SMPD4 regulates mitotic nuclear envelope dynamics and its loss causes microcephaly and diabetes.

Biallelic loss-of-function variants in SMPD4 cause a rare and severe neurodevelopmental disorder with progressive congenital microcephaly and early death. SMPD4 encodes a sphingomyelinase that hydrolyses sphingomyelin into ceramide at neutral pH and can thereby affect membrane lipid homeostasis. SMPD4 localizes to the membranes of the endoplasmic reticulum and nuclear envelope and interacts with nuclear pore complexes (NPC). We refine the clinical phenotype of loss-of-function SMPD4 variants by describing five individuals from three unrelated families with longitudinal data due to prolonged survival. All individuals surviving beyond infancy developed insulin-dependent diabetes, besides presenting with a severe neurodevelopmental disorder and microcephaly, making diabetes one of the most frequent age-dependent non-cerebral abnormalities. We studied the function of SMPD4 at the cellular and organ levels. Knock-down of SMPD4 in human neural stem cells causes reduced proliferation rates and prolonged mitosis. Moreover, SMPD4 depletion results in abnormal nuclear envelope breakdown and reassembly during mitosis and decreased post-mitotic NPC insertion. Fibroblasts from affected individuals show deficient SMPD4-specific neutral sphingomyelinase activity, without changing (sub)cellular lipidome fractions, which suggests a local function of SMPD4 on the nuclear envelope. In embryonic mouse brain, knockdown of Smpd4 impairs cortical progenitor proliferation and induces premature differentiation by altering the balance between neurogenic and proliferative progenitor cell divisions. We hypothesize that, in individuals with SMPD4-related disease, nuclear envelope bending, which is needed to insert NPCs in the nuclear envelope, is impaired in the absence of SMPD4 and interferes with cerebral corticogenesis and survival of pancreatic beta cells.

Acid sphingomyelinase is a key regulator of cytotoxic granule secretion by primary T lymphocytes.

Granule-mediated cytotoxicity is the main effector mechanism of cytotoxic CD8+ T cells. We report that CD8+ T cells from acid sphingomyelinase (ASMase)-deficient (ASMase-KO) mice are defective in exocytosis of cytolytic effector molecules; this defect resulted in attenuated cytotoxic activity of ASMase-KO CD8+ T cells and delayed elimination of lymphocytic choriomeningitis virus from ASMase-KO mice. Cytolytic granules of ASMase-KO and wild-type CD8+ T cells were equally loaded with granzymes and perforin, and correctly directed to the immunological synapse. In wild-type CD8+ T cells, secretory granules underwent shrinkage by 82% after fusion with the plasma membrane. In ASMase-KO CD8+ T cells, the contraction of secretory granules was markedly impaired. Thus, ASMase is required for contraction of secretory granules and expulsion of cytotoxic effector molecules.

BID-dependent release of mitochondrial SMAC dampens XIAP-mediated immunity against Shigella.

The X-linked inhibitor of apoptosis protein (XIAP) is a potent caspase inhibitor, best known for its anti-apoptotic function in cancer. During apoptosis, XIAP is antagonized by SMAC, which is released from the mitochondria upon caspase-mediated activation of BID. Recent studies suggest that XIAP is involved in immune signaling. Here, we explore XIAP as an important mediator of an immune response against the enteroinvasive bacterium Shigella flexneri, both in vitro and in vivo. Our data demonstrate for the first time that Shigella evades the XIAP-mediated immune response by inducing the BID-dependent release of SMAC from the mitochondria. Unlike apoptotic stimuli, Shigella activates the calpain-dependent cleavage of BID to trigger the release of SMAC, which antagonizes the inflammatory action of XIAP without inducing apoptosis. Our results demonstrate how the cellular death machinery can be subverted by an invasive pathogen to ensure bacterial colonization.

Riboflavin (vitamin B2 ) deficiency impairs NADPH oxidase 2 (Nox2) priming and defense against Listeria monocytogenes.

