CN111467493A - Human REV 3L protein cleavage inhibitor and application thereof - Google Patents

Human REV 3L protein cleavage inhibitor and application thereof Download PDF

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CN111467493A
CN111467493A CN201910065565.0A CN201910065565A CN111467493A CN 111467493 A CN111467493 A CN 111467493A CN 201910065565 A CN201910065565 A CN 201910065565A CN 111467493 A CN111467493 A CN 111467493A
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李夏璐
王凤亭
李苗苗
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Capital Normal University
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Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a human REV 3L protein cleavage inhibitor and an application thereof, wherein the human REV 3L protein cleavage inhibitor is a medicine for improving the DNA damage sensitivity of tumor cells and controlling the drug resistance of the tumor cells, and the action target of the medicine is a position-specific cleavage event of human REV 3L protein mediated by protease TASP 1.

Description

Human REV 3L protein cleavage inhibitor and application thereof
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a human REV 3L protein cleavage inhibitor and application thereof.
Background
In tumor treatment, many chemotherapeutic drugs are effective initially, but over time, develop resistance, resulting in tumor recurrence. Secondary drug resistance of tumor cells has become a major cause of failure of tumor chemotherapy. With the development of genome-wide sequencing studies of tumor cells, it is increasingly recognized that genomic changes, in the form of DNA mutations as the major form, are the molecular basis for the development of acquired chemotherapy resistance.
DNA polymerase ζ is the major polymerase involved in low-fidelity, cross-damaged DNA synthesis in eukaryotic cells, whose catalytic subunit REV 3L loses its corrective function due to lack of 3 'to 5' exonuclease activity in yeast cells, polymerase ζ does not affect cell growth and proliferation, but most (> 90%) of yeast cells are mutated by DNA damage, and the formation of more than half of spontaneous mutations depends on polymerase ζ DNA synthesis activity.
Disclosure of Invention
The invention aims to provide an inhibitor for human REV 3L protein cleavage.
Still another object of the present invention is the use of inhibitors of human REV 3L protein cleavage.
Inhibitors of human REV 3L protein cleavage according to embodiments of the invention include small molecule compounds or polypeptides that block cleavage of the full-length human REV 3L protein to produce cleaved protein.
According to the human REV 3L protein cleavage inhibitor of the embodiment of the invention, the small molecule compound or the polypeptide has a target of action on a sequence element which is depended on by the post-translational cleavage of the human REV 3L protein, and is used for blocking the sequence element, and the amino acid sequence of the sequence element is shown as SEQ ID No. 1.
SEQ ID No.1：QLDGTAD
The small molecule compound or polypeptide may block a cleavage-dependent sequence element of human REV 3L protein, or block a site of interaction with TASP1 on REV 3L protein, thereby inhibiting cleavage of REV 3L, e.g., a polypeptide that strongly interacts with a cleavage-dependent sequence element or a compound that achieves the same purpose.
The amino acid sequence of the human REV 3L protein is shown as SEQ ID No. 2:
MFSVRIVTADYYMASPLQGLDTCQSPLTQAPVKKVPVVRVFGATPAGQKTCLHLHGIFPYLYVPYDGYGQQPESYLSQMAFSIDRALNVALGNPSSTAQHVFKVSLVSGMPFYGYHEKERHFMKIYLYNPTMVKRICELLQSGAIMNKFYQPHEAHIPYLLQLFIDYNLYGMNLINLAAVKFRKARRKSNTLHATGSCKNHLSGNSLADTLFRWEQDEIPSSLILEGVEPQSTCELEVDAVAADILNRLDIEAQIGGNPGLQAIWEDEKQRRRNRNETSQMSQPESQDHRFVPATESEKKFQKRLQEILKQNDFSVTLSGSVDYSDGSQEFSAELTLHSEVLSPEMLQCTPANMVEVHKDKESSKGHTRHKVEEALINEEAILNLMENSQTFQPLTQRLSESPVFMDSSPDEALVHLLAGLESDGYRGERNRMPSPCRSFGNNKYPQNSDDEENEPQIEKEEMELSLVMSQRWDSNIEEHCAKKRSLCRNTHRSSTEDDDSSSGEEMEWSDNSLLLASLSIPQLDGTADENSDNPLNNENSRTHSSVIATSKLSVKPSIFHKDAATLEPSSSAKITFQCKHTSALSSHVLNKEDLIEDLSQTNKNTEKGLDNSVTSFTNESTYSMKYPGSLSSTVHSENSHKENSKKEILPVSSCESSIFDYEEDIPSVTRQVPSRKYTNIRKIEKDSPFIHMHRHPNENTLGKNSFNFSDLNHSKNKVSSEGNEKGNSTALSSLFPSSFTENCELLSCSGENRTMVHSLNSTADESGLNKLKIRYEEFQEHKTEKPSLSQQAAHYMFFPSVVLSNCLTRPQKLSPVTYKLQPGNKPSRLKLNKRKLAGHQETSTKSSETGSTKDNFIQNNPCNSNPEKDNALASDLTKTTRGAFENKTPTDGFIDCHFGDGTLETEQSFGLYGNKYTLRAKRKVNYETEDSESSFVTHNSKISLPHPMEIGESLDGTLKSRKRRKMSKKLPPVIIKYIIINRFRGRKNMLVKLGKIDSKEKQVILTEEKMELYKKLAPLKDFWPKVPDSPATKYPIYPLTPKKSHRRKSKHKSAKKKTGKQQRTNNENIKRTLSFRKKRSHAILSPPSPSYNAETEDCDLNYSDVMSKLGFLSERSTSPINSSPPRCWSPTDPRAEEIMAAAEKEAMLFKGPNVYKKTVNSRIGKTSRARAQIKKSKAKLANPSIVTKKRNKRNQTNKLVDDGKKKPRAKQKTNEKGTSRKHTTLKDEKIKSQSGAEVKFVLKHQNVSEFASSSGGSQLLFKQKDMPLMGSAVDHPLSASLPTGINAQQKLSGCFSSFLESKKSVDLQTFPSSRDDLHPSVVCNSIGPGVSKINVQRPHNQSAMFTLKESTLIQKNIFDLSNHLSQVAQNTQISSGMSSKIEDNANNIQRNYLSSIGKLSEYRNSLESKLDQAYTPNFLHCKDSQQQIVCIAEQSKHSETCSPGNTASEESQMPNNCFVTSLRSPIKQIAWEQKQRGFILDMSNFKPERVKPRSLSEAISQTKALSQCKNRNVSTPSAFGEGQSGLAVLKELLQKRQQKAQNANTTQDPLSNKHQPNKNISGSLEHNKANKRTRSVTSPRKPRTPRSTKQKEKIPKLLKVDSLNLQNSSQLDNSVSDDSPIFFSDPGFESCYSLEDSLSPEHNYNFDINTIGQTGFCSFYSGSQFVPADQNLPQKFLSDAVQDLFPGQAIEKNEFLSHDNQKCDEDKHHTTDSASWIRSGTLSPEIFEKSTIDSNENRRHNQWKNSFHPLTTRSNSIMDSFCVQQAEDCLSEKSRLNRSSVSKEVFLSLPQPNNSDWIQGHTRKEMGQSLDSANTSFTAILSSPDGELVDVACEDLELYVSRNNDMLTPTPDSSPRSTSSPSQSKNGSFTPRTANILKPLMSPPSREEIMATLLDHDLSETIYQEPFCSNPSDVPEKPREIGGRLLMVETRLANDLAEFEGDFSLEGLRLWKTAFSAMTQNPRPGSPLRSGQGVVNKGSSNSPKMVEDKKIVIMPCKCAPSRQLVQVWLQAKEEYERSKKLPKTKPTGVVKSAENFSSSVNPDDKPVVPPKMDVSPCILPTTAHTKEDVDNSQIALQAPTTGCSQTASESQMLPPVASASDPEKDEDDDDNYYISYSSPDSPVIPPWQQPISPDSKALNGDDRPSSPVEELPSLAFENFLKPIKDGIQKSPCSEPQEPLVISPINTRARTGKCESLCFHSTPIIQRKLLERLPEAPGLSPLSTEPKTQKLSNKKGSNTDTLRRVLLTQAKNQFAAVNTPQKETSQIDGPSLNNTYGFKVSIQNLQEAKALHEIQNLTLISVELHARTRRDLEPDPEFDPICALFYCISSDTPLPDTEKTELTGVIVIDKDKTVFSQDIRYQTPLLIRSGITGLEVTYAADEKALFHEIANIIKRYDPDILLGYEIQMHSWGYLLQRAAALSIDLCRMISRVPDDKIENRFAAERDEYGSYTMSEINIVGRITLNLWRIMRNEVALTNYTFENVSFHVLHQRFPLFTFRVLSDWFDNKTDLYRWKMVDHYVSRVRGNLQMLEQLDLIGKTSEMARLFGIQFLHVLTRGSQYRVESMMLRIAKPMNYIPVTPSVQQRSQMRAPQCVPLIMEPESRFYSNSVLVLDFQSLYPSIVIAYNYCFSTCLGHVENLGKYDEFKFGCTSLRVPPDLLYQVRHDITVSPNGVAFVKPSVRKGVLPRMLEEILKTRFMVKQSMKAYKQDRALSRMLDARQLGLKLIANVTFGYTSANFSGRMPCIEVGDSIVHKARETLERAIKLVNDTKKWGARVVYGDTDSMFVLLKGATKEQSFKIGQEIAEAVTATNPKPVKLKFEKVYLPCVLQTKKRYVGYMYETLDQKDPVFDAKGIETVRRDSCPAVSKILERSLKLLFETRDISLIKQYVQRQCMKLLEGKASIQDFIFAKEYRGSFSYKPGACVPALELTRKMLTYDRRSEPQVGERVPYVIIYGTPGVPLIQLVRRPVEVLQDPTLRLNATYYITKQILPPLARIFSLIGIDVFSWYHELPRIHKATSSSRSEPEGRKGTISQYFTTLHCPVCDDLTQHGICSKCRSQPQHVAVILNQEIRELERQQEQLVKICKNCTGCFDRHIPCVSLNCPVLFKLSRVNRELSKAPYLRQLLDQF
the human REV 3L protein comprises 3130 amino acids in total.
