Mixed lineage kinase domain-like (MLKL) is a component of the “necrosome,” the multiprotein complex that triggers tumor necrosis factor (TNF)-induced cell death by necroptosis. To define the specific ...role and molecular mechanism of MLKL action, we generated MLKL-deficient mice and solved the crystal structure of MLKL. Although MLKL-deficient mice were viable and displayed no hematopoietic anomalies or other obvious pathology, cells derived from these animals were resistant to TNF-induced necroptosis unless MLKL expression was restored. Structurally, MLKL comprises a four-helical bundle tethered to the pseudokinase domain, which contains an unusual pseudoactive site. Although the pseudokinase domain binds ATP, it is catalytically inactive and its essential nonenzymatic role in necroptotic signaling is induced by receptor-interacting serine-threonine kinase 3 (RIPK3)-mediated phosphorylation. Structure-guided mutation of the MLKL pseudoactive site resulted in constitutive, RIPK3-independent necroptosis, demonstrating that modification of MLKL is essential for propagation of the necroptosis pathway downstream of RIPK3.
•The crystal structure of MLKL reveals an unusual pseudoactive site•The MLKL pseudokinase domain binds ATP but is not catalytically active•MLKL-deficient cells are resistant to TNF-induced necroptosis•MLKL pseudoactive site mutations cause caspase- and RIPK3-independent necroptosis
Identification of peptides by analysis of data acquired by the two established methods for bottom-up proteomics, DDA and DIA, relies heavily on the fragment spectra. In DDA, peptide features detected ...in mass spectrometry data are identified by matching their fragment spectra with a peptide database. In DIA, a peptide's fragment spectra are targeted for extraction and matched with observed spectra. Although fragment ion matching is a central aspect in most peptide identification strategies, the precursor ion in the MS1 data reveals important characteristics as well, including charge state, intensity, monoisotopic m/z, and apex in retention time. Most importantly, the precursor's mass is essential in determining the potential chemical modification state of the underlying peptide sequence. In the timsTOF, with its additional dimension of collisional cross-section, the data representing the precursor ion also reveals the peptide's peak in ion mobility. However, the availability of tools to survey precursor ions with a wide range of abundance in timsTOF data across the full mass range is very limited. Here we present a de novo feature detector called three-dimensional intensity descent (3DID). 3DID can detect and extract peptide features down to a configurable intensity level, and finds many more features than several existing tools. 3DID is written in Python and is freely available with an open-source MIT license to facilitate experimentation and further improvement (DOI 10.5281/zenodo.6513126). The dataset used for validation of the algorithm is publicly available (ProteomeXchange identifier PXD030706).
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.
Mixed lineage kinase domain‐like (MLKL) is the executioner in the caspase‐independent form of programmed cell death called necroptosis. Receptor‐interacting serine/threonine protein kinase 3 (RIPK3) ...phosphorylates MLKL, triggering MLKL oligomerization, membrane translocation and membrane disruption. MLKL also undergoes ubiquitylation during necroptosis, yet neither the mechanism nor the significance of this event has been demonstrated. Here, we show that necroptosis‐specific multi‐mono‐ubiquitylation of MLKL occurs following its activation and oligomerization. Ubiquitylated MLKL accumulates in a digitonin‐insoluble cell fraction comprising organellar and plasma membranes and protein aggregates. Appearance of this ubiquitylated MLKL form can be reduced by expression of a plasma membrane‐located deubiquitylating enzyme. Oligomerization‐induced MLKL ubiquitylation occurs on at least four separate lysine residues and correlates with its proteasome‐ and lysosome‐dependent turnover. Using a MLKL‐DUB fusion strategy, we show that constitutive removal of ubiquitin from MLKL licences MLKL auto‐activation independent of necroptosis signalling in mouse and human cells. Therefore, in addition to the role of ubiquitylation in the kinetic regulation of MLKL‐induced death following an exogenous necroptotic stimulus, it also contributes to restraining basal levels of activated MLKL to avoid unwanted cell death.
SYNOPSIS
RIPK3 phosphorylates the necroptotic effector molecule MLKL leading to its oligomerization and translocation to the plasma membrane to kill cells. MLKL becomes multi mono‐ubiquitylated in an oligomerization dependent manner. Forced de‐ubiquitylation of MLKL increases MLKL's cytotoxic potential and confers RIPK3 and necroptotic stimulus independent activation and cell death.
UbiCRest analysis shows that MLKL becomes multi mono‐ubiquitylated contemporaneously with activating phosphorylation and translocation to membranes.
Analysis of gain and loss of function MLKL mutants indicates that MLKL oligomerization is required for necroptosis induced ubiquitylation.
MLKL‐deubiquitylating enzyme (DUB) fusions kill cells more rapidly than MLKL‐catalytically dead DUB fusions and can induce necroptosis without a necroptotic stimulus, suggesting that ubiquitylation serves as a brake on MLKL's cytotoxic potential.
Mono‐ubiquitylation of the necroptotic effector molecule MLKL promotes its proteasome‐ and lysosome‐mediated turnover to restrains its necroptosis‐inducing activity.
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.
