Struktur und Funktion des menschlichen Autophagieproteins ATG16L1

Autophagy is a conserved eukaryotic mechanism for the sequestration and degradation of cellular components. Apart from its classical role, autophagy is involved in human immunity, especially in capturing and clearance of intracellular pathogens. One of the key players in autophagy, ATG16L1, recently came into focus of interest due to mutations in the gene locus that result in increased susceptibility to the inflammatory bowel disease Morbus Crohn. ATG16L1, in complex with autophagy-related proteins ATG5 and ATG12, localizes on the autophagosome and has also been shown to be recruited by the cytosolic pattern recognition receptors NOD1 and NOD2 to the entry site of cell invading bacteria, thus linking autophagy with the intracellular bacterial sensing. Similarly to the yeast ATG16, human ATG16L1 has an ATG5 binding domain and a coiled coil domain, which are essential and sufficient for functional canonical autophagy. In addition, human ATG16L1 has a C-terminal domain which is absent in its yeast homologue. Precisely this domain was shown to interact with NOD1 and NOD2, in addition to TLR2, TMEM59 and DDB1. Under the experimental conditions tested in the course of this work, these interactions could not be detected. Further work is needed to identify possible factors missing in the experimental set up and detect mediating factors essential for macromolecular interactions with the C-terminal domain of the human ATG16L1. C-terminal domain of human ATG16L1 was used for crystallographic studies and a high resolution structure of this domain was solved. Structure of the C-terminal domain of human ATG16L1 revealed that it folds into a typical WD-40 propeller with seven blades, each composed of four anti-parallel beta-strands. The WD-40 proteins are a large family found in all eukaryotes and usually serve as interaction platform for peptides, nucleic acids and other proteins. Detailed analysis of the structure identified a region on the WD-40 domain of ATG16L1 with high flexibility and a possible functional relevance. Additionally, two symmetry related molecules of the WD-40 domain of ATG16L1 bury a large surface area, which might be physiologically important in mediating dimerization of the WD-40 domains. Based on these observations, the WD-40 domain of ATG16L1 might form a WD-40/WD-40 interaction surface for other macromolecules and peptides, making this domain an interesting candidate for an intersection between autophagy and other cellular processes.Autophagie ist ein konservierter eukaryotischer Mechanismus für die Sequestrierung und den Abbau von intrazellulären Komponenten. Außerdem ist Autophagie an zahlreichen Prozessen im Kontext der menschlichen Immunität beteiligt, insb. an der Erkennung und Beseitigung von intrazellulären Pathogenen. Einer der wichtigsten Akteure in Autophagie, ATG16L1, rückte vor kurzem in den Fokus des Interesses aufgrund von Mutationen im Gen-Locus, welche zu einer erhöhten Anfälligkeit für die entzündliche Darmerkrankung Morbus Crohn führen. ATG16L1, im Komplex mit den autophagieverwandten Proteinen ATG5 und ATG12, wird durch die cytosolischen Muster erkennenden Rezeptoren NOD1 und NOD2 zur Eintrittstelle von invasiven Bakterien rekrutiert, was eine direkte Verbindung zwischen Autophagie und der Erkennung intrazellulärer Bakterien aufzeigt. Ähnlich wie Hefe-ATG16 hat menschliches ATG16L1 eine N-terminale ATG5 Bindungsdomäne und eine Coiled-Coil-Domäne, welche für eine funktionierende kanonische Autophagie notwendig und ausreichend sind. Zusätzlich besitzt menschliches ATG16L1 eine C-terminale Domäne, die im Hefe-ATG16 fehlt. Gerade bei dieser Domäne wurde eine Interaktion mit NOD1 und NOD2, neben TLR2, TMEM59 und DDB1 nachgewiesen. Unter den verwendeten Versuchsbedingungen konnten diese Wechselwirkungen jedoch nicht bestätigt werden. Zusätzlich wurde die Struktur der C-terminale Domäne des menschlichen ATG16L1 aufgeklärt. Diese hochaufgelöste Struktur zeigt, dass die Faltung der Domäne der eines WD-40 Propeller mit sieben Propellerblättern entspricht, wobei sich jedes Blatt aus vier antiparallelen beta-Strängen zusammensetzt. Die WD-40-Proteine sind eine große Familie, die in allen Eukaryoten präsent ist und in der Regel als Interaktionsplattform für Peptide, Nukleinsäuren und andere Proteine dient. Durch eine Detailanlayse der Struktur wurde eine Region auf der WD-40-Domäne des ATG16L1 mit hoher Flexibilität und einer möglichen funktionellen Bedeutung identifiziert. Zusätzlich wurde eine durch symmetrie-verwandte Moleküle der WD-40-Domäne generierte Interaktionsfläche identifiziert, die eine Dimerisierung der WD-40 vermitteln und von physiologischer Relevanz sein könnte. Basierend hierauf könnte die WD-40 Domäne von ATG16L1 eine WD-40/WD-40 Interaktionsfläche für andere Makromoleküle und Peptide bilden, so dass diese Domäne einen interessanten Kandidaten für die Funktion als Bindeglied zwischen Autophagie und anderen zellulären Prozessen darstellt

UL36 Rescues Apoptosis Inhibition and In vivo Replication of a Chimeric MCMV Lacking the M36 Gene.

