Sensing of mitochondrial DNA by ZBP1 promotes RIPK3-mediated necroptosis and ferroptosis in response to diquat poisoning.

Diquat (DQ) poisoning is a severe medical condition associated with life-threatening implications and multiorgan dysfunction. Despite its clinical significance, the precise underlying mechanism remains inadequately understood. This study elucidates that DQ induces instability in the mitochondrial genome of endothelial cells, resulting in the accumulation of Z-form DNA. This process activates Z-DNA binding protein 1 (ZBP1), which then interacts with receptor-interacting protein kinase 3 (RIPK3), ultimately leading to RIPK3-dependent necroptotic and ferroptotic signaling cascades. Specific deletion of either Zbp1 or Ripk3 in endothelial cells simultaneously inhibits both necroptosis and ferroptosis. This dual inhibition significantly reduces organ damage and lowers mortality rate. Notably, our investigation reveals that RIPK3 has a dual role. It not only phosphorylates MLKL to induce necroptosis but also phosphorylates FSP1 to inhibit its enzymatic activity, promoting ferroptosis. The study further shows that deletion of mixed lineage kinase domain-like (Mlkl) and the augmentation of ferroptosis suppressor protein 1 (FSP1)-dependent non-canonical vitamin K cycling can provide partial protection against DQ-induced organ damage. Combining Mlkl deletion with vitamin K treatment demonstrates a heightened efficacy in ameliorating multiorgan damage and lethality induced by DQ. Taken together, this study identifies ZBP1 as a crucial sensor for DQ-induced mitochondrial Z-form DNA, initiating RIPK3-dependent necroptosis and ferroptosis. These findings suggest that targeting the ZBP1/RIPK3-dependent necroptotic and ferroptotic pathways could be a promising approach for drug interventions aimed at mitigating the adverse consequences of DQ poisoning.

Dear-DIAXMBD: Deep Autoencoder Enables Deconvolution of Data-Independent Acquisition Proteomics.

Data-independent acquisition (DIA) technology for protein identification from mass spectrometry and related algorithms is developing rapidly. The spectrum-centric analysis of DIA data without the use of spectra library from data-dependent acquisition data represents a promising direction. In this paper, we proposed an untargeted analysis method, Dear-DIAXMBD, for direct analysis of DIA data. Dear-DIAXMBD first integrates the deep variational autoencoder and triplet loss to learn the representations of the extracted fragment ion chromatograms, then uses the k-means clustering algorithm to aggregate fragments with similar representations into the same classes, and finally establishes the inverted index tables to determine the precursors of fragment clusters between precursors and peptides and between fragments and peptides. We show that Dear-DIAXMBD performs superiorly with the highly complicated DIA data of different species obtained by different instrument platforms. Dear-DIAXMBD is publicly available at https://github.com/jianweishuai/Dear-DIA-XMBD.

Evaluation of DDA Library-Free Strategies for Phosphoproteomics and Ubiquitinomics Data-Independent Acquisition Data.

Phosphoproteomics and ubiquitinomics data-independent acquisition (DIA) mass spectrometry (MS) data is typically analyzed by using a data-dependent acquisition (DDA) spectral library. The performance of various library-free strategies for analyzing phosphoproteomics and ubiquitinomics DIA MS data has not been evaluated. In this study, we systematically compare four commonly used DDA library-free approaches including Spectronaut's directDIA, DIA-Umpire, DIA-MSFragger, and in silico-predicted library for analysis of phosphoproteomics SWATH, DIA, and diaPASEF data as well as ubiquitinomics diaPASEF data. Spectronaut's directDIA shows the highest sensitivity for phosphopeptide detection not only in synthetic phosphopeptide samples but also in phosphoproteomics SWATH-MS and DIA data from real biological samples, when compared to the other three library-free strategies. For phosphoproteomics diaPASEF data, Spectronaut's directDIA and the in silico-predicted library based on DIA-NN identify almost the same number of phosphopeptides as a project-specific DDA spectral library. However, only about 30% of the total phosphopeptides are commonly identified, suggesting that the library-free strategies for phospho-diaPASEF data need further improvement in terms of sensitivity. For ubiquitinomics diaPASEF data, the in silico-predicted library performs the best among the four workflows and detects ∼50% more K-GG peptides than a project-specific DDA spectral library. Our results demonstrate that Spectronaut's directDIA is suitable for the analysis of phosphoproteomics SWATH-MS and DIA MS data, while the in silico-predicted library based on DIA-NN shows substantial advantages for ubiquitinomics diaPASEF MS data.

