CN112162096B - 用于检测细胞线粒体自噬的双荧光蛋白定位检测系统及应用 - Google Patents
用于检测细胞线粒体自噬的双荧光蛋白定位检测系统及应用 Download PDFInfo
- Publication number
- CN112162096B CN112162096B CN202010148310.3A CN202010148310A CN112162096B CN 112162096 B CN112162096 B CN 112162096B CN 202010148310 A CN202010148310 A CN 202010148310A CN 112162096 B CN112162096 B CN 112162096B
- Authority
- CN
- China
- Prior art keywords
- protein
- fluorescent protein
- mitochondrial autophagy
- autophagy
- fluorescent
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 230000004900 autophagic degradation Effects 0.000 title claims abstract description 84
- 230000002438 mitochondrial effect Effects 0.000 title claims abstract description 69
- 108091006047 fluorescent proteins Proteins 0.000 title claims abstract description 56
- 102000034287 fluorescent proteins Human genes 0.000 title claims abstract description 56
- 238000001514 detection method Methods 0.000 title claims abstract description 24
- 230000010856 establishment of protein localization Effects 0.000 title abstract description 9
- 210000004027 cell Anatomy 0.000 claims abstract description 118
- 102000037865 fusion proteins Human genes 0.000 claims abstract description 46
- 108020001507 fusion proteins Proteins 0.000 claims abstract description 46
- 238000000034 method Methods 0.000 claims abstract description 30
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 26
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 24
- 210000003470 mitochondria Anatomy 0.000 claims abstract description 16
- 108010042653 IgA receptor Proteins 0.000 claims abstract description 11
- 102100034014 Prolyl 3-hydroxylase 3 Human genes 0.000 claims abstract description 11
- 239000003086 colorant Substances 0.000 claims abstract description 10
- 210000004957 autophagosome Anatomy 0.000 claims abstract description 4
- 230000008045 co-localization Effects 0.000 claims description 11
- 230000026447 protein localization Effects 0.000 claims description 11
- 238000012258 culturing Methods 0.000 claims description 10
- 239000000411 inducer Substances 0.000 claims description 8
- 230000009977 dual effect Effects 0.000 claims description 7
- 101001129610 Homo sapiens Prohibitin 1 Proteins 0.000 claims description 5
- 102100031169 Prohibitin 1 Human genes 0.000 claims description 5
- 238000012360 testing method Methods 0.000 claims description 5
- 108010048367 enhanced green fluorescent protein Proteins 0.000 claims description 4
- 239000002773 nucleotide Substances 0.000 claims 2
- 125000003729 nucleotide group Chemical group 0.000 claims 2
- 238000011160 research Methods 0.000 abstract description 7
- 238000013459 approach Methods 0.000 abstract description 2
- 239000013589 supplement Substances 0.000 abstract description 2
- 230000004927 fusion Effects 0.000 description 40
- 239000013613 expression plasmid Substances 0.000 description 33
- 239000013612 plasmid Substances 0.000 description 26
- 230000014509 gene expression Effects 0.000 description 20
- 238000012163 sequencing technique Methods 0.000 description 17
- 239000013598 vector Substances 0.000 description 16
- 239000000975 dye Substances 0.000 description 14
- 239000012634 fragment Substances 0.000 description 13
- 108020004707 nucleic acids Proteins 0.000 description 12
- 102000039446 nucleic acids Human genes 0.000 description 12
- 150000007523 nucleic acids Chemical class 0.000 description 12
- 239000002245 particle Substances 0.000 description 12
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 11
- 108020004414 DNA Proteins 0.000 description 10
- 230000000694 effects Effects 0.000 description 10
- 239000005090 green fluorescent protein Substances 0.000 description 10
- 239000002609 medium Substances 0.000 description 9
- 102100024177 Microtubule-associated proteins 1A/1B light chain 3B Human genes 0.000 description 8
- 108091033319 polynucleotide Proteins 0.000 description 8
- 102000040430 polynucleotide Human genes 0.000 description 8
- 239000002157 polynucleotide Substances 0.000 description 8
- 238000010186 staining Methods 0.000 description 8
- 101710171597 Microtubule-associated proteins 1A/1B light chain 3B Proteins 0.000 description 6
- 238000000246 agarose gel electrophoresis Methods 0.000 description 6
- 238000000799 fluorescence microscopy Methods 0.000 description 6
- 230000000149 penetrating effect Effects 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 5
- XCVSDCPWJRHMSZ-UHFFFAOYSA-M [6-(diethylamino)-9-[2-(4-methyl-2-oxochromen-7-yl)oxycarbonylphenyl]xanthen-3-ylidene]-diethylazanium;chloride Chemical group [Cl-].C1=CC(=[N+](CC)CC)C=C2OC3=CC(N(CC)CC)=CC=C3C(C=3C(=CC=CC=3)C(=O)OC=3C=C4OC(=O)C=C(C)C4=CC=3)=C21 XCVSDCPWJRHMSZ-UHFFFAOYSA-M 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 210000004962 mammalian cell Anatomy 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 108010054624 red fluorescent protein Proteins 0.000 description 5
- 238000001890 transfection Methods 0.000 description 5
- 208000010507 Adenocarcinoma of Lung Diseases 0.000 description 4
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 4
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 4
- 241000282414 Homo sapiens Species 0.000 description 4
- 229930040373 Paraformaldehyde Natural products 0.000 description 4
- 229920004890 Triton X-100 Polymers 0.000 description 4
- 239000013504 Triton X-100 Substances 0.000 description 4
- 150000001413 amino acids Chemical group 0.000 description 4
- 210000003855 cell nucleus Anatomy 0.000 description 4
- 238000004140 cleaning Methods 0.000 description 4
- 239000002299 complementary DNA Substances 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 230000018109 developmental process Effects 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 235000003642 hunger Nutrition 0.000 description 4
- 230000003834 intracellular effect Effects 0.000 description 4
- 201000005249 lung adenocarcinoma Diseases 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 235000016709 nutrition Nutrition 0.000 description 4
- 229920002866 paraformaldehyde Polymers 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 230000037351 starvation Effects 0.000 description 4
- 108020004705 Codon Proteins 0.000 description 3
- 102000012410 DNA Ligases Human genes 0.000 description 3
- 108010061982 DNA Ligases Proteins 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 3
- 101001052512 Homo sapiens Microtubule-associated proteins 1A/1B light chain 3B Proteins 0.000 description 3
- FADYJNXDPBKVCA-UHFFFAOYSA-N L-Phenylalanyl-L-lysin Natural products NCCCCC(C(O)=O)NC(=O)C(N)CC1=CC=CC=C1 FADYJNXDPBKVCA-UHFFFAOYSA-N 0.000 description 3
- 241000880493 Leptailurus serval Species 0.000 description 3
- XMBSYZWANAQXEV-UHFFFAOYSA-N N-alpha-L-glutamyl-L-phenylalanine Natural products OC(=O)CCC(N)C(=O)NC(C(O)=O)CC1=CC=CC=C1 XMBSYZWANAQXEV-UHFFFAOYSA-N 0.000 description 3
- 108010002311 N-glycylglutamic acid Proteins 0.000 description 3
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 3
- 229960000723 ampicillin Drugs 0.000 description 3
- 108010047857 aspartylglycine Proteins 0.000 description 3
- 238000007664 blowing Methods 0.000 description 3
- 238000010367 cloning Methods 0.000 description 3
- 238000005520 cutting process Methods 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 230000029087 digestion Effects 0.000 description 3
- 238000001976 enzyme digestion Methods 0.000 description 3
- 238000002073 fluorescence micrograph Methods 0.000 description 3
- 108010042598 glutamyl-aspartyl-glycine Proteins 0.000 description 3
- 238000000265 homogenisation Methods 0.000 description 3
- 238000010166 immunofluorescence Methods 0.000 description 3
- 108010034529 leucyl-lysine Proteins 0.000 description 3
- 230000025608 mitochondrion localization Effects 0.000 description 3
- 238000012795 verification Methods 0.000 description 3
- YWWATNIVMOCSAV-UBHSHLNASA-N Ala-Arg-Phe Chemical compound NC(=N)NCCC[C@H](NC(=O)[C@@H](N)C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 YWWATNIVMOCSAV-UBHSHLNASA-N 0.000 description 2
- MJIJBEYEHBKTIM-BYULHYEWSA-N Asn-Val-Asn Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CC(=O)N)N MJIJBEYEHBKTIM-BYULHYEWSA-N 0.000 description 2
- 108091026890 Coding region Proteins 0.000 description 2
- MTAOBYXRYJZRGQ-WDSKDSINSA-N Glu-Gly-Asp Chemical compound OC(=O)CC[C@H](N)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(O)=O MTAOBYXRYJZRGQ-WDSKDSINSA-N 0.000 description 2
- BTEMNFBEAAOGBR-BZSNNMDCSA-N Leu-Tyr-Lys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CCCCN)C(=O)O)N BTEMNFBEAAOGBR-BZSNNMDCSA-N 0.000 description 2
- LCMWVZLBCUVDAZ-IUCAKERBSA-N Lys-Gly-Glu Chemical compound [NH3+]CCCC[C@H]([NH3+])C(=O)NCC(=O)N[C@H](C([O-])=O)CCC([O-])=O LCMWVZLBCUVDAZ-IUCAKERBSA-N 0.000 description 2
- GODBLDDYHFTUAH-CIUDSAMLSA-N Met-Asp-Glu Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CCC(O)=O GODBLDDYHFTUAH-CIUDSAMLSA-N 0.000 description 2
- LBSWWNKMVPAXOI-GUBZILKMSA-N Met-Val-Ser Chemical compound CSCC[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(O)=O LBSWWNKMVPAXOI-GUBZILKMSA-N 0.000 description 2
- YBAFDPFAUTYYRW-UHFFFAOYSA-N N-L-alpha-glutamyl-L-leucine Natural products CC(C)CC(C(O)=O)NC(=O)C(N)CCC(O)=O YBAFDPFAUTYYRW-UHFFFAOYSA-N 0.000 description 2
- RBRNEFJTEHPDSL-ACRUOGEOSA-N Phe-Phe-Lys Chemical compound C([C@@H](C(=O)N[C@@H](CCCCN)C(O)=O)NC(=O)[C@@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 RBRNEFJTEHPDSL-ACRUOGEOSA-N 0.000 description 2
- 108010004729 Phycoerythrin Proteins 0.000 description 2
- NMELOOXSGDRBRU-YUMQZZPRSA-N Pro-Glu-Gly Chemical compound OC(=O)CNC(=O)[C@H](CCC(=O)O)NC(=O)[C@@H]1CCCN1 NMELOOXSGDRBRU-YUMQZZPRSA-N 0.000 description 2
- KHRLUIPIMIQFGT-AVGNSLFASA-N Pro-Val-Leu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O KHRLUIPIMIQFGT-AVGNSLFASA-N 0.000 description 2
- 102100031156 Prohibitin-2 Human genes 0.000 description 2
- 108020004511 Recombinant DNA Proteins 0.000 description 2
- ILRCGYURZSFMEG-UHFFFAOYSA-N Salidroside Natural products OC1C(O)C(O)C(CO)OC1OCCC1=CC=C(O)C=C1 ILRCGYURZSFMEG-UHFFFAOYSA-N 0.000 description 2
- GZSZPKSBVAOGIE-CIUDSAMLSA-N Ser-Lys-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(O)=O GZSZPKSBVAOGIE-CIUDSAMLSA-N 0.000 description 2
- 108010005233 alanylglutamic acid Proteins 0.000 description 2
- 108010004469 allophycocyanin Proteins 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 108010013835 arginine glutamate Proteins 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 210000003743 erythrocyte Anatomy 0.000 description 2
