Cristea - IFI16 Oligomerization

Cristea - IFI16 Oligomerization
IFI16 pyrin oligomerization mediates antiviral actions
  • Organism: Homo sapiens
  • Instrument: Q Exactive HF
  • SpikeIn: No
  • Keywords: IFI16, oligomerization, innate immunity, herpesvirus, IP-MS
  • Submitter: Krystal Lum
Abstract
The formation of multimerized protein assemblies has emerged as a core component of immune signal amplification, yet the biochemical basis of this phenomenon remains unclear for many mammalian proteins within host defense pathways. The interferon-inducible protein 16 (IFI16) is a viral DNA sensor that oligomerizes upon binding to nuclear viral DNA and induces downstream antiviral responses. Here, we identify the pyrin domain (PYD) residues that mediate IFI16 oligomerization in a charge-dependent manner. Based on structure modeling, these residues are predicted to be surface-exposed within distinct α-helices. By generating oligomerization-deficient mutants, we demonstrate that IFI16 homotypic clustering is necessary for its assembly onto parental viral genomes at the nuclear periphery upon herpes simplex virus 1 (HSV-1) infection. Preventing oligomerization severely hampered the capacity of IFI16 to induce antiviral cytokine expression, suppress viral protein levels, and restrict viral progeny production. Restoring oligomerization via residue-specific charge mimics partially rescued IFI16 antiviral roles. We show that pyrin domains from PYHIN proteins are functionally interchangeable, facilitating cooperative assembly with the IFI16 HINs, highlighting an inherent role for pyrin domains in antiviral response. Using immunoaffinity purification and targeted mass spectrometry, we establish that oligomerization promotes IFI16 interactions with proteins involved in transcriptional regulation, including PAF1C, UBTF, and ND10 bodies. We further discover PAF1C as an HSV-1 restriction factor. Altogether, our study uncovers intrinsic properties that govern IFI16 oligomerization, which serves as a signal amplification platform to activate innate immune responses and to recruit transcriptional regulatory proteins that suppress HSV-1 replication.
Experiment Description
Fibroblast cells expressing IFI16-GFP were lysed using detergent lysis. Immunoaffinity purifications were performed targeting GFP with GFP-Trap_MA beads (Chromotek). IP eluates were reduced and alkylated then subjected to tryptic digestion with suspension trapping columns (S-Trap, Protifi) for 1 hour. Peptide eluates were resuspended in 1% FA, 1% ACN. Peptides were analyzed by nano-liquid chromatography coupled to tandem mass spectrometry with a Q Exactive HF Hybrid Quadrupole-Orbitrap intrument (Thermo Scientific) using parallel-reaction monitoring (PRM) mode. Peptides (2 µL injections) were separated with a 3% solvent B to 30% solvent B gradient (solvent A: 0.1% FA, solvent B: 0.1% FA, 97% ACN) over 60 min at a flow rate of 250 nL/min on an EASYSpray C18 column (75 µm x 50 cm) heated to 50 °C. The instrument was set to 15,000 resolution, AGC target was set to 1E5, MIT of 100 ms, 0.8 m/z isolation window, and NCE set to 27. An MS1 scan was acquired after every 20 PRM scans. MS1 acquisition was performed at 15,000 resolution, 25 ms MIT, and full scan range of 350-1800 m/z.
Sample Description
CRISPR/Cas9 was used to knock out IFI16 from human fibroblasts (HFF-1s) which were then transduced to express IFI16 mutants tagged with GFP or GFP alone.
