Death Domain Research Articles

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.

Asparagine-rich protein (NRP) mediates stress response by regulating biosynthesis of plant secondary metabolites in Arabidopsis

ABSTRACT The plant-specific stress response protein NRP (asparagine-rich protein) is characterized by an asparagine-rich domain at its N-terminus and a conserved development and cell death (DCD) domain at its C-terminus. Previous transcriptional studies and phenotypic analyses have demonstrated the involvement of NRP in response to severe stress conditions, such as high salt and ER Endoplasmic reticulum-stress. We have recently identified distinct roles for NRP in biotic- and abiotic-stress signaling pathways, in which NRP interacts with different signaling proteins to change their subcellular localizations and stability. Here, to further explore the function of NRP, a transcriptome analysis was carried out on nrp1nrp2 knock-out lines at different life stages or under different growing conditions. The most significant changes in the transcriptome at both stages and conditions turned out to be the induction of the synthesis of secondary metabolites (SMs). Such an observation implicates that NRP is a general stress-responsive protein involved in various challenges faced by plants during their life cycle, which might involve a broad alteration in the distribution of SMs.

Comparative transcriptome analysis reveals immunoregulation mechanism in the spleen of large yellow croaker (Larimichthys crocea) in response to Vibrio harveyi infection

Large yellow croaker (Larimichthys crocea) is a commercially important fish species worldwide, but high-density cultivation has led to serious diseases especially by Vibrio harveyi. Understanding the molecular response of large yellow croaker to V. harveyi infection is vital for the development of effective measures to combat the disease. In this study, RNA-Seq was performed on the spleen of uninfected and V. harveyi-infected large yellow croaker. A total of 7527 differentially expressed genes (DEGs) were identified, with 4413 up-regulated and 3114 down-regulated genes. Functional enrichment analysis revealed that several immune-related pathways were highlighted, including Toll-like receptors (TLRs) signaling pathway, apoptosis, p53 signaling pathway, and lysosome. In the TLRs signaling pathway, up-regulated genes included IL1b, bactericidal permeability-increasing protein, and IL12b, while down-expressed genes included tlr3 and irf3. The apoptosis pathway showed up-regulated genes such as casp6, bcl-2-like protein, and cytochrome c, and down-expressed genes such as granzyme B, sptan1, and FADD-Fas (tnfrsf6)-associated via death domain. These results suggest that V. harveyi infection induced inflammatory response and apoptotic cell death in the spleen of large yellow croaker. This study provides valuable insights into the molecular mechanisms underlying the immune response of large yellow croaker to V. harveyi infection

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 novel fish CRADD inhibits Singapore grouper iridovirus (SGIV) infection by mediating apoptosis and modulating SGIV CARD (VP48)-caspase-2 interaction

The bipartite linker protein CRADD is a molecule that contains death domain (DD)- and caspase recruitment domain (CARD), and remarkably assists in inducing apoptosis and activating caspase-2. This study describes a novel fish CRADD (EcCRADD) isolated from the orange-spotted grouper (Epinephelus coioides), meanwhile demonstrating its inhibitory effect in viral replication. Our findings suggest that EcCRADD can form long-chain filamentous structures that may be involved in apoptosis triggered by nuclear fragmentation. EcCRADD engages the pro-apoptotic protein EcRIP1 (receptor interacting protein 1) through the DD domain and activates caspase-2 through the CARD domain. This bridging allows EcCRADD to promote Singapore grouper iridovirus (SGIV)-induced apoptosis and synergistically inhibits SGIV replication in vitro. SGIV contains a CARD protein encoded by ORF048L (VP48). In contrast to EcCRADD, the overexpression of VP48 significantly promotes SGIV replication and inhibits SGIV-induced apoptosis. Furthermore, we found that VP48 may utilize caspase-2 as a scaffolding molecule to activate NF-κB and promote cell survival. However, EcCRADD could activate p53 in the presence of caspase-2 and inhibit this VP48-induced NF-κB activity in a dose-dependent manner. Lastly, EcCRADD inhibits the nuclear expression of VP48 via caspase-2. EcCRADD competes with VP48 to bind with caspase-2. Our study provides insights into host–virus interactions in fish, specifically that EcCRADD inhibits virus replication via the caspase-2-activated p53 pathway and interferes with the VP48-activated NF-κB signaling pathway.

