Νεότερα δεδομένα στα εξωσωματικά συστήματα υποστήριξης ηπατικής λειτουργίας. Συστηματική Ανασκόπηση

Εισαγωγή. Το ήπαρ είναι ένα σημαντικό όργανο του ανθρώπινου οργανισμού που ρυθμίζει την ομοιόσταση, τη σύνθεση πρωτεϊνών και την αποβολή τοξικών μεταβολιτών. Η ανεπάρκεια του μπορεί να αποβεί μοιραία και για το λόγο αυτό στις αρχές του αιώνα εφευρέθηκαν διάφορες συσκευές (συνθετικές και βιοσυνθετικές) για την υποστήριξη της λειτουργίας του. Σκοπός. Η ανάδειξη παράτασης της επιβίωσης και άλλων οφελών σε ασθενείς με ηπατική ανεπάρκεια που υποβλήθηκαν σε εξωσωματική υποστήριξη της ηπατικής λειτουργίας σε σχέση με τη συνηθισμένη θεραπεία. Υλικό και μέθοδος. Χρησιμοποιήθηκαν 18 προοπτικές τυχαιοποιημένες μελέτες από τις βάσεις δεδομένων (PubMed, Medline, Cochrane Library, Google scholar, Embase) από τον 1ο/1999 έως τον 12ο/2018. Επιπλέον αποκλείστηκαν όλες οι περιπτώσεις ασθενών που δεν έπασχαν από ηπατική ανεπάρκεια. Αποτελέσματα-συμπεράσματα. Η χρησιμοποίηση των μεθόδων εξωσωματικής υποστήριξης της ηπατικής λειτουργίας δε φάνηκε να αυξάνει το προσδόκιμο της επιβίωσης στους ασθενείς. Ωστόσο σαφή είναι τα οφέλη στην κάθαρση τοξικών μεταβολιτών όπως σαφής είναι και η ασφάλεια στη χρήση τους. Περαιτέρω έρευνες με καλύτερο σχεδιασμό πρέπει να διεξαχθούν για τη αποφυγή πιθανών σφαλμάτων και την διαπίστωση των οφελών για τους ασθενείς. Τέλος είναι απαραίτητη η ανεύρεση νέων μεθόδων που να υποκαθιστούν και τη συνθετική λειτουργία του ήπατος.Background. Liver is an important organ in the human body which is capable of regulating homeostasis, protein synthesis and detoxification of blood. It’s failure most of the times can be fatal, that’s why methods of extracorporeal liver assist (artificial and bioartificial) have been introduced since the start of the century. Objective. To show possible prolongation of survival along with other benefits in patients that were cured with extracorporeal liver assist devices in contrary with standard of care treatment. Methods. 18 randomized controlled trials from medical databases (PubMed, Medline, Cochrane Library, Google scholar, Embase) through the start of 1999 until 12/2018 have been used to extract the data. Patients that did not have liver impairment were excluded from the study. Results-conclusion. The use of extracorporeal liver assist devices did not end in prolongation of survival in the selected patients. Although, the devices are safe to use and have particularly good results in detoxifying the plasma. More studies with better and thorough planning need to be conducted to eradicate bias and determine the clinical profits of these devices. At last it is important to find new ways to replace protein synthesis in patients with liver failure

Investigation of the 3D structure, dynamics and interaction of proteins contributing to the ubiquitination pathway of ARKADIA protein, through nuclear magnetic resonance (NMR) spectroscopy

