Structural and Functional Insights into Host Death Domains Inactivation by the Bacterial Arginine GlcNAcyltransferase Effector.

Enteropathogenic E. coli NleB and related type III effectors catalyze arginine GlcNAcylation of death domain (DD) proteins to block host defense, but the underlying mechanism is unknown. Here we solve crystal structures of NleB alone and in complex with FADD-DD, UDP, and Mn2+ as well as NleB-GlcNAcylated DDs of TRADD and RIPK1. NleB adopts a GT-A fold with a unique helix-pair insertion to hold FADD-DD; the interface contacts explain the selectivity of NleB for certain DDs. The acceptor arginine is fixed into a cleft, in which Glu253 serves as a base to activate the guanidinium. Analyses of the enzyme-substrate complex and the product structures reveal an inverting sugar-transfer reaction and a detailed catalytic mechanism. These structural insights are validated by mutagenesis analyses of NleB-mediated GlcNAcylation in vitro and its function in mouse infection. Our study builds a structural framework for understanding of NleB-catalyzed arginine GlcNAcylation of host death domain.

Identification of a 10-mer peptide from the death domain of MyD88 which attenuates inflammation and insulin resistance and improves glucose metabolism.

Insulin resistance (IR) is the key pathophysiological cause of type 2 diabetes, and inflammation has been implicated in it. The death domain (DD) of the adaptor protein, MyD88 plays a crucial role in the transduction of TLR4-associated inflammatory signal. Herein, we have identified a 10-residue peptide (M10), from the DD of MyD88 which seems to be involved in Myddosome formation. We hypothesized that M10 could inhibit MyD88-dependent TLR4-signaling and might have effects on inflammation-associated IR. Intriguingly, 10-mer M10 showed oligomeric nature and reversible self-assembly property indicating the peptide's ability to recognize its own amino acid sequence. M10 inhibited LPS-induced nuclear translocation of NF-κB in L6 myotubes and also reduced LPS-induced IL-6 and TNF-α production in peritoneal macrophages of BALB/c mice. Remarkably, M10 inhibited IL-6 and TNF-α secretion in diabetic, db/db mice. Notably, M10 abrogated IR in insulin-resistant L6 myotubes, which was associated with an increase in glucose uptake and a decrease in Ser307-phosphorylation of IRS1, TNF-α-induced JNK activation and nuclear translocation of NF-κB in these cells. Alternate day dosing with M10 (10 and 20 mg/kg) for 30 days in db/db mice significantly lowered blood glucose and improved glucose intolerance after loading, 3.0 g/kg glucose orally. Furthermore, M10 increased insulin and adiponectin secretion in db/db mice. M10-induced glucose uptake in L6 myotubes involved the activation of PI3K/AKT/GLUT4 pathways. A scrambled M10-analog was mostly inactive. Overall, the results show the identification of a 10-mer peptide from the DD of MyD88 with anti-inflammatory and anti-diabetic properties, suggesting that targeting of TLR4-inflammatory pathway, could lead to the discovery of molecules against IR and diabetes.

Calloso-adreno-scrotal agenesis associated with biallelic MAPK-activating death domain protein (MADD) variant: Further phenotypic delineation of MADD deficiency.

MAPK-activating death domain protein (MADD) deficiency is associated with a broad clinical spectrum ranging from mild developmental impairment to fatal multisystem disorder. We report an additional case of severe form with some overlapping and unreported systemic features in a growth-restricted full-term male newborn. The novel findings include corpus callosum agenesis, bilateral adrenal agenesis, scrotal aplasia, and abnormal skin pigmentation. Microscopic changes are only remarkable in thyroid gland that shows decreased, variously sized follicles with absent or non-vacuolated pale colloid. This unique constellation of birth defects is associated with a novel homozygous in-frame MADD gene deletion (NM_003682.4: c.4853_4855delGCT:p.Cys1618del). This case report expands the phenotypic and genetic spectrum of MADD deficiency.

Cellular Dynamics of Fas-Associated Death Domain in the Regulation of Cancer and Inflammation.

