US20210208141A1 - Cysteine-reactive ligand discovery in proteomes - Google Patents
Cysteine-reactive ligand discovery in proteomes Download PDFInfo
- Publication number
- US20210208141A1 US20210208141A1 US17/021,260 US202017021260A US2021208141A1 US 20210208141 A1 US20210208141 A1 US 20210208141A1 US 202017021260 A US202017021260 A US 202017021260A US 2021208141 A1 US2021208141 A1 US 2021208141A1
- Authority
- US
- United States
- Prior art keywords
- proteins
- protein
- zak
- hne
- electrophile
- 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.)
- Abandoned
Links
- 108010026552 Proteome Proteins 0.000 title abstract description 65
- 239000003446 ligand Substances 0.000 title description 6
- JVJFIQYAHPMBBX-UHFFFAOYSA-N 4-hydroxynonenal Chemical compound CCCCCC(O)C=CC=O JVJFIQYAHPMBBX-UHFFFAOYSA-N 0.000 claims abstract description 201
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 104
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 104
- 235000018102 proteins Nutrition 0.000 claims abstract description 100
- 102100033116 Mitogen-activated protein kinase kinase kinase 20 Human genes 0.000 claims abstract description 79
- 101001018157 Homo sapiens Mitogen-activated protein kinase kinase kinase 20 Proteins 0.000 claims abstract description 78
- 235000018417 cysteine Nutrition 0.000 claims abstract description 77
- 239000012039 electrophile Substances 0.000 claims abstract description 59
- 238000006243 chemical reaction Methods 0.000 claims abstract description 36
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 claims abstract description 26
- 230000004048 modification Effects 0.000 claims abstract description 25
- 238000012986 modification Methods 0.000 claims abstract description 25
- 230000009257 reactivity Effects 0.000 claims abstract description 24
- 150000002632 lipids Chemical class 0.000 claims abstract description 20
- 238000000034 method Methods 0.000 claims abstract description 18
- 125000000151 cysteine group Chemical class N[C@@H](CS)C(=O)* 0.000 claims abstract description 13
- 239000000523 sample Substances 0.000 claims description 53
- 108091000080 Phosphotransferase Proteins 0.000 claims description 29
- 102000020233 phosphotransferase Human genes 0.000 claims description 29
- 230000000155 isotopic effect Effects 0.000 claims description 27
- 239000003550 marker Substances 0.000 claims description 24
- -1 azido compound Chemical class 0.000 claims description 8
- PGLTVOMIXTUURA-UHFFFAOYSA-N iodoacetamide Chemical compound NC(=O)CI PGLTVOMIXTUURA-UHFFFAOYSA-N 0.000 claims description 8
- BUVSBIKCBLHNCG-UFLZEWODSA-N 5-[(3as,4s,6ar)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoic acid;azide Chemical compound [N-]=[N+]=[N-].N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 BUVSBIKCBLHNCG-UFLZEWODSA-N 0.000 claims description 5
- 210000004962 mammalian cell Anatomy 0.000 claims description 5
- 238000010461 azide-alkyne cycloaddition reaction Methods 0.000 claims description 4
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 claims description 3
- 238000007259 addition reaction Methods 0.000 claims 1
- 230000002860 competitive effect Effects 0.000 abstract description 30
- 230000004913 activation Effects 0.000 abstract description 26
- 230000037361 pathway Effects 0.000 abstract description 20
- 241000282414 Homo sapiens Species 0.000 abstract description 13
- 230000036542 oxidative stress Effects 0.000 abstract description 10
- 150000001875 compounds Chemical class 0.000 abstract description 9
- 102000004190 Enzymes Human genes 0.000 abstract description 7
- 108090000790 Enzymes Proteins 0.000 abstract description 7
- 230000006870 function Effects 0.000 abstract description 7
- 230000005764 inhibitory process Effects 0.000 abstract description 5
- 238000013459 approach Methods 0.000 abstract description 2
- 230000008713 feedback mechanism Effects 0.000 abstract 1
- 238000004445 quantitative analysis Methods 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 84
- 150000001945 cysteines Chemical class 0.000 description 51
- 108010055717 JNK Mitogen-Activated Protein Kinases Proteins 0.000 description 36
- 102000019145 JUN kinase activity proteins Human genes 0.000 description 36
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 33
- 238000002372 labelling Methods 0.000 description 30
- 238000002474 experimental method Methods 0.000 description 22
- 230000000694 effects Effects 0.000 description 21
- VHRUMKCAEVRUBK-GODQJPCRSA-N 15-deoxy-Delta(12,14)-prostaglandin J2 Chemical compound CCCCC\C=C\C=C1/[C@@H](C\C=C/CCCC(O)=O)C=CC1=O VHRUMKCAEVRUBK-GODQJPCRSA-N 0.000 description 17
- YCRUVTMZPHEOAM-UHFFFAOYSA-N n-hex-5-ynyl-2-iodoacetamide Chemical compound ICC(=O)NCCCCC#C YCRUVTMZPHEOAM-UHFFFAOYSA-N 0.000 description 17
- 230000035945 sensitivity Effects 0.000 description 13
- 108090000765 processed proteins & peptides Proteins 0.000 description 12
- 102000043136 MAP kinase family Human genes 0.000 description 10
- 108091054455 MAP kinase family Proteins 0.000 description 10
- 230000001419 dependent effect Effects 0.000 description 10
- 238000000338 in vitro Methods 0.000 description 10
- 102000004196 processed proteins & peptides Human genes 0.000 description 10
- 206010028980 Neoplasm Diseases 0.000 description 9
- 230000019491 signal transduction Effects 0.000 description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 8
- 238000003556 assay Methods 0.000 description 8
- 201000011510 cancer Diseases 0.000 description 8
- 238000011161 development Methods 0.000 description 8
- 238000011065 in-situ storage Methods 0.000 description 8
- 239000002609 medium Substances 0.000 description 8
- 239000000758 substrate Substances 0.000 description 8
- 238000004458 analytical method Methods 0.000 description 7
- 230000036515 potency Effects 0.000 description 7
- 206010061218 Inflammation Diseases 0.000 description 6
- 108010090804 Streptavidin Proteins 0.000 description 6
- 241000723792 Tobacco etch virus Species 0.000 description 6
- 238000012512 characterization method Methods 0.000 description 6
- 230000004054 inflammatory process Effects 0.000 description 6
- 239000003112 inhibitor Substances 0.000 description 6
- 230000003993 interaction Effects 0.000 description 6
- 239000008188 pellet Substances 0.000 description 6
- 230000004481 post-translational protein modification Effects 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 101710137189 Amyloid-beta A4 protein Proteins 0.000 description 5
- 102100022704 Amyloid-beta precursor protein Human genes 0.000 description 5
- 101710151993 Amyloid-beta precursor protein Proteins 0.000 description 5
- CIUUIPMOFZIWIZ-UHFFFAOYSA-N Bropirimine Chemical compound NC1=NC(O)=C(Br)C(C=2C=CC=CC=2)=N1 CIUUIPMOFZIWIZ-UHFFFAOYSA-N 0.000 description 5
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 5
- 101150115146 EEF2 gene Proteins 0.000 description 5
- 102100031334 Elongation factor 2 Human genes 0.000 description 5
- 108010007457 Extracellular Signal-Regulated MAP Kinases Proteins 0.000 description 5
- 101000727472 Homo sapiens Reticulon-4 Proteins 0.000 description 5
- 102000001291 MAP Kinase Kinase Kinase Human genes 0.000 description 5
- 102100024193 Mitogen-activated protein kinase 1 Human genes 0.000 description 5
- 108030005453 Mitogen-activated protein kinase kinase kinases Proteins 0.000 description 5
- 102100029831 Reticulon-4 Human genes 0.000 description 5
- 239000011324 bead Substances 0.000 description 5
- 239000000872 buffer Substances 0.000 description 5
- 239000006166 lysate Substances 0.000 description 5
- 239000012528 membrane Substances 0.000 description 5
- 102000002574 p38 Mitogen-Activated Protein Kinases Human genes 0.000 description 5
- 108010068338 p38 Mitogen-Activated Protein Kinases Proteins 0.000 description 5
- 238000011002 quantification Methods 0.000 description 5
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 5
- KISWVXRQTGLFGD-UHFFFAOYSA-N 2-[[2-[[6-amino-2-[[2-[[2-[[5-amino-2-[[2-[[1-[2-[[6-amino-2-[(2,5-diamino-5-oxopentanoyl)amino]hexanoyl]amino]-5-(diaminomethylideneamino)pentanoyl]pyrrolidine-2-carbonyl]amino]-3-hydroxypropanoyl]amino]-5-oxopentanoyl]amino]-5-(diaminomethylideneamino)p Chemical compound C1CCN(C(=O)C(CCCN=C(N)N)NC(=O)C(CCCCN)NC(=O)C(N)CCC(N)=O)C1C(=O)NC(CO)C(=O)NC(CCC(N)=O)C(=O)NC(CCCN=C(N)N)C(=O)NC(CO)C(=O)NC(CCCCN)C(=O)NC(C(=O)NC(CC(C)C)C(O)=O)CC1=CC=C(O)C=C1 KISWVXRQTGLFGD-UHFFFAOYSA-N 0.000 description 4
- 101001026998 Homo sapiens Ketosamine-3-kinase Proteins 0.000 description 4
- 102100037378 Ketosamine-3-kinase Human genes 0.000 description 4
- 102000004142 Trypsin Human genes 0.000 description 4
- 108090000631 Trypsin Proteins 0.000 description 4
- 230000008901 benefit Effects 0.000 description 4
- 230000001413 cellular effect Effects 0.000 description 4
- 239000013068 control sample Substances 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 230000009467 reduction Effects 0.000 description 4
- 230000002829 reductive effect Effects 0.000 description 4
- 230000004044 response Effects 0.000 description 4
- 102200061079 rs776498025 Human genes 0.000 description 4
- 238000000527 sonication Methods 0.000 description 4
- 230000035882 stress Effects 0.000 description 4
- 239000012588 trypsin Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 238000001262 western blot Methods 0.000 description 4
- 206010020751 Hypersensitivity Diseases 0.000 description 3
- 239000004472 Lysine Substances 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 102220613017 Mitogen-activated protein kinase kinase kinase 20_K45M_mutation Human genes 0.000 description 3
- 102000001253 Protein Kinase Human genes 0.000 description 3
- 238000010847 SEQUEST Methods 0.000 description 3
- 229940024606 amino acid Drugs 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 3
- 230000003197 catalytic effect Effects 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 238000006352 cycloaddition reaction Methods 0.000 description 3
- 238000009826 distribution Methods 0.000 description 3
- 239000012091 fetal bovine serum Substances 0.000 description 3
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 3
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 3
- 239000012160 loading buffer Substances 0.000 description 3
- 229960003646 lysine Drugs 0.000 description 3
- 230000000269 nucleophilic effect Effects 0.000 description 3
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 3
- 235000020777 polyunsaturated fatty acids Nutrition 0.000 description 3
- 108060006633 protein kinase Proteins 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- XZWYTXMRWQJBGX-VXBMVYAYSA-N FLAG peptide Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](NC(=O)[C@@H](N)CC(O)=O)CC1=CC=C(O)C=C1 XZWYTXMRWQJBGX-VXBMVYAYSA-N 0.000 description 2
- 108010020195 FLAG peptide Proteins 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 101000977270 Homo sapiens MMS19 nucleotide excision repair protein homolog Proteins 0.000 description 2
- 101000950669 Homo sapiens Mitogen-activated protein kinase 9 Proteins 0.000 description 2
- 101001055085 Homo sapiens Mitogen-activated protein kinase kinase kinase 9 Proteins 0.000 description 2
- 101001106090 Homo sapiens Receptor expression-enhancing protein 5 Proteins 0.000 description 2
- BVHLGVCQOALMSV-JEDNCBNOSA-N L-lysine hydrochloride Chemical compound Cl.NCCCC[C@H](N)C(O)=O BVHLGVCQOALMSV-JEDNCBNOSA-N 0.000 description 2
- 238000013051 Liquid chromatography–high-resolution mass spectrometry Methods 0.000 description 2
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 2
- 108010066373 MLK-like mitogen-activated protein triple kinase Proteins 0.000 description 2
- 102100023474 MMS19 nucleotide excision repair protein homolog Human genes 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 108090000744 Mitogen-Activated Protein Kinase Kinases Proteins 0.000 description 2
- 102000004232 Mitogen-Activated Protein Kinase Kinases Human genes 0.000 description 2
- 102100037809 Mitogen-activated protein kinase 9 Human genes 0.000 description 2
- 102000047918 Myelin Basic Human genes 0.000 description 2
- 101710107068 Myelin basic protein Proteins 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 102100021077 Receptor expression-enhancing protein 5 Human genes 0.000 description 2
- 238000000692 Student's t-test Methods 0.000 description 2
- 239000006180 TBST buffer Substances 0.000 description 2
- PZBFGYYEXUXCOF-UHFFFAOYSA-N TCEP Chemical compound OC(=O)CCP(CCC(O)=O)CCC(O)=O PZBFGYYEXUXCOF-UHFFFAOYSA-N 0.000 description 2
- 108010076818 TEV protease Proteins 0.000 description 2
- DKGAVHZHDRPRBM-UHFFFAOYSA-N Tert-Butanol Chemical compound CC(C)(C)O DKGAVHZHDRPRBM-UHFFFAOYSA-N 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 235000001014 amino acid Nutrition 0.000 description 2
- 230000006907 apoptotic process Effects 0.000 description 2
- YZXBAPSDXZZRGB-DOFZRALJSA-N arachidonic acid Chemical compound CCCCC\C=C/C\C=C/C\C=C/C\C=C/CCCC(O)=O YZXBAPSDXZZRGB-DOFZRALJSA-N 0.000 description 2
- 230000003915 cell function Effects 0.000 description 2
- 238000007385 chemical modification Methods 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 230000021615 conjugation Effects 0.000 description 2
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 2
- 229910000366 copper(II) sulfate Inorganic materials 0.000 description 2
- 239000010432 diamond Substances 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 229940042399 direct acting antivirals protease inhibitors Drugs 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 238000013467 fragmentation Methods 0.000 description 2
- 238000006062 fragmentation reaction Methods 0.000 description 2
- 230000014509 gene expression Effects 0.000 description 2
- 238000003384 imaging method Methods 0.000 description 2
- 238000001114 immunoprecipitation Methods 0.000 description 2
- 238000003674 kinase activity assay Methods 0.000 description 2
- 238000013507 mapping Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000002207 metabolite Substances 0.000 description 2
- 230000002018 overexpression Effects 0.000 description 2
- 230000003647 oxidation Effects 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- 230000001590 oxidative effect Effects 0.000 description 2
- 238000002203 pretreatment Methods 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- 230000001177 retroviral effect Effects 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 239000004017 serum-free culture medium Substances 0.000 description 2
- 230000010473 stable expression Effects 0.000 description 2
- 238000003153 stable transfection Methods 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 238000012353 t test Methods 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- 239000013598 vector Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 239000002676 xenobiotic agent Substances 0.000 description 2
- JVJFIQYAHPMBBX-FNORWQNLSA-N (E)-4-hydroxynon-2-enal Chemical compound CCCCCC(O)\C=C\C=O JVJFIQYAHPMBBX-FNORWQNLSA-N 0.000 description 1
- WKGZJBVXZWCZQC-UHFFFAOYSA-N 1-(1-benzyltriazol-4-yl)-n,n-bis[(1-benzyltriazol-4-yl)methyl]methanamine Chemical compound C=1N(CC=2C=CC=CC=2)N=NC=1CN(CC=1N=NN(CC=2C=CC=CC=2)C=1)CC(N=N1)=CN1CC1=CC=CC=C1 WKGZJBVXZWCZQC-UHFFFAOYSA-N 0.000 description 1
- YMZPQKXPKZZSFV-CPWYAANMSA-N 2-[3-[(1r)-1-[(2s)-1-[(2s)-2-[(1r)-cyclohex-2-en-1-yl]-2-(3,4,5-trimethoxyphenyl)acetyl]piperidine-2-carbonyl]oxy-3-(3,4-dimethoxyphenyl)propyl]phenoxy]acetic acid Chemical compound C1=C(OC)C(OC)=CC=C1CC[C@H](C=1C=C(OCC(O)=O)C=CC=1)OC(=O)[C@H]1N(C(=O)[C@@H]([C@H]2C=CCCC2)C=2C=C(OC)C(OC)=C(OC)C=2)CCCC1 YMZPQKXPKZZSFV-CPWYAANMSA-N 0.000 description 1
- SNHKWJJUUBOBAX-UHFFFAOYSA-N 3-oxo-4,5,6,7-tetrahydro-[1,2]oxazolo[4,5-c]pyridine-6-carboxylic acid Chemical compound C1NC(C(=O)O)CC2=C1C(O)=NO2 SNHKWJJUUBOBAX-UHFFFAOYSA-N 0.000 description 1
- YQGYQWLJYKBMRM-WBGPXRNDSA-N 5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]-N-(1-hydrazinyl-1-oxohexan-2-yl)pentanamide Chemical compound C(CCCC[C@@H]1SC[C@@H]2NC(=O)N[C@H]12)(=O)NC(C(=O)NN)CCCC YQGYQWLJYKBMRM-WBGPXRNDSA-N 0.000 description 1
- 102100021308 60S ribosomal protein L23 Human genes 0.000 description 1
- 102100035322 60S ribosomal protein L24 Human genes 0.000 description 1
- 102100028324 ADP-ribose glycohydrolase MACROD1 Human genes 0.000 description 1
- 101100433757 Arabidopsis thaliana ABCG32 gene Proteins 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- KWTQSFXGGICVPE-WCCKRBBISA-N Arginine hydrochloride Chemical compound Cl.OC(=O)[C@@H](N)CCCN=C(N)N KWTQSFXGGICVPE-WCCKRBBISA-N 0.000 description 1
- 239000005552 B01AC04 - Clopidogrel Substances 0.000 description 1
- 101001042041 Bos taurus Isocitrate dehydrogenase [NAD] subunit beta, mitochondrial Proteins 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 102100032405 Chromatin accessibility complex protein 1 Human genes 0.000 description 1
- 108020004635 Complementary DNA Proteins 0.000 description 1
- 102100028629 Cytoskeleton-associated protein 4 Human genes 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 102100023401 Dual specificity mitogen-activated protein kinase kinase 6 Human genes 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 description 1