O-159 Impact of open and closed methods of vitrification on oocyte mitochondria function and RNAseq transcriptome profile: a preclinical screening study

Abstract Study question Do current methods of open vitrification (OV) and closed vitrification (CV) impact on oocyte mitochondria function and transcriptome? Summary answer Oocyte vitrification confers unique mitochondria distribution patterns and alters the expression of stress response genes and mitochondria function relative to control (fresh) oocytes. What is known already Mitochondria are essential for maintenance of metabolic function and developmental competence at physiological conditions. Conversely, open and closed methods of oocyte vitrification are associated with physical, chemical and non-physiological changes, and exposures to cryoprotecting agents which could be harmful at extremely high and low temperatures. Study design, size, duration Ninety-four unfertilised Metaphase II (MII) oocytes were donated to research between May 2018 and August 2021. Fifty-seven oocytes were randomly allocated to the different study groups. Similarly, thirty-seven oocytes were allocated to the different study control groups. Participants/materials, setting, methods Fourteen women underdoing IVF/ICSI treatment cycles were involved in the study. cDNA was obtained for qPCR and the Smart-Seq2 protocol and single-cell RNA-Seq was performed (illumina NextSeq2000). Mitochondrial staining on live-oocytes was performed using Mito-tracker Deep Red stain, and images evaluated using fluorescence microscopy. Differentially expressed genes were identified using the R package edgeR and functional gene ontology was performed using an over-representation analysis via WebGestalt. Correction for multiple testing was calculated using Benjamini-Hochberg method Main results and the role of chance Mean mitochondrial intensity/density showed similarities between open (OV) and closed vitrification (CV) and control (fresh oocytes FO): 25.8 (12.7-44.9, standard deviation [SD] 10.2) for FO oocytes (n = 15), 30.3 (16.2-44.8, SD 10.1) for OV oocytes (n = 11), 26.6 (13.3-64.6, SD 15.3) for CV oocytes (n = 12) (p = 0.617). Distinct mitochondria distribution patterns (cytoplasmic and peripheral) were associated with OV and CV oocytes. Twice as many peripheral to cytoplasmic patterns were observed in FO and OV oocytes in contrast to CV oocytes (p = 037). Mean expression of the gene SDHB, which is associated with the TCA cycle and the mitochondrial electron transport chain was reduced in vitrified oocytes (n = 13) (0.0879 ± 0.0216 vs 0.0593 ± 0.0479), as was the anti-apoptotic gene BCL2 (p = 0.0061), relative to control oocytes (n = 13), (p = 0.01). Conversely, genes associated with apoptosis and stress response (BAX (p = 0.002) and BCL2L1 (p = 0.016)) were significantly enriched relative to control oocytes. RNAseq transcriptome analysis revealed a total of 71 differentially expressed genes following vitrification relative to control oocytes. Genes associated with critical metabolic processes (death domain binding, BCL-2 homology 3 (BH3) domains, and exopolyphosphatatse activities) were enriched in vitrified oocytes relative to the control group (FDR>0.05). Limitations, reasons for caution The use of unfertilised metaphase II (MII) oocytes on day 1 as a preclinical application screening model for vitrification may not reflect the molecular status and integrity of clinical grade MII oocytes on day 0. Wider implications of the findings This study emphasizes the inherent impact of vitrification on oocyte mitochondria function and transcriptome relative to control oocytes. Importantly, unintended perturbations may be detrimental to oocyte health and developmental competence, suggesting the need for a continuous safety screening and monitoring of ART methods of cryopreservation and offspring long-term health. Trial registration number National Institute for Health Research (NIHR). Grant Reference: ICA-CDRF-2015-01-068

