Expression of pattern recognition receptors by oral epithelial cell lines

Sublinguale Immuntherapie (SLIT) ist eine sichere und effektive Möglichkeit der Behandlung von Typ I Allergien. Die dieser Behandlung zugrunde liegenden Immunmechanismen sind derzeit noch nicht gänzlich aufgeklärt. Detailliertes Wissen über mögliche Immunreaktionen in der Mundhöhle trägt zur Aufklärung der Immunmechanismen von SLIT bei. Ziel der vorliegenden Masterarbeit war daher die Analyse der Expression von diversen Pathogen-recognition Rezeptoren (PRR) auf bukkalen und sublingualen Epithelzellen. Hierfür wurden zwei humane Zelllinien (HO-1-N-1 bzw. HO-1-u-1) eingesetzt. Zusätzlich wurden sämtliche Experimente mit den gängigen intestinalen Epithelzelllinien Caco-2/15 und Caco-2 A9 durchgeführt. Mittels semiquantitativer Polymerase-Kettenreaktion (polymerase chain reaction, PCR) und quantitativer „real-time“ PCR (qPCR) wurde die mRNA Expression der Toll-like Rezeptoren (TLR) 1-10, C-type Lectin Rezeptoren DC-SIGN, Dectin-1 und Dectin-2 sowie der NOD-like Rezeptoren NOD1 bzw. NOD2 bestimmt. Des Weiteren wurden die Zellen mit Rezeptor-spezifischen Liganden stimuliert, und die im Falle einer Aktivierung der Signaltransduktionswege erhöhte Menge an ausgeschiedenem IL-8 mittels Enyzme-linked immunosorbent Assay (ELISA) bestimmt. Die bukkale Zelllinie HO-1-N-1 exprimierte mRNA für TLR1, TLR2, TLR4 und TLR6 sowie für NOD1 und NOD2 und reagierte auf Stimulation dieser Rezeptoren mit erhöhter IL-8 Sekretion. Die Resultate der qPCR ließen weiters die Expression von TLR3, TLR8, TLR10 und Dectin-1 vermuten, doch die HO-1-N-1 Zellen reagierten nicht auf die entsprechenden Liganden. Die sublinguale Zelllinie HO-1-u-1 exprimierte mRNA für TLR1, TLR3, TLR4, TLR5 und TLR6 sowie für NOD1. Die Resultate der qPCR ließen weiters auf Expression von TLR2, TLR7, TLR8, TLR10 und Dectin-1 schließen. Erhöhte IL-8 Ausschüttung konnte jedoch nur nach Stimulation mit TLR3-, TLR5- und NOD1-spezifischen Liganden beobachtet werden. Die intestinalen Caco-2/15 und Caco-2 A9 Zelllinien exprimierten mRNA für alle 10 bekannten humanen TLRs sowie für NOD1. Zusätzlich konnte bei der Caco-2/15 Zelllinie Dectin-2 mRNA nachgewiesen werden. Erhöhte IL-8 Produktion wurde nach Stimulation mit TLR1/2-, TLR2/6- und TLR5-spezifischen Liganden gemessen. Zusammenfassend konnte beobachtet werden, dass bukkale und sublinguale Epithelzellen unterschiedliche Mustererkennungsrezeptoren exprimieren und daher auf unterschiedliche Liganden reagieren. Das Liganden-Spektrum der Zelllinie HO-1-N-1 deutet darauf hin, dass bukkale Epithelzellen hauptsächlich auf die Erkennung von Bakterien spezialisiert sind. Sublinguale Epithelzellen reagieren hingegen auf eine geringere Anzahl an Liganden, dafür waren diese sowohl bakteriellen als auch viralen Ursprungs.Sublingual immunotherapy (SLIT) is a safe and effective option for treatment of type I allergy. Still, the immune mechanisms underlying SLIT are not completely understood. In this context, the aim of the master thesis was to analyse the expression of toll-like receptor (TLR) 1-10, C-type lectin receptor DC-SIGN, Dectin-1 and Dectin-2 and NOD-like receptor NOD1 and NOD2 by human epithelial cells. For this purpose, a buccal mucosa cell line, HO-1-N-1, a sublingual epithelial cell line, HO-1-u-1, and two intestinal Caco-2 cell lines were employed. mRNA expression of the different receptors was analysed by reverse-transcription PCR (RT-PCR) and quantitative real-time RT-PCR (qPCR). Moreover, all epithelial cell lines were stimulated with ligands specific for the respective receptors. IL-8 secretion as a readout for the activation of their signalling pathways was determined by ELISA. HO-1-N-1 cells expressed mRNA for TLR1, TLR2, TLR4, TLR6 as well as NOD1 and NOD2 and responded with increased IL-8 synthesis to ligands specific for these receptors. qPCR also indicated the expression of TLR3, TLR8, TLR10 and Dectin-1 in HO-1-N-1. However, the cells were not activated by ligands specific for these receptors. Analysing the HO-1-u-1 cell line, mRNA coding for TLR1, TLR3, TLR4, TLR5, TLR6 and NOD1 was detected. qPCR also indicated expression of TLR2, TLR7, TLR8, TLR10 and Dectin-1. However, HO-1-u-1 cells only responded to ligands targeting TLR3, TLR5 and NOD1. In addition to the epithelial cell lines in the mouth, all experiments were performed with the well-established colorectal epithelial cell lines Caco-2/15 and Caco-2 A9. These cells expressed mRNA for all 10 currently known human TLRs as well as for NOD1. Additionally, mRNA coding for Dectin-2 was detected in Caco-2/15 by qPCR. Analysing the functional response, Caco-2 cells showed IL-8 production upon stimulation with ligands for TLR1/2, TLR2/6 and for TLR5. In conclusion, we found that sublingual and buccal cells show differences in the expression of pattern recognition receptors and respond to stimulation with microbial ligands in a tissue-specific fashion. The spectrum of ligands activating the buccal cell line HO-1-N-1 indicates that these cells are basically specialised in recognising bacterial compounds. The sublingual epithelial cell line HO-1-u-1 responded to fewer ligands but within a broader spectrum, comprising viral dsRNA, bacterial flagellin and peptidoglycan

