1H, 13C, 15N backbone resonance assignment of apo and ADP-ribose bound forms of the macro domain of Hepatitis E virus through solution NMR spectroscopy

International audienceAbstract The genome of Hepatitis E virus (HEV) is 7.2 kilobases long and has three open reading frames. The largest one is ORF1, encoding a non-structural protein involved in the replication process, and whose processing is ill-defined. The ORF1 protein is a multi-modular protein which includes a macro domain (MD). MDs are evolutionarily conserved structures throughout all kingdoms of life. MDs participate in the recognition and removal of ADP-ribosylation, and specifically viral MDs have been identified as erasers of ADP-ribose moieties interpreting them as important players at escaping the early stages of host-immune response. A detailed structural analysis of the apo and bound to ADP-ribose state of the native HEV MD would provide the structural information to understand how HEV MD is implicated in virus-host interplay and how it interacts with its intracellular partner during viral replication. In the present study we present the high yield expression of the native macro domain of HEV and its analysis by solution NMR spectroscopy. The HEV MD is folded in solution and we present a nearly complete backbone and sidechains assignment for apo and bound states. In addition, a secondary structure prediction by TALOS + analysis was performed. The results indicated that HEV MD has a α/β/α topology very similar to that of most viral macro domains

Structural and functional study of the E3 ubiquitin ligase Arkadia

Arkadia/RNF111 is the first E3 ubiquitin ligase that has been found to positively regulate the TGF-beta pathway by targeting for ubiquitin dependent degradatation the Smad7, SnoN (SkiL) and SKI negative regulators. E3 ubiquitin ligase activity of Arkadia depends on its C-terminal RING-H2 domain. Three dimensional structure determination and interaction studies of the RING-H2 domain of Arkadia with the E2 enzyme UbcH5b revealed highly conserved amino acids among the RING-H2 domains that are implicated in the interaction with the E2 enzyme. Moreover, using deep sequencing analysis of human mRNA from tumors of colorectal cancer (CRC) patients led to identification of point mutations in the C-terminus that reduce Arkadia functions. In this thesis the impact of the mutation of the conserved amino acids of Phe964 and Trp972, that are found in the interacting surface of Arkadia RING domain, was studied. Moreover, subject of the study was the impact of the Arg957Cys point mutation, which was found in tumors of patients with CRC. The impact of each point mutation in the structure and the interaction ability with the E2 enzyme UbcH5b was estimated through Nuclear Magnetic Resonance (NMR) spectroscopy. Additionally, a polypeptide that comprises the 124-residues of the C-terminal of Arkadia was studied via NMR spectroscopy and sequences outside the RING that bind UbcH5b enzyme through distinct interfaces, resulting in higher affinity, were identified. The E3 ubiquitin ligase function of the polypeptides was estimated through auto-ubiquitination and luciferase assays.Η πρωτεΐνη Arkadia/RNF111 είναι η πρώτη Ε3 λιγάση ουβικιτίνης η οποία αναγνωρίστηκε ότι ρυθμίζει θετικά το TGF-β μονοπάτι σηματοδοτώντας για αποικοδόμηση τους αρνητικούς ρυθμιστές του μονοπατιού Smad7, SnoN (SkiL) και SKI. Η Arkadia επιτελεί τη δράση της μέσω της RING-Η2 επικράτειά της την οποία φέρει στο C-τελικό της άκρο. Ο προσδιορισμός της δομής και η μελέτη της αλληλεπίδρασης με το Ε2 ένζυμο UbcH5b του πολυπεπτιδικού τμήματος των 68 καταλοίπων, το οποίο έφερε τη RING επικράτεια, ανέδειξε συντηρημένα κατάλοιπα για τις RING-Η2 Ε3 λιγάσες ουβικιτίνης τα οποία εμπλέκονται στην αλληλεπίδραση με το Ε2 ένζυμο UbcH5b. Επιπλέον μελέτες αλληλούχισης γονιδιώματος (deep sequencing) του mRNA ασθενών με καρκίνο του παχέος εντέρου υπέδειξαν την ύπαρξη μεταλλάξεων εντός της RING επικράτειας της Arkadia όπως η μετάλλαξη της Arg σε Cys στη θέση 957 της ακολουθίας της. Στην παρούσα διδακτορική διατριβή πραγματοποιήθηκε η μελέτη της επίδρασης μεταλλάξεων σε συντηρημένα κατάλοιπα όπως η Trp στη θέση 972 και η Phe στη θέση 964 της ακολουθίας. Επιπρόσθετα, πραγματοποιήθηκε η μελέτη της επίδρασης της μετάλλαξης Arg957Cys η οποία έχει ανιχνευθεί σε ιστούς ασθενών με καρκίνο του παχέος εντέρου. Η αποτίμηση της επίδρασης των μεταλλάξεων στη δομή καθώς και οι μελέτες αλληλεπίδρασης των μεταλλαγμάτων με το Ε2 ένζυμο UbcH5b πραγματοποιήθηκαν με τη χρήση φασματοσκοπίας Πυρηνικού Μαγνητικού Συντονισμού. Επιπρόσθετα, μελετήθηκε μέσω φασματοσκοπίας Πυρηνικού Μαγνητικού Συντονισμού το πολυπεπτιδικό τμήμα των 124 καταλοίπων το οποίο αποδείχθηκε ότι φέρει ακολουθίες σημαντικές για την αλληλεπίδραση της πρωτεΐνης με το UbcH5b και τη λειτουργία της ως λιγάση στο μονοπάτι της ουβικιτινίωσης. Η λειτουργικότητα των μελετώμενων πολυπεπτιδίων της Arkadia ως Ε3 λιγάσες ουβικιτίνης ελέγχθηκε μέσω πειραμάτων αυτο-ουβικιτινίωσης και δοκιμασίας λουσιφεράσης

