supplementary materials for · 1/27/2016 · labeled at the end of treatment with 50 ci of...
TRANSCRIPT
www.sciencemag.org/content/351/6269/aad3876/suppl/DC1
Supplementary Materials for
Translation from the 5′ untranslated region shapes the integrated stress response
Shelley R. Starck,* Jordan C. Tsai, Keling Chen, Michael Shodiya, Lei Wang,
Kinnosuke Yahiro, Manuela Martins-Green, Nilabh Shastri,* Peter Walter*
* Corresponding author. E-mail: [email protected] (S.R.S.); [email protected] (N.S.); [email protected] (P.W.)
Published 29 January 2016 in Science 351, aad3876 (2016)
DOI: 10.1126/science.aad3876
This PDF file includes
Materials and Methods Figs. S1 to S15 References
2
Materials and Methods
Plasmid constructs
Human ATF4-luciferase reporter vector (pATF4.1) was prepared by PCR
amplifying human 5’ UTR ATF4-luciferase from pCAX-ATF4-FLuc (51) with forward
(5’-GTACGCGGATCCTTTCTACTTTGCCCGCCCAC-3’) and reverse (5’-
CACTAGGTCGACTTACACGGCGATCTTTCCGC-3’) primers and cloned into the
pcDNA1-based CCC[YL8] plasmid (58) using the BamHI and SalI sites. Tracer peptide
insertions (pATF4.2 – pATF4.5) and other mutations (pATF4.9) were carried out using
the QuikChange Site-Directed Mutagenesis Kit (Stratagene).
Human 5’ UTR BiP-FLAG vectors were prepared from cDNA of total HeLa-K
b
RNA (SuperScript III; Invitrogen), amplified by PCR with forward (5’-
GTACGCGGATCCGGGCTGGGGGAGGGTATATAAG-3’) and reverse (containing
FLAG sequence) (5’-
CACTAGCTCGAGCTACAACTCATCTTTTTCCTTGTCATCGTCATCCTTGTAATC
TGCTGTATCCTCTTCACCAGTTGG-3’) primers and cloned into pcDNA1 using the
BamHI and XhoI sites. Tracer peptide insertions and other mutations were carried out
using the QuikChange Site-Directed Mutagenesis Kit (Stratagene) as described in detail
below.
Lentiviral reporter vectors (pATF4.6 and pATF4.7) were based on pSicoR (88).
Vectors contain a CMV promoter driving expression of ATF4-luciferase were sub-cloned
using an In-Fusion Cloning Kit (Clontech Laboratories). Human ATF4-luciferase
constructs were amplified by PCR, as described in detail below, and cloned into a
pMK1163 backbone linearized by PCR with forward (5’-
GAATTCCGCCCCTCTCCCTC-3’ ) and reverse (5’-
TGGGCGGGCAAAGTAGAAACTC-3’) primers.
Plasmid construct Sequences
ATF4-luciferase Plasmids; pATF4
pATF4.1: pcDNA1 ATF4-luciferase
Wild-type ATF4-luciferase (Human); parent plasmid - modified pcDNA1: CCC-
YL8 (58)
5’: BamHI and 3’: SalI (in red); uORF1 (blue); uORF2 (orange)
5’-
CCAAGCTTGGTACCGAGCTCGGATCCTTTCTACTTTGCCCGCCCACAGATGT
AGTTTTCTCTGCGCGTGTGCGTTTTCCCTCCTCCCCGCCCTCAGGGTCCACGG
CCACCATGGCGTATTAGGGGCAGCAGTGCCTGCGGCAGCATTGGCCTTTGC
AGCGGCGGCAGCAGCACCAGGCTCTGCAGCGGCAACCCCCAGCGGCTTAAG
CCATGGCGCTTCTCACGGCATTCAGCAGCAGCGTTGCTGTAACCGACAA
3
AGACACCTTCGAATTAAGCACATTCCTCGATTCCAGCAAAGCACCGCAA
CATGACCGAAATGAGCTTCCTGAGCAGCGAGGTGTTGGTGGGGGACTTG
ATGTCCCCCTTCGACCAGTCGGGTTTGGGGGCTGAAAGATCTGAAGACGC
CAAAAACATAAAGAAAGGCCCGGCGCCATTCTATCCGCTGGAAGATGGAAC
CGCTGGAGAGCAACTGCATAAGGCTATGAAGAGATACGCCCTGGTTCCTGGA
ACAATTGCTTTTACAGATGCACATATCGAGGTGGACATCACTTACGCTGAGTA
CTTCGAAATGTCCGTTCGGTTGGCAGAAGCTATGAAACGATATGGGCTGAAT
ACAAATCACAGAATCGTCGTATGCAGTGAAAACTCTCTTCAATTCTTTATGCC
GGTGTTGGGCGCGTTATTTATCGGAGTTGCAGTTGCGCCCGCGAACGACATTT
ATAATGAACGTGAATTGCTCAACAGTATGGGCATTTCGCAGCCTACCGTGGT
GTTCGTTTCCAAAAAGGGGTTGCAAAAAATTTTGAACGTGCAAAAAAAGCTC
CCAATCATCCAAAAAATTATTATCATGGATTCTAAAACGGATTACCAGGGAT
TTCAGTCGATGTACACGTTCGTCACATCTCATCTACCTCCCGGTTTTAATGAA
TACGATTTTGTGCCAGAGTCCTTCGATAGGGACAAGACAATTGCACTGATCAT
GAACTCCTCTGGATCTACTGGTCTGCCTAAAGGTGTCGCTCTGCCTCATAGAA
CTGCCTGCGTGAGATTCTCGCATGCCAGAGATCCTATTTTTGGCAATCAAATC
ATTCCGGATACTGCGATTTTAAGTGTTGTTCCATTCCATCACGGTTTTGGAAT
GTTTACTACACTCGGATATTTGATATGTGGATTTCGAGTCGTCTTAATGTATA
GATTTGAAGAAGAGCTGTTTCTGAGGAGCCTTCAGGATTACAAGATTCAAAG
TGCGCTGCTGGTGCCAACCCTATTCTCCTTCTTCGCCAAAAGCACTCTGATTG
ACAAATACGATTTATCTAATTTACACGAAATTGCTTCTGGTGGCGCTCCCCTC
TCTAAGGAAGTCGGGGAAGCGGTTGCCAAGAGGTTCCATCTGCCAGGTATCA
GGCAAGGATATGGGCTCACTGAGACTACATCAGCTATTCTGATTACACCCGA
GGGGGATGATAAACCGGGCGCGGTCGGTAAAGTTGTTCCATTTTTTGAAGCG
AAGGTTGTGGATCTGGATACCGGGAAAACGCTGGGCGTTAATCAAAGAGGCG
AACTGTGTGTGAGAGGTCCTATGATTATGTCCGGTTATGTAAACAATCCGGA
AGCGACCAACGCCTTGATTGACAAGGATGGATGGCTACATTCTGGAGACATA
GCTTACTGGGACGAAGACGAACACTTCTTCATCGTTGACCGCCTAAAGTCTCT
GATTAAGTACAAAGGCTATCAGGTGGCTCCCGCTGAATTGGAATCCATCTTG
CTCCAACACCCCCACATCTTCGACGCAGGTGTCGCAGGTCTTCCCGACGATG
ACGCCGGTGAACTTCCCGCCGCCGTTGTTGTTTTGGAGCACGGAAAGACGAT
GACGGAAAAAGAGATCGTGGATTACGTCGCCAGTCAAGTAACAACCGCGAA
AAAGTTGCGCGGAGGAGTTGTGTTTGTGGACGAAGTACCGAAAGGTCTTACC
GGAAAACTCGACGCAAGAAAAATCAGAGAGATCCTCATAAAGGCCAAGAAG
GGCGGAAAGATCGCCGTGTAAGTCGACCCCCACCTTCAACTACCGGA-3’
pATF4.2: uORF1(MYL8)
Based on pATF4.1; tracer peptide MYL8 (underlined) inserted into uORF1
5’-CCACGGCCACCATGACCTTCAACTACCGGAATCTCTAGGGGCAGCA
pATF4.3: uORF2(KOVAK)
Based on pATF4.1; tracer peptide KOVAK (underlined) inserted into uORF2
5’-GCAGCAGCGTT---
AAATCGATAATCAACTTTGAACACCTCAAATTAAGCACATT
pATF4.4: uORF1(WI9)
4
Based on pATF4.1; tracer peptide WI9 (underlined) inserted into uORF1
5’-
CCACGGCCACCATGTGGATGCACCACAACATGGACCTCATATAGGGGCAG
CA
pATF4.5: uORF1(WI9)-uORF2(KOVAK)
Based on pATF4.1; contains the tracer peptides from both pATF4.4 and from pATF4.3
pATF4.6: WT ATF4-luciferase stable HeLa-Kb
Amplified insert from pATF4.1 using forward (5’- TTTCTACTTTGCCCGCCCACAG -
3’) and reverse (5’-AGAGGGGCGGAATTCTTACACGGCGATCTT-3’) primers and
inserted into pMK1163 backbone.
pATF4.7: uORF2(KOVAK) stable HeLa-Kb
Amplified insert from pATF4.3 using forward (5’- TTTCTACTTTGCCCGCCCACAG -
3’) and reverse (5’-AGAGGGGCGGAATTCTTACACGGCGATCTT-3’) primers and
inserted into pMK1163 backbone.
pATF4.8: Constitutively active ATF4-luciferase
Based on pATF4.1; T>A mutation and deleted C in uORF2 (49). This resulted in fusion
of uORF2 to the coding sequence of ATF4-luciferase.
