LC Domain-Mediated Coalescence Is Essential for Otu Enzymatic Activity to Extend Drosophila Lifespan.

In eukaryotic cells, RNA-binding proteins (RBPs) interact with RNAs to form ribonucleoprotein complexes (RNA granules) that have long been thought to regulate RNA fate or activity. Emerging evidence suggests that some RBPs not only bind RNA but also possess enzymatic activity related to ubiquitin regulation, raising important questions of whether these RBP-formed RNA granules regulate ubiquitin signaling and related biological functions. Here, we show that Drosophila Otu binds RNAs and coalesces to membrane-less biomolecular condensates via its intrinsically disordered low-complexity domain, and coalescence represents a functional state for Otu exerting deubiquitinase activity. Notably, coalescence-mediated enzymatic activity of Otu is positively regulated by its bound RNAs and co-partner Bam. Further genetic analysis reveals that the Otu/Bam deubiquitinase complex and dTraf6 constitute a feedback loop to maintain intestinal immune homeostasis during aging, thereby controlling longevity. Thus, regulated biomolecular condensates may represent a mechanism that controls dynamic enzymatic activities and related biological processes.

Splicing factor ESRP1 derived circ_0068162 promotes the progression of oral squamous cell carcinoma via the miR-186/JAG axis.

OBJECTIVES: Oral squamous cell carcinoma (OSCC) is a common malignancy in the oral and maxillofacial regions with an increasing incidence rate. Circular RNA (circRNA) is a recently discovered long-chain non-coding RNA family member. The objective of this study was to analyze the role of circ_0068162 in OSCC development. METHODS: We downloaded sample data GSE145608 from the Gene Expression Omnibus database. Online databases Starbase, TargetScan and miRDB were used to predict the target microRNAs (miRNAs) and genes. Cell viability and proliferation were assessed using the CCK-8 and EdU assays, respectively. Cell migration and invasion abilities were detected using transwell assay. The double luciferase reporter and RNA immunoprecipitation (RIP) assays were performed to verify the interaction relationship between the identified target molecules. RNase R and actinomycin D treatment were performed to analyze the stability of circ_0068162. RESULTS: We found that circ_0068162 was overexpressed in the cytoplasm of OSCC cells and clinical OSCC tissues. Knockdown of circ_0068162 inhibited the growth, migration and invasion of OSCC cells. We also identified miR-186 as the target miRNA of circ_0068162, and JAG1 and JAG2 as the target genes of miR-186. The miR-186 inhibitor rescued the effects of sh-circ_0068162 and JAG1/JAG2 overexpression rescued the effects of miR-186 mimic in OSCC cells. Furthermore, ESRP1 promoted the biosynthesis of circ_0068162. CONCLUSIONS: The circ_0068162/miR-186/JAGs/ESRP1 feedback loop is closely related to OSCC development.

Single-cell transcriptome analysis reveals tumoral microenvironment heterogenicity and hypervascularization in human carotid body tumor.

Carotid body tumor (CBT) is a rare neck tumor located at the adventitia of the common carotid artery bifurcation. The prominent pathological features of CBT are high vascularization and abnormal proliferation. However, single-cell transcriptome analysis of the microenvironment composition and molecular complexity in CBT has yet to be performed. In this study, we performed single-cell RNA sequencing (scRNA-seq) analysis on human CBT to define the cells that contribute to hypervascularization and chronic hyperplasia. Unbiased clustering analysis of transcriptional profiles identified 16 distinct cell populations including endothelial cells (ECs), smooth muscle cells (SMCs), neuron cells, macrophage cells, neutrophil cells, and T cells. Within the ECs population, we defined subsets with angiogenic capacity plus clear signs of later endothelial progenitor cells (EPCs) to normal ECs. Two populations of macrophages were detectable in CBT, macrophage1 showed enrichment in hypoxia-inducible factor-1 (HIF-1) and as well as an early EPCs cell-like population expressing CD14 and vascular endothelial growth factor. In addition to HIF-1-related transcriptional protein expression, macrophages1 also display a neovasculogenesis-promoting phenotype. SMCs included three populations showing platelet-derived growth factor receptor beta and vimentin expression, indicative of a cancer-associated fibroblast phenotype. Finally, we identified three types of neuronal cells, including chief cells and sustentacular cells, and elucidated their distinct roles in the pathogenesis of CBT and abnormal proliferation of tumors. Overall, our study provided the first comprehensive characterization of the transcriptional landscape of CBT at scRNA-seq profiles, providing novel insights into the mechanisms underlying its formation.

