PEA-15 engages in allosteric interactions using a common scaffold in a phosphorylation-dependent manner

Phosphoprotein enriched in astrocytes, 15 kDa (PEA-15) is a death-effector domain (DED) containing protein involved in regulating mitogen-activated protein kinase and apoptosis pathways. In this molecular dynamics study, we examined how phosphorylation of the PEA-15 C-terminal tail residues, Ser-104 and Ser-116, allosterically mediates conformational changes of the DED and alters the binding specificity from extracellular-regulated kinase (ERK) to Fas-associated death domain (FADD) protein. We delineated that the binding interfaces between the unphosphorylated PEA-15 and ERK2 and between the doubly phosphorylated PEA-15 and FADD are similarly composed of a scaffold that includes both the DED and the C-terminal tail residues of PEA-15. While the unphosphorylated serine residues do not directly interact with ERK2, the phosphorylated Ser-116 engages in strong electrostatic interactions with arginine residues on FADD DED. Upon PEA-15 binding, FADD repositions its death domain (DD) relative to the DED, an essential conformational change to allow the death-inducing signaling complex (DISC) assembly.

PITA, a pre-bilaterian p75NTR, is the evolutionary ancestor of TNF receptors

Tumor necrosis factor receptors (TNFRs) regulate a diverse array of biological functions, including adaptive immunity, neurodevelopment, and many others. Although TNFRs are expressed in all metazoan phyla, a coherent model of the molecular origins of mammalian TNFRs—and how they relate to TNFRs in other phyla—has remained elusive. To address this, we executed a large-scale, systematic Basic Local Alignment Search Tool (BLAST)-based approach to trace the evolutionary ancestry of all 29 human TNFRs. We discovered that all human TNFRs are descendants of a single pre-bilaterian TNFR with strong sequence similarity to the p75 neurotrophin receptor (p75NTR), which we designate as PITA for ‘ p75NTR is the TNFR Ancestor’ . A distinct subset of human TNFRs—including EDAR, XEDAR and TROY—share a unique history as descendants of EDAR-XEDAR-TROY (EXT), which diverged from PITA in a bilaterian ancestor. Most PITA descendants possess a death domain (DD) within their intracellular domain (ICD) but EXTs do not. PITA descendants are expressed in all bilaterian phyla and Cnidaria, but not in non-planulozoan ParaHoxozoa, suggesting that PITA originated in an ancestral planulozoan. Drosophila melanogaster TNFRs (Wengen (Wgn) and Grindelwald (Grnd)) were identified as divergent PITA descendants, providing the first evolutionary link between this model TNFR system and the mammalian TNFR superfamily. This study reveals PITA as the ancestor to human and Drosophila TNFR systems and describes an evolutionary model that will facilitate deciphering TNF-TNFR functions in health and disease.

Long-chain PUFA ameliorate ETEC-induced intestinal inflammation and cell injury by modulating pyroptosis and necroptosis signaling pathways in IPEC-1 cells

This study was aimed to investigate whether eicosapentaenoic acid (EPA) and arachidonic acid (ARA), the representative n-3 or n-6 polyunsaturated fatty acids (PUFA), could alleviate enterotoxigenic Escherichia coli (ETEC) K88-induced inflammation and injury of intestinal porcine epithelial cells 1 (IPEC-1) by modulating pyroptosis and necroptosis signaling pathways. IPEC-1 cells were cultured with or without EPA or ARA in the presence or absence of ETEC K88. EPA and ARA reduced ETEC K88 adhesion and endotoxin content in the supernatant. EPA and ARA increased transepithelial electrical resistance (TEER) and decreased permeability of fluorescein isothiocyanate-labeled dextran (FD4), and increased membrane protein expression of occludin, ZO-1 and claudin-1, and relieved disturbed distribution of these proteins. EPA and ARA also reduced cell necrosis ratio. EPA or ARA reduced mRNA and concentration of tumor necrosis factor-α (TNF-α), interleukin (IL)-6 and IL-8, and decreased mRNA abundances of intestinal toll-like receptors 4 (TLR4) and its downstream signals. Moreover, EPA and ARA downregulated mRNA expression of nod-like receptor protein 3 (NLRP3), caspase 1 and IL-18, and inhibited protein expression of NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), gasdermin D and caspase-1. Finally, EPA and ARA reduced mRNA expression of fas-associated death domain protein (FADD), caspase 8, receptor interacting protein kinase (RIP) 1, mixed lineage kinase-like protein (MLKL), phosphoglycerate mutase 5 (PGAM5), motility related protein 1 (Drp1) and high mobility protein 1 (HMGB1), and inhibited protein expression of phosphorylated-RIP1 (p-RIP1), p-RIP3, p-MLKL and HMGB1. These data demonstrate that EPA and ARA prevent ETEC K88-induced cell inflammation and injury, which is partly through inhibiting pyroptosis and necroptosis signaling pathways.