5’-GACTTGATGACCCC-TTCGAC
BiP-FLAG Plasmids pBiP
pBiP.1: BiP-FLAG (Human); parent plasmid: pcDNA1 (Invitrogen)
5’: BamHI and 3’: XhoI (red); FLAG (cyan)
5’-
CCAAGCTTGGTACCGAGCTCGGATCCGGGCTGGGGGAGGGTATATAAGCCG
AGTAGGCGACGGTGAGGTCGACGCCGGCCAAGACAGCACAGACAGATTGAC
CTATTGGGGTGTTTCGCGAGTGTGAGAGGGAAGCGCCGCGGCCTGTATTTCT
AGACCTGCCCTTCGCCTGGTTCGTGGCGCCTTGTGACCCCGGGCCCCTGCCGC
CTGCAAGTCGGAAATTGCGCTGTGCTCCTGTGCTACGGCCTGTGGCTGGACTG
CCTGCTGCTGCCCAACTGGCTGGCAAGATGAAGCTCTCCCTGGTGGCCGCGA
TGCTGCTGCTGCTCAGCGCGGCGCGGGCCGAGGAGGAGGACAAGAAGGAGG
ACGTGGGCACGGTGGTCGGCATCGACCTGGGGACCACCTACTCCTGCGTCGG
CGTGTTCAAGAACGGCCGCGTGGAGATCATCGCCAACGATCAGGGCAACCGC
ATCACGCCGTCCTATGTCGCCTTCACTCCTGAAGGGGAACGTCTGATTGGCGA
TGCCGCCAAGAACCAGCTCACCTCCAACCCCGAGAACACGGTCTTTGACGCC
AAGCGGCTCATCGGCCGCACGTGGAATGACCCGTCTGTGCAGCAGGACATCA
AGTTCTTGCCGTTCAAGGTGGTTGAAAAGAAAACTAAACCATACATTCAAGT
TGATATTGGAGGTGGGCAAACAAAGACATTTGCTCCTGAAGAAATTTCTGCC
ATGGTTCTCACTAAAATGAAAGAAACCGCTGAGGCTTATTTGGGAAAGAAGG
TTACCCATGCAGTTGTTACTGTACCAGCCTATTTTAATGATGCCCAACGCCAA
GCAACCAAAGACGCTGGAACTATTGCTGGCCTAAATGTTATGAGGATCATCA
ACGAGCCTACGGCAGCTGCTATTGCTTATGGCCTGGATAAGAGGGAGGGGGA
GAAGAACATCCTGGTGTTTGACCTGGGTGGCGGAACCTTCGATGTGTCTCTTC
5
TCACCATTGACAATGGTGTCTTCGAAGTTGTGGCCACTAATGGAGATACTCAT
CTGGGTGGAGAAGACTTTGACCAGCGTGTCATGGAACACTTCATCAAACTGT
ACAAAAAGAAGACGGGCAAAGATGTCAGGAAAGACAATAGAGCTGTGCAGA
AACTCCGGCGCGAGGTAGAAAAGGCCAAACGGGCCCTGTCTTCTCAGCATCA
AGCAAGAATTGAAATTGAGTCCTTCTATGAAGGAGAAGACTTTTCTGAGACC
CTGACTCGGGCCAAATTTGAAGAGCTCAACATGGATCTGTTCCGGTCTACTAT
GAAGCCCGTCCAGAAAGTGTTGGAAGATTCTGATTTGAAGAAGTCTGATATT
GATGAAATTGTTCTTGTTGGTGGCTCGACTCGAATTCCAAAGATTCAGCAACT
GGTTAAAGAGTTCTTCAATGGCAAGGAACCATCCCGTGGCATAAACCCAGAT
GAAGCTGTAGCGTATGGTGCTGCTGTCCAGGCTGGTGTGCTCTCTGGTGATCA
AGATACAGGTGACCTGGTACTGCTTGATGTATGTCCCCTTACACTTGGTATTG
AAACTGTGGGAGGTGTCATGACCAAACTGATTCCAAGGAACACAGTGGTGCC
TACCAAGAAGTCTCAGATCTTTTCTACAGCTTCTGATAATCAACCAACTGTTA
CAATCAAGGTCTATGAAGGTGAAAGACCCCTGACAAAAGACAATCATCTTCT
GGGTACATTTGATCTGACTGGAATTCCTCCTGCTCCTCGTGGGGTCCCACAGA
TTGAAGTCACCTTTGAGATAGATGTGAATGGTATTCTTCGAGTGACAGCTGAA
GACAAGGGTACAGGGAACAAAAATAAGATCACAATCACCAATGACCAGAAT
CGCCTGACACCTGAAGAAATCGAAAGGATGGTTAATGATGCTGAGAAGTTTG
CTGAGGAAGACAAAAAGCTCAAGGAGCGCATTGATACTAGAAATGAGTTGG
AAAGCTATGCCTATTCTCTAAAGAATCAGATTGGAGATAAAGAAAAGCTGGG
AGGTAAACTTTCCTCTGAAGATAAGGAGACCATGGAAAAAGCTGTAGAAGA
AAAGATTGAATGGCTGGAAAGCCACCAAGATGCTGACATTGAAGACTTCAAA
GCTAAGAAGAAGGAACTGGAAGAAATTGTTCAACCAATTATCAGCAAACTCT
ATGGAAGTGCAGGCCCTCCCCCAACTGGTGAAGAGGATACAGCAGATTACA
AGGATGACGATGACAAGGAAAAAGATGAGTTGTAGCTCGAGCATGCATCT
AGAGGGCCCTA-3’
pBiP.2: BiP-FLAG(L416D)
Based on pBiP.1; Leu416Asp mutation
5’-CTGGTAGATCTTGAT
pBiP.3: -190 UUG(LYL8)
Based on pBiP.1; tracer peptide LYL8 (underlined) inserted into the -190 UUG uORF
5’-AGATTGACCTTCAACTACCGGAATCTC---TGA
pBiP.4: -190 UUG…UAG(LYL8)
Based on pBiP.3; codon mutated to a UAG stop codon
5’-AGATTGACCTTCAACTAGCGGAATCTC---TGA
pBiP.5: -61 CUG(KOVAK)
Based on pBiP.1; tracer peptide KOVAK (underlined) inserted into the -61 CUG uORF
5’-
CTGTGCTCCTGTGCTACGGCCTGTAAATCGATAATCAACTTTGAACACCTC
AAATGGCAA
pBiP.6: -61 CUG…UGA(KOVAK)
6
Based on pBiP.5; codon mutated to a UGA stop codon
5’-
CTGTGCTCCTGTGCTACGGCCTGAAAATCGATAATCAACTTTGAACACCTC
AAATGGCAA
pBiP.7: -61 CUG(KOVAK) fusion
Based on pBiP.1; tracer peptide KOVAK (underlined) inserted into the -61 CUG uORF,
removed a T to fuse -61 CUG uORF with BiP-FLAG CDS
5’-GCCTGTAAATCGATAATCAACTTTGAACACCTCAAA-GGCAAG
pBiP.8: -61 CUG. . .UGA(KOVAK) fusion
Based on pBiP.1; codon mutated to a UGA stop codon
5’-GCCTGAAAATCGATAATCAACTTTGAACACCTCAAA-GGCAAG
pBiP.9: uORF mutant
Based on pBiP.1; mutated -190 UUG, -182 UUG, -164 GUG, and -143 CUG to non-start
codons (33) and introduced additional mutations to prevent off-target uORF translation.
5’-
AGACCGACCTACCGGGGCGTTTCGCGAGTTTCAGAGGGAAGCGCCGCGGCC
CGCATTTCCAGACCTGCCC
pBiP.10: +1 ATG BiP-FLAG
Based on pBiP.1; mutated +1 ATG to GCC
pBiP.11: +24 ATG BiP-FLAG
Based on pBiP.1; mutated signal sequence +24 ATG to TTA
pBiP.12: +1 ATG;+24 ATG BiP-FLAG
Based on pBiP.1; mutated +1 ATG to GCC and +24 ATG to TTA
pBiP.13: -12 CTG BiP-FLAG
Based on pBiP.1; mutated -12 CTG to GCC
Integrated Stress Response Inducers
Subtilase cytotoxin (Mutant SubAB or SubAB) preparation: Escherichia coli
BL21(DE3) producing recombinant His-tagged subtilase cytotoxin (SubAB) or
catalytically inactivate mutant SubAS272AB (Mut SubAB) were used as the source of
toxins by purification according to the published procedure (89). Tunicamycin was
obtained from Calbiochem EMD Bioscience. Sodium Arsenite, thapsigargin,
cycloheximide ready-made solution in DMSO, anisomycin, and lipopolysaccharide from
Salmonella typhosa (LPS; L7895) were obtained from Sigma-Aldrich. ISRIB was
synthesized in house (51), polyinosinic:polycytidylic acid (Poly I:C) was purchased from
InvivoGen, and NSC119893 and bruceantin (NSC165563) were obtained from the
NCI/DTP Open Chemical Repository (http://dtp.nci.nih.gov).