- 210000003527 eukaryotic cell Anatomy 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 231100000221 frame shift mutation induction Toxicity 0.000 description 2
- 230000037433 frameshift Effects 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 108010057821 leucylproline Proteins 0.000 description 2
- 108010054155 lysyllysine Proteins 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 201000001441 melanoma Diseases 0.000 description 2
- 230000004898 mitochondrial function Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 210000004940 nucleus Anatomy 0.000 description 2
- 230000035764 nutrition Effects 0.000 description 2
- 230000002018 overexpression Effects 0.000 description 2
- 108010028138 prohibitin Proteins 0.000 description 2
- 102000016670 prohibitin Human genes 0.000 description 2
- 230000001681 protective effect Effects 0.000 description 2
- 239000001044 red dye Substances 0.000 description 2
- ILRCGYURZSFMEG-RQICVUQASA-N salidroside Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)OC1OCCC1=CC=C(O)C=C1 ILRCGYURZSFMEG-RQICVUQASA-N 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 108010069117 seryl-lysyl-aspartic acid Proteins 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- LGQPPBQRUBVTIF-JBDRJPRFSA-N Ala-Ala-Ile Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O LGQPPBQRUBVTIF-JBDRJPRFSA-N 0.000 description 1
- JBVSSSZFNTXJDX-YTLHQDLWSA-N Ala-Ala-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@H](C)N JBVSSSZFNTXJDX-YTLHQDLWSA-N 0.000 description 1
- FXKNPWNXPQZLES-ZLUOBGJFSA-N Ala-Asn-Ser Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(O)=O FXKNPWNXPQZLES-ZLUOBGJFSA-N 0.000 description 1
- LZRNYBIJOSKKRJ-XVYDVKMFSA-N Ala-Asp-His Chemical compound C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N LZRNYBIJOSKKRJ-XVYDVKMFSA-N 0.000 description 1
- RXTBLQVXNIECFP-FXQIFTODSA-N Ala-Gln-Gln Chemical compound C[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O RXTBLQVXNIECFP-FXQIFTODSA-N 0.000 description 1
- NWVVKQZOVSTDBQ-CIUDSAMLSA-N Ala-Glu-Arg Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O NWVVKQZOVSTDBQ-CIUDSAMLSA-N 0.000 description 1
- KXEVYGKATAMXJJ-ACZMJKKPSA-N Ala-Glu-Asp Chemical compound C[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O KXEVYGKATAMXJJ-ACZMJKKPSA-N 0.000 description 1
- HXNNRBHASOSVPG-GUBZILKMSA-N Ala-Glu-Leu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O HXNNRBHASOSVPG-GUBZILKMSA-N 0.000 description 1
- OMMDTNGURYRDAC-NRPADANISA-N Ala-Glu-Val Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O OMMDTNGURYRDAC-NRPADANISA-N 0.000 description 1
- MPLOSMWGDNJSEV-WHFBIAKZSA-N Ala-Gly-Asp Chemical compound [H]N[C@@H](C)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(O)=O MPLOSMWGDNJSEV-WHFBIAKZSA-N 0.000 description 1
- LMFXXZPPZDCPTA-ZKWXMUAHSA-N Ala-Gly-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@H](C)N LMFXXZPPZDCPTA-ZKWXMUAHSA-N 0.000 description 1
- AWZKCUCQJNTBAD-SRVKXCTJSA-N Ala-Leu-Lys Chemical compound C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CCCCN AWZKCUCQJNTBAD-SRVKXCTJSA-N 0.000 description 1
- PMQXMXAASGFUDX-SRVKXCTJSA-N Ala-Lys-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@H](C)N)CCCCN PMQXMXAASGFUDX-SRVKXCTJSA-N 0.000 description 1
- VCSABYLVNWQYQE-UHFFFAOYSA-N Ala-Lys-Lys Natural products NCCCCC(NC(=O)C(N)C)C(=O)NC(CCCCN)C(O)=O VCSABYLVNWQYQE-UHFFFAOYSA-N 0.000 description 1
- MDNAVFBZPROEHO-UHFFFAOYSA-N Ala-Lys-Val Natural products CC(C)C(C(O)=O)NC(=O)C(NC(=O)C(C)N)CCCCN MDNAVFBZPROEHO-UHFFFAOYSA-N 0.000 description 1
- YYAVDNKUWLAFCV-ACZMJKKPSA-N Ala-Ser-Gln Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(O)=O YYAVDNKUWLAFCV-ACZMJKKPSA-N 0.000 description 1
- LYILPUNCKACNGF-NAKRPEOUSA-N Ala-Val-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](C)N LYILPUNCKACNGF-NAKRPEOUSA-N 0.000 description 1
- NEZONWMXZKDMKF-JTQLQIEISA-N Alkannin Chemical compound C1=CC(O)=C2C(=O)C([C@@H](O)CC=C(C)C)=CC(=O)C2=C1O NEZONWMXZKDMKF-JTQLQIEISA-N 0.000 description 1
- VKKYFICVTYKFIO-CIUDSAMLSA-N Arg-Ala-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCCN=C(N)N VKKYFICVTYKFIO-CIUDSAMLSA-N 0.000 description 1
- NABSCJGZKWSNHX-RCWTZXSCSA-N Arg-Arg-Thr Chemical compound NC(N)=NCCC[C@@H](C(=O)N[C@@H]([C@H](O)C)C(O)=O)NC(=O)[C@@H](N)CCCN=C(N)N NABSCJGZKWSNHX-RCWTZXSCSA-N 0.000 description 1
- ITVINTQUZMQWJR-QXEWZRGKSA-N Arg-Asn-Val Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C(C)C)C(O)=O ITVINTQUZMQWJR-QXEWZRGKSA-N 0.000 description 1
- LMPKCSXZJSXBBL-NHCYSSNCSA-N Arg-Gln-Val Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(O)=O LMPKCSXZJSXBBL-NHCYSSNCSA-N 0.000 description 1
- CRCCTGPNZUCAHE-DCAQKATOSA-N Arg-His-Ser Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CO)C(O)=O)CC1=CN=CN1 CRCCTGPNZUCAHE-DCAQKATOSA-N 0.000 description 1
- OOIMKQRCPJBGPD-XUXIUFHCSA-N Arg-Ile-Leu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(O)=O OOIMKQRCPJBGPD-XUXIUFHCSA-N 0.000 description 1
- GMFAGHNRXPSSJS-SRVKXCTJSA-N Arg-Leu-Gln Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O GMFAGHNRXPSSJS-SRVKXCTJSA-N 0.000 description 1
- YLVGUOGAFAJMKP-JYJNAYRXSA-N Arg-Met-Tyr Chemical compound CSCC[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N YLVGUOGAFAJMKP-JYJNAYRXSA-N 0.000 description 1
- VUGWHBXPMAHEGZ-SRVKXCTJSA-N Arg-Pro-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CCCN=C(N)N VUGWHBXPMAHEGZ-SRVKXCTJSA-N 0.000 description 1
- VENMDXUVHSKEIN-GUBZILKMSA-N Arg-Ser-Arg Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O VENMDXUVHSKEIN-GUBZILKMSA-N 0.000 description 1
- YNSUUAOAFCVINY-OSUNSFLBSA-N Arg-Thr-Ile Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O YNSUUAOAFCVINY-OSUNSFLBSA-N 0.000 description 1
- QCTOLCVIGRLMQS-HRCADAONSA-N Arg-Tyr-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC2=CC=C(C=C2)O)NC(=O)[C@H](CCCN=C(N)N)N)C(=O)O QCTOLCVIGRLMQS-HRCADAONSA-N 0.000 description 1
- MEFGKQUUYZOLHM-GMOBBJLQSA-N Asn-Arg-Ile Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O MEFGKQUUYZOLHM-GMOBBJLQSA-N 0.000 description 1
- FAEFJTCTNZTPHX-ACZMJKKPSA-N Asn-Gln-Ala Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(O)=O FAEFJTCTNZTPHX-ACZMJKKPSA-N 0.000 description 1
- WQLJRNRLHWJIRW-KKUMJFAQSA-N Asn-His-Tyr Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)O)NC(=O)[C@H](CC2=CN=CN2)NC(=O)[C@H](CC(=O)N)N)O WQLJRNRLHWJIRW-KKUMJFAQSA-N 0.000 description 1
- WUQXMTITJLFXAU-JIOCBJNQSA-N Asn-Thr-Pro Chemical compound C[C@H]([C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CC(=O)N)N)O WUQXMTITJLFXAU-JIOCBJNQSA-N 0.000 description 1
- RTFXPCYMDYBZNQ-SRVKXCTJSA-N Asn-Tyr-Asn Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(N)=O)C(O)=O RTFXPCYMDYBZNQ-SRVKXCTJSA-N 0.000 description 1
- QNNBHTFDFFFHGC-KKUMJFAQSA-N Asn-Tyr-Lys Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC(=O)N)N)O QNNBHTFDFFFHGC-KKUMJFAQSA-N 0.000 description 1
- HPNDBHLITCHRSO-WHFBIAKZSA-N Asp-Ala-Gly Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(=O)NCC(O)=O HPNDBHLITCHRSO-WHFBIAKZSA-N 0.000 description 1
- FANQWNCPNFEPGZ-WHFBIAKZSA-N Asp-Asp-Gly Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(O)=O FANQWNCPNFEPGZ-WHFBIAKZSA-N 0.000 description 1
- FMWHSNJMHUNLAG-FXQIFTODSA-N Asp-Cys-Arg Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CS)C(=O)N[C@H](C(O)=O)CCCN=C(N)N FMWHSNJMHUNLAG-FXQIFTODSA-N 0.000 description 1
- WBDWQKRLTVCDSY-WHFBIAKZSA-N Asp-Gly-Asp Chemical compound OC(=O)C[C@H](N)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(O)=O WBDWQKRLTVCDSY-WHFBIAKZSA-N 0.000 description 1
- KHGPWGKPYHPOIK-QWRGUYRKSA-N Asp-Gly-Phe Chemical compound [H]N[C@@H](CC(O)=O)C(=O)NCC(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O KHGPWGKPYHPOIK-QWRGUYRKSA-N 0.000 description 1
- LNENWJXDHCFVOF-DCAQKATOSA-N Asp-His-Met Chemical compound CSCC[C@@H](C(=O)O)NC(=O)[C@H](CC1=CN=CN1)NC(=O)[C@H](CC(=O)O)N LNENWJXDHCFVOF-DCAQKATOSA-N 0.000 description 1
- SPWXXPFDTMYTRI-IUKAMOBKSA-N Asp-Ile-Thr Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(O)=O SPWXXPFDTMYTRI-IUKAMOBKSA-N 0.000 description 1
- XLILXFRAKOYEJX-GUBZILKMSA-N Asp-Leu-Gln Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O XLILXFRAKOYEJX-GUBZILKMSA-N 0.000 description 1
- USENATHVGFXRNO-SRVKXCTJSA-N Asp-Tyr-Asp Chemical compound OC(=O)C[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CC(O)=O)C(O)=O)CC1=CC=C(O)C=C1 USENATHVGFXRNO-SRVKXCTJSA-N 0.000 description 1
- 108010083946 Asp-Tyr-Leu-Lys Proteins 0.000 description 1
- BPAUXFVCSYQDQX-JRQIVUDYSA-N Asp-Tyr-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)NC(=O)[C@H](CC(=O)O)N)O BPAUXFVCSYQDQX-JRQIVUDYSA-N 0.000 description 1
- QOJJMJKTMKNFEF-ZKWXMUAHSA-N Asp-Val-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CC(O)=O QOJJMJKTMKNFEF-ZKWXMUAHSA-N 0.000 description 1
- 108010077544 Chromatin Proteins 0.000 description 1
- CHRCKSPMGYDLIA-SRVKXCTJSA-N Cys-Phe-Ser Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(O)=O CHRCKSPMGYDLIA-SRVKXCTJSA-N 0.000 description 1
- NAPULYCVEVVFRB-HEIBUPTGSA-N Cys-Thr-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@@H](N)CS NAPULYCVEVVFRB-HEIBUPTGSA-N 0.000 description 1
- 230000006820 DNA synthesis Effects 0.000 description 1
- WQWMZOIPXWSZNE-WDSKDSINSA-N Gln-Asp-Gly Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(O)=O WQWMZOIPXWSZNE-WDSKDSINSA-N 0.000 description 1
- MADFVRSKEIEZHZ-DCAQKATOSA-N Gln-Gln-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CCC(=O)N)N MADFVRSKEIEZHZ-DCAQKATOSA-N 0.000 description 1
- DRDSQGHKTLSNEA-GLLZPBPUSA-N Gln-Glu-Thr Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O DRDSQGHKTLSNEA-GLLZPBPUSA-N 0.000 description 1
- LTXLIIZACMCQTO-GUBZILKMSA-N Gln-His-Asp Chemical compound C1=C(NC=N1)C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)[C@H](CCC(=O)N)N LTXLIIZACMCQTO-GUBZILKMSA-N 0.000 description 1
- ZBKUIQNCRIYVGH-SDDRHHMPSA-N Gln-Leu-Pro Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CCC(=O)N)N ZBKUIQNCRIYVGH-SDDRHHMPSA-N 0.000 description 1
- YPMDZWPZFOZYFG-GUBZILKMSA-N Gln-Leu-Ser Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O YPMDZWPZFOZYFG-GUBZILKMSA-N 0.000 description 1
- KUBFPYIMAGXGBT-ACZMJKKPSA-N Gln-Ser-Ala Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(O)=O KUBFPYIMAGXGBT-ACZMJKKPSA-N 0.000 description 1
- WIMVKDYAKRAUCG-IHRRRGAJSA-N Gln-Tyr-Glu Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)O)NC(=O)[C@H](CCC(=O)N)N)O WIMVKDYAKRAUCG-IHRRRGAJSA-N 0.000 description 1
- GCYFUZJHAXJKKE-KKUMJFAQSA-N Glu-Arg-Tyr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O GCYFUZJHAXJKKE-KKUMJFAQSA-N 0.000 description 1
- DYFJZDDQPNIPAB-NHCYSSNCSA-N Glu-Arg-Val Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C(C)C)C(O)=O DYFJZDDQPNIPAB-NHCYSSNCSA-N 0.000 description 1