Created on 4/1/19, 12:49 PM
Clustergrammer Heatmap
 
Download
NV0001_Mouse-Skin_mProphet_Panorama_2024-03-09_19-20-18.sky.zip2024-03-10 20:30:291,6595,7905,79028,90434
XW0008_Cas9Myc_DIAassayLIB_OmBcells_17Nov2023_2024-02-24_08-51-18.sky.zip2024-02-24 12:56:485,20383,67483,675605,04024
XW0009_DIAassayLIB_OmBcells_17Nov2023_2024-02-23_18-35-50.sky.zip2024-02-23 22:06:575,20383,64583,647604,72019
AutoQC-lumos-SysS-MouAD-PFC-C2-B5-B7.sky.zip2024-02-20 07:53:561889414
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C2_B07.sky.zip2024-02-18 11:31:099,778127,624127,624966,34712
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C2_B06.sky.zip2024-02-18 10:45:259,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C2_B05.sky.zip2024-02-18 09:51:569,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C2_B04.sky.zip2024-02-18 01:14:219,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C2_B03.sky.zip2024-02-18 00:22:039,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C2_B02.sky.zip2024-02-17 23:29:529,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C2_B01.sky.zip2024-02-17 18:20:009,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B28.sky.zip2024-02-17 17:30:039,778127,624127,624966,3476
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B27.sky.zip2024-02-17 16:57:559,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B26.sky.zip2024-02-17 15:06:069,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B25.sky.zip2024-02-17 14:11:069,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B24.sky.zip2024-02-17 13:17:049,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B23.sky.zip2024-02-17 10:45:369,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B22.sky.zip2024-02-17 09:52:589,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B21.sky.zip2024-02-17 09:01:129,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B20.sky.zip2024-02-17 01:24:329,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B19.sky.zip2024-02-17 00:31:539,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B18.sky.zip2024-02-16 23:42:139,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B17.sky.zip2024-02-16 21:59:109,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B16.sky.zip2024-02-16 21:08:449,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B15.sky.zip2024-02-16 19:45:379,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B14.sky.zip2024-02-16 18:50:509,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B13.sky.zip2024-02-16 17:05:369,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B12.sky.zip2024-02-16 16:13:309,778127,624127,624966,34716
XW0008-Myc248_DIAassayLIB_OmBcells_17Nov2023_2024-02-16_10-02-13.sky.zip2024-02-16 15:02:065,20383,67483,675605,04024
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B11.sky.zip2024-02-16 11:03:589,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B10.sky.zip2024-02-16 10:07:519,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B09.sky.zip2024-02-16 09:14:539,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B08.sky.zip2024-02-16 08:20:059,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B07.sky.zip2024-02-16 01:08:409,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B06.sky.zip2024-02-16 00:17:379,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B05.sky.zip2024-02-15 23:29:389,778127,624127,624966,34716
XW0008_nanos3_DIAassayLIB_OmBcells_17Nov2023_2024-02-15_17-02-46.sky.zip2024-02-15 21:13:165,20383,67483,675605,04024
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B04.sky.zip2024-02-15 16:37:369,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B03.sky.zip2024-02-15 14:42:299,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B02.sky.zip2024-02-15 13:44:359,778127,624127,624966,34716
Lumos-Jax-Cortex-DIA-ind-8mz-ovlp-400to1000-C1_B01.sky.zip2024-02-15 12:45:409,778127,624127,624966,34716
AutoQC-lumos-PCs-MouAD-PFC-C2-B5-B7.sky.zip2024-02-14 16:42:502141417344
AutoQC-lumos-PCs-MouAD-PFC-C2-B1-B4.sky.zip2024-02-14 16:42:332141417364
AutoQC-lumos-PCs-MouAD-PFC-C1-B9-B12.sky.zip2024-02-14 16:42:152141417364
AutoQC-lumos-PCs-MouAD-PFC-C1-B4-B8.sky.zip2024-02-14 16:42:002141417380
AutoQC-lumos-PCs-MouAD-PFC-C1-B25-B28.sky.zip2024-02-14 16:41:372141417354
AutoQC-lumos-PCs-MouAD-PFC-C1-B21-B24.sky.zip2024-02-14 16:41:002141417364
AutoQC-lumos-PCs-MouAD-PFC-C1-B17-B20.sky.zip2024-02-14 16:40:442141417365
