Executing T-REX in mammalian cells

Transcription

1 Supplementary Figure 1 Executing T-REX in mammalian cells HEK-293 cells cultured (a) in 2x 55 cm 2 adherent cell culture plates, and (b and c) in a 48-well multi-well adherent cell culture plate. No cover was placed on the plates during photo-uncaging. See Main Text for detailed experimental conditions and equipment specifications. Also see Supplementary Videos 1 2

2 Supplementary Figure 2 Evaluation of time-dependent redox signal release in cells in T-REX method and validation that HaloTag does not react with HNE. (a) Measurements of HNE release efficiency in cells. HEK293T cells expressing HaloTag alone treated under standard T-REX conditions with Ht-PreHNE were exposed to UV light (0.3 mw/cm 2, 365 nm) for the indicated time periods at which point the cells were harvested, lysed, and subjected to Click coupling and in-gel fluorescence analysis followed by western blot. Error bars designate SD (N=3). (b) Controls to show that HaloTag does not react with HNE. Purified recombinant HaloTag was treated with either the photocaged precursor Ht-PreHNE (Figure 2, inset, and Figure 10) (2 equiv., lane a, positive control), or directly with reactive electrophile HNE (Figure 10) (0, 2, 4, 8, 16 equiv., lane b, c, d, e, f, respectively). After 20-min incubation, the samples were analyzed by in-gel fluorescence. M, molecular weight ladder.

3 Supplementary Figure 3 UV light exposure employed in T-REX is non-invasive: representative data for γ-h2ax 1 and NF-κB 2, markers for DNA damage and inflammatory signaling, respectively. (a) HEK293T cells were exposed to UV light (0.3 mw/cm 2, 340 nm) for the indicated time periods. Mitomycin C (10 μg/ml for 24 h) 3 and aphidicolin (10 μg/ml for 36 h) 4 serve as positive controls. After 12 hours post the end of UV illumination, cells were fixed and analyzed by standard immunofluorescence imaging method using γ-h2ax antibody (Millipore at 1:1000 dilution). Data show mean +/- S.D. N > 50 cells. (b) HEK293T cells stably expressing NRE-inducible firefly luciferase 5 were transfected with the respective plasmids encoding indicated transgene (empty vector, HaloTag alone, or Halo-Keap1 and Renilla luciferase) under constitutive CMV promoters. 24 hours post transfection, half of the plates were exposed to UV light (0.3 mw/cm 2, 365 nm) over 20 min. Phorbol 12-myristate 13-acetate (PMA) (10 ng/ml, 18 h) was used as a positive control for NRE activation 5. NRE activation was measured after 18 hrs. Error bars designate S.D. (N = 8 biological replicates).

4 Supplementary Figure 4 T-REX screen of Halo ORF clones for the discovery of novel electrophile-sensitive targets and pulldown validation of expressed proteins exemplified by zebrafish HSPB7. (a) T-REX-enabled gel-based screen for bona fide HNE-sensitive targets using Halo-ORFeome library (Promega). Individual wells in a 48-well plate contained live HEK-293 cells ectopically expressing a unique HaloTagged gene of interest. The cells were subjected to T-REX-HNE(alkyne) targeting on demand. Post cell lysis, all samples were treated with TEV protease and subsequently subjected to Click coupling reaction with Cy5 azide. Probing with Halo antibody allowed evaluation of expression level (and/or solubility under the lysis conditions used). The hit bands on Cy5-fluorescent gel were judged against Halo protein level revealed by western blot. For example, RRM1, PRKCD, p53r2, and Keap1 (positive control) had roughly similar expression levels. Only RRM1 and Keap1 were HNE-sensitive although all four targets have been previously identified to be potentially redox/hne-sensitive See Main Text for discussion., a non-specific band. Also see Figure 4 and procedural details in Main Text. (b) Zebrafish HSPB7 expression and protein ID of the band shown in Figure 4a was validated by enrichment from HEK-293 cells ectopically expressing Halo-HSPB7 with the use of HaloTag PEG-Biotin ligand (Promega G8592) and streptavidin sepharose beads (GE Healthcare, cat. no ), and subsequent on-bead TEV-protease cleavage followed by gel electrophoresis analysis. Theoretical MW of HSPB7 ~ 18 kda. L, MW ladder.