- NTBDVNJIWCKURJ-ACZMJKKPSA-N Glu-Asp-Asn Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O NTBDVNJIWCKURJ-ACZMJKKPSA-N 0.000 description 1
- DSPQRJXOIXHOHK-WDSKDSINSA-N Glu-Asp-Gly Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(O)=O DSPQRJXOIXHOHK-WDSKDSINSA-N 0.000 description 1
- CJWANNXUTOATSJ-DCAQKATOSA-N Glu-Gln-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CCC(=O)O)N CJWANNXUTOATSJ-DCAQKATOSA-N 0.000 description 1
- PXXGVUVQWQGGIG-YUMQZZPRSA-N Glu-Gly-Arg Chemical compound OC(=O)CC[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCCN=C(N)N PXXGVUVQWQGGIG-YUMQZZPRSA-N 0.000 description 1
- OGNJZUXUTPQVBR-BQBZGAKWSA-N Glu-Gly-Glu Chemical compound OC(=O)CC[C@H](N)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(O)=O OGNJZUXUTPQVBR-BQBZGAKWSA-N 0.000 description 1
- HPJLZFTUUJKWAJ-JHEQGTHGSA-N Glu-Gly-Thr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(O)=O HPJLZFTUUJKWAJ-JHEQGTHGSA-N 0.000 description 1
- VGUYMZGLJUJRBV-YVNDNENWSA-N Glu-Ile-Glu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(O)=O)C(O)=O VGUYMZGLJUJRBV-YVNDNENWSA-N 0.000 description 1
- QXDXIXFSFHUYAX-MNXVOIDGSA-N Glu-Ile-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H](N)CCC(O)=O QXDXIXFSFHUYAX-MNXVOIDGSA-N 0.000 description 1
- VSRCAOIHMGCIJK-SRVKXCTJSA-N Glu-Leu-Arg Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O VSRCAOIHMGCIJK-SRVKXCTJSA-N 0.000 description 1
- VGBSZQSKQRMLHD-MNXVOIDGSA-N Glu-Leu-Ile Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O VGBSZQSKQRMLHD-MNXVOIDGSA-N 0.000 description 1
- IVGJYOOGJLFKQE-AVGNSLFASA-N Glu-Leu-Lys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CCC(=O)O)N IVGJYOOGJLFKQE-AVGNSLFASA-N 0.000 description 1
- QDMVXRNLOPTPIE-WDCWCFNPSA-N Glu-Lys-Thr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(O)=O QDMVXRNLOPTPIE-WDCWCFNPSA-N 0.000 description 1
- JZJGEKDPWVJOLD-QEWYBTABSA-N Glu-Phe-Ile Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O JZJGEKDPWVJOLD-QEWYBTABSA-N 0.000 description 1
- GMVCSRBOSIUTFC-FXQIFTODSA-N Glu-Ser-Glu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(O)=O GMVCSRBOSIUTFC-FXQIFTODSA-N 0.000 description 1
- SYAYROHMAIHWFB-KBIXCLLPSA-N Glu-Ser-Ile Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O SYAYROHMAIHWFB-KBIXCLLPSA-N 0.000 description 1
- QRWPTXLWHHTOCO-DZKIICNBSA-N Glu-Val-Tyr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O QRWPTXLWHHTOCO-DZKIICNBSA-N 0.000 description 1
- QIZJOTQTCAGKPU-KWQFWETISA-N Gly-Ala-Tyr Chemical compound [NH3+]CC(=O)N[C@@H](C)C(=O)N[C@H](C([O-])=O)CC1=CC=C(O)C=C1 QIZJOTQTCAGKPU-KWQFWETISA-N 0.000 description 1
- KKBWDNZXYLGJEY-UHFFFAOYSA-N Gly-Arg-Pro Natural products NCC(=O)NC(CCNC(=N)N)C(=O)N1CCCC1C(=O)O KKBWDNZXYLGJEY-UHFFFAOYSA-N 0.000 description 1
- BGVYNAQWHSTTSP-BYULHYEWSA-N Gly-Asn-Ile Chemical compound [H]NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O BGVYNAQWHSTTSP-BYULHYEWSA-N 0.000 description 1
- KQDMENMTYNBWMR-WHFBIAKZSA-N Gly-Asp-Ala Chemical compound [H]NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(O)=O KQDMENMTYNBWMR-WHFBIAKZSA-N 0.000 description 1
- GNPVTZJUUBPZKW-WDSKDSINSA-N Gly-Gln-Ser Chemical compound [H]NCC(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(O)=O GNPVTZJUUBPZKW-WDSKDSINSA-N 0.000 description 1
- XTQFHTHIAKKCTM-YFKPBYRVSA-N Gly-Glu-Gly Chemical compound NCC(=O)N[C@@H](CCC(O)=O)C(=O)NCC(O)=O XTQFHTHIAKKCTM-YFKPBYRVSA-N 0.000 description 1
- STVHDEHTKFXBJQ-LAEOZQHASA-N Gly-Glu-Ile Chemical compound [H]NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O STVHDEHTKFXBJQ-LAEOZQHASA-N 0.000 description 1
- OLPPXYMMIARYAL-QMMMGPOBSA-N Gly-Gly-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)CNC(=O)CN OLPPXYMMIARYAL-QMMMGPOBSA-N 0.000 description 1
- TVDHVLGFJSHPAX-UWVGGRQHSA-N Gly-His-Arg Chemical compound NC(N)=NCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CC1=CN=CN1 TVDHVLGFJSHPAX-UWVGGRQHSA-N 0.000 description 1
- ORXZVPZCPMKHNR-IUCAKERBSA-N Gly-His-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CC1=CNC=N1 ORXZVPZCPMKHNR-IUCAKERBSA-N 0.000 description 1
- LPCKHUXOGVNZRS-YUMQZZPRSA-N Gly-His-Ser Chemical compound [H]NCC(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CO)C(O)=O LPCKHUXOGVNZRS-YUMQZZPRSA-N 0.000 description 1
- ITZOBNKQDZEOCE-NHCYSSNCSA-N Gly-Ile-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)CN ITZOBNKQDZEOCE-NHCYSSNCSA-N 0.000 description 1
- PAWIVEIWWYGBAM-YUMQZZPRSA-N Gly-Leu-Ala Chemical compound NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(O)=O PAWIVEIWWYGBAM-YUMQZZPRSA-N 0.000 description 1
- LRQXRHGQEVWGPV-NHCYSSNCSA-N Gly-Leu-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)CN LRQXRHGQEVWGPV-NHCYSSNCSA-N 0.000 description 1
- MHXKHKWHPNETGG-QWRGUYRKSA-N Gly-Lys-Leu Chemical compound [H]NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(O)=O MHXKHKWHPNETGG-QWRGUYRKSA-N 0.000 description 1
- MHZXESQPPXOING-KBPBESRZSA-N Gly-Lys-Phe Chemical compound [H]NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O MHZXESQPPXOING-KBPBESRZSA-N 0.000 description 1
- BMWFDYIYBAFROD-WPRPVWTQSA-N Gly-Pro-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)CN BMWFDYIYBAFROD-WPRPVWTQSA-N 0.000 description 1
- POJJAZJHBGXEGM-YUMQZZPRSA-N Gly-Ser-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)CN POJJAZJHBGXEGM-YUMQZZPRSA-N 0.000 description 1
- CQMFNTVQVLQRLT-JHEQGTHGSA-N Gly-Thr-Gln Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(N)=O)C(O)=O CQMFNTVQVLQRLT-JHEQGTHGSA-N 0.000 description 1
- NGRPGJGKJMUGDM-XVKPBYJWSA-N Gly-Val-Gln Chemical compound NCC(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O NGRPGJGKJMUGDM-XVKPBYJWSA-N 0.000 description 1
- JBCLFWXMTIKCCB-UHFFFAOYSA-N H-Gly-Phe-OH Natural products NCC(=O)NC(C(O)=O)CC1=CC=CC=C1 JBCLFWXMTIKCCB-UHFFFAOYSA-N 0.000 description 1
- KYMUEAZVLPRVAE-GUBZILKMSA-N His-Asn-Glu Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O KYMUEAZVLPRVAE-GUBZILKMSA-N 0.000 description 1
- UZZXGLOJRZKYEL-DJFWLOJKSA-N His-Asn-Ile Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O UZZXGLOJRZKYEL-DJFWLOJKSA-N 0.000 description 1
- IGBBXBFSLKRHJB-BZSNNMDCSA-N His-Lys-Phe Chemical compound C([C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CN=CN1 IGBBXBFSLKRHJB-BZSNNMDCSA-N 0.000 description 1
- WYSJPCTWSBJFCO-AVGNSLFASA-N His-Met-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CCSC)NC(=O)[C@H](CC1=CN=CN1)N WYSJPCTWSBJFCO-AVGNSLFASA-N 0.000 description 1
- SVVULKPWDBIPCO-BZSNNMDCSA-N His-Phe-Leu Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(C)C)C(O)=O SVVULKPWDBIPCO-BZSNNMDCSA-N 0.000 description 1
- LPBWRHRHEIYAIP-KKUMJFAQSA-N His-Tyr-Asp Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(O)=O)C(O)=O LPBWRHRHEIYAIP-KKUMJFAQSA-N 0.000 description 1
- 101001129654 Homo sapiens Prohibitin-2 Proteins 0.000 description 1
- HERITAGIPLEJMT-GVARAGBVSA-N Ile-Ala-Tyr Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 HERITAGIPLEJMT-GVARAGBVSA-N 0.000 description 1
- HGNUKGZQASSBKQ-PCBIJLKTSA-N Ile-Asp-Phe Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)N HGNUKGZQASSBKQ-PCBIJLKTSA-N 0.000 description 1
- KFVUBLZRFSVDGO-BYULHYEWSA-N Ile-Gly-Asp Chemical compound CC[C@H](C)[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CC(O)=O KFVUBLZRFSVDGO-BYULHYEWSA-N 0.000 description 1
- DMSVBUWGDLYNLC-IAVJCBSLSA-N Ile-Ile-Phe Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 DMSVBUWGDLYNLC-IAVJCBSLSA-N 0.000 description 1
- OUUCIIJSBIBCHB-ZPFDUUQYSA-N Ile-Leu-Asp Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O OUUCIIJSBIBCHB-ZPFDUUQYSA-N 0.000 description 1
- GVKKVHNRTUFCCE-BJDJZHNGSA-N Ile-Leu-Ser Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)O)N GVKKVHNRTUFCCE-BJDJZHNGSA-N 0.000 description 1
- ADDYYRVQQZFIMW-MNXVOIDGSA-N Ile-Lys-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N ADDYYRVQQZFIMW-MNXVOIDGSA-N 0.000 description 1
- YSGBJIQXTIVBHZ-AJNGGQMLSA-N Ile-Lys-Leu Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(O)=O YSGBJIQXTIVBHZ-AJNGGQMLSA-N 0.000 description 1
- VEPIBPGLTLPBDW-URLPEUOOSA-N Ile-Phe-Thr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(=O)O)N VEPIBPGLTLPBDW-URLPEUOOSA-N 0.000 description 1
- OWSWUWDMSNXTNE-GMOBBJLQSA-N Ile-Pro-Asp Chemical compound CC[C@H](C)[C@@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(=O)O)C(=O)O)N OWSWUWDMSNXTNE-GMOBBJLQSA-N 0.000 description 1
- AKQFLPNANHNTLP-VKOGCVSHSA-N Ile-Pro-Trp Chemical compound CC[C@H](C)[C@@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC2=CNC3=CC=CC=C32)C(=O)O)N AKQFLPNANHNTLP-VKOGCVSHSA-N 0.000 description 1
- YKZAMJXNJUWFIK-JBDRJPRFSA-N Ile-Ser-Ala Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(=O)O)N YKZAMJXNJUWFIK-JBDRJPRFSA-N 0.000 description 1
- KBDIBHQICWDGDL-PPCPHDFISA-N Ile-Thr-Leu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)O)N KBDIBHQICWDGDL-PPCPHDFISA-N 0.000 description 1
- NURNJECQNNCRBK-FLBSBUHZSA-N Ile-Thr-Thr Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O NURNJECQNNCRBK-FLBSBUHZSA-N 0.000 description 1
- DGTOKVBDZXJHNZ-WZLNRYEVSA-N Ile-Thr-Tyr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)O)N DGTOKVBDZXJHNZ-WZLNRYEVSA-N 0.000 description 1
- YWCJXQKATPNPOE-UKJIMTQDSA-N Ile-Val-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N YWCJXQKATPNPOE-UKJIMTQDSA-N 0.000 description 1
- 108010065920 Insulin Lispro Proteins 0.000 description 1
- XBBKIIGCUMBKCO-JXUBOQSCSA-N Leu-Ala-Thr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O XBBKIIGCUMBKCO-JXUBOQSCSA-N 0.000 description 1
- KSZCCRIGNVSHFH-UWVGGRQHSA-N Leu-Arg-Gly Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(O)=O KSZCCRIGNVSHFH-UWVGGRQHSA-N 0.000 description 1
- WUFYAPWIHCUMLL-CIUDSAMLSA-N Leu-Asn-Ala Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(O)=O WUFYAPWIHCUMLL-CIUDSAMLSA-N 0.000 description 1
- DZQMXBALGUHGJT-GUBZILKMSA-N Leu-Glu-Ala Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O DZQMXBALGUHGJT-GUBZILKMSA-N 0.000 description 1
- VBZOAGIPCULURB-QWRGUYRKSA-N Leu-Gly-His Chemical compound CC(C)C[C@@H](C(=O)NCC(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N VBZOAGIPCULURB-QWRGUYRKSA-N 0.000 description 1
- AVEGDIAXTDVBJS-XUXIUFHCSA-N Leu-Ile-Arg Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O AVEGDIAXTDVBJS-XUXIUFHCSA-N 0.000 description 1
- JNDYEOUZBLOVOF-AVGNSLFASA-N Leu-Leu-Gln Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O JNDYEOUZBLOVOF-AVGNSLFASA-N 0.000 description 1
- QNBVTHNJGCOVFA-AVGNSLFASA-N Leu-Leu-Glu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CCC(O)=O QNBVTHNJGCOVFA-AVGNSLFASA-N 0.000 description 1
- XVZCXCTYGHPNEM-UHFFFAOYSA-N Leu-Leu-Pro Natural products CC(C)CC(N)C(=O)NC(CC(C)C)C(=O)N1CCCC1C(O)=O XVZCXCTYGHPNEM-UHFFFAOYSA-N 0.000 description 1