- 108091006010 FLAG-tagged proteins Proteins 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 229920000209 Hexadimethrine bromide Polymers 0.000 description 1
- 101000675833 Homo sapiens 60S ribosomal protein L23 Proteins 0.000 description 1
- 101000660926 Homo sapiens 60S ribosomal protein L24 Proteins 0.000 description 1
- 101000578912 Homo sapiens ADP-ribose glycohydrolase MACROD1 Proteins 0.000 description 1
- 101000943248 Homo sapiens Chromatin accessibility complex protein 1 Proteins 0.000 description 1
- 101000766853 Homo sapiens Cytoskeleton-associated protein 4 Proteins 0.000 description 1
- 101000624426 Homo sapiens Dual specificity mitogen-activated protein kinase kinase 6 Proteins 0.000 description 1
- 101001082060 Homo sapiens Interferon-induced protein with tetratricopeptide repeats 3 Proteins 0.000 description 1
- 101000960234 Homo sapiens Isocitrate dehydrogenase [NADP] cytoplasmic Proteins 0.000 description 1
- 101001024703 Homo sapiens Nck-associated protein 5 Proteins 0.000 description 1
- 101001073417 Homo sapiens Peflin Proteins 0.000 description 1
- 101000711369 Homo sapiens Probable ribosome biogenesis protein RLP24 Proteins 0.000 description 1
- 101001136986 Homo sapiens Proteasome subunit beta type-8 Proteins 0.000 description 1
- 101000710817 Homo sapiens Protein canopy homolog 3 Proteins 0.000 description 1
- 101000779418 Homo sapiens RAC-alpha serine/threonine-protein kinase Proteins 0.000 description 1
- 101000798015 Homo sapiens RAC-beta serine/threonine-protein kinase Proteins 0.000 description 1
- 101000835696 Homo sapiens T-complex protein 1 subunit theta Proteins 0.000 description 1
- 101000954800 Homo sapiens WD repeat domain phosphoinositide-interacting protein 3 Proteins 0.000 description 1
- 206010021143 Hypoxia Diseases 0.000 description 1
- 102100027302 Interferon-induced protein with tetratricopeptide repeats 3 Human genes 0.000 description 1
- 102100039905 Isocitrate dehydrogenase [NADP] cytoplasmic Human genes 0.000 description 1
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 1
- 239000007993 MOPS buffer Substances 0.000 description 1
- 238000006845 Michael addition reaction Methods 0.000 description 1
- 102100026909 Mitogen-activated protein kinase kinase kinase 9 Human genes 0.000 description 1
- 102100036946 Nck-associated protein 5 Human genes 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 102100035845 Peflin Human genes 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 1
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 1
- 101710093543 Probable non-specific lipid-transfer protein Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 1
- 102100035760 Proteasome subunit beta type-8 Human genes 0.000 description 1
- 102100033856 Protein canopy homolog 3 Human genes 0.000 description 1
- 102100033810 RAC-alpha serine/threonine-protein kinase Human genes 0.000 description 1
- 102100032315 RAC-beta serine/threonine-protein kinase Human genes 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 101000761953 Schizosaccharomyces pombe (strain 972 / ATCC 24843) Protein kinase byr2 Proteins 0.000 description 1
- 229940124639 Selective inhibitor Drugs 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- 102100026311 T-complex protein 1 subunit theta Human genes 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 102100037049 WD repeat domain phosphoinositide-interacting protein 3 Human genes 0.000 description 1
- PPZYBFUYKJPWBY-UHFFFAOYSA-N acetylene azide Chemical compound C#C.[N-]=[N+]=[N-] PPZYBFUYKJPWBY-UHFFFAOYSA-N 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- ULXXDDBFHOBEHA-CWDCEQMOSA-N afatinib Chemical compound N1=CN=C2C=C(O[C@@H]3COCC3)C(NC(=O)/C=C/CN(C)C)=CC2=C1NC1=CC=C(F)C(Cl)=C1 ULXXDDBFHOBEHA-CWDCEQMOSA-N 0.000 description 1
- 229960001686 afatinib Drugs 0.000 description 1
- 230000029936 alkylation Effects 0.000 description 1
- 238000005804 alkylation reaction Methods 0.000 description 1
- 208000026935 allergic disease Diseases 0.000 description 1
- 102000012005 alpha-2-HS-Glycoprotein Human genes 0.000 description 1
- 108010075843 alpha-2-HS-Glycoprotein Proteins 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 229940114079 arachidonic acid Drugs 0.000 description 1
- 235000021342 arachidonic acid Nutrition 0.000 description 1
- 229960003121 arginine Drugs 0.000 description 1
- 229960003589 arginine hydrochloride Drugs 0.000 description 1
- 239000012131 assay buffer Substances 0.000 description 1
- DHCLVCXQIBBOPH-UHFFFAOYSA-N beta-glycerol phosphate Natural products OCC(CO)OP(O)(O)=O DHCLVCXQIBBOPH-UHFFFAOYSA-N 0.000 description 1
- GHRQXJHBXKYCLZ-UHFFFAOYSA-L beta-glycerolphosphate Chemical compound [Na+].[Na+].CC(CO)OOP([O-])([O-])=O GHRQXJHBXKYCLZ-UHFFFAOYSA-L 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 230000007248 cellular mechanism Effects 0.000 description 1
- 230000005754 cellular signaling Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 208000037976 chronic inflammation Diseases 0.000 description 1
- 230000006020 chronic inflammation Effects 0.000 description 1
- GKTWGGQPFAXNFI-HNNXBMFYSA-N clopidogrel Chemical compound C1([C@H](N2CC=3C=CSC=3CC2)C(=O)OC)=CC=CC=C1Cl GKTWGGQPFAXNFI-HNNXBMFYSA-N 0.000 description 1
- 229960003009 clopidogrel Drugs 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 230000001120 cytoprotective effect Effects 0.000 description 1
- 229940127089 cytotoxic agent Drugs 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000024531 detection of redox state Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 229940000406 drug candidate Drugs 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- DEFVIWRASFVYLL-UHFFFAOYSA-N ethylene glycol bis(2-aminoethyl)tetraacetic acid Chemical compound OC(=O)CN(CC(O)=O)CCOCCOCCN(CC(O)=O)CC(O)=O DEFVIWRASFVYLL-UHFFFAOYSA-N 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000007429 general method Methods 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- 230000036433 growing body Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 102000048600 human MAP3K20 Human genes 0.000 description 1
- 102000043337 human MAP3K9 Human genes 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 230000009610 hypersensitivity Effects 0.000 description 1
- 230000007954 hypoxia Effects 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000001948 isotopic labelling Methods 0.000 description 1
- 238000000021 kinase assay Methods 0.000 description 1
- 229940043355 kinase inhibitor Drugs 0.000 description 1
- 238000001499 laser induced fluorescence spectroscopy Methods 0.000 description 1
- 230000029226 lipidation Effects 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 238000007069 methylation reaction Methods 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 230000006667 mitochondrial pathway Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000002887 multiple sequence alignment Methods 0.000 description 1
- 230000004770 neurodegeneration Effects 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- SBQLYHNEIUGQKH-UHFFFAOYSA-N omeprazole Chemical compound N1=C2[CH]C(OC)=CC=C2N=C1S(=O)CC1=NC=C(C)C(OC)=C1C SBQLYHNEIUGQKH-UHFFFAOYSA-N 0.000 description 1
- 229960000381 omeprazole Drugs 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 239000007800 oxidant agent Substances 0.000 description 1
- 230000004792 oxidative damage Effects 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000001991 pathophysiological effect Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 238000005502 peroxidation Methods 0.000 description 1
- 230000002085 persistent effect Effects 0.000 description 1
- 229940080469 phosphocellulose Drugs 0.000 description 1
- 239000003757 phosphotransferase inhibitor Substances 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- UQOQENZZLBSFKO-POPPZSFYSA-N prostaglandin J2 Chemical compound CCCCC[C@H](O)\C=C\[C@@H]1[C@@H](C\C=C/CCCC(O)=O)C=CC1=O UQOQENZZLBSFKO-POPPZSFYSA-N 0.000 description 1
- 230000019639 protein methylation Effects 0.000 description 1
- 230000009145 protein modification Effects 0.000 description 1
- 230000009822 protein phosphorylation Effects 0.000 description 1
- 230000017854 proteolysis Effects 0.000 description 1
- 238000000575 proteomic method Methods 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 150000003254 radicals Chemical class 0.000 description 1
- 239000003642 reactive oxygen metabolite Substances 0.000 description 1
- 238000006479 redox reaction Methods 0.000 description 1
- 239000013037 reversible inhibitor Substances 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 230000004137 sphingolipid metabolism Effects 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- KLJFYXOVGVXZKT-CCEZHUSRSA-N trans-hexadec-2-enal Chemical compound CCCCCCCCCCCCC\C=C\C=O KLJFYXOVGVXZKT-CCEZHUSRSA-N 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 241001430294 unidentified retrovirus Species 0.000 description 1
Images
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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
-
- 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
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6842—Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
- C12Y207/11—Protein-serine/threonine kinases (2.7.11)
- C12Y207/11025—Mitogen-activated protein kinase kinase kinase (2.7.11.25), i.e. MAPKKK or MAP3K
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/91—Transferases (2.)
- G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- G01N2333/91205—Phosphotransferases in general
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/10—Screening for compounds of potential therapeutic value involving cells
Definitions
- PTM post-translational modification
- PTMs include direct (non-enzymatic) oxidative or electrophilic modification of nucleophilic residues, such as cysteines, in proteins by reactive small molecules that are products of cellular redox reactions 5,6 .
- nucleophilic residues such as cysteines
- PUFAs polyunsaturated fatty acids
- LDEs endogenous lipid-derived electrophiles
- HNE 4-Hydroxynonenal
- HNE is a major product generated when free radicals initiate the non-enzymatic fragmentation of PUFAs in biological membranes 5,11 .
- 15-deoxy- ⁇ 12,14-prostaglandin J2 (15d-PGJ2) is another LDE produced by a set of enzymes that metabolize arachidonic acid 14 .
- 15d-PGJ2 exhibits anti-inflammatory and cytoprotective properties and has therefore been designated as a pro-resolving signal 14 .
- a third example is the LDE 2-trans-hexadecenal (2-HD), which is a product of sphingolipid metabolism and has recently been shown to function as a protein-modifying cofactor that promotes mitochondrial pathways for apoptosis 15 . Understanding the protein targets of LDEs is critical for elucidating their cellular functions and mechanisms of action.
- 2-HD 2-trans-hexadecenal
- cysteine residues including omeprazole, clopidogrel, and afatinib.
- isoTOP-ABPP isotopic Tandem Orthogonal Proteolysis-ABPP
- its use to quantify the intrinsic reactivity of cysteine residues in cell and tissue proteomes has been previously described by certain of the inventors herein 30 .
- IsoTOP-ABPP measures cysteine reactivity by: 1) treating proteomes with an alkynylated electrophilic iodoacetamide (IA) probe at various concentrations (or for various time periods), 2) conjugation of reactions with isotopically-differentiated azide-biotin tags containing a Tobacco Etch Virus (TEV) cleavage sequence using copper-catalyzed azide-alkyne cycloaddition (CuAAC or click 31 ) chemistry, and 3) enrichment, release, and identification/quantitation of IA-labeled cysteine-containing peptides by streptavidin chromatography, TEV protease treatment, and liquid chromatography-high-resolution mass spectrometry (LC-MS), respectively.
- IA alkynylated electrophilic iodoacetamide
- TEV Tobacco Etch Virus
- isoTOP-ABPP has been advanced to discover and quantify reactions between cysteines and electrophilic metabolites in proteomes.
- ‘competitive’ version of isoTOP-ABPP FIG. 1 a
- a proteome is treated with an electrophile (experimental sample) or DMSO (control sample). Both proteomic samples are then labeled with the IA probe and conjugated by CuAAC to light and heavy azide-biotin tags, respectively.
- the light and heavy samples are then mixed and subjected to the previously described isoTOP-ABPP protocol for enrichment, identification, and quantification of IA-labeled cysteines 30 .
- Electrophile-modified cysteines are quantified by measuring the MS1 chromatographic peak ratios (R values) for heavy (DMSO-treated) over light (electrophile-treated) samples, with higher R values reflecting greater sensitivity to the electrophile.
- R values MS1 chromatographic peak ratios
- competitive isoTOP-ABPP can assay electrophiles against 1000+ cysteines in parallel directly in native proteomes without requiring any chemical modification to the electrophiles themselves.
- the invention in various embodiments, is directed to a competitive isoTOP-ABPP method for identifying a protein target of selective cysteine modification by an electrophile, from among a set of proteins of a proteome, cell, tissue, or organism, comprising:
- identifying the protein target and sites of modification of the electrophile by comparing the abundance of the first isotopic marker and the second isotopic marker for each protein of the set, wherein a target protein for the electrophile possess a relatively higher ratio of the second isotopic marker to the first isotopic marker, compared to an average ratio of second isotopic marker to first isotopic marker among the set of proteins of the combined sample.
- the set of proteins can include one or more proteins, such as kinases.
- a target protein for the lipid-derived electrophiles identified by use of the inventive method has been found to be ZAK kinase.
- the electrophile can be a stress-induced lipid-derived electrophile such as 4-hydroxynonenal (HNE) or 15-deoxy- ⁇ 12,14-prostaglandin J2.
- such stress-induced lipid-derived electrophiles act as messenger molecules that modulate the response of signaling pathways to extracellular stimuli or stress; accordingly the identification of the electrophile-targeted protein(s) from among the large number of proteins in a proteome of a cell can serve to identify cellular components that can then be used in the development of modulators for the identified protein, of which ZAK kinase is an example.
- modulators can be used in the control of signaling pathways, such as the activation of mitogen-activated protein kinase (MAPK) pathways including the JNK, ERK, and p38 MAPK pathways that play roles in cancer and inflammation.
- MAPK mitogen-activated protein kinase
- HNE selectively targets a cysteine residue of ZAK kinase, a mitogen-activated protein kinase kinase kinase (MAP3K) enzyme, in such a way as to confer sensitivity of the MAPK signaling pathways to lipid oxidation products.
- MAP3K mitogen-activated protein kinase kinase kinase
- the identification of ZAK kinase by the method of the invention serves to identify a molecular target for development of ZAK kinase modulators, which can be used to modulate the activity of an MAPK-activating enzyme. Such modulators are believed to have potential as medicinal agents in the treatment of cancer and inflammation.
- FIG. 1 depicts an embodiment of a competitive isoTOP-ABPP for quantitative mapping of cysteine-reactive, lipid-derived electrophile (LDE) reactions in proteomes.
- (a) Competitive isoTOP-ABPP involves treatment of proteomes with DMSO or LDE, proteome labeling with an iodoacetamide-alkyne (IA) probe, CuAAC-based incorporation of isotopically-labeled, TEV protease-cleavable biotin tags, enrichment with streptavidin, and sequential on-bead protease digestions to afford probe-labeled peptides for MS analysis.
- IA iodoacetamide-alkyne
- FIG. 2 depicts results of quantitative profiling of LDE-cysteine reactions in proteomes.
- R values are shown by a dashed line to mark cysteines that exhibit high sensitivity to LDEs, and proteins with cysteines showing the strongest competitive reactivity with LDEs are labeled in green.
- FIG. 3 shows results of determining the potency of HNE-cysteine reactions in proteomes and in cells.
- the IA-labeling of cysteines from ZAK, EEF2, RTN4, and FN3KRP exhibit exceptional sensitivity to HNE competition compared to the rest of the cysteines in the proteome.
- FIG. 4 depicts the functional characterization of HNE modification of ZAK kinase.
- P-loop kinase's ATP binding loop
- FIG. 5 shows that HNE modification of ZAK suppresses JNK pathway activation in cells.
- WT-ZAK-transfected HEK-293T cells show higher basal JNK activation compared to mock-, C22A-ZAK-, or K45M-ZAK-transfected.
- (b) Western blots (b) and normalized phosphorylated JNK levels (c) showing that H 2 O 2 treatment (1 mM, 30 min) increases JNK activation in WT- and C22A-ZAK cells and this increase is blocked or amplified in WT- and C22A-ZAK cells, respectively by pre-treatment with HNE (100 ⁇ M, 30 min).
- a competitive isotopic Tandem Orthogonal Proteolysis Activity-Based Protein Profiling (isoTOP-ABPP) method for quantifying the reactivity of electrophilic compounds against 1000+ proteins comprising reactive cysteines in parallel in the human proteome is disclosed and claimed herein.
