Revisiting the intraperoxisomal pathway of mammalian PEX7

Newly synthesized peroxisomal proteins containing a cleavable type 2 targeting signal (PTS2) are transported to the peroxisome by a cytosolic PEX5-PEX7 complex. There, the trimeric complex becomes inserted into the peroxisomal membrane docking/translocation machinery (DTM), a step that leads to the translocation of the cargo into the organelle matrix. Previous work suggests that PEX5 is retained at the DTM during all the steps occurring at the peroxisome but whether the same applies to PEX7 was unknown. By subjecting different pre-assembled trimeric PEX5-PEX7-PTS2 complexes to in vitro co-import/export assays we found that the export competence of peroxisomal PEX7 is largely determined by the PEX5 molecule that transported it to the peroxisome. This finding suggests that PEX7 is also retained at the DTM during the peroxisomal steps and implies that cargo proteins are released into the organelle matrix by DTM-embedded PEX7. The release step does not depend on PTS2 cleavage. Rather, our data suggest that insertion of the trimeric PEX5-PEX7-PTS2 protein complex into the DTM is probably accompanied by conformational alterations in PEX5 to allow release of the PTS2 protein into the organelle matrix.This work was funded by FEDER funds through the Operational Competitiveness Programme, COMPETE,and by National Funds through FCT, Fundacao para a Ciencia e a Tecnologia, under the project FCOMP-01-0124-FEDER-022718 (Pest-C/SAU/LA0002/2011) and FCOMP-01-0124-FEDER-019731 (PTDC/BIABCM/118577/2010). T.A.R. and C.P.G were supported by Fundacao para a Ciencia e a Tecnologia,Programa Operacional Potencial Humano do QREN and Fundo Social Europeu

Evaluation of the activity and substrate specificity of the human SENP family of SUMO proteases

Protein modification with the small ubiquitin-like modifier (SUMO) is a reversible process regulating many central biological pathways. The reversibility of SUMOylation is ensured by SUMO proteases many of which belong to the sentrin/SUMO-specific protease (SENP) family. In recent years, many advances have been made in allocating SENPs to specific biological pathways. However, due to difficulties in obtaining recombinant full-length active SENPs for thorough enzymatic characterization, our knowledge on these proteases is still limited. In this work, we used in vitro synthesized full-length human SENPs to perform a side-by-side comparison of their activities and substrate specificities. ProSUMO1/2/3, RanGAP1-SUMO1/2/3 and polySUMO2/3 chains were used as substrates in these analyses. We found that SENP1 is by far the most versatile and active SENP whereas SENP3 stands out as the least active of these enzymes. Finally, a comparison between the activities of full-length SENPs and their catalytic domains suggests that in some cases their non-catalytic regions influence their activity.We thank Dr. Frauke Melchior (University of Heidelberg, Germany), Dr. Guy Salvesen (Sanford-Burnham Medical Research Institute, USA), Dr. Hidde Ploegh (Whitehead Institute, USA) and Dr. Joanna Morris (University of Birmingham, UK) for kindly providing plasmids. This work was funded by FEDER (Fundo Europeu de Desenvolvimento Regional) funds through the Operational Competitiveness Programme COMPETE and by National Funds through FCT - Fundação para a Ciência e a Tecnologia under the project FCOMP-01-0124-FEDER-027627 (EXPL/BEX-BCM/0320/2012) and 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. A. V. M. was supported by project FCOMP-01-0124-FEDER-027627-EXPL/BEX-BCM/0320/2012. C. P. G. (SFRH/BPD/64388/2009)and M. P. P. (SFRH/BPD/47447/2008)were supported by FCT, COMPETE, Programa Operacional Potencial Humano (POPH) do QREN, FEDER and Fundo Social Europeu (FSE)

The first minutes in the life of a peroxisomal matrix protein

In the field of intracellular protein sorting, peroxisomes are most famous by their capacity to import oligomeric proteins. The data supporting this remarkable property are abundant and, understandably, have inspired a variety of hypothetical models on how newly synthesized (cytosolic) proteins reach the peroxisome matrix. However, there is also accumulating evidence suggesting that many peroxisomal oligomeric proteins actually arrive at the peroxisome still as monomers. In support of this idea, recent data suggest that PEX5, the shuttling receptor for peroxisomal matrix proteins, is also a chaperone/holdase, binding newly synthesized peroxisomal proteins in the cytosol and blocking their oligomerization. Here we review the data behind these two different perspectives and discuss their mechanistic implications on this protein sorting pathway. This article is part of a Special Issue entitled: Peroxisomes edited by Ralf Erdmann.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 FEDER funds within the partnership agreement PT2020 related with the research unit number 4293; and by the project FCOMP-01-0124-FEDER-019731 (PTDC/BIABCM/118577/2010) funded by national funds through FCT and co-funded by Fundo Europeu de Desenvolvimento Regional (FEDER) through the Operation- alCompetitiveness Programme(COMPETE).A. F.D., T.F., T.A.R. and C. P. G. were supported by FCT, Programa Operacional Potencial Humano (POPH) do Quadro de Referência Estratégico Nacional (QREN), and Fundo Social Europeu (FSE)

