Suchergebnisse

Protein ubiquitination, a major post-translational modification in eukaryotes, requires an adequate pool of free ubiquitin. Cells maintain this pool by two pathways, both involving deubiquitinases (DUBs): recycling of ubiquitin from ubiquitin conjugates and processing of ubiquitin precursors synthesized de novo. Although many advances have been made in recent years regarding ubiquitin recycling, our knowledge on ubiquitin precursor processing is still limited, and questions such as when are these precursors processed and which DUBs are involved remain largely unanswered. Here we provide data suggesting that two of the four mammalian ubiquitin precursors, UBA 52 and UBA 80 , are processed mostly post-translationally whereas the other two, UBB and UBC, probably undergo a combination of co-and post-translational processing. Using an unbiased biochemical approach we found that UCHL 3 , USP 9 X, USP 7 , USP 5 and Otulin/Gumby/FAM 105 b are by far the most active DUBs acting on these precursors. The identification of these DUBs together with their properties suggests that each ubiquitin precursor can be processed in at least two different manners, explaining the robustness of the ubiquitin de novo synthesis pathway. ; We are grateful to Dr. Cheryl Arrowsmith (University of Toronto, Canada) for providing the plasmids pET28a-LIC-USP5 (Addgene plasmid 25299) and pET28a-LIC-USP5(C335A). We thank Dr. João M. Cabral (IBMC, University of Porto, Portugal) for critically reading the manuscript. This work was supported by national funds through FCT - Fundação para a Ciência e a Tecnologia/MEC – Ministério da Educação e Ciência and when applicable co-funded by Fundo de Desenvolvimento Regional (FEDER) funds within the partnership agreement PT2020 related with the research unit number 4293; by Project "NORTE-07-0124-FEDER-000003 -Cell homeotasis tissue organization and organism biology", co-funded by Programa Operacional Regional do Norte (ON.2—O Novo Norte), under the Quadro de Referência Estratégico Nacional (QREN), through FEDER and by FCT; by Portuguese National Mass Spectrometry Network (RNEM) through the project REDE/1504/REM/2005; and by Química Orgânica, Produtos Naturais e Agroalimentares (QOPNA) research unit funds provided by FCT, European Union, QREN, FEDER and Operational Competitiveness Programme (COMPETE) under the projects PEst-C/QUI/UI0062/2013 and FCOMP-01-0124-FEDER-037296. C.P.G. and M.P.P. were supported by FCT, COMPETE and Fundo Social Europeu. A.V.M. was supported by the project FCOMP-01-0124-FEDER-027627-EXPL/BEX-BCM/0320/2012 financed by national funds from FCT/Ministério da Educação e Ciência (PIDDAC) and co-funded by FEDER through COMPETE—Programa Operacional Factores de Competitividade (POFC).

The occurrence of protein synthesis errors (mistranslation) above the typical mean mistranslation level of 10−4 is mostly deleterious to yeast, zebrafish and mammal cells. Previous yeast studies have shown that mistranslation affects fitness and deregulates genes related to lipid metabolism, but there is no experimental proof that such errors alter yeast lipid profiles. We engineered yeast strains to misincorporate serine at alanine and glycine sites on a global scale and evaluated the putative effects on the lipidome. Lipids from whole cells were extracted and analysed by thin layer chromatography (TLC), liquid chromatography-mass spectrometry(LC-MS) and gas chromatography (GC). Oxidative damage, fatty acid desaturation and membrane fluidity changes were screened to identify putative alterations in lipid profiles in both logarithmic (fermentative) and post-diauxic shift (respiratory) phases. There were alterations in several lipid classes, namely lyso-phosphatidylcholine, phosphatidic acid, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, and triglyceride, and in the fatty acid profiles, namely C16:1, C16:0, C18:1 and C18:0. Overall, the relative content of lipid species with saturated FA increased in detriment of those with unsaturated fatty acids. The expression of the OLE1 mRNA was deregulated, but phospholipid fluidity changes were not observed. These data expand current knowledge of mistranslation biology and highlight its putative roles in human diseases. ; This work was supported by FCT(Fundação para a Ciência e a Tecnologia), the European Union, QREN, POPH, FEDER and COMPETE [FCT-ANR/IMI-MIC/0041/2012; PTDC/IMI-MIC/5350/2014; PTDC/BEX-BCM/2121/2014] through the iBiMED (UID/BIM/04501/2013) and QOPNA research units [PTDC/QUI-BIQ/104968/2008; UID QUI/UI0062/2013, FCOMP-01-0124- FEDER-037296], and RNEM [LISBOA-01-0145-FEDER-402-022125 that concerns the Portuguese Mass Spectrometry Network), REDE/1504/REM/2005]. This work was also financed by the European Regional Development Fund (ERDF), through the Centro 2020 Regional Operational Programme under project CENTRO-01-0145-FEDER-000008: BrainHealth 2020, and through the COMPETE 2020 - Operational Programme for Competitiveness and Internationalisation and Portuguese national funds via FCT - Fundação para a Ciência e a Tecnologia, I.P., under projects POCI-01-0145-FEDER-016390:CANCEL STEM and POCI-01-0145-FEDER-007440. Ana R. D. Araújo [SFRH/BD/78835/2011], Catarina Morais [SFRH/BD/79077/2011], Elisabete Maciel [SFRH/BPD/104165/2014], Helena Oliveira [SFRH/BPD/111736/2015], and Tânia Melo [BPD/UI51/5388/2017] were supported by FCT grants. Clara Pereira was supported by Fundo Social Europeu and Programa Operacional Potencial Humano through FCT investigator grant IF/00889/2015.