Fas-associated death domain (FADD) is an adaptor protein that predominantly transduces the apoptosis signal from the death receptor (DR) to activate caspases, leading to the initiation of apoptotic signaling and the coordinated removal of damaged, infected, or unwanted cells. In addition to its apoptotic functions, FADD is involved in signaling pathways related to autophagy, cell proliferation, necroptosis, and cellular senescence, indicating its versatile role in cell survival and proliferation. The subcellular localization and intracellular expression of FADD play a crucial role in determining its functional outcomes, thereby highlighting the importance of spatiotemporal mechanisms and regulation. Furthermore, FADD has emerged as a key regulator of inflammatory signaling, contributing to immune responses and cellular homeostasis. This review provides a comprehensive summary and analysis of the cellular dynamics of FADD in regulating programmed cell death and inflammation through distinct molecular mechanisms associated with various signaling pathways.

Death domain-associated protein DAXX regulates noncoding RNA transcription at the centromere through the transcription regulator ZFAT.

The centromere is an essential chromosomal structure for faithful chromosome segregation during cell division. No protein-coding genes exist at the centromeres, but centromeric DNA is actively transcribed into noncoding RNA (ncRNA). This centromeric transcription and its ncRNA products play important roles in centromere functions. We previously reported that the transcriptional regulator ZFAT (zinc-finger protein with AT hook) plays a pivotal role in ncRNA transcription at the centromere; however, it was unclear how ZFAT involvement was regulated. Here, we show that the death domain-associated protein (DAXX) promotes centromeric localization of ZFAT to regulate ncRNA transcription at the centromere. Coimmunoprecipitation analysis of endogenous proteins clearly reveals that DAXX interacts with ZFAT. In addition, we show that ectopic coexpression of ZFAT with DAXX increases the centromeric levels of both ZFAT and ncRNA, compared with ectopic expression of ZFAT alone. On the other hand, we found that siRNA-mediated depletion of DAXX decreases the centromeric levels of both ZFAT and ncRNA in cells ectopically expressing ZFAT. These results suggest that DAXX promotes the centromeric localization of ZFAT and ZFAT-regulated centromeric ncRNA transcription. Furthermore, we demonstrate that depletion of endogenous DAXX protein is sufficient to cause a decrease in the ncRNA levels at the centromeres of chromosomes 17 and X in which ZFAT regulates the transcription, indicating a physiological significance of DAXX in ZFAT-regulated centromeric ncRNA transcription. Taken together, these results demonstrate that DAXX regulates centromeric ncRNA transcription through ZFAT.

Structural basis of NF-κB signaling by the p75 neurotrophin receptor interaction with adaptor protein TRADD through their respective death domains.

The p75 neurotrophin receptor (p75NTR) is a critical mediator of neuronal death and tissue remodeling and has been implicated in various neurodegenerative diseases and cancers. The death domain (DD) of p75NTR is an intracellular signaling hub and has been shown to interact with diverse adaptor proteins. In breast cancer cells, binding of the adaptor protein TRADD to p75NTR depends on nerve growth factor and promotes cell survival. However, the structural mechanism and functional significance of TRADD recruitment in neuronal p75NTR signaling remain poorly understood. Here we report an NMR structure of the p75NTR-DD and TRADD-DD complex and reveal the mechanism of specific recognition of the TRADD-DD by the p75NTR-DD mainly through electrostatic interactions. Furthermore, we identified spatiotemporal overlap of p75NTR and TRADD expression in developing cerebellar granule neurons (CGNs) at early postnatal stages and discover the physiological relevance of the interaction between TRADD and p75NTR in the regulation of canonical NF-κB signaling and cell survival in CGNs. Our results provide a new structural framework for understanding how the recruitment of TRADD to p75NTR through DD interactions creates a membrane-proximal platform, which can be efficiently regulated by various neurotrophic factors through extracellular domains of p75NTR, to propagate downstream signaling in developing neurons.

Non-cell-autonomous function of DR6 in Schwann cell proliferation.