The de novo synthesis of ubiquitin: Identification of deubiquitinases acting on ubiquitin precursors

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)

Factors involved in ubiquitination and deubiquitination of PEX5, the peroxisomal shuttling receptor

Peroxisomal matrix proteins are synthesized on cytosolic ribosomes and post-translationally targeted to the organelle by the soluble factor PEX5. Besides a role as a receptor, and probably as a chaperone, PEX5 also holds the key to the matrix of the organelle. Indeed, the available data suggest that PEX5 itself pushes these proteins across the peroxisomal membrane using as driving force the strong protein-protein interactions that it establishes with components of the peroxisomal membrane docking/translocation module (DTM). In recent years, much has been learned on how this transport system is reset and kept fine-tuned. Notably, this involves covalent modification of PEX5 with ubiquitin. Two types of PEX5 ubiquitination have been characterized: monoubiquitination at a conserved cysteine, a mandatory event for the extraction of PEX5 from the DTM; and polyubiquitination, probably the result of a quality control mechanism aiming at clearing the DTM from entangled PEX5 molecules. Monoubiquitination of PEX5 is transient in nature and the factors that reverse this modification have recently been identified.This work was funded by FEDER funds through the Operational Competitiveness Programme — COMPETE and by National Funds through FCT — Fundação para a Ciência e a Tecnologia under the project FCOMP-01-0124-FEDER-019731 (PTDC/BIA-BCM/118577/2010). T. A. R., T. F., M. P. P. and C. P. G. are supported by Fundação para a Ciência e a Tecnologia, Programa Operacional Potencial Humano do QREN, and Fundo Social Europeu. A. F. C. is supported by Programa Ciência, funded by Programa Operacional Potencial Humano do QREN, Tipologia 4.2, Promoção do Emprego Científico, by Fundo Social Europeu and by national funds from Ministério da Ciência, Tecnologia e Ensino Superior

Ubiquitin in the peroxisomal protein import pathway

PEX5 is the shuttling receptor for newly synthesized peroxisomal matrix proteins. Alone, or with the help of an adaptor protein, this receptor binds peroxisomal matrix proteins in the cytosol and transports them to the peroxisomal membrane docking/translocation module (DTM). The interaction between cargo-loaded PEX5 and the DTM ultimately results in its insertion into the DTM with the concomitant translocation of the cargo protein across the organelle membrane. PEX5 is not consumed in this event; rather it is dislocated back into the cytosol so that it can promote additional rounds of protein transportation. Remarkably, the data collected in recent years indicate that dislocation is preceded by monoubiquitination of PEX5 at a conserved cysteine residue. This mandatory modification is not the only type of ubiquitination occurring at the DTM. Indeed, several findings suggest that defective receptors jamming the DTM are polyubiquitinated and targeted to the proteasome for degradation.This work was funded by FEDER funds through the Operational Competitiveness Programme e COMPETE and by National Funds through FCT e Fundação para a Ciência e a Tecnologia under the project FCOMP-01-0124-FEDER-019731 (PTDC/BIA-BCM/118577/2010). T.F., T.A.R., M.P.P. and C.P.G. are supported by Fundação para a Ciência e a Tecnologia, Programa Operacional Potencial Humano do QREN, and Fundo Social Europeu. A.F.C. is supported by Programa Ciência, funded by Programa Operacional Potencial Humano do QREN, Tipologia 4.2, Promoção do Emprego Científico, by Fundo Social Europeu and by National Funds from Ministério da Ciência, Tecnologia e Ensino Superior

A cell-free organelle-based in vitro system for studying the peroxisomal protein import machinery