Quantitative proteomic analysis of exosomes from umbilical cord mesenchymal stem cells and rat bone marrow stem cells.

Exosomes derived from mesenchymal stem cells (MSCs) have been used for cancer treatment, however, an in-depth analysis of the exosomal proteomes is lacking. In this manuscript, we use the diaPASEF (parallel accumulation serial fragmentation combined with the data-independent acquisition) method to quantify exosomes derived from human umbilical cord mesenchymal stem cells (UCMSCs) and rat bone marrow stem cells (BMSCs), resulting in identification of 4200 human proteins and 5362 rat proteins. Comparison of human exosomal proteins and total cellular proteins reveals that some proteins exist in the exosomes exclusively that can be served as potential markers for exosomes. Quantitative proteomic analysis of exosomes from different passages of BMSCs shows that the proteins involved in TGF-ß signaling pathway are regulated in abundance, which could be markers for the therapeutic ability of BMSC exosomes. Collectively, the data presented by this study can be a resource for further study of exosome research.

Apical anchorage and stabilization of subpellicular microtubules by apical polar ring ensures Plasmodium ookinete infection in mosquito.

Morphogenesis of many protozoans depends on a polarized establishment of cortical cytoskeleton containing the subpellicular microtubules (SPMTs), which are apically nucleated and anchored by the apical polar ring (APR). In malaria parasite Plasmodium, APR emerges in the host-invading stages, including the ookinete for mosquito infection. So far, the fine structure and molecular components of APR as well as the underlying mechanism of APR-mediated apical positioning of SPMTs are largely unknown. Here, we resolve an unprecedented APR structure composed of a top ring plus approximate 60 radiating spines. We report an APR-localizing and SPMT-binding protein APR2. APR2 disruption impairs ookinete morphogenesis and gliding motility, leading to Plasmodium transmission failure in mosquitoes. The APR2-deficient ookinetes display defective apical anchorage of APR and SPMT due to the impaired integrity of APR. Using protein proximity labeling, we obtain a Plasmodium ookinete APR proteome and validate ten undescribed APR proteins. Among them, APRp2 and APRp4 directly interact with APR2 and also mediate the apical anchorage of SPMTs. This study sheds light on the molecular basis of APR in the organization of Plasmodium ookinete SPMTs.

Inner membrane complex proteomics reveals a palmitoylation regulation critical for intraerythrocytic development of malaria parasite.

Malaria is caused by infection of the erythrocytes by the parasites Plasmodium. Inside the erythrocytes, the parasites multiply via schizogony, an unconventional cell division mode. The inner membrane complex (IMC), an organelle located beneath the parasite plasma membrane, serving as the platform for protein anchorage, is essential for schizogony. So far, the complete repertoire of IMC proteins and their localization determinants remain unclear. Here we used biotin ligase (TurboID)-based proximity labeling to compile the proteome of the schizont IMC of the rodent malaria parasite Plasmodium yoelii. In total, 300 TurboID-interacting proteins were identified. 18 of 21 selected candidates were confirmed to localize in the IMC, indicating good reliability. In light of the existing palmitome of Plasmodium falciparum, 83 proteins of the P. yoelii IMC proteome are potentially palmitoylated. We further identified DHHC2 as the major resident palmitoyl-acyl-transferase of the IMC. Depletion of DHHC2 led to defective schizont segmentation and growth arrest both in vitro and in vivo. DHHC2 was found to palmitoylate two critical IMC proteins CDPK1 and GAP45 for their IMC localization. In summary, this study reports an inventory of new IMC proteins and demonstrates a central role of DHHC2 in governing the IMC localization of proteins during the schizont development.