Death receptor 6 (DR6) is an orphan member of the TNF receptor superfamily and controls cell death and differentiation in a cell-autonomous manner in different cell types. Here, we report an additional non-cell-autonomous function for DR6 in the peripheral nervous system (PNS). DR6-knockout (DR6 KO) mice showed precocious myelination in the PNS Using an in vitro myelination assay, we demonstrate that neuronal DR6 acts in trans on Schwann cells (SCs) and reduces SC proliferation and myelination independently of its cytoplasmic death domain. Mechanistically, DR6 was found to be cleaved in neurons by "a disintegrin and metalloprotease 10" (ADAM10), releasing the soluble DR6 ectodomain (sDR6). Notably, in the in vitro myelination assay, sDR6 was sufficient to rescue the DR6 KO phenotype. Thus, in addition to the cell-autonomous receptor function of full-length DR6, the proteolytically released sDR6 can unexpectedly also act as a paracrine signaling factor in the PNS in a non-cell-autonomous manner during SC proliferation and myelination. This new mode of DR6 signaling will be relevant in future attempts to target DR6 in disease settings.

The Ancient Origins of Death Domains Support the 'Original Sin' Hypothesis for the Evolution of Programmed Cell Death.

The discovery of caspase homologs in bacteria highlighted the relationship between programmed cell death (PCD) evolution and eukaryogenesis. However, the origin of PCD genes in prokaryotes themselves (bacteria and archaea) is poorly understood and a source of controversy. Whether archaea also contain C14 peptidase enzymes and other death domains is largely unknown because of a historical dearth of genomic data. Archaeal genomic databases have grown significantly in the last decade, which allowed us to perform a detailed comparative study of the evolutionary histories of PCD-related death domains in major archaeal phyla, including the deepest branching phyla of Candidatus Aenigmarchaeota, Candidatus Woesearchaeota, and Euryarchaeota. We identified death domains associated with executioners of PCD, like the caspase homologs of the C14 peptidase family, in 321 archaea sequences. Of these, 15.58% were metacaspase type I orthologues and 84.42% were orthocaspases. Maximum likelihood phylogenetic analyses revealed a scattered distribution of orthocaspases and metacaspases in deep-branching bacteria and archaea. The tree topology was incongruent with the prokaryote 16S phylogeny suggesting a common ancestry of PCD genes in prokaryotes and subsequent massive horizontal gene transfer coinciding with the divergence of archaea and bacteria. Previous arguments for the origin of PCD were philosophical in nature with two popular propositions being the "addiction" and 'original sin' hypotheses. Our data support the 'original sin' hypothesis, which argues for a pleiotropic origin of the PCD toolkit with pro-life and pro-death functions tracing back to the emergence of cellular life-the Last Universal Common Ancestor State.

FADD at the Crossroads between Cancer and Inflammation.

Initially described as an adaptor molecule for death receptor (DR)-mediated apoptosis, Fas-associated death domain (FADD) was later implicated in nonapoptotic cellular processes. During the last decade, FADD has been shown to participate and regulate most of the signalosome complexes, including necrosome, FADDosome, innateosome, and inflammasome. Given the role of these signaling complexes, FADD has emerged as a new actor in innate immunity, inflammation, and cancer development. Concomitant to these new roles, a surprising number of mechanisms deemed to regulate FADD functions have been identified, including post-translational modifications of FADD protein and FADD secretion. This review focuses on recent knowledge of the biological roles of FADD, a pleiotropic molecule having multiple partners, and its impact in cancer, innate immunity, and inflammation.

SPOP inhibits mice pancreatic stellate cell activation by promoting FADD degradation in cerulein-induced chronic pancreatitis.

Pancreatic stellate cells (PSCs) have been recognized as key mediators of pancreatic fibrosis, a characteristic feature of chronic pancreatitis (CP). As a cullin-based E3 ubiquitin ligase, speckle-type POZ protein (SPOP) has been identified to participate in tumorigenesis and organ development. However, its biological role in CP remains unknown. Therefore, this study sought to investigate the changed expression of SPOP in CP and to examine the effect on mice PSCs activation of SPOP. We found that SPOP was downregulated in the pancreatic tissues of cerulein-induced CP mice. siRNA-mediated knockdown of SPOP led to significant promotion in primary PSCs activity by activating the nuclear factor-kappaB (NF-κB)/interleukin-6 (IL-6) signaling pathway. In addition, we examined the effects of Fas-associated death domain (FADD), a proven SPOP substrate that activates NF-κB, on the regulation of PSCs activation. We found that FADD was downregulated by SPOP via interaction-mediated degradation, and was upregulated during PSCs activation. The promotion of PSCs activation in knocking down SPOP with siSPOP-1 were counteracted by knocking down FADD. The results suggest that the SPOP-induced inhibition of PSCs activation partially depended on FADD. These results highlight the importance of SPOP in CP and provide a potential target for therapeutic intervention.