Riboflavin, also known as vitamin B2 , is converted by riboflavin kinase (RFK) into flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), which are essential cofactors of dehydrogenases, reductases, and oxidases including the phagocytic NADPH oxidase 2 (Nox2). Riboflavin deficiency is common in young adults and elderly individuals, who are at the coincidental risk for listeriosis. To address the impact of acute riboflavin deficiency on host defense against Listeria monocytogenes (L.m.), we generated conditional RFK knockout (KO) strains of mice. Phagocyte-specific RFK KO impaired the capability of phagocytes to control intracellular L.m., which corresponded to a greater susceptibility of mice to in vivo challenge with L.m. The oxidative burst of RFK-deficient phagocytes in response to L.m. infection was significantly reduced. Mechanistically, TNF-induced priming of Nox2, which is needed for oxidative burst, was defective in RFK-deficient phagocytes. Lack of riboflavin in wild-type macrophages for only 6 h shut down TNF-induced, RFK-mediated de novo FMN/FAD generation, which was accompanied by diminished ROS production and impaired anti-listerial activity. Vice versa, ROS production by riboflavin-deprived macrophages was rapidly restored by riboflavin supplementation. Our results suggest that acute riboflavin deficiency immediately impairs priming of Nox2, which is of crucial relevance for an effective phagocytic immune response in vivo.

Riboflavin kinase couples TNF receptor 1 to NADPH oxidase.

Reactive oxygen species (ROS) produced by NADPH oxidase function as defence and signalling molecules related to innate immunity and various cellular responses. The activation of NADPH oxidase in response to plasma membrane receptor activation depends on the phosphorylation of cytoplasmic oxidase subunits, their translocation to membranes and the assembly of all NADPH oxidase components. Tumour necrosis factor (TNF) is a prominent stimulus of ROS production, but the molecular mechanisms by which TNF activates NADPH oxidase are poorly understood. Here we identify riboflavin kinase (RFK, formerly known as flavokinase) as a previously unrecognized TNF-receptor-1 (TNFR1)-binding protein that physically and functionally couples TNFR1 to NADPH oxidase. In mouse and human cells, RFK binds to both the TNFR1-death domain and to p22(phox), the common subunit of NADPH oxidase isoforms. RFK-mediated bridging of TNFR1 and p22(phox) is a prerequisite for TNF-induced but not for Toll-like-receptor-induced ROS production. Exogenous flavin mononucleotide or FAD was able to substitute fully for TNF stimulation of NADPH oxidase in RFK-deficient cells. RFK is rate-limiting in the synthesis of FAD, an essential prosthetic group of NADPH oxidase. The results suggest that TNF, through the activation of RFK, enhances the incorporation of FAD in NADPH oxidase enzymes, a critical step for the assembly and activation of NADPH oxidase.

Not interferon, but interleukin-6 controls early gene expression in hepatitis B virus infection.

UNLABELLED: With about 350 million virus carriers, hepatitis B virus (HBV) infection remains a major health problem. HBV is a noncytopathic virus causing persistent infection, but it is still unknown whether host recognition of HBV may activate an innate immune response. We describe that upon infection of primary human liver cells, HBV is recognized by nonparenchymal cells of the liver, mainly by liver macrophages (Kupffer cells), although they are not infected. Within 3 hours, this recognition leads to the activation of nuclear factor kappa B (NF-kappaB) and subsequently to the release of interleukin-6 (IL-6) and other proinflammatory cytokines (IL-8, TNF-alpha, IL-1beta), but does not induce an interferon response. The activation of proinflammatory cytokines, however, is transient, and even inhibits responsiveness toward a subsequent challenge. IL-6 released by Kupffer cells after activation of NF-kappaB controls HBV gene expression and replication in hepatocytes at the level of transcription shortly after infection. Upon binding to its receptor complex, IL-6 activates the mitogen-activated protein kinases exogenous signal-regulated kinase 1/2, and c-jun N-terminal kinase, which inhibit expression of hepatocyte nuclear factor (HNF) 1alpha and HNF 4alpha, two transcription factors essential for HBV gene expression and replication. CONCLUSION: Our results demonstrate recognition of HBV patterns by nonparenchymal liver cells, which results in IL-6-mediated control of HBV infection at the transcriptional level. Thus, IL-6 ensures early control of the virus, limiting activation of the adaptive immune response and preventing death of the HBV-infected hepatocyte. This pattern recognition may be essential for a virus, which infects a new host with only a few virions. Our data also indicate that therapeutic neutralization of IL-6 for treatment of certain diseases may represent a risk if the patient is HBV-infected.

T cells redirected against hepatitis B virus surface proteins eliminate infected hepatocytes.

BACKGROUND & AIMS: The final goal in hepatitis B therapy is eradication of the hepatitis B virus (HBV) replication template, the so-called covalently closed circular DNA (cccDNA). Current antiviral treatment of chronic hepatitis B depends on interferon alpha or nucleoside analogues inhibiting the viral reverse transcriptase. Despite treatment, cccDNA mostly persists in the host cell nucleus, continues to produce hepatitis B surface antigen (HBsAg), and causes relapsing disease. We therefore aimed at eliminating persistently infected hepatocytes carrying HBV cccDNA by redirecting cytolytic T cells toward HBsAg-producing cells. METHODS: We designed chimeric T-cell receptors directed against HBV surface proteins present on HBV-infected cells and used them to graft primary human T cells with antibody-like specificity. The receptors were composed of a single chain antibody fragment directed against HBV S or L protein fused to intracellular signalling domains of CD3xi and the costimulatory CD28 molecule. RESULTS: Our results show that these chimeric receptors, when retrovirally delivered and expressed on the cell surface, enable primary human T cells to recognize HBsAg-positive hepatocytes, release interferon gamma and interleukin 2, and, most importantly, lyse HBV replicating cells. When coincubated with HBV-infected primary human hepatocytes, these engineered, antigen-specific T cells selectively eliminated HBV-infected and thus cccDNA-positive target cells. CONCLUSIONS: Elimination of HBV cccDNA-positive hepatocytes following antiviral therapy is a major therapeutic goal in chronic hepatitis B, and adoptive transfer of grafted T cells provides a promising novel therapeutic approach. However, T-cell therapy may also cause liver damage and therefore needs further preclinical evaluation.

Emergence of AnnexinVpos CD31neg CD42blow/neg extracellular vesicles in plasma of humans at extreme altitude.

BACKGROUND: Hypobaric hypoxia has been reported to cause endothelial cell and platelet dysfunction implicated in the formation of microvascular lesions, and in its extremes may contribute to vascular leakage in high altitude pulmonary edema or blood brain barrier disruption leading to cerebral micro-hemorrhage (MH). Platelet function in the development of microvascular lesions remained ill defined, and is still incompletely understood. In this study platelet- and endothelial cell-derived extracellular vesicles (PEV and EEV, respectively) and cell adhesion molecules were characterized in plasma samples of members of a high altitude expedition to delineate the contribution of platelets and endothelial cells to hypobaric hypoxia-induced vascular dysfunction. METHODS AND FINDINGS: In this observational study, platelet and endothelial cell-derived extracellular vesicles were analysed by flow-cytometry in plasma samples from 39 mountaineers participating in a medical research climbing expedition to Himlung Himal, Nepal, 7,050m asl. Megakaryocyte/platelet-derived AnnexinVpos, PECAM-1 (CD31) and glycoprotein-1b (GP1b, CD42b) positive extracellular vesicles (PEV) constituted the predominant fraction of EV in plasma samples up to 6,050m asl. Exposure to an altitude of 7,050m led to a marked decline of CD31pos CD42neg EEV as well as of CD31pos CD42bpos PEV at the same time giving rise to a quantitatively prevailing CD31neg CD42blow/neg subpopulation of AnnexinVpos EV. An almost hundredfold increase in the numbers of this previously unrecognized population of CD31neg CD42blow/neg EV was observed in all participants reaching 7,050m asl. CONCLUSIONS: The emergence of CD31neg CD42blow/neg EV was observed in all participants and thus represents an early hypoxic marker at extreme altitude. Since CD31 and CD42b are required for platelet-endothelial cell interactions, these hypobaric hypoxia-dependent quantitative and phenotypic changes of AnnexinVpos EV subpopulations may serve as early and sensitive indicators of compromised vascular homeostasis.

Mitochondrial reactive oxygen species enable proinflammatory signaling through disulfide linkage of NEMO.

A major function of macrophages during infection is initiation of the proinflammatory response, leading to the secretion of cytokines that help to orchestrate the immune response. Here, we identify reactive oxygen species (ROS) as crucial mediators of proinflammatory signaling leading to cytokine secretion in Listeria monocytogenes-infected macrophages. ROS produced by NADPH oxidases (Noxes), such as Nox2, are key components of the macrophage response to invading pathogens; however, our data show that the ROS that mediated proinflammatory signaling were produced by mitochondria (mtROS). We identified the inhibitor of κB (IκB) kinase (IKK) complex regulatory subunit NEMO [nuclear factor κB (NF-κB) essential modulator] as a target for mtROS. Specifically, mtROS induced intermolecular covalent linkage of NEMO through disulfide bonds formed by Cys54 and Cys347, which was essential for activation of the IKK complex and subsequent signaling through the extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) and NF-κB pathways that eventually led to the secretion of proinflammatory cytokines. We thus identify mtROS-dependent disulfide linkage of NEMO as an essential regulatory step of the proinflammatory response of macrophages to bacterial infection.

The ß2 Integrin Mac-1 Induces Protective LC3-Associated Phagocytosis of Listeria monocytogenes.

The intracellular pathogen Listeria monocytogenes (L.m.) is targeted by the autophagic machinery, but the molecular mechanisms involved and consequences for anti-listerial immunity remain enigmatic. Here, we demonstrate that L.m. infection of macrophages in vivo exclusively evokes LC3-associated phagocytosis (LAP), but not canonical autophagy, and that targeting of L.m. by LAP is required for anti-listerial immunity. The pathway leading to LAP induction in response to L.m. infection emanates from the ß2 integrin Mac-1 (CR3, integrin αMß2), a receptor recognizing diverse microbial ligands. Interaction of L.m. with Mac-1 induces acid sphingomyelinase-mediated changes in membrane lipid composition that facilitate assembly and activation of the phagocyte NAPDH oxidase Nox2. Nox2-derived reactive oxygen species then trigger LC3 recruitment to L.m.-containing phagosomes by LAP. By promoting fusion of L.m.-containing phagosomes with lysosomes, LAP increases exposure of L.m. to bactericidal acid hydrolases, thereby enhancing anti-listerial activity of macrophages and immunity of mice.

PtdIns(4,5)P-restricted plasma membrane localization of FAN is involved in TNF-induced actin reorganization.

The WD-repeat protein factor associated with nSMase activity (FAN) is a member of the family of TNF receptor adaptor proteins that are coupled to specific signaling cascades. However, the precise functional involvement of FAN in specific cellular TNF responses remain unclear. Here, we report the involvement of FAN in TNF-induced actin reorganization and filopodia formation mediated by activation of Cdc42. The pleckstrin-homology (PH) domain of FAN specifically binds to phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P), which targets FAN to the plasma membrane. Site-specific mutagenesis revealed that the ability of FAN to mediate filopodia formation was blunted either by the destruction of the PtdIns(4,5)P binding motif, or by the disruption of intramolecular interactions between the PH domain and the adjacent beige and Chediak-Higashi (BEACH) domain. Furthermore, FAN was shown to interact with the actin cytoskeleton in TNF-stimulated cells via direct filamentous actin (F-actin) binding. The results of this study suggest that PH-mediated plasma membrane targeting of FAN is critically involved in TNF-induced Cdc42 activation and cytoskeleton reorganization.

NF-kappaB-independent down-regulation of XIAP by bortezomib sensitizes HL B cells against cytotoxic drugs.

The proteasome inhibitor bortezomib has been shown to possess promising antitumor activity and significant efficacy against a variety of malignancies. Different studies demonstrated that bortezomib breaks the chemoresistance in different tumor cells basically by altering nuclear factor-kappaB (NF-kappaB) activity. NF-kappaB has been shown to be constitutively active in most primary Hodgkin-Reed-Sternberg (H-RS) cells in lymph node sections and in Hodgkin lymphoma (HL) cell lines and was suggested to be a central molecular switch in apoptosis resistance in HL. Here we report a bimodal effect of bortezomib in HL cells. Whereas high-dose bortezomib induced direct cytotoxicity that correlated with decreased NF-kappaB activity, low-dose bortezomib sensitized HL cells against a variety of cytotoxic drugs without altering NF-kappaB action. Strikingly, bortezomib induced marked XIAP down-regulation at the posttranslational level that was independent of the NF-kappaB status. Similarly, RNA interference (RNAi)-mediated XIAP down-regulation generated susceptibility to cytostatic agents. The results identify XIAP as an NF-kappaB-independent target of bortezomib action that controls the chemoresistant phenotype of HL cells.

Novel tumor necrosis factor-responsive mammalian neutral sphingomyelinase-3 is a C-tail-anchored protein.

Two genes encoding neutral sphingomyelinases-1 and -2 (sphingomyelin phosphodiesterases-2 and -3) have been recently identified that hydrolyze sphingomyelin to phosphorylcholine and ceramide. Data bank searches using a peptide sequence derived from a previously purified bovine neutral sphingomyelinase (nSMase) allowed us to identify a cDNA encoding a novel human sphingomyelinase, nSMase3, that shows only a little homology to nSMase1 and -2. nSMase3 was biochemically characterized by overexpression in a yeast strain, JK9-3ddeltaIsc1p, lacking endogenous SMase activity. Similar to nSMase2, nSMase3 is Mg2+-dependent and shows optimal activity at pH 7, which is enhanced in the presence of phosphatidylserine and inhibited by scyphostatin. nSMase3 is ubiquitously expressed as a 4.6-kb mRNA species. nSMase3 lacks an N-terminal signal peptide, yet contains a 23-amino-acid transmembrane domain close to the C terminus, which is indicative for the family of C-tail-anchored integral membrane proteins. Cellular localization studies with hemagglutinin-tagged nSMase3 demonstrated colocalization with markers of the endoplasmic reticulum as well as with Golgi markers. Tumor necrosis factor stimulates rapid activation of nSMase3 in MCF7 cells with peak activity at 1.5 min, which was impaired by expression of dominant negative FAN.

Targeting of HIV-1 Tat traffic and function by transduction-competent single chain antibodies.

Human immunodeficiency virus type 1-encoded Tat protein is a transactivating factor essentially required for viral replication. Tat binds specifically to the transactivation response RNA stem loop, which is formed at the 5' end of all viral transcripts. The TAR binding motif of Tat also contains a protein transduction domain, PTD that mediates not only nuclear localization of Tat but is also capable of transducing cargo across cellular membranes. In order to target a Tat antagonist directly to the TAR binding site in the nucleus, we engineered a chimeric protein consisting of the Tat-derived PTD fused to the anti-Tat single chain antibody scFvtat1 that binds intracellularly to Tat. Recombinant scFvtat1-PTD(TAT) fusion antibody retained both, anti-Tat specificity and PTD(TAT)-mediated transduction-competence leading to its nuclear accumulation within living cells. Incubation of Jurkat T cells with scFvtat1-PTD(TAT) suppressed Tat-dependent transcription of a HIV-1 reporter gene by >80%. Transfection of a scFvtat1-PTD(TAT) expression plasmid in HEK293 cells resulted in diffuse cytoplasmic and nuclear expression. ScFvtat1-PTD(TAT) did not inhibit HIV-1 Tat translocation to the nucleus, yet showed increased inhibition of 78%, indicating a nuclear site of scFvtat1-PTD(TAT) action. Strikingly, the PTD(TAT) alone showed 55% inhibition in the HIV-1 luciferase reporter assay, indicating competition with HIV-1 Tat binding to the TAR element. The results of this study suggest that Tat traffic can only marginally be affected by anti-Tat antibodies and that effective inhibition of Tat function requires both competition with HIV Tat for TAR binding mediated by PTD(TAT) and steric hindrance mediated by the scFvtat1 moiety.

Acid sphingomyelinase is indispensable for UV light-induced Bax conformational change at the mitochondrial membrane.

Ultraviolet light-induced apoptosis can be caused by DNA damage but also involves immediate-early cell death cascades characteristic of death receptor signaling. Here we show that the UV light-induced apoptotic signaling pathway is unique, targeting Bax activation at the mitochondrial membrane independent of caspase-8 or cathepsin D activity. Cells deficient in acid sphingomyelinase (ASMase) do not show UV light-induced Bax activation, cytochrome c release, or apoptosis. In ASMase-deficient cells, the apoptotic UV light response is restored by stable or transient expression of human ASMase. Bax conformational change in ASMase(-/-) cells is also caused by synthetic C(16)-ceramide acting on intact cells or isolated mitochondria. The results suggest that UV light-triggered ASMase activation is essentially required for Bax conformational change leading to mitochondrial release of pro-apoptotic factors like cytochrome c and Smac.

Crystal structure of the BEACH domain reveals an unusual fold and extensive association with a novel PH domain.

The BEACH domain is highly conserved in a large family of eukaryotic proteins, and is crucial for their functions in vesicle trafficking, membrane dynamics and receptor signaling. However, it does not share any sequence homology with other proteins. Here we report the crystal structure at 2.9 A resolution of the BEACH domain of human neurobeachin. It shows that the BEACH domain has a new and unusual polypeptide backbone fold, as the peptide segments in its core do not assume regular secondary structures. Unexpectedly, the structure also reveals that the BEACH domain is in extensive association with a novel, weakly conserved pleckstrin-homology (PH) domain. Consistent with the structural analysis, biochemical studies show that the PH and BEACH domains have strong interactions, suggesting they may function as a single unit. Functional studies in intact cells demonstrate the requirement of both the PH and the BEACH domains for activity. A prominent groove at the interface between the two domains may be used to recruit their binding partners.

Interaction with factor associated with neutral sphingomyelinase activation, a WD motif-containing protein, identifies receptor for activated C-kinase 1 as a novel component of the signaling pathways of the p55 TNF receptor.

Factor associated with neutral sphingomyelinase activation (FAN) represents a p55 TNFR (TNF-R55)-associated protein essential for the activation of neutral sphingomyelinase. By means of the yeast interaction trap system, we have identified the scaffolding protein receptor for activated C-kinase (RACK)1 as an interaction partner of FAN. Mapping studies in yeast revealed that RACK1 is recruited to the C-terminal WD-repeat region of FAN and binds to FAN through a domain located within WD repeats V to VII of RACK1. Our data indicate that binding of both proteins is not mediated by linear motifs but requires folding into a secondary structure, such as the multibladed propeller characteristic of WD-repeat proteins. The interaction of FAN and RACK1 was verified in vitro by glutathione S-transferase-based coprecipitation assays as well as in eukaryotic cells by coimmunoprecipitation experiments. Colocalization studies in transfected cells suggest that TNF-R55 forms a complex with FAN and that this complex recruits RACK1 to the plasma membrane. Furthermore, activation of N-SMase by TNF was strongly enhanced when RACK1, FAN, and a noncytotoxic TNF-R55 mutant were expressed concurrently, suggesting RACK1 as a modulator of N-SMase activation. Together, these findings implicate RACK1 as a novel component of the signaling pathways of TNF-R55.