- BGZCJDGBBUUBHA-KKUMJFAQSA-N Leu-Lys-Leu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(O)=O BGZCJDGBBUUBHA-KKUMJFAQSA-N 0.000 description 1
- QNTJIDXQHWUBKC-BZSNNMDCSA-N Leu-Lys-Phe Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O QNTJIDXQHWUBKC-BZSNNMDCSA-N 0.000 description 1
- WMIOEVKKYIMVKI-DCAQKATOSA-N Leu-Pro-Ala Chemical compound [H]N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C)C(O)=O WMIOEVKKYIMVKI-DCAQKATOSA-N 0.000 description 1
- QMKFDEUJGYNFMC-AVGNSLFASA-N Leu-Pro-Arg Chemical compound CC(C)C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCN=C(N)N)C(O)=O QMKFDEUJGYNFMC-AVGNSLFASA-N 0.000 description 1
- BMVFXOQHDQZAQU-DCAQKATOSA-N Leu-Pro-Asp Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(=O)O)C(=O)O)N BMVFXOQHDQZAQU-DCAQKATOSA-N 0.000 description 1
- XWEVVRRSIOBJOO-SRVKXCTJSA-N Leu-Pro-Gln Chemical compound [H]N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(O)=O XWEVVRRSIOBJOO-SRVKXCTJSA-N 0.000 description 1
- CHJKEDSZNSONPS-DCAQKATOSA-N Leu-Pro-Ser Chemical compound [H]N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O CHJKEDSZNSONPS-DCAQKATOSA-N 0.000 description 1
- IWMJFLJQHIDZQW-KKUMJFAQSA-N Leu-Ser-Phe Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 IWMJFLJQHIDZQW-KKUMJFAQSA-N 0.000 description 1
- SBANPBVRHYIMRR-UHFFFAOYSA-N Leu-Ser-Pro Natural products CC(C)CC(N)C(=O)NC(CO)C(=O)N1CCCC1C(O)=O SBANPBVRHYIMRR-UHFFFAOYSA-N 0.000 description 1
- PPGBXYKMUMHFBF-KATARQTJSA-N Leu-Ser-Thr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(O)=O PPGBXYKMUMHFBF-KATARQTJSA-N 0.000 description 1
- LFSQWRSVPNKJGP-WDCWCFNPSA-N Leu-Thr-Glu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@H](C(O)=O)CCC(O)=O LFSQWRSVPNKJGP-WDCWCFNPSA-N 0.000 description 1
- FGZVGOAAROXFAB-IXOXFDKPSA-N Leu-Thr-His Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](CC(C)C)N)O FGZVGOAAROXFAB-IXOXFDKPSA-N 0.000 description 1
- ODRREERHVHMIPT-OEAJRASXSA-N Leu-Thr-Phe Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 ODRREERHVHMIPT-OEAJRASXSA-N 0.000 description 1
- HGLKOTPFWOMPOB-MEYUZBJRSA-N Leu-Thr-Tyr Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 HGLKOTPFWOMPOB-MEYUZBJRSA-N 0.000 description 1
- UFPLDOKWDNTTRP-ULQDDVLXSA-N Leu-Tyr-Met Chemical compound CSCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CC(C)C)CC1=CC=C(O)C=C1 UFPLDOKWDNTTRP-ULQDDVLXSA-N 0.000 description 1
- XZNJZXJZBMBGGS-NHCYSSNCSA-N Leu-Val-Asn Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O XZNJZXJZBMBGGS-NHCYSSNCSA-N 0.000 description 1
- VKVDRTGWLVZJOM-DCAQKATOSA-N Leu-Val-Ser Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(O)=O VKVDRTGWLVZJOM-DCAQKATOSA-N 0.000 description 1
- QESXLSQLQHHTIX-RHYQMDGZSA-N Leu-Val-Thr Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O QESXLSQLQHHTIX-RHYQMDGZSA-N 0.000 description 1
- 241001071917 Lithospermum Species 0.000 description 1
- PNPYKQFJGRFYJE-GUBZILKMSA-N Lys-Ala-Glu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(O)=O PNPYKQFJGRFYJE-GUBZILKMSA-N 0.000 description 1
- GQUDMNDPQTXZRV-DCAQKATOSA-N Lys-Arg-Asp Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(O)=O GQUDMNDPQTXZRV-DCAQKATOSA-N 0.000 description 1
- OVIVOCSURJYCTM-GUBZILKMSA-N Lys-Asp-Glu Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CCC(O)=O OVIVOCSURJYCTM-GUBZILKMSA-N 0.000 description 1
- AAORVPFVUIHEAB-YUMQZZPRSA-N Lys-Asp-Gly Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(O)=O AAORVPFVUIHEAB-YUMQZZPRSA-N 0.000 description 1
- WTZUSCUIVPVCRH-SRVKXCTJSA-N Lys-Gln-Arg Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(O)=O)CCCN=C(N)N WTZUSCUIVPVCRH-SRVKXCTJSA-N 0.000 description 1
- RZHLIPMZXOEJTL-AVGNSLFASA-N Lys-Gln-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CCCCN)N RZHLIPMZXOEJTL-AVGNSLFASA-N 0.000 description 1
- QQUJSUFWEDZQQY-AVGNSLFASA-N Lys-Gln-Lys Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(O)=O)CCCCN QQUJSUFWEDZQQY-AVGNSLFASA-N 0.000 description 1
- NDORZBUHCOJQDO-GVXVVHGQSA-N Lys-Gln-Val Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(O)=O NDORZBUHCOJQDO-GVXVVHGQSA-N 0.000 description 1
- DUTMKEAPLLUGNO-JYJNAYRXSA-N Lys-Glu-Phe Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O DUTMKEAPLLUGNO-JYJNAYRXSA-N 0.000 description 1
- ISHNZELVUVPCHY-ZETCQYMHSA-N Lys-Gly-Gly Chemical compound NCCCC[C@H](N)C(=O)NCC(=O)NCC(O)=O ISHNZELVUVPCHY-ZETCQYMHSA-N 0.000 description 1
- MXMDJEJWERYPMO-XUXIUFHCSA-N Lys-Ile-Arg Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O MXMDJEJWERYPMO-XUXIUFHCSA-N 0.000 description 1
- JYXBNQOKPRQNQS-YTFOTSKYSA-N Lys-Ile-Ile Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O JYXBNQOKPRQNQS-YTFOTSKYSA-N 0.000 description 1
- SKRGVGLIRUGANF-AVGNSLFASA-N Lys-Leu-Glu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O SKRGVGLIRUGANF-AVGNSLFASA-N 0.000 description 1
- WRODMZBHNNPRLN-SRVKXCTJSA-N Lys-Leu-Ser Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O WRODMZBHNNPRLN-SRVKXCTJSA-N 0.000 description 1
- OIQSIMFSVLLWBX-VOAKCMCISA-N Lys-Leu-Thr Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O OIQSIMFSVLLWBX-VOAKCMCISA-N 0.000 description 1
- YXPJCVNIDDKGOE-MELADBBJSA-N Lys-Lys-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCCCN)NC(=O)[C@H](CCCCN)N)C(=O)O YXPJCVNIDDKGOE-MELADBBJSA-N 0.000 description 1
- YKBSXQFZWFXFIB-VOAKCMCISA-N Lys-Thr-Lys Chemical compound NCCCC[C@H](N)C(=O)N[C@@H]([C@H](O)C)C(=O)N[C@@H](CCCCN)C(O)=O YKBSXQFZWFXFIB-VOAKCMCISA-N 0.000 description 1
- CAVRAQIDHUPECU-UVOCVTCTSA-N Lys-Thr-Thr Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O CAVRAQIDHUPECU-UVOCVTCTSA-N 0.000 description 1
- XGZDDOKIHSYHTO-SZMVWBNQSA-N Lys-Trp-Glu Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@@H](N)CCCCN)C(=O)N[C@@H](CCC(O)=O)C(O)=O)=CNC2=C1 XGZDDOKIHSYHTO-SZMVWBNQSA-N 0.000 description 1
- BWECSLVQIWEMSC-IHRRRGAJSA-N Lys-Val-His Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](CCCCN)N BWECSLVQIWEMSC-IHRRRGAJSA-N 0.000 description 1
- TXTZMVNJIRZABH-ULQDDVLXSA-N Lys-Val-Phe Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 TXTZMVNJIRZABH-ULQDDVLXSA-N 0.000 description 1
- RIPJMCFGQHGHNP-RHYQMDGZSA-N Lys-Val-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CCCCN)N)O RIPJMCFGQHGHNP-RHYQMDGZSA-N 0.000 description 1
- 102100026639 MICOS complex subunit MIC60 Human genes 0.000 description 1
- 101710128942 MICOS complex subunit MIC60 Proteins 0.000 description 1
- YRAWWKUTNBILNT-FXQIFTODSA-N Met-Ala-Ala Chemical compound CSCC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(O)=O YRAWWKUTNBILNT-FXQIFTODSA-N 0.000 description 1
- VHGIWFGJIHTASW-FXQIFTODSA-N Met-Ala-Asp Chemical compound CSCC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CC(O)=O VHGIWFGJIHTASW-FXQIFTODSA-N 0.000 description 1
- QGQGAIBGTUJRBR-NAKRPEOUSA-N Met-Ala-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCSC QGQGAIBGTUJRBR-NAKRPEOUSA-N 0.000 description 1
- OBVHKUFUDCPZDW-JYJNAYRXSA-N Met-Arg-Phe Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 OBVHKUFUDCPZDW-JYJNAYRXSA-N 0.000 description 1
- NCVJJAJVWILAGI-SRVKXCTJSA-N Met-Gln-Lys Chemical compound CSCC[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CCCCN)C(=O)O)N NCVJJAJVWILAGI-SRVKXCTJSA-N 0.000 description 1
- YORIKIDJCPKBON-YUMQZZPRSA-N Met-Glu-Gly Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(O)=O YORIKIDJCPKBON-YUMQZZPRSA-N 0.000 description 1
- NHXXGBXJTLRGJI-GUBZILKMSA-N Met-Pro-Ser Chemical compound [H]N[C@@H](CCSC)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O NHXXGBXJTLRGJI-GUBZILKMSA-N 0.000 description 1
- CULGJGUDIJATIP-STQMWFEESA-N Met-Tyr-Gly Chemical compound CSCC[C@H](N)C(=O)N[C@H](C(=O)NCC(O)=O)CC1=CC=C(O)C=C1 CULGJGUDIJATIP-STQMWFEESA-N 0.000 description 1
- KZNQNBZMBZJQJO-UHFFFAOYSA-N N-glycyl-L-proline Natural products NCC(=O)N1CCCC1C(O)=O KZNQNBZMBZJQJO-UHFFFAOYSA-N 0.000 description 1
- AJHCSUXXECOXOY-UHFFFAOYSA-N N-glycyl-L-tryptophan Natural products C1=CC=C2C(CC(NC(=O)CN)C(O)=O)=CNC2=C1 AJHCSUXXECOXOY-UHFFFAOYSA-N 0.000 description 1
- NEHSHYOUIWBYSA-DCPHZVHLSA-N Phe-Ala-Trp Chemical compound C[C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)NC(=O)[C@H](CC3=CC=CC=C3)N NEHSHYOUIWBYSA-DCPHZVHLSA-N 0.000 description 1
- CGOMLCQJEMWMCE-STQMWFEESA-N Phe-Arg-Gly Chemical compound NC(N)=NCCC[C@@H](C(=O)NCC(O)=O)NC(=O)[C@@H](N)CC1=CC=CC=C1 CGOMLCQJEMWMCE-STQMWFEESA-N 0.000 description 1
- XMPUYNHKEPFERE-IHRRRGAJSA-N Phe-Asp-Arg Chemical compound NC(N)=NCCC[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CC1=CC=CC=C1 XMPUYNHKEPFERE-IHRRRGAJSA-N 0.000 description 1
- MPFGIYLYWUCSJG-AVGNSLFASA-N Phe-Glu-Asp Chemical compound OC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CC1=CC=CC=C1 MPFGIYLYWUCSJG-AVGNSLFASA-N 0.000 description 1
- CDQCFGOQNYOICK-IHRRRGAJSA-N Phe-Glu-Gln Chemical compound NC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CC1=CC=CC=C1 CDQCFGOQNYOICK-IHRRRGAJSA-N 0.000 description 1
- APJPXSFJBMMOLW-KBPBESRZSA-N Phe-Gly-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CC1=CC=CC=C1 APJPXSFJBMMOLW-KBPBESRZSA-N 0.000 description 1
- VJLLEKDQJSMHRU-STQMWFEESA-N Phe-Gly-Met Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)NCC(=O)N[C@@H](CCSC)C(O)=O VJLLEKDQJSMHRU-STQMWFEESA-N 0.000 description 1
- INHMISZWLJZQGH-ULQDDVLXSA-N Phe-Leu-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC1=CC=CC=C1 INHMISZWLJZQGH-ULQDDVLXSA-N 0.000 description 1
- MJAYDXWQQUOURZ-JYJNAYRXSA-N Phe-Lys-Gln Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(N)=O)C(O)=O MJAYDXWQQUOURZ-JYJNAYRXSA-N 0.000 description 1
- APKRGYLBSCWJJP-FXQIFTODSA-N Pro-Ala-Asp Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](C)C(=O)N[C@@H](CC(O)=O)C(O)=O APKRGYLBSCWJJP-FXQIFTODSA-N 0.000 description 1
- OBVCYFIHIIYIQF-CIUDSAMLSA-N Pro-Asn-Glu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O OBVCYFIHIIYIQF-CIUDSAMLSA-N 0.000 description 1
- KIGGUSRFHJCIEJ-DCAQKATOSA-N Pro-Asp-His Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CC2=CN=CN2)C(=O)O KIGGUSRFHJCIEJ-DCAQKATOSA-N 0.000 description 1
- LQZZPNDMYNZPFT-KKUMJFAQSA-N Pro-Gln-Phe Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O LQZZPNDMYNZPFT-KKUMJFAQSA-N 0.000 description 1
- MGDFPGCFVJFITQ-CIUDSAMLSA-N Pro-Glu-Asp Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O MGDFPGCFVJFITQ-CIUDSAMLSA-N 0.000 description 1
- UREQLMJCKFLLHM-NAKRPEOUSA-N Pro-Ile-Ser Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(O)=O UREQLMJCKFLLHM-NAKRPEOUSA-N 0.000 description 1
- MCWHYUWXVNRXFV-RWMBFGLXSA-N Pro-Leu-Pro Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@@H]2CCCN2 MCWHYUWXVNRXFV-RWMBFGLXSA-N 0.000 description 1