Modulation of CD95-mediated signaling by post-translational modifications: towards understanding CD95 signaling networks.

CD95 is a member of the death receptor family and is well-known to promote apoptosis. However, accumulating evidence indicates that in some context CD95 has not only the potential to induce apoptosis but also can trigger non-apoptotic signal leading to cell survival, proliferation, cancer growth and metastasis. Despite extensive investigations focused on alterations in the expression level of CD95 and associated signal molecules, very few studies, however, have investigated the effects of post-translational modifications such as glycosylation, phosphorylation, palmitoylation, nitrosylation and glutathionylation on CD95 function. Post-translational modifications of CD95 in mammalian systems are likely to play a more prominent role than anticipated in CD95 induced cell death. In this review we will focus on the alterations in CD95-mediated signaling caused by post-translational modifications of CD95.

Identification and analysis of dominant negative mutants of RIP1 DD that disrupt RIPoptosome core formation.

The RIPoptosome, composed of RIP1 and caspase-8, plays an important role in the regulation of apoptosis and necroptosis; however, the mechanism of complex formation by oligomerization and how the caspase-activating process and necroptosis are mediated by the formation of the RIPoptosome is not well-understood. This study revealed that the assembly mechanism of the RIPoptosome core is dependent on salt concentration and not on pH and time. In addition, we demonstrated that three RIP1 mutations, E626K, M637K, and S657K, have dominant negative effects. These dominant negative mutations in RIP1 may have potential applications in therapeutic intervention.

Identification and expression of alternatively spliced novel isoforms of cancer associated MYD88 lacking death domain in mouse.

MYD88 is an adaptor protein known to involve in activation of NF-κB through IL-1 receptor and TLR stimulation. It consists of N-terminal death domain and C-terminal Toll/IL-R homology domain that mediates its interaction with IL-1R associated kinase and IL-1R/TLR, respectively. MYD88 contributes to various types of carcinogenesis due to its involvement in oncogene induced inflammation. In the present study, we have recognized two new alternatively spliced variants of MyD88 gene in mouse using bioinformatics tools and molecular biology techniques in combination. The newly identified non-coding exon (NE-1) from 5' upstream region alternatively splices with either exon E-2 or exon E-5 to produce two novel transcript variants MyD88N1 and MyD88N2 respectively. The transcript variant MyD88N1 was expressed in several tissues studied while the variant MyD88N2 was found to be expressed only in the brain. The analysis of the upstream region of novel exon by in silico approach revealed new promoter region PN, which possess potential signature sequences for diverse transcription factors, suggesting complex gene regulation. Studies of post translational modifications of conceptualized amino acid sequences of these isoforms revealed diversity in properties. Western blot analysis further confirmed the expression of protein isoform MYD88N1.

The death domain-associated protein suppresses porcine epidemic diarrhea virus replication by interacting with signal transducer and activator of transcription 1 and inducing downstream ISG15 expression.

