Proximity-labeling chemoproteomics defines the subcellular cysteinome and inflammation-responsive mitochondrial redoxome - PubMed (original) (raw)

Proximity-labeling chemoproteomics defines the subcellular cysteinome and inflammation-responsive mitochondrial redoxome

Tianyang Yan et al. bioRxiv. 2023.

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Abstract

Proteinaceous cysteines function as essential sensors of cellular redox state. Consequently, defining the cysteine redoxome is a key challenge for functional proteomic studies. While proteome-wide inventories of cysteine oxidation state are readily achieved using established, widely adopted proteomic methods such as OxiCat, Biotin Switch, and SP3-Rox, they typically assay bulk proteomes and therefore fail to capture protein localization-dependent oxidative modifications. To obviate requirements for laborious biochemical fractionation, here, we develop and apply an unprecedented two step cysteine capture method to establish the Local Cysteine Capture (Cys-LoC), and Local Cysteine Oxidation (Cys-LOx) methods, which together yield compartment-specific cysteine capture and quantitation of cysteine oxidation state. Benchmarking of the Cys-LoC method across a panel of subcellular compartments revealed more than 3,500 cysteines not previously captured by whole cell proteomic analysis. Application of the Cys-LOx method to LPS stimulated murine immortalized bone marrow-derived macrophages (iBMDM), revealed previously unidentified mitochondria-specific inflammation-induced cysteine oxidative modifications including those associated with oxidative phosphorylation. These findings shed light on post-translational mechanisms regulating mitochondrial function during the cellular innate immune response.

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Conflict of interest statement

Conflicts of Interest

The authors declare no financial or commercial conflict of interest.

Figures

Figure 1.

Figure 1.. Establishment of Local Cysteine Capture (Cys-LoC) method.

A) Scheme of Cys-LoC Workflow. B) Cysteines identified with Cys-LoC from five compartments aggregated compared to those identified from whole proteome in HEK293T cells. C) Cysteines identified with Mito-Cys-LoC compared to those identified from the whole proteome in HEK293T cells. D) Scheme of database generation with aggregated protein localization annotations from Human Protein Atlas, UniprotKB and CellWhere. PK and FK represented primary key and foreign key, respectively. E) Number of proteins annotated as localized in cytosol (cyto), endoplasmic reticulum (ER), golgi, mitochondrial (mito), and nucleus (nuc). F) Subcellular specificity of cysteines identified with Cys-LoC or with whole lysate [cite sp3 paper]. G) Number of cysteines identified as compartment specific with Cys-LoC or with whole lysate in HEK293T cells. Statistical significance was calculated with unpaired Student’s t-tests, * p<0.05, NS p>0.05. Experiments were performed in duplicates in HEK293T cells for penal B, C, F, G. All MS data can be found in Table S1.

Figure 2.

Figure 2.. Pinpointing sources of non-compartment specific TurboID biotinylation.

A) Scheme of TurboID based proximity labeling protein enrichment. B) Subcellular specificity of proteins identified with TurboID or with whole lysate. C) Number of proteins by number of localization annotations. D) Percentage of proteins within each subcellular compartment containing greater than 1 annotated localization. E) Number of mitochondrial annotated proteins and non-mitochondrial annotated proteins identified with transiently or stably expressed mito-TurboID. F) Absolute quantification of biotin detected for the whole cell, the whole cell with 500 μM exogenous biotin added, and the mitochondrial fraction with 500 μM exogenous biotin added. G) Number of PSMs of mitochondrial annotated proteins and non-mitochondrial annotated proteins identified with mito-TurboID with 50 μM or 500 μM exogenous biotin. H) Proteome analysis of the proteins enriched with mito-TurboID. SA-bkg indicated proteins identified in streptavidin background proteome. I) Localization of biotinylation in cells with mito-TurboID indicated by signals of streptavidin-rhodamine (SA-Rho). J) Localization of mito-TurboID overlaid with Mito-TMRE. Statistical significance was calculated with unpaired Student’s t-tests, * p<0.05, NS p>0.05. Experiments were performed in duplicates in HEK293T cells for panel B, E, F, G, H. All MS data can be found in Table S2.

Figure 3.

Figure 3.. Translation arrest improves the subcellular specificity of proteins enriched by TurboID and cysteines captured by Cys-LoC.

