TLR21 is involved in the NF-κB and IFN-ß pathways in largemouth bass (Micropterus salmoides) and interacts with TRIF but not with the Myd88 adaptor.

Toll-like receptors (TLRs) are pattern recognition receptors that trigger host immune responses against various pathogens by detecting evolutionarily conserved pathogen-associated molecular patterns (PAMPs). TLR21 is a member of the Toll-like receptor family, and emerging data suggest that it recognises unmethylated CpG DNA and is considered a functional homologue of mammalian TLR9. However, little is known regarding the role of TLR21 in the fish immune response. In the present study, we isolated the cDNA sequence of TLR21 from the largemouth bass (Micropterus salmoides) and termed it MsTLR21. The MsTLR21 gene contained an open reading frame (ORF) of 2931 bp and encodes a polypeptide of 976 amino acids. The predicted MsTLR21 protein has two conserved domains, a conserved leucine-rich repeats (LRR) domain and a C-terminal Toll-interleukin (IL) receptor (TIR) domain, similar to those of other fish and mammals. In healthy largemouth bass, the TLR21 transcript was broadly expressed in all the examined tissues, with the highest expression levels in the gills. After challenge with Nocardia seriolae and polyinosinic polycytidylic acid (Poly[I:C]), the expression of TLR21 mRNA was upregulated or downregulated in all tissues tested. Overexpression of TLR21 in 293T cells showed that it has a positive regulatory effect on nuclear factor-kappaB (NF-κB) and interferons-ß (IFN-ß) activity. Subcellular localisation analysis showed that TLR21 was expressed in the cytoplasm. We performed pull-down assays and determined that TLR21 did not interact with myeloid differentiation primary response gene 88 (Myd88); however, it interacted with TIR domain-containing adaptor inducing interferon-ß (TRIF). Taken together, these findings suggest that MsTLR21 plays important roles in TLR/IL-1R signalling pathways and the immune response to pathogen invasion.

Structural Dissection of Epsin-1 N-Terminal Helical Peptide: The Role of Hydrophobic Residues in Modulating Membrane Curvature.

Spatiotemporal structural alterations in cellular membranes are the hallmark of many vital processes. In these cellular events, the induction of local changes in membrane curvature often plays a pivotal role. Many amphiphilic peptides are able to modulate membrane curvature, but there is little information on specific structural factors that direct the curvature change. Epsin-1 is a representative protein thought to initiate invagination of the plasma membrane upon clathrin-coated vesicles formation. Its N-terminal helical segment (EpN18) plays a key role in inducing positive membrane curvature. This study aimed to elucidate the essential structural features of EpN18 in order to better understand general curvature-inducing mechanisms, and to design effective tools for rationally controlling membrane curvature. Structural dissection of peptides derived from EpN18 revealed the decisive contribution of hydrophobic residues to (i) enhancing membrane interactions, (ii) helix structuring, (iii) inducing positive membrane curvature, and (iv) loosening lipid packing. The strongest effect was obtained by substitution with leucine residues, as this EpN18 analog showed a marked ability to promote the influx of octa-arginine cell-penetrating peptides into living cells.

Cyclic ADP ribose isomers: Production, chemical structures, and immune signaling.

Cyclic adenosine diphosphate (ADP)-ribose (cADPR) isomers are signaling molecules produced by bacterial and plant Toll/interleukin-1 receptor (TIR) domains via nicotinamide adenine dinucleotide (oxidized form) (NAD+) hydrolysis. We show that v-cADPR (2'cADPR) and v2-cADPR (3'cADPR) isomers are cyclized by O-glycosidic bond formation between the ribose moieties in ADPR. Structures of 2'cADPR-producing TIR domains reveal conformational changes that lead to an active assembly that resembles those of Toll-like receptor adaptor TIR domains. Mutagenesis reveals a conserved tryptophan that is essential for cyclization. We show that 3'cADPR is an activator of ThsA effector proteins from the bacterial antiphage defense system termed Thoeris and a suppressor of plant immunity when produced by the effector HopAM1. Collectively, our results reveal the molecular basis of cADPR isomer production and establish 3'cADPR in bacteria as an antiviral and plant immunity-suppressing signaling molecule.