- HNE histone deacetylase
- 15d-PGJ2 one of these proteins, ZAK kinase, is labeled (alkylated) by HNE on a conserved, active site-proximal cysteine residue, which inhibits the enzyme and suppresses the activation of JNK pathways by oxidative stress in cancer cells.
- the invention provides, in various embodiments, a competitive isoTOP-ABPP method for identifying a protein target of selective cysteine modification by an electrophile, from among a set of proteins of a proteome, cell, tissue, or organism, comprising:
- identifying the protein target and sites of modification of the electrophile by comparing the abundance of the first isotopic marker and the second isotopic marker for each protein of the set, wherein a target protein for the electrophile possess a relatively higher ratio of the second isotopic marker to the first isotopic marker, compared to an average ratio of second isotopic marker to first isotopic marker among the set of proteins of the combined sample.
- the set of proteins can include one or more proteins, such as ZAK kinase.
- the electrophile can be a lipid derived electrophile, such as a stress-induced electrophile, wherein the protein target of the electrophile is a kinase.
- the kinase can comprise a cysteine residue that is alkylated by the electrophile, and when the electrophile comprises an ⁇ , ⁇ -unsaturated carbonyl group, such as 4-hydroxynonenal or 15-deoxy- ⁇ 12,14-prostaglandin J2, the electrophile can react with the cysteine by a Michael conjugate addition.
- the protein target of the electrophile can be used as a substrate for identification of further inhibitors thereof, by screening a plurality of candidate compounds for modulation of the kinase protein target to identify one or more selective kinase modulator.
- a selective kinase modulator is a potential medicinal compound for treatment of a condition wherein modulation, e.g., inhibition, of the kinase activity is medically indicated.
- a selective modulator of the kinase protein target so identified can be a reversible inhibitor of the kinase protein target, which can be suitable for development as a medicament for treatment of the condition in human beings, e.g., for treatment of cancer or of inflammation.
- the invention also provides, in various embodiments, a protein identified as a target of an electrophile by the method of the invention.
- the protein can be a kinase, such as ZAK kinase, and can be used for the development of kinase modulators, e.g., a ZAK kinase inhibitor suitable for administration to a human subject suffering from a condition such as cancer or inflammation wherein modulation of the kinase is medically indicated.
- cysteine is unique owing to its intrinsically high nucleophilicity, which renders its sensitivity to modification by endogenous electrophiles and oxidants 6 , as well as electrophilic xenobiotics and candidate therapeutics 23,24 .
- Cysteine reactions with electrophilic metabolites have been characterized for purified proteins. 25,26 and, on a global scale in cells and tissues using mass spectrometry-based chemoproteomic 5,16-22 and imaging methods 27 .
- isoTOP-ABPP isotopic Tandem Orthogonal Proteolysis-ABPP
- isoTOP-ABPP measures cysteine reactivity by: 1) treating proteomes with an alkynylated electrophilic iodoacetamide (IA) probe at various concentrations (or for various time periods), 2) conjugation of reactions with isotopically-differentiated azide-biotin tags containing a Tobacco Etch Virus (TEV) cleavage sequence using copper-catalyzed azide-alkyne cycloaddition (CuAAC or click 31 ) chemistry, and 3) enrichment, release, and identification/quantitation of IA-labeled cysteine-containing peptides by streptavidin chromatography, TEV protease treatment, and liquid chromatography-high-resolution mass spectrometry (LC-MS), respectively.
- IA alkynylated electrophilic iodoacetamide
- TEV Tobacco Etch Virus
- isoTOP-ABPP could be advanced to discover and quantify reactions between cysteines in proteomes and any electrophilic compound.
- a proteome is treated with an electrophile (experimental sample) or DMSO (control sample). Both proteomic samples are then labeled with the IA probe and conjugated by CuAAC to light and heavy azide-biotin tags, respectively. The light and heavy samples are then mixed and subjected to our described isoTOP-ABPP protocol for enrichment, identification, and quantification of IA-labeled cysteines 30 .
- Electrophile-modified cysteines are quantified by measuring the MS1 chromatographic peak ratios (R values) for heavy (DMSO-treated) over light (electrophile-treated) samples, with higher R values reflecting greater sensitivity to the electrophile.
- R values MS1 chromatographic peak ratios
- competitive isoTOP-ABPP can assay electrophiles against 1000+ cysteines in parallel directly in native proteomes without requiring any chemical modification to the electrophiles themselves.
- a total of 1400 cysteine reactivities were quantified across the aggregate data set, with at least 900 cysteine reactivities quantified for each LDE ( FIG. 2 a ) more than 750 of which were quantified for all the three electrophiles. Most of the cysteine reactivities (>98%) were unaffected or only marginally affected by LDE treatment (R ⁇ 5); however, a select subgroup showed marked reductions in their IA-probe reactivities (R>5) following exposure to one or more LDEs ( FIG. 2 a ).
- these profiles identified ⁇ 1100 IA-labeled cysteines, many of which showed reduced labeling signals in the presence of HNE, including 8 of the 14 HNE-modified cysteines identified in a previous proteomic study that used a biotinamidohexanoic acid hydrazide probe to enrich and identify (but not to quantify) HNE-modified cysteines 17 .
- ZAK kinase also known as MLK7 or MLTK
- MAPK kinase MAPK kinase kinase
- MAP3K MAPK kinase kinase 32,33
- MAP2Ks MAPK kinases
- HEK-293T cells were stably transfected with cDNAs for WT-ZAK, C22A-ZAK, or K45M-ZAK and the activation state of their MAPK signaling pathways was monitored by western blotting with anti-phosphoprotein antibodies.
- WT-ZAK-expressing cells, but not C22A- or K45M-ZAK-expressing cells showed significantly increased JNK and, to a lesser extent, p38 and ERK pathway activation compared to mock-transfected cells ( FIG. 5 a, b ).
- HNE is also known to itself promote oxidative stress 43,44 that likely adds to the effects of H 2 O 2 and, in the context of an HNE-resistant C22A-ZAK mutant, would serve to further augment activation of the JNK pathway.
- HNE-ZAK interaction acts as a negative-feedback loop that tempers activation of the JNK pathway under high and/or persistent levels of oxidative stress ( FIG. 5 d ).
- LDEs Long viewed as biomarkers of oxidative damage, LDEs have more recently gained attention as second messengers that can regulate diverse cellular processes 8,9 . These findings have inspired the advent of chemoproteomic methods to globally map LDE-protein interactions 5,16-19 . To date, these large-scale profiling efforts have focused on the qualitative inventorying of LDE-reactive proteins in cell and tissue proteomes, generating lists of many candidate targets and pathways for LDE action. Considering, however, that the signaling and pathophysiological functions of LDEs may differ across the endogenous concentration ranges found for these compounds, it is imperative to understand the potencies of LDE-protein interactions in biological systems.
- HNE modification of ZAK may limit the extent of JNK activation caused by oxidative stress, which could help certain cell types, such as tumor and immune cells, survive in the presence of high levels of reactive oxygen species.
- Further studies of ZAK function would benefit from the development of selective inhibitors for this enzyme.
- covalent inhibitors have recently been introduced for many kinases 24,45-47 . These inhibitors often target cysteine residues in or near kinase active sites, which leads us to speculate that the C22-HNE interaction discovered herein may offer a medicinal chemistry starting point for the development of ZAK inhibitors.
- competitive ABPP methods should offer a useful strategy to assess inhibitor target engagement and selectivity 48 .
- Table 1 shows peptide sequences, parental protein names, and R values for IA-labeled cysteines that exhibit IA-labeling competed by one or more LDE with R values>5. HNE-competed cysteines are shaded in pink and 15d-PGJ2-competed cysteines are shaded in green. Note that REEP5 displays significant competition by both LDEs.
- the isoTOP-ABPP method ABPP for quantitative mapping of cysteine-reactive, lipid-derived electrophile (LDE) reactions in proteomes, cells, tissues, or organisms can be used to determine if one or more proteins in the set of proteins therefrom possesses one or more domains, each comprising a reactive cysteine residue, has at least a low affinity (e.g., high micromolar affinity) for any selected structural module, and serves to identify what protein has that affinity.
- a low affinity e.g., high micromolar affinity
- Identification of a high affinity ligand can serve as a structural lead in the development of compounds targeting a particular protein, which can lead to the development of medicinal compounds. Similarly, identification of the protein target of the ligand can be of value in determining a possibly unknown function for the targeted protein.
- MDA-MB-231 cells were grown in L15 media supplemented with 10% fetal bovine serum at 37° C. in a CO 2 -free incubator.
- cells were grown to 100% confluency, washed three times with PBS and scraped in cold PBS.
- Cell pellets were isolated by centrifugation at 1400 ⁇ g for 3 min, and the cell pellets stored at ⁇ 80° C. until further use.
- the harvested cell pellets were lysed by sonication in PBS buffer and fractionated by centrifugation (100,000 ⁇ g, 45 min.) to yield soluble and membrane proteomes.
- the proteomes were prepared fresh from the frozen cell pallets prior to each experiment.
- HNE was purchased from EMD biosciences
- 15d-PGJ2 was purchased from Cayman Chemicals
- 2-HD was purchased from Santa Cruz Biotechnology.
- Proteome samples were diluted to a 4 mg protein/mL solution in PBS.
- one aliquot of the proteome sample (0.5 mL) was treated with 100 ⁇ M of LDE using 5 ⁇ L of a 10 mM stock and the other aliquot was treated with 5 ⁇ L of either ethanol (for HNE and 15d-PGJ2) or DMSO (for 2-HD) as control.
- Each of the control and LDE-treated proteome samples ( ⁇ 2 mg protein/mL in 1 mL volume) was treated with 100 ⁇ M of IA-probe using 10 ⁇ L of a 10 mM stock in DMSO. The labeling reactions were incubated at room temperature for 1 hour.
- Click chemistry (acetylene-azide cycloaddition) was performed by the addition of 100 ⁇ M of either the Heavy-TEV-Tag (for the control sample) or Light-TEV-Tag (for the LDE-treated sample) (20 ⁇ L of a 5 mM stock), 1 mM TCEP (fresh 50 ⁇ stock in water), 100 ⁇ M ligand (17 ⁇ stock in DMSO:t-Butanol 1:4) and 1 mM CuSO 4 (50 ⁇ stock in water). Samples were allowed to react at room temperature for 1 hour. After the click chemistry step, the light and heavy-labeled samples were mixed together and centrifuged (5900 ⁇ g, 4 min, 4° C.) to pellet the precipitated proteins.
- the pellets were washed twice in cold MeOH, after which the pellet was solubilized in PBS containing 1.2% SDS via sonication and heating (5 min, 80° C.). Samples were subjected to streptavidin enrichment of probe-labeled proteins, sequential on-bead trypsin and TEV digestion, and liquid chromatography-mass spectrometry (LC-MS) analysis according to the published isoTOP-ABPP protocol 30 .
- LC-MS liquid chromatography-mass spectrometry
- IA-probe labeled peptides were identified by SEQUEST2 and DTASelect3, and the quantification of heavy/light ratios (isoTOP-ABPP ratios, R) was performed by an in-house software (CIMAGE) as previously described.
- the software was advanced to be able to detect and quantify cases where near complete LDE blockade of IA-probe labeling was achieved (e.g., very small or no light peak) and assign an empirical ratio cut-off of 15 to each of such cases.
- Each experiment consisted of multiple LC/LCMS/MS runs: either HNE, 15d-PGJ2 and 2-HD 100 ⁇ M competition, or HNE competition at different concentrations. All runs were searched using SEQUEST and filtered with DTASelect as described above.
- ZAK-C22A and ZAK-K45M mutants were generated by QuikChange site-directed mutagenesis using the primer 5′-atttgatgacttgcagtttttttgaaacgccggtggaggaagttttg-3′ (SEQ ID NO:1) and 5′-ggacaaggaggtggctgtaatgaagctcctcaaatagag-3′ (SEQ ID NO:2) and their complements.
- Retrovirus was prepared by taking 3.0 ⁇ g of each of pCLNCX and pCL-Ampho vectors and 18 ⁇ L of FuGENE HD reagent (Roche) to transfect 60% confluent HEK-293T cells. Medium was replaced after 1 day of transfection and on the next day virus-containing supernatant was collected, filter sterilized and stored at ⁇ 70° C. 1 mL of virus-containing medium was used to infect target cells in presence of 8 ⁇ g/mL of polybrene for 72 hours and infected cells were selected in medium containing 100 ⁇ g/mL of hygromycin. Surviving cells after the selection were expanded and cultured in regular DMEM medium with 10% FCS.
- HEK-293T cells with stable expression of wild-type or mutant ZAK were grown to 100% confluency on a 10 cm plate.
- Cells were collected, washed with cold PBS (2 ⁇ 10 mL) and lysed in 1 mL of PBS supplemented with 1 ⁇ complete EDTA-free protease inhibitor cocktails by sonication.
- Cell lysates were fractionated by centrifugation (100,000 ⁇ g, 45 min.) and the soluble fraction was incubated with 50 ⁇ L of Anti-FLAG M2 affinity gel (Sigma-Aldrich) at 4° C. for 3 hours.
- FLAG-tagged wild-type and C22A mutant ZAK were immunoprecipitated from HEK-293T cells (1 ⁇ 107). After washing with PBS, the beads were suspended in 100 ⁇ L of PBS buffer and labeled with 250 nM of IA-rhodamine (add 1 ⁇ L of 25 ⁇ M probe stock in DMSO). After 1 hour of labeling at 4° C., 50 ⁇ L of 4 ⁇ gel loading buffer was added and the beads were boiled for 5 min to elute the bound proteins. Gel samples were separated by SDS-PAGE (50 ⁇ L of sample/lane) and visualized in-gel using a Hitachi FMBio II flatbed laser-induced fluorescence scanner (MiraiBio, Alameda, Calif.).
- soluble lysate of HEK-293T overexpressing WT-ZAK were incubated with 10, 50 and 100 ⁇ M of HNE (add 2 ⁇ L of 5, 25 and 50 mM stock) for 30 mins and then subjected to immunoprecipitation.
- soluble lysate (1 mg/mL in PBS) of HEK-239T cells transfected with mock ZAKWT and ZAK-C22A was labeled with 10 ⁇ M of HNEyne4 (Cayman Chemicals, 1 ⁇ L of 500 ⁇ M stock in ethanol) for 1 hour at room temperature. Cycloaddition was performed with 200 ⁇ M rhodamine-azide, 1 mM TCEP, 100 ⁇ M TBTA ligand and 1 mM CuSO 4 . The reaction was allowed to proceed at room temperature for 1 hour before quenching with 20 ⁇ L of 4 ⁇ SDS-PAGE loading buffer (reducing).
- kinase activity assay protocol was adapted from Yu et al 5 .
- Kinase assay buffers, Myeilin Basic Protein (MBP) substrate, and ATP stock solution were purchased from SignalChem.
- Radio-labeled [ 33 P]-ATP was purchased from PerkinElmer. 10 mg of soluble lysate of HEK-293T cells transfected with each of wild-type, C22A and K45M of ZAK were immunoprecipitated and then eluted with 2 ⁇ 300 ⁇ L FLAG-peptide buffer.
- Each sample was concentrated to 100 ⁇ L using an Amicon centrifugal filter (30 kDa cutoff) and exchanged to the assay kinase buffer (5 mM MOPS, pH7.2, 2.5 mM ⁇ -glycerol-phosphate, 5 mM MgCl2, 1 mM EGTA, 0.4 mM EDTA, 0.05 mM DTT and 40 ng/ ⁇ L BSA) to a final volume around 10 ⁇ L.
- the assay kinase buffer 5 mM MOPS, pH7.2, 2.5 mM ⁇ -glycerol-phosphate, 5 mM MgCl2, 1 mM EGTA, 0.4 mM EDTA, 0.05 mM DTT and 40 ng/ ⁇ L BSA
- 4 reactions were set up and each reaction starts with mixing 10 ⁇ L of immunoprecipiated ZAK, 5 ⁇ L of MBP (1 mg/mL) and 5 ⁇ L of HNE (10 or 100 ⁇ M) or H 2
- No-enzyme and no-substrate controls were prepared in parallel.
- the mixed samples were incubated on ice for 15 min and 5 ⁇ L of [ 33 P]-ATP assay cocktail (250 ⁇ M, 167 ⁇ Ci/mL) was then added to initiate the kinase reaction.
- Each reaction mixture was incubated in 30° C. for 15 min and the reaction was terminated by spotting 20 ⁇ L of the reaction mixture onto individual pre-cut strips of phosphocellulose P81 paper.
- the spotted P81 strips were air dried and then washed with 10 mL of 1% phosphoric acid for 3 ⁇ 10 min.
- ZAK activity was measured by counting the radioactivity on the P81 paper in the presence of scintillation fluid in a scintillation counter after subtracting the value obtained from the corresponding no substrate control, and was normalized to that of ZAK-WT without HNE treatment. Experiments were performed in triplicates. 10 ⁇ L of each ZAK variant used in setting up the kinase reaction were run on a SDS-PAGE gel and immunoblotted with an anti-FLAG antibody to ensure that they are enriched at similar levels.
- HEK-293T cells with stable expression of wild-type ZAK were passaged six times in DMEM medium minus 1-lysine and 1-arginine (Thermo) supplemented 10% dialyzed FBS (Gemini), 1% PSQ (1% vol/vol 10,000 units penicillin, 10 mg streptomycin, 29.2 mg lglutamate solution) and 100 ⁇ g/mL [ 13 C 6 , 15 N 4 ] l-arginine-HCl and [ 13 C 6 , 15 N 2 ] l-lysine-HCl (heavy) or 1-arginine-HCl and l-lysine-HCl (light) (Sigma-Aldrich).