GGC repeat expansion in NOTCH2NLC induces dysfunction in ribosome biogenesis and translation.

GGC repeat expansion in the 5' untranslated region (UTR) of NOTCH2NLC is associated with a broad spectrum of neurological disorders, especially neuronal intranuclear inclusion disease (NIID). Studies have found that GGC repeat expansion in NOTCH2NLC induces the formation of polyglycine (polyG)-containing protein, which is involved in the formation of neuronal intranuclear inclusions. However, the mechanism of neurotoxicity induced by NOTCH2NLC GGC repeats is unclear. Here, we used NIID patient-specific induced pluripotent stem cell (iPSC)-derived 3D cerebral organoids (3DCOs) and cellular models to investigate the pathophysiological mechanisms of NOTCH2NLC GGC repeat expansion. IPSC-derived 3DCOs and cellular models showed the deposition of polyG-containing intranuclear inclusions. The NOTCH2NLC GGC repeats could induce the upregulation of autophagic flux, enhance integrated stress response and activate EIF2α phosphorylation. Bulk RNA sequencing for iPSC-derived neurons and single-cell RNA sequencing (scRNA-seq) for iPSC-derived 3DCOs revealed that NOTCH2NLC GGC repeats may be associated with dysfunctions in ribosome biogenesis and translation. Moreover, NOTCH2NLC GGC repeats could induce the NPM1 nucleoplasm translocation, increase nucleolar stress, impair ribosome biogenesis and induce ribosomal RNA sequestration, suggesting dysfunction of membraneless organelles in the NIID cellular model. Dysfunctions in ribosome biogenesis and phosphorylated EIF2α and the resulting increase in the formation of G3BP1-positive stress granules may together lead to whole-cell translational inhibition, which may eventually cause cell death. Interestingly, scRNA-seq revealed that NOTCH2NLC GGC repeats may be associated with a significantly decreased proportion of immature neurons while 3DCOs were developing. Together, our results underscore the value of patient-specific iPSC-derived 3DCOs in investigating the mechanisms of polyG diseases, especially those caused by repeats in human-specific genes.

Mst1 shuts off cytosolic antiviral defense through IRF3 phosphorylation.

Cytosolic RNA/DNA sensing elicits primary defense against viral pathogens. Interferon regulatory factor 3 (IRF3), a key signal mediator/transcriptional factor of the antiviral-sensing pathway, is indispensible for interferon production and antiviral defense. However, how the status of IRF3 activation is controlled remains elusive. Through a functional screen of the human kinome, we found that mammalian sterile 20-like kinase 1 (Mst1), but not Mst2, profoundly inhibited cytosolic nucleic acid sensing. Mst1 associated with IRF3 and directly phosphorylated IRF3 at Thr75 and Thr253. This Mst1-mediated phosphorylation abolished activated IRF3 homodimerization, its occupancy on chromatin, and subsequent IRF3-mediated transcriptional responses. In addition, Mst1 also impeded virus-induced activation of TANK-binding kinase 1 (TBK1), further attenuating IRF3 activation. As a result, Mst1 depletion or ablation enabled an enhanced antiviral response and defense in cells and mice. Therefore, the identification of Mst1 as a novel physiological negative regulator of IRF3 activation provides mechanistic insights into innate antiviral defense and potential antiviral prevention strategies.

Meningeal immunity and neurological diseases: new approaches, new insights.