According to the human REV 3L protein cleavage inhibitor of the specific embodiment of the invention, the small molecule compound or the polypeptide is an expression interference agent or activity inhibitor of protease TASP1, and the amino acid sequence of the protease TASP1 is shown as SEQ ID No. 3.
The amino acid sequence of the protease TASP1 is shown in SEQ ID No. 3:
MTMEKGMSSGEGLPSRSSQVSAGKITAKELETKQSYKEKRGGFVLVHAGAGYHSESKAKEYKHVCKRACQKAIEKLQAGALATDAVTAALVELEDSPFTNAGMGSNLNLLGEIECDASIMDGKSLNFGAVGALSGIKNPVSVANRLLCEGQKGKLSAGRIPPCFLVGEGAYRWAVDHGIPSCPPNIMTTRFSLAAFKRNKRKLELAERVDTDFMQLKKRRQSSEKENDSGTLDTVGAVVVDHEGNVAAAVSSGGLALKHPGRVGQAALYGCGCWAENTGAHNPYSTAVSTSGCGEHLVRTILARECSHALQAEDAHQALLETMQNKFISSPFLASEDGVLGGVIVLRSCRCSAEPDSSQNKQTLLVEFLWSHTTESMCVGYMSAQDGKAKTHISRLPPGAVAGQSVAIEGGVCRLESPVN
the protease TASP1 contains 420 amino acids.
The protease TASP1 gene sequence is shown in SEQ ID No. 4:
GGGTGACTACTTCCGGTGCAGTGAAGGCTCGGGGCTGAAGCGGGGTAATTCCTCTCCTGCAATTACTTTTGGATGGAAGTATGCCCCTTTCTCAGTAGAAGATGGTAATCTTGGAGAATGACCATGGAGAAGGGGATGAGTTCTGGAGAAGGGCTGCCTTCCAGATCATCTCAGGTTTCGGCTGGTAAAATAACAGCCAAAGAGTTGGAAACAAAGCAGTCCTATAAAGAGAAACGAGGAGGCTTTGTGTTGGTGCATGCAGGTGCAGGTTATCATTCTGAATCCAAAGCCAAGGAGTATAAACATGTATGCAAACGAGCTTGTCAGAAGGCAATTGAAAAGCTGCAGGCCGGTGCTCTTGCAACTGACGCAGTCACTGCAGCACTGGTGGAACTTGAGGATTCTCCTTTTACAAATGCAGGAATGGGATCTAATCTAAATCTGTTAGGTGAAATTGAGTGTGATGCCAGCATAATGGATGGAAAATCCTTAAATTTTGGAGCAGTTGGAGCACTGAGTGGAATCAAGAACCCAGTCTCGGTTGCCAACAGACTCTTATGTGAAGGGCAGAAGGGCAAGCTCTCGGCTGGCAGAATTCCTCCCTGCTTTTTAGTTGGAGAAGGAGCCTACAGATGGGCAGTAGATCATGGAATACCCTCTTGCCCTCCTAACATCATGACCACAAGATTCAGTTTAGCTGCATTTAAAAGAAACAAGAGGAAACTAGAGCTGGCAGAAAGGGTGGACACAGATTTTATGCAACTAAAGAAAAGAAGACAATCAAGTGAGAAGGAAAATGACTCAGGCACTTTGGACACGGTAGGCGCTGTGGTTGTGGACCACGAAGGGAATGTTGCTGCTGCTGTCTCCAGTGGAGGCTTGGCCTTGAAACATCCGGGGAGAGTTGGGCAGGCTGCTCTTTATGGATGTGGCTGCTGGGCTGAAAATACTGGAGCTCATAACCCCTACTCCACAGCTGTGAGTACCTCAGGATGTGGAGAGCATCTTGTGCGCACCATACTGGCTAGAGAATGTTCACATGCTTTACAAGCTGAGGATGCTCACCAAGCCCTGTTGGAGACTATGCAAAACAAGTTTATCAGTTCACCTTTCCTTGCCAGTGAAGATGGCGTGCTTGGCGGAGTGATTGTCCTCCGTTCATGCAGATGTTCTGCCGAGCCTGACTCCTCCCAAAATAAGCAGACACTTCTAGTGGAATTTCTGTGGAGCCACACGACGGAGAGCATGTGTGTCGGATATATGTCAGCCCAGGATGGGAAAGCCAAGACTCACATTTCAAGACTTCCTCCTGGTGCGGTGGCAGGACAGTCTGTGGCAATCGAAGGTGGGGTGTGCCGCCTGGAGAGCCCAGTGAACTGACCCTTCAGGCTGAGTGTGAAGCGTCTCAGAGGCATTTCAGAACCTGAGCTTTTGGGGGTTTTTAACTGAAGTTGGTTGTTTTATCTTTCTTGTTTTATAATTCCTATTGCAACCTCGTGCACTGCTCGAGACACAAGTGCTGCTGTAGTTAGCGCTTAGTGACACGCGGGCCTTTGGTGGGTGAGCGGGACTGTGTGTGAGTGTGTGCGCGTATGTGCGCACATATGTGTATGTGTGGAGTATGTGTGTTTGCTTCTCCGTGGATGAAATAGAAACTCCTCATTGTGTGACCAGGAATGGTTAAATCATCTTTACAAAATGTGTGCTTTAACTGTTTACAAGTAAAACCTAAAGTTGCAGGAAACATTTTTTATTTCGTAAAGAGGTACCAACTGTCGCTGATGTGATATGTCAGAACTGAAGAGTAAATCTACTTGTTTAAATGACTTGACAGTGGTAGTGCTCCATTTAATAACAGTAATAAGTAATAAAGTGTTTTTATTTGTTAACCAGTTTAAGTGGATCCTGTGGTAACTTAAACTGTTGTTCTCATCCCTTATATGGGGCATTTTTCTTTAACAAAGAATGGTTTCAGTGAAACAATCTAGCAGAGAATTAATGTCAGAACCTTTTTAAATAATAGTCTGATTGATACAGTTTGTACTTATTTCATCAAGCTTTTCTAAGCTTAAATATTGCATAGCTTCGAGCTGTATGGACTATATTATGAAAGAATATGTAAAGAGAACATACAGTAATGCACAGTCCTTAATTTGTGTATAATGGAAAGTTATTTACAATATAACACTGTAAATAAGAAAGCAAAGTTTATGGGAAAATTCAATATTATCTTTGTTTTTGTTTAAATATATTTTTAAGATAAAGGCACAAAAATAAAAGAAGCGTATTACTGGGTATAGTATGTGACTCCTCTTCTCAGACTAATAAATTATCTTTTGAATCCTTGGTTAAAAAAAAAAAAAAAAAA
the sequence of the human TASP1 gene contains 2368 nucleotides.
The REV 3L-N70 protein is obtained after human REV 3L protein is cut, and the amino acid sequence of the protein is shown as SEQ ID No. 5:
MFSVRIVTADYYMASPLQGLDTCQSPLTQAPVKKVPVVRVFGATPAGQKTCLHLHGIFPYLYVPYDGYGQQPESYLSQMAFSIDRALNVALGNPSSTAQHVFKVSLVSGMPFYGYHEKERHFMKIYLYNPTMVKRICELLQSGAIMNKFYQPHEAHIPYLLQLFIDYNLYGMNLINLAAVKFRKARRKSNTLHATGSCKNHLSGNSLADTLFRWEQDEIPSSLILEGVEPQSTCELEVDAVAADILNRLDIEAQIGGNPGLQAIWEDEKQRRRNRNETSQMSQPESQDHRFVPATESEKKFQKRLQEILKQNDFSVTLSGSVDYSDGSQEFSAELTLHSEVLSPEMLQCTPANMVEVHKDKESSKGHTRHKVEEALINEEAILNLMENSQTFQPLTQRLSESPVFMDSSPDEALVHLLAGLESDGYRGERNRMPSPCRSFGNNKYPQNSDDEENEPQIEKEEMELSLVMSQRWDSNIEEHCAKKRSLCRNTHRSSTEDDDSSSGEEMEWSDNSLLLASLSIPQLD
the human REV 3L-N70 protein comprises 525 amino acids in total.