Ectodysplasin A/Ectodysplasin A Receptor System and Their Roles in Multiple Diseases

Ectodysplasin A (EDA) is a member of the tumor necrosis factor (TNF) family of ligands that was initially reported to induce the formation of various ectodermal derivatives during normal prenatal development. EDA exerts its biological activity as two splice variants, namely, EDA-A1 and EDA-A2. The former binds to the EDA receptor (EDAR), resulting in the recruitment of the intracellular EDAR-associated death domain (EDARADD) adapter protein and the activation of the NF-κB signaling pathway, while the latter binds to a different receptor, EDA2R, also known as X-linked ectodermal dysplasia receptor (XEDAR). Inactivation mutation of the EDA gene or the genes coding for its receptors can result in hypohidrosis ectodermal dysplasia (HED), a condition that is characterized by oligotrichosis, edentulosis or oligodontia, and oligohidrosis or anhidrosis. Recently, as a new liver factor, EDA is gradually known and endowed with some new functions. EDA levels were observed to be upregulated in several metabolic diseases, such as non-alcoholic fatty liver disease (NAFLD), obesity, and insulin resistance. In addition, EDA and its receptors have been implicated in tumor pathogenesis through the regulation of tumor cell proliferation, apoptosis, differentiation, and migration. Here, we first review the role of EDA and its two-receptor system in various signaling pathways and then discuss the physiological and pathological roles of EDA and its receptors.

De novo variants in H3-3A and H3-3B are associated with neurodevelopmental delay, dysmorphic features, and structural brain abnormalities

The histone H3 variant H3.3, encoded by two genes H3-3A and H3-3B, can replace canonical isoforms H3.1 and H3.2. H3.3 is important in chromatin compaction, early embryonic development, and lineage commitment. The role of H3.3 in somatic cancers has been studied extensively, but its association with a congenital disorder has emerged just recently. Here we report eleven de novo missense variants and one de novo stop-loss variant in H3-3A (n = 6) and H3-3B (n = 6) from Baylor Genetics exome cohort (n = 11) and Matchmaker Exchange (n = 1), of which detailed phenotyping was conducted for 10 individuals (H3-3A = 4 and H3-3B = 6) that showed major phenotypes including global developmental delay, short stature, failure to thrive, dysmorphic facial features, structural brain abnormalities, hypotonia, and visual impairment. Three variant constructs (p.R129H, p.M121I, and p.I52N) showed significant decrease in protein expression, while one variant (p.R41C) accumulated at greater levels than wild-type control. One H3.3 variant construct (p.R129H) was found to have stronger interaction with the chaperone death domain-associated protein 6.

Downregulating expression of OPTN elevates neuroinflammation via AIM2 inflammasome- and RIPK1-activating mechanisms in APP/PS1 transgenic mice

Background Neuroinflammation is thought to be a cause of Alzheimer’s disease (AD), which is partly caused by inadequate mitophagy. As a receptor of mitophagy, we aimed to reveal the regulatory roles of optineurin (OPTN) on neuroinflammation in the pathogenesis of AD. Methods BV2 cells and APP/PS1 transgenic (Tg) mice were used as in vitro and in vivo experimental models to determine the regulatory roles of OPTN in neuroinflammation of AD. Sophisticated molecular technologies including quantitative (q) RT-PCR, western blot, enzyme linked immunosorbent assay (ELISA), co-immunoprecipitation (Co-IP) and immunofluorescence (IF) were employed to reveal the inherent mechanisms. Results As a consequence, key roles of OPTN in regulating neuroinflammation were identified by depressing the activity of absent in melanoma 2 (AIM2) inflammasomes and receptor interacting serine/threonine kinase 1 (RIPK1)-mediated NF-κB inflammatory mechanisms. In detail, we found that expression of OPTN was downregulated, which resulted in activation of AIM2 inflammasomes due to a deficiency in mitophagy in APP/PS1 Tg mice. By ectopic expression, OPTN blocks the effects of Aβ oligomer (Aβo) on activating AIM2 inflammasomes by inhibiting mRNA expression of AIM2 and apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC), leading to a reduction in the active form of caspase-1 and interleukin (IL)-1β in microglial cells. Moreover, RIPK1 was also found to be negatively regulated by OPTN via ubiquitin protease hydrolysis, resulting in the synthesis of IL-1β by activating the transcriptional activity of NF-κB in BV2 cells. As an E3 ligase, the UBAN domain of OPTN binds to the death domain (DD) of RIPK1 to facilitate its ubiquitination. Based on these observations, ectopically expressed OPTN in APP/PS1 Tg mice deactivated microglial cells and astrocytes via the AIM2 inflammasome and RIPK-dependent NF-κB pathways, leading to reduce neuroinflammation. Conclusions These results suggest that OPTN can alleviate neuroinflammation through AIM2 and RIPK1 pathways, suggesting that OPTN deficiency may be a potential factor leading to the occurrence of AD.