MSSort-DIAXMBD: A deep learning classification tool of the peptide precursors quantified by OpenSWATH.

OpenSWATH is an analysis toolkit commonly used for data independent acquisition (DIA). Although the output of OpenSWATH is controlled at 1% false discovery rate (FDR), the output report still contains many peptide precursors with low similarity fragments. At the last step of OpenSWATH for peptide quantification, researchers usually need to manually check the similarity of the extracted ion chromatograms (XICs) of fragments to distinguish the high confidence and the low confidence peptide precursors. Here we developed an algorithm with a Graphic User Interface named MSSort-DIAXMBD, which combines the deep convolutional neural network (CNN) and the double-threshold segmentation process, to automatically recognize the high confidence precursors and low confidence precursors. To train the model of MSSort-DIAXMBD, we built a database contained about 50,000 manually classified peptide precursors acquired from different instrument platforms and different species. With the double-threshold segmentation strategy, MSSort-DIAXMBD can reduce the number of the low confidence peptides required for manual inspections to less than 10% and be used as the last step of OpenSWATH to visualize and classify the MS/MS data of peptide precursors. SIGNIFICANCE: Although the output of OpenSWATH is controlled at 1% false discovery rate (FDR), the output report still contains many peptide precursors with low similarity fragments. At the last step of OpenSWATH for peptide quantification, researchers usually need to manually check the similarity of fragment XICs to distinguish the high confidence and the low confidence peptide precursors. However, manual inspection is inefficient. For instance, it takes about 50 h to sort even a small dataset of 1000 MS/MS spectra manually. In this paper we developed a software named MSSort-DIAXMBD to automatically recognize the high confidence precursors. We manually classify 50,000 peptide precursors as training set to train a convolutional neural network. After training finished, MSSort-DIAXMBD takes only a few minutes to automatically classify 20,000 peptide precursors, leaving a small portion of fuzzy ones for manual inspection. On the benchmarked dataset, MSSort-DIAXMBD can significantly improve the efficiency and accuracy of recognition of high confidence peptide precursors.

Investigation of Effects of the Spectral Library on Analysis of diaPASEF Data.

Targeted analysis of data-independent acquisition (DIA) data needs a spectral library, which is generated by data-dependent acquisition (DDA) experiments or directly from DIA data. A comparison of the DDA library and DIA library in analyzing DIA data has been reported. However, the effects of different spectral libraries on the analysis of diaPASEF data have not been investigated. Here, we generate different spectral libraries with varying proteome coverage to analyze parallel accumulation-serial fragmentation (diaPASEF) data. Besides, we also employ the library-free strategy. The library, constructed by extensive fractionation DDA experiments, produces the highest numbers of precursors and proteins but with a high percentage of missing values. The library-free strategy identifies 10-20% fewer proteins than the library-based method but with a high degree of data completeness. A further study shows that the library-free strategy, although it identifies fewer proteins than the library-based method, leads to similar biological conclusions as the library-based method.

Deep representation features from DreamDIAXMBD improve the analysis of data-independent acquisition proteomics.