- GMJDSFYVTAMIBF-FXQIFTODSA-N Pro-Ser-Asp Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(O)=O GMJDSFYVTAMIBF-FXQIFTODSA-N 0.000 description 1
- AJJDPGVVNPUZCR-RHYQMDGZSA-N Pro-Thr-Lys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@@H]1CCCN1)O AJJDPGVVNPUZCR-RHYQMDGZSA-N 0.000 description 1
- FZXSYIPVAFVYBH-KKUMJFAQSA-N Pro-Tyr-Glu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCC(O)=O)C(O)=O FZXSYIPVAFVYBH-KKUMJFAQSA-N 0.000 description 1
- OQSGBXGNAFQGGS-CYDGBPFRSA-N Pro-Val-Ile Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O OQSGBXGNAFQGGS-CYDGBPFRSA-N 0.000 description 1
- ZMLRZBWCXPQADC-TUAOUCFPSA-N Pro-Val-Pro Chemical compound CC(C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@@H]2CCCN2 ZMLRZBWCXPQADC-TUAOUCFPSA-N 0.000 description 1
- 101710098085 Prohibitin-1 Proteins 0.000 description 1
- 101710098058 Prohibitin-2 Proteins 0.000 description 1
- ILRCGYURZSFMEG-RKQHYHRCSA-N Salidroside Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OCCC1=CC=C(O)C=C1 ILRCGYURZSFMEG-RKQHYHRCSA-N 0.000 description 1
- IYCBDVBJWDXQRR-FXQIFTODSA-N Ser-Ala-Met Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](CCSC)C(O)=O IYCBDVBJWDXQRR-FXQIFTODSA-N 0.000 description 1
- NRCJWSGXMAPYQX-LPEHRKFASA-N Ser-Arg-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CO)N)C(=O)O NRCJWSGXMAPYQX-LPEHRKFASA-N 0.000 description 1
- MESDJCNHLZBMEP-ZLUOBGJFSA-N Ser-Asp-Asp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O MESDJCNHLZBMEP-ZLUOBGJFSA-N 0.000 description 1
- LALNXSXEYFUUDD-GUBZILKMSA-N Ser-Glu-Leu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O LALNXSXEYFUUDD-GUBZILKMSA-N 0.000 description 1
- RJHJPZQOMKCSTP-CIUDSAMLSA-N Ser-His-Asn Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC(N)=O)C(O)=O RJHJPZQOMKCSTP-CIUDSAMLSA-N 0.000 description 1
- IFPBAGJBHSNYPR-ZKWXMUAHSA-N Ser-Ile-Gly Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(O)=O IFPBAGJBHSNYPR-ZKWXMUAHSA-N 0.000 description 1
- PPNPDKGQRFSCAC-CIUDSAMLSA-N Ser-Lys-Asp Chemical compound NCCCC[C@H](NC(=O)[C@@H](N)CO)C(=O)N[C@@H](CC(O)=O)C(O)=O PPNPDKGQRFSCAC-CIUDSAMLSA-N 0.000 description 1
- GYDFRTRSSXOZCR-ACZMJKKPSA-N Ser-Ser-Glu Chemical compound OC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCC(O)=O GYDFRTRSSXOZCR-ACZMJKKPSA-N 0.000 description 1
- BMKNXTJLHFIAAH-CIUDSAMLSA-N Ser-Ser-Leu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O BMKNXTJLHFIAAH-CIUDSAMLSA-N 0.000 description 1
- PCMZJFMUYWIERL-ZKWXMUAHSA-N Ser-Val-Asn Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O PCMZJFMUYWIERL-ZKWXMUAHSA-N 0.000 description 1
- JGUWRQWULDWNCM-FXQIFTODSA-N Ser-Val-Ser Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(O)=O JGUWRQWULDWNCM-FXQIFTODSA-N 0.000 description 1
- HSWXBJCBYSWBPT-GUBZILKMSA-N Ser-Val-Val Chemical compound CC(C)[C@H](NC(=O)[C@@H](NC(=O)[C@@H](N)CO)C(C)C)C(O)=O HSWXBJCBYSWBPT-GUBZILKMSA-N 0.000 description 1
- CAGTXGDOIFXLPC-KZVJFYERSA-N Thr-Arg-Ala Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](C)C(O)=O)CCCN=C(N)N CAGTXGDOIFXLPC-KZVJFYERSA-N 0.000 description 1
- JBHMLZSKIXMVFS-XVSYOHENSA-N Thr-Asn-Phe Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O JBHMLZSKIXMVFS-XVSYOHENSA-N 0.000 description 1
- QILPDQCTQZDHFM-HJGDQZAQSA-N Thr-Gln-Arg Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O QILPDQCTQZDHFM-HJGDQZAQSA-N 0.000 description 1
- WLDUCKSCDRIVLJ-NUMRIWBASA-N Thr-Gln-Asp Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CC(=O)O)C(=O)O)N)O WLDUCKSCDRIVLJ-NUMRIWBASA-N 0.000 description 1
- VGYBYGQXZJDZJU-XQXXSGGOSA-N Thr-Glu-Ala Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O VGYBYGQXZJDZJU-XQXXSGGOSA-N 0.000 description 1
- XPNSAQMEAVSQRD-FBCQKBJTSA-N Thr-Gly-Gly Chemical compound C[C@@H](O)[C@H](N)C(=O)NCC(=O)NCC(O)=O XPNSAQMEAVSQRD-FBCQKBJTSA-N 0.000 description 1
- RFKVQLIXNVEOMB-WEDXCCLWSA-N Thr-Leu-Gly Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)O)N)O RFKVQLIXNVEOMB-WEDXCCLWSA-N 0.000 description 1
- KZSYAEWQMJEGRZ-RHYQMDGZSA-N Thr-Leu-Val Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(O)=O KZSYAEWQMJEGRZ-RHYQMDGZSA-N 0.000 description 1
- QHUWWSQZTFLXPQ-FJXKBIBVSA-N Thr-Met-Gly Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCSC)C(=O)NCC(O)=O QHUWWSQZTFLXPQ-FJXKBIBVSA-N 0.000 description 1
- PELIQFPESHBTMA-WLTAIBSBSA-N Thr-Tyr-Gly Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@H](C(=O)NCC(O)=O)CC1=CC=C(O)C=C1 PELIQFPESHBTMA-WLTAIBSBSA-N 0.000 description 1
- NXJZCPKZIKTYLX-XEGUGMAKSA-N Trp-Glu-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)N NXJZCPKZIKTYLX-XEGUGMAKSA-N 0.000 description 1
- IKUMWSDCGQVGHC-UMPQAUOISA-N Trp-Pro-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@@H]1CCCN1C(=O)[C@H](CC2=CNC3=CC=CC=C32)N)O IKUMWSDCGQVGHC-UMPQAUOISA-N 0.000 description 1
- LGEYOIQBBIPHQN-UWJYBYFXSA-N Tyr-Ala-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 LGEYOIQBBIPHQN-UWJYBYFXSA-N 0.000 description 1
- QUILOGWWLXMSAT-IHRRRGAJSA-N Tyr-Gln-Gln Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O QUILOGWWLXMSAT-IHRRRGAJSA-N 0.000 description 1
- JAGGEZACYAAMIL-CQDKDKBSSA-N Tyr-Lys-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC1=CC=C(C=C1)O)N JAGGEZACYAAMIL-CQDKDKBSSA-N 0.000 description 1
- PMHLLBKTDHQMCY-ULQDDVLXSA-N Tyr-Lys-Val Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(O)=O PMHLLBKTDHQMCY-ULQDDVLXSA-N 0.000 description 1
- KLOZTPOXVVRVAQ-DZKIICNBSA-N Tyr-Val-Gln Chemical compound NC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 KLOZTPOXVVRVAQ-DZKIICNBSA-N 0.000 description 1
- GOPQNCQSXBJAII-ULQDDVLXSA-N Tyr-Val-Lys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)N GOPQNCQSXBJAII-ULQDDVLXSA-N 0.000 description 1
- UDNYEPLJTRDMEJ-RCOVLWMOSA-N Val-Asn-Gly Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)NCC(=O)O)N UDNYEPLJTRDMEJ-RCOVLWMOSA-N 0.000 description 1
- NWDOPHYLSORNEX-QXEWZRGKSA-N Val-Asn-Met Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CCSC)C(=O)O)N NWDOPHYLSORNEX-QXEWZRGKSA-N 0.000 description 1
- QGFPYRPIUXBYGR-YDHLFZDLSA-N Val-Asn-Phe Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)N QGFPYRPIUXBYGR-YDHLFZDLSA-N 0.000 description 1
- YCMXFKWYJFZFKS-LAEOZQHASA-N Val-Gln-Asp Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CC(=O)O)C(=O)O)N YCMXFKWYJFZFKS-LAEOZQHASA-N 0.000 description 1
- VFOHXOLPLACADK-GVXVVHGQSA-N Val-Gln-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](C(C)C)N VFOHXOLPLACADK-GVXVVHGQSA-N 0.000 description 1
- ZEVNVXYRZRIRCH-GVXVVHGQSA-N Val-Gln-Lys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CCCCN)C(=O)O)N ZEVNVXYRZRIRCH-GVXVVHGQSA-N 0.000 description 1
- XGJLNBNZNMVJRS-NRPADANISA-N Val-Glu-Ala Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O XGJLNBNZNMVJRS-NRPADANISA-N 0.000 description 1
- GBESYURLQOYWLU-LAEOZQHASA-N Val-Glu-Asp Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CC(=O)O)C(=O)O)N GBESYURLQOYWLU-LAEOZQHASA-N 0.000 description 1
- ROLGIBMFNMZANA-GVXVVHGQSA-N Val-Glu-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](C(C)C)N ROLGIBMFNMZANA-GVXVVHGQSA-N 0.000 description 1
- VHRLUTIMTDOVCG-PEDHHIEDSA-N Val-Ile-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)O)NC(=O)[C@H](C(C)C)N VHRLUTIMTDOVCG-PEDHHIEDSA-N 0.000 description 1
- HPANGHISDXDUQY-ULQDDVLXSA-N Val-Lys-Phe Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)N HPANGHISDXDUQY-ULQDDVLXSA-N 0.000 description 1
- SVFRYKBZHUGKLP-QXEWZRGKSA-N Val-Met-Asn Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(=O)N)C(=O)O)N SVFRYKBZHUGKLP-QXEWZRGKSA-N 0.000 description 1
- UJMCYJKPDFQLHX-XGEHTFHBSA-N Val-Ser-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](C(C)C)N)O UJMCYJKPDFQLHX-XGEHTFHBSA-N 0.000 description 1
- CEKSLIVSNNGOKH-KZVJFYERSA-N Val-Thr-Ala Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](C)C(=O)O)NC(=O)[C@H](C(C)C)N)O CEKSLIVSNNGOKH-KZVJFYERSA-N 0.000 description 1
- HTONZBWRYUKUKC-RCWTZXSCSA-N Val-Thr-Val Chemical compound CC(C)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(O)=O HTONZBWRYUKUKC-RCWTZXSCSA-N 0.000 description 1
- GTACFKZDQFTVAI-STECZYCISA-N Val-Tyr-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)C(C)C)CC1=CC=C(O)C=C1 GTACFKZDQFTVAI-STECZYCISA-N 0.000 description 1
- NLNCNKIVJPEFBC-DLOVCJGASA-N Val-Val-Glu Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CCC(O)=O NLNCNKIVJPEFBC-DLOVCJGASA-N 0.000 description 1
- AEFJNECXZCODJM-UWVGGRQHSA-N Val-Val-Gly Chemical compound CC(C)[C@H]([NH3+])C(=O)N[C@@H](C(C)C)C(=O)NCC([O-])=O AEFJNECXZCODJM-UWVGGRQHSA-N 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 238000007792 addition Methods 0.000 description 1
- 108010008685 alanyl-glutamyl-aspartic acid Proteins 0.000 description 1
- 108010047495 alanylglycine Proteins 0.000 description 1
- 108010011559 alanylphenylalanine Proteins 0.000 description 1
- 108010070783 alanyltyrosine Proteins 0.000 description 1
- UNNKKUDWEASWDN-UHFFFAOYSA-N alkannin Natural products CC(=CCC(O)c1cc(O)c2C(=O)C=CC(=O)c2c1O)C UNNKKUDWEASWDN-UHFFFAOYSA-N 0.000 description 1
- KOSRFJWDECSPRO-UHFFFAOYSA-N alpha-L-glutamyl-L-glutamic acid Natural products OC(=O)CCC(N)C(=O)NC(CCC(O)=O)C(O)=O KOSRFJWDECSPRO-UHFFFAOYSA-N 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 108010062796 arginyllysine Proteins 0.000 description 1
- 108010040443 aspartyl-aspartic acid Proteins 0.000 description 1
- 108010069205 aspartyl-phenylalanine Proteins 0.000 description 1
- 108010038633 aspartylglutamate Proteins 0.000 description 1
- 108010092854 aspartyllysine Proteins 0.000 description 1
- 230000002238 attenuated effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000019522 cellular metabolic process Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 229930002875 chlorophyll Natural products 0.000 description 1
- 235000019804 chlorophyll Nutrition 0.000 description 1
- ATNHDLDRLWWWCB-AENOIHSZSA-M chlorophyll a Chemical compound C1([C@@H](C(=O)OC)C(=O)C2=C3C)=C2N2C3=CC(C(CC)=C3C)=[N+]4C3=CC3=C(C=C)C(C)=C5N3[Mg-2]42[N+]2=C1[C@@H](CCC(=O)OC\C=C(/C)CCC[C@H](C)CCC[C@H](C)CCCC(C)C)[C@H](C)C2=C5 ATNHDLDRLWWWCB-AENOIHSZSA-M 0.000 description 1
- 210000003483 chromatin Anatomy 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 108010063718 gamma-glutamylaspartic acid Proteins 0.000 description 1
- 108010055341 glutamyl-glutamic acid Proteins 0.000 description 1
- XBGGUPMXALFZOT-UHFFFAOYSA-N glycyl-L-tyrosine hemihydrate Natural products NCC(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 XBGGUPMXALFZOT-UHFFFAOYSA-N 0.000 description 1
- 108010072405 glycyl-aspartyl-glycine Proteins 0.000 description 1
- 108010023364 glycyl-histidyl-arginine Proteins 0.000 description 1