Porcine epidemic diarrhea virus (PEDV) is an enteric coronavirus that causes acute enteric disease in piglets and severely threatens the pig industry all over the world. Death domain-associated protein (DAXX) is a classical chaperone protein involved in multiple biological processes, such as cell apoptosis, transcriptional regulation, DNA damage repair, and host innate immunity. However, whether DAXX functions in the anti-PEDV innate immune responses remains unclear. In this study, we found that PEDV infection upregulated DAXX expression and induced its nucleocytoplasmic translocation in IPEC-J2 cells. Furthermore, we found that DAXX overexpression was inhibitory to PEDV replication, while downregulation of DAXX by RNA interference facilitated PEDV replication. The antiviral activity of DAXX was due to its positive effect on IFN-λ3-STAT1 signaling, as DAXX positively regulated STAT1 activation through their interaction in cytoplasm and enhancing the downstream ISG15 expression. Mutation of tryptophan at 621 to alanine in DAXX increased its abundance in the cytoplasm, leading to the upregulation of STAT1 phosphorylation and ISG15 expression. It indicated that cytoplasmic fraction of DAXX was advantageous for the STAT1-ISG15 signaling axis and PEDV inhibition. In summary, these results show that DAXX inhibits PEDV infection by increasing IFN-λ3-induced STAT1 phosphorylation and the downstream ISG15 expression.

Equilibrium between monomers and dimers of the death domain of the p75 neurotrophin receptor in solution.

p75 neurotrophin receptor (p75NTR) contains a C-terminal globular protein module known as the death domain (DD), which plays a central role in apoptotic and inflammatory signaling through the formation of oligomeric protein complexes. A monomeric state of the p75NTR-DD also exists depending on its chemical environment in vitro. However, studies on the oligomeric states of the p75NTR-DD have produced conflicting findings and sparked great controversy. Here we present new evidence from biophysical and biochemical studies to demonstrate the coexistence of symmetric and asymmetric dimers of the p75NTR-DD, which may equilibrate with the monomeric form in solution and in the absence of any other protein. The reversible close-open solution behavior may be important for the p75NTR-DD to serve as an intracellular signaling hub. This result supports an intrinsic ability of the p75NTR-DD to self-associate, in congruence with the oligomerization properties of all members of the DD superfamily.

A Bacterial Quorum Sensing Regulated Protease Inhibits Host Immune Responses by Cleaving Death Domains of Innate Immune Adaptors.

Innate immune adaptor proteins are critical components of the innate immune system that propagate pro-inflammatory responses from their upstream receptors, and lead to pathogen clearance from the host. Bacterial pathogens have developed strategies to survive inside host cells without triggering the innate immune surveillance in ways that are still not fully understood. Here, it is reported that Pseudomonas aeruginosa induces its quorum sensing mechanism after macrophage engulfment. Further investigation of its secretome identified a quorum sensing regulated product, LasB, is responsible for innate immune suppression depending on the MyD88-mediated signaling. Moreover, it is showed that this specific type of pathogen-mediated innate immune suppression is due to the enzymatic digestion of the death domains of the innate immune adaptors, mainly MyD88, and attributed to LasB's large substrate binding groove. Lastly, it is demonstrated that the secretion of LasB from P. aeruginosa directly contributed to MyD88 degradation within macrophages. Hence, it is discovered an example of bacterial quorum sensing-regulated cellular innate immune suppression by direct cleavage of immune adaptors.

A new variant of the ectodysplasin A receptor death domain gene associated with anhidrotic ectodermal dysplasia in a Turkish family and its simple diagnosis by restriction fragment length polymorphism.

Ectodermal dysplasia (ED), which exhibits a wide range of clinical symptoms, may be classified into three major types: hypohidrotic, anhidrotic, and hidrotic. A male child (proband) showing anhidrotic dysplasia was used as the subject of this study. The biopsy of the big toe revealed that the male child had no sweat glands. Genetic analysis of the patient revealed a mutation caused by a homozygous nucleotide substitution in the EDAR-associated death domain (EDARADD) (rs114632254) gene c.439G>A (p.Gly147Arg). Phenotypically, his teeth were sharp, but eight teeth were missing (oligodontia). The patient had normal nails with dry skin, sparse hair, everted lower lip vermilion, hyperpigmented eyelids, and abnormal nasal bridge morphology around the eyes. There is also a homozygous dominant (healthy) female and a heterozygous male in this family, who are cousins (aunt children) to the heterozygous parents. The daughter of the patient was also heterozygous. This mutation represents homozygous recessive inheritance, which we describe for the first time. Furthermore, we demonstrated that this genetic disorder can be readily diagnosed using the restriction fragment length polymorphism (RFLP) method after digestion with MnII restriction endonuclease.