7
Cell-based Expression Assays
HeLa-Kb, L-cell fibroblasts expressing H2-K
b (K
b-L cells; K89 cells) or L-cell
fibroblasts expressing H2-Db (D
b-L cells), and mouse bone-marrow-derived dendritic cell
assays (BM-dendritic cells; see preparation description below) were cultured in RPMI
containing 10% FBS (Life Technologies), 10 mM HEPES (Life Technologies), 1 mM
Sodium Pyruvate (Life Technologies), 2 mM L-glutamine (Sigma-Aldrich), 55 M beta-mercaptoethanol (Life Technologies), Penicillin-Streptomycin (Sigma-Aldrich), a human
retinal pigment epithelial (RPE-19) cell line (ARPE-19 originally from ATCC) was
cultured in DMEM:F12 (Life Technologies) containing 10% FBS (Life Technologies)
and Penicillin-Streptomycin (Sigma-Aldrich), and Human microvascular endothelial cell
line, HMVEC-1 (obtained as a gift from the Centers for Disease Control and Prevention;
Atlanta, GA) were cultured in DMEM with 10% FBS, L-glutamine, and Penicillin-
Streptomycin. Cells were usually plated in 6-well plates (60-80% confluence) 12–18 h
prior to transfection. Cells were transfected with 0.5–2 g plasmid DNA/well with
Lipofectamine 2000 (Invitrogen) for 12-18 h. For integrated stress response (ISR)
treatments, cells were either treated directly on 6-well plates or in suspension in 1.5 mL
tubes (typically 0.5–1 × 105 cells in 500 L). For [
35S]Met/Cys labeling, cells were
labeled at the end of treatment with 50 Ci of [35
S]Met/Cys/sample (EasyTag™
EXPRESS [35
S] Protein Labeling Mix; Perkin Elmer) for 5–10 min. The cells were
collected, rinsed with 1X PBS, and lysed in buffer containing 25 mM Tris-HCl (pH 8.0),
8 mM MgCl2, 1 mM DTT, 1% Triton™ X-100, 15% glycerol, 1X Halt™ Protease
Inhibitor Cocktail (Life Technologies), and 1X Halt™ Phosphatase Inhibitor Cocktail
(Life Technologies) on ice for 30 min. After removing cellular debris by centrifugation
at 14,000 x g for 10 min, the cell lysate was combined with SDS gel-loading buffer (50
mM Tris-HCl (pH 6.8), 2% SDS, 0.1% bromophenol blue, 10% glycerol, 100 mM dithiotheitol), heated to 95°C for 5 min, and resolved using ANY-KD SDS-PAGE
(Invitrogen) or directly combined with ONE-Glo™ Luciferase Assay Substrate
(Promega) for luminescence measurements on a luminometer. Protein was transferred to
Hybond ECL nitrocellulose membranes (Amersham) and probed with the following
antibodies in either 3% milk or 5% BSA (Sigma-Aldrich; #A7906) in PBS + 0.1%
Tween: 1:1000 mouse monoclonal anti-GFP antibody (Clones 7.1 and 13.1; Boehringer
Mannheim); 1:400 rabbit anti--actin (C-11) (Santa Cruz Biotechnology; #SC-1615);
1:1000 rabbit anti-GADPH (Abcam; #ab9485); 1:600 to 1:1000 rabbit anti-BiP
(Proteintech; #11587-1-AP); 1:1000 rabbit anti-eIF2A (Proteintech; #11233-1-AP);
1:1000 rabbit anti-PERK (Cell Signaling; #3192); 1:1000 rabbit anti-eIF2-P (S51) (Cell Signaling; #9721); 1:1000 rabbit anti-ATF4 (Santa Cruz Biotechnology; (anti-CREB)
#SC-200); and 1:1000 to 1:5000 mouse monoclonal anti-FLAG-M2 (Sigma-Aldrich;
#F1804). Secondary antibodies were horseradish peroxidase-conjugated anti-mouse or
anti-rabbit IgG, respectively (GE Healthcare Life Sciences or Promega) or horseradish
peroxidase-conjugated donkey anti-goat (Santa Cruz Biotechnology; #SC-2056). The
blots were developed with SuperSignal (Thermo Scientific) and imaged for
Chemluminescence detection using standard film or a ChemiDoc™ XRS+ Imaging
System (Bio-Rad). Immunoblots for endogenous BiP or BiP-FLAG expression were
primarily visualized using the ChemiDoc™ XRS+ Imaging System to prevent signal
saturation from the high levels of BiP present in the ER lumen of cells.
8
BiP Immunoprecipitation (IP)
HeLa-Kb cells (1×10
5–2×10
6 cells per IP) were transfected as described above.
Cells were either treated directly on the plate or aliquoted into 1.5 mL tubes for treatment
(typically 1×105 cells/500 L for suspension treatment). For radioactive IP experiments,
cells were treated for 1 h and then 50-100 Ci [35
S]Met/Cys was added directly to each
sample for an additional 1 h incubation. Cells were rinsed with 1X PBS and lysed in 150
L of RIPA buffer (25 mM HEPES-KOH (pH 7.4), 150 mM NaCl, 1% Triton™ X-100, 0.5% sodium deoxycholate (prepared fresh), 0.05% SDS, 1X Halt™ Protease Inhibitor
Cocktail, and 1X Halt™ Phosphatase Inhibitor Cocktail) alternating between incubation
on ice with 15 sec disruption by vortexing for a total of 15 min. After removing cellular
debris by centrifugation at 14,000 x g for 20 min, the cell lysate was combined with 5 g
of primary antibody (either rabbit anti-BiP; Proteintech or mouse monoclonal anti-
FLAG-M2; Sigma-Aldrich) and rotated for 1 h at 4ºC. To the cell lysate-primary antibody mixture, a 1:1 mixture of Dynabeads
® Protein-A and Dynabeads
® Protein-G was
added to each sample and rotated for 2 h at 4ºC. Beads were collected with a magnetic
and washed twice with RIPA buffer, and twice with RIPA buffer (without protease or
phosphastase inhibitors). To the samples was added SDS gel-loading buffer (50 mM
Tris-HCl (pH 6.8), 2% SDS, 0.1% bromophenol blue, 10% glycerol, 100 mM DTT) and
heated to 95°C for 5 min. Samples were resolved using ANY-KD SDS-PAGE
(Invitrogen). Gels were stained with Coomassie and dried [35
S]Met/Cys gels were
visualized by exposure to a PhosphoImager screen followed by analysis using a on a
Typhoon PhosphoImager (Molecular Dynamics) with ImageQuant software.
Tracing Translation by T cells (3T)
HeLa-Kb, D
b-L cells, or K
b-L cells were plated in 6-well plates (60-80%
confluence) 12–18 h prior to transfection. Cells were transfected with 0.5–1 g plasmid DNA (per well) using Lipofectamine 2000 (Invitrogen) for 12–18 h and titrated in 96-
well plates after treatment with ISR activators. Lac Z-inducible T cell hybridomas (1 ×
105/well) were added followed by overnight incubation at 37˚C for 16–20 h. The pMHC
I induced accumulation of intracellular β-galactosidase in the hybridoma and was
measured with the conversion of the substrate chlorophenol red-β-D-galactopyranoside
with a 96-well plate reader at 595 nm and 655 nm as the reference wavelength (90).
T cell hybridomas: tracer peptide KSIINFEHLK (KOVAK) processed to
SIINFEHL (SHL8) is recognized by Kb-L cells by the B3Z hybridoma (46, 91); tracer
peptide MTFNYRNL (MYL8) or LTFNYRNL (LYL8) is recognized by Kb-L cells by
the BCZ103 hybridoma (92); tracer peptide WI9 translated from an AUG start codon to
yield MWMHHNMDLI (Met-WI9) processed to WMHHNMDLI (WI9) is recognized
by Db-L cells by the 11p9Z hybridoma (93).
For peptide extracts, cells were rinsed after treatments with 1X PBS, resuspended
in 10% acetic acid, and placed in boiling water for 10 min. Cell debris was removed by
centrifugation and filtered through a 10-kilodalton-cutoff filter (Millipore). The extract
9
was vacuum concentrated, resuspended in 1X PBS containing 25 g/mL phenol red or
RPMI (no additives), and the pH was adjusted to 7 with 0.1 N NaOH. The cell extract or
SHL8 synthetic peptide was titrated in 96-well plates and Kb-expressing cells (K
b-L cells
or HeLa-Kb) were added at 5×10
4 cells/per well together with 1×10
5 B3Z Lac Z-inducible
T cell hybridoma. The T cell response is proportional to the relative amount of peptide
translated from the uORF2(KOVAK)-ATF4-luciferase construct. Synthetic SHL8
peptide was synthesized by D. King (University of California, Berkeley) and the structure
was confirmed by mass spectrometry.
Stable Cell Generation
To produce lentivirus, HEK293T cells were transfected with standard packaging
vectors and pATF4.6 or pATF4.7 using TransIT-LT1 (Mirus). Viral supernatant was
harvested at 72 h and filtered through a 0.45 M PVDF syringe filter. HeLa-Kb were
transduced by centrifugation at 1000 x g for 2 h at 33°C in the presence of the viral
supernatant and 8 g/ml polybrene. Following centrifugation, the viral supernatant was
removed from HeLa-Kb, fresh RPMI media was added, and cells were allowed to recover
for 72 h before expansion and subsequent Luciferase and Tracer peptide assays.
siRNA knock-down experiments
In 6-well plates, 60% confluent HeLa-Kb cells were transfected with 200
pmol/well of either Control siRNA-A or human eIF2A siRNAs (Santa Cruz
Biotechnology) using Lipofectamine 2000 (Invitrogen) for 24–48 h. Cells were
transfected for 12–18 h with plasmid DNAs (0.5–1 g/well; increased signal to noise
observed with 1 g/well) containing BiP-FLAG and GFP Transfection controls: EBNA-
alpha-GFP or EBNA-delta-GFP (94). For transfection controls, cells were analyzed with
a BD LSR II flow cytometer and data were analyzed with BD FACSDiva software to calculate mean fluorescence intensity (MFI). To assess the levels of eIF2A knock-down,
cells were analyzed by immunoblot as described above with an anti-eIF2A antibody
(Proteintech). Expression from the BiP5’ UTR -190 uORF was assessed by titrating
siRNA/plasmid DNA-transfected cells with T cell hybridomas as described in the
Tracing Translation by T cells (3T) section. Full-length BiP levels were assessed by
immunoblot as described in the Cell-based Expression Assay section.
Animal work
Mouse bone-marrow-derived dendritic cells (BM-dendritic cells) were cultured
for 5–8 days with recombinant GM-CSF (10 ng/mL) (Peprotech, Inc) to yield BM-
dendritic cells. All mice were housed within the animal facilities at the University of
California at Berkeley according to IACUC guidelines.