The protease TASP1 expression interference agent or activity inhibitor refers to the interference of TASP1 expression or protease activity inhibition REV 3L cutting small molecular compounds or polypeptides, TASP1 expression interference agent, including but not limited to TASP1 gene mRNA siRNA molecules or shRNA expression vector, protease activity inhibitor including but not limited to the known TASP1 inhibitor 4- [ (4-aniline) methyl ] phenylarsonic acid (4- [ (4-arsophenyl) methyl ] phenyl ] arsonic acid, protease TASP1 activity inhibitor can also be a closed TASP1 protein interaction with REV 3L sequence, thereby specifically inhibiting TASP1 cutting REV 3L.
The application of the human REV 3L protein cleavage inhibitor according to the embodiment of the invention comprises the aspects of preparing an anti-tumor drug, preparing an anti-tumor drug resistance inhibitor, preparing an anti-tumor drug synergist and the like, in the aspect of preparing the anti-tumor drug synergist, the sensitivity of tumor cells to the anti-cancer drugs including but not limited to Cisplatin anticancer drugs can be improved by interfering the REV 3L protein cleavage event mediated by TASP1 protease.
Based on the findings, the invention provides an application for regulating the abundance of REV 3L protein and low-fidelity cross-damage DNA synthesis activity in human cells by controlling the post-translational cleavage of human REV 3L protein so as to realize the enhancement of the sensitivity of tumor cells to DNA damage treatment, the reduction of gene mutation caused by DNA damage induction in the treatment, and has important significance for improving the curative effect of tumor chemotherapy drugs and reducing the drug resistance of the tumor cells.
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FIG. 1 shows the effect of reducing TASP1 expression on REV 3L protein cleavage, wherein A shows the effect of reducing TASP1 expression on REV 3L protein cleavage in He L a cells using siRNA-mediated RNA interference and B shows the effect of reducing TASP1 expression on REV 3L protein cleavage in HCT116 cells using shRNA-mediated RNA interference;
FIG. 2 shows the expression and cleavage effect of REV 3L protein in wild-type and cleavage-deficient mutants, wherein A is the total protein amount of REV 3L in the cleavage-deficient mutant cells and wild-type cells, and B is the mRNA level of REV 3L gene in the cleavage-deficient mutant cells and wild-type cells;
FIG. 3 shows the protein stability of uncut REV 3L-Mut full-length protein in cut-deficient REV 3L-Mut mutant cells and of cut product REV 3L-N70 in wild-type cells, where A is the stability of human REV 3L-Mut full-length protein and cut product REV 3L-N70 after translation of the inhibitor protein (Cycloheximide (CHX)), and B is the stability of human REV 3L-MUT mutant protein and cut product REV 3L-N70 after inhibition of proteasome-mediated protein degradation by MG 132;
FIG. 4 shows REV 3L-MUT mutant and wild-type K-48 polyubiquitination modification, wherein A shows that K-48 polyubiquitination modification exists on full-length wild-type REV 3L protein, and B shows that His-HA-Ubiquitin can capture full-length wild-type REV 3L protein;
FIG. 5 shows that a cleavage defect of REV 3L significantly increases the sensitivity of human rectal cancer cells to DNA damage;
figure 6 shows that REV 3L cleavage site mutation significantly inhibited the formation of DNA mutations in human rectal cancer cells.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.
Example 1 confirmation of post-translational cleavage dependent sequence elements
After inserting a F L AG tag coding sequence 5' upstream of the endogenous REV 3L gene coding region of human HCT116 cells, the cells express a protein with an about 70kDa F L AG tag, and the protein with the 70kDa size and the F L AG tag is finally confirmed to be the REV 3L protein with C-terminal deletion through anti-F L AG immune purification coupling mass spectrum identification under the denaturation condition.
By using a series of REV 3L deletion mutants transiently expressing a series of N-terminal and C-terminal fusion of F L AG and HA tags in HEK293 cells, the 70kDa C-terminal deletion REV 3L protein was found to be generated by a position specific post-translational protein cleavage event, and the cleavage product was named REV 3L-N70 with the amino acid sequence shown in SEQ ID No. 5.
Through further deletion mutant and alanine substitution mutant expression experiments, the cleavage event is finally confirmed to be dependent on the Q L DGTAD sequence element shown in SEQ ID No.1 on the human REV 3L protein.
Example 2 verification of the relationship of the sequence element, protease TASP1 and human REV 3L protein
In human HEK293 cells expressing REV 3L N-terminal fragment containing Q L DGTAD cleavage sequence, the protein cleavage efficiency of REV 3L N-terminal fragment can be obviously improved by over-expressing wild type TASP1, and meanwhile, the over-expressing TASP1-T234A mutant (TASP1-Mut) without protease activity has no corresponding effect.
A stable cell line expressing the N-terminal fragment of REV 3L containing the Q L DGTAD cleavage sequence was constructed in He L a cells in which siRNA-mediated RNA interference was used to reduce the expression of TASP1, and as a result, cleavage of the N-terminal fragment of REV 3L was significantly inhibited as shown in A in FIG. 1.
In HCT116 cells derived from human colon cancer, TASP1 expression is reduced by RNA interference mediated by shRNA, and the result is shown in B in figure 1, the amount of 3F-REV 3L-N70 protein which is the cleavage product of endogenous REV 3L protein is remarkably reduced, and the cleavage of endogenous REV 3L protein is inhibited.
The cleavage experiment by using the reconstruction of the recombinant protein expressed and purified in vitro shows that the TASP1 can directly and specifically mediate the cleavage of the REV 3L protein, and the inactive TASP1-T234A mutant (TASP1-Mut) cannot cleave the recombinant REV 3L protein under the same condition.
Example 3 Effect of post-translational sequence-dependent cleavage of REV 3L by TASP1 protease on the stability of REV 3L protein and DNA polymerase ζ DNA Synthesis Activity
The method is characterized in that site-directed mutation is introduced into an endogenous REV 3L gene of HCT116 of a human rectal cancer cell by utilizing a CRISPR/Cas 9-mediated homologous recombination strategy, so that alanine substitution appears in a cleavage-dependent sequence element of an encoded REV 3L mutant, and a mutant cell line expressing a cleavage-defective REV 3L-Mut protein is obtained.
1. Uncleaved full-length human REV 3L protein can undergo proteasome-mediated protein degradation via K48-polyubiquitination
a) Comparing the cleavage-deficient REV 3L-Mut mutant cells with wild-type cells, as shown in FIG. 2A, the total protein amount of REV 3L in the mutant cells was about 16% of that in the wild-type cells, whereas the level of REV 3L mRNA in the two cells was not significantly different, as shown in FIG. 2B;
b) the amount of uncut full-length REV 3L-Mut protein in cut-defective REV 3L-Mut mutant cells and the amount of protein of cut products REV 3L-N70 in wild-type cells are detected by Western blot experiments at different times after the translation of CHX inhibitor protein, as shown in A in figure 3, the half-life period of uncut full-length human REV 3L-Mut protein in cells is found to be remarkably lower than that of cut products REV 3L-N70, which shows that the post-translational sequence-dependent cutting of REV 3L by TASP1 protease influences the stability of REV 3L protein;
c) the proteasome inhibitor MG132 can effectively improve the stability of uncleaved human REV 3L-MUT mutant protein, but the same treatment does not affect the protein amount of a cleavage product REV 3L-N70 in wild-type cells, and the result is shown as B in FIG. 3, which indicates that the posttranslational sequence-dependent cleavage event of TASP1 protease on REV 3L is closely related to the stability of the full-length protein of human REV 3L, and the cleaved protein REV 3L-N70 has good stability;
d) k-48 polyubiquitination modification exists on REV 3L-MUT mutant obtained by Anti-F L AG immune purification, K-48 polyubiquitination modification exists on full-length wild-type REV 3L protein obtained by Anti-F L AG immune purification, as shown in A in figure 4, and His-HA-Ubiquitin can capture (Pull-down) full-length wild-type REV 3L protein, as shown in B in figure 4.
The above results indicate that the TASP1 protease-mediated REV 3L protein cleavage event maintains the intracellular stability of REV 3L protein by avoiding K48 polyubiquitination-mediated protein degradation of REV 3L protein.