#3220 Z-DNA BINDING PROTEIN 1 AGGRAVATED THE SEPSIS-INDUCED ACUTE KIDNEY INJURY VIA PANOPTOSIS

Abstract Background and Aims Sepsis-induced acute kidney injury (SI-AKI) is a severe complication of critical patients with high morbidity and mortality. Apoptosis, pyroptosis, and necroptosis, regulated by a common pathway - PANoptosis, play essential roles in SI-AKI without the clarified mechanisms. Method We first established the SI-AKI model by intraperitoneal injection of the lipopolysaccharide (LPS) (O111:B4) in the C57B/L6J mice with increased Serum creatinine (Scr), blood urea nitrogen (BUN), and kidney injury biomarkers (NGAL, KIM-1) in renal tissue. The expression of crucial molecules in PANoptosis were examined by quantitative real-time PCR (qPCR) and Western blotting. RIPK1 inhibitor-necrostatin-1(Nec-1) (1.65mg/kg) and pan-caspase inhibitor- Emricasan (10mg/kg) were further employed to mice with LPS. Potential interacting proteins of panoptosome were detected by co-immunoprecipitation. We also analyzed the methylation level of ZBP1 in cell-free DNA among sepsis-non-AKI and SI-AKI patients. Results The expression of crucial molecules in PANoptosis, including IL-1β(0.82 vs. 0.35, P = 0.003), mixed lineage kinase domain-like (MLKL) (0.67 vs. 0.41, P = 0.014), protein kinase 1 (RIPK1) (1.07 vs. 0.72, P = 0.002), cleaved-caspase 3 (1.13 vs. 0.59, P < 0.001), and FAS-associated death domain protein (FADD) (0.78 vs. 0.43 P < 0.001) elevated in the SI-AKI group. Z-DNA binding protein 1 (ZBP1), a typical sensor of PANoptosis, is dramatically activated in the SI-AKI mice. Co-immunoprecipitation showed that caspase 8, ZBP1, peptidoglycan recognition protein 1 (pglyrp1), and RIPK1 are potential components of PANoptosome. Nec-1 and Emricasan could attenuate the LPS-induced increases in the concentrations of BUN and inflammatory cytokine (TNF-a and IL-6), as well as the methylation level of ZBP1 in cell-free DNA decreased in SI-AKI patients verse sepsis-none-AKI (P = 1.60E-06). Conclusion ZBP1 exacerbated SI-AKI via activating PANoptosis with the potencial PANoptosome of ZBP1, caspase 8, pglyrp1, and RIPK1.

A systems biology approach to define mechanisms, phenotypes, and drivers in PanNETs with a personalized perspective

Pancreatic neuroendocrine tumors (PanNETs) are a rare tumor entity with largely unpredictable progression and increasing incidence in developed countries. Molecular pathways involved in PanNETs development are still not elucidated, and specific biomarkers are missing. Moreover, the heterogeneity of PanNETs makes their treatment challenging and most approved targeted therapeutic options for PanNETs lack objective responses. Here, we applied a systems biology approach integrating dynamic modeling strategies, foreign classifier tailored approaches, and patient expression profiles to predict PanNETs progression as well as resistance mechanisms to clinically approved treatments such as the mammalian target of rapamycin complex 1 (mTORC1) inhibitors. We set up a model able to represent frequently reported PanNETs drivers in patient cohorts, such as Menin-1 (MEN1), Death domain associated protein (DAXX), Tuberous Sclerosis (TSC), as well as wild-type tumors. Model-based simulations suggested drivers of cancer progression as both first and second hits after MEN1 loss. In addition, we could predict the benefit of mTORC1 inhibitors on differentially mutated cohorts and hypothesize resistance mechanisms. Our approach sheds light on a more personalized prediction and treatment of PanNET mutant phenotypes.

Erianin as a Promising Novel Agent in the Treatment of Neuroblastoma: The Anticancer Effects and Underlying Molecular Mechanisms.