HLA prediction
10
HLA binding prediction was assessed using three independent programs: 1)
SYFPEITHI: http://www.syfpeithi.de/ (61). This algorithm was used to assess the
known HLA peptides identified from (85) and compared to the peptides generated from
translation of the BiP 5’ UTR. 2) MHC I binding predictions were made using the IEDB
analysis resource consensus tool (87) which combines predictions from ANN, aka
NetMHC (3.4) (95, 96), SMM (97) and Comblib (98). 3) NetMHC 3.4 Server (95, 96).
mRNA transfection
pATF4.1: pcDNA1 ATF4-luciferase and pATF4.3: U2( KOVAK) plasmid DNA
was linearized with HpaI and used as templates for transcription by T7 RNA polymerase
(mMessage mMachine T7; Ambion) to yield wild-type- and uORF2(KOVAK)-ATF4-
luciferase mRNAs. Transcription reactions contained m7GTP or ARCA m
7GTP cap
analog (Ambion) to yield naturally capped mRNAs. The Poly(A) Tailing Kit (Ambion)
was used to add poly(A) tails onto mRNAs. Transcribed mRNA was purified using 1:1
phenol:chloroform extraction, followed by chloroform extraction and purification using illustra Microspin G-25 columns (GE Healthcare Life Sciences). Prior to mRNA
transfection (12–15 h), HeLa-Kb cells were plated (80% confluence) in 6-well plates.
Cells were transfected with mRNA (2 g/well) for 3 h using TransMessenger
Transfection Regent (Qiagen). For mouse bone-marrow-derived dendritic cell assays
(BM-dendritic cells), bone-marrow cells (kind gift from Soo Jung Yang, University of
California, Berkeley) were cultured for 5–8 days with recombinant GM-CSF (10 ng/mL)
(Peprotech, Inc) to yield BM-dendritic cells. BM-dendritic cells (2–4 x106 cells) were
transfected with mRNA for 3 h as described above. Transfected HeLa-Kb or BM-
dendritic cells were titrated in 96-well plates and 1 × 105 B3Z Lac Z-inducible T cell
hybridoma were added and assayed as described in the Tracing Translation by T cells
section.
Fluorescence recovery after bleach (FRAP); TAP-FRAP Assay
Mel JuSo cells stably expressing TAP1-GFP (62) were maintained in Dulbecco's
modified Eagle's medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% FBS
(Invitrogen), 1 mM sodium pyruvate, 100 U/ml penicillin, and 100 μg/ml streptomycin.
The cells were grown in 35mm glass-bottom cell culture dishes coated with collagen
(MatTek Corp., Ashland, MA, USA) and imaged as live cells. Images were obtained
with a Zeiss LSM 710 Axio Observer Inverted confocal microscope (Carl Zeiss Inc.,
Thornwood, N.Y.) equipped with an incubation chamber set to a condition of 37°C and
5% CO2. The 488 nm Argon laser was used to provide high-intensity pulses of light for
photobleaching of GFP in a circular spot (radius = 0.82 µm) in the endoplasmic reticulum
of the cell. Prior to and directly after the laser pulse, images were obtained using a time-
lapse imaging provided by Zeiss LSM software. Recovery of GFP fluorescence was
monitored every second for 20 sec. The fluorescence intensity of unbleached spot in the
endoplasmic reticulum within the same cell was used as baseline in analysis. To block
translation, cells were pre-incubated at 37°C in the presence of 50–100 g/mL cycloheximide for 30 min. To block standard translation, cells were incubated with 25
µM NSC119893 during imaging. Quantitative analyses of fluorescence intensities were
11
performed using EMBL Frap-analysis software with Soumpasis Diffusion model. The
obtained fluorescence recovery curves were normalized to the unbleached fluorescence.
Data was determined using 8 cells per experiment and depicted as mean ± SEM.
MTFNYRNLMAY-----MAY-----MAH-----MAL-----
ATF4 uORF1 Sequence Alignment
ATF4 uORF2 Sequence Alignment
Nested uORF2(KOVAK) Tracer PeptideHumanMouse
CowChicken
10MALLTAFSSSVKSIINFEHLKLSTFLD-SSKAPQHDRNELPEQRGVGGGLDVPLRPVGFGGMALLTAFSSSVA-VTDKDTFELSTFLDSS-KAPQHDRNELPEQRGVGGGLDVPLRPVGFGGMALFTKSSSSVA-VTDKDTFELSTFLESS-KAPQHDRDELPEQRSVGGGLDVPLRPVGFGGMALFTAFSSSLA-VTDKDIFELSTFLELSTKTSQHGRDELSEQRGVRGGLRVPLRPVGFGGMAFFKAPHSSTA-VVDKKTLSLSTSFDVK--ALKPDQDEPLEQRDAVGG-VLPLQPAVFGG
uORF1(MYL8) Tracer PeptideHumanMouse
CowChicken
20 30 40 50 60
Fig. S1: Conserved amino acid sequence alignment of uORF1 of human ATF4 with uORF1
tracer peptide MTFNYRNL (called MYL8) (top) and uORF2 of human ATF4 with nested
uORF2 tracer peptide SIINFEHL (called SHL8) flanked by K residues (called KOVAK)
(bottom).
12
BCZ1
03 T
Cel
l res
pons
e (A
595)
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8 uORF1(MYL8)Kb- L cells
uORF2(MYL8)uORF2(KOVAK)uORF1(WI9)
Cell Number102101 103 104 105
uORF1
ATF4 luciferase
uORF2
tracerpeptide
uORF1
ATF4 luciferase
uORF2
tracerpeptide
Fig. S2: Simultaneous detection of uORF peptides from ATF4 using 3T during normal growth
conditions. The tracer peptide constructs uORF1(MYL8)-ATF4-luciferase, uORF2(MYL8)-
ATF4-luciferase, uORF2(KOVAK)-ATF4-luciferase, or uORF1(WI9)-ATF4-luciferase were
transfected into Kb-L-cells. The tracer peptide MYL8 presented by K
b was detected by the
BCZ103 T cell hybridoma, while neither WI9 nor KOVAK tracer peptides were detected (data
are presented as mean ± SD of two biological replicates and are representative of n = 3).
13
WT ATF4
NS
uORF1(WI9)
uORF1(MYL8)
uORF1(WI9)-uORF2(KOVAK)
uORF2(KOVAK)
Relative ATF4-luciferase to WT (RLU)0 1 2 3 4 5 6 7 8 9 10 11 12
Constitutive ATF4
*
**
uORF1
ATF4 luciferase
uORF2
tracerpeptide
uORF1
ATF4 luciferase
uORF2
tracerpeptide
uORF1
ATF4 luciferase
uORF2
tracerpeptide
uORF1
ATF4 luciferase
uORF2
tracerpeptide
uORF1
ATF4 luciferase
uORF2
uORF1 uORF2 fusion
ATF4 luciferase
Fig. S3: Expression of ATF4-luciferase from constructs with uORF tracer peptides during
normal growth conditions. ATF4-luciferase was measured from constructs with various tracer
peptide insertions or a constitutively active variant and compared to wild-type-ATF4-luciferase
expression (from Fig. 2A) with additional tracer peptide insertion constructs (mean ± SEM; n =
3-4). Statistical significance was evaluated with the unpaired t test (NS, not significant; *P <
0.05; **P < 0.01).
14
A B C
0
0.5
1
1.5
2
2.5
Mut SubAB
SubAB SubAB+ ISRIB
1 2 3 4
[35S]
Met
/Cys
Wild
-typ
e AT
F4-lu
cife
rase
Mut SubAB
Mut SubAB
Mut SubAB + IS
RIBSubAB + IS
RIB
SubAB + ISRIB
SubAB
SubAB
eIF2α-P
eIF2α-P
BiPNS
β-actin
β-actin
PERK-PPERK
ATF4*
*
Fig. S4: Endogenous and transgene expression of ATF4 from cells used for 3T. (A) HeLa-Kb
cells treated for 1 h with Mutant SubAB (0.2 μg/mL) or SubAB (0.2 μg/mL), and ISRIB (200
nM) and labeled with [35
S]-Met/Cys prior to harvesting (n = 3). HeLa-Kb cells transfected with
wildt-ATF4-luciferase plasmid were treated as in (A) except for 3 h and analyzed by (B)
immunoblot (n = 3) or (C) for luciferase activity (mean ± SEM; n = 3). Statistical significance
was evaluated with the unpaired t test (NS, not significant; *P < 0.05).
15
UT ISRIBNaAsO 2
NaAsO 2 + IS
RIB
eIF2α-P
ATF4
1h Treatment
*
[35S]
Met
/Cys
Coom
assie
Fig. S5: Endogenous expression of ATF4 is induced with NaAsO2. (A) HeLa-Kb cells were treated for 1 h with NaAsO2 (10 μM) and/or ISRIB (200 nM) and analyzed by immunoblot (n = 3) (top) or pulsed with [35S]-Met/Cys prior to harvesting (bottom). * = non-specific anti-ATF4 antibody signal.
16
A B
0.2
0.4
0.6
0.8
1.0
Cell Equivalents
0.2
0.3
0.4
0.5
B3Z
Resp
onse
(A59
5)
B3Z
Resp
onse
(A59
5)
untreatedNaAsO2 (3h)no tracer peptide
Tracer Peptide Titrationtracer peptide (SHL8)no tracer peptide
104 105Tracer Peptide (SHL8, pM)
102 103 10410-2 10-1 100 101
Fig. S6: ATF4 uORF2 tracer peptide detection after peptide extraction. (A) Peptide extracts from uORF2(KOVAK)-ATF4-luciferase transfected HeLa-Kb cells after treatment for 3 h with NaAsO2 (10 μM) were prepared using 10% acetic acid with boiling and pH adjustment with NaOH, titrated onto either untransfected HeLa-Kb cells or Kb-L cells followed by addition of B3Z T cell hybridoma (mean ± SD from two biological replicates and are representative of n = 2). (B) Peptide titration of the processed uORF2(KOVAK) peptide (SHL8) used to estimate the number of peptides per cell presented from the uORF2(KOVAK)-ATF4-luciferase reporter: 7209 ± 3833.