Here we describe a protocol to dissect the peroxisomal matrix protein import pathway using a cell-free in vitro system. The system relies on a postnuclear supernatant (PNS), which is prepared from rat/mouse liver, to act as a source of peroxisomes and cytosolic components. A typical in vitro assay comprises the following steps: (i) incubation of the PNS with an in vitro-synthesized 35 S-labeled reporter protein; (ii) treatment of the organelle suspension with a protease that degrades reporter proteins that have not associated with peroxisomes; and (iii) SDS-PAGE/autoradiography analysis. To study transport of proteins into peroxisomes, it is possible to use organelle-resident proteins that contain a peroxisomal targeting signal (PTS) as reporters in the assay. In addition, a receptor (PEX5L/S or PEX5L.PEX7) can be used to report the dynamics of shuttling proteins that mediate the import process. Thus, different but complementary perspectives on the mechanism of this pathway can be obtained. We also describe strategies to fortify the system with recombinant proteins to increase import yields and block specific parts of the machinery at a number of steps. The system recapitulates all the steps of the pathway, including mono-ubiquitination of PEX5L/S at the peroxisome membrane and its ATP-dependent export back into the cytosol by PEX1/PEX6. An in vitro import(/export) experiment can be completed in 24 h.We thank M. Fransen, Katholieke Universiteit-Leuven, for critical comments on the manuscript and for the plasmid encoding histidine-tagged PEX19. We thank P. van Veldhoven, Katholieke Universiteit-Leuven, and P. Maciel, Universidade do Minho, for the expression plasmids encoding prePHYH and GST-Ub, respectively. This work was funded by FEDER—Fundo Europeu de Desenvolvimento Regional through the COMPETE 2020—Operacional Programme for Competitiveness and Internationalization (POCI), Portugal 2020, Portugal’s FCT—Fundação para a Ciência e a Tecnologia/ Ministério da Ciência, Tecnologia e Inovação in the framework of the projects ‘The molecular mechanism of protein import into peroxisomes’ (FCOMP-01-0124-FEDER-019731-PTDC/BIA-BCM/118577/2010), ‘Institute for Research and Innovation in Health Sciences’ (POCI-01-0145-FEDER-007274) and ‘The molecular mechanisms of peroxisome biogenesis’ (PTDC /BEX-BC M/2311/2014) and Norte 2020—Programa Operacional Regional do Norte, under the application of the ‘Porto Neurosciences and Neurologic Disease Research Initiative at i3S (NORTE-01-0145-FEDER-000008)’, awarded to J.E.A. T.A.R., T.F., A.F.D. and C.P.G. were supported by Fundação para a Ciência e a Tecnologia, Programa Operacional Potencial Humano do QREN and Fundo Social Europeu

A PEX7-centered perspective on the peroxisomal targeting signal type 2-mediated protein import pathway

Peroxisomal matrix proteins are synthesized on cytosolic ribosomes and transported to the organelle by shuttling receptors. Matrix proteins containing a type 1 signal are carried to the peroxisome by PEX5, whereas those harboring a type 2 signal are transported by a PEX5-PEX7 complex. The pathway followed by PEX5 during the protein transport cycle has been characterized in detail. In contrast, not much is known regarding PEX7. In this work, we show that PEX7 is targeted to the peroxisome in a PEX5- and cargo-dependent manner, where it becomes resistant to exogenously added proteases. Entry of PEX7 and its cargo into the peroxisome occurs upstream of the first cytosolic ATP-dependent step of the PEX5-mediated import pathway, i.e., before monoubiquitination of PEX5. PEX7 passing through the peroxisome becomes partially, if not completely, exposed to the peroxisome matrix milieu, suggesting that cargo release occurs at the trans side of the peroxisomal membrane. Finally, we found that export of peroxisomal PEX7 back into the cytosol requires export of PEX5 but, strikingly, the two export events are not strictly coupled, indicating that the two proteins leave the peroxisome separately.This work was funded by FEDER funds through the Operational Competitiveness Programme, COMPETE, and by National Funds through FCT, Fundação para a Ciência e a Tecnologia, under the project FCOMP-01-0124-FEDER-022718 (PEst-C/SAU/LA0002/2011) and FCOMP-01-0124-FEDER-019731(PTDC/BIABCM/118577/2010).T.A.R.,I.S.A.,T.F., and C.P.G. were supported by Fundação para a Ciência e a Tecnologia, Programa Operacional Potencial Humano do QREN, and Fundo Social Europeu. M.F. was supported by FWO-Vlaanderen (Onderzoeksproject G.0754.09) and KU Leuven (OT/09/045)

Sp1 phosphorylation by ATM downregulates BER and promotes cell elimination in response to persistent DNA damage.

ATM (ataxia-telangiectasia mutated) is a central molecule for DNA quality control. Its activation by DNA damage promotes cell-cycle delay, which facilitates DNA repair prior to replication. On the other hand, persistent DNA damage has been implicated in ATM-dependent cell death via apoptosis; however, the mechanisms underlying this process remain elusive. Here we find that, in response to persistent DNA strand breaks, ATM phosphorylates transcription factor Sp1 and initiates its degradation. We show that Sp1 controls expression of the key base excision repair gene XRCC1, essential for DNA strand break repair. Therefore, degradation of Sp1 leads to a vicious cycle that involves suppression of DNA repair and further aggravation of the load of DNA damage. This activates transcription of pro-apoptotic genes and renders cells susceptible to elimination via both apoptosis and natural killer cells. These findings constitute a previously unrecognized 'gatekeeper' function of ATM as a detector of cells with persistent DNA damage