Structural and biophysical characterisation of PERK kinase towards understanding ER stress sensing and activation of the Unfolded Protein Response

The unfolded protein response (UPR) is a cellular mechanism that detects the accumulation of misfolded proteins within the endoplasmic reticulum (ER). In mammalian cells, the UPR is mediated by three ER-transmembrane proteins: PERK, IRE1 and ATF6. Early studies in the field provided evidence for the role of BiP, the major ER Hsp70 chaperone, in UPR activation by binding to the luminal domains of PERK, IRE1 and ATF6 and maintaining them in an inactive state. However the underlying mechanism of ER stress sensing and UPR activation is not yet understood. This thesis presents (i) the novel X-ray crystal structure of PERK luminal domain and (ii) a biochemical study of the unconventional interaction between BiP chaperone and the luminal domains of PERK and IRE1 in vitro. Firstly, the structure of PERK luminal domain was solved in two oligomeric states: dimers and tetramers. Compelling evidence is provided for a role of tetramer formation in directing downstream UPR signalling. Secondly, the unprecedented and unconventional direct binding of PERK and IRE1 luminal domains to BiP is demonstrated. The binding surface was mapped to the nucleotide-binding domain (NBD) of BiP. As such, this points away from a substrate-chaperone interaction and rather implies BiP as an explicit UPR signalling component. Upon binding to CH1 unfolded protein, BiP is released from PERK and IRE1 luminal domains. BiP dissociation from PERK and IRE1 is known to lead to their activation. Based on the work presented in this thesis a novel mechanism of ER stress sensing and UPR activation by PERK and IRE1 is proposed. BiP NBD normally interacts with the luminal domains of PERK and IRE1 and represses UPR signalling. During ER stress, binding of unfolded proteins to BiP’s substrate binding domain leads to the dissociation of BiP-luminal domain complexes. As such, the luminal domains are free to intertwine, mediated by an extended α-helix, and form active tetramers competent for cytoplasmic UPR signalling.Open Acces

Crystal structures reveal transient <scp>PERK</scp> luminal domain tetramerization in endoplasmic reticulum stress signaling

Stress caused by accumulation of misfolded proteins within the endoplasmic reticulum (ER) elicits a cellular unfolded protein response (UPR) aimed at maintaining protein-folding capacity. PERK, a key upstream component, recognizes ER stress via its luminal sensor/transducer domain, but the molecular events that lead to UPR activation remain unclear. Here, we describe the crystal structures of mammalian PERK luminal domains captured in dimeric state as well as in a novel tetrameric state. Small angle X-ray scattering analysis (SAXS) supports the existence of both crystal structures also in solution. The salient feature of the tetramer interface, a helix swapped between dimers, implies transient association. Moreover, interface mutations that disrupt tetramer formation in vitro reduce phosphorylation of PERK and its target eIF2α in cells. These results suggest that transient conversion from dimeric to tetrameric state may be a key regulatory step in UPR activation. Synopsis Activation of unfolded protein response (UPR) upon ER stress involves key regulatory roles of ER-luminal sensor/transducer domains in UPR signaling factors. Structural and functional analyses of the PERK luminal domain reveal a novel tetrameric arrangement, whose transient formation may be an important step in UPR activation. Crystal structure of human PERK luminal domain shows a novel tetramer arrangement. Crystal structure of mouse PERK luminal domain is captured in dimeric form. Biophysical analysis confirm that both mouse and human proteins exist as dimers as well as tetramers in solution. Mutations that disrupt tetramerization in solution reduce phosphorylation of PERK and its target eIF2α in cells. Structural and functional analyses of the PERK luminal domain reveal a novel tetrameric arrangement, whose transient formation may be an important step in activation of the unfolded protein response

Estudos de viabilidade do reuso de efluentes liquidos gerados em processos de galvanoplastia por tratamento fisico-quimico