17
WT∆ATG(+1)
∆ATG(+24)∆ATG(+1);∆ATG(+24)
∆CTG(-12)
5’-..CAACUGGCUGGCAAGAUGAAGCUCUCCCUGGUGGCCGCGAUGCUGCUGCUGCUC5’-..CAACUGGCUGGCAAGGCCAAGCUCUCCCUGGUGGCCGCGAUGCUGCUGCUGCUC5’-..CAACUGGCUGGCAAGAUGAAGCUCUCCCUGGUGGCCGCGUUACUGCUGCUGCUC5’-..CAACUGGCUGGCAAGGCCAAGCUCUCCCUGGUGGCCGCGUUACUGCUGCUGCUC5’-..CAAGCCGCUGGCAAGAUGAAGCUCUCCCUGGUGGCCGCGAUGCUGCUGCUGCUC
-12 +1 +24
WT
HeLa-Kb RPE-19 Kb-L Cells (K89)
BiP-FLAG
GAPDH
∆ATG(+1)
∆ATG(+1);∆ATG(+24)
∆CTG(-12)
WT ∆ATG(+1)
∆ATG(+1);∆ATG(+24)
∆CTG(-12)
WT ∆ATG(+1)
∆ATG(+24)
∆ATG(+1);∆ATG(+24)
∆CTG(-12)
BiP-FLAG constructs
Fig. S7: The annotated +1 AUG of BiP is required for full-length CDS expression. Immunoblot
analysis of BiP-FLAG expression from the +1 AUG start codon and nearby in-frame codon
mutants in HeLa-Kb and primary human retinal pigmented epithelium cells (RPE-19) or K
b-L
cells (n = 2). The +24 AUG is present in the BiP signal sequence.
18
B
A C
Mut SubAB
SubAB
BiP-Endo BiP-FLAG BiP(L416D)-FLAG
Mut SubAB
SubAB
1 2 3 4 5 6
PERK-PPERK
BiP-FLAG
C terminal BiP-FLAG
*
[35S]
Met
/Cys
Coom
assie
BiP(L416D)-FLAGMut SubAB
SubABPERK-PPERK
BiP-FLAG
eIF2α-P
β-actin
1 2
Fig. S8: SubAB treatment induces the UPR. (A) SubAB-treated HeLa-Kb cells (2 h) from
BiP(L416D)-FLAG-transfection and analyzed by immunoblot (n = 3). (B) Mutant SubAB (Mut
SubAB) or SubAB treatment (2 h) followed by [35
S]-Met/Cys labeling in RPE-19 cells. (C)
SubAB cleaves endogenous and BiP-FLAG, lanes 1 – 4, analyzed by immunoblot: is full-
length BiP or C-terminal SubAB cleavage product and * is a FLAG antibody non-specific band
used as a loading control. Persistent expression of BiP(L416D)-FLAG is observed with SubAB
treatment (C, lanes 5 and 6). BiP cleavage induces the UPR as shown by activation of PERK
(PERK-P) (n = 2).
19
A
B
BiP 5’ UTR uORF (-190 UUG)
BiP 5’ UTR uORF (-61 CUG)
BiP 5’ UTR uORF (-61 CUG)with nested tracer peptide
BiP 5’ UTR uORF (-190 UUG)with nested tracer peptide
-190
L T Y W G V S R V
L T F N Y R N L
L C S C A T A C G W T A C C C P T G W N
L C S C A T A C K S I I N F E H L K W N
5’-GGG...GACAGAUUGACCUAUUGGGGUGUUUCGCGAGUGUGA
5’-...CUGUGCUCCUGUGCUACGGCCUGUGGCUGGACUGCCUGCUGCUGCCCAACUGGCUGGCAAG...
AUGAAG...
BiP CDS
AUGAAG...
5’-...CUGUGCUCCUGUGCUACGGCCUGUAAAUCGAUAAUCAACUUUGAACACCUCAAAUGGCAAG...
5’-GGG...GACAGAUUGACCUUCAACUACCGGAAUCUC---UGA
UAG
UGA
-61 +1
Fig. S9: Human BiP uORFs present in the 5’ UTR with tracer peptide insertions. (A) The LTFNYRNL tracer peptide (called LYL8) was nested in the -190 UUG uORF. An UAG stop codon (in blue) was inserted in the middle of the -190 UUG uORF and (B) the uORF2 tracer peptide SIINFEHL (SHL8) flanked by K residues (KOVAK) was nested in the -61 CUG uORF. An UGA stop codon (in blue) was inserted after the -61 CUG uORF start codon but before the tracer peptide sequence.
20
A
B
PERK-PPERK
BiP-FLAG
no plasmidDNA BiP-FLAG
-61 CUG N-extended(KOVAK)-BiP-FLAG fusion
5’-...CUGUGCUCCUGUGCUACGGCCUGUAAAUCGAUAAUCAACUUUGAACACCUCAAAGGCAAGAUGAAG...
-190 UUG (MYL8)BiP-FLAG
-61 CUG (KOVAK)BiP-FLAG fusion
β-actin
ATF4*
eIF2α-P
Mut SubABSubAB
UGA
-61 +10.5
0.4
0.3
0.2
102 103 104 105
B3Z
resp
onse
(A59
5)
Cell Number
-61 CUG N-extended(KOVAK) BiP-FLAG fusion-61 CUG...UGAno tracer peptide
Fig. S10: Tracer peptide insertion in the BiP 5’ UTR does not alter expression of the BiP CDS during steady-state and stress conditions. (A) HeLa-Kb cells transfected with BiP-FLAG tracer peptide constructs and treated with Mut SubAB/SubAB (0.2 μg/mL) for 2 h were analyzed by immunoblot (n = 2). (B) Translation of the -61 CUG uORF from -61 CUG uORF(KOVAK)-BiP-FLAG fusion transfected HeLa-Kb cells was assayed with the B3Z T cell hybridoma and compared to cells transfected with a no tracer peptide (BiP-FLAG) construct or a construct with in-frame UGA stop codon (in blue) inserted after the -61 CUG uORF start codon but before the tracer peptide (data are presented as mean ± SD of two biological replicates and are representative of n = 3).
21
A B
[35S]
Met
/Cys
cntrl siRNAMut SubABSubABISRIB
Coom
assie
[35S]
Met
/Cys
Coom
assie
eIF2A siRNA cntrlsiRNA
eIF2AsiRNA
DMSOThapsigargin
Fig. S11: eIF2A knock-down does not alter global protein synthesis. eIF2A siRNA knock-
down (48 h) in (A) mouse Kb-L cells followed by treatment with Mut SubAB/SubAB (0.2
μg/mL), and ISRIB (200 nM) and labeled with [35
S]-Met/Cys prior to harvesting (n = 2) or in (B)
HeLa-Kb cells followed by treatment with thapsigargin (1 μM) (3 h) and labeled with [
35S]-
Met/Cys prior to harvesting (n = 2).
22
Rela
tive
GFP
Pos
itive
Cel
ls
cntrlsiRNA
eIF2AsiRNA
0
20
40
60
80
100
120
Fig. S12: Transfection efficiency after control versus eIF2A siRNA knock-down is equivalent.
Transfection efficiency was determined by flow cytometry analysis of GFP-positive cells after
48h siRNA knock-down in HeLa-Kb cells followed by 16-20 h GFP plasmid (EBNA-GFP and
) transfection (mean ± SD; n = 4).
23
A
B
0
20
40
60
80
100
120
140
160
*
NS
Thapsigargin
cntrl siRNA eIF2A siRNA
cntrlsiRNA
eIF2AsiRNA
ThapsigarginPERK-PPERK
BiP-endogenous
BiP-
endo
geno
us le
vels
β-actin
ATF4*
Fig. S13: eIF2A knock-down deregulates BiP expression during normal growth condition and
ER stress. (A) Immunoblot analysis of endogenous BiP expression after 48 h eIF2A siRNA
knock-down in HeLa-Kb cells followed by treatment with thapsigargin (1 μM) (3 h). (B)
Endogenous BiP expression is enhanced after thapsigargin treatment, while eIF2A siRNA
knock-down depresses basal BiP expression and compromises enhanced expression during
thapsigargin treatment (mean ± SD; n = 3). Statistical significance was evaluated with the
unpaired t test (NS, not significant; *P < 0.05).
24
Human BiP 5’ UTR uORF (-190 UUG)
MH
C I e
pito
pe sc
ore
HLAB*27:05
Known HLAepitopes
-61 CUG peptide 27-35G R D A A A A Q R
otherHLA
HLAB*27:05
LTYWGVSRVA
D
0
5
10
15
20
25
30
******
B
E
0
5
10
15
20
25
Known HLAEpitopes
MH
C I e
pito
pe sc
ore
HLAB*15:16
otherHLA
HLA A*02:01 HLA B*15:16
-190 UUG peptideL T Y W G V S R V
HLAA*02:01
Human BiP 5’ UTR uORF (-61 CUG)
LCSCATACGWTACCCPTGWQDEA
LPGGRDAAAAQRGAGRGGGQEGGRGHGGRHRPGDHLLLRRRVQERP
RGDHRQRSGQPHHAVLCRLHS
C
Peptide
LTYWGVSRV 0.567 108320.678
HLA-A*02:01HLA-A*02:01
WBSBMTYWGVSRV
logscore Affinity (nM) Bind Level Allele
Fig. S14: Translation of the BiP 5’-UTR is predicted to generate human MHC I peptides (HLA epitopes). (A and B) HLA epitope prediction from the BiP 5’ UTR -190 UUG uORF was determined using SYFPEITHI. The predicted -190 UUG peptide epitope (LTYWGVSRV) score was compared to the score for all HLA (except HLA A*02:01 and HLA B*15:16) and to scores for known HLA A*02:01 and HLA B*15:16 epitopes identified from Kowalewski et al. (C and D) HLA epitope prediction from the BiP 5’ UTR -61 CUG uORF was determined using SYFPEITHI. The predicted -61 CUG peptide epitope (GRDAAAAQR, underlined) score was compared to the score for all HLA (except HLA B*27:05) and to scores for known HLA B*27:05 epitopes identified from Kowalewski et al. (E) HLA epitope prediction from the BiP 5’ UTR -190 UUG uORF (LTYWGVSRV or MTYWGVSRV) was determined using the NetMHC 3.4 Server.
