Dataset Information

Proximal interaction network of ARF GTPases

ABSTRACT: The Arf GTPase family is involved in a wide range of cellular regulation including membrane trafficking and organelle structure assembly. Here, we have generated a proximity interaction network for the Arf family using the miniTurboID approach combined with TMT-based quantitative mass spectrometry. Our interactome confirmed known interactions and identifies many novel interactors that provide leads for defining Arf pathway cell biological functions.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Embryo, Epithelial Cell, Early Embryonic Cell, Kidney

DISEASE(S): Disease Free

SUBMITTER: Fulong Li

LAB HEAD: Kun-Liang Guan

PROVIDER: PXD033382 | Pride | 2022-07-20

REPOSITORIES: Pride

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Publications

Defining the proximal interaction networks of Arf GTPases reveals a mechanism for the regulation of PLD1 and PI4KB.

Li Fu-Long FL Wu Zhengming Z Gao Yong-Qi YQ Bowling Forrest Z FZ Franklin J Matthew JM Hu Chongze C Suhandynata Raymond T RT Frohman Michael A MA Airola Michael V MV Zhou Huilin H Guan Kun-Liang KL

The EMBO journal 20220717 17

The Arf GTPase family is involved in a wide range of cellular regulation including membrane trafficking and organelle-structure assembly. Here, we have generated a proximity interaction network for the Arf family using the miniTurboID approach combined with TMT-based quantitative mass spectrometry. Our interactome confirmed known interactions and identified many novel interactors that provide leads for defining Arf pathway cell biological functions. We explored the unexpected finding that phosph ...[more]

PMID: 35844135

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Project description:In the current study, we used a mass spectrometry-based redox proteomics approach to test responses of the cysteine (Cys) proteome to selective disruption of the Trx- and GSH-dependent systems. Auranofin (ARF) was used to inhibit Trx reductase without detectable oxidation of the GSH/GSSG couple, and buthionine sulfoximine (BSO) was used to deplete GSH without detectable oxidation of Trx1. Results for 606 Cys-containing peptides (peptidyl Cys) showed that 36% were oxidized over 1.3-fold by ARF, while BSO-induced oxidation of peptidyl Cys was only 10%. Mean fold oxidation of these peptides was also higher by ARF than BSO treatment. Analysis of potential functional pathways showed that ARF oxidized peptides associated with glycolysis, cytoskeleton remodeling, translation and cell adhesion. Of 60 peptidyl Cys oxidized due to depletion of GSH, 41 were also oxidized by ARF and included proteins of translation and cell adhesion but not glycolysis or cytoskeletal remodeling. Studies to test functional correlates showed that pyruvate kinase activity and lactate levels were decreased with ARF but not BSO, confirming the effects on glycolysis-associated proteins are sensitive to oxidation by ARF. These data show that the Trx system regulates a broader range of proteins than GSH system, support distinct function of Trx and GSH in cellular redox control, and show for the first time in mammalian cells selective targeting peptidyl Cys and biological pathways due to deficient function of the Trx system. ICAT-labeled Cys peptides were analyzed by reverse-phase liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Peptide eluents were monitored in an MS survey scan followed by ten data-dependent MS/MS scans on an LTQ-Orbitrap ion trap mass spectrometer (Thermo Finnigan, San Jose, CA). The LTQ was used to acquire MS/MS spectra (2 m/z isolation width, 35% collision energy, 5,000 AGC target, 200 ms maximum ion time). The Orbitrap was used to collect MS scans (300-1600 m/z, 1,000,000 AGC target, 500 ms maximum ion time, resolution 30,000). All data were converted from .raw files to the .dta format using ExtractMS version 2.0 (Thermo Finnigan, San Jose, CA). The acquired MS/MS spectra were searched against a concatenated target-decoy human RefSeq (release 37 - September 2009 - 38108 target proteins) database of the National Center for Biotechnology Information using the SEQUEST Sorcerer algorithm (version 3.11, SAGE-N) searching parameters included partially tryptic restriction, parent ion mass tolerance (+/- 20 ppm), dynamic modifications of oxidized Met (+15.9949 Da), differential ICAT-modified Cys (+9.0302 Da) and static ICAT modification of Cys (+227.1270 Da). The peptides were classified by charge state and tryptic state (fully and partial) and first filtered by mass accuracy (10 ppm for high-resolution MS), and then dynamically by increasing XCorr and deltaCn values to reduce protein false discovery rate to less than 1%, according to the target-decoy strategy.

Project description:SARS-CoV-2 emerged in China at the end of 2019 and caused the global pandemic of COVID-19, a disease with high morbidity and mortality. While our understanding of this new virus is rapidly increasing, gaps remain in our understanding of how SARS-CoV-2 can effectively suppress host cell antiviral responses. Recent work on other viruses has demonstrated a novel mechanism through which viral proteins can mimic critical regions of human histone proteins. Histone proteins are responsible for governing genome accessibility and their precise regulation is critical for a cell’s ability to control transcription and respond to viral threats. Here, we show that the protein encoded by ORF8 (Orf8) in SARS-CoV-2 functions as a histone mimic of the ARKS motif in histone 3. Orf8 is associated with chromatin, binds to numerous histone-associated proteins, and is itself acetylated within the histone mimic site. Orf8 expression in cells disrupts multiple critical histone post-translational modifications (PTMs) including H3K9ac, H3K9me3, and H3K27me3 and promotes chromatin compaction while Orf8 lacking the histone mimic motif does not. Further, SARS-CoV-2 infection in human cell lines and postmortem patient lung tissue cause these same disruptions to chromatin. However, deletion of the Orf8 gene from SARS-CoV-2 largely blocks its ability to disrupt host-cell chromatin indicating that Orf8 is responsible for these effects. Finally, deletion of the ORF8 gene affects the host-cell transcriptional response to SARS-CoV-2 infection in multiple cell types and decreases the replication of SARS-CoV-2 in human induced pluripotent stem cell-derived lung alveolar type 2 (iAT2) pulmonary cells. These findings demonstrate a novel function for the poorly understood ORF8-encoded protein and a mechanism through which SARS-CoV-2 disrupts host cell epigenetic regulation. Finally, this work provides a molecular basis for the finding that SARS-CoV-2 lacking ORF8 is associated with decreased severity of COVID-19.

Dataset Information

Proximal interaction network of ARF GTPases

Publications

Defining the proximal interaction networks of Arf GTPases reveals a mechanism for the regulation of PLD1 and PI4KB.

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