- 108010038983 glycyl-histidyl-lysine Proteins 0.000 description 1
- 108010028188 glycyl-histidyl-serine Proteins 0.000 description 1
- 108010089804 glycyl-threonine Proteins 0.000 description 1
- 108010015792 glycyllysine Proteins 0.000 description 1
- 108010081551 glycylphenylalanine Proteins 0.000 description 1
- 108010077515 glycylproline Proteins 0.000 description 1
- 108010087823 glycyltyrosine Proteins 0.000 description 1
- 108010037850 glycylvaline Proteins 0.000 description 1
- 108010025306 histidylleucine Proteins 0.000 description 1
- 108010085325 histidylproline Proteins 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 108010044374 isoleucyl-tyrosine Proteins 0.000 description 1
- 238000001638 lipofection Methods 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 210000003712 lysosome Anatomy 0.000 description 1
- 230000001868 lysosomic effect Effects 0.000 description 1
- 108010003700 lysyl aspartic acid Proteins 0.000 description 1
- 108010025153 lysyl-alanyl-alanine Proteins 0.000 description 1
- 108010012988 lysyl-glutamyl-aspartyl-glycine Proteins 0.000 description 1
- 108010038320 lysylphenylalanine Proteins 0.000 description 1
- 108010017391 lysylvaline Proteins 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 210000001700 mitochondrial membrane Anatomy 0.000 description 1
- 230000021125 mitochondrion degradation Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 230000004770 neurodegeneration Effects 0.000 description 1
- 208000015122 neurodegenerative disease Diseases 0.000 description 1
- 230000010627 oxidative phosphorylation Effects 0.000 description 1
- 108010084572 phenylalanyl-valine Proteins 0.000 description 1
- 108010012581 phenylalanylglutamate Proteins 0.000 description 1
- 108010051242 phenylalanylserine Proteins 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 108010015796 prolylisoleucine Proteins 0.000 description 1
- 108010053725 prolylvaline Proteins 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 108010061238 threonyl-glycine Proteins 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 150000003628 tricarboxylic acids Chemical class 0.000 description 1
- 108700004896 tripeptide FEG Proteins 0.000 description 1
- 230000005748 tumor development Effects 0.000 description 1
- 108010051110 tyrosyl-lysine Proteins 0.000 description 1
- 108010020532 tyrosyl-proline Proteins 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 108010073969 valyllysine Proteins 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43595—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from coelenteratae, e.g. medusae
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Organic Chemistry (AREA)
- Analytical Chemistry (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Microbiology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Toxicology (AREA)
- Zoology (AREA)
- Gastroenterology & Hepatology (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Peptides Or Proteins (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明一种用于检测细胞线粒体自噬的双荧光蛋白定位检测系统，所述检测系统至少包括：（1）荧光蛋白A和指示蛋白A组成的融合蛋白；（2）荧光蛋白B和指示蛋白B组成的融合蛋白；所述荧光蛋白A和荧光蛋白B的荧光颜色不同，所述指示蛋白A用于指示自噬小体，所述指示蛋白B用于指示线粒体。本发明为观察线粒体自噬的研究提供了更加快速、便利和准确的途径。是对对线粒体自噬研究的方法学有很好的补充，为线粒体自噬研究方法学开拓了新思路。
Description
技术领域
本发明属于生物技术领域，具体涉及用于检测细胞线粒体自噬的双荧光蛋白定位检测系统及应用。
背景技术
线粒体作为细胞新陈代谢的“能量工厂”，是细胞三羧酸循环和氧化磷酸化的重要场所。线粒体功能正常与否直接关系到细胞、组织乃至机体损伤情况。所以，受损伤的线粒体必须被有效清除，以保证细胞正常生命活动的进行。而线粒体自噬(Mitophagy) 就是细胞选择性清除损伤线粒体以调节胞内线粒体数量和维持线粒体正常功能的一种动态生理平衡过程。
近年来，线粒体自噬已经成为细胞自噬新开拓的研究方向之一。大量的研究报道发现，线粒体自噬的异常可能与神经退行性疾病，糖尿病和肿瘤的发生有密切关系，也可能参与了红细胞(哺乳动物红细胞没有细胞核和线粒体)的正常发生和成熟过程。所以线粒体自噬不仅在疾病治疗和预防方面，而且在生理代谢和机体正常发育过程中都发挥着重要作用。
但是线粒体自噬的研究方法依然不够完善，目前的线粒体自噬最直观的检测方法是通过电镜观察具体的自噬小体形态或者免疫荧光实验观察自噬小体上关键蛋白Lc3b(microtubule associated protein 1light chain 3beta)是否定位在线粒体上，但是电镜检测过程繁琐且周期长，观察区域有限，免疫荧光的抗体效价要求较高，实验周期长、过程繁琐。现在市场上的线粒体染料只对活细胞染色有效果，对于含有细胞固定、通透的免疫荧光等实验效果很不理想。而且抗体和染料的荧光很容易发生猝灭，拍摄效果不佳。
开发一种新的线粒体自噬检测产品尤为重要。
发明内容
为了克服现有技术中所存在的问题，本发明的目的在于提供用于检测细胞线粒体自噬的双荧光蛋白定位检测系统及应用。
为了实现上述目的以及其他相关目的，本发明采用如下技术方案：
本发明的第一方面，提供一种用于检测细胞线粒体自噬的双荧光蛋白定位检测系统，其特征在于，所述检测系统至少包括：(1)荧光蛋白A和指示蛋白A组成的融合蛋白A；(2)荧光蛋白B和指示蛋白B组成的融合蛋白B；
所述荧光蛋白A和荧光蛋白B的荧光颜色不同，所述指示蛋白A用于指示自噬小体，所述指示蛋白B用于指示线粒体。
本发明第二方面，提供一种多核苷酸，能够编码前述的用于检测细胞线粒体自噬的双荧光蛋白定位检测系统中任一种融合蛋白。
本发明第三方面，提供一种核酸构建体，含有前述的多核苷酸。
本发明第四方面，提供一种宿主细胞，含有前述的核酸构建体或基因组中整合有外源的前述的多核苷酸。
本发明第五方面，提供前述用于检测细胞线粒体自噬的双荧光蛋白定位检测系统、前述的多核苷酸、前述的核酸构建体或前述的宿主细胞，在检测细胞线粒体自噬中的应用。
本发明第六方面，提供一种细胞线粒体自噬的检测方法，至少包括如下步骤：
(1)将前述的用于检测细胞线粒体自噬的双荧光蛋白定位检测系统在待测细胞中表达；
(2)培养步骤(1)得到的待测细胞，观察融合蛋白A和融合蛋白B的共定位情况，以判断细胞是否发生线粒体自噬和/或线粒体自噬水平。
与现有技术相比，本发明具有如下有益效果：
本发明的用于检测细胞线粒体自噬的双荧光蛋白定位检测系统及用途，有效解决了目前线粒体自噬研究手段的繁琐操作过程。本发明通过构建融合红色荧光的Mcherry-Lc3b质粒指示自噬小体，同时构建另一种间接参与线粒体自噬过程且融合绿色荧光蛋白的线粒体内膜蛋白PHB1-GFP用于指示线粒体，转染至细胞并观察两种融合荧光蛋白的共定位情况判断细胞是否发生线粒体自噬以及线粒体自噬水平，为观察线粒体自噬的研究提供了更加快速、便利和准确的途径。是对对线粒体自噬研究的方法学有很好的补充，为线粒体自噬研究方法学开拓了新思路。
附图说明
图1-1pcDNA3.1(+)-Mcherry-MCS融合表达质粒图谱。
图1-2pcDNA3.1(+)-Mcherry-MCS融合表达质粒荧光表达图。
图1-3pcDNA3.1(+)-Mcherry-MCS融合表达质粒测序序列比对。
图2-1pcDNA3.1(+)-Mcherry-Lc3b融合表达质粒图谱。
图2-2pcDNA3.1(+)-Mcherry-Lc3b融合表达质粒荧光图(图中，左图DAPI(细胞核染料) 染色显示的是细胞核，中图显示Mcherry-Lc3b的融合蛋白在细胞中的表达情况，右图显示二者叠加后的效果图)。
图2-3pcDNA3.1(+)-Mcherry-Lc3b融合表达质粒测序序列比对。
图3-1pcDNA3.1(+)-PHB1-EGFP融合表达质粒图谱。
图3-2pcDNA3.1(+)-PHB1-EGFP融合表达质粒线粒体定位荧光图(图中，这里左上图DAPI (细胞核染料)染色显示的是细胞核位置，右上图显示PHB1-GFP融合蛋白在细胞中的表达情况，右下图是Mito-Red染色(线粒体染料)显示线粒体的位置，而右下图是前三个图的叠加后的效果图(如图所示红色与绿色完全重合，显示黄色，指示发生了共定位))。图3-3pcDNA3.1(+)-Mcherry-Lc3b融合表达质粒测序序列比对。
图4-1营养条件下Hela细胞内Mcherry-Lc3b融合荧光蛋白表达(图中，这里左上图DAPI (细胞核染料)染色显示的是细胞核位置，右上图显示Mcherry-Lc3b融合蛋白在细胞中的表达情况(即细胞中自噬发生情况)，右下图是二者叠加后的荧光效果图；右下图是整体叠加图的局部放大图(可以比较清晰地看出细胞中的Lc3b颗粒数))。
图4-2饥饿条件下Hela细胞内Mcherry-Lc3b融合荧光蛋白表达(图中，这里左上图DAPI (细胞核染料)染色显示的是细胞核位置，右上图显示Mcherry-Lc3b融合蛋白在细胞中的表达情况(即细胞中自噬发生情况)，右下图是二者叠加后的荧光效果图；右下图是整体叠加图的局部放大图(可以比较清晰地看出细胞中的Lc3b稀释颗粒数))。
图5-1正常培养条件，共转Mcherry-Lc3b和PHB1-EGFP融合表达质粒的Hela细胞内荧光成像图(图中，左上图DAPI(细胞核染料)染色显示的是细胞核位置，右上图显示PHB1-GFP 融合蛋白在细胞中的定位情况(即线粒体定位)，左下图是Mcherry-Lc3b融合蛋白定位情况(即细胞中自噬发生情况)由右下图前三者叠加后的荧光效果图；最下图是整体叠加图的局部放大图)。
图5-2线粒体自噬诱导剂条件下，共转Mcherry-Lc3b和PHB1-EGFP融合表达质粒的Hela 细胞内荧光成像图。(图中，左上图DAPI(细胞核染料)染色显示的是细胞核位置，右上图显示PHB1-GFP融合蛋白在细胞中的定位情况(即线粒体定位)，左下图是Mcherry-Lc3b融合蛋白定位情况(即细胞中自噬发生情况)由右下图前三者叠加后的荧光效果图；最下图是整体叠加图的局部放大图。
具体实施方式
在进一步描述本发明具体实施方式之前，应理解，本发明的保护范围不局限于下述特定的具体实施方案；还应当理解，本发明实施例中使用的术语是为了描述特定的具体实施方案，而不是为了限制本发明的保护范围。下列实施例中未注明具体条件的试验方法，通常按照常规条件，或者按照各制造商所建议的条件。
当实施例给出数值范围时，应理解，除非本发明另有说明，每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。除非另外定义，本发明中使用的所有技术和科学术语与本技术领域技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外，根据本技术领域的技术人员对现有技术的掌握及本发明的记载，还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。
除非另外说明，本发明中所公开的实验方法、检测方法、制备方法均采用本技术领域常规的分子生物学、生物化学、染色质结构和分析、分析化学、细胞培养、重组DNA技术及相关领域的常规技术。
本发明一实施例的用于检测细胞线粒体自噬的双荧光蛋白定位检测系统，至少包括：(1) 荧光蛋白A和指示蛋白A组成的融合蛋白A；(2)荧光蛋白B和指示蛋白B组成的融合蛋白B；
所述荧光蛋白A和荧光蛋白B的荧光颜色不同，所述指示蛋白A用于指示自噬小体，所述指示蛋白B用于指示线粒体。
进一步的，所述指示蛋白A选自Lc3b蛋白。
可选的，所述指示蛋白A的氨基酸序列如SEQ ID NO：1所示；具体的：请给出 MPSEKTFKQRRTFEQRVEDVRLIREQHPTKIPVIIERYKGEKQLPVLDKTKFLVPDHVNMSELIKIIRRRLQLNANQAFFLLVNGHSMVSVSTPISEVYESEKDEDGFLYMVYASQETFGMK LSV
和/或，所述指示蛋白B选自PHB1蛋白。
可选的，所述指示蛋白B的氨基酸序列如SEQ ID NO：2所示；具体的：
MAAKVFESIGKFGLALAVAGGVVNSALYNVDAGHRAVIFDRFRGVQDIVVGEGTHFLIP WVQKPIIFDCRSRPRNVPVITGSKDLQNVNITLRILFRPVASQLPRIFTSIGEDYDERVLPSITTEILKSVVARFDAGELITQRELVSRQVSDDLTERAATFGLILDDVSLTHLTFGKEFTEAVEA KQVAQQEAERARFVVEKAEQQKKAAIISAEGDSKAAELIANSLATAGDGLIELRKLEAAEDIAYQLSRSRNITYLPAGQSVLLQLPQ。
PHB1蛋白是(prohibitin-1)间接作用于自噬蛋白Lc3b(microtubule associatedprotein 1 light chain 3beta)且定位于线粒体内膜上的保守蛋白分子，其本身需要依赖PHB2
(prohibitin-2)作用才会与LC3b(microtubule associated protein 1lightchain 3beta)发生作用，所以不会主动诱发线粒体自噬。
荧光蛋白A、荧光蛋白B可以为各种颜色，采用各种现有技术中的荧光蛋白皆可。例如， GFP、YFP、RFP、藻红蛋白(PE)、别藻蓝蛋白(APC)或紫草素叶绿素(PerCP)等。
优选的，所述荧光蛋白A、荧光蛋白B的颜色优选为互补色或对比色，便于区分。对比色即为在24色色相环中，间隔120度的颜色。互补色即为24色色相环中，间隔180度的颜色。例如橙色和蓝色，红色和绿色，黄色和紫色。
进一步的，所述荧光蛋白A、荧光蛋白B为红色荧光蛋白或绿色荧光蛋白。
优选的，所述荧光蛋白A为红色荧光蛋白，荧光蛋白B为绿色荧光蛋白。色差明显且荧光稳定。
进一步优选的，所述红色荧光蛋白选自Mcherry蛋白，所述绿色荧光蛋白选自EGFP蛋白。
具体的，所述荧光蛋白A的氨基酸序列为如SEQ ID NO：12所示：
MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPL PFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGG HYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELY K。
所述荧光蛋白B的氨基酸序列为SEQ ID NO：13所示：
MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPT LVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQL ADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELY K。
选用Mcherry融合Lc3b(microtubule associated protein 1light chain3beta)，不会因为溶酶体酸性环境而使EGFP荧光衰减，影响观察细胞内真实的自噬情况。根据两种融合荧光蛋白的共定位情况，可以直观地判断细胞是否发生线粒体自噬以及线粒体自噬水平。
本发明一实施例的多核苷酸，能够编码前述的用于检测细胞线粒体自噬的双荧光蛋白定位检测系统中任一种融合蛋白。
本发明一实施例的核酸构建体，含有前述的多核苷酸。
所述核酸构建体可以是将编码前述融合蛋白的基因片段克隆入已知载体获得。
可选的，已知载体为质粒。所述载体可以为哺乳动物细胞过表达质粒。例如，pcDNA3.1- (+)，pcDNA3.1-(-)等。
优选的，本发明实施例所示，所述载体选自pcDNA3.1(+)载体。
本发明一实施例的宿主细胞，含有前述的核酸构建体或基因组中整合有外源的如前所述的多核苷酸。
任何适用于核酸构建体进行表达的细胞都可以作为宿主细胞，例如，高等真核细胞，如哺乳动物细胞等。
前述的用于检测细胞线粒体自噬的双荧光蛋白定位检测系统、多核苷酸、核酸构建体或宿主细胞，可以应用于检测细胞线粒体自噬。
宿主细胞可以是哺乳动物细胞。具体的如CHO、COS.293细胞、Hela细胞、或Bowes黑素瘤细胞的动物细胞等。
本发明一实施例的细胞线粒体自噬的检测方法，至少包括如下步骤：
(1)将前述的用于检测细胞线粒体自噬的双荧光蛋白定位检测系统在待测细胞中表达；
(2)培养步骤(1)得到的待测细胞，观察融合蛋白A和融合蛋白B的共定位情况，以判断细胞是否发生线粒体自噬和/或线粒体自噬水平。
具体的，若融合蛋白A和融合蛋白B发生共定位，则发生线粒体自噬；若融合蛋白A和融合蛋白B未发生共定位，则未发生线粒体自噬。
在一种实施方式中，可以分别构建前述检测系统中的融合蛋白的核酸构建体，将核酸构建体导入宿主细胞中，使融合蛋白表达。
所述核酸构建体可以是将编码前述融合蛋白的基因片段克隆入已知载体获得。
可选的，已知载体为质粒。所述已知载体为能够在真核细胞中表达的载体。具体的，所述载体可以为哺乳动物细胞过表达质粒。例如，pcDNA3.1-(+)，pcDNA3.1-(-)等。
优选的，本发明实施例所示，所述载体选自pcDNA3.1(+)载体。
本领域的技术人员熟知的方法能用于构建所述核酸构建体。这些方法包括重组DNA技术、DNA合成技术等。可将编码所述融合蛋白的DNA有效连接到载体中的多克隆位点上，以指导mRNA合成进而表达蛋白，或者用于同源重组。
可以通过本领域常规的方法将所述核算构建体导入到待测细胞中。例如采用脂质体转染法)、电穿孔法或病毒感染法等。
所述待测细胞是含有线粒体的哺乳动物细胞。具体的如CHO、COS.293细胞、Hela细胞、或Bowes黑素瘤细胞的动物细胞等。
在一种实施方式中，所述方法还包括以下步骤；向所述待测细胞中加入线粒体自噬诱导剂。可以加入到培养所述宿主细胞的培养基中。
线粒体自噬诱导剂可以是Salidroside等。
所述线粒体自噬诱导剂是在转染融合蛋白质粒后加入。诱导16h后观察荧光。表达蛋白需要时间，诱导过程与蛋白表达都在发生。
实施例1
利用pcDNA3.1(+)空载质粒，设计Mcherry引物，PCR产物割胶回收，构建 pcDNA3.1(+)-Mcherry-MCS融合表达质粒，酶切鉴定、测序比对、表达鉴定。
以PcDNA3.1-Tom20-Mcherry质粒的序列为模板(去除TAA终止密码子)，选用pcDNA3.1(+) 空载质粒上的HindⅢ和BamhⅠ两个酶切位点，利用Primer 5软件设计Mcherry引物，并且在正反向引物的5’端加上相应的酶切位点序列(HindⅢ：AAGCTT，BamhⅠ：GGATCC)和相应的保护碱基序列(HindⅢ：CCC，BamhⅠ：CG)，如下：
Mcherry-HindⅢ-F：CCCAAGCTTATGGTGAGCAAGGGCGAGG；(SEQ ID NO：3)
Mcherry-BamhⅠ-R：CGGGATCCCTTGTACAGCTCGTCCATGCC；(SEQ ID NO：4)
PCR反应50μL体系后1％琼脂糖凝胶电泳回收目的片段(725bp)，经HindⅢ和BamhⅠ在37℃水浴双酶切2h后，再次1％琼脂糖凝胶电泳回收目的片段(718bp)。同样 pcDNA3.1(+)空载质粒也经HindⅢ和BamhⅠ在37℃水浴双酶切2h，片段和载体经过T4 DNA连接酶在16℃下连接过夜。将连接好的片段移至DH5α感受态大肠杆菌中，冰浴5min， 42℃水浴1min后立即冰浴5min，加入600μL空白LB培养基在37℃下摇菌1h，1200rpm 离心3min，弃上清，保留20μL吹匀后涂布到氨苄抗性的LB培养板上，37℃过夜培养14h。挑取单菌落进行菌落PCR鉴定，选取阳性克隆菌落扩大培养，抽提质粒，测序比对验证，转染Hela细胞完成表达验证。实验结果显示，pcDNA3.1(+)-Mcherry-MCS融合表达质粒转染后顺利表达红色荧光蛋白，且测序结果比对成功，无突变碱基。具体实验结果详见图1-1。图1-2，100X荧光镜检发现Mcherry红色荧光蛋白在Hela细胞中成功表达。如图1-3所示。通过pcDNA3.1-F通用引物测序，利用NCBI Blast质粒测序序列与原始设计序列信息，结果显示序列完全正确、无突变碱基。
具体的，
pcDNA3.1(+)-Mcherry-MCS融合表达质粒Mcherry序列(去除终止密码子)：