The meninges, membranes surrounding the central nervous system (CNS) boundary, harbor a diverse array of immunocompetent immune cells, and therefore, serve as an immunologically active site. Meningeal immunity has emerged as a key factor in modulating proper brain function and social behavior, performing constant immune surveillance of the CNS, and participating in several neurological diseases. However, it remains to be determined how meningeal immunity contributes to CNS physiology and pathophysiology. With the advances in single-cell omics, new approaches, such as single-cell technologies, unveiled the details of cellular and molecular mechanisms underlying meningeal immunity in CNS homeostasis and dysfunction. These new findings contradict some previous dogmas and shed new light on new possible therapeutic targets. In this review, we focus on the complicated multi-components, powerful meningeal immunosurveillance capability, and its crucial involvement in physiological and neuropathological conditions, as recently revealed by single-cell technologies.

Genetically predicted frailty index and risk of stroke and Alzheimer's disease.

BACKGROUND AND PURPOSE: Previous studies have reported the association between frailty and stroke or Alzheimer's disease (AD). However, the causality remains unclear. The aim of the present study was to evaluate whether genetically predicted frailty is associated with the risk of stroke or AD by a Mendelian randomization (MR) study. METHODS: Genetic variants associated with the frailty index (FI) were obtained from a large genome-wide association study (GWAS). Summary-level data for stroke and AD were adopted from the corresponding large GWAS of individuals of European ancestry. The inverse variance weighted method was used for estimating causal effects. Multivariable analysis was performed for further adjustment. RESULTS: The present MR study indicated a suggestive association between genetically predicted FI and a higher risk of any stroke (odds ratio 1.360, 95% confidence interval 1.006-1.838, p = 0.046). Regarding the subtypes of stroke, genetically predicted FI was associated with a higher risk of large artery atherosclerosis stroke (LAS) (odds ratio 2.487, 95% confidence interval 1.282-4.826, p = 0.007). No causal links were identified between genetically predicted FI and any ischaemic stroke, intracranial haemorrhage, cardioembolic stroke, small artery stroke, AD or AD-by-proxy. Multivariable MR analysis indicated that the association of genetically predicted FI with LAS was attenuated after adjustment for inflammatory bowel disease (p = 0.114). CONCLUSIONS: The MR study suggested that genetically predicted FI may be associated with an increased risk of any stroke. Subgroup analysis indicated a suggestive association between genetically predicted FI and the risk of LAS. The underlying mechanisms need further investigation.

Reciprocal regulation of Akt and Oct4 promotes the self-renewal and survival of embryonal carcinoma cells.

Signaling via the Akt serine/threonine protein kinase plays critical roles in the self-renewal of embryonic stem cells and their malignant counterpart, embryonal carcinoma cells (ECCs). Here we show that in ECCs, Akt phosphorylated the master pluripotency factor Oct4 at threonine 235, and that the levels of phosphorylated Oct4 in ECCs correlated with resistance to apoptosis and tumorigenic potential. Phosphorylation of Oct4 increased its stability and facilitated its nuclear localization and its interaction with Sox2, which promoted the transcription of the core stemness genes POU5F1 and NANOG. Furthermore, in ECCs, unphosphorylated Oct4 bound to the AKT1 promoter and repressed its transcription. Phosphorylation of Oct4 by Akt resulted in dissociation of Oct4 from the AKT1 promoter, which activated AKT1 transcription and promoted cell survival. Therefore, a site-specific, posttranslational modification of the Oct4 protein orchestrates the regulation of its stability, subcellular localization, and transcriptional activities, which collectively promotes the survival and tumorigenicity of ECCs.

Bam-dependent deubiquitinase complex can disrupt germ-line stem cell maintenance by targeting cyclin A.