Erianin is an active dibenzyl compound isolated from Dendrobium officinale and Dendrobium chrysotoxum and there are very few studies on molecular mechanisms and drug targets of erianin. In addition, there is no study investigating the anti-cancer effect of erianin on neuroblastoma cells. The aim of the study is to investigate the anticancer effect of erianin and the underlying mechanism of this effect on SH-SY5Y cells. The effects of erianin on cell viability, invasion and migration were determined by XTT, matrigel chamber and wound healing evaluation, respectively. Expression changes of miRNAs (microRNA) and apoptosis-related genes were evaluated by RT-PCR, and the apoptosis rate was supported by Annexin V evaluation. Erianin significantly decreased cell proliferation, invasion and migration. Erianin administration caused apoptosis by significantly increasing caspase-7, FADD (Fas-associated protein with death domain), BID (BH3 Interacting Domain Death Agonist) and DR5 (Death receptor 5) gene expressions. While the rate of total apoptotic cells was 45.35 ± 6.80% in SH-SY5Y cells treated with erianin, it was 0.133 ± 0.05% in the control group (p = 0.000). In addition, erianin administration significantly decreased the expressions of hsa-miR-155-5p (p = 0.014) and hsa-miR-223-3p (p = 0.004). Also, our study demonstrated for the first time the relationship between erianin and mi-RNAs in a cancer cell. Our study suggests that erianin may be a natural, safe and easily accessible drug candidate that can be used in the treatment of neuroblastoma.

CYD0281, a Bcl-2 BH4 domain antagonist, inhibits tumor angiogenesis and breast cancer tumor growth

BackgroundB-cell lymphoma 2 (Bcl-2) family proteins are key regulators of apoptosis, which possess four conserved Bcl-2 homologies (BH) domains. Among the BH domains, the BH3 domain is considered as a potent ‘death domain’ while the BH4 domain is required for anti-apoptotic activity. Bcl-2 can be converted to a pro-apoptotic molecule through the removal or mutation of the BH4 domain. Bcl-2 is considered as an inducer of angiogenesis, which can promote tumor vascular network formation and further afford nutrients and oxygen to promote tumor progression. However, whether disrupting the function of the BH4 domain to convert Bcl-2 into a pro-apoptotic molecule could make Bcl-2 possess the potential for anti-angiogenic therapy remains to be defined.MethodsCYD0281 was designed and synthesized according to the lead structure of BDA-366, and its function on inducing a conformational change of Bcl-2 was further evaluated via immunoprecipitation (IP) and immunofluorescence (IF) assays. Moreover, the function of CYD0281 on apoptosis of endothelial cells was analyzed via cell viability, flow cytometry, and western blotting assays. Additionally, the role of CYD0281 on angiogenesis in vitro was determined via endothelial cell migration and tube formation assays and rat aortic ring assay. Chick embryo chorioallantoic membrane (CAM) and yolk sac membrane (YSM) models, breast cancer cell xenograft tumor on CAM and in mouse models as well as the Matrigel plug angiogenesis assay were used to explore the effects of CYD0281 on angiogenesis in vivo.ResultsWe identified a novel potent small-molecule Bcl-2-BH4 domain antagonist, CYD0281, which exhibited significant anti-angiogenic effects both in vitro and in vivo, and further inhibited breast cancer tumor growth. CYD0281 was found to induce conformational changes in Bcl-2 through the exposure of the BH3 domain and convert Bcl-2 from an anti-apoptotic molecule into a cell death inducer, thereby resulting in the apoptosis of vascular endothelial cells.ConclusionsThis study has revealed CYD0281 as a novel Bcl-2-BH4 antagonist that induces conformational changes of Bcl-2 to convert to a pro-apoptotic molecule. Our findings indicate that CYD0281 plays a crucial role in anti-angiogenesis and may be further developed as a potential anti-tumor drug candidate for breast cancer. This work also provides a potential anti-angiogenic strategy for breast cancer treatment.