Endocytic protein intersectin1-S shuttles into nucleus to suppress the DNA replication in breast cancer.

Breast cancer is the most common type of cancer worldwide. However, the well-known molecular biomarkers are not enough to meet the needs of precision medicine. In search for novel targets in this regard, we reported ITSN1 (intersectin1) as one of the candidates through mRNA microarray analysis. In the present study, we reported that endocytic protein ITSN1-S exists not only in the cytoplasm but also in nuclei of breast cancer cells. ITSN1-S' functional nuclear localization signal is within its residues 306-312. Its nuclear export signal (NES) resides within its SH3 domains. We also found, the interaction between the CC domain of nuclear ITSN1-S and the NT domain of nuclear DNA helicase II (NDH II) directly suppressed the DNA replication and nascent DNA synthesis by inhibiting the R-loops resolution in breast cancer cells. Furthermore, the interaction between the EH domains of cytoplasmic ITSN1-S and PI3KC2α inhibit cell migration and invasion by inactivating the PI3KC2α-AKT pathway. Our results were confirmed in both ITSN1 gene knockout cells and in vivo assays. Finally, our clinical data showed a potential application of the combined consideration of the cytoplasmic and nuclear ITSN1-S as an independent prognosis factor. In conclusion, our study revealed ITSN1-S' novel positioning in the nuclei of breast cancer cells, its function in suppressing DNA replication, and its potential application in improved breast cancer prognosis.

Sortilin-derived peptides promote pancreatic beta-cell survival through CREB signaling pathway.

Deterioration of insulin secretion and pancreatic beta-cell mass by inflammatory attacks is one of the main pathophysiological features of type 2 diabetes (T2D). Therefore, preserving beta-cell mass and stimulating insulin secretion only in response to glucose for avoiding the hypoglycemia risks, are the most state-of-the-art option for the treatment of T2D. In this study we tested two correlated hypothesis that 1/ the endogenous peptide released from sortilin, known as PE, that stimulates insulin secretion only in response to glucose, protects beta-cells against death induced by cytokines, and 2/ Spadin and Mini-Spadin, two synthetic peptides derived from PE, that mimic the effects of PE in insulin secretion, also provide beneficial effect on beta-cells survival. We show that PE and its derivatives by inducing a rise of intracellular calcium concentration by depolarizing the membrane protect beta-cells against death induced by Interleukin-1ß. Using biochemical, confocal imaging and cell biology techniques, we reveal that the protective effects of PE and its derivatives rely on the activation of the CaM-Kinase pathway, and on the phosphorylation and activation of the transcription factor CREB. In addition, Mini-Spadin promotes beta-cell proliferation, suggesting its possible regenerative effect. This study highlights new possible roles of PE in pancreatic beta-cell survival and its derivatives as pharmacological tools against diabetes.

The multifaceted role of sortilin/neurotensin receptor 3 in human cancer development.

Sortilin (also known as neurotensin receptor 3) is a multitasking protein implicated in numerous pathophysiological processes, including cancer development, cardiovascular impairment, Alzheimer-type dementia, and depression. Although the definitive role of sortilin in human solid and hematological malignancies has been evidenced, few articles reviewed the task. The aim of the current review is to unravel the mechanisms by which sortilin controls oncogenicity and cancer progression; and also to summarize and discuss the original data obtained from international research laboratories on this topic. Questions on how sortilin is involving in the impairment of cell junctions, in exosomes composition and release, as well as in the regulation of epidermal growth factor receptor trafficking are also responded. In addition, we provide a special focus on the regulatory role of sortilin in signal transduction by either neurotrophins or neurotensin in normal and malignant cells. The relevance of sortilin with normal and cancer stem cells is also discussed. The last section provides a general overview of sortilin applications as a diagnostic and prognostic biomarker in the context of cancer detection. Finally, we comment on the future research aspects in which the field of cancer diagnosis, prognosis, and therapy might be developed.

