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The recent revolution in computational protein structure prediction provides folding models for entire proteomes, which can now be integrated with large-scale experimental data. Mass spectrometry (MS)-based proteomics has identified and quantified tens of thousands of posttranslational modifications (PTMs), most of them of uncertain functional relevance. In this study, we determine the structural context of these PTMs and investigate how this information can be leveraged to pinpoint potential regulatory sites. Our analysis uncovers global patterns of PTM occurrence across folded and intrinsically disordered regions. We found that this information can help to distinguish regulatory PTMs from those marking improperly folded proteins. Interestingly, the human proteome contains thousands of proteins that have large folded domains linked by short, disordered regions that are strongly enriched in regulatory phosphosites. These include well-known kinase activation loops that induce protein conformational changes upon phosphorylation. This regulatory mechanism appears to be widespread in kinases but also occurs in other protein families such as solute carriers. It is not limited to phosphorylation but includes ubiquitination and acetylation sites as well. Furthermore, we performed three-dimensional proximity analysis, which revealed examples of spatial coregulation of different PTM types and potential PTM crosstalk. To enable the community to build upon these first analyses, we provide tools for 3D visualization of proteomics data and PTMs as well as python libraries for data accession and processing.

Toxic epidermal necrolysis (TEN) is a fatal drug-induced skin reaction triggered by common medications and is an emerging public health issue 1 – 3 . Patients with TEN undergo severe and sudden epidermal detachment caused by keratinocyte cell death. Although molecular mechanisms that drive keratinocyte cell death have been proposed, the main drivers remain unknown, and there is no effective therapy for TEN 4 – 6 . Here, to systematically map molecular changes that are associated with TEN and identify potential druggable targets, we utilized deep visual proteomics, which provides single-cell-based, cell-type-resolution proteomics 7 , 8 . We analysed formalin-fixed, paraffin-embedded archived skin tissue biopsies of three types of cutaneous drug reactions with varying severity and quantified more than 5,000 proteins in keratinocytes and skin-infiltrating immune cells. This revealed a marked enrichment of type I and type II interferon signatures in the immune cell and keratinocyte compartment of patients with TEN, as well as phosphorylated STAT1 activation. Targeted inhibition with the pan-JAK inhibitor tofacitinib in vitro reduced keratinocyte-directed cytotoxicity. In vivo oral administration of tofacitinib, baricitinib or the JAK1-specific inhibitors abrocitinib or upadacitinib ameliorated clinical and histological disease severity in two distinct mouse models of TEN. Crucially, treatment with JAK inhibitors (JAKi) was safe and associated with rapid cutaneous re-epithelialization and recovery in seven patients with TEN. This study uncovers the JAK/STAT and interferon signalling pathways as key pathogenic drivers of TEN and demonstrates the potential of targeted JAKi as a curative therapy. Cell-type-resolved spatial proteomics of the skin from patients with toxic epidermal necrolysis reveals that it is driven by JAK/STAT signaling, leading to successful treatment of this potentially fatal condition in patients using JAK inhibitors.

Protein ubiquitination is involved in virtually all cellular processes. Enrichment strategies employing antibodies targeting ubiquitin-derived diGly remnants combined with mass spectrometry (MS) have enabled investigations of ubiquitin signaling at a large scale. However, so far the power of data independent acquisition (DIA) with regards to sensitivity in single run analysis and data completeness have not yet been explored. Here, we develop a sensitive workflow combining diGly antibody-based enrichment and optimized Orbitrap-based DIA with comprehensive spectral libraries together containing more than 90,000 diGly peptides. This approach identifies 35,000 diGly peptides in single measurements of proteasome inhibitor-treated cells – double the number and quantitative accuracy of data dependent acquisition. Applied to TNF signaling, the workflow comprehensively captures known sites while adding many novel ones. An in-depth, systems-wide investigation of ubiquitination across the circadian cycle uncovers hundreds of cycling ubiquitination sites and dozens of cycling ubiquitin clusters within individual membrane protein receptors and transporters, highlighting new connections between metabolism and circadian regulation. Protein ubiquitylation is often studied by proteomics but how data independent acquisition (DIA) may advance these studies remains to be explored. Here, the authors show that DIA improves ubiquitylation site identification and quantification, enabling them to characterize the circadian ubiquitinome in human cells.

