Intracellular Sources of ROS/H2O2 in Health and Neurodegeneration: Spotlight on Endoplasmic Reticulum.

Reactive oxygen species (ROS) are produced continuously throughout the cell as products of various redox reactions. Yet these products function as important signal messengers, acting through oxidation of specific target factors. Whilst excess ROS production has the potential to induce oxidative stress, physiological roles of ROS are supported by a spatiotemporal equilibrium between ROS producers and scavengers such as antioxidative enzymes. In the endoplasmic reticulum (ER), hydrogen peroxide (H2O2), a non-radical ROS, is produced through the process of oxidative folding. Utilisation and dysregulation of H2O2, in particular that generated in the ER, affects not only cellular homeostasis but also the longevity of organisms. ROS dysregulation has been implicated in various pathologies including dementia and other neurodegenerative diseases, sanctioning a field of research that strives to better understand cell-intrinsic ROS production. Here we review the organelle-specific ROS-generating and consuming pathways, providing evidence that the ER is a major contributing source of potentially pathologic ROS

Investigating State Restriction in Fluorescent Protein FRET Using Time-Resolved Fluorescence and Anisotropy

Most fluorescent proteins exhibit multiexponential fluorescence decays, indicating a heterogeneous excited state population. FRET between fluorescent proteins should therefore involve multiple energy transfer pathways. We recently demonstrated the FRET pathways between EGFP and mCherry (mC), upon the dimerization of 3-phosphoinositide dependent protein kinase 1 (PDK1), to be highly restricted. A mechanism for FRET restriction based on a highly unfavorable κ(2) orientation factor arising from differences in donor-acceptor transition dipole moment angles in a far from coplanar and near static interaction geometry was proposed. Here this is tested via FRET to mC arising from the association of glutathione (GSH) and glutathione S-transferase (GST) with an intrinsically homogeneous and more mobile donor Oregon Green 488 (OG). A new analysis of the acceptor window intensity, based on the turnover point of the sensitized fluorescence, is combined with donor window intensity and anisotropy measurements which show that unrestricted FRET to mC takes place. However, a long-lived anisotropy decay component in the donor window reveals a GST-GSH population in which FRET does not occur, explaining previous discrepancies between quantitative FRET measurements of GST-GSH association and their accepted values. This reinforces the importance of the local donor-acceptor environment in mediating energy transfer and the need to perform spectrally resolved intensity and anisotropy decay measurements in the accurate quantification of fluorescent protein FRET

Single particle trajectories reveal active endoplasmic reticulum luminal flow

The endoplasmic reticulum (ER), a network of membranous sheets and pipes, supports functions encompassing biogenesis of secretory proteins and delivery of functional solutes throughout the cell[1, 2]. Molecular mobility through the ER network enables these functionalities, but diffusion alone is not sufficient to explain luminal transport across supramicrometre distances. Understanding the ER structure–function relationship is critical in light of mutations in ER morphology-regulating proteins that give rise to neurodegenerative disorders[3, 4]. Here, super-resolution microscopy and analysis of single particle trajectories of ER luminal proteins revealed that the topological organization of the ER correlates with distinct trafficking modes of its luminal content: with a dominant diffusive component in tubular junctions and a fast flow component in tubules. Particle trajectory orientations resolved over time revealed an alternating current of the ER contents, while fast ER super-resolution identified energy-dependent tubule contraction events at specific points as a plausible mechanism for generating active ER luminal flow. The discovery of active flow in the ER has implications for timely ER content distribution throughout the cell, particularly important for cells with extensive ER-containing projections such as neurons.Wellcome Trust - 3-3249/Z/16/Z and 089703/Z/09/Z [Kaminski] UK Demential Research Institute [Avezov] Wellcome Trust - 200848/Z/16/Z, WT: UNS18966 [Ron] FRM Team Research Grant [Holcman] Engineering and Physical Sciences Research Council (EPSRC) - EP/L015889/1 and EP/H018301/1 [Kaminski] Medical Research Council (MRC) - MR/K015850/1 and MR/K02292X/1 [Kaminski

Retarded PDI diffusion and a reductive shift in poise of the calcium depleted endoplasmic reticulum