Type II Binders Targeting the "GLR-Out" Conformation of the Pseudokinase STRADα.

Pseudokinases play important roles in signal transduction and cellular processes similar to those of catalytically competent kinases. However, pseudokinase pharmacological tractability and conformational space accessibility are poorly understood. Pseudokinases have only recently been suggested to adopt "inactive" conformations or interact with conformation-specific kinase inhibitors (e.g., type II compounds). In this work, the heavily substituted pseudokinase STRADα, which possesses a DFG → GLR substitution in the catalytic site that permits nucleotide binding while impairing divalent cation coordination, is used as a test case to demonstrate the potential applicability of conformation-specific, type II compounds to pseudokinase pharmacology. Integrated structural modeling is employed to generate a "GLR-out" conformational ensemble. Likely interacting type II compounds are identified through virtual screening against this ensemble model. Biophysical validation of compound binding is demonstrated through protein thermal stabilization and ATP competition. Localization of a top-performing compound through surface methylation strongly suggests that STRADα can adopt the "GLR-out" conformation and interact with compounds that comply with the standard type II pharmacophore. These results suggest that, despite a loss of catalytic function, some pseudokinases, including STRADα, may retain the conformational switching properties of conventional protein kinases.

Epsins in vascular development, function and disease.

Epsins are a family of adaptor proteins involved in clathrin-dependent endocytosis. In the vasculature, epsins 1 and 2 are functionally redundant members of this family that are expressed in the endothelial cells of blood vessels and the lymphatic system throughout development and adulthood. These proteins contain a number of peptide motifs that allow them to interact with lipid moieties and a variety of proteins. These interactions facilitate the regulation of a wide range of cell signaling pathways. In this review, we focus on the involvement of epsins 1 and 2 in controlling vascular endothelial growth factor receptor signaling in angiogenesis and lymphangiogenesis. We also discuss the therapeutic implications of understanding the molecular mechanisms of epsin-mediated regulation in diseases such as atherosclerosis and diabetes.

HACS1 signaling adaptor protein recognizes a motif in the paired immunoglobulin receptor B cytoplasmic domain.

Hematopoietic adaptor containing SH3 and SAM domains-1 (HACS1) is a signaling protein with two juxtaposed protein-protein interaction domains and an intrinsically unstructured region that spans half the sequence. Here, we describe the interaction between the HACS1 SH3 domain and a sequence near the third immunoreceptor tyrosine-based inhibition motif (ITIM3) of the paired immunoglobulin receptor B (PIRB). From surface plasmon resonance binding assays using a mouse and human PIRB ITIM3 phosphopeptides as ligands, the HACS1 SH3 domain and SHP2 N-terminal SH2 domain demonstrated comparable affinities in the micromolar range. Since the PIRB ITIM3 sequence represents an atypical ligand for an SH3 domain, we determined the NMR structure of the HACS1 SH3 domain and performed a chemical shift mapping study. This study showed that the binding site on the HACS1 SH3 domain for PIRB shares many of the same amino acids found in a canonical binding cleft normally associated with polyproline ligands. Molecular modeling suggests that the respective binding sites in PIRB ITIM3 for the HACS1 SH3 domain and the SHP2 SH2 domain are too close to permit simultaneous binding. As a result, the HACS1-PIRB partnership has the potential to amalgamate signaling pathways that influence both immune and neuronal cell fate.

Structural modification of NADPH oxidase activator (Noxa 1) by oxidative stress: An experimental and computational study.