Orientador: Ruben Bresaola JrDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia CivilResumo: Os despejos industriais dos processos de galvanoplastia causam, em geral, graves problemas de poluição hídrica por conterem metais pesados e utilizarem grandes volumes de água em seus processos. A reciclagem ou reuso dos efluentes para fins diversos pode diminuir a demanda de água dos mananciais e minimizar a quantidade de efluentes lançados nas redes públicas de esgotos ou rios. O presente trabalho tem por objetivo estudar a viabilidade do reuso ou reciclagem das águas residuárias geradas em indústrias de galvanoplastia, por meio de tratamento fisico-químico de coagulação-floculação. Os resultados dos ensaios mostraram que houve remoção de metais acima de 99% utilizando-se NaOH com pH de coagulação igual a 10, com tempo de sedimentação de 90 minutos, ou utilizando-se 30 mg/L de cloreto férrico em pH 10 e também polímero aniônico em pH 9, ambos com tempo de precipitação de 60 minutos. Com isso, conclui-se que, com relação à quantidade de metais, estas águas apresentam teores inferiores aos limites estabelecidos para as águas de Classe 3, de acordo com a legislação CONAMA n. 20, podendo ser reutilizadas para irrigação de culturas arbóreas e forrageiras. Podem, igualmente, ser reutilizadas para lavagem de pisos, em descargas de banheiros e em alguns tanques do primeiro enxágüe das peças. Para reciclagem dessas águas no processo, toma-se necessária a diluição com água limpa para diminuição da condutividade da água tratadaAbstract: The industrial wastes of the galvanotechnick processes cause, in general, serious hydro pollution due to the use of heavy metaIs, besides that, they use great volumes of water in their processes. The recyc1ing or reuse of the waste for many purposes reduces the water demand of the rivers and minimizes the quantity wastes to be launched in the sewers or rivers. Due to these facts, the present work had the objective of studying the feasibility of the reuse or recyc1ing of residuary water generated in galvanotechnick industries by physical-chemical treatment of coagulation-flocculation. The results of the research had shown that there was removal of metaIs above 99% using NaOH with pH equal to 10, with precipitation time of90 minutes, or using FeCh ofpH 10 and also anionic polymer ofpH 9, both with time precipitation of 60 minutes. Therefore, it can be conc1udedthat in relation to the quantity of metaIs, this water can be reused for irrigation of garden culture, for pavement and toilet flush c1eaning. For recyc1ing of these water in process, it becomes necessary the dilution with c1eanwater for decrease of conductivity of treated waterMestradoSaneamentoMestre em Engenharia Civi

Autonomic and circulatory alterations persist despite adequate resuscitation in a 5-day sepsis swine experiment.

Autonomic and vascular failures are common phenotypes of sepsis, typically characterized by tachycardia despite corrected hypotension/hypovolemia, vasopressor resistance, increased arterial stiffness and decreased peripheral vascular resistance. In a 5-day swine experiment of polymicrobial sepsis we aimed at characterizing arterial properties and autonomic mechanisms responsible for cardiovascular homeostasis regulation, with the final goal to verify whether the resuscitation therapy in agreement with standard guidelines was successful in restoring a physiological condition of hemodynamic profile, cardiovascular interactions and autonomic control. Twenty pigs were randomized to polymicrobial sepsis and protocol-based resuscitation or to prolonged mechanical ventilation and sedation without sepsis. The animals were studied at baseline, after sepsis development, and every 24 h during the 3-days resuscitation period. Beat-to-beat carotid blood pressure (BP), carotid blood flow, and central venous pressure were continuously recorded. The two-element Windkessel model was adopted to study carotid arterial compliance, systemic vascular resistance and characteristic time constant τ. Effective arterial elastance was calculated as a simple estimate of total arterial load. Cardiac baroreflex sensitivity (BRS) and low frequency (LF) spectral power of diastolic BP were computed to assess autonomic activity. Sepsis induced significant vascular and autonomic alterations, manifested as increased arterial stiffness, decreased vascular resistance and τ constant, reduced BRS and LF power, higher arterial afterload and elevated heart rate in septic pigs compared to sham animals. This compromised condition was persistent until the end of the experiment, despite achievement of recommended resuscitation goals by administered vasopressors and fluids. Vascular and autonomic alterations persist 3 days after goal-directed resuscitation in a clinically relevant sepsis model. We hypothesize that the addition of these variables to standard clinical markers may better profile patients' response to treatment and this could drive a more tailored therapy which could have a potential impact on long-term outcomes

Blood pressure variability, heart functionality, and left ventricular tissue alterations in a protocol of severe hemorrhagic shock and resuscitation