REV 3L cleavage deletion significantly increases susceptibility of human rectal cancer cells to DNA damage
REV 3L is the catalytic subunit of DNA polymerase zeta, and the degradation of REV 3L protein caused by blocking cleavage inevitably interferes with the activity of DNA polymerase zeta in cells, thus increasing the cytotoxicity of various antineoplastic drugs based on inducing DNA damage.
Comparing the survival rates of wild-type human rectal cancer HCT116 cells treated by different doses of the anti-cancer drug Cisplatin (Cisplatin) at 1 hour with that of REV 3L cleavage-deficient human rectal cancer HCT116 cells, the results are shown in FIG. 5, the inhibition of REV 3L cleavage can obviously improve the sensitivity of human rectal cancer cells to DNA damage type anti-cancer drug Cisplatin, and the survival rate of human rectal cancer HCT116 cells expressing cleavage-deficient REV 3L-Mut after 4 mu MCisplatin treatment is reduced by nearly 50% compared with wild-type human rectal cancer HCT116 cells.
REV 3L cleavage deletion significantly suppresses DNA damage induced mutations in human rectal cancer cells
The Escherichia coli-MBM 7070 strain carries lacZ gene containing premature termination coding seed, full-length L acZ protein with function expressed in the strain depends on mutant tRNA coded by supF gene, shuttle plasmid carrying supF gene is transferred into wild type or mutant human HCT116 cell, after two days, the shuttle plasmid is purified to complete replication in human cell, and the DNA mutation frequency of the shuttle plasmid in the corresponding human cell is quantitatively analyzed by whether the MBM7070 strain can express the phenotype of functional L acZ protein.
Spontaneous and damage-induced DNA mutation frequencies in wild-type HCT116 cells with intact REV 3L cleavage activity (HCT116-REV 3L-WT) and mutant cells with impaired REV 3L cleavage activity (HCT116-REV 3L-Mut) were examined using the supF mutation assay, DNA mutation rates induced by DNA damage in cells were examined using plasmids irradiated with different doses of UV, and spontaneous mutation rates in cells were examined using plasmids not irradiated with UV.
The results of the experiments are shown in FIG. 6, and the mutation rate of DNA induced by spontaneous or damage is obviously increased in HCT116-REV 3L-Mut mutant cells with impaired REV 3L cleavage activity compared with wild-type cells.
Sequence listing
<110> university of capital education
<120> human REV 3L protein cleavage inhibitor and application thereof
<160>5
<170>SIPOSequenceListing 1.0
<210>1
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Gln Leu Asp Gly Thr Ala Asp
1 5
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Met Phe Ser Val Arg Ile Val Thr Ala Asp Tyr Tyr Met Ala Ser Pro
1 5 10 15
Leu Gln Gly Leu Asp Thr Cys Gln Ser Pro Leu Thr Gln Ala Pro Val
20 25 30
Lys Lys Val Pro Val Val Arg Val Phe Gly Ala Thr Pro Ala Gly Gln
35 40 45
Lys Thr Cys Leu His Leu His Gly Ile Phe Pro Tyr Leu Tyr Val Pro
50 55 60
Tyr Asp Gly Tyr Gly Gln Gln Pro Glu Ser Tyr Leu Ser Gln Met Ala
65 7075 80
Phe Ser Ile Asp Arg Ala Leu Asn Val Ala Leu Gly Asn Pro Ser Ser
85 90 95
Thr Ala Gln His Val Phe Lys Val Ser Leu Val Ser Gly Met Pro Phe
100 105 110
Tyr Gly Tyr His Glu Lys Glu Arg His Phe Met Lys Ile Tyr Leu Tyr
115 120 125
Asn Pro Thr Met Val Lys Arg Ile Cys Glu Leu Leu Gln Ser Gly Ala
130 135 140
Ile Met Asn Lys Phe Tyr Gln Pro His Glu Ala His Ile Pro Tyr Leu
145 150 155 160
Leu Gln Leu Phe Ile Asp Tyr Asn Leu Tyr Gly Met Asn Leu Ile Asn
165 170 175
Leu Ala Ala Val Lys Phe Arg Lys Ala Arg Arg Lys Ser Asn Thr Leu
180 185 190
His Ala Thr Gly Ser Cys Lys Asn His Leu Ser Gly Asn Ser Leu Ala
195 200 205
Asp Thr Leu Phe Arg Trp Glu Gln Asp Glu Ile Pro Ser Ser Leu Ile
210 215 220
Leu Glu Gly Val Glu Pro Gln Ser Thr Cys Glu Leu Glu Val Asp Ala
225 230 235240
Val Ala Ala Asp Ile Leu Asn Arg Leu Asp Ile Glu Ala Gln Ile Gly
245 250 255
Gly Asn Pro Gly Leu Gln Ala Ile Trp Glu Asp Glu Lys Gln Arg Arg
260 265 270
Arg Asn Arg Asn Glu Thr Ser Gln Met Ser Gln Pro Glu Ser Gln Asp
275 280 285
His Arg Phe Val Pro Ala Thr Glu Ser Glu Lys Lys Phe Gln Lys Arg
290 295 300
Leu Gln Glu Ile Leu Lys Gln Asn Asp Phe Ser Val Thr Leu Ser Gly
305 310 315 320
Ser Val Asp Tyr Ser Asp Gly Ser Gln Glu Phe Ser Ala Glu Leu Thr
325 330 335
Leu His Ser Glu Val Leu Ser Pro Glu Met Leu Gln Cys Thr Pro Ala
340 345 350
Asn Met Val Glu Val His Lys Asp Lys Glu Ser Ser Lys Gly His Thr
355 360 365
Arg His Lys Val Glu Glu Ala Leu Ile Asn Glu Glu Ala Ile Leu Asn
370 375 380
Leu Met Glu Asn Ser Gln Thr Phe Gln Pro Leu Thr Gln Arg Leu Ser
385 390 395400
Glu Ser Pro Val Phe Met Asp Ser Ser Pro Asp Glu Ala Leu Val His
405 410 415
Leu Leu Ala Gly Leu Glu Ser Asp Gly Tyr Arg Gly Glu Arg Asn Arg
420 425 430
Met Pro Ser Pro Cys Arg Ser Phe Gly Asn Asn Lys Tyr Pro Gln Asn
435 440 445
Ser Asp Asp Glu Glu Asn Glu Pro Gln Ile Glu Lys Glu Glu Met Glu
450 455 460
Leu Ser Leu Val Met Ser Gln Arg Trp Asp Ser Asn Ile Glu Glu His
465 470 475 480
Cys Ala Lys Lys Arg Ser Leu Cys Arg Asn Thr His Arg Ser Ser Thr
485 490 495
Glu Asp Asp Asp Ser Ser Ser Gly Glu Glu Met Glu Trp Ser Asp Asn
500 505 510
Ser Leu Leu Leu Ala Ser Leu Ser Ile Pro Gln Leu Asp Gly Thr Ala
515 520 525
Asp Glu Asn Ser Asp Asn Pro Leu Asn Asn Glu Asn Ser Arg Thr His
530 535 540
Ser Ser Val Ile Ala Thr Ser Lys Leu Ser Val Lys Pro Ser Ile Phe
545 550 555 560
His Lys Asp Ala Ala Thr Leu Glu Pro Ser Ser Ser Ala Lys Ile Thr
565 570 575
Phe Gln Cys Lys His Thr Ser Ala Leu Ser Ser His Val Leu Asn Lys
580 585 590
Glu Asp Leu Ile Glu Asp Leu Ser Gln Thr Asn Lys Asn Thr Glu Lys
595 600 605
Gly Leu Asp Asn Ser Val Thr Ser Phe Thr Asn Glu Ser Thr Tyr Ser