NADPH oxidases 1 (NOX1) derived reactive oxygen species (ROS) play an important role in the progression of cancer through signaling pathways. Therefore, in this paper, we demonstrate the effect of cold atmospheric plasma (CAP) on the structural changes of Noxa1 SH3 protein, one of the regulatory subunits of NOX1. For this purpose, firstly we purified the Noxa1 SH3 protein and analyzed the structure using X-ray crystallography, and subsequently, we treated the protein with two types of CAP reactors such as pulsed dielectric barrier discharge (DBD) and Soft Jet for different time intervals. The structural deformation of Noxa1 SH3 protein was analyzed by various experimental methods (circular dichroism, fluorescence, and NMR spectroscopy) and by MD simulations. Additionally, we demonstrate the effect of CAP (DBD and Soft Jet) on the viability and expression of NOX1 in A375 cancer cells. Our results are useful to understand the structural modification/oxidation occur in protein due to reactive oxygen and nitrogen (RONS) species generated by CAP.

Tks5 SH3 domains exhibit differential effects on invadopodia development.

The Src substrate Tks5 helps scaffold matrix-remodeling invadopodia in invasive cancer cells. Focus was directed here on how the five SH3 domains of Tks5 impact that activity. Mutations designed to inhibit protein-protein interactions were created in the individual SH3 domains of Tks5, and the constructs were introduced into the LNCaP prostate carcinoma cell line, a model system with intrinsically low Tks5 expression and which our lab had previously showed the dependence of Src-dependent Tks5 phosphorylation on invadopodia development. In LNCaP cells, acute increases in wild-type Tks5 led to increased gelatin matrix degradation. A similar result was observed when Tks5 was mutated in its 4th or 5th SH3 domains. This was in contrast to the 1st, 2nd, and 3rd SH3 domain mutations of Tks5 where each had a remarkable accentuating effect on gelatin degradation. Conversely, in the invadopodia-competent Src-3T3 model system, mutations in any one of the first three SH3 domains had a dominant negative effect that largely eliminated the presence of invadopodia, inhibited gelatin degradation activity, and redistributed both Src, cortactin, and Tks5 to what are likely endosomal compartments. A hypothesis involving Tks5 conformational states and the regulation of endosomal trafficking is presented as an explanation for these seemingly disparate results.

G-rich VEGF aptamer as a potential inhibitor of chitin trafficking signal in emerging opportunistic yeast infection.

The alarm is rang for friendly fire; Saccharomyces cerevisiae (S. cerevisiae) newfound as a fungal pathogen with an individual feature. S. cerevisiae has food safety and is not capable of producing infection but, when the host defenses are weakened, there is room for opportunistic S. cerevisiae strains to cause a health issues. Fungal diseases are challenging to treat because, unlike bacteria, the fungal are eukaryotes. Antibiotics only target prokaryotic cells, whereas compounds that kill fungi also harm the mammalian host. Small differences between mammalian and fungal cells regarding genes and proteins sequence and function make finding a drug target more challenging. Recently, Chitin synthase has been considered as a promising target for antifungal drug development as it is absent in mammals. In S. cerevisiae, CHS3, a class IV chitin synthase, produces 90% of the chitin and essential for cell growth. CHS3 from the trans-Golgi network to the plasma membrane requires assembly of the exomer complex (including proteins cargo such as CHS5, CHS6, Bach1, and Arf1). In this work, we performed SELEX (Systematic Evolution of Ligands by EXponential enrichment) as high throughput virtual screening of the RCSB data bank to find an aptamer as potential inhibit of the class IV chitin synthase of S. cerevisiae. Among all the candidates, G-rich VEGF (GVEGF) aptamer (PDB code: 2M53) containing locked sugar parts was observed as potential inhibitor of the assembly of CHS5-CHS6 exomer complex a subsequently block the chitin biosynthesis pathway as an effective anti-fungal. It was suggested from the simulation that an assembly of exomer core should begin CHS5-CHS6, not from CHS5-Bach1. It is notable that secondary structures of CHS6 and Bach1 was observed very similar, but they have only 25% identity at the amino acid sequence that exhibited different features in exomer assembly.

Inhibition of Cdc42-intersectin interaction by small molecule ZCL367 impedes cancer cell cycle progression, proliferation, migration, and tumor growth.