- Soluble proteomes of light and heavy ZAK-WT transfected HEK-293T cells (3 mL each at 7 mg/mL) were treated with 100 ⁇ M of HNE (6 ⁇ L of 50 mM stock) or EtOH for 30 min at room temperature.
- the samples were gel filtrated by PD-10 columns (GE healthcare) to remove unreacted HNE as well as excessive ATP molecules in proteomes.
- Each aliquot of light and heavy proteomes (0.5 mL, 6 mg/mL) was labeled with 20 ⁇ M of acylphosphate-ATP probe (ActivX Biosciences) and then mixed together to proceed with reduction/alkylation, streptavidin enrichment, trypsin digest according to a modified version of the vendor-provided “Xsite Kinase Analysis” protocol6.
- the trypsin digested samples were analysed by LC-MS/MS and enriched kinase peptides were identified by SEQUEST and DTASelect.
- the amounts of probe-labeled kinases with and without HNE treatment were quantified using the CIMAGE module that was developed for quantitative SILAC-ABPP chemoproteomic profiling 7 .
- HEK-293T cells transfected with mock, WT-ZAK, C22A-ZAK and K45M-ZAK were seeded into a 12-well plate with 2.5 ⁇ 10 5 cells per well.
- Cells were grown in regular DMEM medium with 10% FBS for 24 hours and starved in serum-free DMEM medium for another 24 hours. Cells were then treated at 37° C.
Abstract
Cells produce electrophilic products with the potential to modify and affect the function of proteins. Chemoproteomic methods have provided a means to qualitatively inventory proteins targeted by endogenous electrophiles; however, ascertaining the potency and specificity of these reactions to identify the most sensitive sites in the proteome to electrophilic modification requires more quantitative methods. Here, we describe a competitive activity-based profiling method for quantifying the reactivity of electrophilic compounds against 1000+ cysteines in parallel in the human proteome. Using this approach, we identify a select set of proteins that constitute “hot spots” for modification by various lipid-derived electrophiles, including the oxidative stress product 4-hydroxynonenal (HNE). We show that one of these proteins, ZAK kinase, is labeled by HNE on a conserved, active site-proximal cysteine, resulting in enzyme inhibition to create a negative feedback mechanism that can suppress the activation of JNK pathways by oxidative stress.
Description
- The subject patent application is a continuation of U.S. patent application Ser. No. 14/911,316 (filed Feb. 10, 2016; now pending), which is a national stage application of International Application No. PCT/US2014/050828 (filed Aug. 13, 2014; now expired), which claims the benefit of priority to U.S. Provisional Patent Application No. 61/865,165 (filed Aug. 13, 2013; now expired). The full disclosures of the priority applications are incorporated herein by reference in their entirety and for all purposes.
- This invention was made with government support under grant numbers CA087660 and ES020851 awarded by the National Institutes of Health. The government has certain rights in the invention.
- The functional diversity of mammalian proteomes is greatly expanded by the post-translational modification (PTM) of proteins1. A vast and structurally diverse array of PTMs occurs on proteins to regulate their activity, localization, and interactions with other biomolecules. Many PTMs are enzyme-catalyzed, such as protein phosphorylation, glycosylation, lipidation, and methylation. Our understanding of these enzyme-catalyzed PTMs has benefited greatly from chemoproteomic methods for their global profiling and functional characterization in biological systems2-4.
- Another important class of PTMs includes direct (non-enzymatic) oxidative or electrophilic modification of nucleophilic residues, such as cysteines, in proteins by reactive small molecules that are products of cellular redox reactions5,6. When cells, for instance, are subject to various forms of oxidative stress, such as chronic inflammation, hypoxia, or exposure to xenobiotics or environmental pollution, peroxidation of polyunsaturated fatty acids (PUFAs) in the membrane bilayer generates a broad range of secondary products, many of which are electrophilic in nature5. These endogenous lipid-derived electrophiles (LDEs) can modify DNA and proteins to promote cytotoxicity and have been implicated in the pathogenesis of many diseases, including cancer, inflammation, neurodegeneration, and cardiovascular disorders7. More recently, a growing body of studies has also suggested that, at lower and more physiological concentrations, LDEs can serve as messengers that modulate the response of signaling pathways to extracellular stimuli or stress8-10. 4-Hydroxynonenal (HNE), for instance, is a major product generated when free radicals initiate the non-enzymatic fragmentation of PUFAs in biological membranes5,11. The levels of HNE and HNE-protein adducts are elevated in cells and tissues exposed to oxidative stress, and HNE can regulate redox-responsive signaling pathways by still poorly understood mechanisms5,12,13. 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) is another LDE produced by a set of enzymes that metabolize arachidonic acid14. 15d-PGJ2 exhibits anti-inflammatory and cytoprotective properties and has therefore been designated as a pro-resolving signal14. A third example is the LDE 2-trans-hexadecenal (2-HD), which is a product of sphingolipid metabolism and has recently been shown to function as a protein-modifying cofactor that promotes mitochondrial pathways for apoptosis15. Understanding the protein targets of LDEs is critical for elucidating their cellular functions and mechanisms of action.
- Many drugs and drug candidates also act by covalent modification of cysteine residues, including omeprazole, clopidogrel, and afatinib. The discovery of additional cysteine-reactive chemical probes and drugs would benefit from a general method to globally map compound reactivity with cysteines in native biological systems.
- A chemoproteomic method termed isoTOP-ABPP (isotopic Tandem Orthogonal Proteolysis-ABPP) and its use to quantify the intrinsic reactivity of cysteine residues in cell and tissue proteomes has been previously described by certain of the inventors herein30. IsoTOP-ABPP measures cysteine reactivity by: 1) treating proteomes with an alkynylated electrophilic iodoacetamide (IA) probe at various concentrations (or for various time periods), 2) conjugation of reactions with isotopically-differentiated azide-biotin tags containing a Tobacco Etch Virus (TEV) cleavage sequence using copper-catalyzed azide-alkyne cycloaddition (CuAAC or click31) chemistry, and 3) enrichment, release, and identification/quantitation of IA-labeled cysteine-containing peptides by streptavidin chromatography, TEV protease treatment, and liquid chromatography-high-resolution mass spectrometry (LC-MS), respectively.
- In the present invention, isoTOP-ABPP has been advanced to discover and quantify reactions between cysteines and electrophilic metabolites in proteomes. In this advanced, ‘competitive’ version of isoTOP-ABPP (
FIG. 1a ), a proteome is treated with an electrophile (experimental sample) or DMSO (control sample). Both proteomic samples are then labeled with the IA probe and conjugated by CuAAC to light and heavy azide-biotin tags, respectively. The light and heavy samples are then mixed and subjected to the previously described isoTOP-ABPP protocol for enrichment, identification, and quantification of IA-labeled cysteines30. Electrophile-modified cysteines are quantified by measuring the MS1 chromatographic peak ratios (R values) for heavy (DMSO-treated) over light (electrophile-treated) samples, with higher R values reflecting greater sensitivity to the electrophile. In this format, competitive isoTOP-ABPP can assay electrophiles against 1000+ cysteines in parallel directly in native proteomes without requiring any chemical modification to the electrophiles themselves. - The invention, in various embodiments, is directed to a competitive isoTOP-ABPP method for identifying a protein target of selective cysteine modification by an electrophile, from among a set of proteins of a proteome, cell, tissue, or organism, comprising:
- contacting the set of proteins of the proteome and the electrophile to provide an alkylated set of proteins, then,
- contacting the alkylated set with an alkynylated iodoacetamide probe, followed by reaction with an azido compound comprising a first isotopic marker, to provide an isotopically-marked alkylated set, and
- contacting the set of proteins of the proteome, not exposed to the electrophile, with an alkynylated iodoacetamide probe, followed by reaction with an azido compound comprising a second isotopic marker, to provide an isotopically-marked control set, then,
- combining the isotopically-marked alkylated set and the isotopically-marked control set to provide a combined sample, and,
- identifying the protein target and sites of modification of the electrophile by comparing the abundance of the first isotopic marker and the second isotopic marker for each protein of the set, wherein a target protein for the electrophile possess a relatively higher ratio of the second isotopic marker to the first isotopic marker, compared to an average ratio of second isotopic marker to first isotopic marker among the set of proteins of the combined sample.
- The set of proteins can include one or more proteins, such as kinases. A target protein for the lipid-derived electrophiles identified by use of the inventive method has been found to be ZAK kinase. The electrophile can be a stress-induced lipid-derived electrophile such as 4-hydroxynonenal (HNE) or 15-deoxy-Δ12,14-prostaglandin J2. It is believed by the inventors herein that such stress-induced lipid-derived electrophiles act as messenger molecules that modulate the response of signaling pathways to extracellular stimuli or stress; accordingly the identification of the electrophile-targeted protein(s) from among the large number of proteins in a proteome of a cell can serve to identify cellular components that can then be used in the development of modulators for the identified protein, of which ZAK kinase is an example. Such modulators can be used in the control of signaling pathways, such as the activation of mitogen-activated protein kinase (MAPK) pathways including the JNK, ERK, and p38 MAPK pathways that play roles in cancer and inflammation.
- It has been discovered by the inventors herein that HNE selectively targets a cysteine residue of ZAK kinase, a mitogen-activated protein kinase kinase kinase (MAP3K) enzyme, in such a way as to confer sensitivity of the MAPK signaling pathways to lipid oxidation products. The identification of ZAK kinase by the method of the invention serves to identify a molecular target for development of ZAK kinase modulators, which can be used to modulate the activity of an MAPK-activating enzyme. Such modulators are believed to have potential as medicinal agents in the treatment of cancer and inflammation.
-
FIG. 1 depicts an embodiment of a competitive isoTOP-ABPP for quantitative mapping of cysteine-reactive, lipid-derived electrophile (LDE) reactions in proteomes. (a) Competitive isoTOP-ABPP involves treatment of proteomes with DMSO or LDE, proteome labeling with an iodoacetamide-alkyne (IA) probe, CuAAC-based incorporation of isotopically-labeled, TEV protease-cleavable biotin tags, enrichment with streptavidin, and sequential on-bead protease digestions to afford probe-labeled peptides for MS analysis. The IA-probe and the competitive blockade of IA-cysteine reactions by an LDE are shown in the inset. (b) Structures of three LDEs, HNE, 15d-PGJ2 and 2-HD, used in competitive isoTOP-ABPP experiments, with their sites of reactivity marked with asterisks. -
FIG. 2 depicts results of quantitative profiling of LDE-cysteine reactions in proteomes. (a) Distribution of competitive isoTOP-ABPP ratios (R values) quantified from reactions with the human MDA-MB-231 proteome treated with 100 μM HNE (left), 15d-PGJ2 (middle), or 2-HD (right). A cut-off of five-fold or greater blockade of IA-probe labeling (Rvalues>5) is shown by a dashed line to mark cysteines that exhibit high sensitivity to LDEs, and proteins with cysteines showing the strongest competitive reactivity with LDEs are labeled in green. (b) Heat map of cysteines with R values >5 illustrating examples of cysteines that display selectivity for reacting with one of the three tested LDEs (green boxes) and proteins that contain multiple IA-labeled cysteines, only one of which shows sensitivity to LDE competition (red boxes). (c) Representative MS1 profiles for peptides containing cysteines that show selective competition with 15d-PGJ2 (left) or HNE (right). (d) Representative MS1 profiles for multiple cysteine-containing peptides from the same protein, only one of which shows sensitivity to LDE competition. In each example, the LDE-sensitive cysteines is marked in red. -
FIG. 3 shows results of determining the potency of HNE-cysteine reactions in proteomes and in cells. (a) Box-and-whisker plots showing the distribution of R values for ˜1100 cysteines quantified from competitive isoTOP-ABPP experiments with the MDA-MB-231 proteome treated with 5, 10, 50, 100, and 500 μM HNE. The IA-labeling of cysteines from ZAK, EEF2, RTN4, and FN3KRP exhibit exceptional sensitivity to HNE competition compared to the rest of the cysteines in the proteome. (b) Representative MS1 profiles for HNE-sensitive cysteines in ZAK and RTN4 showing concentration-dependent blockade of IA-labeling by HNE. (c) Distribution of R values quantified from competitive isoTOP-ABPP experiments with proteomes from MDA-MB-231 cells treated in situ with DMSO or HNE (100 μM, 60 min), confirming that cysteines in ZAK, EEF2, RTN4 and FN3KRP are also highly sensitive to HNE competition in living cells. (d) Comparison of R values obtained from in vitro versus in situ competitive isoTOP-ABPP experiments. Red and black diamonds mark cysteines that show similar or different in vitro versus in situ R values, respectively. -
FIG. 4 depicts the functional characterization of HNE modification of ZAK kinase. (a) Crystal structure of human MAP3K9 (left, PDB: 3DTC) and multiple sequence alignment of ZAK with other 19 human MAP3Ks (SEQ ID NOs: 27-46) showing the HNE-sensitive cysteine C22 of ZAK is located next to the kinase's ATP binding loop (“P-loop”; note that C22 corresponds to 1150 in MAP3K9) and is unique to ZAK relative to other MAP3K enzymes. (b) Selective IA-labeling of wild-type (WT), but not C22A-ZAK, and concentration-dependent competition of IA-labeling of WT-ZAK by HNE as measured by gel-based ABPP using an IA-rhodamine probe. ZAK were expressed as FLAG-tagged proteins in HEK293T cells by stable transfection and immunoprecipitated prior to IA-probe labeling and analysis. (c) An HNE-alkyne probe (HNEyne16) selectively labels WT-, but not C22A-ZAK in proteomes and in living cells as determined by gel-based ABPP. (d) Catalytic activity of immunoprecipitated WT-, but not C22A-ZAK is inhibited by HNE as measured using a Myelin Basic Protein (MBP) substrate assay. A K45M-ZAK mutant, in which a conserved active-site lysine was mutated, showed no detectable activity and thus served as a catalytically dead control enzyme. All three ZAK variants (WT, C22A, and K45M were expressed at similar levels in transfected HEK293T cells). (e) Quantitative profiling of kinase activities in ZAK-transfected HEK293T proteomes treated with DMSO or HNE (100 μM, 30 min) by SILAC-ABPP using an acylphophate-ATP probe shows that the ATP-binding of ZAK is greatly impaired by HNE modification on C22. Other kinases detected in this assay were, in general unaffected by HNE treatment. For (d) and (e), data are presented as mean values±SEM; N>=3 experiments/group. **, P<0.01, ***, P<0.001, t-test. -
FIG. 5 shows that HNE modification of ZAK suppresses JNK pathway activation in cells. (a) WT-ZAK-transfected HEK-293T cells show higher basal JNK activation compared to mock-, C22A-ZAK-, or K45M-ZAK-transfected. (b), (c) Western blots (b) and normalized phosphorylated JNK levels (c) showing that H2O2 treatment (1 mM, 30 min) increases JNK activation in WT- and C22A-ZAK cells and this increase is blocked or amplified in WT- and C22A-ZAK cells, respectively by pre-treatment with HNE (100 μM, 30 min). (d) A model diagramming ZAK-dependent and ZAK-independent pathways for HNE modulation of JNK activation. Dashed line designates the potential for oxidative stress to generate HNE and initiate a negative feedback loop to limit JNK activation. (e), (f) Western blots (e) and normalized phosphorylated JNK levels (f) showing dramatic, concentration-dependent activation of JNK by HNE (50 or 100 μM, 60 min) in C22A-ZAK cells, but not in WT-ZAK cells. Note that mock- and K45M-ZAK transfected cells also show modest, but significant elevations in JNK activity following HNE treatment, which is consistent with previous studies indicating that HNE can activate JNK by multiple pathways42,43,57 For (a), (c) and (f), data are presented as mean values SEM; N=4 experiments/group. *, P<0.05, **, P<0.01 ##, P<0.01, t-test. - A competitive isotopic Tandem Orthogonal Proteolysis Activity-Based Protein Profiling (isoTOP-ABPP) method for quantifying the reactivity of electrophilic compounds against 1000+ proteins comprising reactive cysteines in parallel in the human proteome is disclosed and claimed herein. Using this approach, we identify select sets of proteins that are preferentially modified by HNE and 15d-PGJ2. We show that one of these proteins, ZAK kinase, is labeled (alkylated) by HNE on a conserved, active site-proximal cysteine residue, which inhibits the enzyme and suppresses the activation of JNK pathways by oxidative stress in cancer cells.
- The invention provides, in various embodiments, a competitive isoTOP-ABPP method for identifying a protein target of selective cysteine modification by an electrophile, from among a set of proteins of a proteome, cell, tissue, or organism, comprising:
- contacting the set of proteins of the proteome and the electrophile to provide an alkylated set of proteins, then,
- contacting the alkylated set with an alkynylated iodoacetamide probe, followed by reaction with an azido compound comprising a first isotopic marker, to provide an isotopically-marked alkylated set, and
- contacting the set of proteins of the proteome, not exposed to the electrophile, with an alkynylated iodoacetamide probe, followed by reaction with an azido compound comprising a second isotopic marker, to provide an isotopically-marked control set, then,
- combining the isotopically-marked alkylated set and the isotopically-marked control set to provide a combined sample, and,
- identifying the protein target and sites of modification of the electrophile by comparing the abundance of the first isotopic marker and the second isotopic marker for each protein of the set, wherein a target protein for the electrophile possess a relatively higher ratio of the second isotopic marker to the first isotopic marker, compared to an average ratio of second isotopic marker to first isotopic marker among the set of proteins of the combined sample.