We developed DreamDIAXMBD (denoted as DreamDIA), a software suite based on a deep representation model for data-independent acquisition (DIA) data analysis. DreamDIA adopts a data-driven strategy to capture comprehensive information from elution patterns of peptides in DIA data and achieves considerable improvements on both identification and quantification performance compared with other state-of-the-art methods such as OpenSWATH, Skyline and DIA-NN. Specifically, in contrast to existing methods which use only 6 to 10 selected fragment ions from spectral libraries, DreamDIA extracts additional features from hundreds of theoretical elution profiles originated from different ions of each precursor using a deep representation network. To achieve higher coverage of target peptides without sacrificing specificity, the extracted features are further processed by nonlinear discriminative models under the framework of positive-unlabeled learning with decoy peptides as affirmative negative controls. DreamDIA is publicly available at https://github.com/xmuyulab/DreamDIA-XMBD for high coverage and accuracy DIA data analysis.

SCASP: A Simple and Robust SDS-Aided Sample Preparation Method for Proteomic Research.

SDS is widely used in sample preparation for proteomic research. However, SDS is incompatible with LC and electrospray ionization. SDS depletion is therefore required ahead of LC-MS analysis. Most of current SDS removal strategies are time consuming, laborious, and have low reproducibility. Here, we describe a method, SDS-cyclodextrin (CD)-assisted sample preparation, by which CD can bind to SDS and form CD-SDS complexes in solutions, allowing for direct tryptic digestion. We demonstrate that SDS-CD-assisted sample preparation is a simple, fast, and robust SDS-based sample preparation method for proteomics application.

RIP1-dependent linear and nonlinear recruitments of caspase-8 and RIP3 respectively to necrosome specify distinct cell death outcomes.

There remains a significant gap in our quantitative understanding of crosstalk between apoptosis and necroptosis pathways. By employing the SWATH-MS technique, we quantified absolute amounts of up to thousands of proteins in dynamic assembling/de-assembling of TNF signaling complexes. Combining SWATH-MS-based network modeling and experimental validation, we found that when RIP1 level is below ~1000 molecules/cell (mpc), the cell solely undergoes TRADD-dependent apoptosis. When RIP1 is above ~1000 mpc, pro-caspase-8 and RIP3 are recruited to necrosome respectively with linear and nonlinear dependence on RIP1 amount, which well explains the co-occurrence of apoptosis and necroptosis and the paradoxical observations that RIP1 is required for necroptosis but its increase down-regulates necroptosis. Higher amount of RIP1 (>~46,000 mpc) suppresses apoptosis, leading to necroptosis alone. The relation between RIP1 level and occurrence of necroptosis or total cell death is biphasic. Our study provides a resource for encoding the complexity of TNF signaling and a quantitative picture how distinct dynamic interplay among proteins function as basis sets in signaling complexes, enabling RIP1 to play diverse roles in governing cell fate decisions.

QuantPipe: A User-Friendly Pipeline Software Tool for DIA Data Analysis Based on the OpenSWATH-PyProphet-TRIC Workflow.

Targeted analysis of data-independent acquisition (DIA) mass spectrometry data requires elegant software tools and strict statistical control. OpenSWATH-PyProphet-TRIC is a widely used DIA data analysis workflow. The OpenSWATH-PyProphet-TRIC workflow is typically executed by running command lines. Here, we present QuantPipe, which is a graphic interface software tool based on the OpenSWATH-PyProphet-TRIC workflow. In addition to OpenSWATH-PyProphet-TRIC functions, QuantPipe can convert the spectral library to the assay library and output peptides and protein intensities. We demonstrated that QuantPipe can be used to analyze SWATH-MS data from TripleTOF 5600 and TripleTOF 6600, phospho-SWATH-MS data, DIA data from Orbitrap instrument, and diaPASEF data from TimsTOF Pro instrument. The executable files, user manual, and source code of QuantPipe are freely available at https://github.com/tachengxmu/QuantPipe/releases.

A Non-canonical PDK1-RSK Signal Diminishes Pro-caspase-8-Mediated Necroptosis Blockade.