Cdc42 is a member of the Rho family of small GTPases that are at the crossroads of major oncogenic signaling pathways involved in both lung and prostate cancers. However, the therapeutic potential of Cdc42 regulation is still unclear due to the lack of pharmacological tools. Herein, we report that ZCL367 is a bona fide Cdc42 inhibitor that suppressed cancer development and ZCL278 can act as a partial Cdc42 agonist. In lung cancer cell lines with varying EGFR and Ras mutations as well as both androgen-independent and androgen-dependent prostate cancer cell lines, ZCL367 impeded cell cycle progression, reduced proliferation, and suppressed migration. ZCL367 decreased Cdc42-intersectin interactions and reduced Cdc42-mediated filopodia formation. ZCL367 showed increased potency and selectivity for Cdc42 when compared to Rac1 and RhoA. ZCL367 reduced A549 tumorigenesis in a xenograft mouse model. Altogether, ZCL367 is a selective Cdc42 inhibitor and an excellent candidate for lead compound optimization for further anticancer studies.

AP180 N-Terminal Homology (ANTH) and Epsin N-Terminal Homology (ENTH) Domains: Physiological Functions and Involvement in Disease.

The AP180 N-terminal homology (ANTH) and Epsin N-terminal homology (ENTH) domains are crucially involved in membrane budding processes. All the ANTH/ENTH-containing proteins share the phosphoinositide-binding activity and can interact with clathrin or its related proteins via multiple binding motifs. Their function also include promotion of clathrin assembly, induction of membrane curvature, and recruitment of various effector proteins, such as those involved in membrane fission. Furthermore, they play a role in the sorting of specific cargo proteins, thereby enabling the cargos to be accurately transported and function at their appropriate locations. As the structural bases underlying these functions are clarified, contrary to their apparent similarity, the mechanisms by which these proteins recognize lipids and proteins have unexpectedly been found to differ from each other. In addition, studies using knockout mice have suggested that their physiological roles may be more complicated than merely supporting membrane budding processes. In this chapter, we review the current knowledge on the biochemical features of ANTH/ENTH domains, their functions predicted from the phenotypes of animals deficient in these domain-containing proteins, and recent findings on the structural basis enabling specific recognition of their ligands. We also discuss the association of these domains with human diseases. Here we focus on CALM, a protein containing an ANTH domain, which is implicated in the pathogenesis of blood cancers and Alzheimer disease, and discuss how alteration of CALM function is involved in these diseases.

Dimerization of sortilin regulates its trafficking to extracellular vesicles.

Extracellular vesicles (EVs) play a critical role in intercellular communication by transferring microRNAs, lipids, and proteins to neighboring cells. Sortilin, a sorting receptor that directs target proteins to the secretory or endocytic compartments of cells, is found in both EVs and cells. In many human diseases, including cancer and cardiovascular disorders, sortilin expression levels are atypically high. To elucidate the relationship between cardiovascular disease, particularly vascular calcification, and sortilin expression levels, we explored the trafficking of sortilin in both the intracellular and extracellular milieu. We previously demonstrated that sortilin promotes vascular calcification via its trafficking of tissue-nonspecific alkaline phosphatase to EVs. Although recent reports have noted that sortilin is regulated by multiple post-translational modifications, the precise mechanisms of sortilin trafficking still need to be determined. Here, we show that sortilin forms homodimers with an intermolecular disulfide bond at the cysteine 783 (Cys783) residue, and because Cys783 can be palmitoylated, it could be shared via palmitoylation and an intermolecular disulfide bond. Formation of this intermolecular disulfide bond leads to trafficking of sortilin to EVs by preventing palmitoylation, which further promotes sortilin trafficking to the Golgi apparatus. Moreover, we found that sortilin-derived propeptide decreased sortilin homodimers within EVs. In conclusion, sortilin is transported to EVs via the formation of homodimers with an intermolecular disulfide bond, which is endogenously regulated by its own propeptide. Therefore, we propose that inhibiting dimerization of sortilin acts as a new therapeutic strategy for the treatment of EV-associated diseases, including vascular calcification and cancer.