Tryptophan alleviates intestinal damage through regulating necroptosis and TLR4/NOD signaling pathways in pigs after lipopolysaccharide challenge

This study aimed to test the hypothesis that necroptosis, toll-like receptor 4 (TLR4)/nucleotide-binding oligomerization domain (NOD) signaling pathway in the jejunum of lipopolysaccharide (LPS)-challenged piglets are involved in the alleviation of intestinal injury and inflammation by tryptophan supplementation. Tryptophan supplementation has improved intestinal morphology. Also, tryptophan has been found to increase the mRNA and protein expression of tight junction proteins and decrease the expression of pro-inflammatory cytokines. Dietary tryptophan decreased the mRNA expression of heat shock protein 70, TLR4, NOD1, NOD2, myeloid differentiation primary response gene 88, interleukin 1 receptor-associated kinase 1, TNF receptor-associated factor 6, receptor-interacting serine/threonine-protein kinase 2-like, nuclear factor-kappaB transcription factor P65 in the jejunum of piglets. Tryptophan alleviated LPS-induced necroptosis and decreased the mRNA expression of mixed lineage kinase domain-like, receptor-interacting serine/threonine kinase 1, receptor-interacting serine/threonine-protein kinase 3-like, Fas (TNFRSF6)-associated via death domain, PGAM family member 5. Collectively, our results suggest that tryptophan supplementation helps in the attenuation of intestinal injury and inflammation by alleviating necroptosis and TLR4/NOD in lipopolysaccharide-challenged pigs.

The adapter protein FADD provides an alternate pathway for entry into the cell cycle by regulating APC/C-Cdh1 E3 ubiquitin ligase activity

The E3 ubiquitin ligase APC/C-Cdh1 maintains the G0/G1 state, and its inactivation is required for cell cycle entry. We reveal a novel role for Fas-associated protein with death domain (FADD) in the cell cycle through its function as an inhibitor of APC/C-Cdh1. Using real-time, single-cell imaging of live cells combined with biochemical analysis, we demonstrate that APC/C-Cdh1 hyperactivity in FADD- deficient cells leads to a G1 arrest despite persistent mitogenic signaling through oncogenic EGFR/KRAS. We further show that FADDWT interacts with Cdh1, while a mutant lacking a consensus KEN-box motif (FADDKEN) fails to interact with Cdh1 and results in a G1 arrest due to it its inability to inhibit APC/C-Cdh1. Additionally, enhanced expression of FADDWT but not FADDKEN, in cells arrested in G1 upon CDK4/6 inhibition, leads to APC/C-Cdh1 inactivation and entry into the cell cycle in the absence of retinoblastoma protein (Rb)- phosphorylation. FADD's function in the cell cycle requires its phosphorylation by CK1α at Ser-194 which promotes its nuclear translocation. Overall, FADD provides a CDK4/6-Rb-E2F independent "bypass" mechanism for cell cycle entry and thus a therapeutic opportunity for CDK4/6 inhibitor resistance.

Connexin 43 dephosphorylation at serine 282 induces spontaneous arrhythmia and increases susceptibility to ischemia/reperfusion injury

BackgroundConnexin 43 (Cx43), the predominant gap junction protein in hearts, is modified by specific (de)phosphorylation events under physiological and pathological states to affect myocardium function and structure. Previously we found that deficiency in Cx43 S282 phosphorylation could impair intercellular communication and contribute to cardiomyocyte apoptosis by activating p38 mitogen-activated protein kinase (p38 MAPK)/factor-associated suicide (Fas)/Fas-associating protein with a novel death domain (FADD) pathway, which is involved in myocardium injury in ischemia/reperfusion (I/R) heart. In addition, mutant at Cx43 S282 substituted with alanine heterozygous mice (S282A+/−) exhibited different degrees of ventricular arrhythmias and only some underwent myocardium apoptosis. In this study, we aimed to investigate the role of Cx43 pS282 in different cardiac pathological phenotypes. MethodsWe examined cardiac function, structure, and relevant protein expression in S282A+/− mice (aged 2, 10 and 30 weeks) by electrocardiograph, echocardiography, histological staining, and co-immunoprecipitation followed by Western blot. Intraperitoneal isoprenaline injection and I/R surgery were applied in S282A+/− mice as external stimulus. 2,3,5-triphenyltetrazolium chloride staining was used for myocardium infarction evaluation. ResultsAdult S282A+/− mice (aged 10 and 30 weeks) still exhibited spontaneous arrhythmia. Unlike neonatal stage (aged around 2 weeks), no apoptosis-related manifestations and the activation of p38 MAPK-Fas-FADD apoptotic pathway were observed in adult S282A+/− hearts. S282A+/− neonatal mice with cardiomyocytes apoptosis exhibited more than 60% dephosphorylation at Cx43 S282 than WT mice, while less than 40% S282 dephosphorylation were found in adult S282A+/− mice. In addition, although S282A+/− mice displayed normal cardiac function, they were highly susceptible to isoproterenol-induced ECG alternans and prone to cardiac injury and deaths upon I/R attack. ConclusionsThese results reinforce that Cx43 S282 dephosphorylation acts as a susceptibility factor in regulating cardiomyocyte survival and cardiac electrical homeostasis in basal conditions and contributes to myocardium injury in the setting of I/R. Cx43 S282 phosphorylation was competent to induce spontaneous arrhythmias, cardiomyocyte apoptosis and deaths based on the degree of S282 dephosphorylation.