Necroptosis induction in vitro often requires caspase-8 (Casp8) inhibition by zVAD because pro-Casp8 cleaves RIP1 to disintegrate the necrosome. It has been unclear how the Casp8 blockade of necroptosis is eliminated naturally. Here, we show that pro-Casp8 within the necrosome can be inactivated by phosphorylation at Thr265 (pC8T265). pC8T265 occurs in vitro in various necroptotic cells and in the cecum of TNF-treated mice. p90 RSK is the kinase of pro-Casp8. It is activated by a mechanism that does not need ERK but PDK1, which is recruited to the RIP1-RIP3-MLKL-containing necrosome. Phosphorylation of pro-Casp8 at Thr265 can substitute for zVAD to permit necroptosis in vitro. pC8T265 mimic T265E knockin mice are embryonic lethal due to unconstrained necroptosis, and the pharmaceutical inhibition of RSK-mediated pC8T265 diminishes TNF-induced cecum damage and lethality in mice by halting necroptosis. Thus, phosphorylation of pro-Casp8 at Thr265 by RSK is an intrinsic mechanism for passing the Casp8 checkpoint of necroptosis.

Data-Driven Modeling Identifies TIRAP-Independent MyD88 Activation Complex and Myddosome Assembly Strategy in LPS/TLR4 Signaling.

TLR4 complexes are essential for the initiation of the LPS-induced innate immune response. The Myddosome, which mainly contains TLR4, TIRAP, MyD88, IRAK1/4 and TRAF6 proteins, is regarded as a major complex of TLR4. Although the Myddosome has been well studied, a quantitative description of the Myddosome assembly dynamics is still lacking. Furthermore, whether some unknown TLR4 complexes exist remains unclear. In this study, we constructed a SWATH-MS data-based mathematical model that describes the component assembly dynamics of TLR4 complexes. In addition to Myddosome, we suggest that a TIRAP-independent MyD88 activation complex is formed upon LPS stimulation, in which TRAF6 is not included. Furthermore, quantitative analysis reveals that the distribution of components in TIRAP-dependent and -independent MyD88 activation complexes are LPS stimulation-dependent. The two complexes compete for recruiting IRAK1/4 proteins. MyD88 forms higher-order assembly in the Myddosome and we show that the strategy to form higher-order assembly is also LPS stimulation-dependent. MyD88 forms a long chain upon weak stimulation, but forms a short chain upon strong stimulation. Higher-order assembly of MyD88 is directly determined by the level of TIRAP in the Myddosome, providing a formation mechanism for efficient signaling transduction. Taken together, our study provides an enhanced understanding of component assembly dynamics and strategies in TLR4 complexes.

Generation of a murine SWATH-MS spectral library to quantify more than 11,000 proteins.

Targeted SWATH-MS data analysis is critically dependent on the spectral library. Comprehensive spectral libraries of human or several other organisms have been published, but the extensive spectral library for mouse, a widely used model organism is not available. Here, we present a large murine spectral library covering more than 11,000 proteins and 240,000 proteotypic peptides, which included proteins derived from 9 murine tissue samples and one murine L929 cell line. This resource supports the quantification of 67% of all murine proteins annotated by UniProtKB/Swiss-Prot. Furthermore, we applied the spectral library to SWATH-MS data from murine tissue samples. Data are available via SWATHAtlas (PASS01441).

Systematic Assessment of the Effect of Internal Library in Targeted Analysis of SWATH-MS.

Targeted analysis of sequential window acquisition of all theoretical mass spectra (SWATH-MS) requires the spectral library, which can be generated by shotgun mass spectrometry (MS) or by the pseudo-spectra files directly obtained from SWATH-MS data. The external library generated by shotgun MS is employed in most SWATH-MS research. However, performance of the internal library, which is constructed by pseudo-spectra files, in the targeted analysis of SWATH-MS has not been systemically evaluated. Here, we show that up to 40% of the peptides detected by the internal library were not overlapped with those detected by the external library for most SWATH-MS data sets. However, the internal library did not identify extra phosphopeptides compared with the external library for phosphoproteomic SWATH-MS data. Therefore, the internal library should be incorporated into the external library for targeted analysis of nonphosphoproteomic SWATH-MS, given that it can significantly increase the number of peptides of SWATH-MS without requiring additional instrument measurement time.