Tumor necrosis factor (TNF) is one of the few cytokines successfully targeted by therapies against inflammatory diseases. However, blocking this well studied and pleiotropic ligand can cause dramatic side-effects. Here, we reason that a systems-level proteomic analysis of TNF signaling could dissect its diverse functions and offer a base for developing more targeted therapies. Therefore, we combine phosphoproteomics time course experiments with subcellular localization and kinase inhibitor analysis to identify functional modules of protein phosphorylation. The majority of regulated phosphorylation events can be assigned to an upstream kinase by inhibiting master kinases. Spatial proteomics reveals phosphorylation-dependent translocations of hundreds of proteins upon TNF stimulation. Phosphoproteome analysis of TNF-induced apoptosis and necroptosis uncovers a key role for transcriptional cyclin-dependent kinase activity to promote cytokine production and prevent excessive cell death downstream of the TNF signaling receptor. This resource of TNF-induced pathways and sites can be explored at http://tnfviewer.biochem.mpg.de/ . Tumor necrosis factor (TNF) has various effects on phosphorylation-mediated cellular signaling. Combining phosphoproteomics, subcellular localization analyses and kinase inhibitor assays, the authors provide systems level insights into TNF signaling and identify modulators of TNF-induced cell death.

Significance The four-helix bundle (4HB) domain of Mixed Lineage Kinase Domain-Like (MLKL) bears two clusters of residues that are required for cell death by necroptosis. Mutations within a cluster centered on the α4 helix of the 4HB domain of MLKL prevented its membrane translocation, oligomerization, and ability to induce necroptosis. This cluster is composed principally of acidic residues and therefore challenges the idea that the 4HB domain engages negatively charged phospholipid membranes via a conventional positively charged interaction surface. The importance of membrane translocation to MLKL-mediated death is supported by our identification of a small molecule that binds the MLKL pseudokinase domain and retards membrane translocation to inhibit necroptotic signaling. Necroptosis is considered to be complementary to the classical caspase-dependent programmed cell death pathway, apoptosis. The pseudokinase Mixed Lineage Kinase Domain-Like (MLKL) is an essential effector protein in the necroptotic cell death pathway downstream of the protein kinase Receptor Interacting Protein Kinase-3 (RIPK3). How MLKL causes cell death is unclear, however RIPK3–mediated phosphorylation of the activation loop in MLKL trips a molecular switch to induce necroptotic cell death. Here, we show that the MLKL pseudokinase domain acts as a latch to restrain the N-terminal four-helix bundle (4HB) domain and that unleashing this domain results in formation of a high-molecular-weight, membrane-localized complex and cell death. Using alanine-scanning mutagenesis, we identified two clusters of residues on opposing faces of the 4HB domain that were required for the 4HB domain to kill cells. The integrity of one cluster was essential for membrane localization, whereas MLKL mutations in the other cluster did not prevent membrane translocation but prevented killing; this demonstrates that membrane localization is necessary, but insufficient, to induce cell death. Finally, we identified a small molecule that binds the nucleotide binding site within the MLKL pseudokinase domain and retards MLKL translocation to membranes, thereby preventing necroptosis. This inhibitor provides a novel tool to investigate necroptosis and demonstrates the feasibility of using small molecules to target the nucleotide binding site of pseudokinases to modulate signal transduction.

The programmed cell death pathway, necroptosis, relies on the pseudokinase, Mixed Lineage Kinase domain-Like (MLKL), for cellular execution downstream of death receptor or Toll-like receptor ligation. Receptor-interacting protein kinase-3 (RIPK3)-mediated phosphorylation of MLKL’s pseudokinase domain leads to MLKL switching from an inert to activated state, where exposure of the N-terminal four-helix bundle (4HB) ‘executioner’ domain leads to cell death. The precise molecular details of MLKL activation, including the stoichiometry of oligomer assemblies, mechanisms of membrane translocation and permeabilisation, remain a matter of debate. Here, we dissect the function of the two ‘brace’ helices that connect the 4HB to the pseudokinase domain of MLKL. In addition to establishing that the integrity of the second brace helix is crucial for the assembly of mouse MLKL homotrimers and cell death, we implicate the brace helices as a device to communicate pseudokinase domain phosphorylation event(s) to the N-terminal executioner 4HB domain. Using mouse:human MLKL chimeras, we defined the first brace helix and adjacent loop as key elements of the molecular switch mechanism that relay pseudokinase domain phosphorylation to the activation of the 4HB domain killing activity. In addition, our chimera data revealed the importance of the pseudokinase domain in conferring host specificity on MLKL killing function, where fusion of the mouse pseudokinase domain converted the human 4HB + brace from inactive to a constitutive killer of mouse fibroblasts. These findings illustrate that the brace helices play an active role in MLKL regulation, rather than simply acting as a tether between the 4HB and pseudokinase domains.