AutoQC-lumos-PCs-MouAD-PFC-C1-B13-B16.sky.zip2024-02-14 16:40:282141417364
AutoQC-lumos-PCs-MouAD-PFC-C1-B1-B3.sky.zip2024-02-14 16:40:082141417347
AutoQC-lumos-SysS-MouAD-PFC-C2-B1-B4.sky.zip2024-02-14 16:10:161889417
AutoQC-lumos-SysS-MouAD-PFC-C1-B9-B12.sky.zip2024-02-14 16:06:251889416
AutoQC-lumos-SysS-MouAD-PFC-C1-B4-B8.sky.zip2024-02-14 16:02:231889422
AutoQC-lumos-SysS-MouAD-PFC-C1-B1-B3.sky.zip2024-02-14 15:59:501889418
AutoQC-lumos-SysS-MouAD-PFC-C1-B17-B20.sky.zip2024-02-14 14:48:381889410
ZipChip_HR_Metabolomics_2024Protocol_2024-02-05_17-24-05.sky.zip2024-02-05 14:24:28100821594
22AminoAcids_Fully13CLabeled_2024-01-29_14-30-52.sky.zip2024-01-29 11:32:1410444936
RBD_M_Glyco_2024-01-25_15-29-41.sky.zip2024-01-26 17:23:2672923972,3829
20240104_Neg_FMT_MCBAs_isoRemove_Cleaned_Final_2024-01-25_21-40-19.sky.zip2024-01-26 16:43:471010030056
20231220_Neg_FMT_BA_Full_reduce_Res50_High_final_2024-01-04_15-44-59.sky.zip2024-01-26 16:43:47405112176
P179_UNCSet1_ACE_v0p3_2024-01-24_22-42-18.sky.zip2024-01-24 19:51:4423034963724
P179_UNCSet2_ACE_v0p3_2024-01-24_22-37-25.sky.zip2024-01-24 19:40:1117021336726
New_iRBD2024-01-15 23:30:5233474794292
Paired_CSF_Plasma_Serum2024-01-15 23:30:523347479460
Initial_Targeted_Proteomics2024-01-15 23:30:5233474794441
TPAD_VL_CSF_PRTC_APOA1_2024-01-07_23-01-46.sky.zip2024-01-07 23:08:493464642412
TPAD-CSF-SP3_1-5.sky.zip2024-01-05 06:03:432,90823,74323,743189,895396
173_peptides_iRTs_chromatogram_library_2023-12-22_00-47-19.sky.zip2023-12-22 01:06:36311833561,0822
Figure_8B_Freiburg_ALG1-CDG-Patients_Comparison_2023-12-22_02-34-55.sky.zip2023-12-22 01:06:2022691284006
Figures_4_5_6_7_8A_Heidelberg_CDG-Patients_2023-12-22_02-32-43.sky.zip2023-12-22 01:06:20206712439014
Figure_S5_Freiburg_ALG11_I-CDG_Natural_Variant_2023-12-22_01-59-41.sky.zip2023-12-22 01:06:2021112146
Figure_9_Freiburg_ALG11_I-CDG_Natural_Variant_2023-12-22_01-53-52.sky.zip2023-12-22 01:06:2021418404
Figures_3_and_S3_HEK_293T_Fibroblasts_HeLa_2023-12-22_01-03-03.sky.zip2023-12-22 01:06:2023701303989
20210301 Calibration Dev_DilutionOil_2023-12-11_10-57-35.sky.zip2023-12-20 00:34:263482654
20210607 Calibration Curve_DilutionDigest_2023-12-11_10-50-40.sky.zip2023-12-20 00:34:2634824108
20210212 Low range exploration 140K-fragmod_Pub_2023-12-08_16-04-13.sky.zip2023-12-20 00:34:263482456
HeatedOilSpike-LowTemp_HighTemp_Combined_Final_2022-05-26_12-00-47.sky.zip2023-12-20 00:34:26591548204
20200715_PeptideSpecificity_SignalRatio_2022-05-25_16-33-02.sky.zip2023-12-20 00:34:2611202212044
20200622_PeptideSpecificityTest_2022-05-25_16-30-20.sky.zip2023-12-20 00:34:2614252713545
20191112_Diff-TempConc_Oil-Spike_24pep_2022-05-25_14-24-35.sky.zip2023-12-20 00:34:2611242715040
20191007_HeatedOilSpike_Extraction_method_24pep_2022-05-25_14-16-21.sky.zip2023-12-20 00:34:2611242715032
20190904_Organic_Aqueous_Extraction_Oil_Spike_24pep_2022-05-25_14-12-26.sky.zip2023-12-20 00:34:2611242715636
September 21 Import V1 (Samples with IS) w Cal Curve_Blanks Deleted_2023-12-01_11-40-59.sky.zip2023-12-02 23:51:3320262687
September 21 Kaylie New Molecule Import v1 (Filtered)_2023-12-01_11-40-01.sky.zip2023-12-02 23:51:3310161698
September 21 Import V1 all samples (Neg mode only)_2023-12-01_11-35-34.sky.zip2023-12-02 23:51:338014514598
THP1_IFN_PRM_Skyline_2023-11-14_14-22-42.sky.zip2023-11-16 13:26:09711771771,5148
CCS_library_v2.sky.zip2023-11-15 14:36:301061,86361,8630
IdentExpression_2023-09-25_14-39-47.sky.zip2023-10-02 20:45:1381581941,16419
2DGel_II_2023-09-25_14-37-39.sky.zip2023-10-02 20:45:13897408805,28034
2DGel_I_2023-09-25_14-36-13.sky.zip2023-10-02 20:45:13252142321,39232
InGelDigest_Der_p_SEA_II_2023-09-18_10-01-52.sky.zip2023-09-21 11:03:445004,0584,42026,52051
InGelDigest_Der_p_SEA_I_2023-09-15_16-14-11.sky.zip2023-09-21 11:03:443272,3822,52115,12652
FASP_Der_p_SEA_2023-09-15_14-24-26.sky.zip2023-09-21 11:03:445504,2964,77128,6261
230504_Myllys_231247ff_2023-09-20_13-22-59.sky.zip2023-09-21 10:31:2410434398
230512MyllysUrea_231247ff_2023-09-21_15-27-13.sky.zip2023-09-21 10:31:24102292
230511Myllys231247ff_AA_Crea_2023-09-20_13-36-24.sky.zip2023-09-21 10:31:242044166126
AutoQC-lumos-SysS-MouAD-C2-B8-10.sky.zip2023-09-17 11:22:321889414
AutoQC-lumos-SysS-MouAD-C2-B5-B7.sky.zip2023-09-17 11:22:301889415
AutoQC-lumos-SysS-MouAD-C2-B1-B4.sky.zip2023-09-17 11:22:281889419
AutoQC-lumos-SysS-MouAD-C1-B9-B12.sky.zip2023-09-17 11:22:261889413