25
[35S]
Met
/Cys
Coom
assie
DMSOBruce
antin
NSC119893
CHX
BiP
A B
GAPDH
Thapsigargin
DMSOAniso
mycin
NSC119893
Fig. S15: BiP expression is unaltered during limiting levels of eIF2•GTP•Met•tRNAi
Met. (A)
Total protein synthesis in HeLa-Kb cells were treated for 8h with the protein synthesis inhibitors
bruceantin (100 nM), NSC119893 (50 μM; replenished every 2h), and cycloheximide (CHX) (100 μg/mL) and pulse labeled with [35S]-Met/Cys prior to harvesting (n = 3). (B) Confluent endothelial human microvascular endothelial cells (HMVEC-1) were treated for 4 h with thapsigargin (4 μM), anisomycin (50 μM), NSC119893 (50 μM; replenished after 2h), or DMSO and analyzed by immunoblot (n = 3).
26
27
REFERENCES AND NOTES
1. P. Walter, D. Ron, The unfolded protein response: From stress pathway to homeostatic regulation. Science 334, 1081–1086 (2011). Medline doi:10.1126/science.1209038
2. Y. Shi, K. M. Vattem, R. Sood, J. An, J. Liang, L. Stramm, R. C. Wek, Identification and characterization of pancreatic eukaryotic initiation factor 2 alpha-subunit kinase, PEK, involved in translational control. Mol. Cell. Biol. 18, 7499–7509 (1998). Medline doi:10.1128/MCB.18.12.7499
3. H. P. Harding, Y. Zhang, D. Ron, Protein translation and folding are coupled by an endoplasmic-reticulum-resident kinase. Nature 397, 271–274 (1999). Medline doi:10.1038/16729
4. J. Galabru, M. G. Katze, N. Robert, A. G. Hovanessian, The binding of double-stranded RNA and adenovirus VAI RNA to the interferon-induced protein kinase. Eur. J. Biochem. 178, 581–589 (1989). Medline doi:10.1111/j.1432-1033.1989.tb14485.x
5. E. Meurs, K. Chong, J. Galabru, N. S. Thomas, I. M. Kerr, B. R. Williams, A. G. Hovanessian, Molecular cloning and characterization of the human double-stranded RNA-activated protein kinase induced by interferon. Cell 62, 379–390 (1990). Medline doi:10.1016/0092-8674(90)90374-N
6. J. J. Berlanga, J. Santoyo, C. De Haro, Characterization of a mammalian homolog of the GCN2 eukaryotic initiation factor 2alpha kinase. Eur. J. Biochem. 265, 754–762 (1999). Medline doi:10.1046/j.1432-1327.1999.00780.x
7. L. Lu, A. P. Han, J. J. Chen, Translation initiation control by heme-regulated eukaryotic initiation factor 2alpha kinase in erythroid cells under cytoplasmic stresses. Mol. Cell. Biol. 21, 7971–7980 (2001). Medline doi:10.1128/MCB.21.23.7971-7980.2001
8. R. J. Jackson, C. U. Hellen, T. V. Pestova, The mechanism of eukaryotic translation initiation and principles of its regulation. Nat. Rev. Mol. Cell Biol. 11, 113–127 (2010). Medline doi:10.1038/nrm2838
9. T. W. Hai, F. Liu, W. J. Coukos, M. R. Green, Transcription factor ATF cDNA clones: An extensive family of leucine zipper proteins able to selectively form DNA-binding heterodimers. Genes Dev. 3 (12B), 2083–2090 (1989). Medline doi:10.1101/gad.3.12b.2083
10. P. D. Lu, H. P. Harding, D. Ron, Translation reinitiation at alternative open reading frames regulates gene expression in an integrated stress response. J. Cell Biol. 167, 27–33 (2004). Medline doi:10.1083/jcb.200408003
11. K. M. Vattem, R. C. Wek, Reinitiation involving upstream ORFs regulates ATF4 mRNA translation in mammalian cells. Proc. Natl. Acad. Sci. U.S.A. 101, 11269–11274 (2004). Medline doi:10.1073/pnas.0400541101
12. A. J. Fornace Jr., I. Alamo Jr., M. C. Hollander, DNA damage-inducible transcripts in mammalian cells. Proc. Natl. Acad. Sci. U.S.A. 85, 8800–8804 (1988). Medline doi:10.1073/pnas.85.23.8800
28
13. D. Ron, J. F. Habener, CHOP, a novel developmentally regulated nuclear protein that dimerizes with transcription factors C/EBP and LAP and functions as a dominant-negative inhibitor of gene transcription. Genes Dev. 6, 439–453 (1992). Medline doi:10.1101/gad.6.3.439
14. J. Han, S. H. Back, J. Hur, Y. H. Lin, R. Gildersleeve, J. Shan, C. L. Yuan, D. Krokowski, S. Wang, M. Hatzoglou, M. S. Kilberg, M. A. Sartor, R. J. Kaufman, ER-stress-induced transcriptional regulation increases protein synthesis leading to cell death. Nat. Cell Biol. 15, 481–490 (2013). Medline doi:10.1038/ncb2738
15. A. G. Hinnebusch, The scanning mechanism of eukaryotic translation initiation. Annu. Rev. Biochem. 83, 779–812 (2014). Medline doi:10.1146/annurev-biochem-060713-035802
16. G. Joslin, W. Hafeez, D. H. Perlmutter, Expression of stress proteins in human mononuclear phagocytes. J. Immunol. 147, 1614–1620 (1991). Medline
17. R. Panniers, Translational control during heat shock. Biochimie 76, 737–747 (1994). Medline doi:10.1016/0300-9084(94)90078-7
18. K. Richter, M. Haslbeck, J. Buchner, The heat shock response: Life on the verge of death. Mol. Cell 40, 253–266 (2010). Medline doi:10.1016/j.molcel.2010.10.006
19. S. D. Der, A. S. Lau, Involvement of the double-stranded-RNA-dependent kinase PKR in interferon expression and interferon-mediated antiviral activity. Proc. Natl. Acad. Sci. U.S.A. 92, 8841–8845 (1995). Medline doi:10.1073/pnas.92.19.8841
20. F. D. Gilfoy, P. W. Mason, West Nile virus-induced interferon production is mediated by the double-stranded RNA-dependent protein kinase PKR. J. Virol. 81, 11148–11158 (2007). Medline doi:10.1128/JVI.00446-07
21. L.-C. Hsu, J. M. Park, K. Zhang, J.-L. Luo, S. Maeda, R. J. Kaufman, L. Eckmann, D. G. Guiney, M. Karin, The protein kinase PKR is required for macrophage apoptosis after activation of Toll-like receptor 4. Nature 428, 341–345 (2004). Medline doi:10.1038/nature02405
22. R. P. Shiu, J. Pouyssegur, I. Pastan, Glucose depletion accounts for the induction of two transformation-sensitive membrane proteins[ ]in Rous sarcoma virus-transformed chick embryo fibroblasts. Proc. Natl. Acad. Sci. U.S.A. 74, 3840–3844 (1977). Medline doi:10.1073/pnas.74.9.3840
23. I. G. Haas, M. Wabl, Immunoglobulin heavy chain binding protein. Nature 306, 387–389 (1983). Medline doi:10.1038/306387a0
24. S. Munro, H. R. Pelham, An Hsp70-like protein in the ER: Identity with the 78 kd glucose-regulated protein and immunoglobulin heavy chain binding protein. Cell 46, 291–300 (1986). Medline doi:10.1016/0092-8674(86)90746-4
25. J. Li, A. S. Lee, Stress induction of GRP78/BiP and its role in cancer. Curr. Mol. Med. 6, 45–54 (2006). Medline doi:10.2174/156652406775574523
26. M. S. Gorbatyuk, O. S. Gorbatyuk, The molecular chaperone GRP78/BiP as a therapeutic target for neurodegenerative disorders: A mini review. J. Genet. Syndr. Gene Ther. 4, 128 (2013). Medline doi:10.4172/2157-7412.1000128
29
27. L. Booth, J. L. Roberts, D. R. Cash, S. Tavallai, S. Jean, A. Fidanza, T. Cruz-Luna, P. Siembiba, K. A. Cycon, C. N. Cornelissen, P. Dent, GRP78/BiP/HSPA5/Dna K is a universal therapeutic target for human disease. J. Cell. Physiol. 230, 1661–1676 (2015). Medline doi:10.1002/jcp.24919
28. C. U. Hellen, P. Sarnow, Internal ribosome entry sites in eukaryotic mRNA molecules. Genes Dev. 15, 1593–1612 (2001). Medline doi:10.1101/gad.891101
29. J. Zhou, J. Wan, X. Gao, X. Zhang, S. R. Jaffrey, S. B. Qian, Dynamic m(6)A mRNA methylation directs translational control of heat shock response. Nature 526, 591–594 (2015). Medline doi:10.1038/nature15377
30. S. E. Calvo, D. J. Pagliarini, V. K. Mootha, Upstream open reading frames cause widespread reduction of protein expression and are polymorphic among humans. Proc. Natl. Acad. Sci. U.S.A. 106, 7507–7512 (2009). Medline doi:10.1073/pnas.0810916106
31. A. M. Resch, A. Y. Ogurtsov, I. B. Rogozin, S. A. Shabalina, E. V. Koonin, Evolution of alternative and constitutive regions of mammalian 5′UTRs. BMC Genomics 10, 162 (2009). Medline doi:10.1186/1471-2164-10-162
32. N. T. Ingolia, L. F. Lareau, J. S. Weissman, Ribosome profiling of mouse embryonic stem cells reveals the complexity and dynamics of mammalian proteomes. Cell 147, 789–802 (2011). Medline doi:10.1016/j.cell.2011.10.002
33. S. Lee, B. Liu, S. Lee, S. X. Huang, B. Shen, S. B. Qian, Global mapping of translation initiation sites in mammalian cells at single-nucleotide resolution. Proc. Natl. Acad. Sci. U.S.A. 109, E2424–E2432 (2012). Medline doi:10.1073/pnas.1207846109