A) Abundance of mito-TurboID in mitochondrial vs non-mitochondrial fractions. Non-mitochondrial fraction refers to the rest of the lysate after crude mitochondria extraction. B) Abundance of mito-TurboID in mitochondrial vs non-mitochondrial fractions with CHX treatment. C) Quantitation of blot data in 3A and 3B. Intensity of the Mito-TurboID lane in the non-mitochondrial fraction is normalized to that in the mitochondrial fraction, which is set to 1.0. D) Expression of TurboID upon treatment of the translational inhibitor CHX. E) Specificity and number of mitochondria localized proteins enriched with mito-TurboID for control condition, dialyzed FBS, CHX treatment or for both. F) Distribution of the proteins enriched with mito-TurboID for control condition, dialyzed FBS, CHX treatment or both. G) Number of PSMs of representative mitochondrial proteins and non-mitochondrial proteins enriched with mito-TurboID for control condition, dialyzed FBS, CHX treatment or with both. H) Difference in intensities of proteins enriched with mito-TurboID with or without dialyzed FBS and CHX treatment. Red dots indicate proteins localized in mitochondria. Black dots indicate proteins localized in organelles other than mitochondria. I) Difference in intensities of cysteines enriched with MitoCys-LoC with or without dialyzed FBS and CHX treatment. Red dots indicate cysteines localized in mitochondria. Black dots indicate cysteines localized in organelles other than mitochondria. J) Specificity of cysteines identified in different subcellular compartments after Cys-LoC with or without dialyzed FBS and CHX treatment and with TurboID optimization. K) Cysteines identified with Cys-LoC targeting different subcellular compartments. Dialyzed FBS (Dia-FBS) treatment was 36 h and CHX treatment was 100 ug/mL for 6 h at 37 °C. Statistical significance was calculated with unpaired Student’s t-tests, * p<0.05, ** p<0.01, *** p<0.005. NS p>0.05. Experiments were performed in triplicates in HEK293T cells. All MS data can be found in Table S3.

Figure 4.

Figure 4.. Establishing the Local Cysteine Oxidation (Cys-LOx) method to analyze basal mitochondrial cysteine oxidation states.

A) Scheme of Cys-LOx method. B) Percent oxidation state of mitochondrial cysteines identified with mito-Cys-LOx. C) Percent oxidation state of cysteines quantified in an exemplary mitochondrial protein. D) Difference in redox states of cysteines quantified with Mito-Cys-LOx with or without dialyzed FBS and CHX treatment. Red dots indicate cysteines localized in mitochondria. Black dots indicate cysteins localized in organelles other than mitochondria. Dialyzed FBS (Dia-FBS) treatment was 36 h and CHX treatment was 100 ug/mL for 6 h at 37 °C. Experiments were performed in triplicate in iBMDM cells. All MS data can be found in Table S4.

Figure 5.

Figure 5.. Cys-LOx outperforms SP3-Rox for quantification of LPS-induced changes of mitochondrial cysteine oxidation states.

A) Oxygen consumption rate (OCR) of mitochondria for control, LPS+IFNγ, CHX or both. This is representative trace of one biological replicate with 5 technical replicates. For some timepoints, the symbols obscure the error bars. B) qCPR analysis of expression of Nos2 with control, LPS+IFNγ, CHX or both. C) Difference of redox states for cysteines quantified with SP3-Rox with or without LPS+IFNγ treatment. Red dots indicate cysteines localized in mitochondria. Black dots indicate cysteins localized in organelles other than mitochondria. D) Different redox states of cysteines quantified with SP3-Rox with or without LPS+IFNγ treatment in exemplary proteins with different splice forms. E) Redox states of cysteines quantified with Mito-Cys-LOx with or without LPS+IFNγ treatment. Statistical significance was calculated with paired Student’s t-tests, **** p<0.001. F) Difference of redox states for cysteines quantified with Mito-Cys-LOx with or without LPS+IFNγ treatment. Red dots indicate cysteines localized in mitochondria. Black dots indicate cysteins localized in organelles other than mitochondria. G) Difference of redox states for cysteines quantified with Mito-Cys-LOx and SP3-Rox with or without LPS+IFNγ treatment. * indicates cysteines in the Safe List we generated that are insensitive to translational arrest. H) GO biological process analysis of mitochondrial cysteines quantified with mito-Cys-ROx that showed more oxidized redox states upon LPS+IFNγ treatment. I) KEGG pathway analysis of mitochondrial cysteines quantified with mito-Cys-ROx that showed more oxidized redox states upon LPS+IFNγ treatment. J) Crystal structure of ISCU with cysteines more oxidized with LPS+IFNγ treatment (PDB 1WFZ). Experiments were performed in triplicates and technical replicates in iBMDM cells. Dialyzed FBS (Dia-FBS) treatment was 36 hrs, CHX treatment was 100 ug/mL for 6 h at 37 °C and LPS+IFNγ treatment was 100 ng/mL LPS and 20 ng/mL IFNγ for 24 h at 37 °C. All MS data can be found in Table S5.

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