Autonomic control of blood pressure (BP) and heart rate (HR) is crucial during bleeding and hemorrhagic shock (HS) to compensate for hypotension and hypoxia. Previous works have observed that at the point of hemodynamic decompensation a marked suppression of BP and HR variability occurs, leading to irreversible shock. We hypothesized that recovery of the autonomic control may be decisive for effective resuscitation, along with restoration of mean BP. We computed cardiovascular indexes of baroreflex sensitivity and BP and HR variability by analyzing hemodynamic recordings collected from five pigs during a protocol of severe hemorrhage and resuscitation; three pigs were sham-treated controls. Moreover, we assessed the effects of severe hemorrhage on heart functionality by integrating the hemodynamic findings with measures of plasma high-sensitivity cardiac troponin T and metabolite concentrations in left ventricular (LV) tissue. Resuscitation was performed with fluids and norepinephrine and then by reinfusion of shed blood. After first resuscitation, mean BP reached the target value, but cardiovascular indexes were not fully restored, hinting at a partial recovery of the autonomic mechanisms. Moreover, cardiac troponins were still elevated, suggesting a persistent myocardial sufferance. After blood reinfusion all the indexes returned to baseline. In the harvested heart, LV metabolic profile confirmed the acute stress condition sensed by the cardiomyocytes. Variability indexes and baroreflex trends can be valuable tools to evaluate the severity of HS, and they may represent a more useful end point for resuscitation in combination with standard measures such as mean values and biological measures. NEW & NOTEWORTHY Autonomic control of blood pressure was highly impaired during hemorrhagic shock, and it was not completely recovered after resuscitation despite global restoration of mean pressures. Moreover, a persistent myocardial sufferance emerged from measured cardiac troponin T and metabolite concentrations of left ventricular tissue. We highlight the importance of combining global mean values and biological markers with measures of variability and autonomic control for a better characterization of the effectiveness of the resuscitation strategy

Shelves of the Iberian Peninsula and the Balearic Islands (I): Morphology and sediment types

Here we present a synthesis of bedforms and sediment types on the shelves surrounding the Iberian Peninsula and the Balearic Islands, after the integration several pieces of bathymetric, morphological and sedimentological datasets. The Iberian and Balearic shelves are divided into segments according to the largescale margin configuration, fluvial sediment supply and hydrodynamic regime. Their geological settings and large-scale sedimentation patterns place the investigated shelves into two broad categories: abrupt, steep and narrow sediment-starved shelves, illustrated by the Cantabrian shelf, and gentle, smooth and wide sediment- fed shelves, such as the shelves off some major flivial systems. An in-depth classification was subsequently attempted, based on morpho-sedimentary types. Under this approach, the Iberian and Balearic shelves can be classified as: (1) storm-dominated shelves, with erosional rocky floors, frequent abrasion surfaces and coarse-grained sediments; (2) current-dominated shelves, showing a good equilibriumbetween sediment fluxes and coastal and shallow ocean circulation, with laterally extensive muddy belts; (3) sediment supply-dominated shelves, where extensive subaqueous deltas develop off river mouths; and (4) wave-dominated shelves that occur off coastal stretches with minor and/or multiple fluvial sediment sources and enhanced littoral current

dosimetry methods in boron neutron capture therapy

Dosimetry studies have been carried out at thermal and epithermal columns of LVR- 15 research reactor for investigating the spatial distribution of gamma dose, fast neutron dose and thermal neutron fluence. Two different dosimetry methods, both based on solid state detectors, have been studied and applied and the accuracy and consistency of the results have been inspected. One method is based on Fricke gel dosimeters that are dilute water solutions and have good tissue equivalence for neutrons and also for all the secondary radiations produced by neutron interactions in tissue or water phantoms. Fricke gel dosimeters give the possibility of separating the various dose contributions, i.e. the gamma dose, the fast neutron dose and the dose due to charged particles generated during thermal neutron reactions by isotopes having high cross section, like 10B. From this last dose, thermal neutron fluence can be obtained by means of the kerma factor. The second method is based on thermoluminescence dosimeters. In particular, the developed method draw advantage from the different heights of the peaks of the glow curve of such phosphors when irradiated with photons or with thermal neutrons. The results show that satisfactory results can be obtained with simple methods, in spite of the complexity of the subject. However, the more suitable dosimeters and principally their utilization and analysis modalities are different for the various neutron beams, mainly depending on the relative intensities of the three components of the neutron field, in particular are different for thermal and epithermal columns

UPR Signal Activation by Luminal Sensor Domains

The unfolded protein response (UPR) is a cell-signaling system that detects the accumulation of unfolded protein within the endoplasmic reticulum (ER) and initiates a number of cellular responses to restore ER homeostasis. The presence of unfolded protein is detected by the ER-luminal sensor domains of the three UPR-transducer proteins IRE1, PERK, and ATF6, which then propagate the signal to the cytosol. In this review, we discuss the various mechanisms of action that have been proposed on how the sensor domains detect the presence of unfolded protein to activate downstream UPR signaling. © 2013 by the authors; licensee MDPI, Basel, Switzerland