- For example, the set of proteins can include one or more proteins, such as ZAK kinase.
- The electrophile can be a lipid derived electrophile, such as a stress-induced electrophile, wherein the protein target of the electrophile is a kinase. The kinase can comprise a cysteine residue that is alkylated by the electrophile, and when the electrophile comprises an α,β-unsaturated carbonyl group, such as 4-hydroxynonenal or 15-deoxy-Δ12,14-prostaglandin J2, the electrophile can react with the cysteine by a Michael conjugate addition.
- By identification of a protein target of an electrophile, the protein target of the electrophile can be used as a substrate for identification of further inhibitors thereof, by screening a plurality of candidate compounds for modulation of the kinase protein target to identify one or more selective kinase modulator. A selective kinase modulator is a potential medicinal compound for treatment of a condition wherein modulation, e.g., inhibition, of the kinase activity is medically indicated. For example, a selective modulator of the kinase protein target so identified can be a reversible inhibitor of the kinase protein target, which can be suitable for development as a medicament for treatment of the condition in human beings, e.g., for treatment of cancer or of inflammation.
- The invention also provides, in various embodiments, a protein identified as a target of an electrophile by the method of the invention. The protein can be a kinase, such as ZAK kinase, and can be used for the development of kinase modulators, e.g., a ZAK kinase inhibitor suitable for administration to a human subject suffering from a condition such as cancer or inflammation wherein modulation of the kinase is medically indicated.
- Among the 20 protein-coding amino acids, cysteine is unique owing to its intrinsically high nucleophilicity, which renders its sensitivity to modification by endogenous electrophiles and oxidants6, as well as electrophilic xenobiotics and candidate therapeutics23,24. Cysteine reactions with electrophilic metabolites have been characterized for purified proteins.25,26 and, on a global scale in cells and tissues using mass spectrometry-based chemoproteomic5,16-22 and imaging methods27. These studies, along with analytical, quantum mechanical, and kinetic work28,29, have, for the most part, confirmed the preferential reactivity that Michael acceptor electrophiles like HNE show for cysteine over other potentially nucleophilic amino acids (e.g., lysine, histidine) in proteomes. We were interested in building on these past findings to determine whether individual cysteines in the proteome display differences in their reactivity with endogenous electrophiles, and, if so, whether potential hot-spots for electrophile modification might constitute key nodes in signaling pathways of redox sensing and response.
- We previously described a chemoproteomic method termed isoTOP-ABPP (isotopic Tandem Orthogonal Proteolysis-ABPP) and its use to quantify the intrinsic reactivity of cysteine residues in cell and tissue proteomes30. isoTOP-ABPP measures cysteine reactivity by: 1) treating proteomes with an alkynylated electrophilic iodoacetamide (IA) probe at various concentrations (or for various time periods), 2) conjugation of reactions with isotopically-differentiated azide-biotin tags containing a Tobacco Etch Virus (TEV) cleavage sequence using copper-catalyzed azide-alkyne cycloaddition (CuAAC or click31) chemistry, and 3) enrichment, release, and identification/quantitation of IA-labeled cysteine-containing peptides by streptavidin chromatography, TEV protease treatment, and liquid chromatography-high-resolution mass spectrometry (LC-MS), respectively.
- Here, we envisioned that isoTOP-ABPP could be advanced to discover and quantify reactions between cysteines in proteomes and any electrophilic compound. In this advanced, ‘competitive’ version of isoTOP-ABPP (
FIG. 1a ), a proteome is treated with an electrophile (experimental sample) or DMSO (control sample). Both proteomic samples are then labeled with the IA probe and conjugated by CuAAC to light and heavy azide-biotin tags, respectively. The light and heavy samples are then mixed and subjected to our described isoTOP-ABPP protocol for enrichment, identification, and quantification of IA-labeled cysteines30. Electrophile-modified cysteines are quantified by measuring the MS1 chromatographic peak ratios (R values) for heavy (DMSO-treated) over light (electrophile-treated) samples, with higher R values reflecting greater sensitivity to the electrophile. In this format, competitive isoTOP-ABPP can assay electrophiles against 1000+ cysteines in parallel directly in native proteomes without requiring any chemical modification to the electrophiles themselves. - We applied competitive isoTOP-ABPP to quantitatively profile the proteome reactivity of three representative endogenous electrophiles—HNE, 15d-PGJ2, and 2-HD, each of which possesses an α,β-unsaturated carbonyl that can react with nucleophilic cysteines via Michael addition (
FIG. 1b ). Competitive isoTOP-ABPP experiments were performed in quadruplicate using the soluble proteome of the human breast cancer cell line MDA-MB-231 cell line. Proteomes were treated with 100 μM HNE, 15d-PGJ2, or 2-HD, for 60 minutes, followed by the IA-probe (100 μM, 60 min). A total of 1400 cysteine reactivities were quantified across the aggregate data set, with at least 900 cysteine reactivities quantified for each LDE (FIG. 2a ) more than 750 of which were quantified for all the three electrophiles. Most of the cysteine reactivities (>98%) were unaffected or only marginally affected by LDE treatment (R<5); however, a select subgroup showed marked reductions in their IA-probe reactivities (R>5) following exposure to one or more LDEs (FIG. 2a ). A closer examination of these LDE-inhibited cysteines revealed a distinct proteome reactivity profile for each tested electrophile, with NE and 15d-PGJ2 both targeting several cysteines in the proteome, the majority of which showed preferential reactivity with one of the two LDEs, and 2-HD exhibiting no detectable high sensitivity (R>5) targets (FIG. 2a ). - The quantitative ranking of cysteines based on the magnitude and selectivity of their inhibition illuminated “hot spots” for LDE reactivity in the proteome (
FIG. 2a and Table 1). Examples included Cys22 of ZAK (or MLTK/MLK7) and Cys848 of MMS19, which were completely blocked with notable selectivity by HNE and 15d-PGJ2, respectively (FIG. 2c ). Competitive isoTOP-ABPP also identified several proteins that possess multiple reactive cysteines, only one of which proved sensitive to competitive blockade by an LDE (FIG. 2d ). These data demonstrate that the quantified R values reflect measurements of individual LDE-cysteine reactions rather than general changes in protein abundance potentially caused by LDE exposure. In this regard, we did not observe any instances of multiple LDE-sensitive cysteines appearing on the same protein (see Table 1, below). We also asked whether the intrinsic reactivity of cysteines, as determined previously by measuring their extents and rates of IA labeling30, might be predictive of sensitivity to LDEs. However, we found that most of the LDE-sensitive cysteines displayed moderate, rather than high IA-reactivity, suggesting that their modification by LDEs depend not only on cysteine nucleophilicity, but also on molecular recognition of the LDEs. - Having found that individual LDEs show markedly distinct cysteine-reactivity profiles, we next focused on identifying the most sensitive sites for LDE reactivity in the proteome by performing a concentration-dependent analysis with HNE. The MDA-MB-231 cell proteome was treated with varying concentrations of HNE (5, 10, 50, 100 and 500 μM) for 60 min and then the IA-labeling profile of each reaction was quantitatively compared to a DMSO-control sample by isoTOP-ABPP. In aggregate, these profiles identified ˜1100 IA-labeled cysteines, many of which showed reduced labeling signals in the presence of HNE, including 8 of the 14 HNE-modified cysteines identified in a previous proteomic study that used a biotinamidohexanoic acid hydrazide probe to enrich and identify (but not to quantify) HNE-modified cysteines17.
- By combining the R values at all 5HNE concentrations, we could extrapolate IC50 values for HNE-blockade of IA probe-labeling for ˜700 of the 1100 cysteines (
FIG. 3a ). This analysis revealed that the vast majority of cysteines were modified by HNE with low potency (IC50 values>100 μM), but a select few cysteines, including C22 of ZAK, C41 of EEF2, C24 of FN3KRP, and C1001 of RTN4, exhibited much higher sensitivities with IC50 values ranging from 6 to 23 μM (FIG. 3a, b ). We next tested whether these hypersensitive cysteines were also inhibited by HNE in situ by treating MDA-MB-231 cells with 50 or 100 μM HNE and then preparing proteomes for analysis by competitive isoTOP-ABPP. These experiments confirmed that the most HNE-sensitive cysteines identified in vitro were also strongly inhibited by HNE in situ (FIG. 3c, d ). We also uncovered another set of cysteines that showed reductions in IA-probe labeling in situ, but not in vitro (FIG. 3d , black diamonds). This finding suggests that certain proteins may preferentially react with HNE in living cells, although we cannot exclude at this point that the reductions in IA probe labeling observed for these proteins reflect a decrease in their overall abundance in HNE-treated cells. - ZAK kinase (also known as MLK7 or MLTK) is part of the mitogen-activated protein kinase (MAPK) network and functions as a MAPK kinase kinase (MAP3K)32,33 There are at least 20 MAP3Ks encoded by the human genome and they are activated by diverse stimuli to phosphorylate and activate downstream MAPK kinases (MAP2Ks) to regulate critical cellular functions, such as differentiation, proliferation and apoptosis34. Previous studies have shown that ZAK can activate all three major MAPK (ERK, JNK, and p38) pathways in mammalian cells,32,35,36 with some preference for JNK32 and is involved in response pathways to stressors such as osmotic shock33, UV radiation37, and chemotherapeutic agents36. Sequence and structure comparisons allowed us to map the HNE-sensitive cysteine in ZAK (C22) to a location proximal to the glycine-rich ATPbinding loop (“P-loop”) (
FIG. 4a ). Interestingly, among all 20 human MAP3Ks, ZAK is the only member that possesses a cysteine at this position (FIG. 4a ), and this cysteine is highly conserved across ZAK orthologues in vertebrates. - This information, combined with the high sensitivity displayed by C22 for HNE (
FIG. 3a ) motivated us to further characterize this interaction and its impact on ZAK activity. We first expressed FLAG-tagged versions of wild type (WT) and a C22A mutant of ZAK by stable transfection in HEK293T cells and found that WT-ZAK showed much stronger IA-rhodamine probe labeling as measured by gel-based ABPP. This result is consistent with our isoTOP-ABPP data sets, which identified C22 as the most IA-reactive cysteine in ZAK30. The gel signals for IA-labeling of WT-ZAK were blocked by pretreatment with HNE over a concentration range that closely matched the HNE-sensitivity profile observed for C22 in competitive isoTOP-ABPP experiments (FIG. 4b , compare toFIG. 3a ). Given that competitive isoTOP-ABPP measures blockade of IA-labeling of cysteines by LDEs, we next used an alkyne-functionalized HNE probe (HNEyne)16 to verify direct labeling of WT-, but not the C22A-ZAK mutant in vitro and in living cells (FIG. 4c ). We then assessed the impact of HNE labeling on ZAK activity using an in vitro Myelin Basic Protein (MBP) substrate assay38 which showed that HNE inhibited WT-, but not C22A-ZAK in a concentration-dependent manner (FIG. 4d ). We note that C22AZAK exhibited reduced basal activity compared to WT-ZAK, but the residual activity of C22-ZAK, which was still much greater than a catalytically dead K45M-ZAK mutant, was insensitive to HNE (FIG. 4d ). Taken together, these data indicate that C22 contributes to the intrinsic catalytic activity of ZAK and reaction of this residue with HNE produces complete inhibition of the kinase. Considering further that C22 is predicted to reside adjacent to the ATP-binding loop of ZAK, we postulated that the HNE-induced loss of kinase activity might be due to blockade of ATP-binding. We tested this hypothesis by performing a competitive SILAC (Stable Isotope Labeling by Amino acids in Cell culture)-ABPP39 experiment using an acylphosphate-ATP probe40, which revealed that probe-labeling of ZAK, but not other kinases, was profoundly reduced in cell proteomes treated with HNE (FIG. 4e ). - We next set out to assess the functional effects of HNE modification of ZAK in human cells. HEK-293T cells were stably transfected with cDNAs for WT-ZAK, C22A-ZAK, or K45M-ZAK and the activation state of their MAPK signaling pathways was monitored by western blotting with anti-phosphoprotein antibodies. WT-ZAK-expressing cells, but not C22A- or K45M-ZAK-expressing cells showed significantly increased JNK and, to a lesser extent, p38 and ERK pathway activation compared to mock-transfected cells (
FIG. 5a, b ). These cellular data are consistent with previous studies showing that overexpression of WT-ZAK in mammalian cells preferentially activates the JNK pathway32 and with our in vitro substrate assay results, which revealed substantially reduced and complete loss of activity for the C22A- and K45M ZAK mutants, respectively (FIG. 4d ). We next treated cells with H2O2 (1 mM, 30 min) to induce oxidative stress, a process that is known to activate the JNK pathway41,42. H2O2 treatment stimulated JNK activity in both WT- and C22A-ZAK-transfected cells (but not K45M-ZAK-transfected cells), with WT-ZAK cells showing the greater level of activation (FIG. 5b,c ). Strikingly, however, pre-treatment with HNE (100 μM, 30 min) produced opposing effects in WT- and C22A-ZAK cells, blocking H2O2-dependent JNK activation in the former cell model, while hyper-activating JNK activity in the latter. We interpret these findings to indicate the existence of both ZAK-dependent and ZAK-independent pathways for HNE modulation of JNK activation (FIG. 5d ). By modifying C22 on ZAK, HNE blocks the contribution that this kinase makes to the activation of the JNK pathway. HNE is also known to itself promote oxidative stress43,44 that likely adds to the effects of H2O2 and, in the context of an HNE-resistant C22A-ZAK mutant, would serve to further augment activation of the JNK pathway. In this model, the HNE-ZAK interaction acts as a negative-feedback loop that tempers activation of the JNK pathway under high and/or persistent levels of oxidative stress (FIG. 5d ). We further tested this idea by evaluating the effects of HNE alone on JNK pathway activity in ZAK-transfected cells. A dramatic concentration-dependent activation of JNK was observed in C22A-ZAK-transfected cells, but not in WT-ZAK-transfected cells, which showed higher basal JNK activation that was mostly unaffected by HNE (FIG. 5e,f ). While we were initially surprised that HNE treatment did not appear to block the basal JNK activation caused by WT-ZAK, we should note that HNE also activated JNK in mock-transfected cells to a level that matched the basal JNK activity observed in WT-ZAK-transfected cells. Thus, the residual JNK activation observed in WT-ZAK-transfected cells may reflect ZAK-independent pathways of JNK activation by HNE (FIG. 5e,f ). That JNK activation was much higher in C22A-ZAK cells compared to the other cell models indicates this HNE-insensitive form of ZAK, which still retains some catalytic activity (seeFIG. 4d ), combines with ZAK-independent, HNE-stimulated pathways to further enhance JNK activation. - Long viewed as biomarkers of oxidative damage, LDEs have more recently gained attention as second messengers that can regulate diverse cellular processes8,9. These findings have inspired the advent of chemoproteomic methods to globally map LDE-protein interactions5,16-19. To date, these large-scale profiling efforts have focused on the qualitative inventorying of LDE-reactive proteins in cell and tissue proteomes, generating lists of many candidate targets and pathways for LDE action. Considering, however, that the signaling and pathophysiological functions of LDEs may differ across the endogenous concentration ranges found for these compounds, it is imperative to understand the potencies of LDE-protein interactions in biological systems. Building on past studies showing that cysteine residues are the principal sites of protein modification by HNE28,29, we created a competitive isoTOP-ABPP platform to quantitatively map LDE reactivity across 1000+ cysteines in parallel directly in native proteomes. The output of this study was the identification of discrete sites of hypersensitivity, or “hot spots”, for LDE modification in the human proteome. Notably, most of these sites show clear preference for reacting with one of the three tested LDEs (HNE, 15d-PGJ2, 2-HD) and moderate, but not extreme levels of intrinsic reactivity. These findings, taken together, indicate that the potency of LDE-protein reactions in the proteome is dictated by a combination of molecular recognition and enhanced cysteine nucleophilicity.