Drosophila germ-line stem cells (GSCs) provide an excellent model to study the regulatory mechanisms of stem cells in vivo. Bag of marbles (bam) has been demonstrated to be necessary and sufficient to promote GSC and cystoblast differentiation. Despite extensive investigation of its regulation and genetic functions, the biochemical nature of the Bam protein has been unknown. Here, we report that Bam is an ubiquitin-associated protein and controls the turnover of cyclin A (CycA). Mechanistically, we found that Bam associated with Otu to form a deubiquitinase complex that stabilized CycA by deubiquitination, thus providing a mechanism to explain how ectopic expression of Bam in GSCs promotes differentiation. Collectively, our findings not only identify a biochemical function of Bam, which contributes to GSC fate determination, but also emphasizes the critical role of proper expression of cyclin proteins mediated by both ubiquitination and deubiquitination pathways in balancing stem cell self-renewal and differentiation.

Smad7 enables STAT3 activation and promotes pluripotency independent of TGF-ß signaling.

Smad7 is a negative feedback product of TGF-ß superfamily signaling and fine tunes a plethora of pleiotropic responses induced by TGF-ß ligands. However, its noncanonical functions independent of TGF-ß signaling remain to be elucidated. Here, we show that Smad7 activates signal transducers and activators of transcription 3 (STAT3) signaling in maintaining mouse embryonic stem cell pluripotency in a manner independent of the TGF-ß receptors, yet dependent on the leukemia inhibitory factor (LIF) coreceptor glycoprotein 130 (gp130). Smad7 directly binds to the intracellular domain of gp130 and disrupts the SHP2-gp130 or SOCS3-gp130 complex, thereby amplifying STAT3 activation. Consequently, Smad7 facilitates LIF-mediated self-renewal of mouse ESCs and is also critical for induced pluripotent stem cell reprogramming. This finding illustrates an uncovered role of the Smad7-STAT3 interplay in maintaining cell pluripotency and also implicates a mechanism involving Smad7 underlying cytokine-dependent regulation of cancer and inflammation.

Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) mediate cell density-dependent proinflammatory responses.

A proper inflammatory response is critical to the restoration of tissue homeostasis after injury or infection, but how such a response is modulated by the physical properties of the cellular and tissue microenvironments is not fully understood. Here, using H358, HeLa, and HEK293T cells, we report that cell density can modulate inflammatory responses through the Hippo signaling pathway. We found that NF-κΒ activation through the proinflammatory cytokines interleukin-1ß (IL-1ß) and tumor necrosis factor α (TNFα) is not affected by cell density. However, we also noted that specific NF-κΒ target genes, such as cyclooxygenase 2 (COX-2), are induced much less at low cell densities than at high cell densities. Mechanistically, we observed that the transcriptional coactivators Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) are localized to the nucleus, bind to TEA domain transcription factors (TEADs), recruit histone deacetylase 7 (HDAC7) to the promoter region of COX-2, and repress its transcription at low cell density and that high cell density abrogates this YAP/TAZ-mediated transcriptional repression. Of note, IL-1ß stimulation promoted cell migration and invasion mainly through COX-2 induction, but YAP inhibited this induction and thus cell migration and invasion. These results suggest that YAP/TAZ-TEAD interactions can repress COX-2 transcription and thereby mediate cell density-dependent modulation of proinflammatory responses. Our findings highlight that the cellular microenvironment significantly influences inflammatory responses via the Hippo pathway.

Ac-LysargiNase Complements Trypsin for the Identification of Ubiquitinated Sites.

Mass spectrometry (MS)-based identification of ubiquitinated sites requires trypsin digestion prior to MS analysis, and a signature peptide was produced with a diglycine residue attached to the ubiquitinated lysine (K-ε-GG peptide). However, the missed cleavage of modified lysines by trypsin results in modified peptides with increased length and charge, whose detection by MS analysis is suppressed by the vast majority of internally unmodified peptides. LysargiNase, the mirrored trypsin, is reported to cleave before lysine and arginine residues and to be favorable for the identification of methylation and phosphorylation, but its digestive characteristics related to ubiquitination are unclear. Herein, we tested the capacity of the in-house developed acetylated LysargiNase (Ac-LysargiNase) with high activity and stability, for cleaving ubiquitinated sites in both the seven types of ubiquitin chains and their corresponding K-ε-GG peptides. Interestingly, Ac-LysargiNase could efficiently cleave the K63-linked chain but had little effect on the other types of chains. Additionally, Ac-LysargiNase had higher exopeptidase activity than trypsin. Utilizing these features of the paired mirror proteases, a workflow of trypsin and Ac-LysargiNase tandem digestion was developed for the identification of ubiquitinated proteins. Through this method, the charge states and ionization capacity of the unmodified peptides were efficiently reduced, and the identification of modified sites was consequently increased by 30% to 50%. Strikingly, approximately 15% of the modified sites were cleaved by Ac-LysargiNase, resulting in shorter K-ε-GG peptides for better identification. The enzyme Ac-LysargiNase is expected to serve as an option for increasing the efficiency of modified site identification in ubiquitome research.