ATGGTGAGCAAGGGCGAGGAGGATAACATGGCCATCATCAAGGAGTTCATGCGCTTCA AGGTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGC GAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGGCCT ACGTGAAGCACCCCGCCGACATCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGG ACTCCTCCCTGCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCTCCG AGCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAGCAGAGGCTGAAGCTGAAGGACGGCGGCCACTACGACGCTGAGGTCAAGACCACCTACAAGGCCAAGAA GCCCGTGCAGCTGCCCGGCGCCTACAACGTCAACATCAAGTTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAACGCGCCGAGGGCCGCCACTCCACC GGCGGCATGGACGAGCTGTACAAG(SEQ ID NO：5)。
实施例2
利用pcDNA3.1(+)-Mcherry-MCS融合表达质粒，并设计LC3B全长引物，以人肺腺癌细胞株A549的cDNA为模板，PCR产物割胶回收，构建pcDNA3.1(+)-Mcherry-Lc3b融合表达质粒，酶切鉴定、测序比对验证、表达鉴定。
参考Homo sapiens microtubule associated protein 1light chain 3beta(NCBI Reference Sequence:NM_022818.5)CDS编码区序列信息，以人的肺腺癌细胞A549的cDNA为模板，选用pcDNA3.1(+)-Mcherry-MCS融合表达质粒上的EcorⅠ和XbaⅠ两个酶切位点，利用 Primer 5软件设计MAP1LC3B引物，并且保证LC3B不会发生移码突变(在正向引物5’端加上碱基T)，并且在正反向引物的5’端加上相应的酶切位点序列(EcorⅠ：GAATTC， XbaⅠ：TCTAGA)和相应的保护碱基序列(EcorⅠ：G，XbaⅠ：GC)，如下：
LC3B-Ecor1-F:GGAATTCTATGCCGTCGGAGAAGACCTTC；(SEQ ID NO：6)
LC3B-Xba1-R:GCTCTAGATTACACTGACAATTTCATCCCGAAC；(SEQ ID NO：7)
PCR反应50μL体系后1％琼脂糖凝胶电泳回收目的片段(394bp)，经EcorⅠ和Xba Ⅰ在37℃水浴双酶切2h后，再次1％琼脂糖凝胶电泳回收目的片段(389bp)。同样 pcDNA3.1(+)空载质粒也经EcorⅠ和XbaⅠ在37℃水浴双酶切2h，片段和载体经过T4 DNA 连接酶在16℃下连接过夜。将连接好的片段移至DH5α感受态大肠杆菌中，冰浴5min，42℃水浴1min后立即冰浴5min，加入600μL空白LB培养基在37℃下摇菌1h，1200rpm离心 3min，弃上清，保留20μL吹匀后涂布到氨苄抗性的LB培养板上，37℃过夜培养14h。挑取单菌落进行菌落PCR鉴定，选取阳性克隆菌落扩大培养，抽提质粒，测序验证，转染Hela 细胞，用4％多聚甲醛室温固定10min，再用1xPBS清洗2遍，再用0.2％Triton X-100通透液通透室温通透10min，再用1xPBS清洗2遍。利用5ug/mL细胞核染料DAPI (Beyotime,C1002)染色5min后弃去，再用1xPBS清洗细2遍。荧光显微镜拍摄荧光图。实验结果如图2-1、2-2和2-3所示，结果显示，pcDNA3.1(+)-Mcherry-Lc3b融合表达质粒测序结果比对成功，无突变碱基，转染后成功表达自噬蛋白颗粒。具体实验结果详见图2-1 和图2-2，图中箭头所示细胞为转染表达Mcherry-Lc3b融合荧光蛋白Hela细胞，Lc3b呈弥散状，部分细胞含有若干个小颗粒，证明了Mcherry-Lc3b融合蛋白质粒的成功构建和表达。如图2-3所示，通过pcDNA3.1-F通用引物测序，利用NCBI Blast质粒测序序列与原始设计序列信息，结果显示序列完全正确、无突变碱基。
具体的，
pcDNA3.1(+)-Mcherry-Lc3b融合表达质粒Mcherry-Lc3b序列：
ATGGTGAGCAAGGGCGAGGAGGATAACATGGCCATCATCAAGGAGTTCATGCGCTTCA AGGTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGTGG CCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGCTT CAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACT TCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAGCAGAGGCTGAAG CTGAAGGACGGCGGCCACTACGACGCTGAGGTCAAGACCACCTACAAGGCCAAGAA GCCCGTGCAGCTGCCCGGCGCCTACAACGTCAACATCAAGTTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAACGCGCCGAGGGCCGCCACTCCACC GGCGGCATGGACGAGCTGTACAAGGGATCCACTAGTCCAGTGTGGTGGAATTCTATGCCGTCGGAGAAGACCTTCAAGCAGCGCCGCACCTTCGAACAAAGAGTAGAAGATGTCC GACTTATTCGAGAGCAGCATCCAACCAAAATCCCGGTGATAATAGAACGATACAAGGGTGAGAAGCAGCTTCCTGTTCTGGATAAAACAAAGTTCCTTGTACCTGACCATGTCAAC ATGAGTGAGCTCATCAAGATAATTAGAAGGCGCTTACAGCTCAATGCTAATCAGGCCTTCTTCCTGTTGGTGAACGGACACAGCATGGTCAGCGTCTCCACACCAATCTCAGAGGTGTATGAGAGTGAGAAAGATGAAGATGGATTCCTGTACATGGTCTATGCCTCCCAGGAGA CGTTCGGGATGAAATTGTCAGTGTAA(SEQ IDNO：8)。
实施例3
利用PcNA3.1-MCS-EGFP融合表达空载质粒，设计PHB1全长引物，以人肺腺癌细胞株A549的cDNA为模板，PCR产物割胶回收，构建pcDNA3.1(+)-PHB1-EGFP融合表达质粒，酶切鉴定、测序比对、表达鉴定。
参考Homo sapiens prohibitin(PHB),transcript variant 2(NCBI ReferenceSequence: NM_002634.4)CDS编码区序列信息(去除终止密码子TGA)，以人的肺腺癌细胞A549的 cDNA为模板，选用pcDNA3.1(+)-MCS-EGFP融合表达空载质粒上的HindⅢ和EcorⅠ两个酶切位点，利用Primer 5软件设计MAP1LC3B引物，并且保证EGFP不会发生移码突变(在反向引物5’端加上碱基C)，并且在正反向引物的5’端加上相应的酶切位点序列(HindⅢ：AAGCTT，EcorⅠ：GAATTC)和相应的保护碱基序列(HindⅢ：CCC，EcorⅠ：G)，如下：
PHB1-Hind3-F：CCCAAGCTTATGGCTGCCAAAGTGTTTGAGTC；(SEQ ID NO：9)
PHB1-Ecor1-R：GGAATTCCCTGGGGCAGCTGGAGGAG；(SEQ ID NO：10)
PCR反应50μL体系后1％琼脂糖凝胶电泳回收目的片段(833bp)，经HindⅢ和EcorⅠ在37℃水浴双酶切2h后，再次1％琼脂糖凝胶电泳回收目的片段(827bp)。同样 pcDNA3.1(+)空载质粒也经HindⅢ和EcorⅠ在37℃水浴双酶切2h，片段和载体经过T4 DNA 连接酶在16℃下连接过夜。将连接好的片段移至DH5α感受态大肠杆菌中，冰浴5min，42℃水浴1min后立即冰浴5min，加入600μL空白LB培养基在37℃下摇菌1h，1200rpm离心 3min，弃上清，保留20μL吹匀后涂布到氨苄抗性的LB培养板上，37℃过夜培养14h。挑取单菌落进行菌落PCR鉴定，选取阳性克隆菌落扩大培养，抽提质粒，测序验证，转染Hela 细胞,利用100nM的Mito-Red染料(Beyotime,C1049)在37℃下孵育25min后弃去，再用 0.1％1xPBST洗涤细胞2遍。再用4％多聚甲醛室温固定10min，再用1xPBS清洗2遍，再用0.2％Triton X-100通透液通透室温通透10min，再用1xPBS清洗2遍。利用5ug/mL细胞核染料DAPI(Beyotime,C1002)染色5min后弃去，再用1xPBS清洗细2遍。荧光显微镜拍摄荧光图。实验结果如图3-1，3-2和3-3，显示，pcDNA3.1(+)-PHB1-EGFP融合表达质粒测序结果比对成功，无突变碱基；且转染后顺利表达PHB1-EGFP融合蛋白。具体实验结果详见图3-1和图3-2，箭头所示Hela细胞中PHB1-EGFP绿色融合荧光蛋白与Mito-Red 线粒体红色荧光准确发生共定位，证明了PHB1-GFP融合蛋白质粒的成功构建和表达，指示该融合蛋白可以用来标记细胞的线粒体位置和形态。图3-3所示，通过pcDNA3.1-F通用引物测序，利用NCBI Blast质粒测序序列与原始设计序列信息，结果显示序列完全正确、无突变碱基。
具体的，
pcDNA3.1(+)-PHB1-EGFP融合表达质粒PHB1-EGFP序列：
ATGGCTGCCAAAGTGTTTGAGTCCATTGGCAAGTTTGGCCTGGCCTTAGCTGTTGCAG GAGGCGTGGTGAACTCTGCCTTATATAATGTGGATGCTGGGCACAGAGCTGTCATCTTTGACCGATTCCGTGGAGTGCAGGACATTGTGGTAGGGGAAGGGACTCATTTTCTCATCC CGTGGGTACAGAAACCAATTATCTTTGACTGCCGTTCTCGACCACGTAATGTGCCAGTC ATCACTGGTAGCAAAGATTTACAGAATGTCAACATCACACTGCGCATCCTCTTCCGGCCTGTCGCCAGCCAGCTTCCTCGCATCTTCACCAGCATCGGAGAGGACTATGATGAGCGT GTGCTGCCGTCCATCACAACTGAGATCCTCAAGTCAGTGGTGGCTCGCTTTGATGCTG GAGAACTAATCACCCAGAGAGAGCTGGTCTCCAGGCAGGTGAGCGACGACCTTACAGAGCGAGCCGCCACCTTTGGGCTCATCCTGGATGACGTGTCCTTGACACATCTGACCTT CGGGAAGGAGTTCACAGAAGCGGTGGAAGCCAAACAGGTGGCTCAGCAGGAAGCAGAGAGGGCCAGATTTGTGGTGGAAAAGGCTGAGCAACAGAAAAAGGCGGCCATCATCT CTGCTGAGGGCGACTCCAAGGCAGCTGAGCTGATTGCCAACTCACTGGCCACTGCAGGGGATGGCCTGATCGAGCTGCGCAAGCTGGAAGCTGCAGAGGACATCGCGTACCAGC TCTCACGCTCTCGGAACATCACCTACCTGCCAGCGGGGCAGTCCGTGCTCCTCCAGCTGCCCCAGGGaattctgcagtcgacggtaccgcgggcccgggatccaccggtcgccaccatggtgagcaagggcgaggagctgtt caccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcg tgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcat cgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacaccccca tcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaa(SEQ ID NO：11)
实施例4
利用已构建的pcDNA3.1(+)-Mcherry-Lc3b融合表达质粒，Lipo2000转染Hela细胞，4-6h 换液，饥饿(无血清培养基)处理24h观察细胞内的荧光点数变化。
在24孔板中每孔加入2x10^4个Hela细胞，培养24h后换成OPTI-MEM培养，分别在两管50μL OPTI-MEM中单独加入pcDNA3.1(+)-Mcherry-Lc3b融合表达质粒500ng、1μLLipo2000，各自室温放置5min，再将含有质粒的MEM加入到含有lip2000的MEM中，轻轻吹匀后37℃孵育30min，每10min混匀一下。将其分为两份均匀铺到24孔板中，培养4-6h 后一孔换成10％FBS的DMEM培养基，另一孔换成无血清的DMEM培养基，37℃、5％CO2 培养24h，用4％多聚甲醛室温固定10min，再用1xPBS清洗2遍，再用0.2％Triton X-100通透液通透室温通透10min，再用1xPBS清洗2遍。利用5ug/mL细胞核染料DAPI (Beyotime,C1002)染色5min后弃去，再用1xPBS清洗细2遍。荧光显微镜拍摄荧光图。荧光显微镜观察拍照两孔细胞内的荧光颗粒数的差异。实验结果如图4-1和图4-2，结果显示，质粒转染后，经饥饿处理的HeLa细胞内的LC3b蛋白表达明显增强，红色颗粒数明显增多，该质粒可以正确指示细胞自噬情况。如图4-1所示，右下方图为左图框出局部放大图，箭头所示Hela细胞在10％FBS营养条件下，胞内Lc3b蛋白颗粒较少，基本呈弥散状均匀分布，显示了营养条件下培养细胞，细胞内的自噬水平，这里用本发明构建的Mcherry-Lc3b 融合蛋白来指示。如图4-2所示，右下方图为左图框出局部放大图，箭头所示Hela细胞在无FBS培养条件下，胞内Lc3b蛋白颗粒较多，基本呈团聚状分布，显示在饥饿条件下培养细胞，细胞内的自噬水平，这里同样用本发明构建的Mcherry-Lc3b融合蛋白来指示。但是对比营养条件下培养细胞的Mcherry-Lc3b荧光图，可以明显看出细胞的自噬的发生水平的差异(已有文献报道饥饿环境能加重自噬)，证明本发明构建的Mcherry-Lc3b融合蛋白确实可以用来观察细胞的自噬水平。
实施例5
利用已构建的pcDNA3.1(+)-PHB1-EGFP融合表达质粒，Lipo2000转染Hela细胞，4-6h 换液，24h后用Mito-Red染色指示线粒体，观察线粒体定位情况。
在24孔板中每孔加入2x10^4个Hela细胞，培养24h后换成OPTI-MEM培养，分别在两管50μL OPTI-MEM中单独加入pcDNA3.1(+)-PHB1-EGFP融合表达质粒250ng、0.5μLLipo2000，各自室温放置5min，再将含有质粒的MEM加入到含有Lipo2000的MEM中，轻轻吹匀后37℃孵育30min，每10min混匀一下。将其分为两份均匀铺到24孔板中，培养 4-6h后换成10％FBS的DMEM培养基，37℃、5％CO2培养24h。利用100nM的Mito-Red 染料(Beyotime,C1049)在37℃下孵育25min后弃去，再用0.1％1xPBST洗涤细胞2遍。再用4％多聚甲醛室温固定10min，再用1xPBS清洗2遍，再用0.2％Triton X-100通透液通透室温通透10min，再用1xPBS清洗2遍。利用5ug/mL细胞核染料DAPI(Beyotime,C1002)染色5min后弃去，再用1xPBS清洗细2遍。荧光显微镜拍摄PHB1-EGFP的定位荧光图。实验结果如图3-1，3-2和3-3，显示，pcDNA3.1(+)-PHB1-EGFP融合表达质粒在HeLa细胞中顺利表达PHB1-EGFP融合蛋白，且与线粒体发生共定位。
实施例6
利用已构建的pcDNA3.1(+)-Mcherry-Lc3b融合表达质粒和已构建的 pcDNA3.1(+)-PHB1-EGFP融合表达质粒，Lipo2000共转Hela细胞，4-6h换液，24h后观察两种荧光蛋白的共定位情况。
在24孔板中每孔加入2x10^4个Hela细胞，培养24h后换成OPTI-MEM培养，分别在一管50μL OPTI-MEM中加入pcDNA3.1(+)-PHB1-EGFP融合表达质粒125ng和 pcDNA3.1(+)-Mcherry-Lc3b融合表达质粒125ng，另一管50μL OPTI-MEM中加入0.5μ Lipo2000，各自室温放置5min，再将两种含有质粒的MEM加入到含有lip2000的MEM中混合，轻轻吹匀后37℃孵育30min，每10min混匀一下。将其分为两份均匀铺到24孔板中，培养4-6h后一孔换成10％FBS的DMEM培养基，另一孔换成无血清的DMEM培养基且加入终浓度为10μM线粒体自噬诱导剂Salidroside(MCE，HY-N0109)，37℃、5％CO2培养16h，利用5ug/mL细胞核染料DAPI(Beyotime,C1002)染色5min后弃去，再用1xPBS 清洗细2遍。荧光显微镜下观察，拍照记录细胞内的PHB1-EGFP和Mcherry-Lc3b的定位情况。实验结果如图5-1和5-2，结果显示，未加线粒体自噬诱导剂的HeLa细胞内自噬水平较低，Lc3b自噬颗粒数较少且不与线粒体发生共定位；而加入线粒体自噬诱导剂的实验组HeLa细胞自噬水平增强，细胞内的Lc3b自噬颗粒数明显增多，且与线粒体发生共定位现象。如图5-1所示，最下方图为框出局部放大图，箭头所示可见Hela细胞内Mcherry-Lc3b 红色荧光颗粒未与PHB1-EGFP绿色荧光发生共定位，提示细胞未发生线粒体自噬。如图 5-2所示，最下方图为框出局部放大图，箭头所示可见Hela细胞内Mcherry-Lc3b红色荧光颗粒与PHB1-EGFP绿色荧光发生大量共定位，提示细胞发生线粒体自噬。
以上所述，仅为本发明的较佳实施例，并非对本发明任何形式上和实质上的限制，应当指出，对于本技术领域的普通技术人员，在不脱离本发明方法的前提下，还将可以做出若干改进和补充，这些改进和补充也应视为本发明的保护范围。凡熟悉本专业的技术人员，在不脱离本发明的精神和范围的情况下，当可利用以上所揭示的技术内容而做出的些许更动、修饰与演变的等同变化，均为本发明的等效实施例；同时，凡依据本发明的实质技术对上述实施例所作的任何等同变化的更动、修饰与演变，均仍属于本发明的技术方案的范围内。
序列表
<110> 上海生物芯片有限公司
<120> 用于检测细胞线粒体自噬的双荧光蛋白定位检测系统及应用
<160> 13
<170> SIPOSequenceListing 1.0
<210> 1
<211> 125
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Met Pro Ser Glu Lys Thr Phe Lys Gln Arg Arg Thr Phe Glu Gln Arg
1 5 10 15
Val Glu Asp Val Arg Leu Ile Arg Glu Gln His Pro Thr Lys Ile Pro
20 25 30
Val Ile Ile Glu Arg Tyr Lys Gly Glu Lys Gln Leu Pro Val Leu Asp
35 40 45
Lys Thr Lys Phe Leu Val Pro Asp His Val Asn Met Ser Glu Leu Ile
50 55 60
Lys Ile Ile Arg Arg Arg Leu Gln Leu Asn Ala Asn Gln Ala Phe Phe
65 70 75 80
Leu Leu Val Asn Gly His Ser Met Val Ser Val Ser Thr Pro Ile Ser
85 90 95
Glu Val Tyr Glu Ser Glu Lys Asp Glu Asp Gly Phe Leu Tyr Met Val
100 105 110
Tyr Ala Ser Gln Glu Thr Phe Gly Met Lys Leu Ser Val
115 120 125
<210> 2
<211> 272
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Met Ala Ala Lys Val Phe Glu Ser Ile Gly Lys Phe Gly Leu Ala Leu
1 5 10 15
Ala Val Ala Gly Gly Val Val Asn Ser Ala Leu Tyr Asn Val Asp Ala
20 25 30
Gly His Arg Ala Val Ile Phe Asp Arg Phe Arg Gly Val Gln Asp Ile
35 40 45
Val Val Gly Glu Gly Thr His Phe Leu Ile Pro Trp Val Gln Lys Pro
50 55 60
Ile Ile Phe Asp Cys Arg Ser Arg Pro Arg Asn Val Pro Val Ile Thr
65 70 75 80
Gly Ser Lys Asp Leu Gln Asn Val Asn Ile Thr Leu Arg Ile Leu Phe
85 90 95
Arg Pro Val Ala Ser Gln Leu Pro Arg Ile Phe Thr Ser Ile Gly Glu
100 105 110
Asp Tyr Asp Glu Arg Val Leu Pro Ser Ile Thr Thr Glu Ile Leu Lys
115 120 125
Ser Val Val Ala Arg Phe Asp Ala Gly Glu Leu Ile Thr Gln Arg Glu
130 135 140
Leu Val Ser Arg Gln Val Ser Asp Asp Leu Thr Glu Arg Ala Ala Thr
145 150 155 160
Phe Gly Leu Ile Leu Asp Asp Val Ser Leu Thr His Leu Thr Phe Gly
165 170 175
Lys Glu Phe Thr Glu Ala Val Glu Ala Lys Gln Val Ala Gln Gln Glu
180 185 190
Ala Glu Arg Ala Arg Phe Val Val Glu Lys Ala Glu Gln Gln Lys Lys
195 200 205
Ala Ala Ile Ile Ser Ala Glu Gly Asp Ser Lys Ala Ala Glu Leu Ile
210 215 220
Ala Asn Ser Leu Ala Thr Ala Gly Asp Gly Leu Ile Glu Leu Arg Lys
225 230 235 240
Leu Glu Ala Ala Glu Asp Ile Ala Tyr Gln Leu Ser Arg Ser Arg Asn
245 250 255
Ile Thr Tyr Leu Pro Ala Gly Gln Ser Val Leu Leu Gln Leu Pro Gln
260 265 270
<210> 3
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
cccaagctta tggtgagcaa gggcgagg 28
<210> 4
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
cgggatccct tgtacagctc gtccatgcc 29
<210> 5
<211> 708
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
atggtgagca agggcgagga ggataacatg gccatcatca aggagttcat gcgcttcaag 60
gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc 120
cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc 180
ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta cgtgaagcac 240
cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa gtgggagcgc 300
gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac 360
ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta 420
atgcagaaga agaccatggg ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc 480
gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgct 540
gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc 600
aacatcaagt tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgaa 660
cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaag 708
<210> 6
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
ggaattctat gccgtcggag aagaccttc 29
<210> 7
<211> 33
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
gctctagatt acactgacaa tttcatcccg aac 33
<210> 8
<211> 1116
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
atggtgagca agggcgagga ggataacatg gccatcatca aggagttcat gcgcttcaag 60
gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc 120
cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc 180
ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta cgtgaagcac 240
cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa gtgggagcgc 300
gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac 360
ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta 420
atgcagaaga agaccatggg ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc 480
gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgct 540
gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc 600
aacatcaagt tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgaa 660
cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaaggg atccactagt 720
ccagtgtggt ggaattctat gccgtcggag aagaccttca agcagcgccg caccttcgaa 780
caaagagtag aagatgtccg acttattcga gagcagcatc caaccaaaat cccggtgata 840
atagaacgat acaagggtga gaagcagctt cctgttctgg ataaaacaaa gttccttgta 900
cctgaccatg tcaacatgag tgagctcatc aagataatta gaaggcgctt acagctcaat 960
gctaatcagg ccttcttcct gttggtgaac ggacacagca tggtcagcgt ctccacacca 1020
atctcagagg tgtatgagag tgagaaagat gaagatggat tcctgtacat ggtctatgcc 1080
tcccaggaga cgttcgggat gaaattgtca gtgtaa 1116
<210> 9
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
cccaagctta tggctgccaa agtgtttgag tc 32
<210> 10
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
ggaattccct ggggcagctg gaggag 26
<210> 11
<211> 1587
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
atggctgcca aagtgtttga gtccattggc aagtttggcc tggccttagc tgttgcagga 60
ggcgtggtga actctgcctt atataatgtg gatgctgggc acagagctgt catctttgac 120
cgattccgtg gagtgcagga cattgtggta ggggaaggga ctcattttct catcccgtgg 180
gtacagaaac caattatctt tgactgccgt tctcgaccac gtaatgtgcc agtcatcact 240
ggtagcaaag atttacagaa tgtcaacatc acactgcgca tcctcttccg gcctgtcgcc 300
agccagcttc ctcgcatctt caccagcatc ggagaggact atgatgagcg tgtgctgccg 360
tccatcacaa ctgagatcct caagtcagtg gtggctcgct ttgatgctgg agaactaatc 420
acccagagag agctggtctc caggcaggtg agcgacgacc ttacagagcg agccgccacc 480
tttgggctca tcctggatga cgtgtccttg acacatctga ccttcgggaa ggagttcaca 540
gaagcggtgg aagccaaaca ggtggctcag caggaagcag agagggccag atttgtggtg 600
gaaaaggctg agcaacagaa aaaggcggcc atcatctctg ctgagggcga ctccaaggca 660
gctgagctga ttgccaactc actggccact gcaggggatg gcctgatcga gctgcgcaag 720
ctggaagctg cagaggacat cgcgtaccag ctctcacgct ctcggaacat cacctacctg 780
ccagcggggc agtccgtgct cctccagctg ccccagggaa ttctgcagtc gacggtaccg 840
cgggcccggg atccaccggt cgccaccatg gtgagcaagg gcgaggagct gttcaccggg 900
gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc 960
ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc 1020
ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc 1080
ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa 1140
ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc 1200
gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc 1260
aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc 1320
tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac 1380
atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac 1440
ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac 1500
cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact 1560
ctcggcatgg acgagctgta caagtaa 1587
<210> 12
<211> 236
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 12
Met Val Ser Lys Gly Glu Glu Asp Asn Met Ala Ile Ile Lys Glu Phe
1 5 10 15
Met Arg Phe Lys Val His Met Glu Gly Ser Val Asn Gly His Glu Phe
20 25 30
Glu Ile Glu Gly Glu Gly Glu Gly Arg Pro Tyr Glu Gly Thr Gln Thr
35 40 45
Ala Lys Leu Lys Val Thr Lys Gly Gly Pro Leu Pro Phe Ala Trp Asp
50 55 60
Ile Leu Ser Pro Gln Phe Met Tyr Gly Ser Lys Ala Tyr Val Lys His
65 70 75 80
Pro Ala Asp Ile Pro Asp Tyr Leu Lys Leu Ser Phe Pro Glu Gly Phe
85 90 95
Lys Trp Glu Arg Val Met Asn Phe Glu Asp Gly Gly Val Val Thr Val
100 105 110
Thr Gln Asp Ser Ser Leu Gln Asp Gly Glu Phe Ile Tyr Lys Val Lys
115 120 125
Leu Arg Gly Thr Asn Phe Pro Ser Asp Gly Pro Val Met Gln Lys Lys
130 135 140
Thr Met Gly Trp Glu Ala Ser Ser Glu Arg Met Tyr Pro Glu Asp Gly
145 150 155 160
Ala Leu Lys Gly Glu Ile Lys Gln Arg Leu Lys Leu Lys Asp Gly Gly
165 170 175
His Tyr Asp Ala Glu Val Lys Thr Thr Tyr Lys Ala Lys Lys Pro Val
180 185 190
Gln Leu Pro Gly Ala Tyr Asn Val Asn Ile Lys Leu Asp Ile Thr Ser
195 200 205
His Asn Glu Asp Tyr Thr Ile Val Glu Gln Tyr Glu Arg Ala Glu Gly
210 215 220
Arg His Ser Thr Gly Gly Met Asp Glu Leu Tyr Lys
225 230 235
<210> 13
<211> 239
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 13
Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu
1 5 10 15
Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly
20 25 30
Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile
35 40 45
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
50 55 60
Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys
65 70 75 80
Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu
85 90 95
Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu
100 105 110
Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly
115 120 125
Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr
130 135 140
Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn
145 150 155 160
Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser
165 170 175
Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly
180 185 190
Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu
195 200 205
Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe
210 215 220
Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys
225 230 235
Claims (4)
1.一种用于检测细胞线粒体自噬的双荧光蛋白定位检测系统，其特征在于，所述检测系统至少包括：(1)荧光蛋白A和指示蛋白A组成的融合蛋白A；(2)荧光蛋白B和指示蛋白B组成的融合蛋白B；
所述荧光蛋白A和荧光蛋白B的荧光颜色不同，所述指示蛋白A用于指示自噬小体，所述指示蛋白B用于指示线粒体；
所述指示蛋白A选自Lc3b蛋白；所述指示蛋白B选自PHB1蛋白；所述荧光蛋白A为Mcherry蛋白；所述荧光蛋白B为EGFP蛋白；
所述融合蛋白A为Mcherry-Lc3b，其核苷酸序列如SEQ ID NO：8所示，所述融合蛋白B为PHB1-GFP，其核苷酸序列如SEQ ID NO：11所示。
2.如权利要求1所述的用于检测细胞线粒体自噬的双荧光蛋白定位检测系统在检测细胞线粒体自噬中的应用。
3.一种细胞线粒体自噬的检测方法，至少包括如下步骤：
(1)将权利要求1所述的用于检测细胞线粒体自噬的双荧光蛋白定位检测系统在待测细胞中表达；
(2)培养步骤(1)得到的待测细胞，观察融合蛋白A和融合蛋白B的共定位情况，以判断细胞是否发生线粒体自噬和/或线粒体自噬水平。
4.如权利要求3所述的细胞线粒体自噬的检测方法，还包括如下步骤：向所述待测细胞中加入线粒体自噬诱导剂。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010148310.3A CN112162096B (zh) | 2020-03-05 | 2020-03-05 | 用于检测细胞线粒体自噬的双荧光蛋白定位检测系统及应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010148310.3A CN112162096B (zh) | 2020-03-05 | 2020-03-05 | 用于检测细胞线粒体自噬的双荧光蛋白定位检测系统及应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112162096A CN112162096A (zh) | 2021-01-01 |
| CN112162096B true CN112162096B (zh) | 2024-07-02 |
Family
ID=73859281
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010148310.3A Active CN112162096B (zh) | 2020-03-05 | 2020-03-05 | 用于检测细胞线粒体自噬的双荧光蛋白定位检测系统及应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112162096B (zh) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113444160A (zh) * | 2021-04-25 | 2021-09-28 | 东北农业大学 | ZmNuC基因及其编码蛋白在抗玉米粗缩病中的应用 |
| CN113234719B (zh) * | 2021-06-08 | 2022-04-29 | 重庆医科大学 | 检测线粒体膜电势的可遗传荧光探针及其应用 |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101863983B (zh) * | 2010-05-25 | 2013-01-23 | 北京大学 | 基于绿色荧光蛋白和红色荧光蛋白的双荧光共定位系统 |
| CN103060421B (zh) * | 2013-01-22 | 2014-06-18 | 上海市内分泌代谢病研究所 | 一种脂肪细胞中自噬的监测方法 |
| CN109576227B (zh) * | 2018-12-27 | 2022-06-10 | 江苏凯基生物技术股份有限公司 | 一种基于荧光素酶报告病毒的自噬细胞系构建方法 |
-
2020
- 2020-03-05 CN CN202010148310.3A patent/CN112162096B/zh active Active
Non-Patent Citations (5)
| Title |
|---|
| microtubule-associated proteins 1A/1B light chain 3B [Macaca mulatta];NCBI;《Genebank》;NP_001180554.1 * |
| Nick J Dolman 等.Tools and techniques to measure mitophagy using fluorescence microscopy.《Autophagy》.2013,第9卷(第11期),第1654-1656页，图2. * |
| prohibitin 1 [Mus musculus];NCBI;《Genebank》;NP_032857.1 * |
| Tools and techniques to measure mitophagy using fluorescence microscopy;Nick J Dolman 等;《Autophagy》;第9卷(第11期);第1654-1656页，图2 * |
| 抗增值蛋白与线粒体自噬的研究进展;胡龙龙 等;《生命的化学》;第37卷(第6期);第1030-1032页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112162096A (zh) | 2021-01-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Kraut-Cohen et al. | Translation-and SRP-independent mRNA targeting to the endoplasmic reticulum in the yeast Saccharomyces cerevisiae | |
| CN103173447B (zh) | 新的β-肌动蛋白和RPS21 启动子及其应用 | |
| CN112162096B (zh) | 用于检测细胞线粒体自噬的双荧光蛋白定位检测系统及应用 | |
| JPH0434559B2 (zh) | ||
| CN104991072B (zh) | 一种昆虫体外蛋白质相互作用检测系统的制备方法及应用 | |
| WO2024012300A1 (zh) | 一种基因编辑的方法和用途 | |
| US20240279630A1 (en) | Isolated cas13 protein and use thereof | |
| Hofhaus et al. | Highly divergent amino termini of the homologous human ALR and yeast scERV1 gene products define species specific differences in cellular localization | |
| CN104004098A (zh) | 一种利用BiFC指示细胞中Wnt信号活性状态的载体组合物及应用 | |
| Kurahashi et al. | Tropomyosin is required for the cell fusion process during conjugation in fission yeast | |
| CN116239703A (zh) | 一种融合蛋白及含有其的高效特异碱基编辑系统和应用 | |
| CN104254603A (zh) | 用于制备xa因子抑制剂的重组解毒剂的方法 | |
| CN110964744A (zh) | 一种能稳定表达Cas9蛋白的人骨肉瘤U-2OS工具细胞株及其制备方法与应用 | |
| CN114075572A (zh) | 一种与门基因电路及获取该与门基因电路的方法 | |
| CN112813049B (zh) | 用于活细胞rna标记的融合蛋白及应用 | |
| WO2024251229A1 (zh) | Cas酶及其系统和应用 | |
| CN113186192B (zh) | Crybb2基因突变体、多肽、试剂盒、构建体及重组细胞 | |
| WO2024041653A1 (zh) | 一种CRISPR-Cas13系统及其应用 | |
| CN109797170A (zh) | 一种检测靶向基因敲除效果的方法 | |
| KR102074590B1 (ko) | 자가포식체 탐침용 프로브 및 이를 이용한 탐침 방법 | |
| CN116218918A (zh) | 一种Jurkat效应细胞及其构建方法和用途 | |
| CN114606208B (zh) | 一种具有高可信度快速的临近蛋白标记系统ScPhastID及标记方法 | |
| CN109504707A (zh) | 基于mitoTALENs的iPSCs线粒体DNA突变位点的修复方法 | |
| CN116855503B (zh) | 过表达mrgprx2的稳转细胞株及其构建方法和应用 | |
| CN118726266A (zh) | 一种braf v600e点突变的肺上皮细胞及其构建方法 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| TG01 | Patent term adjustment | ||
| TG01 | Patent term adjustment |