Role of the AP2 β-Appendage Hub in Recruiting Partners for Clathrin-Coated Vesicle Assembly

Adaptor protein complex 2 α and β-appendage domains act as hubs for the assembly of accessory protein networks involved in clathrin-coated vesicle formation. We identify a large repertoire of β-appendage interactors by mass spectrometry. These interact with two distinct ligand interaction sites on the β-appendage (the “top” and “side” sites) that bind motifs distinct from those previously identified on the α-appendage. We solved the structure of the β-appendage with a peptide from the accessory protein Eps15 bound to the side site and with a peptide from the accessory cargo adaptor β-arrestin bound to the top site. We show that accessory proteins can bind simultaneously to multiple appendages, allowing these to cooperate in enhancing ligand avidities that appear to be irreversible in vitro. We now propose that clathrin, which interacts with the β-appendage, achieves ligand displacement in vivo by self-polymerisation as the coated pit matures. This changes the interaction environment from liquid-phase, affinity-driven interactions, to interactions driven by solid-phase stability (“matricity”). Accessory proteins that interact solely with the appendages are thereby displaced to areas of the coated pit where clathrin has not yet polymerised. However, proteins such as β-arrestin (non-visual arrestin) and autosomal recessive hypercholesterolemia protein, which have direct clathrin interactions, will remain in the coated pits with their interacting receptors

Covalent Protein Modification with ISG15 via a Conserved Cysteine in the Hinge Region

The ubiquitin-like protein ISG15 (interferon-stimulated gene of 15 kDa) is strongly induced by type I interferons and displays antiviral activity. As other ubiquitin-like proteins (Ubls), ISG15 is post-translationally conjugated to substrate proteins by an isopeptide bond between the C-terminal glycine of ISG15 and the side chains of lysine residues in the substrates (ISGylation). ISG15 consists of two ubiquitin-like domains that are separated by a hinge region. In many orthologs, this region contains a single highly reactive cysteine residue. Several hundred potential substrates for ISGylation have been identified but only a few of them have been rigorously verified. In order to investigate the modification of several ISG15 substrates, we have purified ISG15 conjugates from cell extracts by metal-chelate affinity purification and immunoprecipitations. We found that the levels of proteins modified by human ISG15 can be decreased by the addition of reducing agents. With the help of thiol blocking reagents, a mutational analysis and miRNA mediated knock-down of ISG15 expression, we revealed that this modification occurs in living cells via a disulphide bridge between the substrates and Cys78 in the hinge region of ISG15. While the ISG15 activating enzyme UBE1L is conjugated by ISG15 in the classical way, we show that the ubiquitin conjugating enzyme Ubc13 can either be classically conjugated by ISG15 or can form a disulphide bridge with ISG15 at the active site cysteine 87. The latter modification would interfere with its function as ubiquitin conjugating enzyme. However, we found no evidence for an ISG15 modification of the dynamin-like GTPases MxA and hGBP1. These findings indicate that the analysis of potential substrates for ISG15 conjugation must be performed with great care to distinguish between the two types of modification since many assays such as immunoprecipitation or metal-chelate affinity purification are performed with little or no reducing agent present

Das humane Guanylat-bindende Protein 1

Ziel der Arbeit war die Aufklärung des Mechanismus der Hydrolyse von GTP durch das humane Guanylat-bindende Protein 1 (hGBP1). Die Hydrolyse von GTP zu GMP erfolgt unter sequentieller Abspaltung der zwei Phosphatgruppen. Die spezifische Aktivität des Proteins nimmt aufgrund nukleotidabhängiger Oligomerisierung als Funktion der Konzentration zu. Die Dissoziationskonstanten der GuaninnukIeotide liegen im Bereich von 1 - 15 $\mu$M. Die Struktur von nukleotidfreiem hGBP1 und im Komplex mit einem GTP-Analogon wurde aufgeklärt und diente zum gezielten Entwurf von Punkt- und Deletionsmutanten, deren Nukleotidbindung und -hydrolyse untersucht wurde. Anhand der biochemischen und strukturellen Daten wird ein Modell des GTPase-Zyklus aufgestellt und hGBP1 in die Familie der Dynamin-verwandten GTP-bindenden Proteine eingeordnet. Die Identifizierung von dauerhaft aktiven und inaktiven Mutanten bildet den Ausgangspunkt für die Untersuchung der biologischen Funktion von hGBP1.The aim of this thesis was the identification of the mechanism of the hydrolysis of GTP to GMP by the human guanylate binding protein 1 (hGBP1). GTP is hydrolysed to GMP via the sequential cleavage of the two phosphate groups. Due to nucleotide dependent oligomerisation, the protein's specific activity increases with its concentration. The dissociation constants of the guanine nucleotides lie between 1 - 15 $\mu$M. The structure of nucleotide free hGBP1 and in complex with a GTP analogue have been determined and were used for the directed design of both point and deletion mutants. The nucleotide binding and hydrolysis of these mutants were analysed. Based on the biochemical and structural data a model for the GTPase-cycle of hGBP1 is proposed and hGBP1 is grouped into the family of dynamin-related GTP-binding proteins. The identification of permanently active and inactive mutants forms the basis for the identification of the biological function of hGBP1