- Among the most LDE-sensitive cysteines, C22 of ZAK stood out as a particularly intriguing event, given the proposed role that this kinase plays in activating JNK, ERK, and p38 MAPK pathways in both cancer35 and inflammation36. To date, only a handful of studies have investigated ZAK function and its modes of regulation remain poorly understood. Our findings identified ZAK as one of the highest potency targets of HNE in the human proteome. That HNE inhibits human ZAK by modifying an active site-proximal cysteine conserved among ZAK orthologues, but not other MAP3K enzymes, suggests ZAK acts as a special node in MAPK signaling pathways that confers sensitivity to lipid oxidation products. In this way, HNE modification of ZAK may limit the extent of JNK activation caused by oxidative stress, which could help certain cell types, such as tumor and immune cells, survive in the presence of high levels of reactive oxygen species. Further studies of ZAK function would benefit from the development of selective inhibitors for this enzyme. It is noteworthy, in this regard, that covalent inhibitors have recently been introduced for many kinases24,45-47. These inhibitors often target cysteine residues in or near kinase active sites, which leads us to speculate that the C22-HNE interaction discovered herein may offer a medicinal chemistry starting point for the development of ZAK inhibitors. Toward this end, competitive ABPP methods should offer a useful strategy to assess inhibitor target engagement and selectivity48. Beyond ZAK, we also identified several other kinases in our competitive isoTOP-ABPP experiments that possess cysteines that were inhibited by HNE, albeit with lower potencies. Prominent among these was cysteine (C311) in AKT1/2/3, which is an active site-proximal residue implicated in substrate-binding49 and was inhibited by HNE with an IC50 value of ˜60 μM. These proteomic findings nicely confirm recent work showing that recombinant AKT2 is modified by HNE on C31125. Finally, we should emphasize that C22 in ZAK is just one of several hypersensitive sites for LDE modification identified in our competitive isoTOP-ABPP experiments (
FIG. 2 ). We expect that more in-depth biological studies on these high-sensitivity targets of LDEs will reveal additional modes of crosstalk between oxidative stress and signaling pathways in mammalian cells. - From a methodological perspective, we believe that competitive isoTOP-ABPP offers several advantages over more conventional proteomic approaches for the discovery and characterization of protein-small molecule reactions in biological systems. First, quantitative inhibition values are measured in relative terms that are independent of absolute protein abundance. The method is therefore able to sift through signals that span a broad range of intensities to identify reactive sites that are more likely to bear functional consequence. Here, the site-specificity afforded by isoTOP-ABPP is important, since it permits the discovery of potent electrophile-cysteine reactions that may occur on proteins that display several lower-affinity cysteine-electrophile adducts (e.g., EEF2,
FIG. 2d ). Electrophiles also vary considerably in their structures and the stability of the protein adducts that they form. These features can complicate the direct detection of electrophile-protein interactions in proteomic studies. - Table 1, below, shows peptide sequences, parental protein names, and R values for IA-labeled cysteines that exhibit IA-labeling competed by one or more LDE with R values>5. HNE-competed cysteines are shaded in pink and 15d-PGJ2-competed cysteines are shaded in green. Note that REEP5 displays significant competition by both LDEs.
- The isoTOP-ABPP method ABPP for quantitative mapping of cysteine-reactive, lipid-derived electrophile (LDE) reactions in proteomes, cells, tissues, or organisms can be used to determine if one or more proteins in the set of proteins therefrom possesses one or more domains, each comprising a reactive cysteine residue, has at least a low affinity (e.g., high micromolar affinity) for any selected structural module, and serves to identify what protein has that affinity. By combining a plurality of low-affinity structural modules having affinity for a particular protein binding site, a high affinity ligand for that protein can be constructed. Identification of a high affinity ligand can serve as a structural lead in the development of compounds targeting a particular protein, which can lead to the development of medicinal compounds. Similarly, identification of the protein target of the ligand can be of value in determining a possibly unknown function for the targeted protein.
-
TABLE 1 IPI number Name Sequence 4-HNE PGJ2 2-HD IPI00099986.5 FN3KRP ATGHSGGGC*ISQGR (SEQ ID NO: 3) >15 1.62 1.03 IPI00329638.10 ZAK FDDLQFFENC*GGGSFGSVYR (SEQ ID NO: 4) >15 4.24 1.05 IPI00021766.4 RTN4 YSNSALGHVNC*TIK (SEQ ID NO: 5) 11.78 2.93 1.1 IPI00186290.6 EEF2 STLTDSLVC*K (SEQ ID NO: 6) 11.63 3.31 1.06 IPI00141318.2 CKAP4 SSSSSSASAAAAAAAASSSASC*SR 7.73 3.33 1.08 (SEQ ID NO: 7) IPI00024670.5 REEP5 NC*MTDLLAK (SEQ ID NO: 8) 7.08 7.38 1.18 IPI00018235.3 PEF1 QALVNC*NWSSFNDETCLMMINMFDK 5.14 2.9 1.25 (SEQ ID NO: 9) IPI00024673.2 MAPK9 TLEEFQDVYLVMELMDANLC*QVIHMELDHER 5.08 1.78 1.14 (SEQ ID NO: 10) IPI00154451.6 MMS19 LMGLLSDPELGPAAADGFSLLMSDC*TDVLTR 1.78 >15 1.1 (SEQ ID NO: 11) IPI00010158.3 CHRAC1 ATELFVQC*LATYSYR (SEQ ID NO: 12) 1.94 12.32 1.06 IPI00551062.2 TNRC5 QC*DVLVEEFEEVIEDWYR (SEQ ID NO: 13) 1.29 11.77 1.06 IPI00024254.3 IFIT3 GLNPLNAYSDLAEFLETEC*YQTPFNK 1.51 9.43 1.23 (SEQ ID NO: 14) IPI00639841.2 PEC1 WLSDEC*TNAVVNFLSR (SEQ ID NO: 15) 1.86 8.23 1.06 IPI00302925.3 CCT8 IAVYSC*PFDGMITETK (SEQ ID NO: 16) 1.14 7.75 1.06 IPI00155601.1 MACROD1 LEVDAIVNAANSSLLGGGGVDGC*IHR 1.56 7.65 1.02 (SEQ ID NO: 17) IPI00003814.1 MAP2K6 MC*DFGISGYLVDSVAK (SEQ ID NO: 18) 1.9 7.43 1.15 IPI00219103.6 HPCA LLQC*DPSSASQF (SEQ ID NO: 19) 2.17 7.21 1.13 IPI00793696.1 RPL24 C*ESAFLSK (SEQ ID NO: 20) 1.74 6.78 1.25 IPI00027223.2 IDH1 SEGGFIWAC*K (SEQ ID NO: 21) 1.3 6.66 1.13 IPI00021329.3 WDR45L C*NYLALVGGGK (SEQ ID NO: 22) 3.34 6.45 1.06 IPI00640155.1 PSMB8 LLSNMMC*QYR (SEQ ID NO: 23) 1.19 5.86 1.05 IPI00022431.1 AHSG C*DSSPDSAEDVR (SEQ ID NO: 24) 1.35 5.73 1.12 IPI00007675.6 DYNC1L11 VGSFGSSPPGLSSTYTGGPLGNEIASGNGGAAAGD 2.59 5.18 1.1 DEDGQNLWSC*ILSEVSTR (SEQ ID NO: 25) IPI00010153.5 RPL23 ISLGLPVGAVINC*ADNTGAK 1.73 5.06 1.87 (SEQ ID NO: 26) - MDA-MB-231 cells were grown in L15 media supplemented with 10% fetal bovine serum at 37° C. in a CO2-free incubator. For in vitro labeling experiments, cells were grown to 100% confluency, washed three times with PBS and scraped in cold PBS. Cell pellets were isolated by centrifugation at 1400×g for 3 min, and the cell pellets stored at −80° C. until further use. The harvested cell pellets were lysed by sonication in PBS buffer and fractionated by centrifugation (100,000×g, 45 min.) to yield soluble and membrane proteomes. The proteomes were prepared fresh from the frozen cell pallets prior to each experiment.
- HNE was purchased from EMD biosciences, 15d-PGJ2 was purchased from Cayman Chemicals and 2-HD was purchased from Santa Cruz Biotechnology. Proteome samples were diluted to a 4 mg protein/mL solution in PBS. For each profiling experiment, one aliquot of the proteome sample (0.5 mL) was treated with 100 μM of LDE using 5 μL of a 10 mM stock and the other aliquot was treated with 5 μL of either ethanol (for HNE and 15d-PGJ2) or DMSO (for 2-HD) as control. For the concentration-dependent profiling experiments using HNE, aliquots of the proteomes (0.5 mL each) were treated with 5, 10, 50, 100 and 500 μM of HNE using 5 μL of 0.5, 1.0, 5, 10 and 50 mM of stock solution, respectively. After 60 minutes of treatment at room temperature, both the LDE-treated and control aliquots were passed through a NAP-5 Sephadex column (GE healthcare) to remove any unreacted LDE. The volume of each aliquot was increased to 1 mL with the concentration at 2 mg/mL after this step.
- After MDA-MB-231 cells were grown to 100% confluency, the media was removed and replaced with fresh serum-free media containing 100 μM HNE (20 μL of 50 mM stock in 10 mL media). A control flask of cells was treated with 10 mL of serum-free media containing 20 uL of ethanol in parallel. The cells were incubated at 37° C. for 1 hour and harvested as detailed above to prepare HNE-treated and control proteomes, respectively.
- Each of the control and LDE-treated proteome samples (˜2 mg protein/mL in 1 mL volume) was treated with 100 μM of IA-probe using 10 μL of a 10 mM stock in DMSO. The labeling reactions were incubated at room temperature for 1 hour. Click chemistry (acetylene-azide cycloaddition) was performed by the addition of 100 μM of either the Heavy-TEV-Tag (for the control sample) or Light-TEV-Tag (for the LDE-treated sample) (20 μL of a 5 mM stock), 1 mM TCEP (fresh 50× stock in water), 100 μM ligand (17× stock in DMSO:t-Butanol 1:4) and 1 mM CuSO4 (50× stock in water). Samples were allowed to react at room temperature for 1 hour. After the click chemistry step, the light and heavy-labeled samples were mixed together and centrifuged (5900×g, 4 min, 4° C.) to pellet the precipitated proteins.
- The pellets were washed twice in cold MeOH, after which the pellet was solubilized in PBS containing 1.2% SDS via sonication and heating (5 min, 80° C.). Samples were subjected to streptavidin enrichment of probe-labeled proteins, sequential on-bead trypsin and TEV digestion, and liquid chromatography-mass spectrometry (LC-MS) analysis according to the published isoTOP-ABPP protocol30.
- IA-probe labeled peptides were identified by SEQUEST2 and DTASelect3, and the quantification of heavy/light ratios (isoTOP-ABPP ratios, R) was performed by an in-house software (CIMAGE) as previously described. The software was advanced to be able to detect and quantify cases where near complete LDE blockade of IA-probe labeling was achieved (e.g., very small or no light peak) and assign an empirical ratio cut-off of 15 to each of such cases. Each experiment consisted of multiple LC/LCMS/MS runs: either HNE, 15d-PGJ2 and 2-
HD 100 μM competition, or HNE competition at different concentrations. All runs were searched using SEQUEST and filtered with DTASelect as described above. Because the mass-spectrometer was configured for data-dependant fragmentation, peptides are not always identified in every run. In the case of probe-modified peptides that were sequenced in one, but not the other runs, a featured algorithm of CIMAGE was utilized to identify the corresponding peak pairs in the runs without the SEQUEST identification and obtain quantification as previously described. In all cases, the false-positive rate after quantification was found to be less than 1%. - After ratios for unique peptide entries are calculated for each experiment, overlapping peptides with the same labeled cysteine (e.g., same local sequence around the labeled cysteines but different charge states, MudPIT segment numbers, or tryptic termini) are grouped together, and the median ratio from each group is reported as the final ratio (“R”).
- Retroviral Overexpression of FLAG-Tagged ZAK proteins in HEK-293T Cells
- Full-length cDNA encoding human ZAK-β (BC001401) in pOTB7 was purchased from Open BioSystems and subcloned into pFLAG-CMV-6c (Sigma-Aldrich). ZAK-C22A and ZAK-K45M mutants were generated by QuikChange site-directed mutagenesis using the
primer 5′-atttgatgacttgcagttttttgaaaacgccggtggaggaagttttg-3′ (SEQ ID NO:1) and 5′-ggacaaggaggtggctgtaatgaagctcctcaaaatagag-3′ (SEQ ID NO:2) and their complements. Wild-type and mutant ZAK were cloned into a modified pCLNCX retroviral vector. Retrovirus was prepared by taking 3.0 μg of each of pCLNCX and pCL-Ampho vectors and 18 μL of FuGENE HD reagent (Roche) to transfect 60% confluent HEK-293T cells. Medium was replaced after 1 day of transfection and on the next day virus-containing supernatant was collected, filter sterilized and stored at −70° C. 1 mL of virus-containing medium was used to infect target cells in presence of 8 μg/mL of polybrene for 72 hours and infected cells were selected in medium containing 100 μg/mL of hygromycin. Surviving cells after the selection were expanded and cultured in regular DMEM medium with 10% FCS. - HEK-293T cells with stable expression of wild-type or mutant ZAK were grown to 100% confluency on a 10 cm plate. Cells were collected, washed with cold PBS (2×10 mL) and lysed in 1 mL of PBS supplemented with 1× complete EDTA-free protease inhibitor cocktails by sonication. Cell lysates were fractionated by centrifugation (100,000×g, 45 min.) and the soluble fraction was incubated with 50 μL of Anti-FLAG M2 affinity gel (Sigma-Aldrich) at 4° C. for 3 hours. Beads were washed with 5×1 mL of cold PBS (10 min per incubation) and FLAG-ZAK were eluted by either 150 μg/mL of FLAG-peptide solution provided by manufacturer or by 4× gel loading buffer depending on the downstream applications.
- FLAG-tagged wild-type and C22A mutant ZAK were immunoprecipitated from HEK-293T cells (1×107). After washing with PBS, the beads were suspended in 100 μL of PBS buffer and labeled with 250 nM of IA-rhodamine (add 1 μL of 25 μM probe stock in DMSO). After 1 hour of labeling at 4° C., 50 μL of 4× gel loading buffer was added and the beads were boiled for 5 min to elute the bound proteins. Gel samples were separated by SDS-PAGE (50 μL of sample/lane) and visualized in-gel using a Hitachi FMBio II flatbed laser-induced fluorescence scanner (MiraiBio, Alameda, Calif.). For testing HNE blockade on IA labeling of ZAK by gel, soluble lysate of HEK-293T overexpressing WT-ZAK were incubated with 10, 50 and 100 μM of HNE (add 2 μL of 5, 25 and 50 mM stock) for 30 mins and then subjected to immunoprecipitation.
- 50 μL of soluble lysate (1 mg/mL in PBS) of HEK-239T cells transfected with mock ZAKWT and ZAK-C22A was labeled with 10 μM of HNEyne4 (Cayman Chemicals, 1 μL of 500 μM stock in ethanol) for 1 hour at room temperature. Cycloaddition was performed with 200 μM rhodamine-azide, 1 mM TCEP, 100 μM TBTA ligand and 1 mM CuSO4. The reaction was allowed to proceed at room temperature for 1 hour before quenching with 20 μL of 4× SDS-PAGE loading buffer (reducing). Quenched reactions were separated by SDS-PAGE (40 μL of sample/lane) and visualized in-gel using a fluorescence scanner. For the in situ HNEyne labeling, WT- and C22A-ZAK transfected cells were grown in a 6-well plate to 100% confluency and switched into 1 mL of serum-free DMEM medium. Cells were labeled with 5 μM of HNEyne probe (1 μL of 5 mM stock) for 1 hour at 37° C. Cells were then harvested, washed with cold PBS and lysed in 200 μL of PBS with protease inhibitors. 50 μL of soluble lysates were subjected to the cycloaddition protocol as described above and probe labeling was monitored by in-gel fluorescence.
- The kinase activity assay protocol was adapted from Yu et al5. Kinase assay buffers, Myeilin Basic Protein (MBP) substrate, and ATP stock solution were purchased from SignalChem. Radio-labeled [33P]-ATP was purchased from PerkinElmer. 10 mg of soluble lysate of HEK-293T cells transfected with each of wild-type, C22A and K45M of ZAK were immunoprecipitated and then eluted with 2×300 μL FLAG-peptide buffer. Each sample was concentrated to 100 μL using an Amicon centrifugal filter (30 kDa cutoff) and exchanged to the assay kinase buffer (5 mM MOPS, pH7.2, 2.5 mM β-glycerol-phosphate, 5 mM MgCl2, 1 mM EGTA, 0.4 mM EDTA, 0.05 mM DTT and 40 ng/μL BSA) to a final volume around 10 μL. For each ZAK construct, 4 reactions were set up and each reaction starts with mixing 10 μL of immunoprecipiated ZAK, 5 μL of MBP (1 mg/mL) and 5 μL of HNE (10 or 100 μM) or H2O together. No-enzyme and no-substrate controls were prepared in parallel. The mixed samples were incubated on ice for 15 min and 5 μL of [33P]-ATP assay cocktail (250 μM, 167 μCi/mL) was then added to initiate the kinase reaction. Each reaction mixture was incubated in 30° C. for 15 min and the reaction was terminated by spotting 20 μL of the reaction mixture onto individual pre-cut strips of phosphocellulose P81 paper. The spotted P81 strips were air dried and then washed with 10 mL of 1% phosphoric acid for 3×10 min. ZAK activity was measured by counting the radioactivity on the P81 paper in the presence of scintillation fluid in a scintillation counter after subtracting the value obtained from the corresponding no substrate control, and was normalized to that of ZAK-WT without HNE treatment. Experiments were performed in triplicates. 10 μL of each ZAK variant used in setting up the kinase reaction were run on a SDS-PAGE gel and immunoblotted with an anti-FLAG antibody to ensure that they are enriched at similar levels.