Therapeutic efficacy of a synthetic epsin mimetic peptide in glioma tumor model: uncovering multiple mechanisms beyond the VEGF-associated tumor angiogenesis.

Binding of epsin ubiquitin-interacting motif (UIM) with ubiquitylated VEGFR2 is a critical mechanism for epsin-dependent VEGFR2 endocytosis and physiological angiogenesis. Deletion of epsins in vessel endothelium produces uncontrolled tumor angiogenesis and retards tumor growth in animal models. The aim of this study is to test the therapeutic efficacy and targeting specificity of a chemically-synthesized peptide, UPI, which compete for epsin binding sites in VEGFR2 and potentially inhibits Epsin-VEGFR2 interaction in vivo, in an attempt to reproduce an epsin-deficient phenotype in tumor angiogenesis. Our data show that UPI treatment significantly inhibits and shrinks tumor growth in GL261 glioma tumor model. UPI peptide specifically targets VEGFR2 signaling pathway revealed by genetic and biochemical approaches. Furthermore, we demonstrated that UPI peptide treatment caused serious thrombosis in tumor vessels and damages tumor cells after a long-term UPI peptide administration. Besides, we revealed that UPI peptides were unexpectedly targeted cancer cells and induced apoptosis. We conclude that UPI peptide is a potent inhibitor to glioma tumor growth through specific targeting of VEGFR2 signaling in the tumor vasculature and cancer cells, which may offer a potentially novel treatment for cancer patients who are resistant to current anti-VEGF therapies.

Downregulation of Paralemmin-3 Ameliorates Lipopolysaccharide-Induced Acute Lung Injury in Rats by Regulating Inflammatory Response and Inhibiting Formation of TLR4/MyD88 and TLR4/TRIF Complexes.

Previous studies have demonstrated paralemmin-3 (PALM3) participates in Toll-like receptor (TLR) signaling. This study investigated the effect of PALM3 knockdown on lipopolysaccharide (LPS)-induced acute lung injury (ALI) and its underlying mechanisms. We constructed a recombinant adenoviral vector containing short hairpin RNA for PALM3 to knockdown PALM3 expression. A transgene-free adenoviral vector was used as a negative control. The ALI rat model was established by LPS peritoneal injection at 48-h post-transfection. Results showed that downregulation of PALM3 improved the survival rate, attenuated lung pathological changes, alleviated pulmonary edema, lung vascular leakage and neutrophil infiltration, inhibited the production of proinflammatory cytokines and activation of nuclear factor κB and interferon ß regulatory factor 3, and promoted the secretion of anti-inflammatory cytokine interleukin-10 and expression of suppressor of cytokine signaling-3 in the ALI rat model. However, PALM3 knockdown had no effect on TLR4, myeloid differentiation factor 88 (MyD88), and Toll-interleukin-1 receptor domain-containing adaptor inducing interferon ß (TRIF) expression. Moreover, PALM3 knockdown reduced the interaction of TLR4 with MyD88 or TRIF induced by LPS in rat lungs. Therefore, the downregulation of PALM3 protected rats from LPS-induced ALI and its mechanisms were partially associated with the modulation of inflammatory responses and inhibition of TLR4/MyD88 and TLR4/TRIF complex formation.

The perlecan-interacting growth factor progranulin regulates ubiquitination, sorting, and lysosomal degradation of sortilin.

