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- ERO1-independent production of H2O2 within the endoplasmic reticulum fuels Prdx4-mediated oxidative protein foldingPublication . Konno, Tasuku; Melo, Eduardo Pinho; Lopes, Carlos; Mehmeti, Ilir; Lenzen, Sigurd; Ron, David; Avezov, EdwardThe endoplasmic reticulum (ER)-localized peroxiredoxin 4 (PRDX4) supports disulfide bond formation in eukaryotic cells lacking endoplasmic reticulum oxidase 1 (ERO1). The source of peroxide that fuels PRDX4-mediated disulfide bond formation has remained a mystery, because ERO1 is believed to be a major producer of hydrogen peroxide (H2O2) in the ER lumen. We report on a simple kinetic technique to track H2O2 equilibration between cellular compartments, suggesting that the ER is relatively isolated from cytosolic or mitochondria! H2O2 pools. Furthermore, expression of an ER-adapted catalase to degrade lumenal H2O2 attenuated PRDX4-mediated disulfide bond formation in cells lacking ERO1, whereas depletion of H2O2 in the cytosol or mitochondria had no similar effect. ER catalase did not effect the slow residual disulfide bond formation in cells lacking both ERO1 and PRDX4. These observations point to exploitation of a hitherto unrecognized lumenal source of H2O2 by PRDX4 and a parallel slow H2O2-independent pathway for disulfide formation.
- Produção, purificação e estabilidade proteolítica da proteína da priónicaPublication . Lopes, Carlos Rafael dos Reis; Melo, Eduardo Xavier Pinho eAs neuropatologias conhecidas como encefalopatias espongiformes transmissíveis contituem um conjunto de doenças raras, eminentemente fatais, com características patológicas únicas cuja principal expressão é a degenerescência tecidular do sistema nervoso central e periférico. São causadas por um agente proteico denominado prião (PrP). O estudo destes agentes tem sido um factor importantíssimo de evolução científica no domínio da compreensão das proteínas, das interacções proteicas e da engenharia de proteínas. Este trabalho pretendeu aprofundar o conhecimento que existe sobre a PrP nomeadamente sobre a sua estabilidade proteolítica. A proteína possui uma região que é essencial para uma das funções importantíssimas do ponto de vista fisiológico, que se atribui à proteína priónica não infecciosa, que é o transporte de cobre entre o interior neuronal e o espaço pré-sináptico. Esta proteína possui na sua região N-terminal um conjunto de quatro regiões repetidas de oito aminoácidos (octarepeats) a que se atribuem responsabilidades no transporte do cobre neuronal. A mutação ao nível das histidinas destes domínios diminui a interação com o cobre e interfere com a estabilidade molecular da proteína priónica, podendo também desempenhar um papel importante na interacção PrP-PrP. Os restantes objectivos do trabalho prendem-se com a produção de proteína PrP e compreensão dos mecanismos e procedimentos de produção, separação e purificação da proteína priónica.
- Live-cell FRET imaging reveals clustering of the prion protein at the cell surface induced by infectious prionsPublication . Tavares, Evandro; Macedo, J.A.; Paulo, Pedro M. R.; Sousa, Catarina; Lopes, Carlos; Melo, EduardoPrion diseases are associated to the conversion of the prion protein into a misfolded pathological isoform. The mechanism of propagation of protein misfolding by protein templating remains largely unknown. Neuroblastoma cells were transfected with constructs of the prion protein fused to both CFP-GPI-anchored and to YFP-GPI-anchored and directed to its cell membrane location. Live-cell FRET imaging between the prion protein fused to CFP or YFP was measured giving consistent values of 10 +/- 2%. This result was confirmed by fluorescence lifetime imaging microscopy and indicates intermolecular interactions between neighbor prion proteins. In particular, considering that a maximum FRET efficiency of 17 +/- 2% was determined from a positive control consisting of a fusion CFP-YFP-GPI-anchored. A stable cell clone expressing the two fusions containing the prion protein was also selected to minimize cell-to-cell variability. In both, stable and transiently transfected cells, the FRET efficiency consistently increased in the presence of infectious prions - from 4 +/- 1% to 7 +/- 1% in the stable clone and from 10 +/- 2% to 16 +/- 1% in transiently transfected cells. These results clearly reflect an increased clustering of the prion protein on the membrane in the presence of infectious prions, which was not observed in negative control using constructs without the prion protein and upon addition of non-infected brain. Our data corroborates the recent view that the primary site for prion conversion is the cell membrane. Since our fluorescent cell clone is not susceptible to propagate infectivity, we hypothesize that the initial event of prion infectivity might be the clustering of the GPI-anchored prion protein. (C) 2014 Elsevier B.V. All rights reserved.