HTLV-1 Tax Functions as a Ubiquitin E3 Ligase for Direct IKK Activation via Synthesis of Mixed-Linkage Polyubiquitin Chains.

The HTLV-1 oncoprotein Tax plays a key role in CD4+ T cell transformation by promoting cell proliferation and survival, mainly through permanent activation of the NK-κB pathway and induction of many NF-κB target genes. Elucidating the underlying molecular mechanism is therefore critical in understanding HTLV-1-mediated transformation. Current studies have suggested multiple but controversial mechanisms regarding Tax-induced IKK activation mainly due to blending of primary Tax-induced IKK activation events and secondary IKK activation events induced by cytokines secreted by the primary Tax-induced IKK-NF-κB activation events. We reconstituted Tax-stimulated IKK activation in a cell-free system to dissect the essential cellular components for primary IKK activation by Tax and studied the underlying biochemical mechanism. We found that Tax is a putative E3 ubiquitin ligase, which, together with UbcH2, UhcH5c, or UbcH7, catalyzes the assembly of free mixed-linkage polyubiquitin chains. These free mixed-linkage polyubiquitin chains are then responsible for direct IKK activation by binding to the NEMO subunit of IKK. Our studies revealed the biochemical function of Tax in the process of IKK activation, which utilizes the minimal cellular ubiquitination components for NF-κB activation.

Phosphatase holoenzyme PP1/GADD34 negatively regulates TLR response by inhibiting TAK1 serine 412 phosphorylation.

The molecular mechanisms that fine tune TLRs responses need to be fully elucidated. Protein phosphatase-1 (PP1) has been shown to be important in cell death and differentiation. However, the roles of PP1 in TLR-triggered immune response remain unclear. In this study, we demonstrate that PP1 inhibits the activation of the MAPK and NF-κB pathway and the production of TNF-α, IL-6 in macrophages triggered by TLR3, TLR4, and TLR9 in a phosphatase-dependent manner. Conversely, PP1 knockdown increases TLRs-triggered signaling and proinflammatory cytokine production. Tautomycetin, a specific inhibitor of PP1, aggravates LPS-induced endotoxin shock in mice. We further demonstrate that PP1 negatively regulates TLR-triggered signaling by targeting TGF-ß-activated kinase 1 (TAK1) serine 412 (Ser412) phosphorylation, which is required for activation of TAK1-mediated IL-1R and TLR signaling. Mutation of TAK1 Serine 412 to alanine (S412A) significantly inhibits TLR/IL-1R-triggered NF-κB and MAPK activation and induction of proinflammatory cytokines in macrophage and murine embryonic fibroblast cells. DNA damage-inducible protein 34 (GADD34) specifies PP1 to dephosphorylate TAK1 at Ser412. GADD34 depletion abolished the interaction between TAK1 and PP1, and it relieved PP1 overexpression-induced inhibition of TLRs signaling and proinflammatory cytokine production. In addition, knockdown of GADD34 significantly promotes TLR-induced TAK1 Ser412 phosphorylation, downstream NF-κB and MAPK activation, and proinflammatory cytokine production. Therefore, PP1, as a physiologic inhibitor, together with its regulatory subunit GADD34, tightly controls TLR-induced TAK1 Ser412 phosphorylation, preventing excessive activation of TLRs and protecting the host from overwhelmed inflammatory immune responses.

