A real-time analysis of GFP unfolding by the AAA+ unfoldase PAN

Protein quality control systems are essential to maintain a healthy proteome. They often consist of an unfoldase unit, typically an AAA+ ATPase, coupled with a protease unit. In all kingdoms of life, they function to eliminate misfolded proteins, and thus prevent that their aggregates do harm to the cell, and to rapidly regulate protein levels in the presence of environmental changes. Despite the huge progress made in the past two decades in understanding the mechanism of function of protein degradation systems, the fate of the substrate during the unfolding and proteolytic processes remains poorly understood. Here we exploit an NMR-based approach to monitor GFP processing by the archaeal PAN unfoldase and the PAN–20S degradation system in real time. We find that PAN-dependent unfolding of GFP does not involve the release of partially-folded GFP molecules resulting from futile unfolding attempts. In contrast, once stably engaged with PAN, GFP molecules are efficiently transferred to the proteolytic chamber of the 20S subunit, despite the only weak affinity of PAN for the 20S subunit in the absence of substrate. This is essential to guarantee that unfolded but not proteolyzed proteins are not released into solution, where they would form toxic aggregates. The results of our studies are in good agreement with previous results derived from real-time small-angle-neutron-scattering experiments and have the advantage of allowing the investigation of substrates and products at amino-acid resolution

Characterization of a small tRNA-binding protein that interacts with the archaeal proteasome complex

Authors acknowledge financial support from the French Agence Nationale de la Recherche (grant [ANR-18-CE11-0018-01] to B.F. and [ANR-16-CE12-0016-01] to B.C.O). This work used the platforms of the Grenoble Instruct-ERIC Centre (ISBG: UMS3518 CNRS-CEA-UGA-EMBL) with support from FRISBI (ANR-10-INBS-05-02) and GRAL, a project of the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EUR-GS (ANR-17-EURE-0003) within the Grenoble Partnership for Structural Biology. The IBS Electron Microscope facility is supported by the Auvergne Rhône-Alpes Region, the Fonds Feder, the Fondation pour la Recherche Médicale and GIS-IBiSA.The proteasome system allows the elimination of functional or structurally impaired proteins. This includes the degradation of nascent peptides. In Archaea, how the proteasome complex interacts with the translational machinery remains to be described. Here, we characterised a small orphan protein, Q9UZY3 (Uniprot ID) conserved in Thermococcales. The protein was identified in native pull-down experiments using the proteasome regulatory complex (PAN) as bait. X-ray crystallography and SAXS experiments revealed that the protein is monomeric and adopts a β-barrel core structure with an Oligonucleotide/oligosaccharide-Binding (OB) fold, typically found in translation elongation factors. Mobility shift experiment showed that Q9UZY3 displays tRNA binding properties. Pull-downs, co-immunoprecipitation and ITC studies revealed that Q9UZY3 interacts in vitro with PAN. Native pull-downs and proteomic analysis using different versions of Q9UZY3 showed that the protein interacts with the assembled PAN-20S proteasome machinery in Pyrococcus abyssi cellular extracts. The protein was therefore named Pbp11, for Proteasome Binding Protein of 11 kDa. Interestingly, the interaction network of Pbp11 also includes ribosomal proteins, tRNA processing enzymes and exosome subunits dependent on Pbp11's N-terminal domain that was found to be essential for tRNA binding. Together these data suggest that Pbp11 participates in an interface between the proteasome and the translational machinery.Publisher PDFPeer reviewe

Similar effectiveness of dapagliflozin and GLP-1 receptor agonists concerning combined endpoints in routine clinical practice: A multicentre retrospective study

Aims According to cardiovascular outcome trials, some sodium-glucose contransporter-2 inhibitors (SGLT2i) and glucagon-like peptide-1 receptor agonists (GLP-1RA) are recommended for secondary cardiovascular prevention in type 2 diabetes (T2D). In this real-world study, we compared the simultaneous reductions in HbA1c, body weight and systolic blood pressure after initiation of dapagliflozin or GLP-1RA as second or a more advanced line of therapy. Materials and methods DARWIN-T2D was a retrospective multi-centre study conducted at diabetes specialist clinics in Italy that compared T2D patients who initiated dapagliflozin or GLP-1RA (exenatide once weekly or liraglutide). Data were collected at baseline and at the first follow-up visit after 3 to 12 months. The primary endpoint was the proportion of patients achieving a simultaneous reduction in HbA1c, body weight and systolic blood pressure. To reduce confounding, we used multivariable adjustment (MVA) or propensity score matching (PSM). Results Totals of 473 patients initiating dapagliflozin and 336 patients initiating GLP-1RA were included. The two groups differed in age, diabetes duration, HbA1c, weight and concomitant medications. The median follow-up was 6 months in both groups. Using MVA or PSM, the primary endpoint was observed in 30% to 32% of patients, with no difference between groups. Simultaneous reduction of HbA1c, BP and SBP by specific threshold, as well as achievement of final goals, did not differ between groups. GLP-1RA reduced HbA1c by 0.3% more than the reduction achieved with dapagliflozin. Conclusion In routine specialist care, initiation of dapagliflozin can be as effective as initiation of a GLP-1RA for attainment of combined risk factor goals

Des microbes repoussent les limites de la vie

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Etude de Pan A et de Pan B (deux protéines régulatrices du protéasome chez les archaea halophiles)

Les protéasomes sont de larges protéases ATP-dépendante impliquées dans la dégradation des protéines régulatrices et des protéines anormales dans la cellule. Cette thèse porte sur l'étude de deux AAA-ATPases : Pans A et Pans B régulatrices du protéasome chez les archaea halophiles. Une partie de ce manuscrit est consacrée à la caractérisation du mode de régulation des deux Pans chez l'Archaea halophile extrême Halobacterium salinarium. Les expériences montrent l'existence de deux isoformes des protéines Pans dans les cellules. Les modifications portent sur la région N-ter des deux protéines et mettent probablement en jeu l'utilisation alternative de deux départs de traduction. La cartographie des régions 5' non codantes des transcrits révèle l'existence d'une hétérogénéité au niveau des ARNm des pans. Le travail s'intéresse ensuite aux états d'oligomérisation et d'association in cellulo des protéines PANs. Des études de fractionnement par ultracentrifugation sur gradient de sucrose indiquent un état de faible oligomérisation des PANs et l'absence de complexes stables avec le protéasome 20S. La dernière partie du travail porte sur la régulation de l'expression des gènes panA et panB au cours de la réponse aux stress salin et thermique. Ce travail montre que les deux protéines Pans, activatrices potentielles du protéasome, ne sont pas régulées de façon identique en réponse à un stress environnemental.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

Intracellular molecular dynamics studied by neutron scattering

Incoherent neutron scattering experiments have produced important insights into intracellular molecular dynamics in vivo. Selected results highlight the role of water dynamics in cancer and brain cells, as well as cellular adaptation through the evolution of appropriate molecular dynamics, in order to respond to environmental challenges