Necroptotic cell death is mediated by the most terminal known effector of the pathway, MLKL. Precisely how phosphorylation of the MLKL pseudokinase domain activation loop by the upstream kinase, RIPK3, induces unmasking of the N-terminal executioner four-helix bundle (4HB) domain of MLKL, higher-order assemblies, and permeabilization of plasma membranes remains poorly understood. Here, we reveal the existence of a basal monomeric MLKL conformer present in human cells prior to exposure to a necroptotic stimulus. Following activation, toggling within the MLKL pseudokinase domain promotes 4HB domain disengagement from the pseudokinase domain αC helix and pseudocatalytic loop, to enable formation of a necroptosis-inducing tetramer. In contrast to mouse MLKL, substitution of RIPK3 substrate sites in the human MLKL pseudokinase domain completely abrogated necroptotic signaling. Therefore, while the pseudokinase domains of mouse and human MLKL function as molecular switches to control MLKL activation, the underlying mechanism differs between species. RIPK3-mediated phosphorylation of the mixed lineage kinase domain-like (MLKL) pseudokinase is thought to be the trigger for MLKL activation during necroptotic signaling. Here the authors provide evidence that the transition of human MLKL from a monomeric state to a tetramer is essential for necroptosis signalling.

Efferocytosis is a process by which phagocytes remove dead or dying cells. It is considered anti-inflammatory, as the removal process reduces potential inflammatory molecules originating from dead cells and results in the reprogramming of macrophages to an anti-inflammatory state. However, engulfment of infected dead cells, deregulated phagocytosis and perturbed digestion of apoptotic bodies induce inflammatory signalling pathways during efferocytosis. The affected inflammatory signalling molecules and the mechanism of activation are largely unknown. I discuss how the choice of dead cell cargo, the type of ingestion, and the digestion efficiency can influence phagocyte programming in the context of disease. I also present the latest findings, highlight knowledge gaps, and propose selected experimental approaches to fill them.

Fibrosis represents the uncontrolled replacement of parenchymal tissue with extracellular matrix (ECM) produced by myofibroblasts. While genetic fate-tracing and single-cell RNA-Seq technologies have helped elucidate fibroblast heterogeneity and ontogeny beyond fibroblast to myofibroblast differentiation, newly identified fibroblast populations remain ill defined, with respect to both the molecular cues driving their differentiation and their subsequent role in fibrosis. Using an unbiased approach, we identified the metalloprotease ADAMTS12 as a fibroblast-specific gene that is strongly upregulated during active fibrogenesis in humans and mice. Functional in vivo KO studies in mice confirmed that Adamts12 was critical during fibrogenesis in both heart and kidney. Mechanistically, using a combination of spatial transcriptomics and expression of catalytically active or inactive ADAMTS12, we demonstrated that the active protease of ADAMTS12 shaped ECM composition and cleaved hemicentin 1 (HMCN1) to enable the activation and migration of a distinct injury-responsive fibroblast subset defined by aberrant high JAK/STAT signaling.

Inflammation and excess cytokine release are hallmarks of severe COVID-19. While programmed cell death is known to drive inflammation, its role in SARS-CoV-2 pathogenesis remains unclear. Using gene-targeted murine COVID-19 models, we here find that caspase-8 is critical for cytokine release and inflammation. Loss of caspase-8 reduces disease severity and viral load in mice, and this occurs independently of its apoptotic function. Instead, reduction in SARS-CoV-2 pathology is linked to decreased IL-1β levels and inflammation. Loss of pyroptosis and necroptosis mediators in gene-targeted animals provides no additional benefits in mitigating disease outcomes beyond that conferred by loss of caspase-8. Spatial transcriptomic and proteomic analyses of caspase-8-deficient mice confirm that improved outcomes are due to reduced pro-inflammatory responses, rather than changes in cell death signalling. Elevated expression of caspase-8 and cFLIP in infected lungs, alongside caspase-8-mediated cleavage of N4BP1, a suppressor of NF-kB signalling, indicates a role of this signalling axis in pathological inflammation. Collectively, these findings highlight non-apoptotic functions of caspase-8 as a driver of severe COVID-19 through modulation of inflammation, not through the induction of apoptosis. During SARS-CoV-2 infection caspase-8 fuels harmful inflammation independent of apoptosis. Genetic loss of caspase-8 reduces cytokine levels, lowers viral load and mitigates disease severity, revealing non-apoptotic caspase-8 as a key driver of severe COVID-19.