34. N. T. Ingolia, G. A. Brar, N. Stern-Ginossar, M. S. Harris, G. J. Talhouarne, S. E. Jackson, M. R. Wills, J. S. Weissman, Ribosome profiling reveals pervasive translation outside of annotated protein-coding genes. Cell Reports 8, 1365–1379 (2014). Medline doi:10.1016/j.celrep.2014.07.045
35. M. Cazzola, R. C. Skoda, Translational pathophysiology: A novel molecular mechanism of human disease. Blood 95, 3280–3288 (2000). Medline
36. T. Kondo, M. Okabe, M. Sanada, M. Kurosawa, S. Suzuki, M. Kobayashi, M. Hosokawa, M. Asaka, Familial essential thrombocythemia associated with one-base deletion in the 5′-untranslated region of the thrombopoietin gene. Blood 92, 1091–1096 (1998). Medline
37. A. Wiestner, R. J. Schlemper, A. P. van der Maas, R. C. Skoda, An activating splice donor mutation in the thrombopoietin gene causes hereditary thrombocythaemia. Nat. Genet. 18, 49–52 (1998). Medline doi:10.1038/ng0198-49
38. N. Ghilardi, R. C. Skoda, A single-base deletion in the thrombopoietin (TPO) gene causes familial essential thrombocythemia through a mechanism of more efficient translation of TPO mRNA. Blood 94, 1480–1482 (1999). Medline
39. S. A. Slavoff, A. J. Mitchell, A. G. Schwaid, M. N. Cabili, J. Ma, J. Z. Levin, A. D. Karger, B. A. Budnik, J. L. Rinn, A. Saghatelian, Peptidomic discovery of short open reading frame-encoded peptides in human cells. Nat. Chem. Biol. 9, 59–64 (2013). Medline doi:10.1038/nchembio.1120
30
40. S. J. Andrews, J. A. Rothnagel, Emerging evidence for functional peptides encoded by short open reading frames. Nat. Rev. Genet. 15, 193–204 (2014). Medline doi:10.1038/nrg3520
41. V. Olexiouk, J. Crappé, S. Verbruggen, K. Verhegen, L. Martens, G. Menschaert, sORFs.org: A repository of small ORFs identified by ribosome profiling. Nucleic Acids Res. gkv1175 (2015). Medline doi:10.1093/nar/gkv1175
42. A. Pauli, M. L. Norris, E. Valen, G. L. Chew, J. A. Gagnon, S. Zimmerman, A. Mitchell, J. Ma, J. Dubrulle, D. Reyon, S. Q. Tsai, J. K. Joung, A. Saghatelian, A. F. Schier, Toddler: An embryonic signal that promotes cell movement via Apelin receptors. Science 343, 1248636 (2014). Medline doi:10.1126/science.1248636
43. D. E. Andreev, P. B. O’Connor, C. Fahey, E. M. Kenny, I. M. Terenin, S. E. Dmitriev, P. Cormican, D. W. Morris, I. N. Shatsky, P. V. Baranov, Translation of 5′ leaders is pervasive in genes resistant to eIF2 repression. eLife 4, e03971 (2015). Medline doi:10.7554/eLife.03971
44. C. Sidrauski, A. M. McGeachy, N. T. Ingolia, P. Walter, The small molecule ISRIB reverses the effects of eIF2α phosphorylation on translation and stress granule assembly. eLife 4, (2015). Medline doi:10.7554/eLife.05033
45. J. Somers, T. Pöyry, A. E. Willis, A perspective on mammalian upstream open reading frame function. Int. J. Biochem. Cell Biol. 45, 1690–1700 (2013). Medline doi:10.1016/j.biocel.2013.04.020
46. J. Karttunen, S. Sanderson, N. Shastri, Detection of rare antigen-presenting cells by the lacZ T-cell activation assay suggests an expression cloning strategy for T-cell antigens. Proc. Natl. Acad. Sci. U.S.A. 89, 6020–6024 (1992). Medline doi:10.1073/pnas.89.13.6020
47. Materials and methods are available as supplementary materials on Science Online.
48. H. P. Harding, Y. Zhang, H. Zeng, I. Novoa, P. D. Lu, M. Calfon, N. Sadri, C. Yun, B. Popko, R. Paules, D. F. Stojdl, J. C. Bell, T. Hettmann, J. M. Leiden, D. Ron, An integrated stress response regulates amino acid metabolism and resistance to oxidative stress. Mol. Cell 11, 619–633 (2003). Medline doi:10.1016/S1097-2765(03)00105-9
49. L. M. Mielnicki, R. G. Hughes, P. M. Chevray, S. C. Pruitt, Mutated Atf4 suppresses c-Ha-ras oncogene transcript levels and cellular transformation in NIH3T3 fibroblasts. Biochem. Biophys. Res. Commun. 228, 586–595 (1996). Medline doi:10.1006/bbrc.1996.1702
50. A. W. Paton, T. Beddoe, C. M. Thorpe, J. C. Whisstock, M. C. Wilce, J. Rossjohn, U. M. Talbot, J. C. Paton, AB5 subtilase cytotoxin inactivates the endoplasmic reticulum chaperone BiP. Nature 443, 548–552 (2006). Medline doi:10.1038/nature05124
51. C. Sidrauski, D. Acosta-Alvear, A. Khoutorsky, P. Vedantham, B. R. Hearn, H. Li, K. Gamache, C. M. Gallagher, K. K. Ang, C. Wilson, V. Okreglak, A. Ashkenazi, B. Hann, K. Nader, M. R. Arkin, A. R. Renslo, N. Sonenberg, P. Walter, Pharmacological brake-release of mRNA translation enhances cognitive memory. eLife 2, e00498 (2013). Medline doi:10.7554/eLife.00498
52. C. Sidrauski, J. C. Tsai, M. Kampmann, B. R. Hearn, P. Vedantham, P. Jaishankar, M. Sokabe, A. S. Mendez, B. W. Newton, E. L. Tang, E. Verschueren, J. R. Johnson, N. J.
31
Krogan, C. S. Fraser, J. S. Weissman, A. R. Renslo, P. Walter, Pharmacological dimerization and activation of the exchange factor eIF2B antagonizes the integrated stress response. eLife 4, e07314 (2015). Medline doi:10.7554/eLife.07314
53. Y. Sekine, A. Zyryanova, A. Crespillo-Casado, P. M. Fischer, H. P. Harding, D. Ron, Mutations in a translation initiation factor identify the target of a memory-enhancing compound. Science 348, 1027–1030 (2015). Medline doi:10.1126/science.aaa6986
54. K. Zhan, K. M. Vattem, B. N. Bauer, T. E. Dever, J. J. Chen, R. C. Wek, Phosphorylation of eukaryotic initiation factor 2 by heme-regulated inhibitor kinase-related protein kinases in Schizosaccharomyces pombe is important for resistance to environmental stresses. Mol. Cell. Biol. 22, 7134–7146 (2002). Medline doi:10.1128/MCB.22.20.7134-7146.2002
55. J. P. Abastado, P. F. Miller, B. M. Jackson, A. G. Hinnebusch, Suppression of ribosomal reinitiation at upstream open reading frames in amino acid-starved cells forms the basis for GCN4 translational control. Mol. Cell. Biol. 11, 486–496 (1991). Medline
56. K. Gülow, D. Bienert, I. G. Haas, BiP is feed-back regulated by control of protein translation efficiency. J. Cell Sci. 115, 2443–2452 (2002). Medline
57. S. R. Schwab, J. A. Shugart, T. Horng, S. Malarkannan, N. Shastri, Unanticipated antigens: Translation initiation at CUG with leucine. PLOS Biol. 2, e366 (2004). Medline doi:10.1371/journal.pbio.0020366
58. S. R. Starck, V. Jiang, M. Pavon-Eternod, S. Prasad, B. McCarthy, T. Pan, N. Shastri, Leucine-tRNA initiates at CUG start codons for protein synthesis and presentation by MHC class I. Science 336, 1719–1723 (2012). Medline doi:10.1126/science.1220270
59. H. Liang, S. He, J. Yang, X. Jia, P. Wang, X. Chen, Z. Zhang, X. Zou, M. A. McNutt, W. H. Shen, Y. Yin, PTENα, a PTEN isoform translated through alternative initiation, regulates mitochondrial function and energy metabolism. Cell Metab. 19, 836–848 (2014). Medline
60. F. Robert, L. D. Kapp, S. N. Khan, M. G. Acker, S. Kolitz, S. Kazemi, R. J. Kaufman, W. C. Merrick, A. E. Koromilas, J. R. Lorsch, J. Pelletier, Initiation of protein synthesis by hepatitis C virus is refractory to reduced eIF2·GTP·Met-tRNAi
Met ternary complex availability. Mol. Biol. Cell 17, 4632–4644 (2006). Medline doi:10.1091/mbc.E06-06-0478
61. H. Rammensee, J. Bachmann, N. P. Emmerich, O. A. Bachor, S. Stevanović, SYFPEITHI: Database for MHC ligands and peptide motifs. Immunogenetics 50, 213–219 (1999). Medline doi:10.1007/s002510050595
62. E. A. Reits, J. C. Vos, M. Grommé, J. Neefjes, The major substrates for TAP in vivo are derived from newly synthesized proteins. Nature 404, 774–778 (2000). Medline doi:10.1038/35008103
63. M. J. Androlewicz, P. Cresswell, How selective is the transporter associated with antigen processing? Immunity 5, 1–5 (1996). Medline doi:10.1016/S1074-7613(00)80304-0
64. G. E. Hammer, T. Kanaseki, N. Shastri, The final touches make perfect the peptide-MHC class I repertoire. Immunity 26, 397–406 (2007). Medline doi:10.1016/j.immuni.2007.04.003
32
65. K. R. Brandvold, R. I. Morimoto, The chemical biology of molecular chaperones—Implications for modulation of proteostasis. J. Mol. Biol. 427, 2931–2947 (2015). Medline doi:10.1016/j.jmb.2015.05.010