610 615 620
Met Lys Tyr Pro Gly Ser Leu Ser Ser Thr Val His Ser Glu Asn Ser
625 630 635 640
His Lys Glu Asn Ser Lys Lys Glu Ile Leu Pro Val Ser Ser Cys Glu
645 650 655
Ser Ser Ile Phe Asp Tyr Glu Glu Asp Ile Pro Ser Val Thr Arg Gln
660 665 670
Val Pro Ser Arg Lys Tyr Thr Asn Ile Arg Lys Ile Glu Lys Asp Ser
675 680 685
Pro Phe Ile His Met His Arg His Pro Asn Glu Asn Thr Leu Gly Lys
690 695 700
Asn Ser Phe Asn Phe Ser Asp Leu Asn His Ser Lys Asn Lys Val Ser
705 710 715 720
Ser Glu Gly Asn Glu Lys Gly Asn Ser Thr Ala Leu Ser Ser Leu Phe
725 730 735
Pro Ser Ser Phe Thr Glu Asn Cys Glu Leu Leu Ser Cys Ser Gly Glu
740 745 750
Asn Arg Thr Met Val His Ser Leu Asn Ser Thr Ala Asp Glu Ser Gly
755 760 765
Leu Asn Lys Leu Lys Ile Arg Tyr Glu Glu Phe Gln Glu His Lys Thr
770 775 780
Glu Lys Pro Ser Leu Ser Gln Gln Ala Ala His Tyr Met Phe Phe Pro
785 790 795 800
Ser Val Val Leu Ser Asn Cys Leu Thr Arg Pro Gln Lys Leu Ser Pro
805 810 815
Val Thr Tyr Lys Leu Gln Pro Gly Asn Lys Pro Ser Arg Leu Lys Leu
820 825 830
Asn Lys Arg Lys Leu Ala Gly His Gln Glu Thr Ser Thr Lys Ser Ser
835 840 845
Glu Thr Gly Ser Thr Lys Asp Asn Phe Ile Gln Asn Asn Pro Cys Asn
850 855 860
Ser Asn Pro Glu Lys Asp Asn Ala Leu Ala Ser Asp Leu Thr Lys Thr
865 870 875 880
Thr Arg Gly Ala Phe Glu Asn Lys Thr Pro Thr Asp Gly Phe Ile Asp
885 890 895
Cys His Phe Gly Asp Gly Thr Leu Glu Thr Glu Gln Ser Phe Gly Leu
900 905 910
Tyr Gly Asn Lys Tyr Thr Leu Arg Ala Lys Arg Lys Val Asn Tyr Glu
915 920 925
Thr Glu Asp Ser Glu Ser Ser Phe Val Thr His Asn Ser Lys Ile Ser
930 935 940
Leu Pro His Pro Met Glu Ile Gly Glu Ser Leu Asp Gly Thr Leu Lys
945 950 955 960
Ser Arg Lys Arg Arg Lys Met Ser Lys Lys Leu Pro Pro Val Ile Ile
965 970 975
Lys Tyr Ile Ile Ile Asn Arg Phe Arg Gly Arg Lys Asn Met Leu Val
980 985 990
Lys Leu Gly Lys Ile Asp Ser Lys Glu Lys Gln Val Ile Leu Thr Glu
995 1000 1005
Glu Lys Met Glu Leu Tyr Lys Lys Leu Ala Pro Leu Lys Asp Phe Trp
1010 1015 1020
Pro Lys Val Pro Asp Ser Pro Ala Thr Lys Tyr Pro Ile Tyr Pro Leu
1025 1030 1035 1040
Thr Pro Lys Lys Ser His Arg Arg Lys Ser Lys His Lys Ser Ala Lys
1045 1050 1055
Lys Lys Thr Gly Lys Gln Gln Arg Thr Asn Asn Glu Asn Ile Lys Arg
1060 1065 1070
Thr Leu Ser Phe Arg Lys Lys Arg Ser His Ala Ile Leu Ser Pro Pro
1075 1080 1085
Ser Pro Ser Tyr Asn Ala Glu Thr Glu Asp Cys Asp Leu Asn Tyr Ser
1090 1095 1100
Asp Val Met Ser Lys Leu Gly Phe Leu Ser Glu Arg Ser Thr Ser Pro
1105 1110 1115 1120
Ile Asn Ser Ser Pro Pro Arg Cys Trp Ser Pro Thr Asp Pro Arg Ala
1125 1130 1135
Glu Glu Ile Met Ala Ala Ala Glu Lys Glu Ala Met Leu Phe Lys Gly
1140 1145 1150
Pro Asn Val Tyr Lys Lys Thr Val Asn Ser Arg Ile Gly Lys Thr Ser
1155 1160 1165
Arg Ala Arg Ala Gln Ile Lys Lys Ser Lys Ala Lys Leu Ala Asn Pro
1170 1175 1180
Ser Ile Val Thr Lys Lys Arg Asn Lys Arg Asn Gln Thr Asn Lys Leu
1185 1190 1195 1200
Val Asp Asp Gly Lys Lys Lys Pro Arg Ala Lys Gln Lys Thr Asn Glu
1205 1210 1215
Lys Gly Thr Ser Arg Lys His Thr Thr Leu Lys Asp Glu Lys Ile Lys
1220 1225 1230
Ser Gln Ser Gly Ala Glu Val Lys Phe Val Leu Lys His Gln Asn Val
1235 1240 1245
Ser Glu Phe Ala Ser Ser Ser Gly Gly Ser Gln Leu Leu Phe Lys Gln
1250 1255 1260
Lys Asp Met Pro Leu Met Gly Ser Ala Val Asp His Pro Leu Ser Ala
1265 1270 1275 1280
Ser Leu Pro Thr Gly Ile Asn Ala Gln Gln Lys Leu Ser Gly Cys Phe
1285 1290 1295
Ser Ser Phe Leu Glu Ser Lys Lys Ser Val Asp Leu Gln Thr Phe Pro
1300 1305 1310
Ser Ser Arg Asp Asp Leu His Pro Ser Val Val Cys Asn Ser Ile Gly
1315 1320 1325
Pro Gly Val Ser Lys Ile Asn Val Gln Arg Pro His Asn Gln Ser Ala
1330 1335 1340
Met Phe Thr Leu Lys Glu Ser Thr Leu Ile Gln Lys Asn Ile Phe Asp
1345 1350 1355 1360
Leu Ser Asn His Leu Ser Gln Val Ala Gln Asn Thr Gln Ile Ser Ser
1365 1370 1375
Gly Met Ser Ser Lys Ile Glu Asp Asn Ala Asn Asn Ile Gln Arg Asn
1380 1385 1390
Tyr Leu Ser Ser Ile Gly Lys Leu Ser Glu Tyr Arg Asn Ser Leu Glu
1395 1400 1405
Ser Lys Leu Asp Gln Ala Tyr Thr Pro Asn Phe Leu His Cys Lys Asp
1410 1415 1420
Ser Gln Gln Gln Ile Val Cys Ile Ala Glu Gln Ser Lys His Ser Glu
1425 1430 1435 1440
Thr Cys Ser Pro Gly Asn Thr Ala Ser Glu Glu Ser Gln Met Pro Asn
1445 1450 1455
Asn Cys Phe Val Thr Ser Leu Arg Ser Pro Ile Lys Gln Ile Ala Trp
1460 1465 1470
Glu Gln Lys Gln Arg Gly Phe Ile Leu Asp Met Ser Asn Phe Lys Pro
1475 1480 1485
Glu Arg Val Lys Pro Arg Ser Leu Ser Glu Ala Ile Ser Gln Thr Lys
1490 1495 1500
Ala Leu Ser Gln Cys Lys Asn Arg Asn Val Ser Thr Pro Ser Ala Phe
1505 1510 1515 1520
Gly Glu Gly Gln Ser Gly Leu Ala Val Leu Lys Glu Leu Leu Gln Lys
1525 1530 1535
Arg Gln Gln Lys Ala Gln Asn Ala Asn Thr Thr Gln Asp Pro Leu Ser
1540 1545 1550
Asn Lys His Gln Pro Asn Lys Asn Ile Ser Gly Ser Leu Glu His Asn
1555 1560 1565
Lys Ala Asn Lys Arg Thr Arg Ser Val Thr Ser Pro Arg Lys Pro Arg
1570 1575 1580
Thr Pro Arg Ser Thr Lys Gln Lys Glu Lys Ile Pro Lys Leu Leu Lys
1585 1590 1595 1600
Val Asp Ser Leu Asn Leu Gln Asn Ser Ser Gln Leu Asp Asn Ser Val
1605 1610 1615
Ser Asp Asp Ser Pro Ile Phe Phe Ser Asp Pro Gly Phe Glu Ser Cys
1620 1625 1630
Tyr Ser Leu Glu Asp Ser Leu Ser Pro Glu His Asn Tyr Asn Phe Asp
1635 1640 1645
Ile Asn Thr Ile Gly Gln Thr Gly Phe Cys Ser Phe Tyr Ser Gly Ser
1650 1655 1660
Gln Phe Val Pro Ala Asp Gln Asn Leu Pro Gln Lys Phe Leu Ser Asp
1665 1670 1675 1680
Ala Val Gln Asp Leu Phe Pro Gly Gln Ala Ile Glu Lys Asn Glu Phe
1685 1690 1695
Leu Ser His Asp Asn Gln Lys Cys Asp Glu Asp Lys His His Thr Thr
1700 1705 1710
Asp Ser Ala Ser Trp Ile Arg Ser Gly Thr Leu Ser Pro Glu Ile Phe
1715 1720 1725
Glu Lys Ser Thr Ile Asp Ser Asn Glu Asn Arg Arg His Asn Gln Trp
1730 1735 1740
Lys Asn Ser Phe His Pro Leu Thr Thr Arg Ser Asn Ser Ile Met Asp
1745 1750 1755 1760