Regulation of innate immune functions by guanylate-binding proteins

Guanylate-binding proteins (GBP) are a family of dynamin-related large GTPases which are expressed in response to interferons and other pro-inflammatory cytokines. GBPs mediate a broad spectrum of innate immune functions against intracellular pathogens ranging from viruses to bacteria and protozoa. Several binding partners for individual GBPs have been identified and several different mechanisms of action have been proposed depending on the organisms, the cell type and the pathogen used. Many of these anti-pathogenic functions of GBPs involve the recruitment to and the subsequent destruction of pathogen containing vacuolar compartments, the assembly of large oligomeric innate immune complexes such as the inflammasome, or the induction of autophagy. Furthermore, GBPs often cooperate with immunity-related GTPases (IRGs), another family of dynamin-related GTPases, to exert their anti-pathogenic function, but since most IRGs have been lost in the evolution of higher primates, the anti-pathogenic function of human GBPs seems to be IRG-independent. GBPs and IRGs share biochemical and structural properties with the other members of the dynamin superfamily such as low nucleotide affinity and a high intrinsic GTPase activity which can be further enhanced by oligomerisation. Furthermore, GBPs and IRGs can interact with lipid membranes. In the case of three human and murine GBP isoforms this interaction is mediated by C-terminal isoprenylation. Based on cell biological studies, and in analogy to the function of other dynamins in membrane scission events, it has been postulated that both GBPs and IRGs might actively disrupt the outer membrane of pathogen-containing vacuole leading to the detection and destruction of the pathogen by the cytosolic innate immune system of the host. Recent evidence, however, indicates that GBPs might rather function by mediating membrane tethering events similar to the dynaminrelated atlastin and mitofusin proteins, which mediate fusion of the ER and mitochondria, respectively. The aim of this review is to highlight the current knowledge on the function of GBPs in innate immunity and to combine it with the recent progress in the biochemical characterisation of this protein family

Further X−Ray Studies of Mesophase Structure of Hexakis (Alkylsulfono) Benzene (HASBn) and Tribenzocyclononene (II−n) Homologues

Mesophase forming compounds of hexakis(alkylsulfono)benzene compounds (HASBn) are found for n = 7 to 15 carbons per sidechain. We compare X-ray studies for n = 14 with previous results for n = 13. The diffraction patterns indicate a more disordered structure for n = 14, even in the room temperature crystalline phase. The mesophase diffraction pattern is consistent with hexagonal packing of disordered discotic columns, with average lattice repeat a = 29.3 Å. The (10), (11), and (20) reflections are observed with the last one being barely detectible. A fourth broad scattering peak centered about a spacing of about 5.0 Å is attributed to the spacing between molecules within the columns. These numbers are slightly larger than the corresponding numbers for n = 13, as should be expected. Four different homologous series of tribenzocyclononene compounds have been studied. Compounds in series II having 8 to 15 carbon atoms per sidechain exhibit mesomorphism, with structures based on columns of pyramidic or “bowlic” molecules. We compare results obtained from compound II-15 (15 carbon atoms per sidechain) with results obtained from other compounds in this series. The diffraction pattern indicates considerably more disorder for II-15 than for II-13 at all temperatures. Out of five observed features in the hexagonal mesophase, 4 diffraction lines are most simply indexed in terms of a lattice parameter of 59.7 Å. The (11), (20), (22) and (40) reflections are observed but not the (10) reflection

Mechanism and chain specificity of RNF216/TRIAD3, the ubiquitin ligase mutated in Gordon Holmes syndrome

Gordon Holmes syndrome (GDHS) is an adult-onset neurodegenerative disorder characterized by ataxia and hypogonadotropic hypogonadism. GDHS is caused by mutations in the gene encoding the RING-between-RING (RBR)-type ubiquitin ligase RNF216, also known as TRIAD3. The molecular pathology of GDHS is not understood, although RNF216 has been reported to modify several substrates with K48-linked ubiquitin chains, thereby targeting them for proteasomal degradation. We identified RNF216 in a bioinformatical screen for putative SUMO-targeted ubiquitin ligases and confirmed that a cluster of predicted SUMO-interaction motifs (SIMs) indeed recognizes SUMO2 chains without targeting them for ubiquitination. Surprisingly, purified RNF216 turned out to be a highly active ubiquitin ligase that exclusively forms K63-linked ubiquitin chains, suggesting that the previously reported increase of K48-linked chains after RNF216 overexpression is an indirect effect. The linkage-determining region of RNF216 was mapped to a narrow window encompassing the last two Zn-fingers of the RBR triad, including a short C-terminal extension. Neither the SIMs nor a newly discovered ubiquitin-binding domain in the central portion of RNF216 contributes to chain specificity. Both missense mutations reported in GDHS patients completely abrogate the ubiquitin ligase activity. For the R660C mutation, ligase activity could be restored by using a chemical ubiquitin loading protocol that circumvents the requirement for ubiquitin-conjugating (E2) enzymes. This result suggests Arg-660 to be required for the ubiquitin transfer from the E2 to the catalytic cysteine. Our findings necessitate a re-evaluation of the previously assumed degradative role of RNF216 and rather argue for a non-degradative K63 ubiquitination, potentially acting on SUMOylated substrates