Transforming growth factor (TGF)-ß-activated kinase 1 (TAK1) activation requires phosphorylation of serine 412 by protein kinase A catalytic subunit α (PKACα) and X-linked protein kinase (PRKX).

TGF-ß-activated kinase 1 (TAK1) is a key kinase in mediating Toll-like receptors (TLRs) and interleukin-1 receptor (IL-1R) signaling. Although TAK1 activation involves the phosphorylation of Thr-184 and Thr-187 residues at the activation loop, the molecular mechanism underlying the complete activation of TAK1 remains elusive. In this work, we show that the Thr-187 phosphorylation of TAK1 is regulated by its C-terminal coiled-coil domain-mediated dimerization in an autophosphorylation manner. Importantly, we find that TAK1 activation in mediating downstream signaling requires an additional phosphorylation at Ser-412, which is critical for TAK1 response to proinflammatory stimuli, such as TNF-α, LPS, and IL-1ß. In vitro kinase and shRNA-based knockdown assays reveal that TAK1 Ser-412 phosphorylation is regulated by cAMP-dependent protein kinase catalytic subunit α (PKACα) and X-linked protein kinase (PRKX), which is essential for proper signaling and proinflammatory cytokine induction by TLR/IL-1R activation. Morpholino-based in vivo knockdown and rescue studies show that the corresponding site Ser-391 in zebrafish TAK1 plays a conserved role in NF-κB activation. Collectively, our data unravel a previously unknown mechanism involving TAK1 phosphorylation mediated by PKACα and PRKX that contributes to innate immune signaling.

Complex structure of OspI and Ubc13: the molecular basis of Ubc13 deamidation and convergence of bacterial and host E2 recognition.

Ubc13 is an important ubiquitin-conjugating (E2) enzyme in the NF-κB signaling pathway. The Shigella effector OspI targets Ubc13 and deamidates Gln100 of Ubc13 to a glutamic acid residue, leading to the inhibition of host inflammatory responses. Here we report the crystal structure of the OspI-Ubc13 complex at 2.3 Å resolution. The structure reveals that OspI uses two differently charged regions to extensively interact with the α1 helix, L1 loop and L2 loop of Ubc13. The Gln100 residue is bound within the hydrophilic catalytic pocket of OspI. A comparison between Ubc13-bound and wild-type free OspI structures revealed that Ubc13 binding induces notable structural reassembly of the catalytic pocket, suggesting that substrate binding might be involved in the catalysis of OspI. The OspI-binding sites in Ubc13 largely overlap with the binding residues for host ubiquitin E3 ligases and a deubiquitinating enzyme, which suggests that the bacterial effector and host proteins exploit the same surface on Ubc13 for specific recognition. Biochemical results indicate that both of the differently charged regions in OspI are important for the interaction with Ubc13, and the specificity determinants in Ubc13 for OspI recognition reside in the distinct residues in the α1 helix and L2 region. Our study reveals the molecular basis of Ubc13 deamidation by OspI, as well as a convergence of E2 recognition by bacterial and host proteins.

Direct activation of protein kinases by unanchored polyubiquitin chains.

TRAF6 is a ubiquitin ligase that is essential for the activation of NF-kappaB and MAP kinases in several signalling pathways, including those emanating from the interleukin 1 and Toll-like receptors. TRAF6 functions together with a ubiquitin-conjugating enzyme complex consisting of UBC13 (also known as UBE2N) and UEV1A (UBE2V1) to catalyse Lys 63-linked polyubiquitination, which activates the TAK1 (also known as MAP3K7) kinase complex. TAK1 in turn phosphorylates and activates IkappaB kinase (IKK), leading to the activation of NF-kappaB. Although several proteins are known to be polyubiquitinated in the IL1R and Toll-like receptor pathways, it is not clear whether ubiquitination of any of these proteins is important for TAK1 or IKK activation. By reconstituting TAK1 activation in vitro using purified proteins, here we show that free Lys 63 polyubiquitin chains, which are not conjugated to any target protein, directly activate TAK1 by binding to the ubiquitin receptor TAB2 (also known as MAP3K7IP2). This binding leads to autophosphorylation and activation of TAK1. Furthermore, we found that unanchored polyubiquitin chains synthesized by TRAF6 and UBCH5C (also known as UBE2D3) activate the IKK complex. Disassembly of the polyubiquitin chains by deubiquitination enzymes prevented TAK1 and IKK activation. These results indicate that unanchored polyubiquitin chains directly activate TAK1 and IKK, suggesting a new mechanism of protein kinase regulation.