Ser Phe Cys Val Gln Gln Ala Glu Asp Cys Leu Ser Glu Lys Ser Arg
1765 1770 1775
Leu Asn Arg Ser Ser Val Ser Lys Glu Val Phe Leu Ser Leu Pro Gln
1780 1785 1790
Pro Asn Asn Ser Asp Trp Ile Gln Gly His Thr Arg Lys Glu Met Gly
1795 1800 1805
Gln Ser Leu Asp Ser Ala Asn Thr Ser Phe Thr Ala Ile Leu Ser Ser
1810 1815 1820
Pro Asp Gly Glu Leu Val Asp Val Ala Cys Glu Asp Leu Glu Leu Tyr
1825 1830 1835 1840
Val Ser Arg Asn Asn Asp Met Leu Thr Pro Thr Pro Asp Ser Ser Pro
1845 1850 1855
Arg Ser Thr Ser Ser Pro Ser Gln Ser Lys Asn Gly Ser Phe Thr Pro
1860 1865 1870
Arg Thr Ala Asn Ile Leu Lys Pro Leu Met Ser Pro Pro Ser Arg Glu
1875 1880 1885
Glu Ile Met Ala Thr Leu Leu Asp His Asp Leu Ser Glu Thr Ile Tyr
1890 1895 1900
Gln Glu Pro Phe Cys Ser Asn Pro Ser Asp Val Pro Glu Lys Pro Arg
1905 1910 1915 1920
Glu Ile Gly Gly Arg Leu Leu Met Val Glu Thr Arg Leu Ala Asn Asp
1925 1930 1935
Leu Ala Glu Phe Glu Gly Asp Phe Ser Leu Glu Gly Leu Arg Leu Trp
1940 1945 1950
Lys Thr Ala Phe Ser Ala Met Thr Gln Asn Pro Arg Pro Gly Ser Pro
1955 1960 1965
Leu Arg Ser Gly Gln Gly Val Val Asn Lys Gly Ser Ser Asn Ser Pro
1970 1975 1980
Lys Met Val Glu Asp Lys Lys Ile Val Ile Met Pro Cys Lys Cys Ala
1985 1990 1995 2000
Pro Ser Arg Gln Leu Val Gln Val Trp Leu Gln Ala Lys Glu Glu Tyr
2005 2010 2015
Glu Arg Ser Lys Lys Leu Pro Lys Thr Lys Pro Thr Gly Val Val Lys
2020 2025 2030
Ser Ala Glu Asn Phe Ser Ser Ser Val Asn Pro Asp Asp Lys Pro Val
2035 2040 2045
Val Pro Pro Lys Met Asp Val Ser Pro Cys Ile Leu Pro Thr Thr Ala
2050 2055 2060
His Thr Lys Glu Asp Val Asp Asn Ser Gln Ile Ala Leu Gln Ala Pro
2065 2070 2075 2080
Thr Thr Gly Cys Ser Gln Thr Ala Ser Glu Ser Gln Met Leu Pro Pro
2085 2090 2095
Val Ala Ser Ala Ser Asp Pro Glu Lys Asp Glu Asp Asp Asp Asp Asn
2100 2105 2110
Tyr Tyr Ile Ser Tyr Ser Ser Pro Asp Ser Pro Val Ile Pro Pro Trp
2115 2120 2125
Gln Gln Pro Ile Ser Pro Asp Ser Lys Ala Leu Asn Gly Asp Asp Arg
2130 2135 2140
Pro Ser Ser Pro Val Glu Glu Leu Pro Ser Leu Ala Phe Glu Asn Phe
2145 2150 2155 2160
Leu Lys Pro Ile Lys Asp Gly Ile Gln Lys Ser Pro Cys Ser Glu Pro
2165 2170 2175
Gln Glu Pro Leu Val Ile Ser Pro Ile Asn Thr Arg Ala Arg Thr Gly
2180 2185 2190
Lys Cys Glu Ser Leu Cys Phe His Ser Thr Pro Ile Ile Gln Arg Lys
2195 2200 2205
Leu Leu Glu Arg Leu Pro Glu Ala Pro Gly Leu Ser Pro Leu Ser Thr
2210 2215 2220
Glu Pro Lys Thr Gln Lys Leu Ser Asn Lys Lys Gly Ser Asn Thr Asp
2225 2230 2235 2240
Thr Leu Arg Arg Val Leu Leu Thr Gln Ala Lys Asn Gln Phe Ala Ala
2245 2250 2255
Val Asn Thr Pro Gln Lys Glu Thr Ser Gln Ile Asp Gly Pro Ser Leu
2260 2265 2270
Asn Asn Thr Tyr Gly Phe Lys Val Ser Ile Gln Asn Leu Gln Glu Ala
2275 2280 2285
Lys Ala Leu His Glu Ile Gln Asn Leu Thr Leu Ile Ser Val Glu Leu
2290 2295 2300
His Ala Arg Thr Arg Arg Asp Leu Glu Pro Asp Pro Glu Phe Asp Pro
2305 2310 2315 2320
Ile Cys Ala Leu Phe Tyr Cys Ile Ser Ser Asp Thr Pro Leu Pro Asp
2325 2330 2335
Thr Glu Lys Thr Glu Leu Thr Gly Val Ile Val Ile Asp Lys Asp Lys
2340 2345 2350
Thr Val Phe Ser Gln Asp Ile Arg Tyr Gln Thr Pro Leu Leu Ile Arg
2355 2360 2365
Ser Gly Ile Thr Gly Leu Glu Val Thr Tyr Ala Ala Asp Glu Lys Ala
2370 2375 2380
Leu Phe His Glu Ile Ala Asn Ile Ile Lys Arg Tyr Asp Pro Asp Ile
2385 2390 2395 2400
Leu Leu Gly Tyr Glu Ile Gln Met His Ser Trp Gly Tyr Leu Leu Gln
2405 2410 2415
Arg Ala Ala Ala Leu Ser Ile Asp Leu Cys Arg Met Ile Ser Arg Val
2420 2425 2430
Pro Asp Asp Lys Ile Glu Asn Arg Phe Ala Ala Glu Arg Asp Glu Tyr
2435 2440 2445
Gly Ser Tyr Thr Met Ser Glu Ile Asn Ile Val Gly Arg Ile Thr Leu
2450 2455 2460
Asn Leu Trp Arg Ile Met Arg Asn Glu Val Ala Leu Thr Asn Tyr Thr
2465 2470 2475 2480
Phe Glu Asn Val Ser Phe His Val Leu His Gln Arg Phe Pro Leu Phe
2485 2490 2495
Thr Phe Arg Val Leu Ser Asp Trp Phe Asp Asn Lys Thr Asp Leu Tyr
2500 2505 2510
Arg Trp Lys Met Val Asp His Tyr Val Ser Arg Val Arg Gly Asn Leu
2515 2520 2525
Gln Met Leu Glu Gln Leu Asp Leu Ile Gly Lys Thr Ser Glu Met Ala
2530 2535 2540
Arg Leu Phe Gly Ile Gln Phe Leu His Val Leu Thr Arg Gly Ser Gln
2545 2550 2555 2560
Tyr Arg Val Glu Ser Met Met Leu Arg Ile Ala Lys Pro Met Asn Tyr
2565 2570 2575
Ile Pro Val Thr Pro Ser Val Gln Gln Arg Ser Gln Met Arg Ala Pro
2580 2585 2590
Gln Cys Val Pro Leu Ile Met Glu Pro Glu Ser Arg Phe Tyr Ser Asn
2595 2600 2605
Ser Val Leu Val Leu Asp Phe Gln Ser Leu Tyr Pro Ser Ile Val Ile
2610 2615 2620
Ala Tyr Asn Tyr Cys Phe Ser Thr Cys Leu Gly His Val Glu Asn Leu
2625 2630 2635 2640
Gly Lys Tyr Asp Glu Phe Lys Phe Gly Cys Thr Ser Leu Arg Val Pro
2645 2650 2655
Pro Asp Leu Leu Tyr Gln Val Arg His Asp Ile Thr Val Ser Pro Asn
2660 2665 2670
Gly Val Ala Phe Val Lys Pro Ser Val Arg Lys Gly Val Leu Pro Arg
2675 2680 2685
Met Leu Glu Glu Ile Leu Lys Thr Arg Phe Met Val Lys Gln Ser Met
2690 2695 2700
Lys Ala Tyr Lys Gln Asp Arg Ala Leu Ser Arg Met Leu Asp Ala Arg
2705 2710 2715 2720
Gln Leu Gly Leu Lys Leu Ile Ala Asn Val Thr Phe Gly Tyr Thr Ser
2725 2730 2735
Ala Asn Phe Ser Gly Arg Met Pro Cys Ile Glu Val Gly Asp Ser Ile
2740 2745 2750
Val His Lys Ala Arg Glu Thr Leu Glu Arg Ala Ile Lys Leu Val Asn
2755 2760 2765
Asp Thr Lys Lys Trp Gly Ala Arg Val Val Tyr Gly Asp Thr Asp Ser
2770 2775 2780
Met Phe Val Leu Leu Lys Gly Ala Thr Lys Glu Gln Ser Phe Lys Ile
2785 2790 2795 2800
Gly Gln Glu Ile Ala Glu Ala Val Thr Ala Thr Asn Pro Lys Pro Val
2805 2810 2815
Lys Leu Lys Phe Glu Lys Val Tyr Leu Pro Cys Val Leu Gln Thr Lys
2820 2825 2830
Lys Arg Tyr Val Gly Tyr Met Tyr Glu Thr Leu Asp Gln Lys Asp Pro
2835 2840 2845
Val Phe Asp Ala Lys Gly Ile Glu Thr Val Arg Arg Asp Ser Cys Pro
2850 2855 2860
Ala Val Ser Lys Ile Leu Glu Arg Ser Leu Lys Leu Leu Phe Glu Thr
2865 2870 2875 2880
Arg Asp Ile Ser Leu Ile Lys Gln Tyr Val Gln Arg Gln Cys Met Lys
2885 2890 2895