- TriPer, an optical probe tuned to the endoplasmic reticulum tracks changes in luminal H2O2Publication . Melo, Eduardo; Rafael dos Reis Lopes, Carlos; Gollwitzer, Peter; Lortz, Stephan; Lenzen, Sigurd; Mehmeti, Ilir; Kaminski, Clemens F.; Ron, David; Avezov, EdwardBackground: The fate of hydrogen peroxide (H2O2) in the endoplasmic reticulum (ER) has been inferred indirectly from the activity of ER-localized thiol oxidases and peroxiredoxins, in vitro, and the consequences of their genetic manipulation, in vivo. Over the years hints have suggested that glutathione, puzzlingly abundant in the ER lumen, might have a role in reducing the heavy burden of H2O2 produced by the luminal enzymatic machinery for disulfide bond formation. However, limitations in existing organelle-targeted H2O2 probes have rendered them inert in the thiol-oxidizing ER, precluding experimental follow-up of glutathione's role in ER H2O2 metabolism. Results: Here we report on the development of TriPer, a vital optical probe sensitive to changes in the concentration of H2O2 in the thiol-oxidizing environment of the ER. Consistent with the hypothesized contribution of oxidative protein folding to H2O2 production, ER-localized TriPer detected an increase in the luminal H2O2 signal upon induction of pro-insulin (a disulfide-bonded protein of pancreatic beta-cells), which was attenuated by the ectopic expression of catalase in the ER lumen. Interfering with glutathione production in the cytosol by buthionine sulfoximine (BSO) or enhancing its localized destruction by expression of the glutathione-degrading enzyme ChaC1 in the lumen of the ER further enhanced the luminal H2O2 signal and eroded beta-cell viability. Conclusions: A tri-cysteine system with a single peroxidatic thiol enables H2O2 detection in oxidizing milieux such as that of the ER. Tracking ER H2O2 in live pancreatic beta-cells points to a role for glutathione in H2O2 turnover.
- Stability of protein formulations at subzero temperatures by Isochoric CoolingPublication . Tavares, Evandro; Lopes, Carlos; Silva, Joana G.; Duarte, Andreia; Geraldes, Vitor; Rodrigues, Miguel A.; Melo, Eduardo; Correia, CátiaOptimization of protein formulations at subzero temperatures is required for many applications such as storage, transport, and lyophilization. Using isochoric cooling (constant volume) is possible to reach subzero temperatures without freezing aqueous solutions. This accelerates protein damage as protein may unfold by cold denaturation and diffusional and conformational freedom is still present. The use of isochoric cooling to faster protein formulations was first demonstrated for the biomedical relevant protein disulfide isomerase A1. Three osmolytes, sucrose, glycerol, and l-arginine, significantly increased the stability of protein disulfide isomerase A1 at -20°C with all tested under isochoric cooling within the short time frame of 700 h. The redox green fluorescent protein 2 was used to evaluate the applicability of isochoric cooling for stability analysis of highly stable proteins. This derivative of GFP is 2.6-fold more stable than the highly stable GFP β-barrel structure. Nevertheless, it was possible to denature a fraction of roGFP2 at -20°C and to assign a stabilizing effect to sucrose. Isochoric cooling was further applied to insulin. Protein damage was evaluated through a signaling event elicited on human hepatocyte carcinoma cells. Insulin at -20°C under isochoric cooling lost 22% of its function after 15 days and 0.6M sucrose prevented insulin deactivation.
- A conformational-dependent interdomain redox relay at the core of protein disulfide isomerase activityPublication . Pinho Melo, Eduardo; El-Guendouz, Soukaina; Correia, Cátia; Teodoro Duarte Garcia Morais, Fernando Jorge; Lopes, CarlosProtein disulfide isomerases (PDIs) are a family of molecular chaperones resident in the endoplasmic reticulum (ER) emerging as important factors in disease. In addition to an holdase function, some members catalyse disulfide bond formation and isomerization, a crucial step for native folding and prevention of aggregation of misfolded proteins. PDIs are characterized by a modular arrangement of thioredoxin-like domains, with the canonical, first identified PDIA1, organized as four thioredoxin-like domains forming a horseshoe with two active sites at the extremities. Using two fluorescent redox sensors, roGFP2 and HyPer, as client substrates either unfolded or native, and the in vitro reconstitution of the full pathways of oxidative protein in the ER, we clarified important aspects underlying the catalytic cycle of PDIA1. The N-terminal a active site is the main oxidant of thiols and can transfer electrons to the C-terminal a´ active site relying on the redox-dependent conformational flexibility of PDIA1 that allows the formation of an interdomain disulfide bond. The a´ active site act then as a crossing point to redirect electrons to the ER downstream oxidases or back to client proteins. The two active sites of PDIA1 work cooperatively as an interdomain redox relay that explains PDIA1 oxidative activity to form native disulfides and PDIA1 reductase activity to resolve scrambled disulfides. Moreover, this mechanism reveals a new rational for shutting perpetuity for this down oxidative protein folding under ER redox imbalance or when the levels of unfolded proteins and folding intermediates exceed the folding capacity of the system.