Novel NFkB Variant V213E: Phenotypic Description & Molecular Modeling

Abstract BACKGROUND: Mutations of NFkB1 are a known cause of inborn errors of immunity resulting in immunodeficiency due to the role of NFkB1 as a transcriptional regulator of immunomodulating proteins. We present a patient with a diagnosis of common variable immunodeficiency (CVID), cytopenias with massive splenomegaly, and nodular regenerative hyperplasia of the liver. Our proband, a 22-year-old male, presents with a clinical picture of immunodeficiency, and has a maternal cousin with a similar gene mutation but differing phenotype. Next generation panel sequencing (NGPS) was utilized to discover a novel, single point mutation variant in NFkB1, V213E, that is present in our proband. METHODS: The novel single-point mutation V213E in the NFkB gene was modeled in-silico using the I-TASSER homology modeling software. Molecular dynamics simulations of both the wild type and variant mutant form fitted to accommodate the novel mutation were conducted and analyzed to assess for possible pathogenicity of the novel mutation. RESULTS: This variant replaces Valine with Glutamic Acid at position 213 in the transcription regulator’s sequence. We perform molecular modeling to analyze the proteomic impact and difference in molecular dynamics (MDS) which depicted suppressed dynamics in the ankyrin region and death domain of the protein complex. Analysis of the proband’s family showed no significant family history except for the maternal cousin with a mutation in NFkB1. CONCLUSION: This case both highlights the heterogeneity in phenotype of NFkB1 pathogenic variants as shown in this patient, full penetrance of the variant mutation, and suggests the pathogenicity of a new variant through protein modeling techniques and molecular dynamic simulations.

MAP kinase activating death domain deficiency is a novel cause ofimpaired 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.

SWATH proteomics analysis of placental tissue with intrahepatic cholestasis of pregnancy

IntroductionIntrahepatic cholestasis of pregnancy (ICP) usually occurs in the second and third trimesters. The disease's etiology and diagnostic criteria are currently unknown. Based on a sequence window to obtain all theoretical fragment ions (SWATH) proteomic approach, this study sought to identify potential proteins in placental tissue that may be involved in the pathogenesis of ICP and adverse fetal pregnancy outcomes. MethodsThe postpartum placental tissue of pregnant women with ICP were chosen as the case group (ICP group) (subdivided into mild ICP group (MICP group) and severe ICP group (SICP group)), and healthy pregnant women were chosen as the control group (CTR). The hematoxylin-eosin (HE) staining was used to observe the histologic changes of placenta. The SWATH analysis combined with liquid chromatography-tandem mass spectrometry (LC-MS) was used to screen the differentially expressed proteins (DEPs) in ICP and CTR groups, and bioinformatics analysis was used to find out the biological process of these differential proteins. ResultsProteomic studies showed there were 126 DEPs from pregnant women with ICP and healthy pregnant women. Most of the identified proteins were functionally related to humoral immune response, cell response to lipopolysaccharide, antioxidant activity and heme metabolism. A subsequent examination of placentas from patients with mild and severe ICP revealed 48 proteins that were differentially expressed. Through death domain receptors and fibrinogen complexes, these DEPs primarily regulate extrinsic apoptotic signaling pathways, blood coagulation, and fibrin clot formation. The differential expressions of HBD, HPX, PDE3A, and PRG4 were down-regulated by Western blot analysis, which was consistent with proteomics. DiscussionThis preliminary study helps us to understand the changes in the placental proteome of ICP patients, and provides new insights into the pathophysiology of ICP.