Leu Leu Glu Gly Lys Ala Ser Ile Gln Asp Phe Ile Phe Ala Lys Glu
2900 2905 2910
Tyr Arg Gly Ser Phe Ser Tyr Lys Pro Gly Ala Cys Val Pro Ala Leu
2915 2920 2925
Glu Leu Thr Arg Lys Met Leu Thr Tyr Asp Arg Arg Ser Glu Pro Gln
2930 2935 2940
Val Gly Glu Arg Val Pro Tyr Val Ile Ile Tyr Gly Thr Pro Gly Val
2945 2950 29552960
Pro Leu Ile Gln Leu Val Arg Arg Pro Val Glu Val Leu Gln Asp Pro
2965 2970 2975
Thr Leu Arg Leu Asn Ala Thr Tyr Tyr Ile Thr Lys Gln Ile Leu Pro
2980 2985 2990
Pro Leu Ala Arg Ile Phe Ser Leu Ile Gly Ile Asp Val Phe Ser Trp
2995 3000 3005
Tyr His Glu Leu Pro Arg Ile His Lys Ala Thr Ser Ser Ser Arg Ser
3010 3015 3020
Glu Pro Glu Gly Arg Lys Gly Thr Ile Ser Gln Tyr Phe Thr Thr Leu
3025 3030 3035 3040
His Cys Pro Val Cys Asp Asp Leu Thr Gln His Gly Ile Cys Ser Lys
3045 3050 3055
Cys Arg Ser Gln Pro Gln His Val Ala Val Ile Leu Asn Gln Glu Ile
3060 3065 3070
Arg Glu Leu Glu Arg Gln Gln Glu Gln Leu Val Lys Ile Cys Lys Asn
3075 3080 3085
Cys Thr Gly Cys Phe Asp Arg His Ile Pro Cys Val Ser Leu Asn Cys
3090 3095 3100
Pro Val Leu Phe Lys Leu Ser Arg Val Asn Arg Glu Leu Ser Lys Ala
3105 3110 31153120
Pro Tyr Leu Arg Gln Leu Leu Asp Gln Phe
3125 3130
<210>3
<211>420
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>3
Met Thr Met Glu Lys Gly Met Ser Ser Gly Glu Gly Leu Pro Ser Arg
1 5 10 15
Ser Ser Gln Val Ser Ala Gly Lys Ile Thr Ala Lys Glu Leu Glu Thr
20 25 30
Lys Gln Ser Tyr Lys Glu Lys Arg Gly Gly Phe Val Leu Val His Ala
35 40 45
Gly Ala Gly Tyr His Ser Glu Ser Lys Ala Lys Glu Tyr Lys His Val
50 55 60
Cys Lys Arg Ala Cys Gln Lys Ala Ile Glu Lys Leu Gln Ala Gly Ala
65 70 75 80
Leu Ala Thr Asp Ala Val Thr Ala Ala Leu Val Glu Leu Glu Asp Ser
85 90 95
Pro Phe Thr Asn Ala Gly Met Gly Ser Asn Leu Asn Leu Leu Gly Glu
100 105 110
Ile Glu Cys Asp Ala Ser Ile Met Asp Gly Lys Ser Leu Asn Phe Gly
115 120 125
Ala Val Gly Ala Leu Ser Gly Ile Lys Asn Pro Val Ser Val Ala Asn
130 135 140
Arg Leu Leu Cys Glu Gly Gln Lys Gly Lys Leu Ser Ala Gly Arg Ile
145 150 155 160
Pro Pro Cys Phe Leu Val Gly Glu Gly Ala Tyr Arg Trp Ala Val Asp
165 170 175
His Gly Ile Pro Ser Cys Pro Pro Asn Ile Met Thr Thr Arg Phe Ser
180 185 190
Leu Ala Ala Phe Lys Arg Asn Lys Arg Lys Leu Glu Leu Ala Glu Arg
195 200 205
Val Asp Thr Asp Phe Met Gln Leu Lys Lys Arg Arg Gln Ser Ser Glu
210 215 220
Lys Glu Asn Asp Ser Gly Thr Leu Asp Thr Val Gly Ala Val Val Val
225 230 235 240
Asp His Glu Gly Asn Val Ala Ala Ala Val Ser Ser Gly Gly Leu Ala
245 250 255
Leu Lys His Pro Gly Arg Val Gly Gln Ala Ala Leu Tyr Gly Cys Gly
260 265 270
Cys Trp Ala Glu Asn Thr Gly Ala His Asn Pro Tyr Ser Thr Ala Val
275 280 285
Ser Thr Ser Gly Cys Gly Glu His Leu Val Arg Thr Ile Leu Ala Arg
290 295 300
Glu Cys Ser His Ala Leu Gln Ala Glu Asp Ala His Gln Ala Leu Leu
305 310 315 320
Glu Thr Met Gln Asn Lys Phe Ile Ser Ser Pro Phe Leu Ala Ser Glu
325 330 335
Asp Gly Val Leu Gly Gly Val Ile Val Leu Arg Ser Cys Arg Cys Ser
340 345 350
Ala Glu Pro Asp Ser Ser Gln Asn Lys Gln Thr Leu Leu Val Glu Phe
355 360 365
Leu Trp Ser His Thr Thr Glu Ser Met Cys Val Gly Tyr Met Ser Ala
370 375 380
Gln Asp Gly Lys Ala Lys Thr His Ile Ser Arg Leu Pro Pro Gly Ala
385 390 395 400
Val Ala Gly Gln Ser Val Ala Ile Glu Gly Gly Val Cys Arg Leu Glu
405 410 415
Ser Pro Val Asn
420
<210>4
<211>2368
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>4
gggtgactac ttccggtgca gtgaaggctc ggggctgaag cggggtaatt cctctcctgc 60
aattactttt ggatggaagt atgccccttt ctcagtagaa gatggtaatc ttggagaatg 120
accatggaga aggggatgag ttctggagaa gggctgcctt ccagatcatc tcaggtttcg 180
gctggtaaaa taacagccaa agagttggaa acaaagcagt cctataaaga gaaacgagga 240
ggctttgtgt tggtgcatgc aggtgcaggt tatcattctg aatccaaagc caaggagtat 300
aaacatgtat gcaaacgagc ttgtcagaag gcaattgaaa agctgcaggc cggtgctctt 360
gcaactgacg cagtcactgc agcactggtg gaacttgagg attctccttt tacaaatgca 420
ggaatgggat ctaatctaaa tctgttaggt gaaattgagt gtgatgccag cataatggat 480
ggaaaatcct taaattttgg agcagttgga gcactgagtg gaatcaagaa cccagtctcg 540
gttgccaaca gactcttatg tgaagggcag aagggcaagc tctcggctgg cagaattcct 600
ccctgctttt tagttggaga aggagcctac agatgggcag tagatcatgg aataccctct 660
tgccctccta acatcatgac cacaagattc agtttagctg catttaaaag aaacaagagg 720
aaactagagc tggcagaaag ggtggacaca gattttatgc aactaaagaa aagaagacaa 780
tcaagtgaga aggaaaatga ctcaggcact ttggacacgg taggcgctgt ggttgtggac 840
cacgaaggga atgttgctgc tgctgtctcc agtggaggct tggccttgaa acatccgggg 900
agagttgggc aggctgctct ttatggatgt ggctgctggg ctgaaaatac tggagctcat 960
aacccctact ccacagctgt gagtacctca ggatgtggag agcatcttgt gcgcaccata 1020
ctggctagag aatgttcaca tgctttacaa gctgaggatg ctcaccaagc cctgttggag 1080
actatgcaaa acaagtttat cagttcacct ttccttgcca gtgaagatgg cgtgcttggc 1140
ggagtgattg tcctccgttc atgcagatgt tctgccgagc ctgactcctc ccaaaataag 1200
cagacacttc tagtggaatt tctgtggagc cacacgacgg agagcatgtg tgtcggatat 1260
atgtcagccc aggatgggaa agccaagact cacatttcaa gacttcctcc tggtgcggtg 1320
gcaggacagt ctgtggcaat cgaaggtggg gtgtgccgcc tggagagccc agtgaactga 1380
cccttcaggc tgagtgtgaa gcgtctcaga ggcatttcag aacctgagct tttgggggtt 1440
tttaactgaa gttggttgtt ttatctttct tgttttataa ttcctattgc aacctcgtgc 1500
actgctcgag acacaagtgc tgctgtagtt agcgcttagt gacacgcggg cctttggtgg 1560
gtgagcggga ctgtgtgtga gtgtgtgcgc gtatgtgcgc acatatgtgt atgtgtggag 1620
tatgtgtgtt tgcttctccg tggatgaaat agaaactcct cattgtgtga ccaggaatgg 1680
ttaaatcatc tttacaaaat gtgtgcttta actgtttaca agtaaaacct aaagttgcag 1740
gaaacatttt ttatttcgta aagaggtacc aactgtcgct gatgtgatat gtcagaactg 1800
aagagtaaat ctacttgttt aaatgacttg acagtggtag tgctccattt aataacagta 1860
ataagtaata aagtgttttt atttgttaac cagtttaagt ggatcctgtg gtaacttaaa 1920
ctgttgttct catcccttat atggggcatt tttctttaac aaagaatggt ttcagtgaaa 1980
caatctagca gagaattaat gtcagaacct ttttaaataa tagtctgatt gatacagttt 2040