Atg4 proteolytic activity can be inhibited by Atg1 phosphorylation

The biogenesis of autophagosomes depends on the conjugation of Atg8-like proteins with phosphatidylethanolamine. Atg8 processing by the cysteine protease Atg4 is required for its covalent linkage to phosphatidylethanolamine, but it is also necessary for Atg8 deconjugation from this lipid to release it from membranes. How these two cleavage steps are coordinated is unknown. Here we show that phosphorylation by Atg1 inhibits Atg4 function, an event that appears to exclusively occur at the site of autophagosome biogenesis. These results are consistent with a model where the Atg8-phosphatidylethanolamine pool essential for autophagosome formation is protected at least in part by Atg4 phosphorylation by Atg1 while newly synthesized cytoplasmic Atg8 remains susceptible to constitutive Atg4 processing

TECPR1 conjugates LC3 to damaged endomembranes upon detection of sphingomyelin exposure.

Invasive bacteria enter the cytosol of host cells through initial uptake into bacteria-containing vacuoles (BCVs) and subsequent rupture of the BCV membrane, thereby exposing to the cytosol intraluminal, otherwise shielded danger signals such as glycans and sphingomyelin. The detection of glycans by galectin-8 triggers anti-bacterial autophagy, but how cells sense and respond to cytosolically exposed sphingomyelin remains unknown. Here, we identify TECPR1 (tectonin beta-propeller repeat containing 1) as a receptor for cytosolically exposed sphingomyelin, which recruits ATG5 into an E3 ligase complex that mediates lipid conjugation of LC3 independently of ATG16L1. TECPR1 binds sphingomyelin through its N-terminal DysF domain (N'DysF), a feature not shared by other mammalian DysF domains. Solving the crystal structure of N'DysF, we identified key residues required for the interaction, including a solvent-exposed tryptophan (W154) essential for binding to sphingomyelin-positive membranes and the conjugation of LC3 to lipids. Specificity of the ATG5/ATG12-E3 ligase responsible for the conjugation of LC3 is therefore conferred by interchangeable receptor subunits, that is, the canonical ATG16L1 and the sphingomyelin-specific TECPR1, in an arrangement reminiscent of certain multi-subunit ubiquitin E3 ligases

Reconstitution of autophagosome nucleation defines Atg9 vesicles as seeds for membrane formation

Autophagosomes form de novo in a manner that is incompletely understood. Particularly enigmatic are autophagy-related protein 9 (Atg9)-containing vesicles that are required for autophagy machinery assembly but do not supply the bulk of the autophagosomal membrane. In this study, we reconstituted autophagosome nucleation using recombinant components from yeast. We found that Atg9 proteoliposomes first recruited the phosphatidylinositol 3-phosphate kinase complex, followed by Atg21, the Atg2-Atg18 lipid transfer complex, and the E3-like Atg12-Atg5-Atg16 complex, which promoted Atg8 lipidation. Furthermore, we found that Atg2 could transfer lipids for Atg8 lipidation. In selective autophagy, these reactions could potentially be coupled to the cargo via the Atg19-Atg11-Atg9 interactions. We thus propose that Atg9 vesicles form seeds that establish membrane contact sites to initiate lipid transfer from compartments such as the endoplasmic reticulum

Conserved Atg8 recognition sites mediate Atg4 association with autophagosomal membranes and Atg8 deconjugation

Deconjugation of the Atg8/LC3 protein family members from phosphatidylethanolamine (PE) by Atg4 proteases is essential for autophagy progression, but how this event is regulated remains to be understood. Here, we show that yeast Atg4 is recruited onto autophagosomal membranes by direct binding to Atg8 via two evolutionarily conserved Atg8 recognition sites, a classical LC3-interacting region (LIR) at the C-terminus of the protein and a novel motif at the N-terminus. Although both sites are important for Atg4-Atg8 interaction in vivo, only the new N-terminal motif, close to the catalytic center, plays a key role in Atg4 recruitment to autophagosomal membranes and specific Atg8 deconjugation. We thus propose a model where Atg4 activity on autophagosomal membranes depends on the cooperative action of at least two sites within Atg4, in which one functions as a constitutive Atg8 binding module, while the other has a preference toward PE-bound Atg8

Conserved Atg8 recognition sites mediate Atg4 association with autophagosomal membranes and Atg8 deconjugation

Deconjugation of the Atg8/LC3 protein family members from phosphatidylethanolamine (PE) by Atg4 proteases is essential for autophagy progression, but how this event is regulated remains to be understood. Here, we show that yeast Atg4 is recruited onto autophagosomal membranes by direct binding to Atg8 via two evolutionarily conserved Atg8 recognition sites, a classical LC3-interacting region (LIR) at the C-terminus of the protein and a novel motif at the N-terminus. Although both sites are important for Atg4-Atg8 interaction in vivo, only the new N-terminal motif, close to the catalytic center, plays a key role in Atg4 recruitment to autophagosomal membranes and specific Atg8 deconjugation. We thus propose a model where Atg4 activity on autophagosomal membranes depends on the cooperative action of at least two sites within Atg4, in which one functions as a constitutive Atg8 binding module, while the other has a preference toward PE-bound Atg8

How RB1CC1/FIP200 claws its way to autophagic engulfment of SQSTM1/p62-ubiquitin condensates.

Macroautophagy/autophagy mediates the degradation of ubiquitinated aggregated proteins within lysosomes in a process known as aggrephagy. The cargo receptor SQSTM1/p62 condenses aggregated proteins into larger structures and links them to the nascent autophagosomal membrane (phagophore). How the condensation reaction and autophagosome formation are coupled is unclear. We recently discovered that a region of SQSTM1 containing its LIR motif directly interacts with RB1CC1/FIP200, a protein acting at early stages of autophagosome formation. Determination of the structure of the C-terminal region of RB1CC1 revealed a claw-shaped domain. Using a structure-function approach, we show that the interaction of SQSTM1 with the RB1CC1 claw domain is crucial for the productive recruitment of the autophagy machinery to ubiquitin-positive condensates and their subsequent degradation by autophagy. We also found that concentrated Atg8-family proteins on the phagophore displace RB1CC1 from SQSTM1, suggesting an intrinsic directionality in the process of autophagosome formation. Ultimately, our study reveals how the interplay of SQSTM1 and RB1CC1 couples cargo condensation to autophagosome formation

FIP200 Claw Domain Binding to p62 Promotes Autophagosome Formation at Ubiquitin Condensates.

The autophagy cargo receptor p62 facilitates the condensation of misfolded, ubiquitin-positive proteins and their degradation by autophagy, but the molecular mechanism of p62 signaling to the core autophagy machinery is unclear. Here, we show that disordered residues 326-380 of p62 directly interact with the C-terminal region (CTR) of FIP200. Crystal structure determination shows that the FIP200 CTR contains a dimeric globular domain that we designated the "Claw" for its shape. The interaction of p62 with FIP200 is mediated by a positively charged pocket in the Claw, enhanced by p62 phosphorylation, mutually exclusive with the binding of p62 to LC3B, and it promotes degradation of ubiquitinated cargo by autophagy. Furthermore, the recruitment of the FIP200 CTR slows the phase separation of ubiquitinated proteins by p62 in a reconstituted system. Our data provide the molecular basis for a crosstalk between cargo condensation and autophagosome formation

FIP200 Claw Domain Binding to p62 Promotes Autophagosome Formation at Ubiquitin Condensates.

The autophagy cargo receptor p62 facilitates the condensation of misfolded, ubiquitin-positive proteins and their degradation by autophagy, but the molecular mechanism of p62 signaling to the core autophagy machinery is unclear. Here, we show that disordered residues 326-380 of p62 directly interact with the C-terminal region (CTR) of FIP200. Crystal structure determination shows that the FIP200 CTR contains a dimeric globular domain that we designated the "Claw" for its shape. The interaction of p62 with FIP200 is mediated by a positively charged pocket in the Claw, enhanced by p62 phosphorylation, mutually exclusive with the binding of p62 to LC3B, and it promotes degradation of ubiquitinated cargo by autophagy. Furthermore, the recruitment of the FIP200 CTR slows the phase separation of ubiquitinated proteins by p62 in a reconstituted system. Our data provide the molecular basis for a crosstalk between cargo condensation and autophagosome formation