Cell homeostasis is an important feature of cell life that is basically maintainedby the ubiquitination and subsequent degradation of proteins. Ubiquitination is a posttranslationalthree-step enzymatic modification mechanism involving the enzymes E1,E2 and E3, which participate in many cellular functions. The degradation of proteinsby 26S proteasome is a well studied process of ubiquitination pathway fetching E2 inthe heart of this mechanism, which transfers the activated Ub to E3 Ub ligase, theselectivity factor of the pathway, which is bound to the target substrate. Even though abig number of E2-E3 complexes have been studied, not all of them are functionallyactive, indicating a more complex mechanism of selectivity. The mobility of E2~Ubthioester complex seems to play a key role in the transfer of Ub to the substrate, whilebinding of E3 ligase to the E2 enzyme stabilizes the complex to a preferred, for the Ubtransfer, conformation. Among the E3 ligases, RING is the largest category of E3fetching a RING domain with a characteristic “cross-brace” arrangement of Zn2+binding.Arkadia (Akd) and Arkadia2C (Akd2C) are RING E3 ligases that positivelyregulate TGF-β and BMP pathways accordingly, by degrading their negative regulators.Both of them have a RING-H2 domain at their C-terminal, while their substraterecognition and binding motif is located nearby. Akd and Akd2C are involved in theearly development stages of the organism but also taking part in many pathologicalconditions and cancer.Ο έλεγχος της ομοιόστασης του κυττάρου διατηρείται σε μεγάλο βαθμό από τηνουβικιτινίωση και την επακόλουθη αποικοδόμηση των πρωτεϊνών. Η ουβικιτινίωσηείναι ένας μετα-μεταφραστικός ενζυμικός μηχανισμός τριών σταδίων στον οποίοεμπλέκονται τα ένζυμα Ε1, Ε2 και Ε3, τα οποία διαδραματίζουν σημαντικό ρόλο σεπληθώρα κυτταρικών λειτουργιών. Η εκτενέστερα μελετημένη λειτουργία τουμονοπατιού της Ub, η αποικοδόμηση των πρωτεϊνών από το πρωτεάσωμα 26S, έχει στοκέντρο της το Ε2 ένζυμο, το οποίο φέρει την ενεργοποιημένη Ub στον παράγονταεκλεκτικότητας, την Ε3 λιγάση, σε σύμπλοκο με την πρωτεΐνη-στόχο. Αν και πολλάΕ2-Ε3 σύμπλοκα έχουν παρατηρηθεί δεν είναι όλα λειτουργικά, αναδεικνύοντας τηνύπαρξη ενός πολυπλοκότερου μηχανισμού εκλεκτικότητας. Η δυναμική συμπεριφοράτου Ε2~Ub συζεύγματος φαίνεται να διαδραματίζει το σημαντικότερο ρόλο για τηνμεταφορά της Ub στο υπόστρωμα, ενώ η Ε3 λιγάση είναι επιφορτισμένη με τηνσταθεροποιήσή του στην προτιμητέα, για την μεταφορά της Ub, διαμόρφωση. Εκ τωνΕ3 λιγασών, οι RING E3 λιγάσες αποτελούν τη μεγαλύτερη κατηγορία και φέρουν έναRING τομέα με το χαρακτηριστικό “cross-brace” μοτίβο δέσμευσης ιόντων Zn2+.Οι πρωτεΐνες Arkadia (Akd) και Arkadia2C (Akd2C) είναι RING E3 λιγάσες οιοποίες ρυθμίζουν θετικά το TGF-β και BMP σηματοδοτικό μονοπάτι αντίστοιχα, μέσωτης αποικοδόμησης των αρνητικών ρυθμιστών τους. Φέρουν ένα RING-H2 τομέα στοC-τελικό τους άκρο μέσω του οποίου αλληλεπιδρούν με τα Ε2 ένζυμα, ενώ κοντά σεαυτόν υπάρχει και το σημείο πρόσδεσης των υποστρωμάτων τους. Οι πρωτεΐνες αυτέςαν και εμπλέκονται στα αρχικά αναπτυξιακά στάδια των οργανισμών, έχουνσυσχετιστεί με αρκετές παθολογικές καταστάσεις και καρκινογένεση

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

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

NMR study of non-structural proteins-part II: H-1, C-13, N-15 backbone and side-chain resonance assignment of macro domain from Venezuelan equine encephalitis virus (VEEV)

International audienceno abstrac

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

Losartan Interactions with 2-Hydroxypropyl-β-CD

Losartan potassium salt (LSR) is a well-known antihypertensive drug with proven beneficial effects on human health. Its formulation with the non-toxic 2-hydroxypropyl-β-cyclodextrin (2-HP-β-CD) could improve its pharmacological profile. Thus, its molecular interactions are studied using a combination of Differential Scanning Calorimetry (DSC), Nuclear Magnetic Resonance (NMR) and Molecular Dynamics (MD). First, its complexation is shown through Differential Scanning Calorimetry as lyophilization provided distinct thermal properties in comparison to the mixture. The complexation is further proved by utilizing the chemical shift changes in the complexation and T1 values. Furthermore, the reversible favorable complexation was shown by MD calculations. Such physical chemical properties provide evidence that this formulation must be further explored through biological experiments

A combined NMR and molecular dynamics simulation study to determine the conformational properties of rat/mouse 35-55 myelin oligodendrocyte glycoprotein epitope implicated in the induction of experimental autoimmune encephalomyelitis

A combined NMR and molecular dynamics simulation study to determine the conformational properties of rat/mouse 35-55 myelin oligodendrocyte glycoprotein epitope implicated in the induction of experimental autoimmune encephalomyeliti