When nanoparticles interact with human blood, a multitude of plasma components adsorb onto the surface of the nanoparticles, forming a biomolecular corona. Corona composition is known to be ...influenced by the chemical composition of nanoparticles. In contrast, the possible effects of variations in the human blood proteome between healthy individuals on the formation of the corona and its subsequent interactions with immune cells in blood are unknown. Herein, we prepared and examined a matrix of 11 particles (including organic and inorganic particles of three sizes and five surface chemistries) and plasma samples from 23 healthy donors to form donor-specific biomolecular coronas (personalized coronas) and investigated the impact of the personalized coronas on particle interactions with immune cells in human blood. Among the particles examined, poly(ethylene glycol) (PEG)-coated mesoporous silica (MS) particles, irrespective of particle size (800, 450, or 100 nm in diameter), displayed the widest range (up to 60-fold difference) of donor-dependent variance in immune cell association. In contrast, PEG particles (after MS core removal) of 860, 518, or 133 nm in diameter displayed consistent stealth behavior (negligible cell association), irrespective of plasma donor. For comparison, clinically relevant PEGylated doxorubicin-encapsulated liposomes (Doxil) (74 nm in diameter) showed significant variance in association with monocytes and B cells across all plasma donors studied. An in-depth proteomic analysis of each biomolecular corona studied was performed, and the results were compared against the nanoparticle–blood cell association results, with individual variance in the proteome driving differential association with specific immune cell types. We identified key immunoglobulin and complement proteins that explicitly enriched or depleted within the corona and which strongly correlated with the cell association pattern observed across the 23 donors. This study demonstrates how plasma variance in healthy individuals significantly influences the blood immune cell interactions of nanoparticles.
The detection of aberrant cells by natural killer (NK) cells is controlled by the integration of signals from activating and inhibitory ligands and from cytokines such as IL-15. We identified ...cytokine-inducible SH2-containing protein (CIS, encoded by Cish) as a critical negative regulator of IL-15 signaling in NK cells. Cish was rapidly induced in response to IL-15, and deletion of Cish rendered NK cells hypersensitive to IL-15, as evidenced by enhanced proliferation, survival, IFN-γ production and cytotoxicity toward tumors. This was associated with increased JAK-STAT signaling in NK cells in which Cish was deleted. Correspondingly, CIS interacted with the tyrosine kinase JAK1, inhibiting its enzymatic activity and targeting JAK for proteasomal degradation. Cish(-/-) mice were resistant to melanoma, prostate and breast cancer metastasis in vivo, and this was intrinsic to NK cell activity. Our data uncover a potent intracellular checkpoint in NK cell-mediated tumor immunity and suggest possibilities for new cancer immunotherapies directed at blocking CIS function.
Mass spectrometry (MS) enables high-throughput identification and quantification of proteins in complex biological samples and can provide insights into the global function of biological systems. ...Label-free quantification is cost-effective and suitable for the analysis of human samples. Despite rapid developments in label-free data acquisition workflows, the number of proteins quantified across samples can be limited by technical and biological variability. This variation can result in missing values which can in turn challenge downstream data analysis tasks. General purpose or gene expression-specific imputation algorithms are widely used to improve data completeness. Here, we propose an imputation algorithm designated for label-free MS data that is aware of the type of missingness affecting data. On published datasets acquired by data-dependent and data-independent acquisition workflows with variable degrees of biological complexity, we demonstrate that the proposed missing value estimation procedure by barycenter computation competes closely with the state-of-the-art imputation algorithms in differential abundance tasks while outperforming them in the accuracy of variance estimates of the peptide abundance measurements, and better controls the false discovery rate in label-free MS experiments. The barycenter estimation procedure is implemented in the msImpute software package and is available from the Bioconductor repository.
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•msImpute provides imputation that is aware of the type of missingness in data•More-accurate estimates of variance and better control of the false discovery rate•The msImpute software package is available from the Bioconductor repository
The number of proteins quantified across samples by label-free mass-spectrometry (MS) is limited by technical and biological variability resulting in missing values that challenge downstream analysis. We present an imputation algorithm for label-free MS data that is aware of the type of missingness affecting data. Missing value estimation by msImpute outperforms state-of-the-art imputation methods in the accuracy of variance estimates for peptide abundance and better controls the false discovery rate in MS experiments. msImpute is available from the Bioconductor repository.
Interleukin-1β (IL-1β) is activated by inflammasome-associated caspase-1 in rare autoinflammatory conditions and in a variety of other inflammatory diseases. Therefore, IL-1β activity must be ...fine-tuned to enable anti-microbial responses whilst limiting collateral damage. Here, we show that precursor IL-1β is rapidly turned over by the proteasome and this correlates with its decoration by K11-linked, K63-linked and K48-linked ubiquitin chains. The ubiquitylation of IL-1β is not just a degradation signal triggered by inflammasome priming and activating stimuli, but also limits IL-1β cleavage by caspase-1. IL-1β K133 is modified by ubiquitin and forms a salt bridge with IL-1β D129. Loss of IL-1β K133 ubiquitylation, or disruption of the K133:D129 electrostatic interaction, stabilizes IL-1β. Accordingly, Il1b
mice have increased levels of precursor IL-1β upon inflammasome priming and increased production of bioactive IL-1β, both in vitro and in response to LPS injection. These findings identify mechanisms that can limit IL-1β activity and safeguard against damaging inflammation.
RNA-binding proteins are customarily regarded as important facilitators of gene expression. In recent years, RNA-protein interactions have also emerged as a pervasive force in the regulation of ...homeostasis. The compendium of proteins with provable RNA-binding function has swelled from the hundreds to the thousands astride the partnership of mass spectrometry-based proteomics and RNA sequencing. At the foundation of these advances is the adaptation of RNA-centric capture methods that can extract bound protein that has been cross-linked in its native environment. These methods reveal snapshots in time displaying an extensive network of regulation and a wealth of data that can be used for both the discovery of RNA-binding function and the molecular interfaces at which these interactions occur. This review will focus on the impact of these developments on our broader perception of post-transcriptional regulation, and how the technical features of current capture methods, as applied in mammalian systems, create a challenging medium for interpretation by systems biologists and target validation by experimental researchers.
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