Diverse sequence determinants control human and mouse receptor interacting protein 3 (RIP3) and mixed lineage kinase domain-like (MLKL) interaction in necroptotic signaling.

Receptor interacting protein 3 (RIP3) is a protein kinase essential for TNF-induced necroptosis. Phosphorylation on Ser-227 in human RIP3 (hRIP3) is required for its interaction with human mixed lineage kinase domain-like (MLKL) in the necrosome, a signaling complex induced by TNF stimulation. RIP1 and RIP3 mediate necrosome aggregation leading to the formation of amyloid-like signaling complexes. We found that TNF induces Thr-231 and Ser-232 phosphorylation in mouse RIP3 (mRIP3) and this phosphorylation is required for mRIP3 to interact with mMLKL. Ser-232 in mRIP3 corresponds to Ser-227 in hRIP3, whereas Thr-231 is not conserved in hRIP3. Although the RIP3-MLKL interaction is required for necroptosis in both human and mouse cells, hRIP3 does not interact with mMLKL and mRIP3 cannot bind to hMLKL. The species specificity of the RIP3-MLKL interaction is primarily determined by the sequence differences in the phosphorylation sites and the flanking sequence around the phosphorylation sites in hRIP3 and mRIP3. It appears that the RIP3-MLKL interaction has been selected as an evolutionarily conserved mechanism in mediating necroptosis signaling despite that differing structural and mechanistic bases for this interaction emerged simultaneously in different organisms. In addition, we further revealed that the interaction of RIP3 with MLKL prevented massive abnormal RIP3 aggregation, and therefore should be crucial for formation of the amyloid signaling complex of necrosomes. We also found that the interaction between RIP3 and MLKL is required for the translocation of necrosomes to mitochondria-associated membranes. Our data demonstrate the importance of the RIP3-MLKL interaction in the formation of functional necrosomes and suggest that translocation of necrosomes to mitochondria-associated membranes is essential for necroptosis signaling.

Type I Interferon Signalling and Ischemic Stroke: Mechanisms and Therapeutic Potentials.

Type I interferon (IFN-I) signalling is intricately involved in the pathogenesis of multiple infectious diseases, autoimmune diseases, and neurological diseases. Acute ischemic stroke provokes overactivation of IFN-I signalling within the injured brain, particularly in microglia. Following cerebral ischemia, damage-associated molecular patterns (DAMPs) released from injured neural cells elicit marked proinflammatory episodes within minutes. Among these, self-nucleic acids, including nuclear DNA and mitochondrial DNA (mtDNA), have been recognized as a critical alarm signal to fan the flames of neuroinflammation, predominantly via inducing IFN-I signalling activation in microglia. The concept of interferon-responsive microglia (IRM), marked by upregulation of a plethora of IFN-stimulated genes, has been emergingly elucidated in ischemic mouse brains, particularly in aged ones. Among the pattern recognition receptors responsible for IFN-I induction, cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) plays integral roles in potentiating microglia-driven neuroinflammation and secondary brain injury after cerebral ischemia. Here, we aim to provide an up-to-date review on the multifaceted roles of IFN-I signalling, the detailed molecular and cellular mechanisms leading to and resulting from aberrant IFN-I signalling activation after cerebral ischemia, and the therapeutic potentials. A thorough exploration of these above points will inform our quest for IFN-based therapies as effective immunomodulatory therapeutics to complement the limited repertoire of thrombolytic agents, thereby facilitating the translation from bench to bedside.