Vitamin D deficiency triggers intrinsic apoptosis by impairing SPP1-dependent antiapoptotic signaling in chronic hematogenous osteomyelitis

Chronic hematogenous osteomyelitis (CHOM) is a common bone disease characterized by the development of sequestra after bacterial infection. Emerging evidence has shown that vitamin D (VD) deficiency raises the risk of osteomyelitis, but the underlying mechanisms remain obscure. Here, we establish a CHOM model in VD diet-deficient mice by intravenous inoculation of Staphylococcus aureus. Whole-genome microarray analyses using osteoblast cells isolated from sequestra reveal significant downregulation of SPP1 (secreted phosphoprotein 1). Molecular basis investigations show that VD sufficiency activates the VDR/RXR (VD receptor/retinoid X receptor) heterodimer to recruit NCOA1 (nuclear receptor coactivator 1) and transactivate SPP1 in healthy osteoblast cells. Secreted SPP1 binds to the cell surface molecule CD40 to activate serine/threonine-protein kinase Akt1, which then phosphorylates forkhead box O3a (FOXO3a), blocking FOXO3a-mediated transcription. By contrast, VD deficiency impairs the NCOA1-VDR/RXR-mediated overexpression of SPP1, leading to the inactivation of Akt1 and the accumulation of FOXO3a. FOXO3a then upregulates the expression of the apoptotic genes BAX (Bcl2-associated X-protein), BID (BH3 interacting death domain), and BIM (Bcl2-interacting mediator of cell death), to induce apoptosis. Administration of the NCOA1 inhibitor gossypol to the CHOM mice also promotes the occurrence of sequestra. VD supplementation can reactivate the SPP1-dependent antiapoptotic signaling and improve the outcomes of CHOM. Collectively, our data reveal that VD deficiency promotes bone destruction in CHOM by the removal of SPP1-dependent antiapoptotic signaling.

Inhibition of TRADD ameliorates chondrocyte necroptosis and osteoarthritis by blocking RIPK1-TAK1 pathway and restoring autophagy

Osteoarthritis (OA) is an age-related disease characterized by cartilage degeneration. TNFR1-associated death domain protein (TRADD) is a key upstream molecule of TNF-α signals but its role in OA pathogenesis is unknown. This study aimed to verify that whether inhibition of TRADD could protect against chondrocyte necroptosis and OA, and further elucidate the underlying mechanism. We demonstrated that TNF-α-related OA-like phenotypes including inflammation response, extracellular matrix degradation, apoptosis, and necroptosis in chondrocytes were inhibited by TRADD deficiency. Furthermore, TRADD interacted with TRAF2 and knockdown of TRADD suppressed the activation of RIPK1-TAK1-NF-κB signals and restored impaired autophagy. ICCB-19, the selective inhibitor of TRADD, also attenuated necroptosis in chondrocytes. Mechanismly, ICCB-19 blocked the phosphorylation of TAK1-NF-κB signals and restored impaired autophagy, whereas inhibiting autophagic process with 3-Methyladenine compromised these effects of ICCB-19. The in vivo study showed that the intra-articular injection of ICCB-19 rescued the expression of collagen alpha-1(II) chain and LC3, and mitigated the cartilage degeneration of OA mice. This study demonstrates that TRADD mediates TNF-α-induced necroptosis and OA-like phenotypes of chondrocytes and suggests that ICCB-19 suppresses chondrocyte damage and cartilage degeneration by inhibiting TNF-α-TRADD-mediated signals and dysregulation of autophagy in chondrocytes. ICCB-19 may serve as an important option for OA therapy.