66. B. Wu, C. Hunt, R. Morimoto, Structure and expression of the human gene encoding major heat shock protein HSP70. Mol. Cell. Biol. 5, 330–341 (1985). Medline doi:10.1128/MCB.5.2.330
67. X. Zhang, X. Gao, R. A. Coots, C. S. Conn, B. Liu, S. B. Qian, Translational control of the cytosolic stress response by mitochondrial ribosomal protein L18. Nat. Struct. Mol. Biol. 22, 404–410 (2015). Medline
68. W. L. Zoll, L. E. Horton, A. A. Komar, J. O. Hensold, W. C. Merrick, Characterization of mammalian eIF2A and identification of the yeast homolog. J. Biol. Chem. 277, 37079–37087 (2002). Medline doi:10.1074/jbc.M207109200
69. J. H. Kim, S. M. Park, J. H. Park, S. J. Keum, S. K. Jang, eIF2A mediates translation of hepatitis C viral mRNA under stress conditions. EMBO J. 30, 2454–2464 (2011). Medline doi:10.1038/emboj.2011.146
70. I. Ventoso, M. A. Sanz, S. Molina, J. J. Berlanga, L. Carrasco, M. Esteban, Translational resistance of late alphavirus mRNA to eIF2alpha phosphorylation: A strategy to overcome the antiviral effect of protein kinase PKR. Genes Dev. 20, 87–100 (2006). Medline doi:10.1101/gad.357006
71. D. G. Macejak, P. Sarnow, Internal initiation of translation mediated by the 5′ leader of a cellular mRNA. Nature 353, 90–94 (1991). Medline doi:10.1038/353090a0
72. Y. K. Kim, S. K. Jang, Continuous heat shock enhances translational initiation directed by internal ribosomal entry site. Biochem. Biophys. Res. Commun. 297, 224–231 (2002). Medline doi:10.1016/S0006-291X(02)02154-X
73. M. C. Lai, Y. H. Lee, W. Y. Tarn, The DEAD-box RNA helicase DDX3 associates with export messenger ribonucleoproteins as well as tip-associated protein and participates in translational control. Mol. Biol. Cell 19, 3847–3858 (2008). Medline doi:10.1091/mbc.E07-12-1264
74. V. P. Pisareva, A. V. Pisarev, A. A. Komar, C. U. Hellen, T. V. Pestova, Translation initiation on mammalian mRNAs with structured 5′UTRs requires DExH-box protein DHX29. Cell 135, 1237–1250 (2008). Medline doi:10.1016/j.cell.2008.10.037
75. M. A. Skabkin, O. V. Skabkina, V. Dhote, A. A. Komar, C. U. Hellen, T. V. Pestova, Activities of Ligatin and MCT-1/DENR in eukaryotic translation initiation and ribosomal recycling. Genes Dev. 24, 1787–1801 (2010). Medline doi:10.1101/gad.1957510
76. M. Oyama, C. Itagaki, H. Hata, Y. Suzuki, T. Izumi, T. Natsume, T. Isobe, S. Sugano, Analysis of small human proteins reveals the translation of upstream open reading frames of mRNAs. Genome Res. 14 (10B), 2048–2052 (2004). Medline doi:10.1101/gr.2384604
77. M. A. Purbhoo, D. J. Irvine, J. B. Huppa, M. M. Davis, T cell killing does not require the formation of a stable mature immunological synapse. Nat. Immunol. 5, 524–530 (2004). Medline doi:10.1038/ni1058
33
78. D. F. Hunt, R. A. Henderson, J. Shabanowitz, K. Sakaguchi, H. Michel, N. Sevilir, A. L. Cox, E. Appella, V. H. Engelhard, Characterization of peptides bound to the class I MHC molecule HLA-A2.1 by mass spectrometry. Science 255, 1261–1263 (1992). Medline doi:10.1126/science.1546328
79. H. Escobar, D. K. Crockett, E. Reyes-Vargas, A. Baena, A. L. Rockwood, P. E. Jensen, J. C. Delgado, Large scale mass spectrometric profiling of peptides eluted from HLA molecules reveals N-terminal-extended peptide motifs. J. Immunol. 181, 4874–4882 (2008). Medline doi:10.4049/jimmunol.181.7.4874
80. V. H. Engelhard, The contributions of mass spectrometry to understanding of immune recognition by T lymphocytes. Int. J. Mass Spectrom. 259, 32–39 (2007). Medline doi:10.1016/j.ijms.2006.08.009
81. B. Vanderperre, J. F. Lucier, C. Bissonnette, J. Motard, G. Tremblay, S. Vanderperre, M. Wisztorski, M. Salzet, F. M. Boisvert, X. Roucou, Direct detection of alternative open reading frames translation products in human significantly expands the proteome. PLOS ONE 8, e70698 (2013). Medline doi:10.1371/journal.pone.0070698
82. V. L. Crotzer, R. E. Christian, J. M. Brooks, J. Shabanowitz, R. E. Settlage, J. A. Marto, F. M. White, A. B. Rickinson, D. F. Hunt, V. H. Engelhard, Immunodominance among EBV-derived epitopes restricted by HLA-B27 does not correlate with epitope abundance in EBV-transformed B-lymphoblastoid cell lines. J. Immunol. 164, 6120–6129 (2000). Medline doi:10.4049/jimmunol.164.12.6120
83. G. Lubec, L. Afjehi-Sadat, Limitations and pitfalls in protein identification by mass spectrometry. Chem. Rev. 107, 3568–3584 (2007). Medline doi:10.1021/cr068213f
84. S. R. Starck, N. Shastri, Non-conventional sources of peptides presented by MHC class I. Cell. Mol. Life Sci. 68, 1471–1479 (2011). Medline doi:10.1007/s00018-011-0655-0
85. D. J. Kowalewski, H. Schuster, L. Backert, C. Berlin, S. Kahn, L. Kanz, H. R. Salih, H. G. Rammensee, S. Stevanovic, J. S. Stickel, HLA ligandome analysis identifies the underlying specificities of spontaneous antileukemia immune responses in chronic lymphocytic leukemia (CLL). Proc. Natl. Acad. Sci. U.S.A. 112, E166–E175 (2015). Medline
86. G. L. Law, A. Raney, C. Heusner, D. R. Morris, Polyamine regulation of ribosome pausing at the upstream open reading frame of S-adenosylmethionine decarboxylase. J. Biol. Chem. 276, 38036–38043 (2001). Medline
87. A. Ventura, A. Meissner, C. P. Dillon, M. McManus, P. A. Sharp, L. Van Parijs, R. Jaenisch, T. Jacks, Cre-lox-regulated conditional RNA interference from transgenes. Proc. Natl. Acad. Sci. U.S.A. 101, 10380–10385 (2004). Medline doi:10.1073/pnas.0403954101
88. N. Morinaga, K. Yahiro, G. Matsuura, M. Watanabe, F. Nomura, J. Moss, M. Noda, Two distinct cytotoxic activities of subtilase cytotoxin produced by shiga-toxigenic Escherichia coli. Infect. Immun. 75, 488–496 (2007). Medline doi:10.1128/IAI.01336-06
89. S. Sanderson, N. Shastri, LacZ inducible, antigen/MHC-specific T cell hybrids. Int. Immunol. 6, 369–376 (1994). Medline doi:10.1093/intimm/6.3.369
34
90. J. Kunisawa, N. Shastri, The group II chaperonin TRiC protects proteolytic intermediates from degradation in the MHC class I antigen processing pathway. Mol. Cell 12, 565–576 (2003). Medline doi:10.1016/j.molcel.2003.08.009
91. S. Malarkannan, P. P. Shih, P. A. Eden, T. Horng, A. R. Zuberi, G. Christianson, D. Roopenian, N. Shastri, The molecular and functional characterization of a dominant minor H antigen, H60. J. Immunol. 161, 3501–3509 (1998). Medline
92. S. Malarkannan, L. M. Mendoza, N. Shastri, Generation of antigen-specific, lacZ-inducible T-cell hybrids. Methods Mol. Biol. 156, 265–272 (2001). Medline
93. S. Cardinaud, S. R. Starck, P. Chandra, N. Shastri, The synthesis of truncated polypeptides for immune surveillance and viral evasion. PLOS ONE 5, e8692 (2010). Medline doi:10.1371/journal.pone.0008692
94. Y. Kim, J. Ponomarenko, Z. Zhu, D. Tamang, P. Wang, J. Greenbaum, C. Lundegaard, A. Sette, O. Lund, P. E. Bourne, M. Nielsen, B. Peters, Immune epitope database analysis resource. Nucleic Acids Res. 40 (W1), W525–W530 (2012). Medline doi:10.1093/nar/gks438
95. M. Nielsen, C. Lundegaard, P. Worning, S. L. Lauemøller, K. Lamberth, S. Buus, S. Brunak, O. Lund, Reliable prediction of T-cell epitopes using neural networks with novel sequence representations. Protein Sci. 12, 1007–1017 (2003). Medline doi:10.1110/ps.0239403
96. C. Lundegaard, K. Lamberth, M. Harndahl, S. Buus, O. Lund, M. Nielsen, NetMHC-3.0: Accurate web accessible predictions of human, mouse and monkey MHC class I affinities for peptides of length 8-11. Nucleic Acids Res. 36 (Web Server), W509–W512 (2008). Medline doi:10.1093/nar/gkn202
97. B. Peters, A. Sette, Generating quantitative models describing the sequence specificity of biological processes with the stabilized matrix method. BMC Bioinformatics 6, 132 (2005). Medline doi:10.1186/1471-2105-6-132
98. J. Sidney, E. Assarsson, C. Moore, S. Ngo, C. Pinilla, A. Sette, B. Peters, Quantitative peptide binding motifs for 19 human and mouse MHC class I molecules derived using positional scanning combinatorial peptide libraries. Immunome Res. 4, 2 (2008). Medline doi:10.1186/1745-7580-4-2