MAP kinase activating death domain deficiency is a novel cause of impaired lymphocyte cytotoxicity.

Most hereditary forms of hemophagocytic lymphohistiocytosis (HLH) are caused by defects of cytotoxicity, including the vesicle trafficking disorder Griscelli syndrome type 2 (GS2, RAB27A deficiency). Deficiency of the mitogen-activated protein kinase activating death domain protein (MADD) results in a protean syndrome with neurological and endocrinological involvement. MADD acts as a guanine nucleotide exchange factor for small guanosine triphosphatases, including RAB27A. A homozygous splice site mutation in MADD was identified in a female infant with syndromic features, secretory diarrhea, and features of HLH. Aberrant splicing caused by this mutation leads to an in-frame deletion of 30 base pairs and favors other aberrant variants. Patient natural killer (NK) cells and cytotoxic T cells showed a severe degranulation defect leading to absent perforin-mediated cytotoxicity. Platelets displayed defective adenosine triphosphate secretion, similar to that in GS2. To prove causality, we introduced a CRISPR/Cas9-based MADD knockout in the NK cell line NK-92mi. MADD-deficient NK-92mi cells showed a degranulation defect and impaired cytotoxicity similar to that of the patient. The defect of cytotoxicity was confirmed in another patient with MADD deficiency. In conclusion, RAB27A-interacting MADD is involved in vesicle release by cytotoxic cells and platelets. MADD deficiency causes a degranulation defect and represents a novel disease predisposing to an HLH phenotype.

Death domain fold proteins in immune signaling and transcriptional regulation.

Death domain fold (DDF) superfamily comprises of the death domain (DD), death effector domain (DED), caspase activation recruitment domain (CARD), and pyrin domain (PYD). By utilizing a conserved mode of interaction involving six distinct surfaces, a DDF serves as a building block that can densely pack into homomultimers or filaments. Studies of immune signaling components have revealed that DDF-mediated filament formation plays a central role in mediating signal transduction and amplification. The unique ability of DDFs to self-oligomerize upon external signals and induce oligomerization of partner molecules underlies key processes in many innate immune signaling pathways, as exemplified by RIG-I-like receptor signalosome and inflammasome assembly. Recent studies showed that DDFs are not only limited to immune signaling pathways, but also are involved with transcriptional regulation and other biological processes. Considering that DDF annotation still remains a challenge, the current list of DDFs and their functions may represent just the tip of the iceberg within the full spectrum of DDF biology. In this review, we discuss recent advances in our understanding of DDF functions, structures, and assembly architectures with a focus on CARD- and PYD-containing proteins. We also discuss areas of future research and the potential relationship of DDFs with biomolecular condensates formed by liquid-liquid phase separation (LLPS).

Roles of the adaptor protein tumor necrosis factor receptor type 1-associated death domain protein (TRADD) in human diseases.

Cells communication in response to extracellular or biophysical stimulus relies on elaborated systems of signal transduction. In the course of most signal pathway, the cascades involve signal protein complexes, which are often assembled by adaptor proteins. Tumor necrosis factor receptor type 1-associated death domain protein (TRADD) is an adaptor molecule involved in various signal pathways and mediating multiple biological activities, including cell survival, cell proliferation, cell differentiation, apoptosis, necroptosis and inflammation. TRADD contains an N terminal tumor necrosis factor receptor-associated factor 2 (TRAF2) binding domain and a C terminal death domain (DD) for interacting with multiple DD-containing proteins. Following activation of specific receptors, such as tumor necrosis factor receptor 1 (TNFR1), death receptor 3 (DR3), tumor necrosis factor-related apoptosis-inducing ligand receptor 1 (TRAILR1, DR4), TRAILR1 (DR5), DR6 and p75 neurotrophin receptor (p75NTR)，TRADD can bind to the receptors, serving as a platform for the recruitment of the downstream molecules for signal propagating and thus mediating various physiological and pathological processes. In this review, we provide a brief overview of the current knowledge on TRADD and discuss the roles of TRADD in infectious and inflammatory diseases, cardiovascular diseases, central nervous system diseases, cancer, endometriosis, hepatocyte proliferation, preterm birth and perinatal development.