E2 Partner Tunes the Ubiquitylation Specificity of Arkadia E3 Ubiquitin Ligase

Arkadia (RNF111) is a positive regulator of the TGF-β signaling that mediates the proteasome-dependent degradation of negative factors of the pathway. It is classified as an E3 ubiquitin ligase and a SUMO-targeted ubiquitin ligase (STUBL), implicated in various pathological conditions including cancer and fibrosis. The enzymatic (ligase) activity of Arkadia is located at its C-terminus and involves the RING domain. Notably, E3 ligases require E2 enzymes to perform ubiquitylation. However, little is known about the cooperation of Arkadia with various E2 enzymes and the type of ubiquitylation that they mediate. In the present work, we study the interaction of Arkadia with the E2 partners UbcH5B and UbcH13, as well as UbcH7. Through NMR spectroscopy, we found that the E2–Arkadia interaction surface is similar in all pairs examined. Nonetheless, the requirements and factors that determine an enzymatically active E2–Arkadia complex differ in each case. Furthermore, we revealed that the cooperation of Arkadia with different E2s results in either monoubiquitylation or polyubiquitin chain formation via K63, K48, or K11 linkages, which can determine the fate of the substrate and lead to distinct biological outcomes

Unveiling the Essential Role of Arkadia’s Non-RING Elements in the Ubiquitination Process

Arkadia is a positive regulator of the TGFβ-SMAD2/3 pathway, acting through its C-terminal RING-H2 domain and targeting for degradation of its negative regulators. Here we explore the role of regions outside the RING domain (non-RING elements) of Arkadia on the E2-E3 interaction. The contribution of the non-RING elements was addressed using Arkadia RING 68 aa and Arkadia 119 aa polypeptides. The highly conserved NRGA (asparagine-arginine-glycine-alanine) and TIER (threonine-isoleucine-glutamine-arginine) motifs within the 119 aa Arkadia polypeptide, have been shown to be required for pSMAD2/3 substrate recognition and ubiquitination in vivo. However, the role of the NRGA and TIER motifs in the enzymatic activity of Arkadia has not been addressed. Here, nuclear magnetic resonance interaction studies with the E2 enzyme, UBCH5B, C85S UBCH5B-Ub oxyester hydrolysis, and auto-ubiquitination assays were used to address the role of the non-RING elements in E2-E3 interaction and in the enzymatic activity of the RING. The results support that the non-RING elements including the NRGA and TIER motifs are required for E2-E3 recognition and interaction and for efficient auto-ubiquitination. Furthermore, while Arkadia isoform-2 and its close homologue Arkadia 2C are known to interact with free ubiquitin, the results here showed that Arkadia isoform-1 does not interact with free ubiquitin

E2 Partner Tunes the Ubiquitylation Specificity of Arkadia E3 Ubiquitin Ligase

Arkadia (RNF111) is a positive regulator of the TGF-β signaling that mediates the proteasome-dependent degradation of negative factors of the pathway. It is classified as an E3 ubiquitin ligase and a SUMO-targeted ubiquitin ligase (STUBL), implicated in various pathological conditions including cancer and fibrosis. The enzymatic (ligase) activity of Arkadia is located at its C-terminus and involves the RING domain. Notably, E3 ligases require E2 enzymes to perform ubiquitylation. However, little is known about the cooperation of Arkadia with various E2 enzymes and the type of ubiquitylation that they mediate. In the present work, we study the interaction of Arkadia with the E2 partners UbcH5B and UbcH13, as well as UbcH7. Through NMR spectroscopy, we found that the E2–Arkadia interaction surface is similar in all pairs examined. Nonetheless, the requirements and factors that determine an enzymatically active E2–Arkadia complex differ in each case. Furthermore, we revealed that the cooperation of Arkadia with different E2s results in either monoubiquitylation or polyubiquitin chain formation via K63, K48, or K11 linkages, which can determine the fate of the substrate and lead to distinct biological outcomes

Conformational plasticity of the VEEV macro domain is important for binding of ADP-ribose

International audienceVenezuelan equine encephalitis virus (VEEV) is a new world alphavirus which can be involved in several central nervous system disorders such as encephalitis and meningitis. The VEEV genome codes for 4 non-structural proteins (nsP), of which nsP3 contains a Macro domain. Macro domains (MD) can be found as stand-alone proteins or embedded within larger proteins in viruses, bacteria and eukaryotes. Their most common feature is the binding of ADP-ribose (ADPr), while several macro domains act as ribosylation writers, erasers or readers. Alphavirus MD erase ribosylation but their precise contribution in viral replication is still under investigation. NMR-driven titration experiments of ADPr in solution with the VEEV macro domain (in apo- and complex state) show that it adopts a suitable conformation for ADPr binding. Specific experiments indicate that the flexibility of the loops β5-α3 and α3-β6 is critical for formation of the complex and assists a wrapping mechanism for ADPr binding. Furthermore, along with this sequence of events, the VEEV MD undergoes a conformational exchange process between the apo state and a low-populated "dark" conformational state