13 p.-6 fig.-1 tab. ; Endothelial dysfunction has been widely associated with oxidative stress, glucotoxicity and lipotoxicity and underlies the development of cardiovascular diseases (CVDs), atherosclerosis and diabetes. In such pathological conditions, lipids are emerging as mediators of signalling pathways evoking key cellular responses as expression of proinflammatory genes, proliferation and apoptosis. Hence, the assessment of lipid profiles in endothelial cells (EC) can provide valuable information on the molecular alterations underlying CVDs, atherosclerosis and diabetes. We performed a lipidomic approach based on hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) for the analysis of the phospholipidome of bovine aortic EC (BAEC) exposed to oxidative (H2O2), glycative (glucose), or lipoxidative (4-hydroxynonenal, HNE) stress. The phospholipid (PL) profile was evaluated for the classes PC, PE, PS, PG, PI, SM, LPC and CL. H2O2 induced a more acute adaptation of the PL profile than glucose or HNE. Unsaturated PL molecular species were up-regulated after 24h incubation with H2O2, while an opposite trend was observed in glucose- and HNE-treated cells. This study compared, for the first time, the adaptation of the phospholipidome of BAEC upon different induced biochemical stresses. Although further biological studies will be necessary, our results unveil specific lipid signatures in response to characteristic types of stress. ; This work was supported by funding from European Commission's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement number 675132 (MSCA-ITN-ETN MASSTRPLAN) to University of Aveiro and CSIC. Thanks are due to University of Aveiro, FCT/MEC, European Union, QREN, COMPETE for the financial support to the QOPNA (FCT UID/QUI/00062/2013), through national funds and where applicable co-financed by the FEDER, within the PT2020 Partnership Agreement, to the Portuguese Mass Spectrometry Network (LISBOA-01-0145-FEDER-402-022125). Tânia Melo is grateful for her Post-Doc grant (BPD/UI 51/5388/2017). Work at D.P.S. laboratory is also supported by grants from Spanish MINECO/FEDER, SAF2015-68590-R, and Instituto de Salud Carlos III/FEDER, RETIC Aradyal,RD16/0006/0021. The stay of M.M.L. at University of Aveiro was part of COST Action CM1001. ; Peer reviewed

Peroxisomal matrix proteins are synthesized on cytosolic ribosomes and transported by the shuttling receptor PEX5 to the peroxisomal membrane docking/translocation machinery, where they are translocated into the organelle matrix. Under certain experimental conditions this protein import machinery has the remarkable capacity to accept already oligomerized proteins, a property that has heavily influenced current models on the mechanism of peroxisomal protein import. However, whether or not oligomeric proteins are really the best and most frequent clients of this machinery remain unclear. In this work, we present three lines of evidence suggesting that the peroxisomal import machinery displays a preference for monomeric proteins. First, in agreement with previous findings on catalase, we show that PEX5 binds newly synthesized (monomeric) acyl-CoA oxidase 1 (ACOX1) and urate oxidase (UOX), potently inhibiting their oligomerization. Second, in vitro import experiments suggest that monomeric ACOX1 and UOX are better peroxisomal import substrates than the corresponding oligomeric forms. Finally, we provide data strongly suggesting that although ACOX1 lacking a peroxisomal targeting signal can be imported into peroxisomes when co-expressed with ACOX1 containing its targeting signal, this import pathway is inefficient. ; This work was funded by Fundo Europeu de Desenvolvimento Regional (FEDER) through the Operational Competitiveness Programme (COMPETE); by National Funds through Fundação para a Ciência e a Tecnologia (FCT) under the project FCOMP-01–0124-FEDER-022718 (PEst-C/SAU/LA0002/2011) and FCOMP-01–0124-FEDER-019731 (PTDC/BIABCM/118577/2010); by Portuguese National Mass Spectrometry Network (RNEM) through the project REDE/1504/REM/2005; and by Química Orgânica, Produtos Naturais e Agroalimentares (QOPNA) research unit funds provided by FCT, European Union, QREN, FEDER and COMPETE under the projects PEst-C/QUI/UI0062/2013 and FCOMP-01–0124-FEDER-037296. M.O.F., T.F., T.A.R., M.P.P. and C.P.G. were supported by FCT, Programa Operacional Potencial Humano (POPH) do Quadro de Referência Estratégico Nacional (QREN) and Fundo Social Europeu. The work done in Leuven was supported by grants from the 'Fonds voor Wetenschappelijk Onderzoek-Vlaanderen (Onderzoeksproject G.0754.09)' and the KU Leuven (OT/14/100).