Despite extensive clinical and experimental studies over the past decades, the pathogenesis and progression to the castration-resistant stage of prostate cancer remains largely unknown. Progranulin, a secreted growth factor, strongly binds the heparin-sulfate proteoglycan perlecan, and counteracts its biological activity. We established that progranulin acts as an autocrine growth factor and promotes prostate cancer cell motility, invasion, and anchorage-independent growth. Progranulin was overexpressed in prostate cancer tissues vis-à-vis non-neoplastic tissues supporting the hypothesis that progranulin may play a key role in prostate cancer progression. However, progranulin's mode of action is not well understood and proteins regulating progranulin signaling have not been identified. Sortilin, a single-pass type I transmembrane protein of the Vps10 family, binds progranulin in neurons and targets progranulin for lysosomal degradation. Significantly, in DU145 and PC3 cells, we detected very low levels of sortilin associated with high levels of progranulin production and enhanced motility. Restoring sortilin expression decreased progranulin levels, inhibited motility and anchorage-independent growth and destabilized Akt. These results demonstrated a critical role for sortilin in regulating progranulin and suggest that sortilin loss may contribute to prostate cancer progression. Here, we provide the novel observation that progranulin downregulated sortilin protein levels independent of transcription. Progranulin induced sortilin ubiquitination, internalization via clathrin-dependent endocytosis and sorting into early endosomes for lysosomal degradation. Collectively, these results constitute a regulatory feed-back mechanism whereby sortilin downregulation ensures sustained progranulin-mediated oncogenesis.

TIR-domain-containing adapter-inducing interferon-ß (TRIF) forms filamentous structures, whose pro-apoptotic signalling is terminated by autophagy.

The formation of amyloid-like protein structures has recently emerged as a feature in signal transduction, particularly in innate immunity. These structures appear to depend on defined domains for their formation but likely also require dedicated ways to terminate signalling. We, here, define the innate immunity protein/Toll-like receptor adaptor TIR-domain-containing adapter-inducing interferon-ß (TRIF) as a novel platform of fibril formation and probe signal initiation through TRIF as well as its termination in Toll-like receptor 3 (TLR3)-stimulated melanoma cells. A main signalling pathway triggered by TLR3 caused apoptosis, which was controlled by inhibitor of apoptosis proteins and was dependent on RIPK1 and independent of TNF. Using correlative electron/fluorescence microscopy, we visualised fibrillar structures formed through both Toll/interleukin-1 receptor and RIP homotypic interacting motif regions of TRIF. We provide evidence that these fibrillary structures are active signalling platforms whose activity is terminated by autophagy. TRIF-signalling enhanced autophagy, and fibrillary structures were partly contained within autophagosomes. Inhibition of autophagy increased levels of pro-apoptotic TRIF complexes, leading to the accumulation of active caspase-8 and enhanced apoptosis while stimulation of autophagy reduced TRIF-dependent death. We conclude that pro-death signals through TRIF are regulated by autophagy and propose that pro-apoptotic signalling through TRIF/RIPK1/caspase-8 occurs in fibrillary platforms.

Structural Characterization of the p75 Neurotrophin Receptor: A Stranger in the TNFR Superfamily.

Although p75 neurotrophin receptor (p75NTR) was the founding member of the tumor necrosis factor (TNF) receptor superfamily (TNFRSF), it is an atypical TNFRSF protein. p75NTR like TNF-R1 and Fas-R contain an extracellular domain with four cysteine-rich domains (CRD) and a death domain (DD) in the intracellular region. While TNFRSF proteins are activated by trimeric TNFSF ligands, p75NTR forms dimers activated by dimeric neurotrophins that are structurally unrelated to TNFSF proteins. In addition, although p75NTR shares with other members the interaction with the TNF receptor-associated factors to activate the NF-κB and cell death pathways, p75NTR does not interact with the DD-containing proteins FADD, TRADD, or MyD88. By contrast, the DD of p75NTR is able to recruit several protein interactors via a full catalog of DD interactions not described before in the TNFRSF. p75-DD forms homotypic symmetrical DD-DD complexes with itself and with the related p45-DD; forms heterotypic DD-CARD interactions with the RIP2-CARD domain, and forms a new interaction between a DD and RhoGDI. All these features, in addition to its promiscuous interactions with several ligands and coreceptors, its processing by α- and γ-secretases, the dimeric nature of its transmembrane domain and its "special" juxtamembrane region, make p75NTR a truly stranger in the TNFR superfamily. In this chapter, I will summarize the known structural aspects of p75NTR and I will analyze from a structural point of view, the similitudes and differences between p75NTR and the other members of the TNFRSF.