Apoptosis is an important defense mechanism mounted by the immune system to control virus replication. Hence, cytomegaloviruses (CMV) evolved and acquired numerous anti-apoptotic genes. The product of the human CMV (HCMV) UL36 gene, pUL36 (also known as vICA), binds to pro-caspase-8, thus inhibiting death-receptor apoptosis and enabling viral replication in differentiated THP-1 cells. In vivo studies of the function of HCMV genes are severely limited due to the strict host specificity of cytomegaloviruses, but CMV orthologues that co-evolved with other species allow the experimental study of CMV biology in vivo. The mouse CMV (MCMV) homolog of the UL36 gene is called M36, and its protein product (pM36) is a functional homolog of vICA that binds to murine caspase-8 and inhibits its activation. M36-deficient MCMV is severely growth impaired in macrophages and in vivo. Here we show that pUL36 binds to the murine pro-caspase-8, and that UL36 expression inhibits death-receptor apoptosis in murine cells and can replace M36 to allow MCMV growth in vitro and in vivo. We generated a chimeric MCMV expressing the UL36 ORF sequence instead of the M36 one. The newly generated MCMV(UL36) inhibited apoptosis in macrophage lines RAW 264.7, J774A.1, and IC-21 and its growth was rescued to wild type levels. Similarly, growth was rescued in vivo in the liver and spleen, but only partially in the salivary glands of BALB/c and C57BL/6 mice. In conclusion, we determined that an immune-evasive HCMV gene is conserved enough to functionally replace its MCMV counterpart and thus allow its study in an in vivo setting. As UL36 and M36 proteins engage the same molecular host target, our newly developed model can facilitate studies of anti-viral compounds targeting pUL36 in vivo

Autophagy proteins stabilize pathogen-containing phagosomes for prolonged MHC II antigen processing

Antigen preservation for presentation is a hallmark of potent antigen-presenting cells. In this paper, we report that in human macrophages and dendritic cells, a subset of phagosomes gets coated with Atg8/LC3, a component of the molecular machinery of macroautophagy, and maintains phagocytosed antigens for prolonged presentation on major histocompatibility complex class II molecules. These Atg8/LC3-positive phagosomes are formed around the antigen with TLR2 agonists and require reactive oxygen species production by NOX2 for their generation. A deficiency in the NOX2-dependent formation of these antigen storage phagosomes could contribute to compromise antifungal immune control in chronic granulomatous disease patients

UL36 Rescues Apoptosis Inhibition and In vivo Replication of a Chimeric MCMV Lacking the M36 Gene

Apoptosis is an important defense mechanism mounted by the immune system to control virus replication. Hence, cytomegaloviruses (CMV) evolved and acquired numerous anti-apoptotic genes. The product of the human CMV (HCMV) UL36 gene, pUL36 (also known as vICA), binds to pro-caspase-8, thus inhibiting death-receptor apoptosis and enabling viral replication in differentiated THP-1 cells. In vivo studies of the function of HCMV genes are severely limited due to the strict host specificity of cytomegaloviruses, but CMV orthologues that co-evolved with other species allow the experimental study of CMV biology in vivo. The mouse CMV (MCMV) homolog of the UL36 gene is called M36, and its protein product (pM36) is a functional homolog of vICA that binds to murine caspase-8 and inhibits its activation. M36-deficient MCMV is severely growth impaired in macrophages and in vivo. Here we show that pUL36 binds to the murine pro-caspase-8, and that UL36 expression inhibits death-receptor apoptosis in murine cells and can replace M36 to allow MCMV growth in vitro and in vivo. We generated a chimeric MCMV expressing the UL36 ORF sequence instead of the M36 one. The newly generated MCMVUL36 inhibited apoptosis in macrophage lines RAW 264.7, J774A.1, and IC-21 and its growth was rescued to wild type levels. Similarly, growth was rescued in vivo in the liver and spleen, but only partially in the salivary glands of BALB/c and C57BL/6 mice. In conclusion, we determined that an immune-evasive HCMV gene is conserved enough to functionally replace its MCMV counterpart and thus allow its study in an in vivo setting. As UL36 and M36 proteins engage the same molecular host target, our newly developed model can facilitate studies of anti-viral compounds targeting pUL36 in vivo