gtacttattt catcaagctt ttctaagctt aaatattgca tagcttcgag ctgtatggac 2100
tatattatga aagaatatgt aaagagaaca tacagtaatg cacagtcctt aatttgtgta 2160
taatggaaag ttatttacaa tataacactg taaataagaa agcaaagttt atgggaaaat 2220
tcaatattat ctttgttttt gtttaaatat atttttaaga taaaggcaca aaaataaaag 2280
aagcgtatta ctgggtatag tatgtgactc ctcttctcag actaataaat tatcttttga 2340
atccttggtt aaaaaaaaaa aaaaaaaa 2368
<210>5
<211>525
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>5
Met Phe Ser Val Arg Ile Val Thr Ala Asp Tyr Tyr Met Ala Ser Pro
1 5 10 15
Leu Gln Gly Leu Asp Thr Cys Gln Ser Pro Leu Thr Gln Ala Pro Val
20 25 30
Lys Lys Val Pro Val Val Arg Val Phe Gly Ala Thr Pro Ala Gly Gln
35 40 45
Lys Thr Cys Leu His Leu His Gly Ile Phe Pro Tyr Leu Tyr Val Pro
50 55 60
Tyr Asp Gly Tyr Gly Gln Gln Pro Glu Ser Tyr Leu Ser Gln Met Ala
65 70 75 80
Phe Ser Ile Asp Arg Ala Leu Asn Val Ala Leu Gly Asn Pro Ser Ser
85 90 95
Thr Ala Gln His Val Phe Lys Val Ser Leu Val Ser Gly Met Pro Phe
100 105 110
Tyr Gly Tyr His Glu Lys Glu Arg His Phe Met Lys Ile Tyr Leu Tyr
115 120 125
Asn Pro Thr Met Val Lys Arg Ile Cys Glu Leu Leu Gln Ser Gly Ala
130 135 140
Ile Met Asn Lys Phe Tyr Gln Pro His Glu Ala His Ile Pro Tyr Leu
145 150 155 160
Leu Gln Leu Phe Ile Asp Tyr Asn Leu Tyr Gly Met Asn Leu Ile Asn
165 170 175
Leu Ala Ala Val Lys Phe Arg Lys Ala Arg Arg Lys Ser Asn Thr Leu
180 185 190
His Ala Thr Gly Ser Cys Lys Asn His Leu Ser Gly Asn Ser Leu Ala
195 200 205
Asp Thr Leu Phe Arg Trp Glu Gln Asp Glu Ile Pro Ser Ser Leu Ile
210 215 220
Leu Glu Gly Val Glu Pro Gln Ser Thr Cys Glu Leu Glu Val Asp Ala
225 230 235 240
Val Ala Ala Asp Ile Leu Asn Arg Leu Asp Ile Glu Ala Gln Ile Gly
245 250 255
Gly Asn Pro Gly Leu Gln Ala Ile Trp Glu Asp Glu Lys Gln Arg Arg
260 265 270
Arg Asn Arg Asn Glu Thr Ser Gln Met Ser Gln Pro Glu Ser Gln Asp
275 280 285
His Arg Phe Val Pro Ala Thr Glu Ser Glu Lys Lys Phe Gln Lys Arg
290 295 300
Leu Gln Glu Ile Leu Lys Gln Asn Asp Phe Ser Val Thr Leu Ser Gly
305 310 315 320
Ser Val Asp Tyr Ser Asp Gly Ser Gln Glu Phe Ser Ala Glu Leu Thr
325 330 335
Leu His Ser Glu Val Leu Ser Pro Glu Met Leu Gln Cys Thr Pro Ala
340 345 350
Asn Met Val Glu Val His Lys Asp Lys Glu Ser Ser Lys Gly His Thr
355 360 365
Arg His Lys Val Glu Glu Ala Leu Ile Asn Glu Glu Ala Ile Leu Asn
370 375 380
Leu Met Glu Asn Ser Gln Thr Phe Gln Pro Leu Thr Gln Arg Leu Ser
385 390 395 400
Glu Ser Pro Val Phe Met Asp Ser Ser Pro Asp Glu Ala Leu Val His
405 410 415
Leu Leu Ala Gly Leu Glu Ser Asp Gly Tyr Arg Gly Glu Arg Asn Arg
420 425 430
Met Pro Ser Pro Cys Arg Ser Phe Gly Asn Asn Lys Tyr Pro Gln Asn
435 440 445
Ser Asp Asp Glu Glu Asn Glu Pro Gln Ile Glu Lys Glu Glu Met Glu
450 455 460
Leu Ser Leu Val Met Ser Gln Arg Trp Asp Ser Asn Ile Glu Glu His
465 470 475 480
Cys Ala Lys Lys Arg Ser Leu Cys Arg Asn Thr His Arg Ser Ser Thr
485 490 495
Glu Asp Asp Asp Ser Ser Ser Gly Glu Glu Met Glu Trp Ser Asp Asn
500 505 510
Ser Leu Leu Leu Ala Ser Leu Ser Ile Pro Gln Leu Asp
515 520 525

Claims (10)

1. An inhibitor of human REV 3L protein cleavage, wherein the inhibitor comprises a small molecule compound or polypeptide that prevents cleavage of full-length human REV 3L protein to produce a cleaved protein.
2. The inhibitor for human REV 3L protein cleavage according to claim 1, wherein the small molecule compound or polypeptide has a target of action on a sequence element dependent on post-translational cleavage of human REV 3L protein, and is used for blocking the sequence element, and the amino acid sequence of the sequence element is shown as SEQ ID No. 1.
3. The human REV 3L protein cleavage inhibitor according to claim 1, wherein the small molecule compound or polypeptide is an inhibitor or an inhibitor of the activity of the expression of the protease TASP1, and the amino acid sequence of the protease TASP1 is shown in seq id No. 3.
4. The human REV 3L protein cleavage inhibitor according to claim 3, wherein the interfering agent expressed by the protease TASP1 is an siRNA molecule or shRNA expression vector directed against mRNA of the gene encoding the protease TASP 1.
5. The inhibitor of human REV 3L protein cleavage according to claim 3, wherein the activity inhibitor is a compound that inhibits the activity of the protease TASP1, or blocks sequences on the TASP1 protein that interact with REV 3L.
6. Use of the human REV 3L protein cleavage inhibitor according to claim 1 in the preparation of an anti-tumor medicament.
7. Use of the human REV 3L protein cleavage inhibitor of claim 1 for the preparation of a tumor drug resistance inhibitor.
8. Use of the human REV 3L protein cleavage inhibitor of claim 1 in the preparation of a potentiator of an anti-tumor drug.
9. The use according to claim 8, wherein the anti-neoplastic agent is a DNA damage inducing molecular based anti-neoplastic agent.
10. The use according to claim 8, wherein the antineoplastic drug is cisplatin.
CN201910065565.0A 2019-01-23 2019-01-23 Human REV 3L protein cleavage inhibitor and application thereof Pending CN111467493A (en)

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PCT/CN2020/073459 WO2020151697A1 (en) 2019-01-23 2020-01-21 Sequence element that human rev3l protein post-translational cleavage depends upon and use thereof

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CN112553274B (en) * 2020-12-02 2023-06-13 江南大学 Method for cutting DNA by soybean extract Bowman-Birk inhibitor
CN114392344A (en) * 2022-01-17 2022-04-26 首都师范大学 Rad51 inhibitor and application thereof
CN114392344B (en) * 2022-01-17 2023-09-05 首都师范大学 Rad51 inhibitor and application thereof

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