- HEK-293T cells with stable expression of wild-type ZAK were passaged six times in DMEM medium minus 1-lysine and 1-arginine (Thermo) supplemented 10% dialyzed FBS (Gemini), 1% PSQ (1% vol/vol 10,000 units penicillin, 10 mg streptomycin, 29.2 mg lglutamate solution) and 100 μg/mL [13C6, 15N4] l-arginine-HCl and [13C6, 15N2] l-lysine-HCl (heavy) or 1-arginine-HCl and l-lysine-HCl (light) (Sigma-Aldrich). Soluble proteomes of light and heavy ZAK-WT transfected HEK-293T cells (3 mL each at 7 mg/mL) were treated with 100 μM of HNE (6 μL of 50 mM stock) or EtOH for 30 min at room temperature. The samples were gel filtrated by PD-10 columns (GE healthcare) to remove unreacted HNE as well as excessive ATP molecules in proteomes. Each aliquot of light and heavy proteomes (0.5 mL, 6 mg/mL) was labeled with 20 μM of acylphosphate-ATP probe (ActivX Biosciences) and then mixed together to proceed with reduction/alkylation, streptavidin enrichment, trypsin digest according to a modified version of the vendor-provided “Xsite Kinase Analysis” protocol6. The trypsin digested samples were analysed by LC-MS/MS and enriched kinase peptides were identified by SEQUEST and DTASelect. The amounts of probe-labeled kinases with and without HNE treatment were quantified using the CIMAGE module that was developed for quantitative SILAC-ABPP chemoproteomic profiling7. As internal controls, light and heavy proteomes were trypsin digested without probe labeling and streptavidin enrichment, and analysed by LC-MS/MS to quantify the basic level of each kinase between light and heavy samples. The normalized ratio, for each identified kinase, was computed by dividing the ratio from the probe-labeling experiment by that from the unenriched experiment, and these ratios (from four replicates) were used to calculate the means and standard deviations that were reported in
FIG. 4 e. - Mouse and rabbit monoclonal antibodies against phospho-ERK 1/2 (Thr202/Tyr204), phosphor-SAPK/JNK (Thr183/Thr185), phosphor-p38 MAPK (Thr180/Thr182) and total ERK 1/2, SAPK/JNK and p38 MAPK were purchased from Cell Signaling Technology. HEK-293T cells transfected with mock, WT-ZAK, C22A-ZAK and K45M-ZAK were seeded into a 12-well plate with 2.5×105 cells per well. Cells were grown in regular DMEM medium with 10% FBS for 24 hours and starved in serum-free DMEM medium for another 24 hours. Cells were then treated at 37° C. either with 100 μM of HNE (2 μL of 50 mM stock) for 30 min followed by 1 mM of H2O2 for 30 min, or with 50 and 100 μM of HNE alone for 60 min. After the treatment, cells were harvested, washed with 2×1 mL of cold PBS, and then lysed by sonication in 100 μL of PBS buffer supplemented with 1× complete protease inhibitors cocktail and 1×PhosSTOP phosphatase inhibitors cocktail (Roche). 30 μg of soluble lysate of each sample was separated by SDS-PAGE, transferred to nitrocellulose membrane, blocked in 5% milk TBST and blotted against the primary antibodies (1:2000) listed above for 16 hours at 4° C. After washing in TBST (3×10 minutes), membranes were blotted with IRDye secondary antibodies (1:10,000) for 1 hour at room temperature and scanned by an Odyssey imaging system (LI-COR). Protein band intensities were quantified by ImageJ8 and ratios of phosphor-MAPK over total MAPK were computed. Experiments were repeated in at least four replicates.
-
- 1. Walsh, C. T. Posttranslational Modification of Proteins. Expanding Nature's Inventory., (Roberts & Company, Greenwood Village, Colo., 2005).
- 2. Leitner, A. & Lindner, W. Chemistry meets proteomics: the use of chemical tagging reactions for MS-based proteomics. Proteomics 6, 5418-34 (2006).
- 3. Tate, E. W. Recent advances in chemical proteomics: exploring the posttranslational proteome. J Chem Biol 1, 17-26 (2008).
- 4. Hang, H. C. & Linder, M. E. Exploring protein lipidation with chemical biology. Chem Rev 111, 6341-58 (2011).
- 5. Jacobs, A. T. & Marnett, L. J. Systems analysis of protein modification and cellular responses induced by electrophile stress. Acc Chem Res 43, 673-83 (2010).
- 6. Leonard, S. E. & Carroll, K. S. Chemical ‘omics’ approaches for understanding protein cysteine oxidation in biology. Curr
Opin Chem Biol 15, 88-102 (2011). - 7. Gueraud, F., Atalay, M., Bresgen, N., Cipak, A., Eckl, P. M., Huc, L., Jouanin, I., Siems, W. & Uchida, K. Chemistry and biochemistry of lipid peroxidation products. Free Radic Res 44, 1098-124 (2010).
- 8. Dubinina, E. E. & Dadali, V. A. Role of 4-hydroxy-trans-2-nonenal in cell functions. Biochemistry (Mosc) 75, 1069-87 (2010).
- 9. Fritz, K. S. & Petersen, D. R. An overview of the chemistry and biology of reactive aldehydes. Free Radic Biol Med (2012).
- 10. Rudolph, T. K. & Freeman, B. A. Transduction of redox signaling by electrophile-protein reactions.
Sci Signal 2, re7 (2009). - 11. Fritz, K. S. & Petersen, D. R. Exploring the biology of lipid peroxidation-derived protein carbonylation.
Chem Res Toxicol 24, 1411-9 (2011). - 12. Leonarduzzi, G., Robbesyn, F. & Poli, G. Signaling kinases modulated by 4-hydroxynonenal. Free Radic Biol Med 37, 1694-702 (2004).
- 13. Jacobs, A. T. & Marnett, L. J. Heat shock factor 1 attenuates 4-Hydroxynonenal mediated
apoptosis: critical role for heat shock protein 70 induction and
stabilization of Bcl-XL. J Biol Chem 282, 33412-20 (2007). - 14. Surh, Y. J., Na, H. K., Park, J. M., Lee, H. N., Kim, W., Yoon, I. S. & Kim, D. D. 15-Deoxy-Delta(1)(2),(1)(4)-prostaglandin J(2), an electrophilic lipid mediator of antiinflammatory
and pro-resolving signaling. Biochem Pharmacol 82, 1335-51
(2011). - 15. Chipuk, J. E., McStay, G. P., Bharti, A., Kuwana, T., Clarke, C. J., Siskind, L. J., Obeid, L. M. & Green, D. R. Sphingolipid metabolism cooperates with BAK and BAX to promote the mitochondrial pathway of apoptosis. Cell 148, 988-1000
(2012). - 16. Vila, A., Tallman, K. A., Jacobs, A. T., Liebler, D. C., Porter, N. A. & Marnett, L. J. Identification of protein targets of 4-hydroxynonenal using click chemistry for ex vivo biotinylation of azido and alkynyl derivatives. Chem Res Toxicol 21, 432-44 (2008).
- 17. Codreanu, S. G., Zhang, B., Sobecki, S. M., Billheimer, D. D. & Liebler, D. C. Global analysis of protein damage by the lipid electrophile 4-hydroxy-2-nonenal.
Mol Cell Proteomics 8, 670-80 (2009). - 18. Han, B., Hare, M., Wickramasekara, S., Fang, Y. & Maier, C. S. A comparative ‘bottom up’ proteomics strategy for the site-specific identification and quantification of protein modifications by electrophilic lipids. J Proteomics 75, 5724-33 (2012).
- 19. Roe, M. R., Xie, H., Bandhakavi, S. & Griffin, T. J. Proteomic mapping of 4-hydroxynonenal protein modification sites by solid-phase hydrazide chemistry and mass spectrometry. Anal Chem 79, 3747-56 (2007).
- 20. Kim, H. Y., Tallman, K. A., Liebler, D. C. & Porter, N. A. An azido-biotin reagent for use in the isolation of protein adducts of lipid-derived electrophiles by streptavidin catch and photorelease.
Mol Cell Proteomics 8, 2080-9 (2009). - 21. Perluigi, M., Coccia, R. & Butterfield, D. A. 4-Hydroxy-2-nonenal, a reactive product of lipid peroxidation, and neurodegenerative diseases: a toxic combination illuminated by redox proteomics studies. Antioxid Redox Signal 17, 1590-609 (2012).
- 22. Aldini, G., Carini, M., Vistoli, G., Shibata, T., Kusano, Y., Gamberoni, L., Dalle-Donne, I., Milzani, A. & Uchida, K. Identification of actin as a 15-deoxy-Delta12,14-prostaglandin J2 target in neuroblastoma cells: mass spectrometric, computational, and functional approaches to investigate the effect on cytoskeletal derangement. Biochemistry 46, 2707-18 (2007).
- 23. Singh, J., Petter, R. C., Baillie, T. A. & Whitty, A. The resurgence of covalent drugs. Nat
Rev Drug Discov 10, 307-17 (2011). - 24. Liu, Q., Sabnis, Y., Zhao, Z., Zhang, T., Buhrlage, S. J., Jones, L. H. & Gray, N. S. Developing irreversible inhibitors of the protein kinase cysteinome.
Chem Biol 20, 146-59 (2013). - 25. Shearn, C. T., Fritz, K. S., Reigan, P. & Petersen, D. R. Modification of Akt2 by 4-hydroxynonenal inhibits insulin-dependent Akt signaling in HepG2 cells.
Biochemistry 50, 3984-96 (2011). - 26. Bennaars-Eiden, A., Higgins, L., Hertzel, A. V., Kapphahn, R. J., Ferrington, D. A. & Bernlohr, D. A. Covalent modification of epithelial fatty acid-binding protein by 4-hydroxynonenal in vitro and in vivo. Evidence for a role in antioxidant biology. J Biol Chem 277, 50693-702 (2002).
- 27. Higdon, A. N., Dranka, B. P., Hill, B. G., Oh, J. Y., Johnson, M. S., Landar, A. & Darley-Usmar, V. M. Methods for imaging and detecting modification of proteins by reactive lipid species. Free Radic Biol Med 47, 201-12 (2009).
- 28. LoPachin, R. M., Gavin, T., Petersen, D. R. & Barber, D. S. Molecular mechanisms of 4-hydroxy-2-nonenal and acrolein toxicity: nucleophilic targets and adduct formation. Chem Res Toxicol 22, 1499-508 (2009).
- 29. Doom, J. A. & Petersen, D. R. Covalent modification of amino acid nucleophiles by the lipid peroxidation products 4-hydroxy-2-nonenal and 4-oxo-2-nonenal.
Chem Res Toxicol 15, 1445-50 (2002). - 30. Weerapana, E., Wang, C., Simon, G. M., Richter, F., Khare, S., Dillon, M. B., Bachovchin, D. A., Mowen, K., Baker, D. & Cravatt, B. F. Quantitative reactivity profiling predicts functional cysteines in proteomes. Nature 468, 790-5 (2010).
- 31. Rostovtsev, V. V., Green, J. G., Fokin, V. V. & Sharpless, K. B. A stepwise Huisgen cycloaddition process: copper(I)-catalyzed regioselective “ligation” of azides and terminal alkynes. Angew. Chem. Int. Ed. Engl. 41, 2596-2599 (2002).
- 32. Bloem, L. J., Pickard, T. R., Acton, S., Donoghue, M., Beavis, R. C., Knierman, M. D. & Wang, X. Tissue distribution and functional expression of a cDNA encoding a novel mixed lineage kinase. J Mol Cell Cardiol 33, 1739-50 (2001).
- 33. Gotoh, I., Adachi, M. & Nishida, E. Identification and characterization of a novel MAP kinase kinase kinase, MLTK. J Biol Chem 276, 4276-86 (2001).
- 34. Keshet, Y. & Seger, R. The MAP kinase signaling cascades: a system of hundreds of components regulates a diverse array of physiological functions. Methods Mol Biol 661, 3-38 (2010).
- 35. Yang, J. J., Lee, Y. J., Hung, H. H., Tseng, W. P., Tu, C. C., Lee, H. & Wu, W. J. ZAK inhibits human lung cancer cell growth via ERK and JNK activation in an AP-1-dependent manner. Cancer Sci 101, 1374-81 (2010).
- 36. Wong, J., Smith, L. B., Magun, E. A., Engstrom, T., Kelley-Howard, K., Jandhyala, D. M., Thorpe, C. M., Magun, B. E. & Wood, L. J. Small molecule kinase inhibitors block the ZAK-dependent inflammatory effects of doxorubicin. Cancer Biol Ther 14, 56-63 (2013).
- 37. Wang, X., Mader, M. M., Toth, J. E., Yu, X., Jin, N., Campbell, R. M., Smallwood, J. K., Christe, M. E., Chatterjee, A., Goodson, T., Jr., Vlahos, C. J., Matter, W. F. & Bloem, L. J. Complete inhibition of anisomycin and UV radiation but not cytokine induced JNK and p38 activation by an aryl-substituted dihydropyrrolopyrazole quinoline and mixed lineage kinase 7 small interfering RNA. J Biol Chem 280, 19298-305 (2005).
- 38. Yu, X. & Bloem, L. J. Effect of C-terminal truncations on MLK7 catalytic activity and JNK activation. Biochem Biophys Res Commun 310, 452-7 (2003).
- 39. Bachovchin, D. A., Mohr, J. T., Speers, A. E., Wang, C., Berlin, J. M., Spicer, T. P., Fernandez-Vega, V., Chase, P., Hodder, P. S., Schurer, S. C., Nomura, D. K., Rosen, H., Fu, G. C. & Cravatt, B. F. Academic cross-fertilization by public screening yields a remarkable class of protein phosphatase methylesterase-1 inhibitors. Proc Natl Acad Sci USA 108, 6811-6 (2011).
- 40. Patricelli, M. P., Szardenings, A. K., Liyanage, M., Nomanbhoy, T. K., Wu, M., Weissig, H., Aban, A., Chun, D., Tanner, S. & Kozarich, J. W. Functional interrogation of the kinome using nucleotide acyl phosphates. Biochemistry 46, 350-8 (2007).
- 41. Shen, H. M. & Liu, Z. G. JNK signaling pathway is a key modulator in cell death mediated by reactive oxygen and nitrogen species. Free
Radic Biol Med 40, 928-39 (2006). - 42. Forman, H. J. Reactive oxygen species and alpha,beta-unsaturated aldehydes as second messengers in signal transduction. Ann N Y Acad Sci 1203, 35-44 (2010).
- 43. Kutuk, O. & Basaga, H. Apoptosis signalling by 4-hydroxynonenal: a role for JNK-c-Jun/AP-1 pathway. Redox Rep 12, 30-4 (2007).
- 44. Uchida, K. 4-Hydroxy-2-nonenal: a product and mediator of oxidative stress. Prog Lipid Res 42, 318-43 (2003).
- 45. Gushwa, N. N., Kang, S., Chen, J. & Taunton, J. Selective targeting of distinct active site nucleophiles by irreversible SRC-family kinase inhibitors. J Am Chem Soc 134, 20214-7 (2012).
- 46. Cohen, M. S., Zhang, C., Shokat, K. M. & Taunton, J. Structural bioinformaticsbased design of selective, irreversible kinase inhibitors. Science 308, 1318-21 (2005).
- 47. Zhou, W., Hur, W., McDermott, U., Dutt, A., Xian, W., Ficarro, S. B., Zhang, J., Sharma, S. V., Brugge, J., Meyerson, M., Settleman, J. & Gray, N. S. A structure16 guided approach to creating covalent FGFR inhibitors. Chem Biol 17, 285-95 (2010).
- 48. Simon, G. M., Niphakis, M. J. & Cravatt, B. F. Determining target engagement in living systems. Nat Chem Biol 9, 200-5 (2013).
- 49. Huang, X., Begley, M., Morgenstern, K. A., Gu, Y., Rose, P., Zhao, H. & Zhu, X. Crystal structure of an inactive Akt2 kinase domain.
Structure 11, 21-30 (2003). - 50. Carbone, D. L., Doom, J. A., Kiebler, Z., Ickes, B. R. & Petersen, D. R. Modification of heat shock protein 90 by 4-hydroxynonenal in a rat model of chronic alcoholic liver disease. J Pharmacol Exp Ther 315, 8-15 (2005).
- 51. Carbone, D. L., Doom, J. A., Kiebler, Z., Sampey, B. P. & Petersen, D. R. Inhibition of Hsp72-mediated protein refolding by 4-hydroxy-2-nonenal. Chem Res Toxicol 17, 1459-67 (2004).
- 52. Deng, X., Weerapana, E., Ulanovskaya, O., Sun, F., Liang, H., Ji, Q., Ye, Y., Fu, Y., Zhou, L., Li, J., Zhang, H., Wang, C., Alvarez, S., Hicks, L. M., Lan, L., Wu, M., Cravatt, B. F. & He, C. Proteome-wide Quantification and Characterization of Oxidation-Sensitive Cysteines in Pathogenic Bacteria. Cell Host Microbe 13, 358-70 (2013).
- 53. Wang, T., Kartika, R. & Spiegel, D. A. Exploring post-translational arginine modification using chemically synthesized methylglyoxal hydroimidazolones. J Am Chem Soc 134, 8958-67 (2012).
- 54. Weerapana, E., Simon, G. M. & Cravatt, B. F. Disparate proteome reactivity profiles of carbon electrophiles.
Nat Chem Biol 4, 405-7 (2008). - 55. Ban, H., Gavrilyuk, J. & Barbas, C. F., 3rd. Tyrosine bioconjugation through aqueous ene-type reactions: a click-like reaction for tyrosine. J Am Chem Soc 132, 1523-5 (2010).
- 56. Fujishima, S. H., Yasui, R., Miki, T., Ojida, A. & Hamachi, I. Ligand-directed acyl imidazole chemistry for labeling of membrane-bound proteins on live cells. J Am Chem Soc 134, 3961-4 (2012).
- 57. Parola, M., Robino, G., Marra, F., Pinzani, M., Bellomo, G., Leonarduzzi, G., Chiarugi, P., Camandola, S., Poli, G., Waeg, G., Gentilini, P. & Dianzani, M. U. HNE interacts directly with JNK isoforms in human hepatic stellate cells. J Clin Invest 102, 1942-50 (1998).
- All patents and publications referred to herein are incorporated by reference herein to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference in its entirety.
- The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.