Background: Endoplasmic reticulum (ER) lumenal protein thiol redox balance resists dramatic variation in unfolded protein load imposed by diverse physiological challenges including compromise in the key upstream oxidases. Lumenal calcium depletion, incurred during normal cell signaling, stands out as a notable exception to this resilience, promoting a rapid and reversible shift towards a more reducing poise. Calcium depletion induced ER redox alterations are relevant to physiological conditions associated with calcium signaling, such as the response of pancreatic cells to secretagogues and neuronal activity. The core components of the ER redox machinery are well characterized; however, the molecular basis for the calcium-depletion induced shift in redox balance is presently obscure. Results: In vitro, the core machinery for generating disulfides, consisting of ERO1 and the oxidizing protein disulfide isomerase, PDI1A, was indifferent to variation in calcium concentration within the physiological range. However, ER calcium depletion in vivo led to a selective 2.5-fold decline in PDI1A mobility, whereas the mobility of the reducing PDI family member, ERdj5 was unaffected. In vivo, fluorescence resonance energy transfer measurements revealed that declining PDI1A mobility correlated with formation of a complex with the abundant ER chaperone calreticulin, whose mobility was also inhibited by calcium depletion and the calcium depletion-mediated reductive shift was attenuated in cells lacking calreticulin. Measurements with purified proteins confirmed that the PDI1A-calreticulin complex dissociated as Ca2+ concentrations approached those normally found in the ER lumen ([Ca2+] K-0.5max = 190 mu M). Conclusions: Our findings suggest that selective sequestration of PDI1A in a calcium depletion-mediated complex with the abundant chaperone calreticulin attenuates the effective concentration of this major lumenal thiol oxidant, providing a plausible and simple mechanism for the observed shift in ER lumenal redox poise upon physiological calcium depletion.Wellcome Trust [Wellcome 084812/Z/08/Z]; European Commission (EU FP7 Beta-Bat) [277713]; Fundacao para a Ciencia e Tecnologia, Portugal [PTDC/QUI-BIQ/119677/2010]info:eu-repo/semantics/publishedVersio

A novel signalling screen demonstrates that CALR mutations activate essential MAPK signalling and facilitate megakaryocyte differentiation.

Most MPN patients lacking JAK2 mutations harbour somatic CALR mutations that are thought to activate cytokine signalling although the mechanism is unclear. To identify kinases important for survival of CALR-mutant cells we developed a novel strategy (KISMET) which utilises the full range of kinase selectivity data available from each inhibitor and thus takes advantage of off-target noise that limits conventional siRNA or inhibitor screens. KISMET successfully identified known essential kinases in haematopoietic and non-haematopoietic cell lines and identified the MAPK pathway as required for growth of the CALR-mutated MARIMO cells. Expression of mutant CALR in murine or human haematopoietic cell lines was accompanied by MPL-dependent activation of MAPK signalling, and MPN patients with CALR mutations showed increased MAPK activity in CD34-cells, platelets and megakaryocytes. Although CALR mutations resulted in protein instability and proteosomal degradation, mutant CALR was able to enhance megakaryopoiesis and pro-platelet production from human CD34+ progenitors. These data link aberrant MAPK activation to the MPN phenotype and identify it as a potential therapeutic target in CALR-mutant positive MPNs.Leukemia accepted article preview online, 14 October 2016. doi:10.1038/leu.2016.280.Work in the Green lab is supported by Leukemia and Lymphoma Research, Cancer Research UK, the NIHR Cambridge Biomedical Research Centre, the Cambridge Experimental Cancer Medicine Centre and the Leukemia & Lymphoma Society of America. WW is supported by the Austrian Science Foundation (J 3578-B21). CGA is supported by Kay Kendall Leukaemia Fund clinical research fellowship. UM is supported by a Cancer Research UK Clinician Scientist Fellowship. Work in the Huntly lab is supported by the European Research Council, the MRC (UK), Bloodwise, the Cambridge NIHR funded BRC, KKLF and a WT/MRC Stem Cell centre grant. Work in the Green and Huntly Labs is supported by core support grants by the Wellcome Trust to the Cambridge Institute for Medical Research (100140/z/12/z) and Wellcome Trust-MRC Cambridge Stem Cell Institute (097922/Z/11/Z)

CMS physics technical design report : Addendum on high density QCD with heavy ions

Peer reviewe

FICD acts bifunctionally to AMPylate and de-AMPylate the endoplasmic reticulum chaperone BiP

Protein folding homeostasis in the endoplasmic reticulum (ER) is defended by an unfolded protein response that matches ER chaperone capacity to the burden of unfolded proteins. As levels of unfolded proteins decline, a metazoan-specific FIC-domain-containing ER-localized enzyme (FICD) rapidly inactivates the major ER chaperone BiP by AMPylating T518. Here we show that the single catalytic domain of FICD can also release the attached AMP, restoring functionality to BiP. Consistent with a role for endogenous FICD in de-AMPylating BiP, FICD$_{-/-}$ hamster cells are hypersensitive to introduction of a constitutively AMPylating, de-AMPylation-defective mutant FICD. These opposing activities hinge on a regulatory residue, E234, whose default state renders FICD a constitutive de-AMPylase $\textit{in vitro}$. The location of E234 on a conserved regulatory helix and the mutually antagonistic activities of FICD $\textit{in vivo}$, suggest a mechanism whereby fluctuating unfolded protein load actively switches FICD from a de-AMPylase to an AMPylase.Supported by Wellcome Trust Principal Research Fellowship to D.R. (Wellcome 200848/Z/16/Z), a UK Medical Research Council PhD studentship to L.A.P. and a Wellcome Trust Strategic Award to the Cambridge Institute for Medical Research (Wellcome 100140)