Ginsenoside Rb1 promotes the activation of PPARα pathway via inhibiting FADD to ameliorate heart failure

PurposeGinsenoside Rb1 (GRb1), a dammarane-type triterpene saponin compound mainly distributed in ginseng (Panax ginseng), has been demonstrated to ameliorate cardiovascular diseases. However, it remains unclear whether GRb1 alleviates heart failure (HF) by maintaining cardiac energy metabolism balance. Therefore, this work aimed to investigate the cardiac benefits of GRb1 against cardiac energy deficit and explore its mechanism of action. Methods and resultsIsoproterenol (ISO) induced HF Sprague–Dawley rats were administrated with GRb1 or fenofibrate for 6 weeks. ISO-induced primary neonatal rat cardiomyocytes (NRCMs) were used as the in vitro model. In vivo, GRb1 significantly improved the structural and metabolic disorder, as demonstrated by the restoration of cardiac function, inhibition of cardiac hypertrophy and fibrosis, and increased adenosine triphosphate (ATP) generation. In vitro, GRb1 effectively protected mitochondrial function and scavenged excessive reactive oxygen species. Moreover, in ISO-induced NRCMs, GRb1 significantly inhibited the abnormal upregulation of Fas-associated death domain (FADD), promoted transcriptional activation of peroxisome proliferator-activated receptor-alpha (PPARα), improved the aberrant expression of cardiac energy metabolism-related enzymes and cardiac fatty acid oxidation, and subsequently increased the synthesis of ATP. Noticeably, GRb1 could inhibit the increased binding between FADD and PPARα, which contributed to the activation of PPARα. Furthermore, GRb1 strengthened the thermal stabilization of FADD and might bind to FADD directly. ConclusionsCollectively, it’s part of the in-depth mechanism of GRb1’s cardio-protection that GRb1 could directly bind to FADD and counteract its negative role in the transcription of PPARα thus ameliorating cardiac energy derangement and HF.

SODD Promotes Lung Cancer Tumorigenesis by Activating the PDK1/AKT and RAF/MEK/ERK Signaling.

The Bcl2-associated athanogene4 (BAG4/SODD) protein could be identified as a tumor marker for several malignancies and plays a major role in the occurrence, development, and drug resistance of tumors. However, the role of Silencer of death domains (SODD) in lung carcinogenesis is still elusive. To illuminate the effect of SODD on the proliferation, migration, invasion, and apoptosis of lung cancer cells and tumor growth in vivo and explore the corresponding mechanism. The expression of SODD in tumor and normal tissues was determined and compared via western blot. SODD gene knockout lung cancer cells (H1299 cells) were established through a CRISPR/Cas9 gene deleting system, and a transient SODD overexpression of H1299 cells was also constructed. Then, cell proliferation and invasion were assessed through colony formation and cell counting kit-8 assays, transwell migration assays, and wound healing assays. Cell drug sensitivity is also analyzed by Cell Counting Kit-8 assay. The flow cytometer was used to perform cell circle and apoptosis analysis. The interaction of SODD and RAF-1 was confirmed by co-immunoprecipitation, and the phosphorylated level of Phosphatidylinositol 3-kinase (PI3K), Serine/threonine-protein kinase (AKT), Rapidly accelerated fibrosarcoma (RAF)-1,and extracellular signal regulated kinase (ERK) in cells was examined by western blot to evaluate the activation of PI3K/PDK1/AKT and RAF/MEK/ERK pathways. In vivo, Xenograft tumor assay of SODD knockout H1299 cells was used to evaluate further the role of SODD on the proliferation of H1299 cells. SODD binds to RAF-1 and is over-expressed in lung tissues, and promotes the proliferation, migration, invasion, and drug sensitivity of H1299 cells. The reduced cells in the S phase and increased cells arrested in the G2/M phase were found in SODD knockout H1299 cells, and more cells got apoptosis. The expression of 3-phosphoinositide-dependent protein kinase 1(PDK1) protein in SODD knockout H1299 cells decreases distinctively, and the phosphorylated level of AKT, RAF-1, and ERK-1 kinase in SODD knockout H1299 cells is also less than that in normal H1299 cells. In contrast, SODD overexpression significantly increases the phosphorylation of AKT. In vivo, SODD promotes the tumorigenicity of H1299 cells in nude mice. SODD is overexpressed in lung tissues and plays a considerable role in the development and progression of lung cancer by regulating the PI3K/PDK1/AKT and RAF/MEK/ERK pathways.