Claims (12)
1-15. (canceled)
16. A protein identified as a target of a lipid-derived electrophile by the method comprising:
(a) contacting a first set of proteins of a mammalian cell with the lipid-derived electrophile, wherein the lipid-derived electrophile is a cysteine-reactive lipid-derived electrophile to generate an alkylated set of proteins;
(b) contacting the alkylated set of proteins with an alkynylated iodoacetamide probe, followed by reaction with an azido compound comprising a first isotopic marker, to provide an isotopically-marked alkylated set of proteins, wherein the reaction with the azido compound is carried out using a copper-catalyzed azide-alkyne cycloaddition reaction;
(c) contacting the first set of proteins of a mammalian cell of step (a), not exposed to the lipid-derived electrophile, with an alkynylated iodoacetamide probe, followed by reaction with an azido compound comprising a second isotopic marker, to provide an isotopically-marked control set of proteins, wherein the reaction with the azido compound is carried out using a copper-catalyzed azide-alkyne cycloaddition reaction;
(d) combining the isotopically-marked alkylated set of proteins and the isotopically-marked control set of proteins to provide a combined sample;
(e) quantifying reactivities of the isotopically-marked alkylated set of proteins and the isotopically-marked control set of proteins to identify a protein target by comparing the abundance of the first isotopic marker and the abundance of the second isotopic marker for each protein of the combined sample; and
(g) identifying the protein target and sites of modification of the electrophile.
17. The protein of claim 16 , wherein the first set of proteins includes two or more proteins.
18. The protein of claim 16 , wherein the lipid-derived electrophile is a stress-induced electrophile.
19. The protein of claim 16 , wherein lipid-derived electrophile comprises a Michael acceptor having an α,β-unsaturated carbonyl group.
20. The protein of claim 16 , wherein the lipid-derived electrophile undergoes a Michael conjugate addition reaction with a cysteine residue of one or more proteins of the first set of proteins.
21. The protein of claim 16 , wherein the lipid-derived electrophile is 4-hydroxynonenal (HNE) or 15-deoxy-Al 2,14-prostaglandin J2 (15d-PGJ2).
22. The protein of claim 16 , wherein the first isotopic marker and the second isotopic marker are isotopically-differentiated azide-biotin tags.
23. The protein of claim 16 wherein the protein target possess at least 5-fold or higher ratio of the second isotopic marker to the first isotopic marker among the proteins of the combined sample.
24. The protein of claim 16 wherein the site of cysteine modification by a lipid-derived electrophile is at a non-active site.
25. The protein of claim 16 wherein the protein is a kinase.
26. The protein of claim 25 , wherein the kinase is ZAK kinase.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US17/021,260 US20210208141A1 (en) | 2013-08-13 | 2020-09-15 | Cysteine-reactive ligand discovery in proteomes |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201361865165P | 2013-08-13 | 2013-08-13 | |
PCT/US2014/050828 WO2015023724A1 (en) | 2013-08-13 | 2014-08-13 | Cysteine-reactive ligand discovery in proteomes |
US201614911316A | 2016-02-10 | 2016-02-10 | |
US17/021,260 US20210208141A1 (en) | 2013-08-13 | 2020-09-15 | Cysteine-reactive ligand discovery in proteomes |
Related Parent Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2014/050828 Continuation WO2015023724A1 (en) | 2013-08-13 | 2014-08-13 | Cysteine-reactive ligand discovery in proteomes |
US14/911,316 Continuation US10782295B2 (en) | 2013-08-13 | 2014-08-13 | Cysteine-reactive ligand discovery in proteomes |
Publications (1)
Publication Number | Publication Date |
---|---|
US20210208141A1 true US20210208141A1 (en) | 2021-07-08 |
Family
ID=52468649
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US14/911,316 Active 2034-12-17 US10782295B2 (en) | 2013-08-13 | 2014-08-13 | Cysteine-reactive ligand discovery in proteomes |
US17/021,260 Abandoned US20210208141A1 (en) | 2013-08-13 | 2020-09-15 | Cysteine-reactive ligand discovery in proteomes |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US14/911,316 Active 2034-12-17 US10782295B2 (en) | 2013-08-13 | 2014-08-13 | Cysteine-reactive ligand discovery in proteomes |
Country Status (3)
Country | Link |
---|---|
US (2) | US10782295B2 (en) |
EP (1) | EP3033625B1 (en) |
WO (1) | WO2015023724A1 (en) |
Families Citing this family (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US10782295B2 (en) | 2013-08-13 | 2020-09-22 | The Scripps Research Institute | Cysteine-reactive ligand discovery in proteomes |
EP3079668A1 (en) | 2013-12-09 | 2016-10-19 | Durect Corporation | Pharmaceutically active agent complexes, polymer complexes, and compositions and methods involving the same |
EP3274712A4 (en) | 2015-03-27 | 2019-01-23 | The Scripps Research Institute | Lipid probes and uses thereof |
US10670605B2 (en) | 2015-10-22 | 2020-06-02 | The Scripps Research Institute | Cysteine reactive probes and uses thereof |
US11535597B2 (en) | 2017-01-18 | 2022-12-27 | The Scripps Research Institute | Photoreactive ligands and uses thereof |
AU2018215447A1 (en) * | 2017-02-03 | 2019-08-08 | The Regents Of The University Of California | Compositions and methods for inhibiting reticulon 4 |
US10807951B2 (en) | 2017-10-13 | 2020-10-20 | The Regents Of The University Of California | mTORC1 modulators |
JP2021523367A (en) * | 2018-05-10 | 2021-09-02 | ブリストル−マイヤーズ スクイブ カンパニーBristol−Myers Squibb Company | Assay for determining in vivo receptor occupancy |
CN112940071B (en) * | 2021-02-03 | 2023-06-23 | 南京工业大学 | Method for realizing alkynyl functionalization of cysteine and polypeptide thereof by utilizing microchannel reactor |
CN114354733B (en) * | 2021-12-31 | 2022-11-25 | 北京大学 | Method for quantitative chemical proteomics screening target based on DIA |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2002042773A2 (en) * | 2000-11-21 | 2002-05-30 | Sunesis Pharmaceuticals, Inc. | An extended tethering approach for rapid identification of ligands |
US20090068107A1 (en) * | 2006-10-02 | 2009-03-12 | The Scripps Research Institute | Enzyme regulating ether lipid signaling pathways |
Family Cites Families (31)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4469863A (en) | 1980-11-12 | 1984-09-04 | Ts O Paul O P | Nonionic nucleic acid alkyl and aryl phosphonates and processes for manufacture and use thereof |
US5235033A (en) | 1985-03-15 | 1993-08-10 | Anti-Gene Development Group | Alpha-morpholino ribonucleoside derivatives and polymers thereof |
US5034506A (en) | 1985-03-15 | 1991-07-23 | Anti-Gene Development Group | Uncharged morpholino-based polymers having achiral intersubunit linkages |
US5216141A (en) | 1988-06-06 | 1993-06-01 | Benner Steven A | Oligonucleotide analogs containing sulfur linkages |
US5602240A (en) | 1990-07-27 | 1997-02-11 | Ciba Geigy Ag. | Backbone modified oligonucleotide analogs |
US5386023A (en) | 1990-07-27 | 1995-01-31 | Isis Pharmaceuticals | Backbone modified oligonucleotide analogs and preparation thereof through reductive coupling |
US5644048A (en) | 1992-01-10 | 1997-07-01 | Isis Pharmaceuticals, Inc. | Process for preparing phosphorothioate oligonucleotides |
US5637684A (en) | 1994-02-23 | 1997-06-10 | Isis Pharmaceuticals, Inc. | Phosphoramidate and phosphorothioamidate oligomeric compounds |
US6344330B1 (en) | 1998-03-27 | 2002-02-05 | The Regents Of The University Of California | Pharmacophore recombination for the identification of small molecule drug lead compounds |
WO2000077184A1 (en) | 1999-06-10 | 2000-12-21 | Pharmacia & Upjohn Company | Caspase-8 crystals, models and methods |
WO2005118833A2 (en) | 2004-06-01 | 2005-12-15 | Bayer Healthcare Ag | Diagnostics and therapeutics for diseases associated with sterile-alpha motif and leucine zipper containing kinase (zak) |
US7348437B2 (en) | 2004-06-01 | 2008-03-25 | The Scripps Research Institute | Proteomic analysis |
US7935479B2 (en) | 2004-07-19 | 2011-05-03 | Cell Biosciences, Inc. | Methods and devices for analyte detection |
WO2006112841A1 (en) | 2005-04-19 | 2006-10-26 | The Scripps Research Institute | Methods for metabolite profiling |
US20100179118A1 (en) | 2006-09-08 | 2010-07-15 | Dainippon Sumitomo Pharma Co., Ltd. | Cyclic aminoalkylcarboxamide derivative |
CN101219219B (en) | 2007-01-10 | 2013-02-13 | 北京普罗吉生物科技发展有限公司 | Complex containing vascellum chalone or fragment, preparation method and application thereof |
EP1947193A1 (en) | 2007-01-17 | 2008-07-23 | Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. | Screening method for anti-diabetic compounds |
US8778302B2 (en) | 2007-03-09 | 2014-07-15 | The University Of British Columbia | Procaspase 8-mediated disease targeting |
US8846053B2 (en) | 2008-09-26 | 2014-09-30 | Sdg, Inc. | Orally bioavailable lipid-based constructs |
US20110097720A1 (en) | 2008-01-02 | 2011-04-28 | Alnylam Pharmaceuticals, Inc. | Screening method for selected amino lipid-containing compositions |
EP2269064B1 (en) | 2008-03-27 | 2016-11-30 | Promega Corporation | Protein labeling with cyanobenzothiazole conjugates |
US20100203647A1 (en) | 2008-11-21 | 2010-08-12 | The Rockefeller University | Chemical Reporters of Protein Acylation |
US20110020837A1 (en) | 2009-07-07 | 2011-01-27 | Universiteit Utrecht Holding B.V. | Method for isolating or identifying a target protein interacting with a lipid in a cell |
US8669065B1 (en) | 2011-08-28 | 2014-03-11 | Scott B Hansen | Methods for identifying molecules that modulate lipid binding sites of ion channels |
JP2015503330A (en) | 2011-12-22 | 2015-02-02 | アベオ ファーマシューティカルズ, インコーポレイテッド | Identification of multigene biomarkers |
EP3004141A4 (en) | 2013-06-03 | 2017-05-31 | Acetylon Pharmaceuticals, Inc. | Histone deacetylase ( hdac) biomarkers in multiple myeloma |
US10782295B2 (en) | 2013-08-13 | 2020-09-22 | The Scripps Research Institute | Cysteine-reactive ligand discovery in proteomes |
US10034892B2 (en) | 2014-08-21 | 2018-07-31 | Srx Cardio, Llc | Composition and methods of use of small molecules as binding ligands for the modulation of proprotein convertase subtilisin/kexin type 9(PCSK9) protein activity |
EP3274712A4 (en) | 2015-03-27 | 2019-01-23 | The Scripps Research Institute | Lipid probes and uses thereof |
US10670605B2 (en) | 2015-10-22 | 2020-06-02 | The Scripps Research Institute | Cysteine reactive probes and uses thereof |
US11535597B2 (en) | 2017-01-18 | 2022-12-27 | The Scripps Research Institute | Photoreactive ligands and uses thereof |
-
2014
- 2014-08-13 US US14/911,316 patent/US10782295B2/en active Active
- 2014-08-13 EP EP14836595.0A patent/EP3033625B1/en active Active
- 2014-08-13 WO PCT/US2014/050828 patent/WO2015023724A1/en active Application Filing
-
2020
- 2020-09-15 US US17/021,260 patent/US20210208141A1/en not_active Abandoned
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2002042773A2 (en) * | 2000-11-21 | 2002-05-30 | Sunesis Pharmaceuticals, Inc. | An extended tethering approach for rapid identification of ligands |
US20090068107A1 (en) * | 2006-10-02 | 2009-03-12 | The Scripps Research Institute | Enzyme regulating ether lipid signaling pathways |
Non-Patent Citations (11)
Title |
---|
Bischoff et al., Amino Acids: Chemistry, Functionality and Selected Non-Enzymatic Post-Translational Modifications, Journal of Proteomics, 2012, 75, 2275-2296. (Year: 2012) * |
Chalker et al., Chemical Modifications of Proteins at Cysteine: Opportunities in Chemistry and Biology, Chem. Asian J., 2009, 4, 630-640. (Year: 2009) * |
Gu et al., Chemical Proteomics with Sulfonyl Fluoride Probes Reveals Selective Labeling of Functional Tyrosines in Glutathione Transferases, Chemistry & Biology, 2013, 20, 541-548. (Year: 2013) * |
Hashimoto et al., Activation and Role of MAP Kinases in 15d-PG2-Induced Apoptosis in the Human Pancreatic Cancer Cell Line MIA PaCa-2, Pancreas, 2004, 28(2), 153-159. (Year: 2004) * |
Jacob et al., Control of Oxidative Posttranslational Cysteine Modifications: From Intricate Chemistry to Widespread Biological and Medical Applications, Chemical Research in Toxicology, 2012, 25, 588-604. (Year: 2012) * |
Jacobs et al., Systems Analysis of Protein Modification and Cellular Responses Induced by Electrophile Stress, Accounts of Chemical Research, 2010, 43(5), 673-683. (Year: 2010) * |
Liu et al., Developing Irreversible Inhibitors of the Protein Kinase Cysteinome, Chemistry & Biology Review, 2013, 20, 146-159. (Year: 2013) * |
Wang et al., A Chemoproteomic Platform to Quantitatively Map Targets of Lipid-Derived Electrophiles, Nature Methods, epub Dec 1 2013, 11(1), 79-87. (Year: 2013) * |
Weerapana et al., Quantitative Reactivity Profiling Predicts Functional Cysteines in Proteomes, Nature, 2010, 468, 790-797. (Year: 2010) * |
Weerapana et al., Quantitative Reactivity Profiling Predicts Functional Cysteines in Proteomes, Supplementary Information, Nature, 2010, 468, 1-263. (Year: 2010) * |
Weerapana et al., Supplemental Information, Quantitative Reactivity Profiling Predicts Functional Cysteines in Proteomes, Nature, 2010, 468, 1-263. (Year: 2010) * |
Also Published As
Publication number | Publication date |
---|---|
EP3033625B1 (en) | 2020-01-22 |
US10782295B2 (en) | 2020-09-22 |
US20160252509A1 (en) | 2016-09-01 |
WO2015023724A1 (en) | 2015-02-19 |
EP3033625A4 (en) | 2017-07-05 |
EP3033625A1 (en) | 2016-06-22 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20210208141A1 (en) | Cysteine-reactive ligand discovery in proteomes | |
Abbasov et al. | A proteome-wide atlas of lysine-reactive chemistry | |
Martín‐Gago et al. | Arylfluorosulfate‐based electrophiles for covalent protein labeling: a new addition to the arsenal | |
Cotto-Rios et al. | Deubiquitinases as a signaling target of oxidative stress | |
Park et al. | SIRT2-mediated deacetylation and tetramerization of pyruvate kinase directs glycolysis and tumor growth | |
Saline et al. | AMPK and AKT protein kinases hierarchically phosphorylate the N-terminus of the FOXO1 transcription factor, modulating interactions with 14-3-3 proteins | |
Greco et al. | Nuclear import of histone deacetylase 5 by requisite nuclear localization signal phosphorylation | |
Michaloglou et al. | The tyrosine phosphatase PTPN14 is a negative regulator of YAP activity | |
Mardin et al. | Components of the Hippo pathway cooperate with Nek2 kinase to regulate centrosome disjunction | |
Formosa et al. | Characterization of mitochondrial FOXRED1 in the assembly of respiratory chain complex I | |
Long et al. | Akt3 is a privileged first responder in isozyme-specific electrophile response | |
Mackeen et al. | Small-molecule-based inhibition of histone demethylation in cells assessed by quantitative mass spectrometry | |
Siepi et al. | HIPK2 catalytic activity and subcellular localization are regulated by activation-loop Y354 autophosphorylation | |
Reinhardt et al. | Purification of CK1 by affinity chromatography on immobilised axin | |
Mooney et al. | Cancer/testis antigen PAGE4, a regulator of c-Jun transactivation, is phosphorylated by homeodomain-interacting protein kinase 1, a component of the stress-response pathway | |
Elias et al. | Identification and characterization of phosphorylation sites within the pregnane X receptor protein | |
Iliuk et al. | Chemical visualization of phosphoproteomes on membrane | |
Shi et al. | Activity based high-throughput screening for novel O-GlcNAc transferase substrates using a dynamic peptide microarray | |
Kodama et al. | Selective involvement of p130Cas/Crk/Pyk2/c-Src in endothelin-1-induced JNK activation | |
Niinae et al. | Identification of endogenous kinase substrates by proximity labeling combined with kinase perturbation and phosphorylation motifs | |
Jansen et al. | Inhibition of prenylated KRAS in a lipid environment | |
Garcia et al. | Unbiased proteomics identifies plasminogen activator inhibitor-1 as a negative regulator of endothelial nitric oxide synthase | |
Budayeva et al. | Human sirtuin 2 localization, transient interactions, and impact on the proteome point to its role in intracellular trafficking | |
Yuan et al. | Thiol‐based redox proteomics in cancer research | |
Kovářová et al. | High molecular weight forms of mammalian respiratory chain complex II |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
STPP | Information on status: patent application and granting procedure in general |
Free format text: APPLICATION DISPATCHED FROM PREEXAM, NOT YET DOCKETED |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: FINAL REJECTION MAILED |
|
STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION |