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Most lncRNAs display species-specific expression patterns suggesting that animal models of cancer may only incompletely recapitulate the regulatory crosstalk between lncRNAs and oncogenic pathways in humans. Among these pathways, Sonic Hedgehog (SHH) signaling is aberrantly activated in several human cancer entities. We unravel that aberrant expression of the primate-specific lncRNA HedgeHog Interacting Protein-AntiSense 1 (HHIP-AS1) is a hallmark of SHH-driven tumors including medulloblastoma and atypical teratoid/rhabdoid tumors. HHIP-AS1 is actively transcribed from a bidirectional promoter shared with SHH regulator HHIP. Knockdown of HHIP-AS1 induces mitotic spindle deregulation impairing tumorigenicity in vitro and in vivo. Mechanistically, HHIP-AS1 binds directly to the mRNA of cytoplasmic dynein 1 intermediate chain 2 (DYNC1I2) and attenuates its degradation by hsa-miR-425-5p. We uncover that neither HHIP-AS1 nor the corresponding regulatory element in DYNC1I2 are evolutionary conserved in mice. Taken together, we discover an lncRNA-mediated mechanism that enables the pro-mitotic effects of SHH pathway activation in human tumors.

International audience; APO2L/TRAIL (TNF-related apoptosis-inducing ligand) induces death of tumor cells through two agonist receptors, TRAIL-R1 and TRAIL-R2. We demonstrate here that N-linked glycosylation (N-glyc) plays also an important regulatory role for TRAIL-R1-mediated and mouse TRAIL receptor (mTRAIL-R)-mediated apoptosis, but not for TRAIL-R2, which is devoid of N-glycans. Cells expressing N-glyc-defective mutants of TRAIL-R1 and mouse TRAIL-R were less sensitive to TRAIL than their wild-type counterparts. Defective apoptotic signaling by N-glyc-deficient TRAIL receptors was associated with lower TRAIL receptor aggregation and reduced DISC formation, but not with reduced TRAIL-binding affinity. Our results also indicate that TRAIL receptor N-glyc impacts immune evasion strategies. The cytomegalovirus (CMV) UL141 protein, which restricts cell-surface expression of human TRAIL death receptors, binds with significant higher affinity TRAIL-R1 lacking N-glyc, suggesting that this sugar modification may have evolved as a counterstrategy to prevent receptor inhibition by UL141. Altogether our findings demonstrate that N-glyc of TRAIL-R1 promotes TRAIL signaling and restricts virus-mediated inhibition.

The adipocyte plasma membrane-associated protein APMAP is expressed in the brain where it associates with γ-secretase, a protease responsible for the generation of the amyloid-β peptides (Aβ) implicated in the pathogenesis of Alzheimer’s disease (AD). In this study, behavioral investigations revealed spatial learning and memory deficiencies in our newly generated mouse line lacking the protein APMAP. In a mouse model of AD, the constitutive deletion of APMAP worsened the spatial memory phenotype and led to increased Aβ production and deposition into senile plaques. To investigate at the molecular level the neurobiological functions of APMAP (memory and Aβ formation) and a possible link with the pathological hallmarks of AD (memory impairment and Aβ pathology), we next developed a procedure for the high-grade purification of cellular APMAP protein complexes. The biochemical characterization of these complexes revealed a series of new APMAP interactomers. Among these, the heat shock protein HSPA1A and the cation-dependent mannose-6-phosphate receptor (CD-M6PR) negatively regulated APP processing and Aβ production, while clusterin, calnexin, arginase-1, PTGFRN and the cation-independent mannose-6-phosphate receptor (CI-M6PR/IGF2R) positively regulated APP and Aβ production. Several of the newly identified APMAP interactomers contribute to the autophagy-lysosome system, further supporting an emergent agreement that this pathway can modulate APP metabolism and Aβ generation. Importantly, we have also demonstrated increased alternative splicing of APMAP and lowered levels of the Aβ controllers HSPA1A and CD-M6PR in human brains from neuropathologically verified AD cases. Electronic supplementary material The online version of this article (10.1186/s40478-019-0660-3) contains supplementary material, which is available to authorized users.

Microbial pathogens commonly escape the human immune system by varying surface proteins. We investigated the mechanisms used for that purpose by Pneumocystis jirovecii . This uncultivable fungus is an obligate pulmonary pathogen that in immunocompromised individuals causes pneumonia, a major life-threatening infection. Long-read PacBio sequencing was used to assemble a core of subtelomeres of a single P. jirovecii strain from a bronchoalveolar lavage fluid specimen from a single patient. A total of 113 genes encoding surface proteins were identified, including 28 pseudogenes. These genes formed a subtelomeric gene superfamily, which included five families encoding adhesive glycosylphosphatidylinositol (GPI)-anchored glycoproteins and one family encoding excreted glycoproteins. Numerical analyses suggested that diversification of the glycoproteins relies on mosaic genes created by ectopic recombination and occurs only within each family. DNA motifs suggested that all genes are expressed independently, except those of the family encoding the most abundant surface glycoproteins, which are subject to mutually exclusive expression. PCR analyses showed that exchange of the expressed gene of the latter family occurs frequently, possibly favored by the location of the genes proximal to the telomere because this allows concomitant telomere exchange. Our observations suggest that (i) the P. jirovecii cell surface is made of a complex mixture of different surface proteins, with a majority of a single isoform of the most abundant glycoprotein, (ii) genetic mosaicism within each family ensures variation of the glycoproteins, and (iii) the strategy of the fungus consists of the continuous production of new subpopulations composed of cells that are antigenically different. IMPORTANCE Pneumocystis jirovecii is a fungus causing severe pneumonia in immunocompromised individuals. It is the second most frequent life-threatening invasive fungal infection. We have studied the mechanisms of antigenic variation used by this pathogen to escape the human immune system, a strategy commonly used by pathogenic microorganisms. Using a new DNA sequencing technology generating long reads, we could characterize the highly repetitive gene families encoding the proteins that are present on the cellular surface of this pest. These gene families are localized in the regions close to the ends of all chromosomes, the subtelomeres. Such chromosomal localization was found to favor genetic recombinations between members of each gene family and to allow diversification of these proteins continuously over time. This pathogen seems to use a strategy of antigenic variation consisting of the continuous production of new subpopulations composed of cells that are antigenically different. Such a strategy is unique among human pathogens.

Reactive oxygen species (ROS) participate in the pathogenesis of emphysema. Among ROS-producing enzymes, NOX NADPH oxidases are thought to be responsible for tissue injury associated with several lung pathologies. To determine whether NOX2 and/or NOX1 participate in the development of emphysema, their expression patterns were first studied by immunohistochemistry in the lungs of emphysematous patients. Subsequently, we investigated their contribution to elastase-induced emphysema using NOX2- and NOX1-deficient mice. In human lung, NOX2 was mainly detected in macrophages of control and emphysematous lungs, while NOX1 was expressed in alveolar epithelium and bronchial cells. We observed an elevated number of NOX2-positive cells in human emphysematous lungs, as well as increased NOX2 and NOX1 mRNA expression in mouse lungs following elastase exposure. Elastase-induced alveolar airspace enlargement and elastin degradation were prevented in NOX2-deficient mice, but not in NOX1-deficient mice. This protection was independent of inflammation and correlated with reduced ROS production. Concomitantly, an elevation of sirtuin 1 (SIRT1) level and a decrease of matrix metalloproteinase-9 (MMP-9) expression and activity were observed in alveolar macrophages and neutrophils. We addressed the specific role of macrophage-restricted functional NOX2 in elastase-induced lung emphysema using Ncf1 mutant mice and Ncf1 macrophage rescue mice (Ncf1 mutant mice with transgenic expression of Ncf1 only in CD68-positive mononuclear phagocytes; the MN mouse). Compared to WT mice, the lack of functional NOX2 led to decreased elastase-induced ROS production and protected against emphysema. In contrast, ROS production was restored specifically in macrophages from Ncf1 rescue mice and contributes to emphysema. Taken together, our results demonstrate that NOX2 is involved in the pathogenesis of human emphysema and macrophage-specific NOX2 participates in elastase-induced emphysema through the involvement of SIRT1/MMP-9 pathways in mice.

During infection, plasma membrane (PM) blebs protect host cells against bacterial pore-forming toxins (PFTs), but were also proposed to promote pathogen dissemination. However, the details and impact of blebbing regulation during infection remained unclear. Here, we identify the endoplasmic reticulum chaperone Gp96 as a novel regulator of PFT-induced blebbing. Gp96 interacts with non-muscle myosin heavy chain IIA (NMHCIIA) and controls its activity and remodelling, which is required for appropriate coordination of bleb formation and retraction. This mechanism involves NMHCIIA-Gp96 interaction and their recruitment to PM blebs and strongly resembles retraction of uropod-like structures from polarized migrating cells, a process that also promotes NMHCIIA-Gp96 association. Consistently, Gp96 and NMHCIIA not only protect the PM integrity from listeriolysin O (LLO) during infection by Listeria monocytogenes but also affect cytoskeletal organization and cell migration. Finally, we validate the association between Gp96 and NMHCIIA in vivo and show that Gp96 is required to protect hosts from LLO-dependent killing. We are grateful to M.T. Almeida and M. Martins who made preliminary observations on the Gp96-NMHCIIA interaction. We are thankful to P. Cossart from Institut Pasteur, Paris, France; O.V. Vieira from CEDOC, Nova Medical School Lisbon, Portugal; and G. Van der Goot from Ecole Polytechnique Federale de Lausanne, Switzerland, for the kind gift of purified LLO, SLO and aerolysin, respectively. We thank C. Leitao (AFCU), P. Sampaio (ALM) and R. Fernandes (HEMS) from IBMC facilities for technical assistance and J. Bessa and J. Marques for guidance in zebrafish experiments. This work was supported by national funds through FCT-Fundacao para a Ciencia e a Tecnologia/MEC-Ministerio da Educacao e Ciencia and co-funded by Fundo Europeu de Desenvolvimento Regional (FEDER) within the partnership agreement PT2020 related to the research unit number 4293 and through the Operational Competitiveness Programme (COMPETE) under the project "NORTE-07-0124-FEDER-000002-Host-Pathogen Interactions" co-funded by Programa Operacional Regional do Norte (ON.2-O Novo Norte), under the Quadro de Referencia Estrategico Nacional (QREN), through the FEDER and by FCT. It also received support from a Research Grant 2014 by European Society of Clinical Microbiology and Infectious Diseases (ESCMID) (to SS) and from PT2020 research project Infect-ERA/0001/2013 PROANTILIS. Work in the SM Laboratory is supported by a Wellcome Trust Research Career Development Fellowship (WT097411MA) and the Lister Institute of Preventive Medicine. FSM was funded through an EMBO Long-term Postdoctoral Fellowship (EMBO ALTF 864-2012) and a FCT Post-Doctoral Fellowship (SFRH/BPD/94458/2013) through FCT/MEC co-funded by QREN and POPH (Programa Operacional Potencial Humano). CB and JCP received FCT Doctoral Fellowships (SFRH/BD/112217/2015 and SFRH/BD/86871/2012, respectively). SS was supported by FCT Investigator Programme (COMPETE, POPH and FCT).

The gut microbiota is involved in metabolic and immune disorders associated with obesity and type 2 diabetes. We previously demonstrated that prebiotic treatment may significantly improve host health by modulating bacterial species related to the improvement of gut endocrine, barrier and immune functions. An analysis of the gut metagenome is needed to determine which bacterial functions and taxa are responsible for beneficial microbiota-host interactions upon nutritional intervention. We subjected mice to prebiotic (Pre) treatment under physiological (control diet: CT) and pathological conditions (high-fat diet: HFD) for 8 weeks and investigated the production of intestinal antimicrobial peptides and the gut microbiome. HFD feeding significantly decreased the expression of regenerating islet-derived 3-gamma (Reg3g) and phospholipase A2 group-II (PLA2g2) in the jejunum. Prebiotic treatment increased Reg3g expression (by ∼50-fold) and improved intestinal homeostasis as suggested by the increase in the expression of intectin, a key protein involved in intestinal epithelial cell turnover. Deep metagenomic sequencing analysis revealed that HFD and prebiotic treatment significantly affected the gut microbiome at different taxonomic levels. Functional analyses based on the occurrence of clusters of orthologous groups (COGs) of proteins also revealed distinct profiles for the HFD, Pre, HFD-Pre and CT groups. Finally, the gut microbiota modulations induced by prebiotics counteracted HFD-induced inflammation and related metabolic disorders. Thus, we identified novel putative taxa and metabolic functions that may contribute to the development of or protection against the metabolic alterations observed during HFD feeding and HFD-Pre feeding.

The integral membrane glycoprotein BARP associates with voltage-gated calcium channel Cavβ subunits, disrupting their association with Cavα1 subunits and suppressing overall channel activity., Voltage-gated calcium channels (VGCCs) are key regulators of cell signaling and Ca2+-dependent release of neurotransmitters and hormones. Understanding the mechanisms that inactivate VGCCs to prevent intracellular Ca2+ overload and govern their specific subcellular localization is of critical importance. We report the identification and functional characterization of VGCC β-anchoring and -regulatory protein (BARP), a previously uncharacterized integral membrane glycoprotein expressed in neuroendocrine cells and neurons. BARP interacts via two cytosolic domains (I and II) with all Cavβ subunit isoforms, affecting their subcellular localization and suppressing VGCC activity. Domain I interacts at the α1 interaction domain–binding pocket in Cavβ and interferes with the association between Cavβ and Cavα1. In the absence of domain I binding, BARP can form a ternary complex with Cavα1 and Cavβ via domain II. BARP does not affect cell surface expression of Cavα1 but inhibits Ca2+ channel activity at the plasma membrane, resulting in the inhibition of Ca2+-evoked exocytosis. Thus, BARP can modulate the localization of Cavβ and its association with the Cavα1 subunit to negatively regulate VGCC activity.

Synaptic rearrangements during critical periods of postnatal brain development rely on the correct formation, strengthening, and elimination of synapses and associated dendritic spines to form functional networks. The correct balance of these processes is thought to be regulated by synapse-specific changes in the subunit composition of NMDA-type glutamate receptors (NMDARs). Among these, the nonconventional NMDAR subunit GluN3A has been suggested to play a role as a molecular brake in synaptic maturation. We tested here this hypothesis using confocal time-lapse imaging in rat hippocampal organotypic slices and assessed the role of GluN3A-containing NMDARs on spine dynamics. We found that overexpressing GluN3A reduced spine density over time, increased spine elimination, and decreased spine stability. The effect of GluN3A overexpression could be further enhanced by using an endocytosis-deficient GluN3A mutant and reproduced by silencing the adaptor protein PACSIN1, which prevents the endocytosis of endogenous GluN3A. Conversely, silencing of GluN3A reduced spine elimination and favored spine stability. Moreover, reexpression of GluN3A in more mature tissue reinstated an increased spine pruning and a low spine stability. Mechanistically, the decreased stability in GluN3A overexpressing neurons could be linked to a failure of plasticity-inducing protocols to selectively stabilize spines and was dependent on the ability of GluN3A to bind the postsynaptic scaffold GIT1. Together, these data provide strong evidence that GluN3A prevents the activity-dependent stabilization of synapses thereby promoting spine pruning, and suggest that GluN3A expression operates as a molecular signal for controlling the extent and timing of synapse maturation.

Hematopoietic stem cells (HSCs) reside in specialized bone marrow (BM) niches regulated by the sympathetic nervous system (SNS). Here, we have examined whether mononuclear phagocytes modulate the HSC niche. We defined three populations of BM mononuclear phagocytes that include Gr-1hi monocytes (MOs), Gr-1lo MOs, and macrophages (MΦ) based on differential expression of Gr-1, CD115, F4/80, and CD169. Using MO and MΦ conditional depletion models, we found that reductions in BM mononuclear phagocytes led to reduced BM CXCL12 levels, the selective down-regulation of HSC retention genes in Nestin+ niche cells, and egress of HSCs/progenitors to the bloodstream. Furthermore, specific depletion of CD169+ MΦ, which spares BM MOs, was sufficient to induce HSC/progenitor egress. MΦ depletion also enhanced mobilization induced by a CXCR4 antagonist or granulocyte colony-stimulating factor. These results highlight two antagonistic, tightly balanced pathways that regulate maintenance of HSCs/progenitors in the niche during homeostasis, in which MΦ cross talk with the Nestin+ niche cell promotes retention, and in contrast, SNS signals enhance egress. Thus, strategies that target BM MΦ hold the potential to augment stem cell yields in patients that mobilize HSCs/progenitors poorly.

In his letter, Dr. Pick criticizes our use of relative values when representing the NOX2 inhibitory action of a novel small molecule (GSK2795039) in a semi-recombinant NOX2 membrane assay. To address this concern, we performed additional experiments using the superoxide inhibitable assays cytochrome C and water soluble tetrazolium salt (WST-1) reduction. In this letter, we document turnover values between 80 and 100 mol O2(•-)/s/mol cytochrome b558 in our semi-recombinant assay and confirmed that GSK2795039 inhibits the NOX2 isoform in the submicromolar range. Antioxid. Redox Signal. 23, 1251-1253.

Dictyostelium amoebae grow as single cells but upon starvation they initiate multicellular development. Phg2 was characterized previously as a kinase controlling cellular adhesion and the organization of the actin cytoskeleton. Here we report that Phg2 also plays a role during the transition between growth and multicellular development, as evidenced by the fact that phg2 mutant cells can initiate development even in the presence of nutrients. Even at low cell density and in rich medium, phg2 mutant cells express discoidin, one of the earliest predevelopmental markers. Complementation studies indicate that, in addition to the kinase domain, the core region of Phg2 is involved in the initiation of development. In this region, a small domain contiguous with a previously described ras-binding domain was found to interact with the Dictyostelium ortholog of the mammalian adhesion-regulating molecule (ADRM1). In addition, adrm1 knockout cells also exhibit abnormal initiation of development. These results suggest that a Phg2-Adrm1 signaling pathway is involved in the control of the transition from growth to differentiation in Dictyostelium. Phg2 thus plays a dual role in the control of cellular adhesion and initiation of development.

Recent findings have implicated Fas/Fas ligand (FasL) in mediating the death of keratinocytes in spongiotic lesions. We asked whether dying keratinocytes could potentially initiate a protective response of the skin to limit the destruction of the epidermis in the spongiotic areas. In addition to apoptosis, treatment of keratinocyte cultures in vitro with FasL triggers a profound phoshorylation of the epidermal growth factor receptor (EGFR) and of its downstream effectors ERK and protein kinase B (PKB/Akt). Using a variety of inhibitors and blocking antibodies, we demonstrated that: (i) apoptosis is required for the generation of the signal(s) leading to the activation of EGFR, ERK, and Akt; (ii) the activation of EGFR, ERK, and Akt by FasL is indeed mediated by its bona fide receptor Fas; (iii) the activation of EGFR is essential for the subsequent activation of ERK and Akt; and (iv) apoptotic keratinocytes secrete soluble EGFR ligands (including amphiregulin) that are processed from membrane-bound proligand forms by metalloproteinase(s). Our findings demonstrate a potential mechanism for the restriction and repair of spongiotic damage in eczemas.

Lymph node metastasis is a frequent reason for adverse clinical outcome in many epithelial neoplasms, including head and neck squamous cell carcinoma. The mechanisms underlying the capability of epithelial neoplasms to metastasize via lymphatic vessels have not yet been fully elucidated. There is great debate about whether cancer cells can metastasize by expansion and invasion of pre-existing peritumoral lymphatics or by the formation and invasion of new lymphatics within tumours (lymphangiogenesis). In order to investigate this issue, we examined 81 tissue specimens from patients with head and neck squamous cell carcinoma, using immunostaining for the specific lymphatic endothelium marker podoplanin, and assessed intratumoral and peritumoral lymphatic density. We also quantified lymphatic invasion and examined the possible associations of all the above parameters with clinicopathological features and outcome. Finally, we used double staining with podoplanin and the cell proliferation marker Ki-67 in order to evaluate lymphangiogenesis. High intratumoral and peritumoral lymphatic density were both significantly associated with the presence of lymph node metastasis at the time of diagnosis (chi2 test, p < 0.001 and p = 0.007, respectively) and there was a significant correlation between high intratumoral lymphatic density and lymphatic invasion. Patients with higher intratumoral lymphatic density exhibited shorter overall survival (log rank p < 0.001) and this correlation remained significant after multivariate analysis (Cox p = 0.04), indicating that intratumoral lymphatic density is an independent prognostic factor for mortality. Peritumoral lymphatic density had no influence on outcome. Double staining revealed the existence of proliferating intratumoral lymphatics, in which tumour emboli were occasionally observed. These results indicate that lymphangiogenesis indeed occurs in head and neck squamous cell carcinoma; that newly formed vessels are targets of invasion by cancer cells; and that intratumoral lymphatic density might be used as a criterion to separate patients at higher risk of an adverse clinical outcome. J Pathol

Infection of pancreatic necrosis is a severe complication of acute pancreatitis. Because Toll-like receptor 4 (TLR4) has been identified as a receptor necessary to transduct the signal of bacteria-derived lipopolysaccharide into cells, we investigated the role of TLR4 on pancreatic and pulmonary injury in acute pancreatitis and acute pancreatitis associated with endotoxemia in wild-type and TLR4-deficient mice.Laboratory investigation.University laboratory.Heterozygous TLR4 mice.Mice were injected intraperitoneally with a supramaximal dose of cerulein each hour for 10 hrs. To mimic infection, additional groups of mice were injected with lipopolysaccharide in the presence or absence of cerulein injections.The severity of acute pancreatitis was assessed by serum amylase activity, pancreatic edema, acinar cell necrosis, and pancreas myeloperoxidase activity. Lung injury was quantitated by lung microvascular permeability and lung myeloperoxidase activity. Injections of cerulein induced an edematous pancreatitis that was of similar severity in wild-type and TLR4-deficient mice. Lipopolysaccharide alone had no toxic effect on pancreas and lungs and did not worsen the pancreatic injury induced by cerulein in wild-type and TRL4-deficient mice. In contrast, lipopolysaccharide worsened pancreatitis-associated lung injury, and the deficiency in TLR4 fully prevented this aggravation.TLR4 may not play a role in the pancreatitis-associated lung injury but participates in the pulmonary injury mediated by endotoxemia.

Heparan sulfate proteoglycans (HSPGs) are known to participate in a wide range of biological events, including cellular trafficking. In this study we report that in situ cerebral blood vessels highly express HSPGs. Of the syndecan family, syndecan-2 is highly expressed on virtually all brain vessels and syndecan-1 and -3 are only present on larger blood vessels. These endothelial HSPGs have a functional role in monocyte diapedesis across brain endothelium, as assessed in our in vitro adhesion and migration assays. Our data indicate that heparin prevents monocyte adhesion to brain endothelium by interacting solely with the monocyte. Transendothelial migration of monocytes can be prevented by preincubation of brain endothelium with heparin by enzymatic removal of heparan sulphate side chains or by inhibition of cellular sulfation. Blocking of G-protein-dependent signaling in the monocytes prevented monocyte adhesion and migration to similar extent, suggesting that G-dependent signaling may be involved in HSPG-mediated monocyte adhesion and transendothelial migration. Our data demonstrate that brain endothelial HSPGs have a modulatory role in the transendothelial migration of monocytes in a direct and indirect fashion and may therefore contribute to the formation of neuroinflammatory lesions.

Tumor necrosis factor (TNF) ligand and receptor superfamily members play critical roles in diverse developmental and pathological settings. In search for novel TNF superfamily members, we identified a murine chromosomal locus that contains three new TNF receptor-related genes. Sequence alignments suggest that the ligand binding regions of these murine TNF receptor homologues, mTNFRH1, -2 and -3, are most homologous to those of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors. By using a number of in vitro ligand-receptor binding assays, we demonstrate that mTNFRH1 and -2, but not mTNFRH3, bind murine TRAIL, suggesting that they are indeed TRAIL receptors. This notion is further supported by our demonstration that both mTNFRH1:Fc and mTNFRH2:Fc fusion proteins inhibited mTRAIL-induced apoptosis of Jurkat cells. Unlike the only other known murine TRAIL receptor mTRAILR2, however, neither mTNFRH2 nor mTNFRH3 has a cytoplasmic region containing the well characterized death domain motif. Coupled with our observation that overexpression of mTNFRH1 and -2 in 293T cells neither induces apoptosis nor triggers NFkappaB activation, we propose that the mTnfrh1 and mTnfrh2 genes encode the first described murine decoy receptors for TRAIL, and we renamed them mDcTrailr1 and -r2, respectively. Interestingly, the overall sequence structures of mDcTRAILR1 and -R2 are quite distinct from those of the known human decoy TRAIL receptors, suggesting that the presence of TRAIL decoy receptors represents a more recent evolutionary event.

Signet-ring cells are relatively common in breast cancers but are frequently overlooked. Although previously defined as a subtype of mucin producing carcinomas, breast carcinomas with signet-ring cell (SRC) differentiation nowadays are not considered a distinct entity.The objective of the present study was to characterize the morphological features and mucin expression profile of breast carcinomas with SRC differentiation. All breast carcinomas diagnosed at Centro Hospitalar S. Joao between 1996 and 2006 in which the pathology report mentioned the presence of SRCs (n= 11) and four mucinous carcinomas were included in the study. The frequency of SRCs and immunohistochemistry expression of MUC1/MUC2/MUC5AC/MUC6 were evaluated.We confirmed that SRC differentiation can occur in different histological types, including ductal, lobular, mucinous and metaplastic carcinomas. The proportion of SRCs was highly variable (range: 8-70%). Tumors encompassed SRCs of intracytoplasmic lumina and goblet-cell type. A higher percentage of SRCs was associated with lymphovascular invasion (p= 0.047). All tumors expressed cytoplasmic and membranous MUC1. Secretory mucins were more frequent in mucinous carcinomas and in carcinomas with extensive SRC differentiation.We conclude that besides the usefulness of mucin immunodetection for the differential diagnosis of carcinomas with SRC differentiation of breast origin, it is important to report SRC differentiation regardless of histological type because of its intrinsic prognostic value. We especially thank Professor Sobrinho-Simões for the careful review of the manuscript. IPATIMUP integrates the i3S Research Unit, which is partially supported by FCT, the Portuguese Foundation for Science and Technology. This work is funded by FEDER funds through the Operational Program for Competitiveness Factors-COMPETE and National Funds through the FCT-Foundation for Science and Technology , under the projects: PEst-C/SAU/LA0003/2013 and PTDC/BBB-EBI/0786/2012 .

Exocytosis of insulin containing Large Dense Core Vesicles (LDCVs) from pancreatic (β)-cells and derived cell lines is mainly controlled by Ca(2+). Several lines of evidence have demonstrated a role of the Ca(2+)- and phospholipid-binding protein synaptotagmin (syt) in this event. Synaptotagmins form a large protein family with distinct affinities for Ca(2+) determined by their two C(2) domains (C(2)A/B). Except for the well-characterized isoforms I and II, their role is still unclear. We have used here insulin-secreting cells as a model system for LDCV exocytosis to gain insight into the function of synaptotagmins. Immunocytochemical analysis revealed that of the candidate Ca(2+) sensors in LDCV exocytosis, syt III was not expressed in primary (β)-cells, whereas syt IV was only found adjacent to the TGN. However, syt V-VIII isoforms were expressed at different levels in various insulin-secreting cells and in pancreatic islet preparations. In streptolysin-O permeabilized primary (β)-cells the introduction of recombinant peptides (100 nM) corresponding to the C(2) domains of syt V, VII and VIII, but not of syt III, IV or VI, inhibited Ca(2+)-evoked insulin exocytosis by 30% without altering GTP*S-induced release. Our observations demonstrate that syt III and IV are not involved in the exocytosis of LDCVs from primary (β)-cells whereas V, VII and VIII may mediate Ca(2+)-regulation of exocytosis.

Heparan sulfate proteoglycans (HSPGs) have been suggested to play an important role in the formation and persistence of senile plaques and neurofibrillary tangles in dementia of the Alzheimer's type (DAT). We performed a comparative immunohistochemical analysis of the expression of the HSPGs agrin, perlecan, glypican-1, and syndecans 1-3 in the lesions of DAT brain neocortex and hippocampus. Using a panel of specific antibodies directed against the protein backbone of the various HSPG species and against the glycosaminoglycan (GAG) side-chains, we demonstrated the following. The basement membrane-associated HSPG, agrin, is widely expressed in senile plaques, neurofibrillary tangles and cerebral blood vessels, whereas the expression of the other basement membrane-associated HSPG, perlecan, is lacking in senile plaques and neurofibrillary tangles and is restricted to the cerebral vasculature. Glypican and three different syndecans, all cell membrane-associated HSPG species, are also expressed in senile plaques and neurofibrillary tangles, albeit at a lower frequency than agrin. Heparan sulfate GAG side chains are also associated with both senile plaques and neurofibrillary tangles. Our results suggest that glycosaminoglycan side chains of the HSPGs agrin, syndecan, and glypican, but not perlecan, may play an important role in the formation of both senile plaques and neurofibrillary tangles. In addition, we speculate that agrin, because it contains nine protease-inhibiting domains, may protect the protein aggregates in senile plaques and neurofibrillary tangles against extracellular proteolytic degradation, leading to the persistence of these deposits.

Human Fas ligand (L) (CD95L) and tumor necrosis factor (TNF)-α undergo metalloproteinase-mediated proteolytic processing in their extracellular domains resulting in the release of soluble trimeric ligands (soluble [s]FasL, sTNF-α) which, in the case of sFasL, is thought to be implicated in diseases such as hepatitis and AIDS. Here we show that the processing of sFasL occurs between Ser126 and Leu127. The apoptotic-inducing capacity of naturally processed sFasL was reduced by >1,000-fold compared with membrane-bound FasL, and injection of high doses of recombinant sFasL in mice did not induce liver failure. However, soluble FasL retained its capacity to interact with Fas, and restoration of its cytotoxic activity was achieved both in vitro and in vivo with the addition of cross-linking antibodies. Similarly, the marginal apoptotic activity of recombinant soluble TNF-related apoptosis-inducing ligand (sTRAIL), another member of the TNF ligand family, was greatly increased upon cross-linking. These results indicate that the mere trimerization of the Fas and TRAIL receptors may not be sufficient to trigger death signals. Thus, the observation that sFasL is less cytotoxic than membrane-bound FasL may explain why in certain types of cancer, systemic tissue damage is not detected, even though the levels of circulating sFasL are high.

Viruses have evolved many distinct strategies to avoid the host's apoptotic response. Here we describe a new family of viral inhibitors (v-FLIPs) which interfere with apoptosis signalled through death receptors and which are present in several gamma-herpesviruses (including Kaposi's-sarcoma-associated human herpesvirus-8), as well as in the tumorigenic human molluscipoxvirus. v-FLIPs contain two death-effector domains which interact with the adaptor protein FADD, and this inhibits the recruitment and activation of the protease FLICE by the CD95 death receptor. Cells expressing v-FLIPs are protected against apoptosis induced by CD95 or by the related death receptors TRAMP and TRAIL-R. The herpesvirus saimiri FLIP is detected late during the lytic viral replication cycle, at a time when host cells are partially protected from CD95-ligand-mediated apoptosis. Protection of virus-infected cells against death-receptor-induced apoptosis may lead to higher virus production and contribute to the persistence and oncogenicity of several FLIP-encoding viruses.

NADPH oxidases synthesize reactive oxygen species that may participate in fibrosis progression. NOX4 and NOX2 are NADPH oxidases expressed in the kidneys, with the former being the major renal isoform, but their contribution to renal disease is not well understood. Here, we used the unilateral urinary obstruction model of chronic renal injury to decipher the role of these enzymes using wild-type, NOX4-, NOX2-, and NOX4/NOX2-deficient mice. Compared with wild-type mice, NOX4-deficient mice exhibited more interstitial fibrosis and tubular apoptosis after obstruction, with lower interstitial capillary density and reduced expression of hypoxia-inducible factor-1α and vascular endothelial growth factor in obstructed kidneys. Furthermore, NOX4-deficient kidneys exhibited increased oxidative stress. With NOX4 deficiency, renal expression of other NOX isoforms was not altered but NRF2 protein expression was reduced under both basal and obstructed conditions. Concomitant deficiency of NOX2 did not modify the phenotype exhibited by NOX4-deficient mice after obstruction. NOX4 silencing in a mouse collecting duct (mCCD(cl1)) cell line increased TGF-β1-induced apoptosis and decreased NRF2 protein along with expression of its target genes. In addition, NOX4 silencing decreased hypoxia-inducible factor-1α and expression of its target genes in response to hypoxia. In summary, these results demonstrate that the absence of NOX4 promotes kidney fibrosis, independent of NOX2, through enhanced tubular cell apoptosis, decreased microvascularization, and enhanced oxidative stress. Thus, NOX4 is crucial for the survival of kidney tubular cells under injurious conditions.

Next-generation sequencing offers an unprecedented opportunity to jointly analyze cellular and viral transcriptional activity without prerequisite knowledge of the nature of the transcripts. SupT1 cells were infected with a vesicular stomatitis virus G envelope protein (VSV-G)-pseudotyped HIV vector. At 24 h postinfection, both cellular and viral transcriptomes were analyzed by serial analysis of gene expression followed by high-throughput sequencing (SAGE-Seq). Read mapping resulted in 33 to 44 million tags aligning with the human transcriptome and 0.23 to 0.25 million tags aligning with the genome of the HIV-1 vector. Thus, at peak infection, 1 transcript in 143 is of viral origin (0.7%), including a small component of antisense viral transcription. Of the detected cellular transcripts, 826 (2.3%) were differentially expressed between mock- and HIV-infected samples. The approach also assessed whether HIV-1 infection modulates the expression of repetitive elements or endogenous retroviruses. We observed very active transcription of these elements, with 1 transcript in 237 being of such origin, corresponding on average to 123,123 reads in mock-infected samples (0.40%) and 129,149 reads in HIV-1-infected samples (0.45%) mapping to the genomic Repbase repository. This analysis highlights key details in the generation and interpretation of high-throughput data in the setting of HIV-1 cellular infection.

International audience; NOX enzymes are reactive oxygen species (ROS)-generating NADPH oxidases. Several members of the NOX family depend on the p22(phox) subunit, encoded by the CYBA gene. CYBA is highly polymorphic, and has been widely studied as a potential risk factor for various diseases, with conflicting results. In the present study, we used Epstein-Barr (EBV)-transformed B-lymphocytes from 50 healthy unrelated individuals to analyze their CYBA mRNA sequence and NOX2-dependent ROS generation. Seven single-nucleotide polymorphisms (SNPs) were identified (five previously described, two novel). The combination of these SNPs yielded 11 distinct haplotypes, which could be grouped into seven haplogroups (A-G). Haplogroup C (c.214T>C, c.521T>C, and c.(*)24G>A) showed a significantly lower ROS generation, as compared to the most frequent haplogroup, A. CYBA variants from the seven haplogroups were transduced into p22(phox)-deficient B-lymphocytes. The haplogroup C variant showed significantly lower ROS production. c.214T>C and c.521T>C lead to nonsynonymous codon changes, while c.(*)24G>A lies within the 3'UTR. Using a luciferase/3'UTR construct, we showed that the (*)24A allele led to decreased reporter gene activity. These results help to unravel the complex nature of how genetic variations in CYBA influence NOX2 activity, and indicate that haplotypes, rather than individual SNPs, define the effect on ROS generation.

Endogenous retroviruses are implicated in the pathogenesis of systemic lupus erythematosus (SLE). Because four different classes of endogenous retroviruses, i.e., ecotropic, xenotropic, polytropic, or modified polytropic (mPT), are expressed in mice, we investigated the possibility that a particular class of endogenous retroviruses is associated with the development of murine SLE. We observed >15-fold increased expression of mPT env (envelope) RNA in livers of all four lupus-prone mice, as compared with those of nine nonautoimmune strains of mice. This was not the case for the three other classes of retroviruses. Furthermore, we found that in addition to intact mPT transcripts, many strains of mice expressed two defective mPT env transcripts which carry a deletion in the env sequence of the 3′ portion of the gp70 surface protein and the 5′ portion of the p15E transmembrane protein, respectively. Remarkably, in contrast to nonautoimmune strains of mice, all four lupus-prone mice expressed abundant levels of intact mPT env transcripts, but only low or nondetectable levels of the mutant env transcripts. The Sgp3 (serum gp70 production 3) locus derived from lupus-prone mice was responsible for the selective up-regulation of the intact mPT env RNA. Finally, we observed that single-stranded RNA-specific TLR7 played a critical role in the production of anti-gp70 autoantibodies. These data suggest that lupus-prone mice may possess a unique genetic mechanism responsible for the expression of mPT retroviruses, which could act as a triggering factor through activating TLR7 for the development of autoimmune responses in mice predisposed to SLE.

Mannose-6-phosphate receptors (MPRs) transport lysosomal hydrolases from the trans Golgi network (TGN) to endosomes. Recently, the multi-ligand receptor sortilin has also been implicated in this transport, but the transport carriers involved herein have not been identified. By quantitative immuno-electron microscopy, we localized endogenous sortilin of HepG2 cells predominantly to the TGN and endosomes. In the TGN, sortilin colocalized with MPRs in the same clathrin-coated vesicles. In endosomes, sortilin and MPRs concentrated in sorting nexin 1 (SNX1)-positive buds and vesicles. SNX1 depletion by small interfering RNA resulted in decreased pools of sortilin in the TGN and an increase in lysosomal degradation. These data indicate that sortilin and MPRs recycle to the TGN in SNX1-dependent carriers, which we named endosome-to-TGN transport carriers (ETCs). Notably, ETCs emerge from early endosomes (EE), lack recycling plasma membrane proteins and by three-dimensional electron tomography exhibit unique structural features. Hence, ETCs are distinct from hitherto described EE-derived membranes involved in recycling. Our data emphasize an important role of EEs in recycling to the TGN and indicate that different, specialized exit events occur on the same EE vacuole.

The Ccr4-Not complex is a multifunctional regulatory platform composed of nine subunits that controls diverse cellular events including mRNA degradation, protein ubiquitination, and transcription. In this study, we identified the yeast Saccharomyces cerevisiae osmotic and oxidative stress transcription factor Skn7 as a new target for regulation by the Ccr4-Not complex. Skn7 interacts with Not1 in a two-hybrid assay and coimmunoprecipitates with Not5 in a Not4-dependent manner. Skn7-dependent expression of OCH1 and Skn7 binding to the OCH1 promoter are increased in not4 Δ or not5 Δ mutants. Skn7 purified from wild-type cells but not from not4 Δ cells is associated with the Srb10 kinase. This kinase plays a central role in the regulation of Skn7 by Not4, since increased OCH1 expression in not4 Δ cells requires Srb10. These results reveal a critical role for the Ccr4-Not complex in the mechanism of activation of Skn7 that is dependent upon the Srb10 kinase.

Exocytosis of late endocytic compartments in Dictyostelium has mostly been studied by live microscopy. Here we show that this exocytosis is accompanied by a complete fusion of late endosomes with the plasma membrane resulting in the transient formation of membrane microdomains that can be visualized by immunofluorescence in fixed cells. This permitted to demonstrate that fusion of late endocytic compartments with the cell surface does not occur in regions of the plasma membrane engaged in the formation of pseudopods, macropinosomes or phagosomes. Our results propose that exocytosis of late endosomes and actin-driven membrane remodeling are mutually exclusive processes.

Murine fibroblasts expressing viral receptors and human cyclin T1 allow HIV-1 entry and viral gene expression but do not support efficient assembly. A chimeric HIV-1 carrying a non-homologous matrix (MA) from murine leukemia virus in place of HIV-1 MA can assemble efficiently in murine cells, yet has poor infectivity. Here, we assess the ability of a homologous MA from SIV MAC239 to complement assembly and infection in chimeric viruses designated SHIV(MA). The resulting SHIV(MA) chimeras produce more virus than native HIV-1 when transfected into murine cells. SHIV(MA) exhibits cell-type-specific replication in human T cell lines, replicating well in MT4 cells and poorly in Jurkat cells due to an incompatibility with the HIV-1 Env. The infectivity defects of SHIV(MA) are rescued by pseudotyping with VSV-G but not by truncation of the cytoplasmic tail of Env. Passage of SHIV(MA) in Jurkat cells produces variants with improved Env incorporation and improved replication in Jurkat but not in 3T3 TXC cells. The results indicate that cell-type-specific, or species-specific, host factors interact with MA to modulate the efficiency of assembly and its compatibility with Env. With additional selection, SIV/HIV-1 chimeras may be useful for the development of murine models of lentiviral infection.

Synaptotagmins (Syts) are involved in Ca(2+)-dependent insulin release. However, which Syt isoform is functional in primary beta-cells remains unknown. We demonstrate by electron microscopy of pancreatic islets, the association of Syt 9 with insulin granules. Silencing of Syt 9 by RNA interference adenovirus in islet cells had no effect on the expression of Syt 5, Syt 7 and Syt 3 isoforms. The latter was localized at the plasma membrane of pancreatic polypeptide cells. Insulin release in response to glucose or tolbutamide was strongly inhibited in Syt 9 deficient islets, whereas exocytosis potentiated by raising cAMP levels, was unaltered. Thus, Syt 9 may act as Ca(2+) sensor for beta-cell secretion.

Partitioning of proteins in cholesterol and sphingolipid enriched plasma membrane microdomains, called lipid rafts, is critical for many signal transduction and protein sorting events. Although raft partitioning of many signaling molecules remains to be determined, glycosylphosphatidyl-inositol (GPI)-anchored proteins possess high affinity for lipid rafts and are currently exploited as markers to investigate fundamental mechanisms in protein sorting and signal transduction events. In this study, we demonstrate that two recombinant GPI-anchored green fluorescent proteins (GFP-GPIs) that differ in their GPI signal sequence confer distinct localization in plasma membrane microdomains. GFP fused to the GPI signal of the decay accelerating factor GFP-GPI(DAF) partitioned exclusively in lipid rafts, whereas GFP fused to the GPI signal of TRAIL-R3, GFP-GPI(TRAIL-R3), associated only minimally with microdomains. In addition, we investigated the unique ability of purified GFP-GPIs to insert into membrane microdomains of primary lymphocytes. This cell surface painting allows rapid, stable, and functional association of the GPI-anchored proteins with the target cell plasma membrane. The distinct membrane localization of the two GFP-GPIs was observed irrespective of whether the GPI-anchored molecules were painted or transfected. Furthermore, we show that painted GFP-GPI(DAF) was totally dependent on the GPI anchor and that the membrane insertion was increased by the addition of raft-associated lipids such as cholesterol, sphingomyelin, and dipalmitoyl-phosphatidylethanolamine. Thus, this study provides evidence that different GPI signal sequences lead to distinct membrane microdomain localization and that painted GFP-GPI(DAF) serves as an excellent fluorescent marker for lipid rafts in live cells.

Streptococcus uberis and Serratia marcescens are Gram-positive and Gram-negative bacteria, respectively, that induce clinical mastitis. Once initial host barrier systems have been breached by these pathogens, the innate immune system provides the next level of defense against these infectious agents. The innate immune response is characterized by the induction of pro-inflammatory cytokines, as well as increases in other accessory proteins that facilitate host recognition and elimination of the pathogens. The objective of the current study was to characterize the innate immune response during clinical mastitis elicited by these two important, yet less well-studied, Gram-positive and Gram-negative organisms. The pro-inflammatory cytokine response and changes in the levels of the innate immune accessory recognition proteins, soluble CD14 (sCD14) and lipopolysaccharide (LPS)-binding protein (LBP), were studied. Decreased milk output, induction of a febrile response, and increased acute phase synthesis of LBP were all characteristic of the systemic response to intramammary infection with either organism. Infection with either bacteria similarly resulted in increased milk levels of IL-1 beta, IL-8, IL-10, IL-12, IFN-gamma, TNF-alpha, sCD14, LBP, and the complement component, C5a. However, the duration of and/or maximal changes in the increased levels of these inflammatory markers were significantly different for several of the inflammatory parameters assayed. In particular, S. uberis infection was characterized by the sustained elevation of higher milk levels of IL-1 beta, IL-10, IL-12, IFN-gamma, and C5a, relative to S. marcescens infection. Together, these data demonstrate the variability of the innate immune response to two distinct mastitis pathogens.

Heparan sulfate proteoglycans (HSPGs) are known to participate in a wide range of biological events, including cellular trafficking. In this study we report that in situ cerebral blood vessels highly express HSPGs. Of the syndecan family, syndecan-2 is highly expressed on virtually all brain vessels and syndecan-1 and -3 are only present on larger blood vessels. These endothelial HSPGs have a functional role in monocyte diapedesis across brain endothelium, as assessed in our in vitro adhesion and migration assays. Our data indicate that heparin prevents monocyte adhesion to brain endothelium by interacting solely with the monocyte. Transendothelial migration of monocytes can be prevented by preincubation of brain endothelium with heparin by enzymatic removal of heparan sulphate side chains or by inhibition of cellular sulfation. Blocking of G-protein-dependent signaling in the monocytes prevented monocyte adhesion and migration to similar extent, suggesting that G-dependent signaling may be involved in HSPG-mediated monocyte adhesion and transendothelial migration. Our data demonstrate that brain endothelial HSPGs have a modulatory role in the transendothelial migration of monocytes in a direct and indirect fashion and may therefore contribute to the formation of neuroinflammatory lesions.

The tetraspanin web refers to a network of molecular interactions involving tetraspanins and other molecules. Inside the tetraspanin web, small primary complexes containing only one tetraspanin and one specific partner molecule such as CD151/alpha3beta1 integrin and CD9/CD9P-1 (FPRP) can be observed under particular conditions. Here we demonstrate that when cells are lysed with Brij97, the tetraspanins CD151 and CD9 allow and/or stabilize the interaction of their partner molecules with other tetraspanins and that their two partners associate under conditions maintaining tetraspanin/tetraspanin interactions. The tetraspanins were also found to partition into a detergent-resistant membrane environment to which the integrin alpha3beta1 was relocalized upon expression of CD151.

The membrane-bound form of Fas ligand (FasL) signals apoptosis in target cells through engagement of the death receptor Fas, whereas the proteolytically processed, soluble form of FasL does not induce cell death. However, soluble FasL can be rendered active upon cross-linking. Since the minimal extent of oligomerization of FasL that exerts cytotoxicity is unknown, we engineered hexameric proteins containing two trimers of FasL within the same molecule. This was achieved by fusing FasL to the Fc portion of immunoglobulin G1 or to the collagen domain of ACRP30/adiponectin. Trimeric FasL and hexameric FasL both bound to Fas, but only the hexameric forms were highly cytotoxic and competent to signal apoptosis via formation of a death-inducing signaling complex. Three sequential early events in Fas-mediated apoptosis could be dissected, namely, receptor binding, receptor activation, and recruitment of intracellular signaling molecules, each of which occurred independently of the subsequent one. These results demonstrate that the limited oligomerization of FasL, and most likely of some other tumor necrosis factor family ligands such as CD40L, is required for triggering of the signaling pathways.

In focal adhesions of eukaryotic cells, transmembrane receptors of the integrin family and a large set of adaptor proteins form the physical link between the extracellular substrate and the actin cytoskeleton. During cell migration, nascent focal adhesions within filopodia and lamellipodia make the initial exploratory contacts with the cellular environment, whereas maturing focal adhesions pull the cell forward against the resistance of 'sliding' focal adhesions at the cell rear. Experimental approaches are now available for analysing the dynamics and interior structure of these different focal adhesions. Analysing focal-adhesion dynamics using green-fluorescent-protein-linked integrin leads us to propose that the acto-myosin-controlled density and turnover of integrins in focal adhesions is used to sense the elasticity and spacing of extracellular ligands, regulating cell migration by mechanically transduced signaling.

Increasing evidence suggests that apoptosis plays an important role in the pathogenesis of autoimmune and proliferative thyroid diseases, and that the apoptotic pathways involved are complex and highly regulated. Autoimmune thyroid diseases such as Hashimoto's thyroiditis and Graves' disease have been associated with differential expression of Fas and TRAIL receptor-mediated apoptosis. Thus, the thyroid cell destruction characteristic of autoimmune thyroiditis can be seen as the consequence of inappropriate expression of Fas or TRAIL death pathway molecules and down-regulation of the apoptosis controlling protein Bcl-2, which may be induced by cytokines released locally by infiltrating lymphocytes. In contrast, Graves' thyrocytes are protected from apoptotic death possibly by the anti-apoptotic action of thyrotrophin receptor antibodies or soluble Fas and/or the overexpression of Fas ligand which all create an anti-apoptotic potential for the thyroid cells and favor apoptosis of the infiltrating lymphocytes. On the other hand, an imbalance between thyroid cell proliferation and cell death may be crucial for goiter formation or cancer development and progression. In human thyroid goiter, Fas-mediated apoptosis is suppressed, leading to thyroid cell hyperplasia. Furthermore, malignant thyroid cells may escape immune attack by over expressing Fas ligand and inducing apoptosis in the invading immune cells. However, the exact mechanisms involved in the regulation of apoptosis in thyroid disease remain unclear. Further investigation is needed that may provide new strategies in the prevention and treatment of these diseases. Minerva Med

Death receptors, such as Fas and tumor necrosis factor-related apoptosis-inducing ligand receptors, recruit Fas-associated death domain and pro-caspase-8 homodimers, which are then autoproteolytically activated. Active caspase-8 is released into the cytoplasm, where it cleaves various proteins including pro-caspase-3, resulting in apoptosis. The cellular Fas-associated death domain-like interleukin-1-beta-converting enzyme-inhibitory protein long form (FLIP(L)), a structural homologue of caspase-8 lacking caspase activity because of several mutations in the active site, is a potent inhibitor of death receptor-induced apoptosis. FLIP(L) is proposed to block caspase-8 activity by forming a proteolytically inactive heterodimer with caspase-8. In contrast, we propose that FLIP(L)-bound caspase-8 is an active protease. Upon heterocomplex formation, a limited caspase-8 autoprocessing occurs resulting in the generation of the p43/41 and the p12 subunits. This partially processed form but also the non-cleaved FLIP(L)-caspase-8 heterocomplex are proteolytically active because they both bind synthetic substrates efficiently. Moreover, FLIP(L) expression favors receptor-interacting kinase (RIP) processing within the Fas-signaling complex. We propose that FLIP(L) inhibits caspase-8 release-dependent pro-apoptotic signals, whereas the single, membrane-restricted active site of the FLIP(L)-caspase-8 heterocomplex is proteolytically active and acts on local substrates such as RIP.

TNF receptor family members fused to the constant domain of immunoglobulin G have been widely used as immunoadhesins in basic in vitro and in vivo research and in some clinical applications. In this study, we assemble soluble, high avidity chimeric receptors on a pentameric scaffold derived from the coiled-coil domain of cartilage oligomeric matrix protein (COMP). The affinity of Fas and CD40 (but not TNFR-1 and TRAIL-R2) to their ligands is increased by fusion to COMP, when compared to the respective Fc chimeras. In functional assays, Fas:COMP was at least 20-fold more active than Fas:Fc at inhibiting the action of sFasL, and CD40:COMP could block CD40L-mediated proliferation of B cells, whereas CD40:Fc could not. In conclusion, members of the TNF receptor family can display high specificity and excellent avidity for their ligands if they are adequately multimerized.

Gram-negative bacterial endotoxin (a lipopolysaccharide (LPS)) specifically binds to CD14, a glycosylphosphatidyl inositol (GPI)-anchored surface myeloid glycoprotein. This interaction leads to cell activation, but it also promotes LPS internalization and detoxification. In this work, we investigated the route of LPS and CD14 internalization and the relevance of CD14 GPI anchor in the endocytic pathway. In promonocytic THP-1 cells transfected with a GPI or a chimeric integral form of CD14, we showed by differential buoyancy in sucrose density gradients that these two forms of CD14 were sorted to different plasma membrane subdomains. However, both forms of CD14 associated preferentially with the same surface microfilament-enriched microvilli or ruffles. Electron microscopic studies indicated that CD14 internalized via macropinocytosis, a process resembling that of phagocytosis, different from "classical" receptor-mediated endocytic pathways, such as clathrin-coated pits or caveolae. With cell warming, the CD14-enriched ruffles fused and formed large vesicles. Later, these vacuoles made stacks and condensed into phago-lysosomes. CD14 was specifically associated with all of these structures. Radiolabeled LPS internalization paralleled CD14 internalization. Confocal microscopic studies confirmed the co-localization of LPS and CD14 both at the cell surface and in endosomal compartments. The microfilament-disrupting, macropinocytosis blocking agent cytochalasin D inhibited LPS and CD14 internalization but did not prevent LPS-dependent activation, indicating that these two processes are dissociated.