Quantification of Dynamic Protein Interactions and Phosphorylation in LPS Signaling Pathway by SWATH-MS.

Lipopolysaccharide (LPS)-induced macrophage activation is a prototype of innate immune response. Although key effector proteins in LPS signaling pathway have been revealed, the map of dynamic protein interactions and phosphorylation as well as the stoichiometry of protein complexes are lacking. Here we present a dynamic map of protein interactions and phosphorylation in MyD88, TRAF6 and NEMO complexes obtained by SWATH-MS. The comprehensive MS measurement leads to quantification of up to about 3,000 proteins across about 21-40 IP samples. We detected and quantified almost all known interactors of MyD88, TRAF6 and NEMO. By analyzing these quantitative data, we uncovered differential recruitment of IRAK family proteins to LPS-induced signaling complexes and determined the stoichiometry of the Myddosome complex. In addition, quantitative phosphoproteomics analysis identified a number of unreported high-confidence phosphosites on the key proteins in LPS signaling pathway. Collectively, data of dynamic protein interactions and phosphorylation presented by this study could be a resource for further study of the LPS signaling pathway.

Inactivation of Cyclic AMP Response Element Transcription Caused by Constitutive p38 Activation Is Mediated by Hyperphosphorylation-Dependent CRTC2 Nucleocytoplasmic Transport.

The p38 signal transduction pathway can be activated transiently or constitutively, depending on the contexts in which the activation occurs. However, the biological consequence of constitutive activation of p38 is largely unknown. After screening 300 transcriptional cofactors, we identified CRTC2 as a downstream substrate of constitutively activated p38. Constitutive, rather than transient, activation of p38 led to hyperphosphorylation of CRTC2, resulting in CRTC2 cytosolic relocation and subsequent inactivation of cyclic AMP response element (CRE)-mediated transcription. Interestingly, the cytosolic translocation of CRTC2 depended on phosphorylation accumulation at multiple sites (≥11 phosphoserine/phosphothreonine residues) but not on specific sites. The hyperphosphorylation-driven nucleocytoplasmic transport of CRTC2 may not be a rare case of nuclear export of proteins, as we also observed that constitutively activated p38 promoted FOS nuclear export in a hyperphosphorylation-dependent manner. Collectively, our study uncovered a previously unknown mechanism of inactivation of selected transcription, which results from hyperphosphorylation-driven nucleocytoplasmic transport of cofactors or transcription factors mediated by constitutively active kinase.

RIP3 targets pyruvate dehydrogenase complex to increase aerobic respiration in TNF-induced necroptosis.

Receptor-interacting protein kinase 3 (RIP3)-regulated production of reactive oxygen species (ROS) positively feeds back on tumour necrosis factor (TNF)-induced necroptosis, a type of programmed necrosis. Glutamine catabolism is known to contribute to RIP3-mediated ROS induction, but the major contributor is unknown. Here, we show that RIP3 activates the pyruvate dehydrogenase complex (PDC, also known as PDH), the rate-limiting enzyme linking glycolysis to aerobic respiration, by directly phosphorylating the PDC E3 subunit (PDC-E3) on T135. Upon activation, PDC enhances aerobic respiration and subsequent mitochondrial ROS production. Unexpectedly, mixed-lineage kinase domain-like (MLKL) is also required for the induction of aerobic respiration, and we further show that it is required for RIP3 translocation to meet mitochondria-localized PDC. Our data uncover a regulation mechanism of PDC activity, show that PDC activation by RIP3 is most likely the major mechanism activated by TNF to increase aerobic respiration and its by-product ROS, and suggest that RIP3-dependent induction of aerobic respiration contributes to pathologies related to oxidative stress.