Heterologous Expression and Assembly of Human TLR Signaling Components in Saccharomyces cerevisiae

Toll-like receptor (TLR) signaling is key to detect pathogens and initiating inflammation. Ligand recognition triggers the assembly of supramolecular organizing centers (SMOCs) consisting of large complexes composed of multiple subunits. Building such signaling hubs relies on Toll Interleukin-1 Receptor (TIR) and Death Domain (DD) protein-protein interaction domains. We have expressed TIR domain-containing components of the human myddosome (TIRAP and MyD88) and triffosome (TRAM and TRIF) SMOCs in Saccharomyces cerevisiae, as a platform for their study. Interactions between the TLR4 TIR domain, TIRAP, and MyD88 were recapitulated in yeast. Human TIRAP decorated the yeast plasma membrane (PM), except for the bud neck, whereas MyD88 was found at cytoplasmic spots, which were consistent with endoplasmic reticulum (ER)-mitochondria junctions, as evidenced by co-localization with Mmm1 and Mdm34, components of the ER and Mitochondria Encounter Structures (ERMES). The formation of MyD88-TIRAP foci at the yeast PM was reinforced by co-expression of a membrane-bound TLR4 TIR domain. Mutations in essential residues of their TIR domains aborted MyD88 recruitment by TIRAP, but their respective subcellular localizations were unaltered. TRAM and TRIF, however, did not co-localize in yeast. TRAM assembled long PM-bound filaments that were disrupted by co-expression of the TLR4 TIR domain. Our results evidence that the yeast model can be exploited to study the interactions and subcellular localization of human SMOC components in vivo.

Molecular Evidence of the Inhibitory Potential of Melatonin against NaAsO2-Induced Aging in Male Rats

Arsenic (As) poisoning is widespread due to exposure to pollution. The toxic level of (As) causes oxidative stress-induced aging and tissue damage. Since melatonin (MLT) has anti-oxidant and anti-aging properties, we aimed to evaluate the protective effect of MLT against the toxicity of sodium arsenite (NaAsO2). Healthy male NMRI mice were divided into eight different groups. The control group received a standard regular diet. Other groups were treated with varying diets, including MLT alone, NaAsO2, and NaAsO2 plus MLT. After one month of treatment, biochemical and pathological tests were performed on blood, heart, and lung tissue samples. NaAsO2 increased the levels of TNF-α, 8-hydroxy-2-deoxy guanosine (8OHdG), malondialdehyde (MDA), reactive oxygen species (ROS), and high mobility group box 1 (HMGB1), increased the expression of TNF receptor type 1-associated death domain (TRADD) mRNA and telomerase reverse transcriptase, and decreased the expression of Klotho (KL) mRNA in both plasma and tissues. In contrast, MLT reduced MDA, ROS, HMGB1, lactate, and TNF-α enhanced the mRNA expression of KL, and suppressed the mRNA expression of the TERT and TRADD genes. Thus, MLT confers potent protection against NaAsO2- induced tissue injury and oxidative stress.

FADD Deficiency Mimicking ALPS-FAS: An Expanding Phenotype.

Autoimmune lymphoproliferative syndrome (ALPS) is caused due to defects in the Fas-mediated apoptotic pathway. This disease is characterised by chronic non -malignant lymphoproliferation, autoimmune cytopenias and accumulation of double-negative T cells. FAS is the most commonly affected gene observed in patients with ALPS. There is a paucity of data in other ALPS associated genes. Mutations in the FADD gene is extremely rare, and to date, only five patients with a homozygous mutation in exon 2 and one patient with a compound heterozygous mutation are reported. The clinical spectrum of FADD includes neurological dysfunction, recurrent bacterial and viral infections and liver dysfunction. Here we report two patients with novel FADD mutation with clinical phenotype like ALPS-FAS. This article provides an expanding phenotype of FADD deficiency where patients can solely present with lymphoproliferation and autoimmunity without any features of febrile episodes or neurodevelopmental delay. We identified a novel homozygous FADD mutation by targeted Next-generation sequencing (NGS). We performed a comprehensive immune evaluation along with biomarker estimation, mTOR activity on DNTs and apoptosis assay. We describe two siblings born to third-degree consanguineous marriage with homozygous FADD mutation. The index patient P1 presented with lymphoproliferation, autoimmune manifestations in the form of Evans phenotype, lymphocytosis and elevated DNTs, whereas P2 only had mild lymphoproliferation with no autoimmune manifestations. The mutation in our patients lies in exon 2 of the FADD gene, encoding the death domain. The death domain (DD) of FADD protein interacts directly with the death domains of Fas, and mutation at this site disrupts binding to Fas and abolishes apoptosis. FADD deficiency should also be considered in patients presenting with lymphoproliferation and autoimmunity since they can mimic clinical features of ALPS-FAS.