5 Supplementary Table 1. LC-MS/MS identification of site of electrophilic modification on C- terminal HaloTagged Keap1 ectopically expressed in HEK-293 cells subsequent to T-REX redox targeting. Human Keap1 (100%), 70.0 kda, Mascot Score 769, 47 unique peptides with different modifications, 1 distinct tryptic peptides with added mass of 136 Da for possible modifications of reduced CHE(alkyne) Michael adduct. The modified peptide was found present in corresponding unmodified form of the native peptide with Cys being alkylated by carbamidomethylation. 341/624 amino acids (54% coverage). Matched peptide with CHE related modifications shown in green, other matched peptide shown in red. MQPDPRPSGA GACCRFLPLQ SQCPEGAGDA VMYASTECKA EVTPSQHGNR TFSYTLEDHT KQAFGIMNEL RLSQQLCDVT LQVKYQDAPA AQFMAHKVVL ASSSPVFKAM FTNGLREQGM EVVSIEGIHP KVMERLIEFA YTASISMGEK CVLHVMNGAV MYQIDSVVRA CSDFLVQQLD PSNAIGIANF AEQIGCVELH QRAREYIYMH FGEVAKQEEF FNLSHCQLVT LISRDDLNVR CESEVFHACI NWVKYDCEQR RFYVQALLRA VRCHSLTPNF LQMQLQKCEI LQSDSRCKDY LVKIFEELTL HKPTQVMPCR APKVGRLIYT AGGYFRQSLS YLEAYNPSDG TWLRLADLQV PRSGLAGCVV GGLLYAVGGR NNSPDGNTDS SALDCYNPMT NQWSPCAPMS VPRNRIGVGV IDGHIYAVGG SHGCIHHNSV ERYEPERDEW HLVAPMLTRR IGVGVAVLNR LLYAVGGFDG TNRLNSAECY YPERNEWRMI TAMNTIRSGA GVCVLHNCIY AAGGYDGQDQ LNSVERYDVE TETWTFVAPM KHRRSALGIT VHQGRIYVLG GYDGHTFLDS VECYDPDTDT WSEVTRMTSG RSGVGVAVTM EPCRKQIDQQ NCTC Unique peptide with CHE-alkyne modification MS spectra (asterisk suggests modification site) C613 SGVGVAVTMEPC* R p-value: Mascot Ion Score: 38 Expectation Values: Chemical synthesis and characterization of Pre-CHE(Alkyne) (also known as Ht-PreCHE) was previously reported 11.

6 Supplementary Table 2. LC-MS/MS identification of site of electrophilic modification on C- terminal HaloTagged Keap1 ectopically expressed in HEK-293 cells subsequent to whole-cell CHE treatment. Human Keap1 (100%), 70.0 kda, Mascot Score 1821, 130 unique peptides with different modifications, 1 distinct tryptic peptides with added mass of 136 Da for possible modifications of reduced CHE(alkyne) Michael adduct. The modified peptide was found present in corresponding unmodified form of the native peptide with Cys being alkylated by carbamidomethylation. 414/624 amino acids (66% coverage). Matched peptide with CHE related modifications shown in green, other matched peptide shown in red. MQPDPRPSGA GACCRFLPLQ SQCPEGAGDA VMYASTECKA EVTPSQHGNR TFSYTLEDHT KQAFGIMNEL RLSQQLCDVT LQVKYQDAPA AQFMAHKVVL ASSSPVFKAM FTNGLREQG M EVVSIEGIH PKVMERLIEFA YTASISMGEK CVLHVMNGAV MYQIDSVVRA CSDFLVQQLD PSNAIGIANF AEQIGCVELH QRAREYIYMH FGEVAKQEEF FNLSHCQLVT LISRDDLNVR CESEVFHACI NWVKYDCEQR RFYVQALLRA VRCHSLTPNF LQMQLQKCEI LQSDSRCKDY LVKIFEELTLH KPTQVMPCRA PKVGRLIYTAG GYFRQSLSYL EAYNPSDGT WLRLADLQV PRSGLAGCVV GGLLYAVGGR NNSPDGNTDS SALDCYNPMT NQWSPCAPMS VPRNRIGVGV IDGHIYAVGG SHGCIHHNSV ERYEPERDEW HLVAPMLTRR IGVGVAVLNR LLYAVGGFDG TNRLNSAECY YPERNEWRMI TAMNTIRSGA GVCVLHNCIY AAGGYDGQDQ LNSVERYDVE TETWTFVAPM KHRRSALGIT VHQGRIYVLG GYDGHTFLDS VECYDPDTDT WSEVTRMTSG RSGVGVAVTM EPCRKQIDQQ NCTC Unique peptide with CHE-alkyne modification (asterisk MS spectra suggests modification site) C613 SGVGVAVTMEPC *R p-value: Mascot Ion Score: 31 Expectation Values: Chemical synthesis and characterization of CHE(Alkyne) was previously reported 11.