mutations at different sites in SEQ ID NO:1, and the specific amino acids used for substitution, result in different mutants. Figure 13 It can be seen that when L is used instead of R at position 293 and S is used instead of E at position 335, the resulting mutants have higher activity.

[0171] In addition, from Figure 12 As can be seen, the mutant (R293L) provided in this application exhibits an average activity 14.4 times higher than the only reported active USP14 mutant carrying the S432E mutation. This indicates that the highly active USP14 mutant can be used in vitro for drug screening targeting USP14 as the sole target. Intracellular expression of the highly active USP14 mutant can be used to prepare tumor cell lines, tumor models, or neurodegenerative disease models.

[0172] The above description is merely a preferred embodiment of this application and is not intended to limit the application in any other way. Any person skilled in the art may make changes or modifications to the disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of this application without departing from the scope of the technical solution of this application shall still fall within the protection scope of this application.

Claims

1. A USP14 mutant having an amino acid sequence as shown in SEQ ID NO:2 or 4.

2. A nucleic acid molecule having a sequence encoding the USP14 mutant of claim 1.

3. The nucleic acid molecule according to claim 2, wherein, The nucleic acid molecule is a sequence as shown in SEQ ID NO:3 or 5.

4. An expression vector comprising the nucleic acid molecule of any one of claims 2-3.

5. The expression vector according to claim 4, wherein, The expression vector is a plasmid, a granule, or a viral vector.

6. The expression vector according to claim 5, wherein the expression vector is a plasmid.

7. The expression vector according to claim 5, wherein the expression vector is a bacteriophage.

8. A host cell comprising the expression vector according to any one of claims 4-7.

9. The host cell according to claim 8, wherein, The host cell is a eukaryotic cell or a prokaryotic cell.

10. The host cell according to claim 8, wherein the host cell is a prokaryotic cell.

11. The host cell according to claim 8, wherein the host cell is a bacterial cell.

12. The host cell according to claim 8, wherein the host cell is Escherichia coli.

Images